bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin‐Like growth factor I and protein kinase C activation stimulate pulmonary artery smooth muscle cell proliferation through separate but synergistic pathways
Authors: Dempsey Edward C. , Stenmark Kurt R. , McMurtry Ivan F. , O'Brien Richard F. , Voelkel Norbert F. , Badesch David B.
Published: February 2005

Smooth muscle cell (SMC) hyperplasia is an important component of vascular remodeling in chronic hypoxic pulmonary hypertension. The mechanisms underlying SMC proliferation in the remodeling process are poorly understood, but may involve insulin‐like growth factor I (IGF‐I). This study investigates the potential proliferative effects of IGF‐I on SMC cultured from the pulmonary arteries (PA) of neonatal calves. We hypothesized that IGF‐I stimulates PA SMC proliferation through a protein kinase C (PKC)‐indepenent pathway, but that PKC activation would augment this proliferative response. Incorporation of 3H‐thymidine was used as an index of cellular prohteration, and was correlated with subsequent changes in cell counts. Under serum‐free conditions, IGF‐I (100 ng/ml) induced a 6‐fold increase in thymidine incorporation by quiescent PA SMC. This stimulation was not blocked by dihydrosphingosine, an inhibitor of PKC activation. Phorbol myristate acetate (PMA) (1 nM), a membrane‐permeable PKC activator, induced a 12‐fold increase in thymidine incorporation which was 70% inhibited by dihydrosphingosine. Co‐incubation with IGF‐I and PMA caused a 60‐fold increase in thymidine incorporation, which was 30% inhibited by dihydrosphingosine. This synergistic increase in thymidine incorporation was associated with a subsequent significant increase in cell number. PKC‐downregulated cells (1,000 nM PMA × 30 hr) proliferated in response to IGF‐I but not PMA, and did not demonstrate synergism with the combination of IGF‐I and PMA. The threshold concentrations of IGF‐I and PMA for synergism were approximately 1 ng/ml and 1 pM, respectively. We conclude that IGF‐I stimulates neonatal PA SMC proliferation via a PKC‐independent pathway, and that trace amounts of PKC activators are capable of synergistically augmenting this response. We speculate that the synergistic stimulation of SMC proliferation by IGF‐I and PKC activators may play an important role in hypertensive pulmonary vascular remodeling.
Comparative study of oxygen toxicity in human fibroblasts and endothelial cells
Authors: Michiels Carine , Toussaint Olivier , Remacle José
Published: February 2005

The resistance of human pulmonary fibroblasts (WI‐38) and human umbilical vein endothelial cells to oxygen toxicity (1 atm O2) was compared. Endothelial cells were more sensitive than fibroblasts. They contained also less antioxidant enzymes except for SOD: respectively 132%, 96%, 70%, 59%, and 21% of the SOD, GSH peroxidase, GSH reductase, catalase, and G6PD content of fibroblasts. However, they contained 1.81‐fold more GSH than fibroblasts. Their lower content of antioxidant enzymes can explain their higher sensitivity to oxygen. The efficiency of natural antioxidant molecules and enzymes in the protection of cells incubated 3 days under 1 atm O2 was studied. α‐tocopherol added in the culture medium led to a significant protection, contrary to the result for ascorbic acid. Microinjection of catalase, SOD, and GSH peroxidase directly into the cells was also tested: the protection was concentration dependent for both types of cells but SOD did not protect the endothelial cells. Lower activities of the other enzymes were needed to achieve protection of the endothelial cells, compared to fibroblasts. Since endothelial cells were also shown to display lower antioxidant enzyme activities, it can be hypothesized that their content is optimized for survival in Physiological conditions.
Transforming growth factor β enhances parathyroid hormone stimulation of adenylate cyclase in clonal osteoblast‐like cells
Authors: Gutierrez Gloria E. , Mundy Gregory R. , Katz Michael S. , Manning David R. , Hewlett Erik L.
Published: February 2005

The effects of transforming growth factor β (TGFβ) on parathyroid hormone (PTH)‐responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR‐106) with the osteoblast phenotype. Purified TGFβ incubated with UMR‐106 cells for 48 hr produced a concentration‐dependent increase in PTH stimulation of adenylate cyclase, with maximal increase in PTH response (37%) occurring at 1 ng/ml TGFβ. TGFβ also enhanced receptor‐mediated activation of adenylate cyclase by isoproterenol and prostaglandin E2 (PGE2) and nonreceptormediated enzyme activation by cholera toxin and forskolin. In cells in which PTH‐stimulated adenylate cyclase activity was augmented by treatment with pertussis toxin, the incremental increase in PTH response produced by TGFβ was reduced by 33%. However, TGFβ neither mimicked nor altered the ability of pertussis toxin to catalyze the ADP‐ribosylation of a 41, 000‐Da protein, presumably the α subunit of the inhibitory guanine nucleotide‐binding regulatory component (Gi) of adenylate cyclase, in cholate‐extracted UMR‐106 cell membranes. TGFβ also had no effect on the levels of α or β subunits of Gi, as assessed by immunotransfer blotting. In time course studies, brief (≥ 30 min) exposure of cells to TGFβ during early culture was sufficient to increase PTH response but only after exposed cells were subsquently allowed to grow for prolonged periods. TGFβ enhancement of PTH and isoproterenol responses was blocked by prior treatment of cells with cycloheximide but not indomethacin. The results suggest that TGFβ enhances PTH response in osteoblast‐like cells by action(s) exerted at nonreceptor components of adenylate cyclase. The effect of TGFβ may involve Gi, although in a manner unrelated to either pertussis toxin‐catalyzed ADP‐ribosylation of the α subunit of Gi or changes in levels of Gi subunits. The regulatory action of TGFβ on adenylate cyclase is likely to be mediated by the rapid generation of cellular signals excluding prostaglandins, followed by a prolonged sequence of events involving protein synthesis. These observations suggest a mechanism by which TGFβ may regulate osteoblast responses to systemic hormones.
Anti‐proliferative activity of L‐651,582 correlates with calcium‐mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase
Authors: Hupe Donald J. , Behrens Nancy D. , Boltz Robert
Published: February 2005

