Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity

Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation.
Introduction
The existence of different endocytic mechanisms is becoming of increasing interest, for instance in relation to growth and differentiation, cell adhesion, regulation of the activity of signalling receptors, drug delivery, and entry of pathogens. The most well-studied endocytic mechanism involves clathrin-coated pits and vesicles, and impressive amounts of details are now known about clathrin-dependent endocytosis (Schmid and McMahon 2007; Benmerah and Lamaze 2007; Ungewickell and Hinrichsen 2007). Initially, the idea was that the formation of the clathrin basket was the driving force during membrane internalization, and quantitative studies suggested that the pathway could account for everything endocytosed (Doxsey et al. 1987). With basic ideas such as “biology is simple” and questions like, “how can a vesicle form without clathrin?”, it was difficult and it took time to get acceptance for the existence of alternative endocytic pathways (Montesano et al. 1982; Sandvig et al. 1985, 1987; Moya et al. 1985; Payne et al. 1988; Sandvig and van Deurs 1991, 1994; Damke et al. 1995). Clathrin-dependent endocytosis can be inhibited both by removing clathrin from the plasma membrane by a treatment involving hypotonic shock and depletion of cytosolic potassium (Larkin et al. 1983), and by “freezing” the clathrin coat at the surface by acidification of the cytosol (Sandvig et al. 1987, 1988). The finding that endocytosis of for instance the protein toxin ricin continued under both conditions, suggesting that clathrin-independent endocytosis was responsible for this uptake, was apparently difficult to accept for a number of investigators during a 10 year period (1985–1995). New techniques in molecular biology have been crucial for the elucidation of endocytic processes, and when in 1995 it was published that the dynamin-mutant K44A inhibited clathrin-mediated endocytosis and that the cells still endocytosed (Damke et al. 1995), the complexity of endocytosis started to become apparent to the community in general. Not only did clathrin-independent endocytosis exist, it could also be regulated (Damke et al. 1995). We now know that a number of signalling pathways can regulate clathrin-independent endocytosis (for review, see Sandvig and van Deurs 2005; Marsh and Helenius 2006; Mayor and Pagano 2007). Despite accumulating evidence for several endocytic mechanisms, partly obtained by studies of toxins and viruses (Marsh and Helenius 2006; Sandvig and van Deurs 2002), many text-books in cell biology still operate with only two endocytic mechanisms, uptake from clathrin-coated pits and caveolae. The aim of this brief review is to outline the extreme complexity of clathrin-independent endocytosis as it appears today.
Clathrin-independent endocytosis: expansion of the field
The number of clathrin-independent endocytic pathways revealed has been increasing with time. Mayor and Pagano (Mayor and Pagano 2007) recently subdivided the different mechanisms into four types: RhoA-regulated, cdc42-regulated, and Arf6-dependent mechanisms, and caveolar uptake. Of these, the RhoA-regulated mechanism and caveolar endocytosis also require the GTP-binding protein dynamin (Fig. 1a). However, there are already reports of mechanisms that may not necessarily fit into any of these four categories. For instance, clathrin-independent internalization of proteoglycans seems to depend not only on dynamin, but also on flotillin (Payne et al. 2007), and internalization of Herpes Simplex Virus Protein VP22 is independent of clathrin, but dependent on dynamin and Arf6 (Nishi and Saigo 2007). Also, studies of virus and toxin uptake indicate that there could be even more mechanisms (Marsh and Helenius 2006; Sandvig 2001; Sandvig and van Deurs 2005). Since cells may compensate for loss of one endocytic pathway by upregulating another (Damke et al. 1995), one can of course also question to which extent interfering with the endocytic machinery of physiologically relevant pathways in different ways, might create new pathways or cause upregulation of those normally playing only a minor role. Today, more specific methods such as RNA interference (RNAi) are used to interfere with a given process. However, also RNAi-mediated removal of a given molecule can induce upregulation of related molecules (Utskarpen et al. 2006). Moreover, upon removal of a membrane-associated molecule one may ask: To which extent do we actually know that the endocytic process is blocked? Could the rest of the machinery allow uptake to continue with different kinetics and be interpreted as a “new mechanism”? Similarly, an old objection to the notion that removal of clathrin by potassium-depletion (Larkin et al. 1983; Moya et al. 1985; Madshus et al. 1987; Sandvig et al. 1985) revealed endocytosis normally operating in parallel to the clathrin-dependent pathway, was that the rest of the endocytic machinery originally belonging to the clathrin-dependent machinery was still operating. A similar critical attitude should be applied on today’s interpretations. Importantly, both toxins and viruses can modify endocytic events, in some instances by inducing signalling, even though they may bind to glycolipid receptors (Marsh and Helenius 2006; Lauvrak et al. 2006; Römer et al. 2007).
Fig. 1An overview of a factors involved in different types of endocytosis, and b schematic drawing of polarized MDCK cells where some proteins/enzymes involved in regulation of apical clathrin-independent endocytosis have been listed. It should be noted that apical clathrin-independent endocytosis may consist of more than one mechanism and that polarized MDCK cells have caveolae only at the basolateral surface. For references, see the text
Internalization of membrane components and ligands by endocytosis thus have different requirements when it comes to dependency of dynamin, caveolin, flotillin, and small GTPases, and in addition, cholesterol and other specific lipids play a role (see below). To which extent the different endocytic mechanisms vary depending on cell type, growth conditions and the extent of cell polarization is to a large extent not known, and has to be addressed in future studies. It should be stressed that most of our knowledge about clathrin-independent endocytic mechanisms derives from cells in culture.
The work of combining different results into clearly defined models is far from finished. In 2005 it was published that CtBP3/BARS drives membrane fission in processes where dynamin is not involved (Bonazzi et al. 2005). Interfering with the action of this protein reduces fluid phase uptake, as previously reported for cdc42-dependent uptake (Mayor and Pagano 2007). However, whether cdc42 and CtBP3/BARS affect the same process does not seem to be known. The possibility exists that CtBP3/BARS might indirectly affect endocytosis (Mayor and Pagano 2007). Furthermore, when inhibiting endocytic uptake of a ligand, it can be difficult to measure whether it is movement of the ligand into the membrane invagination giving rise to the endocytic vesicle that is reduced or whether it is the formation of the vesicle that is the step affected. To classify the different mechanisms properly, more information is required (see Fig. 1a, b). It is important to note that polarized cells have a differential regulation of clathrin-independent endocytosis at the two poles, and regulators of apical clathrin-independent endocytosis have been indicated in Fig. 1b (separate chapter below). Interestingly, caveolae are only present on the basolateral side in the polarized epithelial cell line MDCK (Verkade et al. 2000; Vogel et al. 1998). In the following sections we will describe various aspects of the clathrin-independent mechanisms in more detail.
Membrane lipids required for clathrin-independent endocytosis
In addition to a large number of proteins, also membrane lipids are important for clathrin-independent endocytosis. Interfering with sphingolipid synthesis may affect different types of clathrin-independent endocytosis by mechanisms poorly understood (Cheng et al. 2006). Importantly, sphingomyelin seems to be required both for recruitment of RhoA and cdc42 to the membrane and for endocytosis involving these GTP-binding proteins (Cheng et al. 2006). Cdc42-dependent uptake is independent not only of RhoA, but also of Rac, clearly separating it from macropinocytosis which requires Rac activity (for review, see (Hanzal-Bayer and Hancock 2007; Chadda et al. 2007). Interestingly, sphingolipid synthesis, or rather the production of glycosphingolipids was found to be required for transport of caveolin to the plasma membrane and for caveolae formation (Cheng et al. 2006).
A change in the fatty acid composition of membrane lipids can also be expected to change the properties of the membrane and its interaction with cytosolic proteins of importance for endocytosis. It was recently found that incubation of cells with unsaturated fatty acids affect the uptake of the glycolipid-binding Shiga toxin which is internalized both by clathrin-dependent and clathrin-independent endocytosis (Spilsberg et al. 2007). A possible explanation for this finding is that there is a change in the amount and/or size of rafts or their ability to become associated with the endocytic machinery. Lipid rafts play a central role in membrane structure and function (Rajendran and Simons 2005), and it is therefore not surprising that the fatty acid composition may affect endocytosis. Raft-dependent uptake have recently been reviewed (Marsh and Helenius 2006; Lajoie and Nabi 2007; Hanzal-Bayer and Hancock 2007).
One can often see the generalized statement: clathrin-independent endocytosis is cholesterol-dependent (Hanzal-Bayer and Hancock 2007). This is true for processes such as macropinocytosis (Grimmer et al. 2002), uptake from caveolae, Rho-dependent uptake of IL2 (a recent review has been written by Lajoie and Nabi 2007), and cdc42-dependent endocytosis where it was reported that there is a cholesterol-dependent activation of cdc42 which in turn mediates actin polymerization required for endocytosis via this pathway (Chadda et al. 2007). However, a normal concentration of cholesterol does not seem to be required for uptake of all ligands taken in by clathrin-independent endocytosis (Rodal et al. 1999). When enough cholesterol has been extracted from the plasma membrane for the caveolae to disappear and the clathrin-coated pits to flatten, endocytosis of the plant toxin ricin still occurs (Rodal et al. 1999). Furthermore, when Cholera toxin uptake into HeLa cells is reduced by expression of the dynamin-mutant K44A or by induction of antisense to clathrin heavy chain in BHK cells, endocytosis of this toxin can be increased by extraction of cholesterol with methyl-β-cyclodextrin (Sandvig et al. 2004). Similarly, uptake of Shiga toxin is increased by treatment with methyl-β-cyclodextrin after block of clathrin-dependent uptake by induced expression of antisense to clathrin heavy chain in BHK cells (Fig. 2). One possible explanation is that these toxins are retained at the cell surface by being associated with rafts, and that release from these structures by treatment with methyl-β-cyclodextrin actually permits endocytosis of the toxins. Also, uptake of proteoglycan-binding ligands, reported to be clathrin- and caveolin-independent, but dynamin- and flotillin-dependent, were unaffected by filipin and nystatin, drugs known to disrupt lipid rafts (Payne et al. 2007). It should be noted that cholesterol-depletion can inhibit the formation of invaginated clathrin-coated pits by a so far unknown process (Rodal et al. 1999; Subtil et al. 1999), and cholesterol-dependency is therefore not necessarily a tool to easily distinguish between endocytic processes.
