Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo.
Morphological era of discovery (1950s and 1960s)
Visualization of the cell interior by electron microscopy catalysed both morphologists and biochemists to initiate experiments and make observations to identify, define and understand the complex compartmentalization of specialized cells, most notably including the morphologists G.E. Palade, C. DeDuve, D.W. Fawcett, K.R. Porter, J. Rhodin and F.S. Sjostrand. The exocrine pancreatic acinar was a specialized cell type favoured by GE Palade, and it was with this cell type that the first observations on cells with specialized granule compartments were made.
The complex morphology of the cell compartments, and the desire to link a morphological observation with basic biochemical functions and pathways known at that time initiated the monumental effort to couple a biochemical approach with EM observations. Indeed in 1956, Palade observed intracisternal granules in the endoplasmic reticulum (ER) which resembled zymogen granules, and postulated that the material in the intracisternal granules was possibly the same as the zymogen granules, and these granules were related to the granules in the cells in the endocrine pancreas, β-cell granules (Palade 1956). Using the pancreatic acinar cell model, experiments by Siekevitz and Palade between the late 1950s and early 1960s defined the role of the ER and other subcellular compartments in the synthesis of zymogen granule proteins, and resulted in a series of papers published in the newly established Journal of Biophysical and Biochemical Cytology, soon to be renamed Journal of Cell Biology (Siekevitz and Palade 1958, 1962). The concept that proteins are transported from their site of synthesis, the ER, through the Golgi complex was also being recognized and visualized using EM autoradiography in the exocrine pancreas (Caro and Palade 1964), first at a low temporal level of resolution. Direct evidence for transport from ER through Golgi to zymogen granules was then obtained using more precise pulse-chase protocols on tissue slices by Jamieson and Palade (Jamieson and Palade 1967a, b). This technical advance was further exploited by Jamieson who applied subcellular fractionation techniques to pulse-labelled slices. Separation of the rough and smooth ER from condensing vacuoles and zymogen granules allowed a quantitative kinetic analysis and a direct demonstration that proteins are transferred from the ER to zymogen granules via condensing vacuoles (Jamieson and Palade 1967a, b).
Parallel studies in the anterior pituitary gland showed that discrete, small individual granules, called immature secretory granules (ISG) in mammotroph cells originated from the Golgi cisternae. These small ISGs coalesced into aggregates surrounded by a single membrane, finally becoming mature secretory granules (MSGs) (Smith and Farquhar 1966). Additional information about the secretory process in mammotrophs was obtained using high resolution EM autoradiography. ISGs (20–100 nm structures) were found to be maximally labelled after 30 min pulse with [3H]-leucine, and mature over the following 2 h to MSGs. This kinetic of maturation correlated well with the observations in the exocrine pancreas which demonstrated the condensing vacuoles were labelled after 37 min (Jamieson and Palade 1967a). The complexity of the secretory granule compartment was revealed in subsequent studies on dispersed pituitary cells where four types of secretory granules were detected (Salpeter and Farquhar 1981). After 15–55 min of chase the labelled protein was found in small (Type I) ISGs, and subsequently in type II and III polymorphic granules, and then larger Type IV MSGs after 55–185 min of chase (see Fig. 1, and Farquhar et al. 1978). A higher degree of resolution was obtained using newly developed fine-grain emulsion which allowed an accurate detection of small structures (20–100 nm), which combined with a more sophisticated analysis of silver grain distribution, led to the discovery that concentration of secretory protein in the ISG compartment was 200 times that of the adjacent Golgi cisternae (Salpeter and Farquhar 1981).
Fig. 1Mammotroph cell from the anterior pituitary gland of a lactating rat. This micrograph illustrates the morphological complexity of the regulated secretory pathway, and the different types of ISGs. ER endoplasmic reticulum; CM cell membrane; SG secretory granule; SV smooth vesicles; VE vesicle; LB lytic body. Reproduced from the J Cell Biol, 1966, 31:319–347. Copyright 1966 The Rockefeller University Press
During this period Tartakoff and colleagues outlined the concept that secretory cells may have different ways to secrete newly synthesized proteins; cells such as plasma cells, fibroblasts, and macrophages secrete in a “non-regulated” fashion in contrast to “regulated” cells such as exocrine pancreas, the hallmark of which is the storage of the secretory proteins (Tartakoff and Vassalli 1978). The nature of the secretory pathways in such “non-storage” secretory cells was also explored by the use of drugs that perturbed secretion, such as monensin, a Na+/K+ ionophore which causes a neutralization of acidic intracellular compartments, or drugs that alter the energy status of the cell (Tartakoff et al. 1977). The common requirement for energy, cyclic nucleotides, Ca+, and cytoplasmic Na+/K+, in both non-regulated and regulated cells, gave rise to the consensus that there is a common secretory pathway originating in the RER, through the Golgi complex (for review see Palade 1975).
Many of the observations made were obtained using careful morphological approaches and expanded using biochemical techniques, and subcellular fractionation. However, these early biochemical approaches were limited by the inability to manipulate the cell systems in use, typically tissues from mice or rats. The arrival of molecular tools and genetic manipulation provided the next wave of advances bringing the field to our current level of understanding.
Cell line model systems, the molecular age and the sorting problem
Regulated secretory cells, as well as other cell types such as liver cells, have specialized plasma membrane domains, which by definition have a unique composition. Therefore, in addition to classification of regulated versus non-regulated secretion (also called constitutive secretion (Kelly 1985), it was recognized that there must be multiple ways to reach distinct domains from the Golgi complex, in particular for membrane proteins. The first direct demonstration of multiple routes to the plasma membrane was from the work of Gumbiner and Kelly in 1982. Their observations using the AtT20 cell line, a mouse pituitary cell line, showed that the regulated secretory hormone ACTH was secreted with different kinetics from a viral model membrane protein, gp70, a glycoprotein of the endogenous murine leukaemia virus. Surprisingly, the kinetics of the secretion of a proportion of the ACTH precursor, POMC, which escaped the activity of pro-hormone processing enzymes present in the MSG was the same as gp70 (Gumbiner and Kelly 1982). These experiments solidified “the two pathways hypothesis” (Kelly 1985) which defined distinct post-Golgi pathways for proteins targeted for regulated and constitutive secretion. At this time it was also recognized that the mechanism for segregation of cargo proteins (either soluble or membrane associated), or sorting into a particular pathway was an important issue, as was the identification of the precise location for the initiation of the sorting process. A key technological advance in elucidating both sorting and location was the development of immunoelectron microscopy using thin, frozen sections (Tokuyasu 1980) which allowed the identification of proteins within subcellular compartments.
TGN and post-TGN sorting and processing in neuroendocrine cells
It was recognized in mid-1980s that the trans-Golgi network (TGN) might be the key exit point for proteins destined to the plasma membrane, constitutive secretory vesicles (CSV), endosomes, and ISGs (Griffiths and Simons 1986). It was proposed that sorting receptors might function in this compartment to segregate different cargos into different vesicles (Burgess and Kelly 1987). It also became apparent from studies on the mannose-6-phosphate receptor (M6PR), which with its bound lysosomal enzyme is sorted to endosomes, that sorting to endosomes from the TGN utilized clathrin and clathrin-coated vesicles (Geuze et al. 1985). At this time it was also proposed that CSVs were formed without the aid of clathrin coats (Griffiths et al. 1985). However, clathrin-coated regions were detected on the surface of β-cell secretory granules associated with the TGN after monensin treatment (Orci et al. 1984) or on ACTH-containing ISGs in AtT20 cells (Tooze and Tooze 1986) raising the possibility that 1) ISGs formation is through clathrin-coated regions of the TGN, or 2) that the biogenesis of ISGs involved a clathrin-coated vesicle dependent pathway.
Acidification of the TGN and post-TGN secretory compartments was increasingly recognized to be important for both transport (see Tartakoff et al. 1977) and sorting. A key contribution at this time was the direct demonstration, using DAMP labelling (Anderson et al. 1984), that the TGN and post-TGN compartments were acidic, and there was a gradient of acidification in the secretory pathway. It was proposed that acidification could play role in receptor-ligand uncoupling and recycling, thereby providing directionality to transport (Anderson and Pathak 1985). Although the concentration of DAMP, was thought to be proportional to the extent of acidification, later studies provided accurate measures of pH of the TGN and ISG (see review Moore et al. 2002).
Neutralization of acidic compartments caused mis-sorting of regulated secretory proteins to the constitutive pathway (Moore et al. 1983), a result which gave rise to the possibility that regulated secretory proteins may have sorting signals, and could be sorted by a receptor-ligand interaction in the TGN, or in a post-TGN compartment. The most controversial observation in support of this hypothesis was the proposed role for carboxypeptidase E as the sorting receptor (Cool et al. 1997), although this was immediately refuted (Irminger et al. 1997). Evidence in support for a sorting signal was the demonstration that regulated secretory proteins have transferable sorting signals, the first being in 1986 (Moore and Kelly 1986). An alternative hypothesis was that aggregation of regulated secretory proteins, best demonstrated for the Granins but also shown for a variety of other regulated secretory proteins, and favoured by low pH on the TGN, drives lumenal segregation of regulated secretory proteins. The segregation of the regulated secretory proteins maybe further enhanced by a homophilic interaction with membrane associated population of Granins (Gerdes et al. 1989). Furthermore, evidence for formation of multiple secretory granule populations with different hormone content supports the idea that aggregation drives sorting (Hashimoto et al. 1987). Some of these results form the basis for the “sorting for entry” hypothesis, which proposes that regulated secretory proteins are sorted into ISGs in the TGN, while constitutively secreted proteins enter CSVs (see review Tooze 1998).