L‐651,582, 5‐amino‐[4‐(4‐chlorobenzoyl)‐3, 5‐dichlorobenzyl]‐1, 2, 3‐triazole‐4‐carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H‐hypoxanthine, 14C‐adenine, and 14C‐formate incorporation into nucleotide pools in Madin‐Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L‐651, 582 inhibited incorporation of 3H‐hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose‐5‐phosphate. PRPP synthetase was not inhibited directly by the compound, indicating a regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.
Distribution of vinculin in the Z‐disk of striated muscle: Analysis by laser scanning confocal microscopy
Authors: Terracio Louis , Simpson David G. , Hilenski Lula , Carver Wayne , Decker Robert S. , Vinson Nancy , Borg Thomas K.
Published: February 2005

Vinculin is a major cytoskeletal component in striated muscle, where it has been reported to form a rib‐like structure between the cell membrane and the Z‐disk termed a costamere. This arrangement of vinculin has been purported to be involved in the alignment of the myofibrils. However, the three‐dimensional arrangement of vinculin in relation to the Z‐disk of the myofibril was not known. In the present study, we examined the distribution of vinculin in striated muscle with monospecific antibodies using immunofluorescence and laser scanning confocal microscopy. Isolated cardiac and skeletal muscle cells from a variety of species, tissue sections, and neonatal myocytes with developing myofibrils were examined. Optical sectioning in the X‐Y and X‐Z planes demonstrated that vinculin immunoreactivity was heaviest at the periphery of the cell; however, the immunoreactivity was also distributed within the Z‐disk although at a relatively reduced level. This distribution is potentially significant in understanding the physiological significance of vinculin in striated muscle function and in myofi‐brillogenesis.
[D‐Arg1, D‐Phe5,D‐Trp7,9,Leu11] substance P, a neuropeptide antagonist, blocks binding, Ca2+‐mobilizing, and mitogenic effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells
Authors: Fabregat Isabel , Rozengurt Enrique
Published: February 2005

Endothelin (ET1) and vasoactive intestinal contractor (VIC) stimulate quiescent Swiss 3T3 cells to resume DNA synthesis acting synergistically with epidermal growth factors (EGF) and other mitogens. The peptide “D‐Arg1‐D‐Phe5,D‐Trp7,9Leu11” substance P has been identified as a broad spectrum neuropeptide antagonist which blocks the binding and biological effects of the Ca2+‐mobilizing neuropeptides bombesin, vasopressin, and bradykinin. In the present study we show that [D‐Arg1,D‐Phe5,D‐Trp7,9,Leu11] substance P also acts as an ET1/VIC antagonist as judged by the following criteria: (a) inhibition of specific 125l‐labelled ET1 binding to a ET1/VIC receptor in a competitive and dose‐dependent manner; (b) blocking of the rapid increase in the cytosolic Ca2+ concentration promoted by ET1 or VIC; and (c) inhibition of DNA synthesis stimulated by VIC in the presence of EGF. The inhibitory effects of [D‐Arg1,D‐Phe5,D‐Trp7,9,Leu11] substance P on Ca2+ mobilization and DNA synthesis were reversed by increasing the concentration of VIC. This is the first time that a peptide structurally unrelated to ET1 or VIC is shown to block the binding and mitogenic effects of peptides of the endothelin family.
Vitamin D regulates transferrin receptor expression by bone marrow macrophage precursors
Authors: Tanaka Hiroyuki , Teitelbaum Steven L.
Published: February 2005