Fig. 2Shiga toxin endocytosis is strongly increased upon methyl-β-cyclodextrin-treatment of BHK cells induced for expression of antisense to clathrin heavy chain. BHK cells with inducible expression of antisense to clathrin heavy chain was preincubated with methyl-β-cyclodextrin (10 mM) for 30 min. Then biotin- and TAG-labeled Shiga toxin (25 ng/ml) was added and the cells were incubated for 20 min. Surface-bound and endocytosed Shiga toxin was quantified as previously described for Cholera toxin (Torgersen et al. 2001). The data are presented as internalized toxin as percent of total cell-associated toxin (mean ± SD, n = 4). Endocytosis of transferrin was performed in parallel to verify the inhibition of clathrin-dependent endocytosis upon induction of antisense to clathrin heavy chain. Upon induction, transferrin uptake was reduced by 95%
Role of actin in endocytosis
Recruitment of actin to clathrin-coated pits and subsequent actin-driven formation and internalization of endocytic vesicles and the appearance of actin “comet tails” on such vesicles is well-established and appears to be a highly complex process involving several protein systems (McPherson 2002; Kaksonen et al. 2005, 2006; Smythe and Ayscough 2006). Interestingly, a crucial role of actin in clathrin-independent endocytosis is now becoming evident as well (Kaksonen et al. 2006; Yarar et al. 2007; Lanzetti 2007), and it seems likely that actin will turn out to be involved in all clathrin-independent endocytic mechanisms. However, in some cases actin might function to facilitate the process and not be strictly required. It has for instance been reported that there is a difference between cells growing in monolayers and cells in suspension when it comes to actin requirement for clathrin-dependent endocytosis (Fujimoto et al. 2000). One of the most studied examples of a clathrin-independent endocytic mechanism that is based on actin polymerization, is macropinocytosis (see below), a major fluid-phase uptake pathway.
Macropinocytosis
An increasing complexity of the formation of macropinosomes is becoming evident. Thus, it has long been known that GTP-bound Rac stimulates accumulation of actin filaments at the plasma membrane and that it is involved in plasma membrane ruffling induced by growth factors (Ridley et al. 1992) (Fig. 1a). Rac in turn activates the Arp2/3 protein complex and WAVE, a process involved in ruffling (Gao et al. 2007). In addition to Rac, also Ras, Src and phosphoinositide (PI) 3-kinase promote macropinocytosis (Gao et al. 2007), and a role of N-WASP and the sorting nexin 9 (SNX9) in assembly and organization of actin during ruffle formation and macropinocytosis has recently been shown (Yarar et al. 2007). Also, histone deacetylase 6 (HDAC6) is associated with plasma membrane ruffles and formation of macropinosomes upon stimulation with growth factors, processes that were inhibited in HDAC6-deficient cells. Interestingly, a substrate of HDAC6, the heat shock protein Hsp90, seemed to be involved in ruffling and macropinocytosis as well (Gao et al. 2007). Moreover, a clathrin-independent process reminiscent of macropinocytosis has recently been reported (Orth et al. 2006). Here, actin-dependent circular dorsal ruffles or dorsal membrane waves are formed upon growth factor stimulation, leading to significant receptor internalization. Like in ordinary macropinocytosis, PI 3-kinase is involved, but interestingly, also dynamin, which is a part of the actin-polymerizing Arp2/3-N-WASP-cortactin system and a large number of other proteins are required for this internalization mechanism (Orth et al. 2006; Orth and McNiven 2006; McNiven 2006).
RhoA-, cdc42- and CTBP3/BARS-dependent uptake
The RhoA-dependent uptake of IL-2 receptors, γc-cytokine receptor and IgE receptor is a dynamin-dependent process (Mayor and Pagano 2007) (Fig. 1). Whether RhoA is required for the correct sorting of ligand or whether it is involved in the endocytic process as such is however being discussed (Mayor and Pagano 2007). Data from polarized cells showing that ricin uptake and fluid phase endocytosis at the apical side of polarized MDCK cells are also dependent on RhoA (Garred et al. 2001), suggest that it is the endocytic mechanism itself that is RhoA-dependent.
A cdc42-regulated process, which operates independently of dynamin, has been reported to be involved in uptake of glycosylphosphatidylinositol (GPI)-anchored proteins as well as being responsible for a major fraction of fluid-phase uptake in the cell (Kalia et al. 2006). GPI-anchored proteins were found to be internalized into CHO cells by a cdc42-dependent, but Arf6-independent mechanism. The internalized membrane ended up in vesicles without EEA1, Rab4, or Rab5, but these vesicles were found to fuse with normal endosomal compartments via a Rab5/PI3-kinase dependent machinery. That a GPI-anchored protein and its ligand can enter independently of caveolae and dynamin and reach an acidic compartment, was first shown in studies of a GPI-anchored diphtheria toxin receptor (Skretting et al. 1999). The finding that diphtheria toxin bound to this GPI-linked receptor intoxicates cells, demonstrates that the toxin has been transferred to an acidic compartment facilitating low pH-induced conformational change of diphtheria toxin and translocation of the toxin to the cytosol.
The finding that the cdc42-dependent mechanism is responsible for most fluid uptake (Kalia et al. 2006) is surprising, considering all the other uptake mechanisms now reported, not least macropinocytosis. It was recently shown that cdc42 is associated with the recycling endosome and that also Par proteins play a role in the endocytic process (Balklava et al. 2007). To which extent there is an indirect effect on the uptake from the plasma membrane when mutants of cdc42 are studied, is not quite clear. Also, as mentioned earlier, since CtBP3/BARS is involved in dynamin-independent endocytosis (Bonazzi et al. 2005), and also endocytosis regulated by this protein can account for a considerable amount of fluid phase uptake, it should be elucidated to which extent cdc42 and CtBP3/BARS regulated processes are related.
Arf6-dependent uptake
ADP-ribosylation factor 6 (Arf6) has been identified as a key regulator of a clathrin- and dynamin-independent endocytic pathway mainly studied in HeLa and COS-7 cells, and responsible for uptake and recycling of a collection of integral plasma membrane proteins devoid of conventional endocytic signals (see below). The number of cargo proteins reported to be internalized by Arf6-regulated endocytosis in these cells is increasing and includes the plasma membrane proteins Major Histocompatibility Complex Class I (MHCI) and interleukin-2 receptor α-subunit/Tac (Radhakrishna and Donaldson 1997), β1-integrin (Brown et al. 2001), M2 muscarinic acetylcholine receptor (Delaney et al. 2002) and certain GPI-anchored proteins, such as CD59 (Naslavsky et al. 2004). Internalized cargo can recycle to the plasma membrane via the tubular Arf6-positive recycling compartment (Radhakrishna and Donaldson 1997) or, alternatively, be delivered to Rab5 and EEA1 positive early endosomes for further transport to late endosomes and lysosomes for degradation (Naslavsky et al. 2003). Recently, rafts were reported to be recycled by an Arf6-dependent process (Balasubramanian et al. 2007). Interestingly, the Herpes Simplex Virus protein VP22 was in CHO-K1 and HeLa cells shown to be endocytosed by a mechanism dependent on Arf6, dynamin and lipid rafts, but independent of Rho-GTPases (Nishi and Saigo 2007).
A new class of trafficking motifs in non-clathrin-dependent cargo proteins have been identified and suggested to prevent internalization and/or facilitate recycling of proteins carrying these, via the Arf6-regulated pathway by enhancing their association with the Arf6-specific guanine nucleotide exchange factor EFA6 (Gong et al. 2007). In certain cell types, rather than defining a separate endocytic pathway, Arf6 regulates clathrin-dependent uptake. In Hek293 cells, for example, Arf6 regulates uptake of several G-protein coupled receptors by clathrin-dependent endocytosis (Houndolo et al. 2005), and there is also evidence for complex formation between Arf6, the β-subunit of AP2 and the heavy chain of clathrin (Poupart et al. 2007). The data furthermore suggest that Arf6 regulates the angiotensin AA type 1 receptor by recruiting AP2 and clathrin. Arf6 has also in HeLa cells been proposed to have a direct function in clathrin coat recruitment to the plasma membrane. In fact, Arf6-GTP has recently been demonstrated to interact with AP-2 both in vitro and in vivo, and a clathrin-interacting Arf6-specific GTPase activating protein (called SMAP), affecting clathrin-dependent endocytosis only, has also been identified (Paleotti et al. 2005; Tanabe et al. 2005). Interestingly, in polarized MDCK cells, Arf6 associated with the guanine nucleotide exchange factor ARNO regulates endocytosis exclusively at the apical surface by recruiting actin to clathrin-coated pits (Hyman et al. 2006).
The present picture is complicated as many of the roles ascribed to Arf6 within the endocytic system seem to be not only cell type dependent, but also determined by the exchange factor associated with Arf6 (Kalia et al. 2006). The Arf6 GTP exchange factor ARNO has previously been shown to be dependent on inositol-lipids for its membrane recruitment. However, the process seems to be even more complex as small G proteins of the Arl 4 family can function in recruitment of the Arf6 GEF cytohesin to the plasma membrane (Hofmann et al. 2007). Perhaps this might explain some of the apparent differences between cell types.
Caveolae and endocytosis
Caveolae are characteristic, small (50–100 nm), regular invaginations of the plasma membrane as seen in the EM (Fig. 3). Since caveolae have this distinct, easily recognizable ultrastructural morphology, they have been of particular interest in relation to clathrin-independent endocytosis. The distinct shape of caveolae early led to the assumption that caveolae were endocytic structures comparable to clathrin-coated pits and vesicles. Also, in thin sections for EM which are not cut perpendicular to the plasma membrane, one might get the impression of several free, i.e. not surface-connected, caveolar profiles, supporting the idea of caveolar endocytosis and a front of internalized caveolae moving into the cell (Fig. 3). Interestingly, more careful EM examination—for instance using Ruthenium Red as a surface marker—reveal that most if not all such vesicular profiles are indeed surface-connected caveolae (Fig. 4). Also, the observation that e.g. Cholera toxin localized to caveolae supported the idea that caveolae were responsible for the endocytic uptake of Cholera toxin, a notion that is certainly an oversimplification (Torgersen et al. 2001; Shogomori and Futerman 2001; Nichols et al. 2001; see discussion in Hommelgaard et al. 2005; see also Kirkham et al. 2005).
Fig. 3Appearance of caveolae in myoepithelial cells. In (a) is seen a single caveola (Cav) and for comparison a clathrin-coated pit (Cp). b shows a group of caveolae at the plasma membrane; as the section is largely perpendicular to the membrane, most of the caveolae are clearly seen to be surface-connected. In (c) is shown an example of a section which is not perpendicular to the plasma membrane, and here many of the caveolae appears as free (not surface-connected) vesicles. If such “free” vesicles should be unequivocally established as caveolae, they can be immunogold-labeled using an antibody against caveolin, as shown on the ultracryosection in (d) (Cav). An unlabeled, Cp is also seen. Bars 500 nmFig. 4Caveolae are surface-connected structures. a–d show filter-grown MDCK cells postfixed with the electron-dense cell surface marker Ruthenium Red from the basolateral side. In (a) is seen a number of caveolae connected to the basolateral membrane (arrowheads). Also note that three caveolae (arrows) are connected to the plasma membrane (open arrow) via a larger membrane invagination (asterisk). b–d show clusters of caveolae (arrows) associated with elongated “vacuolar” structures (asterisks) apparently freely localized in the cytoplasm. However, the labelling with Ruthenium Red clearly demonstrates that these structure are indeed connected to the basolateral membrane, as revealed in (a). En, early and later endosomes; Nu, nucleus. Bar 500 nm
However, fluorescence recovery after photobleaching (FRAP) analysis of cells expressing GFP-caveolin revealed that caveolae under normal conditions are not involved in endocytosis to any significant degree (Thomsen et al. 2002). Similarly, more recently live cell imaging of cells expressing GFP-caveolin showed that even high concentrations of EGF, which had been reported to lead to internalization of the EGFR via caveolae (Sigismund et al. 2005), do not mobilize caveolae above a low background level (Kazazic et al. 2006). Also, in a careful EM study where the setup allowed to discriminate between caveolin-associated and other vesicles as well as between surface-connected and free vesicles, Kirkham et al. (2005) showed that only about 2% of caveolae bud from the plasma membrane per minute. For comparison, it takes 50–150 s for a clathrin-coated pit to assemble and pinch off to form a free, clathrin-coated vesicle (Ungewickell and Hinrichsen 2007). This means that the entire population of coated pits present at a given time point will have been internalized within approximately 1–2.5 min. Thus, today it appears well-established and generally accepted that under normal, non-stimulating conditions, caveolae are quite stable structures at the plasma membrane that are not involved in endocytosis to any significant degree (van Deurs et al. 2003; Pelkmans et al. 2004; Hommelgaard et al. 2005; Parton et al. 2006; Kirkham et al. 2005; Lajoie and Nabi 2007).