Another model to explain sorting in the regulated pathway was developed from experiments in β-cells, where it was shown that insulin-containing secretory granule formation is driven by the hexamerization and condensation of insulin in the ISGs (Arvan et al. 1991). These studies revealed an essential difference between neuroendocrine cells and β-cell insulin granules, giving rise to the “sorting by retention” hypothesis (see review Arvan and Castle 1998) which proposes that rather than an active sorting in the TGN, the crucial sorting step occurs in the post-TGN ISGs. Both the “sorting for entry” and “sorting by retention” hypothesis agree on that non-regulated (possibly mis-sorted) secretory proteins are removed by ISG-specific clathrin-coated vesicles in a pathway called “constitutive-like secretion”, after secretory granules have formed from the TGN.
Most, if not all of the soluble regulated secretory proteins are processed by the endopeptidases, the pro-hormone convertases (PCs). Their discovery and final molecular characterization was driven by the original pioneering work of Steiner and colleagues working in the β-cell (Steiner et al. 1974), and reviewed by (Seidah et al. 1993). These enzymes are present in ISGs and MSGs, and are subjected to the same sorting machinery as are proteins such as the Granins. As their activity is pH-dependent, a large number of studies used the kinetics of their activation as monitors for studying where and when the sorting of regulated proteins occurred (see also below). A direct demonstration that the site of prohomone processing of proinsulin was the clathrin-coated ISG was obtained by immunocytochemistry (Orci et al. 1985b), which were subsequently shown to be acidic (Orci et al. 1986).
The progress from the 1950s until the early 1990s was the result of many researchers’ effort, most of which have not been covered in this brief review. The reader is directed to comprehensive reviews from the period (Burgess and Kelly 1987; Mains et al. 1987), and more recently (Borgonovo et al. 2006; Dannies 2001; Kim et al. 2006; Meldolesi et al. 2004; Solimena and Gerdes 2003). The main questions which we focus on in the next section are the molecular machinery of secretory granule formation and maturation, as addressed by the work started in the laboratory of W. Huttner and continued in the author’s laboratory which is largely based on in vitro reconstitution assays using isolated TGN, and post-TGN subcellular compartments, in particular ISGs as first developed using Golgi membranes by Rothman and colleagues (Fries and Rothman 1980).
In vitro reconstitution and biochemical analysis of secretory granule biogenesis
Cell-free reconstitution of ISG budding from TGN
While MSGs from a variety of tissues had been extensively characterized morphologically as well as biochemically, little was known about ISGs beyond their morphological appearance (Smith and Farquhar 1966). The cell-free reconstitution of secretory granule budding from the TGN brought new insights to understanding the formation of ISGs (Tooze and Huttner 1990). This assay was based on (1) the high fidelity sorting properties of PC12 cells which, unlike AtT20 cells, target greater than 90% of their regulated secretory protein to ISGs and MSGs, (2) the selective labelling of a regulated secretory marker, secretogranin II (SgII), and a constitutively secreted protein, heparan sulfate proteoglycan (hsPG) with radioative sulfate by sulfotransferases present only in the TGN, and (3) the ability to separate the TGN, ISGs and CSVs from one another by sequential velocity and equilibrium gradients centrifugations. This assay demonstrated that SgII and hsPG were present in two vesicle populations, SgII-containing regulated secretory vesicles (ISGs), and hsPG-containing CSVs. These results provided the first demonstration that regulated secretory proteins and constitutive secretory proteins were sorted into two distinct vesicle populations directly upon exit from the TGN, in support of the “sorting for entry” hypothesis. The formation of both ISGs and CSVs required GTP-binding proteins (Tooze et al. 1990), possibly heterotrimeric G-proteins (Leyte et al. 1992). The rate of formation of the ISGs was indistinguishable from the CSVs, occurring with a t1/2 of 15 min.
In addition, the subcellular fractionation protocol developed to distinguish ISGs and MSGs allowed the biochemical and morphological characterization of ISGs, in comparison to MSGs, isolated from PC12 cells (Tooze et al. 1991). ISGs were shown to have several components of the clathrin-coat machinery, including AP-1 (Dittié et al. 1996), and ARF1 (Austin et al. 2000), in addition to non-granule proteins such as the M6PR and furin (Dittié et al. 1997, 1999). In addition, it was possible to determine the size of ISGs and MSGs, results which provided direct support for the early EM autoradiography (Smith and Farquhar 1966) and became the basis for the experiments directed towards a molecular understanding of the change in size of the ISG through homotypic fusion and subsequent remodelling of the ISG through clathrin coats (see Fig. 2).
Fig. 2Key steps in secretory granule biogenesis. Model based on data discussed above, in addition to contributions from many researchers which have not been mentioned, but whose contributions are noted. Step 1 depicts the formation of ISGs from the TGN, Step 2 the homotypic fusion event between ISGs, and Step 3 the clathrin-mediated membrane remodelling event. See text for abbreviations
Determination of the pH of ISGs using an in vitro approach
While it was known from earlier studies that in endocrine cells MSGs are acidified it was not known what the precise pH of the ISG was, and if the pH of the ISG differed from the TGN. The establishment of a PC12 stable cell line expressing the PC2 allowed us to characterize the endopeptidase PC2 activity during granule maturation (Dittié and Tooze 1995). In this cell line, PC2 is correctly targeted to the ISGs and co-sedimented with SgII in fractions containing ISGs and MSGs. [35S] sulfate labelling demonstrated that SgII was proteolytically processed by PC2 in ISGs after approximately 30 min of chase into several lower-molecular-mass proteins, the major ones being a 18 and 28 kDa sulphated fragment.
As the efficiency of processing of SgII by PC2 was pH dependent, we used the ability to quantitate the extent of processing at different pHs to determine the pH in the ISGs (Urbé et al. 1997). Isolated ISGs and TGN, containing [35S] sulfate pulse-labelled SgII were incubated in presence of ATP at physiological pH and the extent of SgII processing in these compartments was compared to a standard curve prepared using ISGs equilibrated at a defined set of pHs. This allowed us to determine that the ISGs intra-granular pH was 6.3, similar to the TGN pH and clearly higher than the pH of MSGs (pH 5.5–5.0). Interestingly, no processing of SgII could be observed in the membrane fraction highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs. This data represent further evidence that the ISG is indeed a functionally distinct organelle from the TGN. Furthermore, the rate of SgII processing was strongly dependent on the intragranular pH, demonstrating that processing of SgII can be used as a pH indicator for granule interior.
ISG-ISG homotypic fusion
As shown originally by Farquhar (Smith and Farquhar 1966) in the anterior pituitary, and more recently in PC12 cells (Tooze et al. 1991), ISGs increase in size during maturation, and the increase in size was proposed to reflect homotypic fusion of ISG. To demonstrate that ISG–ISG fusion occurred, we developed an assay that provided the first biochemical evidence for such a fusion event and allowed us to dissect the molecular requirements of this process.
The cell-free assay to reconstitute homotypic fusion was performed by mixing two populations of ISGs, one containing the pro-hormone convertase PC2 and the other containing [35S]sulfate-labelled secretogranin II (SgII) (Urbé et al. 1998). The fusion was then measured by quantification of the 18 kDa PC2 cleavage product of SgII. This in vitro reconstitution of ISG-ISG fusion revealed that homotypic fusion is dependent on NSF (Urbé et al. 1998), α-SNAP and on the SNARE Syntaxin 6 but not on Syntaxin 1 or SNAP-25 (Wendler et al. 2001). More recently, Synaptotagmin IV (Syt IV), a member of the Synaptotagmin family of proteins involved in membrane fusion, has also been shown to be required for this step of granule maturation (Ahras et al. 2006).
ISG membrane remodelling
In addition to ISG-ISG homotypic fusion, ISG content and membrane remodelling is another important step during granule maturation. This remodelling is performed via budding of clathrin-coated vesicle from the maturing granule membrane, and is the pathway for non-regulated constitutive-like secretion, or possibly sorting to endosomes. This clathrin-mediated remodelling step is a common feature in neuroendocrine and endocrine secretory granules, and is thought to provide a mechanism for proof-reading the content and membrane composition of the maturing ISG to ensure the production of MSGs which contain biologically active hormones and can undergo efficient exocytosis.
ISGs in endocrine and neuroendocrine cells were shown by morphological techniques to be partially clathrin coated (Orci et al. 1985a; Tooze and Tooze 1986) but the recruitment mechanism and the composition of this clathrin coat were unknown until a biochemical approach was used. Our laboratory developed a fourth cell-free assay whereby the recruitment of the clathrin adaptor complex AP-1 to ISGs was reconstituted by the addition of bovine brain cytosol (Dittié et al. 1996). These experiments showed that AP-1 recruitment to ISGs was ATP-independent but GTP- and, ARF1-dependent. This study demonstrated that it is the AP-1 complex that is involved in the clathrin binding to ISGs. Cross-linking experiments demonstrated the direct interaction of AP-1 and ARF1 on the ISG (Austin et al. 2000). The M6PR and furin are likely part of the cargo targeted for removal from the ISGs by clathrin-coats as they possess sequences in their cytoplasmic domains which interact with AP-1, and are present on ISGs, but absent from MSGs (Dittié et al. 1997, 1999).