1,25‐Dihydroxyvitamin D3 [1,25(OH)2D3] is known to prompt monocytic differentiation of a variety of leukemic lines. We previously extended these observations to non‐transformed bone marrow macrophage precursors by demonstrating that the steroid enhances plasma membrane expression of the macrophage‐specific mannose‐fucose receptor (Clohisy et al., J Biol Chem 262:15922–15929, 1987). Because this membrane protein is involved in non‐opsonin mediated endocytosis, these observations raised the possibility that 1,25(OH)2D3 globally upregulates endocytic receptors. The present study, aimed at addressing this issue, turns to the transferrin receptor as a paradigm for endocytic receptors and explores the impact of 1,25(OH)2D3 on its expression. We found that in contrast to the mannose‐fucose receptor, plasma membrane transferrin receptor expression by bone marrow‐derived macrophage precursors declines by at least 30% in a dose‐dependent fashion with exposure to 1,25(OH)2D3. The effect reflects diminished receptor capacity with no change in Kd, and is independent of cell cycle. Moreover, while Vmax of receptor‐ligand internalization mirrors plasma membrane occupancy, Kuptake remains unaltered in the presence of vitamin D3, indicating that the down‐regulating event does not reflect on enhanced rate of endocytosis. Further, pulse chase experiments show parallel cell surface, intracellular, and medium redistribution of radioligand with time steroid‐treated and control cells. In a similar vein, while total cell‐associated radioligand falls in the presence of vitamin D3, the percentage of intracellular and surface bound counts at equilibrium are constant in both groups. Finally, immunoprecipitation studies reveal that the down‐regulating effects of 1,25(OH)2D3 cannot be explained by inhibition of transferrin receptor synthesis. Thus, the decrease in total cellular transferrin binding sites is likely to represent either enhanced degradation or synthesis of [cryptic] receptors which fail to recognize 125 I‐transferrin.
Studies on the spermatogenic sulfogalactolipid binding protein SLIP 1
Authors: Lingwood Clifford , Nutikka Anita
Published: February 2005

We have purified the testicular sulfogalactolipid binding protein SLIP 1 and shown by photoaffinity labeling that it contains an ATP binding site. Purified SLIP 1 was fluorescently labeled and shown to retain specific Sulfogalactolipid binding function. This probe was used to investigate the topology of SLIP I binding sites on testicular germ cells. The binding pattern precisely coincided with the previously demonstrated asymmetric surface domains of sulfogalactoglycerolipid (SGG). Occasionally these SGG‐containing, SLIP 1‐binding cell surface domains exactly coincided with structural features on the cell surface as detected by differential interference contrast microscopy. These results demonstrate that SLIP 1/SGG interactions could provide an effective intercellular communication network between testicular germ cells within the seminiferous tubule.
Effects of adenosine receptor agonists on volume‐activated ion transport in pig red cells
Authors: Sohn Dong Hwan , Kim Hyun Dju
Published: February 2005

Swelling of pig red cells leads to an increase in a chloride‐dependent K flux which can be potentiated by cAMP, whereas cell shrinking causes a selective increase in Na movement which is mediated by a Na/H exchanger. We examined the influence of adenosine and adenosine receptor agonists on the volume‐sensitive, ouabain‐resistant, chloride‐dependent K flux, referred to as Rb flux and volume‐activated Na/H exchange pathway. It was found that adenosine and adenosine receptor agonists inhibited the Rb flux. N6‐cyclohexyl adenosine (CHA) has been found to be the most potent inhibitor with EC50 of approximately 4.5 μM followed by 2‐chloroadenosine (Cl‐ado) with EC50 of approximately 27 μM and 5′‐(N‐ethyl)‐carboxamido‐adenosine (NECA) with EC50 of approximately 185 μM. CHA also inhibits the cAMP‐stimulated Rb flux. However, CHA does not alter the basal intracellular cAMP level nor the intracellular cAMP content raised by exogenously added cAMP. In contrast to the adenosine agonist action on the Rb flux, Na/H exchange, which is activated upon cell shrinkage, exhibits a slight stimulation in response to CHA. These findings suggest that the presence of A1 adenosine receptors on the surface of red cells influences the regulation of volume‐activated ion transport.
Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures
Authors: Leboy Phoebe S. , Beresford Jon N. , Devlin Carole , Owen Maureen E.
Published: February 2005

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or I μM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25‐dihydroxyvitamin D3 to these dexamethasone‐treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25‐dihydroxyvitamin D3‐treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone‐treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.
Fibroblasts inhibit mineralised bone nodule formation by rat bone marrow stromal cells in vitro
Authors: Ogiso Bunnai , Hughes Francis J. , Melcher Antony H. , McCulloch Christopher A. G.
Published: February 2005

The ability of rat skin fibroblasts (RSF) and human periodontal ligament fibroblasts (HPL) to inhibit the formation of mineralised bone nodules in rat bone marrow stromal cell (BMSC) cultures was studied. Co‐culture of HPL or RSF with BMSC resulted in a large reduction of bone nodule formation when compared with controls. Conditioned medium from HPL or RSF cultures inhibited bone nodule formation in a dose‐dependent manner. HPL‐conditioned medium depressed cell proliferation and alkaline phosphatase expression in BMSC cultures. These effects were not due to increased cytotoxicity or nutrient depletion. Inhibitory activity was recovered in a fraction of less than 1 kD following ultrafiltration and was insensitive to freeze‐thawing. The inhibitory activity was blocked when HPL cultures were grown in the presence of 105M indomethacin. Dose‐dependent inhibition of bone nodule formation was also observed in cultures incubated with prostaglandins E2 (at 10−6M) or F2α(at 10−7). The results indicate that fibroblasts may inhibit osteoblast differentiation and function in part by release of soluble factors including prostaglandins.
The RGD containing site of the mouse laminin A chain is active for cell attachment, spreading, migration and neurite outgrowth
Authors: Tashiro Ken‐Ichiro , Sephel Gregory C. , Greatorex Dave , Sasaki Makoto , Shirashi Norio , Martin George R. , Kleinman Hynda K. , Yamada Yoshihiko
Published: February 2005