On the other hand, it is also evident from the above mentioned studies that a few caveolae may pinch off from the plasma membrane (Thomsen et al. 2002; Kirkham et al. 2005). This could simply be due to a basal turnover of caveolae at the plasma membrane. Normally, caveolae seem to be stabilized at, or anchored to, the plasma membrane by actin filaments (Stahlhut and van Deurs 2000; Hommelgaard et al. 2005; Thomsen et al. 2002; Pelkmans et al. 2002). The phosphatase inhibitor okadaic acid stimulates caveolar clustering and internalization in an actin-dependent manner (Parton et al. 1994; Thomsen et al. 2002). One could therefore also imagine that caveolae might be involved in a very slow, regulated endocytic uptake which under certain circumstances can be speeded up, for instance by controlled reorganization of the actin cytoskeleton. However, this remains speculative.
Interestingly, it was recently reported that coassembly of flotillin-1 and -2 induced membrane curvature and formation of membrane invaginations morphologically similar to caveolae but different from these by not containing caveolin, the major caveolar protein. These flotillin-positive but caveolin-negative structures became internalized in contrast to “classical” caveolae (Frick et al. 2007).
Caveolae perform short range “kiss-and-run” motility very close to the plasma membrane. The process most likely involves continuous internalization and recycling of caveolae immediately beneath the plasma membrane (probably in the narrow 100–200 nm zone between the plasma membrane and the stabilizing actin cytoskeleton) (Thomsen et al. 2002, Fig. 5, online movie; Pelkmans and Zerial 2005). The significance of this process is unclear but it may explain the widely accepted role of caveolae in endothelial transcytosis. Thus, since endothelial cells are often very flat (100–200 nm), caveolae that have pinched off from e.g. the luminal endothelial plasma membrane, could fuse immediately after with the ablumenal plasma membrane or, in cases where the endothelial cell is very flat, the fusion may take place even before the pinching has been completed, and temporary transendothelial channels may arise (van Deurs et al. 2003).
When SV40 virus is added to cells, the virus particles are trapped in caveolae which subsequently become internalized by a process which requires tyrosine phosphorylation, reorganization of the cortical actin cytoskeleton, and recruitment of dynamin (Pelkmans et al. 2001, 2002). Characteristically, the caveolar internalization is a single wave phenomenon and it takes hours before caveolin/caveolae reappear at the plasma membrane. More recently, it was shown that SV40 virus is actually internalized much more efficiently by a caveolin-independent mechanism (Damm et al. 2005). In any case it is questionable whether the downregulation of plasma membrane caveolae induced by SV40 virus represents any normally occurring, inducible clathrin-independent endocytic mechanism. Rather, it might reflect clearance of the plasma membrane for caveolae occupied by cross-linked or aggregated receptors or ligands. Such downregulation of caveolae stimulated by receptor cross-linking is discussed in more detail elsewhere (see Hommelgaard et al. 2005).
In this context, it is important to consider the many non-endocytic functions that have been associated with plasma membrane caveolae (see van Deurs et al. 2003). Thus, endothelial nitric oxide synthase (eNOS) is associated with caveolin-1, and caveolae play an important role in NO signaling, and are also involved in e.g. calcium signalling (Murata et al. 2007). Moreover, caveolae is important for cholesterol transport and homeostasis in adipocytes, and it was recently found that cholesterol-induced transport of caveolin to lipid droplets requires dynamin and PKC and very likely involves caveolar endocytosis (Le et al. 2006). Therefore, rather than considering caveolae in general as structures specialized for clathrin-independent endocytosis, they should be considered multifunctional membrane domains with physiological roles depending on cell type and situation (van Deurs et al. 2003). In addition, a recent report suggests that caveolins may even regulate cell function independently of caveolae (Head and Insel 2007).
Regulation of endocytosis in polarized epithelial cells
Studies of endocytic mechanisms in cell lines do not necessarily reflect the uptake mechanisms operating in vivo. Both cell density, cell adhesion and polarization can be regulators of endocytosis (del Pozo and Schwartz 2007; Sandvig 1978; Sandvig and van Deurs 2005; Eker et al. 1994; Holm et al. 1995; Llorente et al. 2000; Sandvig et al. 2000; Llorente et al. 1996). An early argument against clathrin-independent endocytosis was, not unexpectedly, that this could be a process occurring only in unpolarized cell lines grown on plastic support. However, investigations of polarized cells, such as MDCK I cells grown on filters, revealed that clathrin-independent endocytosis contributed to the endocytic uptake both on the apical and basolateral side (Eker et al. 1994). When using cytosolic acidification to block clathrin-dependent endocytosis (Sandvig et al. 1987), there was still uptake of the plant toxin ricin at both poles (Eker et al. 1994). The fact that there was a larger reduction of uptake at the apical side than at the basolateral side, does, however, not necessarily provide information about the fraction of clathrin-independent endocytosis at the two poles, since the low pH might affect the remaining uptake at the two poles differently. Importantly, apical clathrin-independent endocytosis turns out to be under complex regulation, and can be regulated independently of uptake at the basolateral side. Using either cells permeabilized at the basolaterial side or membrane permeable compounds (for review, see Sandvig and van Deurs 2005), we have shown that apical clathrin-independent endocytosis is regulated by protein kinase A (Eker et al. 1994), protein kinase C (Holm et al. 1995), phospholipase D (Sandvig et al. 2000), cyclooxygenase (Llorente et al. 2000) and calmodulin (Llorente et al. 1996). Interestingly, a Rho family member, apparently RhoA, seems to regulate apical endocytosis in a manner which can be supported by GTPγS (Garred et al. 2001). The RhoA-mediated mechanism is able to mediate uptake of both gold particles, ricin-HRP and HRP.

endocytosis
caveolae
ricin
flotillin
shiga toxin
cholera toxin
rho proteins


Mouse N-acetyltransferase type 2, the homologue of human N-acetyltransferase type 1

There is increasing evidence that human arylamine N-acetyltransferase type 1 (NAT1, EC 2.3.1.5), although first identified as a homologue of a drug-metabolising enzyme, appears to be a marker in human oestrogen receptor positive breast cancer. Mouse Nat2 is the mouse equivalent of human NAT1. The development of mouse models of breast cancer is important, and it is essential to explore the biological role of mouse Nat2. We have therefore produced mouse Nat2 as a recombinant protein and have investigated its substrate specificity profile in comparison with human NAT1. In addition, we have tested the effects of inhibitors on mouse Nat2, including compounds which are endogenous and exogenous steroids. We show that tamoxifen, genistein and diethylstilbestrol inhibit mouse Nat2. The steroid analogue, bisphenol A, also inhibits mouse Nat2 enzymic activity and is shown by NMR spectroscopy, through shifts in proton peaks, to bind close to the active site. A three-dimensional structure for human NAT1 has recently been released, and we have used this crystal structure to generate a model of the mouse Nat2 structure. We propose that a conformational change in the structure is required in order for ligands to bind to the active site of the protein.
1
Introduction
Arylamine N-acetyltransferases (NATs, EC 2.3.1.5) have traditionally been identified as drug-metabolising enzymes responsible for the metabolism of arylamines, arylhydroxylamines and arylhydrazines. NATs were first identified in man and a range of eukaryotes and have had an important part to play in the early years of identification of pharmacogenetic variation in response to drug treatment [1].
The hydrazine drug isoniazid is polymorphically N-acetylated in humans to its therapeutically inactive form by the human isoenzyme now known as human NAT2. There are two human NAT isoenzymes: human NAT2, which metabolises isoniazid, and human NAT1, which does not metabolise isoniazid or other arylhydrazines [2], but does catalyse the acetylation of a distinct but overlapping series of arylamines [2]. The pattern of expression of the two human NAT genes also differs. As a result of genome-wide microarray [3–12] and proteomic studies [13], it is clear that human NAT1 is highly expressed in oestrogen receptor positive breast cancer [14]. There is also evidence that the level of expression of human NAT1 affects the growth of cultured breast cancer cells [13].
The role of the human NAT1 enzyme in breast cancer has not been extensively explored, although it has previously been demonstrated that the anti-oestrogen compound tamoxifen is an inhibitor of human NAT1 [15–17]. It has also been demonstrated that both human NAT1 and the oestrogen receptor are down-regulated in tissues in which p53 is mutated [5]. It appears that human NAT1 is a marker for oestrogen receptor positive breast cancer, although information is still accumulating on the relationship of this marker to others, including the oestrogen receptor itself [14]. However, if the human NAT1 enzyme is to be considered as a target for breast cancer therapy, it is essential that there is a suitable in vivo animal model for testing [18,19]. Mice represent the most convenient animal models of disease. At present, the status of mouse Nat2, the equivalent of human NAT1, in a model of breast cancer has not been explored. We have previously demonstrated that mouse Nat2 is expressed in the epithelial cells lining the mammary ducts [20]. This is the same location as the human NAT1 enzyme in normal breast tissue [13,21].
In order to provide a firm foundation for establishing such a model, we have generated pure recombinant mouse Nat2 and investigated its activity with a wide range of substrates and the effects of a range of potential inhibitors, including endogenous and exogenous steroids.
2
Materials and methods
2.1
Chemicals
All chemicals were purchased from Sigma–Aldrich and all molecular biology reagents were purchased from Promega, unless otherwise stated.