Further investigation into cargo sorting from the ISG showed that VAMP4, a SNARE involved in endosome to TGN vesicle trafficking, which is present on ISGs but removed from the maturing granule membrane, is also able to bind AP-1. The recruitment of AP-1 by VAMP4 is dependent on phosphorylation of the cytoplasmic domain of VAMP4 by the kinase CKII. CKII phosphorylation of VAMP4 allows the recruitment of PACS1 to ISGs enhancing the AP-1 dependent sorting event (Hinners et al. 2003). These data suggest that sorting non-regulated secretory proteins from ISGs, including the SNARE proteins, can be mediated by the recruitment of clathrin coats and may be essential for maturation of ISGs.
More recently, our laboratory demonstrated that the GGAs proteins are also involved in this clathrin-mediated membrane-remodelling step of secretory granule maturation. GGAs (Golgi-associated, γ-ear-containing, ADP-ribosylation factor-binding protein) are involved in recruitment of AP-1 and clathrin coats to membranes (for review see Robinson and Bonifacino 2001). siRNA mediated knock-down of GGA3 and over-expression of dominant negative form of GGA proteins resulted in a retention of the SNAREs Syntaxin 6 and VAMP4 in the MSGs. In addition, an alteration of the PC2 activity was also detected, suggesting that inhibition or alteration of clathrin-coat remodelling inhibited or altered intra-lumenal acidification (Kakhlon et al. 2006). Similar effects on PC2 activity were seen after siRNA mediated knock-down of Syt IV, suggesting that inhibition of either homotypic fusion or remodelling have similar detrimental effects on acidification of ISGs.
It was proposed that secretory granules, like other membranes have lipid micro-domains, or rafts (Tooze et al. 2001), and that the lipid composition of the secretory granule membrane is important for protein sorting during clathrin-coat mediated membrane remodelling. In fact, cholesterol was shown to be an important component in secretory granule formation (Wang et al. 2000). Recent work by Katsumata et al. (2007) has shown that the secretory granule membrane is composed of two kinds of micro-domains and that the proteins expected to be removed during the membrane remodelling (VAMP4 and Syntaxin6) are clustered together in the same micro-domain and separated from the proteins (VAMP2) that stay on the MSG membrane. These experiments validate our observations (see above), as well as the work by H.P. Moore in AtT20 cells (Eaton et al. 2000) and provide new information that opens new avenues for research into the role of lipids in sorting from ISGs.
Recent developments in secretory granule formation and maturation
In this last section we summarize the most recent developments in our understanding of secretory granule biogenesis obtained from biochemical screens, in vivo experiments based on genetic manipulation of cell in culture as well as in vivo transgenic mouse models. Only a small number of recent results are highlighted and readers are directed to recent reviews (Borgonovo et al. 2006; Dannies 2001; Kim et al. 2006; Meldolesi et al. 2004; Solimena and Gerdes 2003). The experiments we highlight show the direction that the field has taken, and the way less tractable questions can be addressed by new biochemical screens and transgenetic mouse models.
Role of cholesterol in secretory granule biogenesis
Secretory granule formation is dependent on lipid raft microdomains that are enriched in cholesterols (Wang et al. 2000). A recent investigation of the role in vivo of cholesterol in secretory granule biogenesis was done using Smith–Lemli–Opitz Syndrome (SLOS) and lathosterolis mouse models (Gondre-Lewis et al. 2006). Both diseases are a result of inborn errors in cholesterol synthesis. SLOS is a disease caused by a defect in the function of the enzyme, 7-dehydrocholesterol reductase (DHCR7), required for the final step of cholesterol biosynthesis. Patients with lathosterolosis lack lathosterol-5-desaturase (SC5D), the enzyme that catalyse the next-to-last step in cholesterol synthesis. Data obtained from DHCR7 and SC5D knock-out mice demonstrated that impairment of the cholesterol biosynthesis results in a significant decrease in secretory granules in pancreas, pituitary and adrenal glands. In addition, abberant zymogen granules were found in the exocrine pancreas, in which there was also observed a decrease in regulated secretion which could be rescued with exogenous cholesterol. The authors conclude that the presence of an elevated quantity of other sterols, 7-DHC and lathosterol, in Dhcr7-/- and Sc5d-/- mice, respectively, could not replace cholesterol in the regulated secretory pathway. They speculate that the abnormal properties of the secretory granule in these mice models may be attributed to the reduced rigidity of membranes containing these sterols instead of cholesterol. Importantly, this study demonstrates that cholesterol is essential during secretory granule biogenesis in vivo.
Regulation of secretory granule maturation
Peptidylglycine α-amidating monooxygenase (PAM) is an essential enzyme in the processing of many bioactive peptides and hormones (Prigge et al. 2000). PAM is targeted to the regulated secretory pathway in neurons and neuroendocrine cells. It is a type I membrane protein containing two enzymatic domains within the lumen of the secretory granule, a transmembrane domain, and a cytosolic domain containing sorting signals. PAM catalyses one of the final steps in peptide biosynthesis, and is retrieved from ISGs and the plasma membrane for re-utilization in newly forming secretory granules (Ferraro et al. 2005). Among the proteins able to interact with PAM are two Rho guanine nucleotide exchange factors (GEFs), Kalirin and Trio, identified by yeast two-hybrid screens using the cytosolic domain of PAM as bait (Alam et al. 1996; Xin et al. 2004).
In a recent study, Kalirin and Trio have been shown to be involved in the maturation of secretory granules (Ferraro et al. 2007). Overexpression of their N-terminal GEF domain enhances secretion from ISGs, reducing regulated secretory protein storage through constitutive-like secretion, in the absence of secretagogue stimulation of regulated exocytosis. Conversely, when GEF activity is inhibited the constitutive-like release is inhibited, resulting in the accumulation of cargo in MSGs above even normal levels. These results indicate that these two Rho-GEFs regulate sorting into secretory granules, constitutive-like secretion from ISGs, and perhaps constitutive secretion, and provide a novel mechanism for the regulation of secretory granule maturation.
Role of Chromogranin A in secretory granule formation
Chromogranin A (CgA) and B (CgB), members of the Granin family, have long been proposed to control the secretory granule biogenesis because of their pH-, calcium- and catecholamine-dependent aggregation properties, and widespread expression pattern. Recent results supports this hypothesis, however other recent results suggest that the regulated phenotype (Day and Gorr 2003; Meldolesi et al. 2004) is not simply conferred by expression of CgA.
In support of the hypothesis, in 2001, Kim et al. demonstrated that specific depletion of CgA, but not of CgB, by siRNA impaired secretory granule production in PC12 cells. In contrast, over-expression of CgA in 6T3 cells lacking CgA and the regulated secretory pathway, or fibroblastic CV-1 cells, not only triggered recovery of secretory granules but also regulated secretion (Kim et al. 2001). However, Malosio et al.(2004) refuted the hypothesis by demonstrating with very similar approaches that the “newly formed” vesicles which contained the CgA were in fact lysosomes, and overexpression of CgA did not induce the appearance of secretory granules, or alter the number of secretory granules.
Three groups used mouse models to determine the role of Chromogranin A in vivo by targeted ablation of the Chromogranin A gene. Mahapatra et al.(2005) confirmed the putative role of CgA in secretory granule biogenesis, in particular a decrease of chromaffin granule size and number. In addition, defects were observed in neurotransmitter storage and release and regulation of blood pressure. Similar results were obtained using anti-sense vectors specific for CgA in transgenic animals which resulted in a reduction of the CgA levels (Kim et al. 2005). In the adrenal medulla of these mice there was a large decrease in the number of secretory granules; in addition it was noted that the secretory granules present appeared to be swollen. This defect may be related to a need to maintain a proportional catecholamine and CgA concentrations.
Lastly, Hendy et al. (2006) found that the CgA null mutant mice had elevated secretion of epinephrine, norepinephrine and dopamine and that mRNA and protein level of other secretory granule proteins were up-regulated. No obvious abnormalities in development or neuronal and endocrine functions have been noticed. The authors suggested then that the increased expression of the other Granin family members is likely to compensate for the CgA deficiency.
Role of prohormone convertases in secretory granule maturation
Both in vitro and ex vivo the activity of the PC enzymes (notably PC1/3 and PC2 which will be the focus of the discussion here) have been extensively studied. PC null mice models have confirmed the in vivo function of the PCs and extended the characterization of their specific substrates and action in different tissues.
The first PC transgenic mice model obtained was the PC2 null mouse (Furuta et al. 1997). Mice lacking PC2 activity develop normally and are fertile, but exhibit a variety of neuroendocrine processing abnormalities in the brain and in pancreatic islets. These mice show elevated levels of pro-insulin (Furuta et al. 1998) and pancreatic islet pro-glucagon processing is completely blocked. Other defects in PC2 null animals include lack of production of α-MSH associated with accumulation of ACTH in the pituitary intermediate lobe (Laurent et al. 2002).