The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD‐containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA‐6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.
Regulation of ornithine decarboxylase mRNA by phorbol esters and insulin in normal and C‐kinase‐deficient rat hepatoma cells
Authors: Butler Andrew P. , Cohn William B. , Mar Penny K. , Montgomery Raechelle L.
Published: February 2005

Tumor‐promoting phorbol esters and insulin produce similar effects in Reuber H35 rat hepatoma cell proliferation, including increased ornithine decarboxylase (ODC) enzyme activity, DNA synthesis, and mitogenesis. We investigated ODC mRNA accumulation in cells treated with either insulin or 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). Both agents caused rapid accumulation of ODC mRNA: for TPA, it was maximal 3 hr after treatment (4–6‐fold greater than control cells) and returned quickly to control levels; for insulin, it was significantly longer, continuing to increase for at least 6 hr. Simultaneous treatment with TPA and insulin led to additive effects on ODC mRNA. Induction of ODC by TPA was blocked by down‐regulation or inhibition of protein kinase C (PKC), consistent with a PKC‐mediated mechanism. In contrast, PKC down‐regulation had little effect on ODC induction by insulin. Furthermore, although both agents stimulated ribosomal S6 protein phosphorylation in cells containing normal amounts of PKC, the response to TPA was abolished in PKC‐depleted cells; the effect of insulin was only slightly inhibited. TPA caused a rapid redistribution of essentially all of the PKC activity from the cytosolic to the membrane fraction of the cells, whereas insulin had no effect on PKC distribution. These results suggest that although insulin and TPA share some common cytoplasmic signalling pathways, their effects on phosphorylation of nuclear proteins and transcription of ODC may be mediated by distinct factors.
Effect of 2% dimethyl sulfoxide on the mitogenic properties of epidermal growth factor and hepatocyte growth factor in primary hepatocyte culture
Authors: Kost Diane P. , Michalopoulos George K.
Published: February 2005

Two percent dimethyl sulfoxide (DMSO) reversibly inhibited DNA synthesis in primary rat hepatocyte cultures maintained with epidermal growth factor (EGF) or hepatocyte growth factor (HGF). These data suggest that, in vitro, DMSO is a non‐specific inhibitor of hepatocyte proliferation, regardless of the stimulating mitogen. In addition, removal of DMSO from mitogen‐free cultures resulted in an increase in DNA synthesis. Protein synthesis gradually but irreversibly declined in all cultures after DMSO removal. The relevance of these findings to regulation of hepatocyte growth is discussed.
Culture perfusion schedules influence the metabolic activity and granulocyte‐macrophage colony‐stimulating factor production rates of human bone marrow stromal cells
Authors: Caldwell Jerry , Palsson Bernhard O. , Locey Betty , Emerson Stephen G.
Published: February 2005

The metabolic function and GM‐CSF production rates of adherent human bone marrow stromal cells were investigated as functions of medium and serum feeding rates. A range of medium exchange schedules was studied, ranging from a typical Dexter culture protocol of one weekly medium exchange to a full media exchange daily, which more closely approximates what bone marrow cells experience in situ. Glucose consumption was found to be significantly higher at full daily exchange rate than at any other exchange schedule examined. However, the lactate yield on glucose was a constant, at 1.8 mol/mol, under all conditions considered. Differential serum vs. medium exchange experiment showed that both serum supply and medium nutrients were responsible for the altered behavior at high exchange rates. Glutamine consumption was found to be insignificant under all culture conditions examined. A change in exchange schedule from 50% daily medium exchange to full daily medium exchange after 14 days of culture was found to result in a transient production of GM‐CSF and a change in metabolic behavior to resemble that of cultures which had full daily exchange from day one. These results suggest that both stromal cell metabolism and GM‐CSF production are sensitive to medium exchange schedules. Taken together, the data presented indicate that attempts to model the function of human bone marrow in vitro may be well served by beginning with medium exchange schedules that more closely mimic the in vivo physiologic state of bone marrow.
Proliferation of cultured fibroblasts is inhibited byL‐Iduronate—containing glycosaminoglycans
Authors: Westergren‐Thorsson Gunilla , Önnervik Per‐Ola , Fransson Lars‐ÅKe , Malmström Anders
Published: February 2005