2.2
Cloning and expression studies of mouse Nat2
The mouse Nat2 open-reading frame [22] was sub-cloned into pET28b(+) (Novagen) using the compatible restriction enzyme sites, NdeI and EcoRI. This allowed the production of recombinant mouse NAT protein with an N-terminal His-tag, for ease of downstream purification. The pET28b(+) plasmid containing mouse Nat2 was transformed into Escherichia coli strain JM109 and further transformed into Rosetta(DE3)pLysS (Novagen) after confirming the correct insert sequence (DNA Sequencing Facility, Biochemistry, University of Oxford). The positive transformant was grown in LB to mid-log phase at 37 °C with shaking (180 rpm) and stored at −80 °C with 10% (v/v) glycerol. Thawed glycerol stock (100 μL) was used to inoculate fresh LB media (100 mL) supplemented with kanamycin (30 μg/mL) and chloramphenicol (34 μg/mL) and the culture was incubated at 37 °C for 16 h with shaking (180 rpm). The starter culture was then diluted 50-fold into fresh LB media (2 L) supplemented with kanamycin (30 μg/mL), which was then incubated at 27 °C with shaking (180 rpm). When the absorbance at 600 nm reached 0.7, the expression of mouse Nat2 was induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The culture was grown for a further 16 h and the cells were harvested by centrifugation (6000 × g, 4 °C, 20 min). The cell pellet was resuspended in 20 mL lysis buffer (300 mM NaCl, 20 mM Tris–HCl (pH 8.0) containing 1× EDTA-free Complete Protease Inhibitor (Roche)) and then stored at −80 °C.
For expression of 15N-labelled mouse Nat2, the starter culture was diluted 50-fold in 15N-labelled Standard-E. coli-OD2 N medium (Silantes) supplemented with kanamycin (30 μg/mL) and grown at 37 °C until the absorbance at 600 nm reached approximately 0.6. The cells were further grown at 27 °C until the absorbance reached 0.7, when the expression was induced with 0.1 mM IPTG. After induction for 17 h, the cells were harvested and stored as described for unlabelled mouse Nat2.
The resuspended frozen cells were thawed at 37 °C and sonicated on ice (25 cycles of 30 s on, 45 s off at 10 μm). The soluble lysate was isolated from the cell debris by centrifugation (12,000 × g, 4 °C, 20 min) and incubated (4 °C, 5 min) with 5 mL of Ni-NTA resin (Qiagen). The resin was sequentially washed with lysis buffer containing increasing imidazole concentrations in a step gradient at 4 °C (2 washes each of 10 mL of 0 mM, 10 mM, 20 mM, 50 mM and 100 mM imidazole). Fractions containing hexa-histidine tagged Nat2 (His-Nat2), were pooled and thrombin (5 U/mg NAT) was added to remove the His-tag. After 16 h incubation at 4 °C, the protein sample was dialysed against 20 mM Tris–HCl (pH 8.0), 1 mM dithiothreitol (DTT), 1 mM EDTA buffer, and concentrated to 1 mg/mL by centrifugation in a 15 mL concentrator (Amicon) after addition of 5% (v/v) glycerol. Protein samples in 100 μL aliquots were frozen in liquid nitrogen and stored at −80 °C.
Recombinant hamster Nat2 was expressed and purified as previously described [23]. 15N-labelled hamster Nat2 was grown and expressed as described for 15N-labelled mouse Nat2 and purified by the same method as for the unlabelled hamster Nat2.
The expression levels and activities of soluble Nat2 were monitored by visualizing the levels of recombinant protein expression on 12% acrylamide Tris–Glycine SDS-PAGE as well as by measuring the rate of acetylation of 4-aminobenzoic acid (PABA) as a substrate (see method below) [24].
2.3
Enzymic assays
2.3.1
Acetylation of arylamines
The rates of arylamine acetylation by NATs were determined colorimetrically as previously described [25], but with minor modifications. Each assay contained the enzyme, arylamine substrate (see each figure legends for concentrations) and AcCoA (400 μM) in a total volume of 100 μL in assay buffer (20 mM Tris–HCl (pH 8.0), 1 mM DTT). Initially, the enzyme and substrate mixtures were pre-incubated at 25 °C for 5 min and AcCoA was added to start the reaction. The reaction was quenched using 20% (w/v) trichloroacetic acid (TCA) at different time intervals. The stopped reaction was centrifuged (16,000 × g rpm, 10 min) to pellet the precipitated proteins. The stopped reaction mixture (200 μL) was added to 800 μL of 5% (w/v) 4-(N,N-dimethylamino)benzaldehyde (DMAB) in 9:1 acetonitrile:water to develop the colour. The absorption at 450 nm was measured with a Hitachi U-2001 UV–vis spectrophotometer, with the amount of residual substrate in the reaction was determined by comparison with a standard curve.
2.3.2
Hydrolysis of AcCoA (free CoA production)
The rate of production of the free thiol Coenzyme A by NAT, in the presence of a range of known NAT acetyl-acceptor substrates, was determined by using Ellman's reagent, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) as previously described [2,26]. The substrate (500 μM) and purified recombinant NAT were pre-incubated (25 °C, 5 min) in 96-well flat-bottomed polystyrene plates (Costar®, Corning Inc.) in 20 mM Tris–HCl (pH 8.0). AcCoA (400 μM) was added to start the reaction in a final volume of 100 μL. The reaction was quenched with the addition of 25 μL of guanidine–HCl solution (6.4 M guanidine–HCl, 0.1 M Tris–HCl (pH 7.3)) containing 5 mM DTNB. The absorbance at 405 nm was measured on a plate-reader (Sunrise, Tecan). Assay buffer was used to replace substrate, AcCoA or NAT for control reactions. The amount of CoA produced in the assay was determined in comparison with a CoA standard curve.
Substrate abbreviations used for the substrate selectivity assays are as follows: aniline (ANL), 4-aminobenzoic acid (PABA), 4-aminosalicylic acid (4AS), 5-aminosalicylic acid (5AS), 4-chloroaniline (CLA), 4-bromoaniline (BRA), 4-iodoaniline (IOA), 4-methoxyanline (ANS), 4-ethoxyaniline (EOA), 4-butoxyanline (BOA), 4-hexyloxyaniline (HOA), 4-phenoxyaniline (POA), 4-aminoveratrole (4AV), 2-aminofluorene (2AF), 4-aminobenzoyl-l-glutamate (pABGlu), sulfamethazine (SMZ), procainamide (PRO), 4-aminopyridine (APY), isoniazid (INH), hydralazine (HDZ) and phenylhydrazine (PHZ). The stock concentration of substrate was 100 mM dissolved in DMSO, such that the final concentration of DMSO was less than 5% in the assays. DMSO only controls were carried out.
2.4
NMR spectroscopy
The 2D 1H-15N HSQC spectrum of mouse Nat2 was collected using a 750 MHz NMR spectrometer (Department of Biochemistry, University of Oxford). The spectrum was collected at 20 °C for a sample containing 30 mg/mL of uniformly 15N-labelled mouse Nat2 in 95% H2O/5% D2O 10 mM Tris buffer at pH 7.0. Spectral widths of 12,500 and 2500 Hz were used in F2 and F1, respectively. The dataset contained 1024 and 128 complex points in t2 and t1, respectively.
1D 1H NMR spectra for hamster and mouse Nat2 were collected at 20 °C using a jump-return sequence. This sequence enabled the observation of HN peaks which exchange to a significant extent with H2O. Spectra were collected using a 600 MHz spectrometer using a sweep width of 12,500 Hz. Unlabelled hamster and mouse Nat2 were used at a concentration of 10 mg/mL in the buffer described above. The titration of mouse Nat2 with bisphenol A was carried out by stepwise addition of small volumes (5–20 μL) of a concentrated solution of bisphenol A (100 mM in deuterated DMSO) to the mouse Nat2 solution in the NMR tube. The highest concentration of DMSO in the sample used was 2.8%. DMSO alone had no effect on the NMR spectrum of mouse Nat2 using concentrations up to 5%.
3
Results
3.1
Cloning and expression of mouse Nat2
The mouse Nat2 was cloned into E. coli expression vector pET28b(+) and expressed in E. coli Rosetta (DE3)pLysS with an N-terminal hexa-histidine tag. The recombinant mouse His-Nat2 was purified using immobilised metal affinity chromatography, using a Ni-NTA column (Novagen), and eluted (Fig. 1a). Pure Nat2 predominantly eluted in the 50 mM and 100 mM imidazole washes. The hexa-histidine tag was readily removed by thrombin digestion (Fig. 1b). The purified recombinant Nat2 was active, readily catalysing the acetylation of PABA, a well-studied arylamine substrate of mouse Nat2 [27] (Table 1). The enzyme was relatively stable, and the enzymic activity was maintained (>85%) after incubation at temperatures ranging between 4 °C and 25 °C over a 72 h period.
3.2
Substrate specificity of mouse Nat2
To further characterise the purified recombinant mouse Nat2, a panel of known NAT substrates (20 compounds) was tested to determine the substrate selectivity of recombinant mouse Nat2. In order to allow direct comparison with substrate selectivity profile for human NAT1 and NAT2, the assay for mouse Nat2 was performed under the same conditions as previously described [2].
Mouse Nat2 shows high specific activities with a broad range of arylamines, including PABA, 5AS, 4AS and 2AF (Fig. 2 and Supplementary Table 1). In contrast, mouse Nat2 shows low specific activities against arylhydrazine substrates such as HDZ, INH, and PHZ, and against arylamine drugs procainamide and sulfamethazine (Fig. 2). This overall trend is in agreement with the substrate selectivity observed in previous studies with impure recombinant mouse Nat2 using a subset of these compounds [22,27,28], although it is much less than the activity observed towards these substrates with human NAT2 (Fig. 2). There was low, yet measurable activity using arylhydrazine substrates with purified mouse Nat2, which has not been observed in previous studies using recombinant mouse Nat2 expressed in a transient expression system [28]. Pure recombinant mouse Nat2 also readily acetylates 2-aminofluorene, the substrate which has been used to identify mouse strains carrying an active form of mouse Nat2—the fast acetylating C57Bl/6 strain, rather than the inactive form as is found in the slow acetylating A/J strain which is unstable and likely to be subject to rapid degradation [29].
The specific activity pattern of human NAT1 [2] overlaps with the mouse Nat2 profile (Fig. 2), reconfirming that mouse Nat2 is very similar to human NAT1 in substrate specificity. Mouse Nat2 shows differences from the activity profile of human NAT1, however. It appears that mouse Nat2 generally has higher inherent specific activity than human NAT1 and also acetylates arylhydrazines, when the acetylation of these compounds by human NAT1 is not detected under the same conditions [2].
3.3
Mouse Nat2 inhibition by steroidogenic compounds
It had been demonstrated, with extracts of tissues and cells now known to express the human NAT1 gene, that tamoxifen is an inhibitor of human NAT1 [15–17]. In view of the relationship between the expression of human NAT1 and oestrogen receptor positivity in breast cancer [14], we wished to investigate the effects of steroidogenic compounds and xenobiotic oestrogenic compounds, including tamoxifen, on the activity of mouse Nat2.