7B2, also known as SgV or SGNE-1, is a small acidic protein exclusively localized to neuroendocrine tissues. This protein has been shown to be associated to PC2 and it functions as a chaperone for PC2 (Braks and Martens 1994), and for review see Mbikay et al. (2001). A 7B2 null transgenic mice model has shown that 7B2 is required for PC2 activation in vivo, having in addition important functions in regulating pituitary hormone secretion (Westphal et al. 1999). 7B2 null mice have no demonstrable PC2 activity, and in general agreement with the PC2 null mouse (Furuta et al. 1997), are deficient in processing islet hormones and display hypoglycemia, hyperinsulinemia, and hypoglucanemia, with generalized islet cell expansion and altered islet cell morphology. However, the most important impairment of PC2-mediated peptide processing in 7B2 nulls involved the synthesis of the corticotrophin ACTH. ACTH is produced by cleavage of POMC through the action of PC1/PC3, further internal cleavage of ACTH occurs by the action of PC2 specifically in the neurointermediate lobe. In 7B2 nulls, ACTH remains intact in the intermediate lobe, resulting in extremely high levels of ACTH in this lobe (Westphal et al. 1999). The overall phenotype suggests that 7B2 may be involved in PC2 activity, as well as secretory granule biogenesis.
Finally, the role of the PC1/3 endopeptidase has also been investigated in a transgenic mouse model. Disruption of gene-encoding mouse PC1/PC3 results in a syndrome of severe post-natal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2 (Zhu et al. 2002). In summary, the analysis of PC2, 7B2 and PC1/3 null mice has confirmed that PCs play a crucial role in the processing of many hormone precursors and the highlight the importance of PC2 and PC1/3 in neuroendocrine tissues, and regulated secretion.
Role of Rab3D in secretory granule maturation
Rab proteins are small GTPases that belong to the Ras protein superfamily and that function in membrane traffic. Rab3A is one of the best-characterized Rab protein. In mammals, three additional isoforms are expressed which are referred to as Rab3B, Rab3C and Rab3D. Rab3A, Rab3B and Rab3C are predominantly expressed in the nervous system, where they are localized to synaptic vesicles (Fischer von Mollard et al. 1994; Martelli et al. 2000). Rab3A-deficient mice studies suggested that Rab3A has a function intimately associated with fusion more than a function in vesicle tethering at the synapse (Geppert et al. 1997).
In contrast to Rab3A, Rab3B and Rab3C, Rab3D is predominantly expressed outside the nervous system, in peripheral tissues where the other isoforms either are expressed at low levels or are lacking. Originally identified in fat cells, Rab3D is present in several additional cell types including secretory cells such as pancreatic and parotid acinar cells, mast cells and peptide-secreting cell lines. In secretory cells Rab3D appears to be predominantly localized to secretory granules, thus mirroring the distribution of Rab3A in neurons and neuroendocrine cells. It was hypothesised that Rab3D could have the same function as Rab3A but in secretory cells.
Riedel et al. (2002) investigated the role of this Rab3 isoform by knocking out the Rab3D gene in mice. Rab3D-deficient mice are viable and fertile and showed no obvious defect. In particular, both kinetics and dose response of secretagogue-induced enzyme secretion of the pancreas were normal. However, these mice show a substantially increased size of secretory granules in both exocrine pancreas and the parotid gland with the volume being doubled, while the intragranular protein concentration appears to be unchanged. These data suggest that Rab3D functions in granule maturation but not in exocytic membrane fusion.
Conclusions and future perspectives
The early morphological and biochemical characterization of the regulated secretory pathway provided key insights to the secretory process. More recent experiments have provided a more detailed understanding of the functional properties, and the regulation of the maturation of neuroendocrine secretory granules. Many important issues need to be resolved, for example how the cytoplasmic events, such as membrane remodelling are coupled with the intralumenal biochemical changes such as PC activation, prohormone cleavage, and acidification. In addition, the biological relevance of the maturation process, as it affects hormone processing and secretion, remains to be determined in light of the differences between endocrine and neuroendocrine granules.

immature secretory granule
mature secretory granule
secretogranin
chromogranin
prohormone convertase


The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans

The nematode, Caenorhabditis elegans, possesses the most extensive known superfamily of cys-loop ligand-gated ion channels (cys-loop LGICs) consisting of 102 subunit-encoding genes. Less than half of these genes have been functionally characterised which include cation-permeable channels gated by acetylcholine (ACh) and γ-aminobutyric acid (GABA) as well as anion-selective channels gated by ACh, GABA, glutamate and serotonin. Following the guidelines set for genetic nomenclature for C. elegans, we have designated unnamed subunits as lgc genes (ligand-gated ion channels of the cys-loop superfamily). Phylogenetic analysis shows that several of these lgc subunits form distinct groups which may represent novel cys-loop LGIC subtypes.
Introduction
Members of the cysteine-loop ligand-gated ion channel (cys-loop LGIC) superfamily are neurotransmitter receptors that mediate synaptic transmission in vertebrates and invertebrates. Cys-loop LGICs are made up of five homologous subunits arranged around a central ion channel (Sine and Engel 2006). The characteristic cys-loop motif is situated in the N-terminal extracellular ligand-binding region of each subunit and consists of two disulphide-bond forming cysteines separated by 13 amino acid residues, several of which are highly conserved to form a signature sequence (Gene Ontology ID GO:0005230). The cys-loop plays roles in receptor assembly (Green and Wanamaker 1997) and gating of the ion channel (Grutter et al. 2005).
The subunit composition determines the functional and pharmacological properties of the cys-loop LGIC, thus receptor diversity is generated by multiple subunit-encoding genes. For instance, the human cys-loop LGIC superfamily consists of 45 genes encoding cation-permeable nicotinic acetylcholine receptors (nAChRs) (Kalamida et al. 2007), cation-permeable serotonin or 5-hydroxytryptamine type 3 (5-HT3) receptors (Reeves and Lummis 2002), anion-permeable γ-aminobutyric acid (GABA) type A and C (GABAA and GABAC) receptors (Bormann 2000; Darlison et al. 2005) and anion-permeable glycine receptors (Kirsch 2006). As demonstrated in the fruit fly (Drosophila melanogaster), honey bee (Apis mellifera) and red flour beetle (Tribolium castaneum), insect cys-loop LGIC superfamilies are more compact, consisting of just over 20 subunits (Jones and Sattelle 2006; Jones and Sattelle 2007; Littleton and Ganetzky 2000). Approximately 15 of these genes are known to encode cation-permeable nAChRs (Sattelle et al. 2005), anion and possibly cation channels gated by GABA (Buckingham et al. 2005), glutamate-gated anion channels (Vassilatis et al. 1997) and histamine-gated anion channels (Gisselmann et al. 2002; Zheng et al. 2002). The largest known cys-loop LGIC superfamily belongs to the simple nematode, Caenorhabditis elegans, where an early analysis of its completed genome revealed 90 ligand-gated ion channel genes (Bargmann 1998). In common with vertebrates, members of this large cys-loop LGIC superfamily include cation-permeable nAChRs (Jones and Sattelle 2004) and anion-permeable GABA receptors (Schuske et al. 2004). As is the case for insects, C. elegans has glutamate-gated anion channels (Wolstenholme and Rogers 2005) but so far no nematode histamine-gated ion channels have been found. However, C. elegans possesses cys-loop LGIC receptors not identified in vertebrates and insects which include anion channels gated by acetylcholine (Putrenko et al. 2005) and serotonin (Ranganathan et al. 2000). The considerable diversity of receptor subtypes in C. elegans may be broadened further as many cys-loop LGIC subunits have yet to be characterised. Here, we present an update on the genes present in the C. elegans cys-loop LGIC superfamily.
Methods
All C. elegans protein sequences bearing motifs particular to cys-loop LGICs were assembled from WormBase (http://www.wormbase.org release WS185 Dec 23 2007). These motifs and their source databases are neurotransmitter-gated ion-channel ligand-binding INTERPRO:IPR006202, neurotransmitter-gated ion-channel INTERPRO:IPR006201, neurotransmitter-gated ion-channel ligand binding domain PFAM:PF02931, neurotransmitter-gated ion-channel transmembrane region PFAM:PF02932 and ligand-gated ion channel KOG3644. Cys-loop and TM2 sequences were identified from an alignment of protein sequences of whole subunits constructed with ClustalX (Thompson et al. 1997) using the slow-accurate mode with a gap opening penalty of 10 and a gap extension penalty of 0.1 as well as applying the Gonnet 250 protein weight matrix (Benner et al. 1994). The protein alignment was viewed using GeneDoc (http://www.nrbsc.org/gfx/genedoc/index.html). The neighbour-joining method (Saitou and Nei 1987), available with the ClustalX program, was used to construct a phylogenetic tree, which was then displayed using the TreeView application (Page 1996).
Results and discussion
Our analysis of the C. elegans genome revealed the largest cys-loop LGIC superfamily so far characterised consisting of 102 genes. As we have previously reported the C. elegans nAChR and orphan subunits (Brown et al. 2006; Jones et al. 2007; Jones and Sattelle 2004; Mongan et al. 1998), we concentrate here on the remaining cys-loop LGIC genes. For unnamed subunit genes, we continued to use the nomenclature first used for orphan nAChR subunits (Jones et al. 2007). Thus, 24 genes were designated lgc for ligand-gated ion channel of the cys-loop LGIC superfamily (Table 1). As shown in Fig. 1, the C. elegans cys-loop LGIC subunits were grouped according to sequence homology. As with the C. elegans nAChR subunits (Mongan et al. 1998), each group was named, if possible, after a well characterised subunit. For a detailed comparison of C. elegans cys-loop LGIC subunits with those of other organisms, see (Dent 2006).