We have modified a method (Gilles et al:Anal. Biochem., 159:109–113, 1986) for measuring cell number, that is basec on the binding of crystal violet to cell nuclei and used it to assay effects of various glycosaminoglycans on growth of human lung fibroblasts. The procedure was modified by growing cells in microcultures (96 well microplates) and by measuring the amount of adsorbed dye using a microplate photometer after solubilisation of the cells with detergent. There was a linear relationship between absorbance and cell number measured by a Coulter Counter. The method is rapid and sensitive and can be used for screening many samples as well as measuring growth rates at low initial cell densities. Even the low growth rates obtained in the absence of serum can be detected. Human lung fibroblasts were growth arrested by serum deprivation and then grown for periods of up to 4 days in the presence of serum and exogenously added glycosaminoglycans (range, 0.1–100 μg/ml). Hyaluronan, chondroitin sulfate, and dextran sulfate were without effects, whereas dermatan sulfate, certain heparan sulfates, and heparin suppressed growth (20%–50% inhibition). The antiproliferative activity of dermatan sulfate increased with increasing iduronate content. Certain heparan sulfates with moderately high sulfate andL‐iduronate contents were better inhibitors than heparin despite the fact that the latter glycan has even higher sulfate andL‐iduronate contents. The antiprolifera‐tive effect of exogenous glycans appeared after a lag period of 3–4 days, suggesting that they interfered with factors produced by the cells. Furthermore, the degree of inhibition was greater when the initial cell density was low. Above a certain level of seeded cells (approx. 10,000 cells/well), there was no inhibition by any of the glycosaminoglycans. It is possible that exogenous glycans cannot overcome endogenous growth‐promoting effects in densely seeded cultures.
Heavy metals induce expression of the TPA‐inducible sequence (TIS) genes
Authors: Epner Daniel E. , Herschman Harvey R.
Published: February 2005

We previously cloned a set of primary response genes, which we call TIS (TPA‐Inducible Sequence) genes, from a cDNA library prepared from Swiss 3T3 cells treated with tetradecanoyl phorbol acetate (TPA) and cyclohexirnide. TPA, polypeptide growth factors, and serum induce TIS gene expression in 3T3 cells. We now report that cadmium and zinc elevate mRNA levels for the TIS genes, including TIS28 (c‐fos), in Swiss 3T3 cells. The time‐Course of TIS gene mRNA accumulation after metal exposure is delayed in comparison to the accumulation of TIS gene mRNA after treatment with TPA and growth factors. Cadmium induction of the TIS gene message accumulation is blocked by actinomycin D. Moreover, cadmium treatment does not significantly stabilize TIS gene messages. TIS gene induction by metal is a primary response; TIS8, which encodes a zinc‐finger transcription factor, and TIS28 (c‐fos), can be induced in the presence of cadmium and cycloheximide, an inhibitor of protein synthesis. Downregulation of protein kinase C does not attenuate TIS gene induction by heavy metals.
Aluminum fluoride stimulates inositol phosphate metabolism and inhibits expression of differentiation markers in mouse keratinocytes
Authors: Lee Edmund , Yuspa Stuart H.
Published: February 2005

Mouse keratinocytes are induced to differentiate in vitro by elevating the level of extracellular calcium from 0.05 mM, where keratinocytes express a basal cell phenotype, to >0.10 mM, where they express the differentiated phenotype. This process has been associated with a rapid, sustained increase in inositol phosphate (InsP) turnover, which precedes the expression of differentiation‐specific proteins. In 0.05 mM Ca2+ medium, aluminum and fluoride salts (AIF), which combine to activate honspecifically heterotrimeric guanine nucleotide‐binding (G) proteins, cause a concentration‐dependent increase in InsP metabolism in kerati‐nocytes and generate elevated intracellular diacylglycerol levels. This is associated with an inhibition of cell growth. Treatment with both AlF and Ca2+ >0.10 mM resulted in an additive increase in InsP turnover, implying the presence of at least two responsive InsP pools. AlF inhibited the expression of differentiation markers induced by Ca2+ >0.10 mM and altered the morphology of keratinocytes from squamous to dendritic, which was reversible upon withdrawal of AlF. Neoplastic keratinocytes, in which basal levels of InsP metabolism are higher than in normal cells, do not differentiate in response to Ca2+. Neoplastic keratinocytes responded to AlF treatment with an even greater rise in InsP metabolism. AIF also inhibited cell growth and reversibly altered morphology in neoplastic keratinocytes. These data suggest that InsP metabolism in keratinocytes is at least partially regulated by a G protein mechanism. Furthermore, an increase in InsP metabolism is not sufficient to stimulate differentiation and may be inhibitory to differentiation if exceeding limited increases. However, these observations cannot exclude the possibility that other AlF‐stimulated pathways involving G or non‐G proteins can also influence keratinocyte biology.
Involvement of second messenger systems in stimulation of angiotensin converting enzyme of bovine endothelial cells
Authors: Dasarathy Yamuna , Fanburg Barry L.
Published: February 2005