From the inhibition study using a broad range of steroidogenic compounds, it has been demonstrated that mouse Nat2 is inhibited selectively by oestrogenic compounds, with 17-hydroxy-β-estradiol identified as the most potent inhibitor (Table 2 and Supplementary Fig. 1). This was substantiated by the inhibition of mouse Nat2 activity by xenobiotic oestrogens, such as tamoxifen, and the IC50 values for these inhibitors are shown in Table 3. The phytoestrogens genistein, alpha-zearalenol and the synthetic oestrogen diethylstilbestrol show similar potency of mouse Nat2 inhibition to 17-hydroxy-β-estradiol, tamoxifen and 4-hydroxy tamoxifen. Bisphenol A was also shown to be an inhibitor of mouse Nat2, albeit with lower potency than some of the other compounds tested. One key practical advantage of using bisphenol A as an inhibitor is the improved solubility of this compound relative to the other phytoestrogens and steroids.
3.4
Structure of mouse Nat2
Until recently, there has been no structural information on eukaryotic NAT enzymes. However, recently the three-dimensional structure of a human NAT1 mutant (Phe/Ser substitution at position 125) was determined by X-ray crystallography (PDB accession code: 2IJA [30]). A non-mutated human NAT1 crystal structure has also been deposited in which the active-site cysteine residue was modified to S-(2-anilino-2-oxoethyl)-cysteine (PDB accession code: 2PQT). It is considered likely that both the mutation of residue 125 and the modification of the active-site cysteine residue stabilise the structure of the human enzyme, because thousands of attempts to generate a crystal from the native human NAT1 protein have been unsuccessful (A. Kawamura, unpublished results). The structure of human NAT1 is illustrated in comparison with the NAT from M. smegmatis (Fig. 3). The eukaryotic proteins contain a loop between the second and third domains. This inter-domain loop is not present in their prokaryotic counterparts [31], as highlighted in Fig. 3. It is interesting to note that in the structure of the human NAT1 enzyme, this inter-domain loop is folded back over the active site. Likewise, the C-terminus is shown to be folded over the active-site cleft. These observations indicate that a conformational change in the protein may be required to allow the substrates to access the active-site cysteine residue. However, a structure of human NAT2 with coenzyme A bound has also been deposited (PDB accession code: 2PFR), and the position of the coenzyme A is such that the C-terminus and inter-domain loops regions are not significantly changed. This is in contrast to the recently reported structure of Mycobacterium marinum NAT in complex with coenzyme A [32]. In this bacterial NAT, the C-terminus is shorter than in the eukaryotic structures, and there is no inter-domain loop, and the ligand is found in the space where these regions are located in the eukaryotic structure.
We have generated a structural model of the mouse Nat2 protein, whose amino acid sequence is over 80% identical to human NAT1, by using the program Modeller 8v2 [33]. Fig. 4 shows the structural model of mouse Nat2 and a comparison of the mouse Nat2 and human NAT1 protein structures. The two structures share 1191 equivalent atoms, over which the root mean squared deviation is 0.75 Å. In mouse Nat2, as in human NAT1, the inter-domain loop and the C-terminus occlude the active site. Despite the evidence for coenzyme A binding to human NAT2 without major conformational rearrangement of the C-terminus and inter-domain loop regions, it has not been possible to perform ligand docking into the mouse Nat2 or human NAT1 structures without disturbing these regions of the protein structure. Therefore, it remains possible that the inter-domain loop regions and C-termini of the eukaryotic NAT proteins are conformationally flexible, in order to accommodate substrate or inhibitor binding.
3.5
NMR studies
NMR studies are ideal for investigating protein interactions with substrates and inhibitors in solution. Therefore, we have generated mouse Nat2 uniformly labelled with 15N to allow 2D NMR investigations of the protein. The 2D 1H-15N HSQC spectrum of mouse Nat2 is shown in Fig. 5; this spectrum contains a peak for each backbone amide (1HN-15N) and additional peaks from the side chains of Asn, Gln and Trp. The HSQC spectrum is well resolved, as expected for a compact globular structure, and some cross peaks are relatively sharp for a 30 kDa protein, this may indicate some flexibility between the domains of mouse Nat2 or the presence of mobile loops. The HSQC spectrum of mouse Nat2 has been compared with the corresponding spectrum from hamster Nat2 for which a full assignment is available [34]. The pattern of peaks in the HSQC spectra is extremely similar for the two homologous proteins; this allows some peaks observed in the mouse Nat2 spectrum to be assigned on the basis of the hamster Nat2 spectrum.
The downfield region of the 1H 1D spectra, collected with a jump-return sequence, of mouse and hamster Nat2 also show a similar pattern of peaks (Fig. 6A and B). The four peaks are compared in Table 4, together with their assignments. Two of the peaks (2 and 3) have been assigned previously for hamster Nat2 and can be assigned in the mouse spectrum by homology; these correspond to strongly hydrogen bonded tryptophan indole groups: the tryptophan associated with the P-loop (Trp132, see Fig. 7) and Trp67, which is located adjacent to the active-site Cys68, and appears to have a structural role. The strong hydrogen bonds are likely to be responsible for the large downfield shifts of these tryptophan peaks compared to random coil values (∼9.5 ppm). The two remaining peaks (1 and 4) are assigned to the imidazole 1HN of histidine on the basis of 15N decoupling experiments; these peaks have not been assigned previously for hamster Nat2. The side chain 1HN groups of histidine are usually not observed by NMR, particularly at pH 7, due to their rapid exchange with solvent protons. The observation of these peaks indicates that the histidine residues are probably buried within the protein and that the side chain 1HN are involved in hydrogen bonds. Analysis of the homology model of mouse Nat2 suggests that at least one of these peaks might arise from the active site histidine, His107. The homology model of mouse Nat 2 indicates that the side chain of His107 is involved in two hydrogen bonds in the active site and is the least accessible to solvent. In addition, recent enzymological studies with hamster Nat2 have confirmed that the active-site cysteine and histidine residues exist as a thiolate-imidazolium ion pair [35], in which the histidine has two tightly bound and H-bonded protons.
3.6
Interaction of mouse Nat2 with exogenous steroid inhibitors
In order to probe the molecular interactions of mouse Nat2 and the exogenous steroid inhibitors, we have investigated the effect of bisphenol A on the 1D 1H NMR spectrum of mouse Nat2 (Figs. 8 and 9). The observation of changes in chemical shift for particular amino acid residues which result from the addition of a ligand are usually interpreted as an indication that these residues are located in close proximity to the ligand-binding site. When bisphenol A was titrated into a solution of mouse Nat2, the two downfield histidine peaks (1 and 4, Table 4) were observed to shift by more than 0.05 ppm (Fig. 9); peak 1 shifts downfield by ∼0.12 ppm and peak 4 shifts upfield by ∼0.08 ppm in the presence of 8 equivalents of bisphenol A. Additional small shifts were also observed for the side chain HN of Trp67 and Trp132 (Fig. 9). The observation of progressive changes in chemical shift upon addition of bisphenol A indicates fast exchange on the NMR timescale; this would be expected given the relatively weak affinity of mouse Nat2 for this ligand (IC50 = 290 ± 10 μM). We have postulated above that at least one of the histidine peaks arises from the active-site histidine, His107; therefore, these results show that binding of bisphenol A is likely to take place in close proximity to the active site of mouse Nat2.
4
Discussion
The possibility that human NAT1 may serve as a marker for sub-dividing populations of different breast cancers is intriguing. It may also be that human NAT1 will find a role as a target for breast cancer therapies. In order to exploit these findings fully, it will be essential to have an animal model. There have been, for a long time, suggestions that human NAT1 has an endogenous role. The mouse equivalent of human NAT1 is mouse Nat2. Genetic deletion of mouse Nat2 has not created an overt phenotype; however, some of these studies in mice indicate that Nat2 deletion gives rise to skewed sex ratios both on mixed [36] and pure [37] genetic backgrounds, indicating a gender-dependent phenotype, discernable at the population level, that is consistent with a role in endocrine function. There is now also clear evidence that mouse Nat2 has a role in vivo in folate catabolism [38]. These studies, together with the data on human NAT1 and breast cancer, are intriguing and are likely to provide clues as to the endogenous role of NAT1. The data presented increases the knowledge of mouse Nat2 in comparison with human NAT1 and reinforce the notion that mouse Nat2 is a good homologue of human NAT1. Mouse Nat2 has a similar substrate specific activity profile to human NAT1.
The inhibition studies show that mouse Nat2 is inhibited by endogenous steroids and, like human NAT1, is inhibited by tamoxifen. Bisphenol A, which is used for making polycarbonates [39], has been reported to activate the oestrogen receptor [40]. Like tamoxifen, it is also an inhibitor of mouse Nat2. We have used bisphenol A to look at the binding of an oestrogenic stimulatory compound to mouse Nat2 and show by NMR that bisphenol A interacts with mouse Nat2 close to the active site region. These studies could not be done directly with tamoxifen because of the high concentrations of protein (and hence ligand) required for NMR experiments under which conditions tamoxifen is insoluble. Both endogenous and xenobiotic oestrogens have been shown to modify breast cancer risk, and tamoxifen is widely used for breast cancer therapy (reviewed in [41]). The effect of different synthetic or natural oestrogens on cell proliferation has been analysed directly, in breast cancer cell lines, and in gene expression studies [41–44]. Although the dose–response curves for different oestrogenic compounds vary with the assay system, bisphenol A is generally less active than tamoxifen, genistein or diethylstilbestrol, which reflects the order of potency of mouse Nat2 inhibition. Circulating plasma concentrations of the phytoestogen genistein have been found to be as high as 18 μM after a soy-based meal [45], and intratumoral levels of tamoxifen can reach micromolar concentrations [46]. Whether the effects on inhibition of Nat2 activity in vitro may be physiologically relevant for breast cancer prevention or therapy will require further analysis.
These studies pave the way for investigation of the role of mouse Nat2, the human NAT1 homologue, in studies of oestrogen receptor positive breast cancer. This is particularly relevant in view of the observation that mouse Nat2 is located histologically in the mammary gland epithelial cells [20]. The structural studies also provide a rational basis for identification of mouse Nat2 inhibitors. We have recently screened a 5000-strong compound library using human NAT1, human NAT2, mouse Nat1 and mouse Nat2; from these studies, several compounds been have identified which are potent and specific inhibitors of human NAT1 and its mouse homologue, Nat2. An accumulation of information on the eukaryotic NAT proteins at a structural level will allow further development of chemical tools for investigation of the role of this marker in breast cancer in humans, and its potential in animal models.

selective estrogen receptor modulator/serm


Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB

Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions.
Introduction
The vascular endothelial growth factor (VEGF) family includes seven members which are structurally homologous, but display molecular and functional diversity [58, 81]. VEGFA, the most well known member of the VEGF family, is a crucial regulator of angiogenesis and vascular permeability in both physiological and pathological conditions such as tumor growth, chronic inflammation, and ischemia [10, 16, 19]. In addition to the unquestioned role in angiogenesis, it has recently been shown that VEGFA has direct trophic effects on neuronal and glial cells in the central nervous system [10, 22, 25, 53].
VEGFB is most closely related to VEGFA [46, 81]; however, the biological function of VEGFB is less well characterized than the function of VEGFA. VEGFB is expressed early during development and appears to have prominent expression in the central nervous system [1, 36]. Additionally, VEGFB has been shown to function as an angiogenic and neuroprotective protein [29, 63, 68] and recent evidence suggests a role for VEGFB in neurogenesis [69, 70].