Table 1C. elegans cys-loop LGIC subunits that have been given names in this report. Amino acid residues preceding TM2 that are important for determining the charge selectivity of the ion channel (Jensen et al. 2005) are given. The cys-loop sequence is shown with the cysteine residues highlighted in black shading while gray shading indicates conserved residues. The presence of a putative second cys-loop (Dent 2006) is also notedFig. 1Tree showing the cys-loop LGIC gene superfamily of C. elegans. This figure focuses on cys-loop LGIC subunits other than nAChRs although representative subunits of the nAChR groups are shown. For the complete C. elegans nAChR gene family, see (Jones et al. 2007). Based on protein sequence homology, the subunits are divided into eight groups with three highly divergent subunits, LGC-32, LGC-33 and LGC-34, not belonging to any group
Ungrouped subunits
Three subunits, LGC-32, LGC-33 and LGC-34, were not placed into any groups as they are highly divergent (Fig. 1), only showing up to 10, 10 and 15% identity respectively with other C. elegans, human and D. melanogaster cys-loop LGICs. These subunits have not been functionally characterised so it is unknown what ligands act on them. The sequence of LGC-34 and a close homologue in the parasitic nematode, Dirofilaria immitis, has been previously described where it has been noted that even though the subunit possesses features common to cys-loop LGICs, such as conserved residues in the N-terminal extracellular region and four transmembrane domains, the cys-loop is absent (Yates and Wolstenholme 2004). Interestingly, a bacterial proton-gated ion channel (Glvi) resembling cys-loop LGICs but also lacking the cys-loop has been recently reported (Bocquet et al. 2007; Tasneem et al. 2005). LGC-34 may thus represent more ancestral members of the cys-loop LGIC superfamily. Unlike Glvi, LGC-34 possesses two cysteine residues which are conserved in glutamate-, histamine- and vertebrate glycine-gated anion channels (Dent 2006; Yates and Wolstenholme 2004). Known as the second cys-loop, these two cysteines flank loop C which is a region that contributes to ligand binding (Corringer et al. 2000).
The EXP-1 Group
This group consists of only two subunits, one of which is EXP-1, a GABA-gated cation channel that mediates enteric muscle contraction (Beg and Jorgensen 2003). While EXP-1 overall resembles more closely anion permeable cys-loop LGICs such as UNC-49 as opposed to cation-permeable nAChRs, it lacks the PAR motif preceding the second transmembrane domain (TM2) which is important for anion selectivity (Jensen et al. 2005). Instead it possesses the residues ETE where the presence of glutamic acid residues has been implicated in determining cation selectivity (Jensen et al. 2005; Wotring and Weiss 2008). The other subunit in this group, LGC-35, also has a glutamate residue preceding TM2 (Table 1) and thus may be a cation channel.
The AVR-14 Group
This group consists of six subunits (Fig. 1) that make up glutamate-gated chloride channels that are targeted by the avermectin and milbemycin anthelmintics (Wolstenholme and Rogers 2005). Examples of GluCl function include AVR-15 and GLC-2 in the inhibition of pharyngeal pumping (Dent et al. 1997; Laughton et al. 1997; Pemberton et al. 2001) and AVR-14, AVR-15, GLC-1 and GLC-3 in regulating locomotion (Cook et al. 2006). Recently, it has been shown GLC-3 plays a role in olfactory behaviour (Chalasani et al. 2007).
The UNC-49 Group
Products of the unc-49 gene form a GABA-gated anion channel that mediates body muscle inhibition during locomotion (Bamber et al. 1999; Richmond and Jorgensen 1999). There are four other subunits included in this group (Fig. 1). With regard to other species, members of the UNC-49 group are clearly most closely related to mammalian and insect GABA-gated anion channels (Dent 2006). GAB-1 forms functional GABA receptors when coexpressed in Xenopus laevis oocytes with either HG1A or HG1E, which are putative GABA receptor subunits from the parasitic nematode Haemonchus contortus (Feng et al. 2002). The remaining subunits have yet to be characterised but it is worth noting that LGC-36 possesses the amino acids AER instead of PAR before TM2 (Table 1). The presence of a glutamic acid residue at this position and the absence of a proline may result in a cation-selective channel (Jensen et al. 2005).
The GGR-1 Group
This group includes six subunits (Fig. 1), the functional characterisation of which has so far not been published. The neurotransmitters/ligands to which GGR-1 group subunits respond to cannot be easily inferred through phylogenetic analysis. For example, when comparing with human cys-loop LGICs they bear closest resemblance to glycine α subunits with approximately 25% identity while LGC-42 is slightly more similar to histamine-gated anion channels with 26% identity when comparing with insect cys-loop LGICs. Members of the GGR-1 group have been previously denoted as “ce_Group I” subunits which most closely resemble insect histamine-gated anion channels (Dent 2006). It would be of interest to determine if members of the GGR-1 group respond to glycine, histamine or other ligands not so far known to act on C. elegans cys-loop LGICs. The subunits of the GGR-1 group are likely to be anion-selective as they all possess the PAR motif before TM2 (Table 1) which is important for anion selectivity (Jensen et al. 2005). The only exception is GGR-1 which instead has the amino acid residues PGR. With the proline still present and an absence of an acidic residue (particularly glutamic acid) in this region, it is likely that GGR-1 will also be anion selective.
The LGC-45 Group
This group consists of three subunits (Fig. 1), the functional characterisation of which has so far not been published. As is the case for members of the GGR-1 group, LGC-43, LGC-44 and LGC-45 most closely resemble glycine α subunits and histamine-gated anion channels when compared with human and insect cys-loop LGICs respectively. However, unlike the GGR-1 group, the LGC-45 group subunits are likely to be cation selective as they lack the PAR motif before TM2 (Table 1) which is important for anion selectivity (Jensen et al. 2005). In particular, LGC-44 and LGC-45 possess glutamic acid instead of the proline residue which is likely to result in a cation-selective channel (Wotring and Weiss 2008). While LGC-44 shows notable sequence identity with other cys-loop LGIC subunits (up to 37% with LGC-45), it lacks the cys-loop (Table 1), thus it may represent an ancestral cys-loop LGIC subunit as has been noted for LGC-34.
The ACC-1 Group
This group includes ACC-1, ACC-2, ACC-3 and ACC-4 which are acetylcholine-gated anion channels (Putrenko et al. 2005). Acetylcholine-gated anion channels have also been characterised from the snail, Lymnae stagnalis, which appear to have evolved from cation channels through amino acid substitutions in the ion channel pore (van Nierop et al. 2005). In contrast, acetylcholine-gated anion channels of the ACC-1 group may have arisen from substitutions in the ligand-binding domain of anion channels (Putrenko et al. 2005; van Nierop et al. 2005).
The MOD-1 Group
MOD-1 modulates locomotory behaviour and is the only known invertebrate cys-loop LGIC to be gated by serotonin (Ranganathan et al. 2000). Unlike mammals which possess cation-permeable serotonin receptors (Reeves and Lummis 2002), MOD-1 is anion selective. One other subunit, LGC-50, is closely related to MOD-1 (Fig. 1).
The GGR-3 Group
This group includes six subunits (Fig. 1), the functional characterisation of which has so far not been published. All six subunits are likely to form anion-selective channels as they have the PAR amino acid motif before TM2 (Table 1) (Jensen et al. 2005). When comparing with human cys-loop LGICs they bear closest resemblance to GABA α, γ and ε subunits with approximately 22% identity while they most closely resemble histamine-gated chloride channels (21% identity) when comparing with insect cys-loop LGICs. As with the GGR-1 group, it would be of interest to determine if the GGR-3 group consists of ion channels responding to ligands not so far known to act on C. elegans cys-loop LGICs.
Cys-loop LGIC superfamilies of other nematode species
Genome sequencing projects are allowing the comparison of cys-loop LGIC superfamilies from different nematode species. For instance, Caenorhabditis briggsae, which is seemingly identical to C. elegans (Gupta et al. 2007; Stein et al. 2003), also has an extensive cys-loop LGIC gene superfamily although there are a few exceptions where there appears to be gene expansions in C. elegans, mainly within the nAChR orphan group. For example, lgc-23, lgc-24 and lgc-28 in C. elegans (Jones et al. 2007) may be paralogues resulting from gene duplication in the elegans species lineage since only one homologue (Cbr-lgc-28) is observed in C. briggsae. In line with this, lgc-23 and lgc-24 are situated close together within 6 kb in the C. elegans genome which may reflect a recent gene duplication event. Likewise, lgc-11 and lgc-12, as well as lgc-25 and lgc-26 have single homologues in C. briggsae, Cbr-lgc-11 and Cbr-lgc-26 respectively. Six C. elegans cys-loop LGIC subunits, lgc-13 to lgc-18, are tightly clustered within 20 kb of the T01H10 cosmid whereas in C. briggsae there are seven similar genes (CBG01219, CBG01221, CBG02706, CBG16212, CBG16226, CBG16227 and CBG16229), all of which are not clustered together. Outside of the nAChR orphan group, avr-15 and glc-1 may be paralogues arising in the elegans lineage as there is only a single homologue (CBG06688) in C. briggsae.