We have demonstrated previously that a variety of agents including corticosteroids, thyroid hormone, cationophores, methylxanthines, and analogues of cAMP–all of which have diversified functions in various tissues–elevate cellular angiotensin converting enzyme (ACE) activity of bovine endothelial cells in culture. In addition to these agents, we have now found that direct and receptor‐mediated stimulators of adenylate cyclase, i.e., forskolin and cholera toxin, increase cellular ACE activity after 48 h incubation in culture. In an attempt to search out a more unifying concept of these stimulatory effects, we have further investigated the roles of second messengers in the stimulatory actions. Ca2+ ionophore, A23187 produced significant increases in both intracellular Ca2+ and ACE of endothelial cells. In contrast to Ca2+ ionophore, agents that transiently mobilize Ca2+ from intracellular reserves such as bradykinin, acetylcholine, and ATP have no effect on the level of cellular ACE. Representative agents that elevate cellular cAMP (e.g., isobutyl methylxanthine [IBMX] and dibutyryl cAMP) elevated cellular ACE, but the slightly increased [Ca2+], produced by these agents did not reach statistical significance. While IBMX, cholera toxin, and forskolin elevated cellular cAMP, other ACE stimulatory agents (hormones and cationophores) had no effect on cAMP. Ca2+ ionophore and the agents that elevated intracellular cAMP potentiated the effect of dexamethasone, thyroid hormone, and aldosterone in elevating cellular ACE activity. Increases in ACE activity produced by all stimulants were inhibited by the presence of 10–50 nM ouabain in the culture medium. Inhibition of ACE elevation by ouabain was reversed by increasing the extracellular [K+], thereby implicating Na+, K+‐ATPase in the ACE regulatory mechanism. These results support the presence of multiple independent mechanisms for the regulation of cellular ACE. In addition to possible involvement of intracellular Ca2+‐ and cAMP‐dependent pathways, ACE is also increased by corticosteroids and thyroid hormone through mechanisms unrelated to Ca2+‐and cAMP.
Modulation of ATP and drug binding by monoclonal antibodies against P‐glycoprotein
Authors: Georges Elias , Zhang Jian‐Ting , Ling Victor
Published: February 2005

The role of P‐glycoprotein in mediating the drug‐resistance phenotype in multidrug resistant cells is now well documented. It is thought to function as an energy‐dependent drug‐efflux pump of broad specificity. Structurally, P‐glycoprotein is an internally duplicated molecule containing two large multi‐spanning transmembrane domains and two cytoplasmic ATP binding domains. In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P‐glycoprotein molecule inhibit ATP binding to P‐glycoprotein in vitro. We also provide direct evidence that both predicted ATP‐binding domains bind ATP and that there is co‐operativity between the two sites. In addition, the capacity of P‐glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies. These observations are the first evidence linking specific perturbations of the P‐glycoprotein molecule with ATP and drug binding.
Fetuin and alpha‐2HS glycoprotein induce alkaline phosphatase in epiphyseal growth plate chondrocytes
Authors: Ishikawa Yoshinori , Wu Licia N. Y. , Valhmu Wilmot B. , Wuthier Roy E.
Published: February 2005

A previously described chondrocyte alkaline phosphatase induction factor (CAP‐IF) for chicken epiphyseal growth plate chondrocytes has been purified to SDS‐PAGE homogeneity from fetal bovine serum by ammonium sulfate precipitation and by dye‐ligand affinity (Affi‐Gel Blue and Reactive Green‐19 agarose) and hybroxyapatite column chromatographies. As determined by immunoprecipitation of [35S]methionine‐labeled cellular proteins after 3 day treatment, this highly purified CAP‐IF increases the level of AP and certain other membrane proteins 2‐ to 3‐fold over control values. The pure protein of apparent 64.5 kDa molecular weight has been identified as fetuin by N‐terminal amino acid sequencing. This was confirmed by the finding that high alkaline phosphatase (AP)‐inducing activity is present in fetuin prepared by the Spiro method. However, fetuins prepared by the Pedersen or Deutsch procedures are inactive. At least half of the CAP‐IF activity of fetuin was irreversibly destroyed by treatment with EDTA and addition of Zn2+ did not reactivate the EDTA‐treated fetuin. Ascorbate synergistically enhanced the effect of fetuin on chondrocyte AP activity by over 8‐fold during 3 day exposure. Because of the very high homology between fetuin and the A‐chain of α2‐HS glycoprotein, we also tested and found that α2HS glycoproteins from human serum and bovine bone are both strong AP inducers. Our findings suggest that the AP‐inducing activity resides in a labile, cystatin/Zn2+‐binding domain common to these related serum glycoproteins. These proteins appear to play a role in enhancing AP expression in normal growth plate cartilage differentiation.
Signal transduction by bFGF, but not TGFβ1, involves arachidonic acid metabolism in endothelial cells
Authors: Fafeur VéRonique , Jiang Zhi Ping , Böhlen Peter
Published: February 2005