The diverse functions of VEGF proteins can be explained by their differential binding to signaling VEGF receptors [VEGFRs; VEGFR-1 (Flt-1), VEGFR-2 (Flk1/KDR), and VEGFR-3 (Flt-4) [50, 81]]. VEGFA binds to VEGFR-1 and VEGFR-2, whereas VEGFB binds specifically to VEGFR-1 and not to VEGFR-2 [81].
The VEGF-signaling pathway, involving both neuronal and glial cells, has been implicated in several neurological disorders, including neurodegeneration, stroke, and cerebral and spinal trauma [10]. In addition, expression of VEGFA is upregulated in neuronal and glial cells after epileptic seizures in rats [13], suggesting a role for VEGFA in seizure disorders. A recent study points to a neuroprotective role for VEGFA following status epilepticus [45]. The relevance of these findings in animal models to human epileptic disorders is uncertain. Using serial analysis of gene expression (SAGE), we recently identified the VEGFB gene to be upregulated in human tissue from a patient with focal cortical dysplasia (FCD) and intractable epilepsy compared to control cortex (Boer et al., unpublished observations). Upregulation of VEGFA and its receptor has also been recently shown in the hippocampus of cases of human temporal lobe epilepsy (TLE) [56]. However, the distribution of VEGFA, VEGFB, and VEGFRs in epilepsy-associated human malformations of cortical development has not yet been defined.
In the present study, we investigated the expression of both VEGFA and VEGFB and their receptors (VEGFR-1 and VEGFR-2) in patients with FCD, which is a developmental disorder known to be a major cause of intractable epilepsy [73]. We report the specific cellular distribution, including both the neuronal and the glial components of the dysplastic cortex, and we discuss the potential role of VEGFA, VEGFB, and their receptors in the histogenesis and epileptogenesis of this developmental lesion.
Materials and methods
Subjects
The cases included in this study were obtained from the databases of the Departments of Neuropathology of the Academic Medical Center (University of Amsterdam; UVA) in Amsterdam and the University Medical Center in Utrecht (UMCU). We examined surgically resected tissue from nine patients undergoing epilepsy surgery for focal cortical dysplasia. Informed consent was obtained for the use of brain tissue and for access to medical records for research purposes. The tissue was obtained and used in a manner compliant with the Declaration of Helsinki.
The classification system proposed by Palmini et al. [48] was used for grading the degree of FCD and only patients with FCD type IIB located in the temporal lobe were included. The clinical characteristics derived from the patient’s medical records are summarized in Table 1. The predominant type of seizure pattern was that of complex partial seizures, which were resistant to maximal doses of antiepileptic drugs (AEDs; carbamazepine, valproic acid, phenytoin, levetiracetam, oxcarbazepine, and clonazepam). Information concerning the exact time of last seizure occurrence prior to surgical resection was not available. However, all the patients included in our series did not have seizure activity in the last 24 h before surgery. The patients underwent presurgical evaluation [74]. Intraoperative ECoG was performed routinely in all operations for tailoring of surgery and we classified the post-operative seizure outcome according to Engel [17]. Follow-up period ranged from 1 to 9 years.
Table 1Summary of clinical findings of patients with focal cortical dysplasiaPatient/sex/age (years)DiagnosisDuration of epilepsy (years)Seizure typeEngel class1/M/11FCD IIB11CPSI2/M/31FCD IIB20CPSI3/F/25FCD IIB9CPSI4/F/22FCD IIB21CPS/SGSI5/M/18FCD IIB14CPSI6/M/17FCD IIB10CPSI7/F/16FCD IIB11CPSI8/M/29FCD IIB21CPSI9/M/28FCD IIB21CPSIFCD focal cortical dysplasia, CPS complex partial seizures, SGS secondary generalized seizures
Normal-appearing control cortex/white matter from temporal region was obtained at autopsy from five adult control patients (male/female: 2/3; mean age 42, range 17–55) without history of neurological diseases. All autopsies were performed within 12 h after death (post mortem delay: 11, 11.5, 9, 8.5, 6). The cause of death was represented by acute myocardial infarction. In addition, four of the nine FCD cases contained sufficient amount of perilesional zone (normal-appearing cortex/white matter adjacent to the lesion), for comparison with the autopsy specimens. This material represents good disease control tissue, since it is exposed to the same seizure activity, drugs, fixation time, and the age and gender are also the same.
Tissue preparation
Tissue was fixed in 10% buffered formalin and embedded in paraffin. Two representative paraffin blocks per case (containing the complete lesion or the largest part of the lesion resected at surgery) were sectioned, stained, and assessed. Paraffin-embedded tissue was sectioned at 6 μm, mounted on organosilane-coated slides (Sigma, St Louis, MO) and used for histological and immunocytochemical reactions as described below. Frozen tissue from control cortex and FCD tissue, stored at −80°C, was used for western blot analysis.
Antibody characterization
To document the presence of a heterogeneous population of cells, we used the following antibodies: glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Glostrup, Denmark; 1:4,000; monoclonal mouse, DAKO; 1:50), vimentin (mouse clone V9, DAKO; 1:1,000), MAP2 (polyclonal rabbit; Chemicon; 1:500), neuronal nuclear protein (NeuN; mouse clone MAB377, Chemicon, Temecula, CA, USA; 1:2,000), non-phosphorylated neurofilament (SMI311; Sternberger monoclonals, Lutherville, MD; 1:1,000), human leukocyte antigen (HLA)-DP, -DQ, -DR (CR3/43; monoclonal mouse, DAKO; 1:400), CD68 (mouse clone PG-M1, DAKO; 1:200) and CD31 (mouse clone JC70A, DAKO; 1:100).
For the detection of VEGFA, VEGFB, and their receptors, the following antibodies (Abs) were used: VEGFA (G153-694, monoclonal mouse; recognizing VEGF 165 and 189 [51], Pharmingen, CA, USA; 1:100), VEGFA (A-20, SC-152, polyclonal rabbit; raised against the N-terminus of VEGFA, recognizing VEGF 121, 165 and 189, Santa Cruz Bio., CA, USA; 1:100), VEGFB (H-70, SC-13083, polyclonal rabbit; raised against amino acids 1–70 of human VEGFB, Santa Cruz Bio.; 1:20), Flt-1 (VEGFR-1; C-17, SC-316, polyclonal rabbit, Santa Cruz Bio.; 1:100), Flk-1 (VEGFR-2; A-3, SC-6251, monoclonal mouse; Santa Cruz Bio.; 1:100). To allow comparative analysis, we used on frozen specimens of normal (n = 3) and FCD tissue (n = 2), two additional antibodies (which are not suitable for staining paraffin-embedded, formalin-fixed tissue): VEGFR-1 (clone Flt-19, 1:400; developed against the recombinant human extracellular domain of VEGFR-1) and VEGFR-2 (clone KDR-1, 1:400; developed against the recombinant human extracellular domain of VEGFR-2), kindly provided by Dr. H. A. Weich (National Research Center for Biotechnology, Braunschweig, Germany), and previously characterized on human tissues [49, 64, 80]. Similar immunoreactivity patterns were observed on paraffin-embedded and frozen tissue.
The specificity of the antibodies used for immunocytochemistry on paraffin-embedded, formalin-fixed tissue (VEGFA, VEGFB, VEGFR-1, VEGFR-2; Santa Cruz Bio.), was further tested by performing western blot analysis of total homogenates of human control cortex. We also include one FCD case of which sufficient frozen material for blot analysis was available (Fig. 1). VEGFR1 and VEGFR-2 receptor proteins were detectable as a band of approximately 180 and 200 kDa, respectively; VEGFB was detectable as a band of approximately 40 kDa; VEGFA labeled a prominent band at approximately 48 kDa and a light band at 21 kDa (reducing conditions), as recently reported in human brain tissue (control hippocampus and FCD; [56]). All immunoreactive bands disappeared after preadsorption with the corresponding peptide.
Fig. 1Representative immunoblot of VEGFA, VEGFB, VEGFR-1, and VEGFR-2 in total homogenates from control cortex and FCD tissue. Expression of β-actin (as reference protein) is shown in the same protein extracts
For immunoblot analysis, human normal cortex (n = 3) and FCD (n = 1) samples were homogenized in lysis buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP-40, Na-orthevanadate (10.4 mg/ml), 5 mM EDTA (pH 8.0), 5 mM NaF, and protease inhibitor cocktail (Boehringer Mannheim, Germany). Protein content was determined using the bicinchoninic acid method [65]. Non-reducing conditions were used to improve the detection of the VEGFA antibody, as previously reported [8]. For electrophoresis, equal amounts of proteins (30 μg/lane) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis in 7.5–12.5% gels. Separated proteins were transferred to nitrocellulose paper for 1 h and 30 min, using a semi-dry electroblotting system (BioRad, Transblot SD, Hercules, CA, USA). Blots were incubated overnight in TTBS (20 mM Tris , 150 mM NaCl, 0.1% Tween, pH 7.5)/5% non-fat dry milk, containing the primary antibody (VEGFA, VEGFR-1, and VEGFR-2, 1:1,000; VEGFB, 1:200). After several washes in TTBS, the membranes were incubated in TTBS/5% non-fat dry milk/1% BSA, containing the goat anti-rabbit coupled to horseradish peroxidase (1:2,500; Dako, Denmark) for 1 h. After washes in TTBS, immunoreactivity was visualized using an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). Expression of β-actin (monoclonal mouse, Sigma, St Louis, MO; 1:50,000) was used as reference protein. Because of the limited availability of frozen material from FCD cases, a complete analysis with statistical comparison between control and FCD by immunoblot could not be performed.
Immunocytochemistry
Sections were deparaffinized, re-hydrated, and incubated for 20 min in 0.3% H2O2 diluted in methanol to quench the endogenous peroxidase activity. Antigen retrieval was performed by incubation for 10 min at 121°C in citrate buffer (0.01 M, pH 6.0); sections were washed with phosphate-buffered saline (PBS), and incubated for 30 min in 10% normal goat serum (Harlan Sera-Lab, Loughborough, Leicestershire, UK). We incubated the sections with the primary antibodies overnight at 4°C. Hereafter, sections were washed in PBS and we used the ready-for-use Powervision peroxidase system (Immunologic, Duiven, The Netherlands) and 3,3′-diaminobenzidine as chromogen to develop the staining. Sections were counterstained with hematoxylin, dehydrated, and coverslipped. Sections incubated without the primary antibody and excess of the antigenic peptide were essentially blank.
For double-labeling studies, after incubation overnight at 4°C with the primary antibodies, sections were incubated for 2 h at RT with Alexa Fluor® 568-conjugated anti-rabbit IgG and Alexa Fluor® 488 anti-mouse IgG (1:1,000; Molecular Probes, The Netherlands). The VEGFR-2 antibody (monoclonal mouse) could only be combined with MAP2 and GFAP (polyclonal rabbit). Sections were analyzed by means of a laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA; MRC1024) equipped with an argon-ion laser.