The cys-loop LGIC gene families of the parasitic nematodes, Brugia malyai and Trichinella spiralis, are much smaller than those of C. elegans and C. briggsae consisting of 30 and 19 subunits respectively (Williamson et al. 2007). The cys-loop LGIC subunits of both parasites have clear orthologous relationships with those of C. elegans with one exception, a nAChR subunit (ACR-26) which so far appears particular to B. malayi. As discussed by Williamson and colleagues, the striking difference in the cys-loop LGIC gene family sizes may reflect a free-living versus a parasitic lifestyle. Thus, the large complement of subunits in C. elegans and C. briggsae may be required for a small nervous system to be able to respond to many environmental cues which may be more restricted in the specific environmental niche occupied by parasitic nematodes. This shows that the extensive cys-loop LGIC gene superfamily observed for C. elegans is not a feature of all nematodes.

ion channel
caenorhabditis elegans
acetylcholine
gaba
glutamate
serotonin


Does the Consumption of Green Tea Reduce the Risk of Lung Cancer Among Smokers?

Experimental and epidemiological studies were reviewed to assess whether the consumption of green tea could reduce the risk of lung cancer in smokers. Articles published since 1990 were located by searching electronic databases PubMed, Ovid and Science Direct, using keywords ‘lung cancer’, ‘tea’ and ‘smoking’ without any restriction on language. After relevant articles had been located, further papers were obtained from their reference lists. Evidence from experimental studies (in vitro animal and human trials) suggested that regular intake of green tea may be protective against tobacco carcinogens. However, the mechanism behind the protective effect is only partly understood. In most of the epidemiological studies reviewed, the green tea exposure was within 5 years of the interview or follow-up, which would coincide with the induction period and latent period of lung cancer. Longer term studies are thus needed to further quantify the cancer risk. There is some evidence suggesting regular intake of green tea at high level (>3 cups per day) may reduce the risk of smokers developing lung cancer. Improvement in measuring green tea intake is required in order to confirm the evidence from epidemiological studies.
Introduction
Lung cancer has become one of the leading causes of death in both developed and developing countries (1,2). Most of the deaths from lung cancer are due to smoking tobacco products. Worldwide in 2000, tobacco smoking contributed 77% of the lung cancer deaths in males aged 30–69 years, and 82% in males over 70 years (1,2). In 2003, for example, the prevalence of smoking in China was estimated to be 58.9% among adult males, while Chinese smokers are four times more likely to die from lung cancer than non-smokers (3).
Although elimination of tobacco smoke exposure is the obvious strategy to control lung cancer, no country has been completely successful in implementing an effective intervention. The efficacy of smoking cessation programs may be enhanced if they are conducted in conjunction with other complementary therapies. Epidemiological and experimental studies have suggested that green tea may be protective against cancer in different sites (4–21). In particular, recent evidence from in vitro, animal and human trial studies indicated the possibility that the consumption of green tea may reduce the risk of lung cancer among smokers (2,22–35), yet findings from epidemiological studies remained inconsistent.
The aim of this review is to assess evidence from experimental and epidemiological studies whether green tea is a risk modifier for those exposed to tobacco smoke. The focus is on the measurement of green tea exposure undertaken in these studies.
Methods
Articles published since 1990 were located by searching electronic databases PubMed, Ovid and Science Direct, using keywords ‘lung cancer’, ‘tea’ and ‘smoking’ without any restriction on language. After relevant articles had been located, further papers were obtained from their reference lists. A total of 78 relevant articles were eventually found.
Results and Discussion
In vitro and Animal Studies
Both in vitro and animal studies have demonstrated that green tea or tea polyphenols have a chemopreventive effect against carcinogens from tobacco and is dependent on both the period and dose of the exposure.
Period of exposure
The period of exposure to green tea or tea polyphenols and the period of exposure to carcinogens must overlap. In one in vitro study, green tea polyphenols were introduced once into the living cell environment 2 h before exposing the cells to a smoke solution for 30 min. Result showed that green tea polyphenols can reduce DNA strand breakage induced by cigarette smoke in cultured human bronchial cells A549 (32). In another in vitro study, human lymphoid cells were exposed to one single dose of green tea extracting solution 2 h before adding benzo(a)pyrene-diol-epoxide (BPDE)—a metabolic production of benzo(a)pyrene (B(a)P), which is a major component of tobacco. The results demonstrated that green tea can significantly reduce DNA damage caused by BPDE (22). Similarly in four other animal studies (23,29,33,34), mice were given green tea prior to or within 48 h of exposure to tobacco carcinogens, and the green tea was continuously administered for 16 weeks or 9 months. Other experimental conditions such as diet, humidity, temperature and dark/light cycle were controlled. Three out of the four studies found a reduction in the incidence and the size of lung cancer tumors due to drinking green tea (23,33,34).
Dose of exposure
The preventive effect of green tea is likely to be related to the concentration of green tea as well as the dose of carcinogens. In experimental studies both cells and animals were often treated with relatively high doses of carcinogens and green tea. For example, in a study which showed a protective effect of green tea, the concentration of tobacco carcinogens used was equivalent to the amount contained in the sidestream smoke from one cigarette dissolved in 24 ml of water, while the concentration of green tea polyphenol was set at 250 μg ml−1, corresponding to 116.5 μg ml−1 of epigallocatechin-3-gallate (EGCG) (32). Moreover, the bioavailability of different green tea polyphenols has been a concern in animal and human studies. Green tea polyphenols are not usually stable, specifically, in water, EGCG can oxidize more than 50% within 12 h, yet green tea used in experimental studies was typically prepared every 48 h (33,34) to every 7 days (29).
In some studies where mice were exposed to both NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] and green tea, the NNK dosage administered was usually equal to that experienced by a smoker in a lifetime (36). In one experiment mice were randomized into different groups and given green tea solution at concentrations of 0.2%, 0.4% and 0.6% as drinking fluid 24 h after treating with NNK. Comparing with the control group that drank only water, the protective effect of green tea was only evident at the 0.6% concentration level (33). In another study (29), mice were exposed to high levels of environmental tobacco smoke for 5 months, while being given 1.25% green tea (2.5 g tea leave mixed with 200 ml boiling water). The results indicated green tea had little effect on reducing tumor incidence or multiplicity of the tumors. However, none of the animals in the green tea treatment group experienced early death while >10% of the mice in the control group experienced early deaths, probably due to an overdose of carcinogens exposure (29). Findings from these experiments suggest that green tea may not be effective to inhibit the formation or growth of lung tumor under high level of carcinogens exposure and over a long duration.
Human Trials
Short-term effect
Because a single dose of green tea intake only produces a short-term antioxidant effect, regular consumption seems necessary. In a human study (37), 21 non-smokers consumed 300 ml of green tea solution or about three cups. Ferric reducing ability of plasma (FRAP-assay) was used to determine the antioxidant activity in plasma. The results of this study showed that green tea consumption produced a significant increase in antioxidant activity within 30–120 min of exposure, but after 120 min the antioxidant activity appeared to decrease. Another human trial with 10 healthy subjects similarly found a short-term increase of plasma antioxidant induced by drinking two to three cups of green tea at one time (38). Such a short-term increase seemed to correspond with the relatively short half-life of tea polyphenols (30). In one pharmacokinetic study (39), 30 healthy participants ingested 200 mg of EGCG equivalent to two cups of green tea. Blood samples were collected at 0.5, 1, 2, 4, 6, 8 and 24 h after the EGCG administration. This experiment found that the mean half-life of EGCG in plasma was only 118 min.
Protective effect from regular consumption
Regular green tea consumption could be protective over an extended period. In one pilot study (35), three non-smokers and three smokers (consumed more than 10 cigarettes per day) drank two cups of green tea in the morning, two cups in the afternoon and one cup at night daily for 4 weeks. Each cup of green tea contained 400–500 mg of green tea extract. Oral keratinocytes were harvested from each subject for analysis. Outcomes included a reduction in smoking-induced DNA damage, increased apoptosis and a further decrease in the number of cells carrying damaged DNA (35). In a randomized control trial (27), 133 smokers who smoked at least 10 cigarettes per day during the past year were randomized into three groups consuming at least four cups daily of either (i) decaffeinated green tea, (ii) decaffeinated black tea, or (iii) water. After 4 months of intervention, smokers who drank decaffeinated green tea had a significant decrease in their urinary 8-hydroxydeoxyguanosine (8-OHdG) level, but no significant changes were observed in both water and black tea groups. Similar reduction in urinary 8-OHdG level was observed among HbsAg positive volunteers who took daily capsules containing green tea polyphenols equivalent to two to four cups of tea for 3 months (40). In an intensive observational study (25), the frequencies of sister-chromatid exchange (SCE) in peripheral lymphocytes were measured as markers of smoking-induced DNA damage. The frequencies of SCE were significantly higher among smokers who were non-tea drinkers, than those of non-smokers and smokers who consumed green tea at least two to three cups per day during the past 6 months. The differences between the latter two groups, however, were not significant. It is possible that consuming high levels of green tea over a long period may reduce the DNA damage caused by tobacco smoking.