We investigated the stimulation of early cellular events resulting from the interaction of the growth factor basic FGF (bFGF) and of the growth inhibitor transforming growth factor beta‐type 1 (TGFβ1), with their specific receptors on bovine endothelial cells. At mitogenic concentrations, bFGF stimulated the rapid release of arachidonic acid and its metabolites from (3H)‐arachidonic acid labeled cells. When arachidonic acid metabolism was stimulated by addition of the calcium ionophore A23187, the effect of bFGF was amplified. Nordihydroguaïaretic acid, an inhibitor of the lipoxygenase pathway of arachidonic acid metabolism, decreased the mitogenic effect of bFGF, whereas indomethacin, an inhibitor of the cyclooxygenase pathway, was ineffective. These findings suggest that metabolism of arachidonic acid to lipoxygenase products may be necessary for the mitogenic effect of bFGF. Basic FGF did not stimulate the production of inositol phosphates from cells labelled with myo‐(2‐3H)‐inositol nor did it induce calcium mobilization, as measured by fura‐2 fluorescence, indicating that bFGF does not activate phosphoinositide specific phospholipase C in endothelial cells, but rather, that bFGF‐induced arachidonic acid metabolism is mediated by another phospholipase. TGFβ1, which inhibits basal and bFGF‐induced endothelial cell growth, had no effect on arachidonic acid matabolism and inositol phosphate formation and did not prevent bFGF‐induced arachidonic acid metabolism. These results suggest that the inhibitory action of TGFβ1 on endothelial cell growth occurs through different mechanisms.
Multiple pathways for ligand internalization in rat hepatocytes II: Effect of hyperosmolarity and contribution of fluid‐phase endocytosis
Authors: Moss Anita L. , Ward Walter F.
Published: February 2005

In a companion report (Moss and Ward: J. Cell. Physiol. 149:313–318, 1991) evidence was presented for multiple pathways for insulin internalization based on differences between the internalization of insulin and that of two other ligands, asialofetuin (Afet) and epidermal growth factor (EGF), in the presence of several perturbations of endocytosis. In the present study we have explored the characteristics of three internalization pathways and the contribution of each to overall insulin uptake. Freshly isolated hepatocytes were incubated with radiolabeled ligands in the presence of hyperosmolar sucrose, treatment that is thought to inhibit the coated pit pathway of endocytosis. Insulin internalization was decreased approximately 39%, but much greater decreases were observed with Afet (86%) and EGF (62%). Competition between uptake of radiolabeled and unlabeled insulin was observed in hyperosmolar‐treated cells, suggestive of endocytosis by a receptor‐mediated noncoated‐pit pathway. Uptake of radiolabeled insulin that persisted in the presence of hyperosmolarity and high concentrations of unlabeled insulin suggested a third uptake pathway: fluid‐phase endocytosis. A rate of fluid‐phase endocytosis of 7.2 μL/hr/106 cells was determined from the uptake of the fluid‐phase marker lucifer yellow. At high insulin concentrations (≥ 250 ng/ml), fluid‐phase endocytosis appears to be the predominant pathway for insulin uptake, but at lower insulin concentrations (physiological) the coated pit and noncoated pit pathways are the primary routes for insulin internalization.
Characterization of spleen colonies derived from mice with mutations at the w locus
Authors: Barker Jane E. , Starr Eleanor
Published: February 2005

Mice with mutations at the W locus have a hemopoietic stem cell defect characterized by an apparent deficiency of spleen colony forming cells (CFU‐S). In the present report, we provide evidence that mutant cells form colonies and we compare the characteristics of the colonies derived from mutant and normal cells. To perform the colony‐derivation studies, marrow cells were transferred into lethally irradiated congenic hosts that differed from the donors in the ubiquitous genetic marker, glucose phosphate isomerase (GPI‐1). Donor GPI‐1 comprised over 50% of the marker in the host spleen and marrow by 12 days post injection, regardless of whether the donor was mutant or normal. To characterize the colonies, serially sectioned host spleens were examined microscopically. Colonies are present by 8 days post‐transplantation regardless of donor genotype, but mutant colonies are distinctly different from normal colonies. The proportion of blast and granulocyte colonies is always greater in W/Wv than in +/+ recipients. Unlike the W/Wv donors, the +/+ donors generate primarily erythrocyte colonies at 8, 10, and 14 days and mixed colonies at 12 days post‐injection. Colonies from the mutant mice are generally smaller but visible colonies do appear by 12 days. The results are consistent with the notion that the anemia in W/Wv mice is caused by the early restriction of differentiating cells to a non‐erythrocyte lineage accompanied by the delayed amplification of mutant hemopoietic cells. Whether this means erythrocyte‐committed cells are absent or are present but unable to respond to the appropriate cytokines is not possible to determine from the current experiments.
Differential regulation of cerebroside sulfotransferase and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase by basic fibroblast growth factor in relation to proliferation in rat oligodendrocyte cultures
Authors: Fressinaud Catherine , Sarliève Louis L. , Dalençon Dominique , Labourdette Gérard
Published: February 2005

Previous results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol., 141:667–674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocere‐broside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet‐derived growth factor, and high insulin concentrations. In contrast, transforming growth factor β or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC +) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20‐day‐old OL cultures grown in chemically defined medium. The dose‐response curve of [125I] lododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA‐C). From 10−8 to 10−5M ARA‐C there was a dose‐dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10−6M ARA‐C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide‐expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly after the arrest of OL precursor division.
Low intracellular pH induces redistribution of fodrin and instabilization of lateral walls in MDCK cells
Authors: Eskelinen Sinikka , Huotari Virva , Sormunen Raija , Palovuori Riitta , Kok Jan Willem , Lehto Veli‐Pekka
Published: February 2005