Evaluation of immunostaining
Semi-quantitative evaluation of immunoreactivity
As previously reported [3, 54], a semi-quantitative analysis was done using an Olympus microscope and examining in each section, high-power non overlapping fields (of 0.0655 × 0.0655 mm width, each corresponding to 4.290 μm2), defined in the center of the lesion using a square grid inserted into the eyepiece. A total microscopical area of 858.050 μm2 was assessed per case. Neuronal cell bodies were differentiated from glia and glia-neuronal balloon cells on the basis of morphology. Balloon cells have eccentric nuclei and ballooned opalescent eosinophilic cytoplasm. The staining intensity was evaluated using a semi-quantitative three-point scale where immunoreactivity was defined as: − absent (0); + moderate (1); ++ strong staining (2); intensity score (Table 2). This score represents the predominant cell staining intensity found in each section for the different cell types (neurons, astrocytes, microglial cells, and balloon cells) as averaged from the selected fields (as previously described [3, 54]).
Table 2VEGFA, VEGFB, VEGFR-1, and VEGFR-2 distribution in different cellular types in cases of FCD (% of cases with immunoreactive cells)Focal cortical dysplasia (n = 9)NeuronsAstrocytesBalloon cells−+++−+++−+++VEGFA022%78%011%89%011%89%VEGFB033%67%78%22%078%22%0VEGFR-1055%45%022%78%033%67%VEGFR-2011%89%55%45% 022% 45%33%FCD focal cortical dysplasia; immunoreactivity: − not present, + moderate, ++ strong
Frequency of cell staining
In each slice, we assessed the number of neurons and astrocytes labeled by a specific Ab on the total number of each cell type within the lesion using an ocular grid [4]. This frequency score was assigned using three distinct categories: (1) <10%, rare; (2) 11–30%, sparse; (3) >30%, high. The product of the intensity and the frequency scores was taken to give the total immunoreactivity score, as previously reported [23, 54].
For statistical analysis of data, SPSS for Windows was used. Data were compared using a non-parametric Kruskal–Wallis test followed by a Mann–Whitney test to assess the difference between groups. P < 0.05 was taken as the level of significance.
Results
Human material and histological features
All nine patients had chronic pharmacoresistant epilepsy and were all seizure-free postoperatively (Engel’s class I; Table 1). The FCD cases included in this study have all the histopathological features of severe (type IIB) FCD, according to the classification of Palmini et al. [48]. The resected specimens consisted of disorganized neocortex containing immature neurons, giant neurons, dysmorphic neurons, and balloon cells. Neurons and balloon cells were also observed in the subcortical white matter and there was a prominent population of reactive astrocytes. Cells of the microglia/macrophage lineage were also observed within the dysplastic cortex, suggesting activation of inflammatory processes in FCD [6].
Expression of VEGF and VEGFR in normal temporal cortex and FCD
Cellular distribution of VEGFA
VEGFA staining was observed within the histologically normal cortex (Fig. 2a, b). The staining was strongest in pyramidal neurons, which displayed somatic staining and staining of the apical dendrites (Fig. 2b). Neuropil staining was weak and resting glial cells did not show VEGFA immunoreactivity (IR). Weak staining was observed in endothelial cells. Autopsy material and the perilesional cortex showed similar IR.
Fig. 2VEGFA immunoreactivity in focal cortical dysplasia type IIB. a VEGFA immunoreactivity (IR) within the histologically normal adult cortex. b High magnification showing somatic staining in pyramidal neurons (inset: vascular staining). c VEGFA in focal cortical dysplasia (FCD; type IIB) showing strong IR within the dysplastic cortex. d High magnification showing VEGFA IR in dysplastic neurons (arrows). e A binucleated VEGFA positive dysplastic neuron. f VEGFA IR within the subcortical dysplastic region. g VEGFA expression in a balloon cell. h, i VEGFA expression in reactive astrocytes (arrows in i indicate perivascular astrocytic end-feet) j–l double-labeling of GFAP (green, j) with VEGFA (red, k) shows co-localization (yellow, l) in astrocytes. m–o Double-labeling of non-phosphorylated neurofilament (SMI311; NF, green, m) with VEGFA (red, n) shows co-localization (yellow, o) in dysplastic neurons. p Merged image showing co-localization of CD68 (green) with VEGFA (red) in macrophages. Scale bar in a a, c, f 400 μm; b, d 120 μm; e, g–i 35 μm; j–o 40 μm; p 18 μm
In the majority of FCD cases, strong VEGFA immunoreactivity (IR) was observed in dysplastic neurons located throughout the dysplastic cortex (Fig. 2c–e; Table 2; Fig. 6). Strong staining was also detected in balloon cells, reactive astrocytes, and in perivascular astrocytic end-feet (Fig. 2f–i; Table 2; Fig. 6). Endothelial IR was weak. Double-labeling experiments confirmed expression in reactive astrocytes, neurons, and in CD68+ macrophages (Fig. 2p). Immunocytochemistry using two different antibodies to VEGF (Pharmingen and Santa Cruz Bio.) demonstrated similar patterns.
Cellular distribution of VEGFB
Histologically normal cortex displayed only weak VEGFB IR (Fig. 3a). Both autopsy and surgical specimens showed light staining in pyramidal neurons and in endothelial cells. Glial cells did not show VEGFB IR.
Fig. 3VEGFB immunoreactivity in focal cortical dysplasia type IIB. a Histologically normal adult cortex, showing neuronal distribution of VEGFB with weak immunoreactivity (IR) in pyramidal cell neurons (high magnification of a pyramidal neuron is shown in the inset aI); weak staining was also observed in blood vessels (inset aII). b VEGFB in focal cortical dysplasia (FCD; type IIB) showing strong IR within the dysplastic cortex. c Strong VEGFB IR in dysplastic neurons of different size and shape (arrows). d Undetectable VEGFB IR in balloon cells (arrows). e–g Absence of co-localization between GFAP (green, e) with VEGFB (red, f) in astrocytes (g, merged image). h–j Double-labeling of non-phosphorylated neurofilament (SMI311; NF, green, h) with VEGFB (red, i) shows co-localization (yellow, j) in dysplastic neurons. k Merged image showing co-localization of CD68 (CD68; green) with VEGFB (red) in macrophages. Scale bar in a a, b 200 μm; c–j 40 μm; k 18 μm
In FCD specimens, moderate to strong VEGFB IR was observed within the dysplastic cortex (Fig. 3b; Table 2; Fig. 6) with strong VEGFB IR in dysplastic neurons (Fig. 3c). In the majority of cases (seven out of nine), balloon cells and reactive astrocytes did not express VEGFB (Fig. 3d; Table 2; Fig. 6). Double-labeling experiments confirmed the absence of VEGFB IR in GFAP-positive cells (astrocytes), whereas co-localization was found with neurofilament in dysplastic neurons (Fig. 3h–j). VEGFB IR was also observed in CD68+ macrophages (Fig. 3k).
Cellular distribution of VEGFR-1
Histologically normal cortex (autopsy and surgical specimens) displayed only weak VEGFR-1 IR, which was restricted to pyramidal neurons (Fig. 4a). IR in blood vessels was weak (Fig. 4b). Glial cells did not show VEGFR-1 IR.
Fig. 4VEGFR-1 immunoreactivity in focal cortical dysplasia type IIB. a, b Histologically normal adult cortex (a) and white matter (b) showing weak immunoreactivity (IR) in neurons (a) and blood vessels (b; arrowheads). c, d VEGFR-1 in focal cortical dysplasia (FCD; type IIB) showing strong IR in dysplastic neurons (c; arrows) and in blood vessels (d; arrows). Inset in c Co-localization between non-phosphorylated neurofilament (SMI311; NF, green) with VEGFR-1 (red) in dysplastic neurons. Inset in d Co-localization between CD31 (endothelial marker; green) with VEGFR-1 (red) in blood vessels. e–g Strong IR in balloon cells of different size (arrows) and glial cells (arrowheads in e). h–j Co-localization between GFAP (green, h) with VEGFR-1 (red, i) in astrocytes (j, merged image). k–m Co-localization of CD68 (CD68; green, k) with VEGFR-1 (red, l) in macrophages (m, merged image). Scale bar in a a–d and h–m 40 μm; e–g 35 μm
In FCD specimens, moderate to strong VEGFR-1 staining was observed within the dysplastic cortex (Fig. 4c–g; Table 2; Fig. 6). VEGFR-1 IR was observed in different cell types, including dysplastic neurons, astrocytes, and endothelial cells. Double-labeling experiments confirmed the co-localization of VEGFR-1 IR with neuronal (inset in Fig. 4c), endothelial (inset in Fig. 4d), and glial (Fig. 4h–j) markers. Additionally, expression of VEGFR-1 was observed in CD68+ macrophages (Fig. 4k–m).
Cellular distribution of VEGFR-2
VEGFR-2 staining was observed within the histologically normal cortex (autopsy and perilesional zone) in pyramidal neurons (Fig. 5a). IR in blood vessels was weak (Fig. 5b). Glial cells did not show VEGFB IR.
Fig. 5VEGFR-2 immunoreactivity in focal cortical dysplasia type IIB. a, b Histologically normal adult cortex (a) and white matter (b) showing moderate immunoreactivity (IR) in pyramidal cells (a); in the white matter, endothelial IR was weak and glial IR was under detection level (b). c, d VEGFR-2 in focal cortical dysplasia (FCD; type IIB) showing strong IR in dysplastic neurons (arrows in c), but weak IR in blood vessels (arrowheads in d). e, f Strong IR in a dysplastic neuron (arrow in e) and in a balloon cell (arrow in f), but weak IR in glial cells (arrowheads in e, f). Inset in e Co-localization between non-phosphorylated neurofilament (SMI311; NF, green) with VEGFR-2 (red) in a dysplastic neuron. g–i Co-localization between MAP-2 (green, g) with VEGFR-2 (red, h) in balloon cells (i, merged image). Scale bar in a a–d and g–i 40 μm; e, f 35 μm
In the large majority of FCD cases (eight out of nine), VEGFR-2 was strongly expressed in dysplastic neurons (Fig. 5c, e; Table 2; Fig. 6). VEGFR-2 IR was also detected in balloon cells, but only three out of nine cases displayed strong staining for VEGFR-2 (Fig. 5f; Table 2). Endothelial expression was weak. In many FCD cases (five out of nine), expression of VEGFR-2 was undetectable in reactive astrocytes (Table 2). Double-labeling experiments confirmed the co-localization of VEGFR-2 IR with neuronal markers (neurofilament or MAP2, Fig. 5e, g–i) within the dysplastic cortex.