Lung tumor treatment
Only one clinical trial used green tea for treatment of lung cancer (41). In this Phase I study, 17 advanced lung cancer patients were given green tea extract at a single dose of 3 g m−2 per day for 4 weeks. The dosage equates to 20 cups of green tea, which has been found to be the maximum tolerated dose. Unfortunately, no effect of the treatment was observed. Therefore, green tea is unlikely to be an effective cytotoxic agent against existing tumor.
Limitation of human trials
Except two studies (27,40), most human intervention studies involved small sample sizes generally less than 50 subjects. In addition to ethical concerns, there were neither direct measurement on the concentration of green tea polyphenols in lung tissue nor on the change of lung tissue after green tea exposure. Instead, the effects of green tea were measured by the change in DNA damage and apoptosis of oral cells (35), changes in biomarkers of DNA damage such as 8-OHdG and SCE (25,27,40), and changes in plasma antioxidant capacity (30,37,38). Trials with sufficient sample size and longer period of follow-up are needed to confirm the protective effect of green tea.
Mechanisms Based on Experimental Studies
As shown in Fig. 1, carcinogens contained in cigarette smoke are activated by Phase I enzymes in the body, leading to the formation of DNA adducts (36). If DNA adducts escape cellular repair mechanisms, it may result in miscoding and eventually a stable mutation. Blocking any step in the pathway would reduce the incidence of lung cancer among smokers (36). Table 1 provides the possible mechanisms of chemoprevention effect of green tea. The evidence suggested that catechins in green tea, especially EGCG, may prevent the formation of a mutated cell.
Figure 1.Mechanism of tobacco carcinogens inducing lung cancer.
Table 1.Possible mechanism of actionPossible mechanism of chemoprevention effect of green teaReferencesActing as antioxidant to prevent DNA damage and reduce the formation of DNA adducts(11,22,25,27,35,40)Increasing apoptosis and preventing the persistence of miscoding DNA (possibly by inducing gene regulation)(11,26,31,33,35,40,43,44)Activating Phase II detoxifying enzymes, and increasing the excretion of carcinogens (possibly by inducing gene regulation)(2,8,28,31)
In both animal and in vitro studies, green tea was found to increase the activities of Phase II enzymes (2,8,28,31). Phase II enzymes transform carcinogens into non-toxic molecules for subsequent excretion (42). The amount of carcinogens that may be further activated, as well as DNA adducts, are thus reduced. The antioxidant properties of green tea have also been demonstrated in trials involving smokers. It reduced the urine concentration of 8-OHdG, a strong biomarker of DNA damage (27). Moreover, green tea extract could inhibit the formation of human DNA adducts (11,22,35).
Once carcinogen DNA adducts have formed, persistent mutation is likely unless cells with DNA adducts are removed by apoptosis (36). Human (35), animal (11,33) and in vitro studies (26,43,44) have shown that green tea can induce apoptosis among injured cells exposed to carcinogens from tobacco smoke. However, smoking may be regarded as a chronic exposure, since long duration of tobacco smoking has greater impact on lung caner risk than the amount smoked per day (45). Therefore, a long exposure to green tea is needed in order to reduce the damage caused by tobacco carcinogens.
Epidemiological Studies
Induction time and latent period of lung carcinoma
Latent period refers to the time between disease occurrence and detection, while tumor induction time commences when the effect of the exposure occurs until all of the component causes involved in one sufficient cause are satisfied—the time of disease occurrence (46). Exposure within the induction and latent periods is irrelevant to the formation of a tumor (47). Moreover, the incidence of lung cancer does not appear to be lower among ex-smokers who quit smoking within 5 years than current smokers (48,49). This indicates that the sum of induction period and latent period of lung cancer caused by tobacco smoking may be longer than 5 years. Logically, no reduction in incidence of lung carcinoma is expected among current smokers who drink green tea for 5 years prior to the calculation of incidence, regardless of their consumption level.
Measurement of green tea exposure and other limitations
Table 2 summarizes epidemiological studies on tea and lung cancer. Three case–control studies (50–52) found a significantly lower risk of lung cancer among non-smokers with a high consumption of green or black tea, but no protective effect was evident among smokers (50,51). However, the period of tea consumption referred to 5 years or less before the diagnosis or interview. Although the subjects might have been drinking tea for a long time, the duration and quantity of tea consumption was not assessed, so the effect of green tea could not be fully justified. Cohort studies, on the other hand, were based on information of tea consumption at the beginning of the study (53–55) and suggested that tea consumption increased with age (54,55). In particular, the Japanese study (53) with a long follow-up period of 14 years showed that people consuming green tea two to four times per day had a relative risk of 0.78 (95% CI = 0.6–1.0), when compared with those drinking once or less per day and after adjustment for potential confounders. But for subjects drinking green tea more than five times per day, the relative risk was only 0.79 (95% CI = 0.59–1.1), the non-significance result probably due to insufficient sample size at this high level of consumption (53).
Table 2.Epidemiology studies on tea and lung cancerCountryStudy designSample sizeTea type and highest consumption levelReference period of intakeSmoking status of subjectsResultsChina (50)Population-based case–control studyCases: 649 Controls: 675Green tea: >1500 g tea leaves per year5 years before interviewSmokers and non-smokersOR = 0.65; 95% CI = 0.45–0.93 for non-smokers OR = 0.94; 95% CI = 0.40–2.22 for smokersCanada (52)Population-based case–control studyCases: 582 Controls: 582Type unspecified: >7 cups per day2 years before studyNon-smokersOR = 0.4; 95% CI = 02–0.7USA (51)Population based case–control studyCases: 161 Controls 483Green tea: >1 cup per day1 year before diagnosis or interviewSmokers and non-smokersOR = 0.9; 95% CI = 0.5–1.6Japan (53)Cohort study (1980–94)Subjects: 38 540 Person-years at risk: unspecified Cases: 436Green tea: >5 times per dayUnspecified; survey taken at start of studySmokers and non-smokersRR = 0.78; 95% CI = 0.6–1.0 for people drinking tea 2–4 times per dayJapan (54)Cohort study (1986–95)Subjects: 8552 Person-years at risk: 71 248.5 Cases: 384Green tea: >10 cups per dayUnspecified; survey taken at start of studySmokers and non-smokersCancer in all sites RR = 0.68 for males; 95% CI = 0.39–1.21 RR = 0.57 for females; 95% CI = 0.33–0.98OR, odds ratio; RR, relative risk; CI, confidence interval.
In summary, there is only limited evidence from epidemiological studies that green tea is protective against lung cancer among smokers. Moreover, green tea exposure was generally taken to be within 5 years of interview or follow-up, which would have little impact on the incidence of lung cancer.
Conclusion
Our review suggests that regular intake of green tea at high levels (>3 cups per day) may offer protection against tobacco carcinogens for smokers, provided that the duration of green tea consumption is sufficiently long to cover the smoking period. For epidemiology studies, improvement in measuring green tea intake is required in order to confirm the chemopreventive effect of green tea observed in in vitro animal and human trials.


Adjacent level discitis after anterior cervical discectomy and fusion (ACDF): a case report

This report describes a case of spondylodiscitis occurring adjacent to levels at which anterior cervical discectomy and fusion was performed. The objective is to describe a rare cause of spondylodiscitis and discuss its successful management. Post-operative discitis involving the same level is a known occurrence. We report an interesting case of spondylodiscitis occurring at the adjacent level of fusion, and to our knowledge this is the first such case reported in literature. A two-level decompression and fusion was performed at C5–6 and C6–7 levels with PEEK cages and anterior cervical plating in a middle-aged gentleman for persistent axial neck pain and left-sided radiculopathy involving C6 and C7 distribution. After 6 weeks, the patient presented to us with complaints of mild paresthesia in the abdomen and extremities. Radiological investigations including plain radiographs and MRI revealed a surprising finding of discitis at C4–5 level with an associated epidural abscess. In view of the patient’s myelopathic symptoms, surgical debridement and decompression of the spinal cord was performed. The plate and screws were removed, the cages were left intact, and the C4–5 disc level was reconstructed with tricortical iliac crest autograft. No further instrumentation was performed. The biopsy specimen from the disc at C4–5 level grew Serratia marcescens. It was contemplated that C4–5 discitis was initiated by inoculation of bacteria at the superior endplate of C5 by contaminated vertebral pins/drill-bit or screws. Adjacent level discitis is a rare but potentially serious complication of anterior cervical fusion. A high index of suspicion of infection is necessary if the patient complains of new symptoms after anterior cervical fusion. Thorough assessment and aggressive treatment is necessary for successful management.
Introduction
Post-operative discitis occurring at the operated level is a known complication of spinal surgery. The most common organisms implicated in the pathogenesis are Staphylococcus aureus and Staphylococcus epidermidis. Degeneration occurring at the adjacent level is also a well-established phenomenon after spinal fusion. We present a unique case of discitis occurring at a level adjacent to the levels that underwent anterior cervical fusion. The case becomes interesting because of numerous factors e.g. the site of involvement, the likely etiology, the organism concerned, the subtle nature of the symptoms, problems with diagnosis and its management.