We have studied the effect of intracellular pH on the establishment and maintenance of the cellular polarity in MDCK cells by utilizing nigericin which causes lowering of the cytoplasmic pH. At pH below 6.5, MDCK cells lost their polarized morphology and became roundish, with an increased apical area and shortened and unstable lateral walls. The lateral wall marker proteins uvomorulin and Na, K‐ATPase remained segregated to the lateral walls in the acidified cells, as shown by immunofluorescence microscopy. Fodrin, on the other hand, was released from its normal basolateral residence and was found in the cytoplasm. Actin, which normally co‐localizes with fodrin along the basolateral walls, showed a dotty distribution in the cytoplasm of acidified cells, while stress fibers remained intact. Microtubular network appeared flattened, but the Golgi complex retained its apical position. The pH change–induced alterations were readily reversible, as the normal basal‐apical polarity (columnar shape, distinct apical and lateral domains with apposing and stiff lateral membranes) was reformed within 10 minutes after restoring the normal pH gradient across the cell membrane. This coincided with the translocation of fodrin from the cytoplasm to the lateral walls. The results show that lowering of intracellular pH leads to temporary segregation of fodrin from the other components of the membrane skeleton assembly, and that association of fodrin with the lateral walls seems to be a prerequisite for their close apposition and for the maintenance of normal basal‐axial polarity.
Degradation of collagen in the bone‐resorbing compartment underlying the osteoclast involves both cysteine‐proteinases and matrix metalloproteinases
Authors: Everts Vincent , Delaissé Jean‐Marie , Korper Wolf , Niehof Anneke , Vaes Gilbert , Beertsen Wouter
Published: February 2005

The site of action of cysteine‐proteinases (CPs) and matrix metalloproteinases (MMPs) in the degradation of bone collagen by osteoclasts was investigated by evaluating the effects of the CP‐inhibitor trans‐epoxy‐succinyl‐L‐leucylamido (4‐guanidino)‐butane (E‐64) and the MMP‐inhibitor N‐(3‐N‐benzyloxycarbonyl amino‐1‐R‐carboxypropyl)‐L‐leucyl‐O‐methyl‐L‐tyrosine N‐methylamide (Cl‐1) in an in vitro model system of PTH‐stimulated mouse calvaria. In the presence of each of the two inhibitors a large area of collagen free of mineral crystallites was seen adjacent to the ruffled border of the osteoclasts. Following a culture period of 24 h this area proved to be about 10 times larger in inhibitor‐treated explants than in controls. Moreover the percentage of osteoclasts in close contact with such demineralized bone areas appeared to be significantly higher in inhibitor‐treated explants than in control specimens (60% and 5%, respectively). These effects were not apparent when the osteoclastic activity was inhibited with calcitonin. No significant differences were found between the effects of the two inhibitors, E‐64 and Cl‐1. Our observations indicate that under the influence of inhibitors of MMPs and CPs demineralization of bone by osteoclasts proceeded up to a certain point whereas matrix degradation was strongly inhibited. It is concluded that within the osteoclastic resorption lacuna both CPs and MMPs participate in the degradation of the collagenous bone matrix.
Subtypes of betaglycan and of type I and type II transforming growth factor‐β (TGF‐β) receptors with different affinities for TGF‐β1 and TGF‐β2 are exhibited by human placental trophoblast cells
Authors: Mitchell E. Jane , Fitz‐Gibbon Linda , O'Connor‐McCourt Maureen D.
Published: February 2005

Transforming growth factor‐β is likely to be an important factor controlling placental activities, including growth, differentiation, invasiveness, hormone production, and immunosuppression. We have used a chemical cross‐linking technique with either 125I‐TGF‐β1 or 125I‐TGF‐β2 and bis(sulfosuccinimidyl) suberate (BS3) to characterize TGF‐β binding components on human placental cells in primary culture. Trophoblast‐enriched primary cultures exhibited a predominant affinity‐labelled complex characteristic of membrane‐anchored betaglycan (formerly termed the Type III TGF‐β receptor) and relatively low levels of the Type I and Type II TGF‐β receptor complexes. The results from affinity labelling saturation and competition experiments with TGF‐β1 and TGF‐β2 suggest the existence of two distinct subtypes of betaglycan: one subtype has a lower capacity and higher affinity, binds both TGF‐β1 and TGF‐β2, yet has a preferential affinity for TGF‐β2; the second subtype has a higher capacity and lower affinity and binds TGF‐β1 exclusively. In contrast, mesenchymal cell‐enriched placental primary cultures possessed only one subtype of the betaglycan component that binds the two TGF‐β isoforms with similar affinities and capacities as observed on most cell lines. These experiments demonstrate that the betaglycan component which exhibits a higher affinity for TGF‐β2 than for TGF‐β1, that we had observed previously on term placental membranes, is actually present on trophoblast cells. In addition to the two distinctive betaglycan subtypes, subtypes of the Type I and II TGF‐β receptors were detected on the trophoblast‐enriched cultures. In competition experiments, when 125I‐TGF‐β1 was used as the radiotracer, the Type I and II TGF‐β receptors show a much higher affinity for TGF‐β1 than for TGF‐β2, as observed with other cell types. However, when 125I‐TGF‐β2 was used, low abundance subtypes of both the Type I and II receptors that show similar affinities for TGF‐β1 and TGF‐β2 were also revealed.