Fig. 6Distribution of immunoreactivity scores (total score; see details in “Materials and methods” section) in neurons and astrocytes of control, perilesional, and FCD specimens. a, e VEGFA; b, f VEGFB; c, g VEGFR-1; d, h VEGFR-2. a–d Neurons; e–h astrocytes. IR scores of VEGFs and VEGFRs in neurons of FCD were greater than IR scores of VEGFs and VEGFRs of control and perilesional cortex; scores of VEGFA and VEGFRs in astrocytes of FCD were greater than IR scores of VEGFA and VEGFRs of control and perilesional cortex (P < 0.05). There were no significant differences in IR scores of VEGFs and VEGFRs between control and perilesional cortex
Discussion
In addition to their role in angiogenesis, VEGF proteins and their receptors have been implicated in several neurological disorders, including epilepsy [13, 44, 56]. In the present study, we demonstrate a prominent expression of VEGFA, VEGFB, and their signaling receptors in FCD type IIB, a malformation of cortical development associated with intractable epilepsy. This is particularly interesting in view of the recently proposed role of VEGFs and their signaling pathways during development and in epilepsy-associated pathologies [13, 26, 33, 69, 76].
Expression of VEGFA and VEGFB in normal temporal cortex
In histologically normal temporal cortex (autopsy and perilesional zone), we have shown weak expression of both VEGFA and VEGFB in cortical neurons. Expression of VEGFA and VEGFB, including both mRNA and protein, has been demonstrated in neurons in adult rodent brain [39, 41, 42, 72]. In human adult brain, only few studies have described neuronal expression of VEGFA in control tissue [8, 78], which was similar to our observed staining pattern in the control temporal specimens.
To our knowledge, previous studies of VEGFB protein expression in human control cortex have not been described. However, VEGFB mRNA was detected in human hippocampal cortex [24], and VEGFB mRNA and protein expression has been described in adult rodent brain [36, 42]. In control rat brain, VEGFB was constitutively expressed in endothelial cells [42]. In our study, we observed only weak endothelial VEGFB IR in blood vessels. In agreement with previous studies [8, 36, 78], immunoreactivity (IR) for both VEGFs was not observed in glial cells within control specimens.
Differential cellular distribution of VEGFA and VEGFB in FCD
In the present study, we provide evidence for a consistent expression of both VEGFA and VEGFB within the dysplastic cortex of patients with FCD. Both VEGFs are highly expressed in dysplastic neurons; however, only the VEGFA protein is prominently expressed in reactive astrocytes. Expression of VEGFA in astrocytes has been shown in several other pathologies associated with reactive gliosis, such as ischemic stroke, traumatic brain injury, neurodegenerative disorders, and the hippocampus following entorhinal deafferentation [8, 27, 59, 78, 79]. In addition, we previously reported upregulation of both neuronal and glial VEGFA expression in patients with hemimegalenchephaly, an epilepsy-associated malformation of cortical development [5, 7]. In the present study, we also observed expression of VEGFA in balloon cells, which are characteristic cell types of severe FCD [48]. Whether these cells are glial or neuronal in nature is still controversial [12].
Induction of both neuronal and astroglial VEGFA expression has been shown in different experimental models of seizures and human temporal lobe epilepsy (TLE) [13, 44, 56]. Rigau et al. [56] showed increased levels of VEGFA in the hippocampus of several cases of TLE, including two cases of focal dysplasia. All TLE cases showed VEGFA expression in pyramidal neurons and granule cells of the hippocampus [56]. However, immunocytochemical analysis of the temporal cortex and the FCD cases was not performed.
The molecular mechanism underlying the induction of VEGFA expression after seizures remains unclear. One possible mechanism, which has been proposed to explain the association between seizure activity and the induction of VEGFA expression, is represented by the stabilization of the hypoxia inducible factor-1α (HIF-1α). HIF-1α is a transcription factor which upregulates VEGFA transcription under hypoxic conditions [21, 38, 61]. Hypoxia may occur during seizures, representing an important trigger in the induction of VEGFA expression, particularly in case of long lasting seizures, such as in status epilepticus models. However, VEGFA expression is already induced after acute seizures [44] and the mechanisms that regulate VEGFA expression are complex. Several transcription factors, including AP-1, HIF-1α, and NF-κB, have been identified to regulate VEGFA expression [31], and recently it has also been shown that inflammatory cytokines, such as interleukin-1β (IL-1β), activate HIF1α and VEGFA gene expression in primary human astrocytes [2]. Interestingly, increased expression of proinflammatory cytokines and related molecules has been reported in both animal models and human epilepsy-associated pathologies, including FCD [3, 15, 54, 77]. In addition, VEGFA has been demonstrated to be a key mediator of the inflammatory process [14, 35]. Thus, we might speculate that the prominent expression of VEGFA within the dysplastic cortex could be a critical component of the complex cascade of events leading to a chronic inflammatory state and the sustained seizure activity [76, 77]. With respect to inflammation, inflammatory cells, such as macrophages, can also release various angiogenic cytokines including VEGFs [71]. Accordingly, we observed expression of both VEGFA and VEGFB in macrophages (CD68-positive cells), as previously shown in animal models of brain ischemia [11, 42, 52].
VEGFA effects can also compromise the integrity of the blood–brain barrier (BBB; [60]). Interestingly, alterations of the BBB permeability have been recently observed in both human and experimental temporal lobe epilepsy with positive correlation between the increased vascular permeability and the occurrence of spontaneous seizures in chronic epileptic rats [55, 56, 75].
In contrast, several studies highlight a dichotomous function of VEGFA, also demonstrating a neuroprotective role [13, 47]. Administration of VEGFA and neuronal expression of VEGFA have been shown to stimulate neurogenesis in vitro and in vivo [9, 28]. In addition, it has been suggested that the neuroprotective effects of VEGFA are mediated by the neuronal VEGFR-2 and the subsequent activation of the PI3K/Akt survival pathway [32, 67].
VEGFB expression is not induced by hypoxia or several transcription factors known to regulate VEGFA expression [18], as the promoter region of VEGFB lacks HIF-1 and AP-1 sites [43, 62]. The regulation of the expression of VEGFB remains unknown. Since all cases examined were associated with epilepsy, we cannot exclude that chronic seizure activity could also contribute to the VEGFB expression in FCD specimens. Alternatively, since VEGFB expression has been shown to be prominent during early brain development [36], the strong neuronal expression of VEGFB could represent an intrinsic and immature feature of the dysplastic neuronal cells that could contribute to their survival. Recent studies using VEGFB knock-out mice demonstrate a neurotrophic and neuroprotective activity of VEGFB, exerting a direct action on neurons, and promoting neurogenesis [68, 69].
This is an observational study and we were, therefore, not able to investigate the spatio-temporal regulation of the VEGF system. Further research in animal models of cortical dysplasia is clearly needed to elucidate the role of VEGFs and their signaling pathways in the histogenesis or epileptogenesis of developmental disorders.
Expression of VEGF receptors in normal temporal cortex
In histologically normal temporal cortex (autopsy and perilesional zone), VEGFR-1 and VEGFR-2 showed a similar pattern of expression, with weak to moderate immunostaining in pyramidal neurons. Neuronal expression of VEGFRs’ mRNA and protein has been reported in adult human and rodent brain, with strong expression in the hippocampus [8, 11, 78, 79]. In agreement with these studies, we did not detect glial VEGFR expression in histologically normal cortex and only weak VEGFR expression was observed in endothelial cells.
Differential cellular distribution of VEGFR-1 and VEGFR-2 in FCD
Consistent expression of both VEGFR-1 and VEGFR-2 was detected within the dysplastic cortex of patients with FCD. Both receptors were upregulated in dysplastic neurons. Increased expression of the VEGFRs and, in particular VEGFR-1, was observed in reactive astrocytes. Upregulation of VEGFRs in neurons and reactive astrocytes has been shown in several other pathological conditions including ischemia, neurodegenerative diseases, and trauma [8, 11, 37, 39, 66, 79]. Recently, increased expression of VEGFR-2 has been shown in several cases of TLE, including two cases of cortical dysplasia [56]. Immunocytochemical analysis demonstrated expression only in endothelial cells, whereas neuronal VEGFR-2 IR was not detected in either control hippocampus or TLE specimens [56]. Differences in the phenotypes of cells expressing VEGFRs have been observed in several other studies [8, 30, 52]. These discrepancies may be caused by differences in experimental methods, tissue processing, or the use of different antibodies that recognize different epitopes.
Our results support the neuronal expression of VEGFR-2 recently reported in human brain [8]. The similar expression pattern of VEGFA and VEGFR-2, with prominent neuronal IR, suggests autocrine/paracrine effects on dysplastic neurons, supporting the hypothesis of a mechanism to protect abnormal neurons from cell death associated with seizures. Autocrine and/or paracrine effects of VEGFA are supported by the observation that administration of VEGFA has been shown to induce mRNA and protein expression of both receptors in adult rat brain [34, 57]. A protective mechanism of VEGFA has been suggested in epileptic rats, showing that VEGFA may reduce spontaneous discharges in epileptic rats [40]. Therefore, upregulation of VEGFA could represent an endogenous compensatory mechanism to reduce excitability and to prevent cell loss after severe seizures. Accordingly, infusion of VEGFA into the hippocampus has been shown to protect against neuronal cell loss after pilocarpine-induced status epilepticus [45].
In the present study, we also provide evidence for the expression of VEGFR-1 in activated cells of the microglia/macrophage lineage, which have been shown to be present in FCD specimens [6]. This is in agreement with previous in vitro and in vivo studies [11, 20] showing VEGFR-1 expression in activated microglial cells. These observations suggest that the microglia/macrophage lineage is also a target for VEGF, which may affect chemotaxis and proliferation of these cells, contributing to the inflammatory state in the epileptic brain.
There is substantial information about the function and the signaling through VEGFR-2; in contrast, signaling through VEGFR-1 remains poorly understood and has been a matter of discussion. A decoy role has been proposed for VEGFR-1, but more recently, functional signaling via VEGFR-1 has been reported (for reviews see [43, 67]). These observations may give rise to new therapeutic strategies focusing on VEGFR-1 specific ligands, such as VEGFB [43].
Although a rapid induction of VEGF and its receptors has been shown in different experimental models of seizures [13, 44, 56], seizures alone cannot account for changes in neuronal and glial expression in FCD since perilesional tissue was exposed to seizures but did not show significant upregulation of VEGFs and/or VEGFRs. Therefore, the lesion per se or the concomitant presence of the lesion and the epileptic activity, are likely to play a role in modulating the VEGF system in these developmental disorders.
Conclusions
Our observed cellular distribution of VEGFA, VEGFB, and their signaling receptors indicate that different cellular components of FCD are involved in VEGF-signaling. In this context, future studies, using both in vivo and in vitro models, will be important to achieve a better understanding of the role of the VEGF-mediated pathways in the histogenesis and epileptogenesis of developmental lesions associated with intractable chronic epilepsy. Presently, signaling via VEGF receptors is not targeted by existing therapies in epileptic patients, but it can be potentially useful in view of its involvement in the regulation of neurogenesis, inflammation, and BBB integrity. However, an effective therapeutic intervention based on modulation of the VEGF system has to take into consideration the specific role of VEGFA and VEGFB and the multiple effects (protective and/or detrimental) reported for VEGFA.

vascular endothelial growth factor
receptors
epilepsy
dysplastic neurons
immunocytochemistry
astrocytes