Case report
A healthy 56-year-old male patient presented with neck pain of 6 months duration and associated radicular pain and numbness in the left upper-limb. The history was negative for fever, night pain or constitutional symptoms. MRI revealed left-sided foraminal stenosis at C5–6 and C6–7 levels secondary to disc herniation. A decision to perform surgical decompression and stabilization was taken after a failure of conservative trial of 12 weeks. A standard anterior cervical discectomy and fusion (ACDF) was performed at both levels using a right-sided approach. Based on anatomical landmarks and radiological guidance, adequate care was taken to expose only the presumed C5–6 and C6–7 disc levels and avoid inadvertent injury to other discs. The Casper distractor system was subsequently utilized throughout the surgery by obtaining anchorage of the Casper vertebral pins in the C5 and C7 vertebral bodies. Iliac crest autograft-packed PEEK (Solis, Stryker, Allendale, NJ, USA) cages were placed at both levels and cervical plating (Orion, Medtronic-Sofamor-Danek, Memphis, TN, USA) was performed. Prophylactic intravenous antiobiotics (cefazolin) was administered 1 h before surgery and continued for 48 h post-surgery. The immediate post-operative period was uneventful and the patient had good recovery from his neurological symptoms.
The patient presented 6 weeks later with mild neck and bilateral shoulder pain associated with paresthesia in his upper limbs, left lower limb and abdomen. On examination, the patient was afebrile and there was no warmth or tenderness in the region of the neck. The neck movements were terminally restricted, but there was no motor weakness. Hyperreflexia could be elicited in both lower limbs, Babinski’s sign was negative and there were no objective sensory deficits. Hematological investigations revealed a mild increase in ESR (30 mm/h), normal CRP (1.1 μg/ml) and a mild increase in total WBC count (8.18×109/l). Radiographs revealed persistently enlarged pre-vertebral soft-tissue shadow, decreased disc height at C4–5 level with erosion of end-plates and segmental C4–5 kyphosis, apart from the presence of halo around the screws as shown in Fig. 1. MRI revealed features of discitis at C4–5 level with associated pre-vertebral and epidural abscesses from C4 to C6 levels as well as artifacts secondary to instrumentation and cages as shown in Fig. 2a and b.
Fig. 1Lateral X-ray of cervical spine at 6 weeks after index surgery. Persistence of pre-vertebral soft-tissue shadow, erosion and irregularity of C4 and C5 end plates apart from segmental kyphosis and loosening of screws can be seenFig. 2a Sagittal T-2 weighted MRI image showing hyperintensity signal at C4–5 disc space. Anteriorly located epidural abscess at the same level can be appreciated. b Axial T-2 weighted MRI image displaying anterior compression of the spinal cord by the epidural abscess
The patient underwent an aggressive surgical debridement and stabilization through a left-sided approach. The loose screws and the plate were removed. A thorough decompression of the epidural abscess along with surgical debridement of C4–5 disc space was performed. No pus or evidence of infection was seen at the C5–6 and C6–7 levels and the cages were left behind as they appeared stable and secure. Tricortical iliac-crest autograft was impacted at the prepared C4–5 disc space and thorough lavage with antibiotic solution (cloxacillin and gentamycin) was carried out. The wound was then closed over a drain and an empirical course of intravenous antibiotic (cefazolin) was initiated. The drain was taken out after 3 days and the culture specimen grew Serratia marcescens, sensitive to ceftriaxone but resistant to the more commonly used antibiotics including cefazolin. Intravenous ceftriaxone was initiated for the first 3 weeks, and then the patient was discharged on oral antibiotics for another 3 weeks. The patient’s condition improved dramatically with complete neurological recovery within the first week. At the 8-month mark, he is asymptomatic and the X-rays performed after 5 months reveal solid fusion at all levels and obliteration of the pre-vertebral soft-tissue shadow as shown in Fig. 3.Fig. 3Lateral X-ray at 5 months after the revision surgery. Solid fusion at all levels and obliteration of the pre-vertebral soft-tissue shadow can be seen
Discussion
Post-operative discitis in cervical spine is not a frequent occurrence due to the rich vascularity and the routine use of antibiotic prophylaxis [5]. The prevalence ranges from 0 to 1.1%. The use of metal implants may increase the possibility of infection [4]. Discitis is a known complication of cervical discography [2, 11]. Occasional cases of cervical discitis secondary to a fishbone lodged in the throat have been reported [4, 7].
A case of post-operative discitis adjacent to the operated level has not been previously reported. The potential causes of adjacent level discitis at C4–5 were scrutinized. One of the possibilities was an accidental inoculation of bacteria into the C4–5 disc space intra-operatively by a contaminated spinal needle that was used as a radiological marker. When we reviewed the intra-operative localizing X-ray, it was evident that the needle was placed into the C5–6 intervertebral disc and hence this possibility was ruled out. The possibility of discitis secondary to an esophageal perforation was ruled out since the patient neither had dysphagia nor the severe systemic symptoms that usually accompany esophageal injury in the post-operative period. Additionally, no esophageal tear was noticed during the second surgery. Discitis secondary to annular injury of C4–5 intrevertebral disc was ruled out since great care was taken to avoid inadvertent exposure of uninvolved levels during surgery. Hematogenous spread is another possibility but the only positive cultures came from the C4–5 intervertebral disc during the operation. Other samples, e.g. blood, sputum, and urine cultures demonstrated no growth and chest X-ray appeared normal. The only other possibility could be inoculation of bacteria into the superior endplate of C5 and initiation of an infective process in the C4–5 peridiscal region. This could be through the contaminated Caspar pins or the drill-bit utilized to create holes for the screws used for anterior instrumentation. This is a possibility since the organism that was cultured (S. marcescens) is known to contaminate and breed in normal saline solution [3, 6], and the operative instruments in question are generally placed in seemingly sterile large bowls filled with normal saline during surgery.
Post-operative spinal infection due to S. marcescens is very rare. In a review of 2,391 consecutive index procedures on the spine, 46 cases of wound infection were identified [8]. Amongst them, only one case of Serratia infection was recognized and that was a part of mixed infection. Serratia spondylodiscitis has been reported recently involving the lumbar spine [3]. One of the patients had discitis at the same level that underwent a micro-discectomy. The second case was interesting in that her discitis occurred at the same level that had earlier undergone a posterior decompression (laminotomies and foraminotomies) but the disc was untouched. These two patients presented acutely within a week of index surgery with a fiery presentation, described as life threatening by the authors. The ESR, WBC counts and CRP levels were elevated and the blood culture grew S. marcescens. However, our patient had a subacute course and was afebrile throughout. The only abnormal features were subtle axial neck pain with subjective paresthesia in the extremities and abdomen. The ESR and WBC count were modestly raised but the CRP was normal. A high index of suspicion along with reliance on radiological investigations was essential to diagnose the condition and institute appropriate treatment. Persistence of pre-vertebral soft-tissue shadow apart from reduction in the C4–5 disc height, segmental kyphosis and irregularity of the endplates at C4–5 pointed towards a diagnosis of C4–5 discitis and prompted us to perform MRI in order to confirm the diagnosis and decide on the further management.
The literature is not very clear with respect to the duration of antibiotics to be given for spinal fusions. The protocols recommended range from no antibiotics for anterior spinal surgery [9] to 3 days of antibiotics for all spinal surgeries [10]. A meta-analysis performed to identify the efficacy of prophylactic antibiotics in spinal surgery found that there was no difference in the efficacies of various antibiotic regimens, provided at least one dose of pre-operative antibiotic with gram-positive coverage was administered [1]. Our protocol for antibiotic prophylaxis for spinal surgeries consists of a pre-operative dose followed by 2-day course of intravenous cefazolin. The protocol followed in our division is based on the fact that the drainage tube as well as the urinary catheter is removed by 48 h and hence the antibiotics are usually continued till then. S. marcescens is highly resistant to cefazolin and sensitive to third-generation cephalosporins [3], and this was evident in the culture sensitivity reports of our patient.
An aggressive approach to debride the C4–5 disc space as well as to decompress the epidural space was necessary in the wake of myelopathic symptoms and in order to isolate the organism. The spine was approached from the opposite side (left side) for the second surgery since inadvertent esophageal or neurovascular injury as a result of post-operative scarring was a possibility on the right side. The issue of leaving the PEEK cages at C5–6 and C6–7 levels behind during the revision surgery is debatable. We did not replace them because of a number of reasons. The infection was principally arising from the C4–5 disc level, the cages were absolutely stable and we wanted to minimize graft site morbidity. Most importantly, we felt that infection would not thrive in the presence of stability. This approach has been advocated by other authors too [8]. In the second surgery, a plate was not utilized for instrumentation for the following reasons. Firstly, the bones were soft as a result of inflammatory edema resulting from infection and hence were not ideal for screw implantation. Secondly, since we were also not sure what the organism was and as most of the times it is Staphylococcus aureus, the risk of persistent infection as a result of glycocalyx membrane formation is high. Additionally, the construct appeared stable after the C4–5 level was impacted with the autologous iliac crest graft and the lower two levels were already stable as aforementioned.
Conclusions
The possibility of infection should be considered in patients with an alteration in the nature of their symptoms after index surgery. Unusual presentations can be a challenge to diagnose and treat and a high index of suspicion and aggressive approach are prudent for a successful outcome.

adjacent level discitis
epidural abscess
serratia marcescens
cervical spine
cervical spondylodiscitis