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P V Bodine

Publications and source records attributed to P V Bodine.

At least 19 recordsLinked to original sources
PubMed1996

Development and characterization of a conditionally transformed adult human osteoblastic cell line.

Many osteoblastic cell lines are currently in use, but these have limitations either in terms of their relevance to adult human biology and disease or in terms of their suitability for biochemical and molecular analyses. Consequently, we undertook the development of conditionally transformed adult human osteoblastic cell lines. Osteoblasts were obtained from a normal explant cancellous bone chip culture. These cells were infected with adenovirus-ori-SV40 tsA 209, which encodes a temperature-sensitive large T-antigen mutant. Cells immortalized with this virus express a transformed phenotype at the permissive temperature of 34 degrees C but revert to a normal phenotype at the nonpermissive temperature of 40 degrees C. Using this approach, we have isolated several cell clones and describe the characterization of one that was designated HOB-02-C1. Immunocytochemistry revealed that > 95% of the cells express the large T-antigen at both temperatures. These cells exponentially proliferate at 34 degrees C with a doubling time of approximately 2 days but irreversibly stop dividing at 40 degrees C. However, cell volume increases > 2-fold when the cells are maintained for 6 days at the higher temperature. This clone expresses alpha 1 type (I) procollagen mRNA and secretes type I procollagen C-peptide at both temperatures, although the levels were slightly elevated at 40 degrees C. The cell line expresses alkaline phosphatase activity at 34 degrees C, and the basal level of this enzyme increases 2- to 6-fold at 40 degrees C. Alkaline phosphatase activity is induced 4- to 8-fold by 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures, but transforming growth factor-beta 1 (TGF-beta 1) suppresses enzyme expression > 90% at 40 degrees C. Vitamin D3 also induces a 10-fold increase in osteocalcin secretion when the clone is maintained at 34 degrees C, and this induction is enhanced > 8-fold at 40 degrees C. Parathyroid hormone and forskolin stimulate a 4- to 6-fold increase in the production of intracellular cyclic AMP (cAMP) by the cells at 34 degrees C, and this stimulation is enhanced 2- to 4-fold at 40 degrees C. In contrast, prostaglandin E2 stimulates a 7- to 8-fold increase in cAMP only when the cells are maintained at 34 degrees C. This cell line secretes TGF-beta 1 and interleukin-6 (IL-6) at 34 degrees C, but only the basal secretion of IL-6 increases 70% at 40 degrees C. Finally, alizarin red-S histochemical staining demonstrates that these cells produce mineralized nodules at both temperatures. In summary, the results of this study indicate that the HOB-02-C1 cells have a mature osteoblastic phenotype. Consequently, this new cell line and others obtained in a similar fashion should be valuable in vitro tools for cellular, biochemical, and molecular studies of adult human osteoblast biology.

Adenoviridae↗
PubMed1996

Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone.

Osteocytes are differentiated forms of osteoblasts that arise upon entrapment within the bone matrix. In this report, we describe the establishment and hormonal regulation of the first conditionally transformed human preosteocytic cell line. Primary adult bone cells were obtained from protease cell line. Primary adult bone cells were obtained from protease digestion of cancellous chips. The cells were infected with adenovirus-ori- SV40 tsA 209, which encodes for a temperature-sensitive large T-antigen. After immortalization, we isolated a clone designated HOB-01-C1. This cell line expressed the mutant T-antigen and proliferated at the permissive temperature (34 C) but stopped dividing at the nonpermissive temperature (39-40 C). Electron microscopy of cells incubated at 39 C demonstrated the presence of preosteocytic cellular processes, some of which appeared to form gap junctions or were rich in microfilaments. The clone expressed alpha 1 type (I) procollagen messenger RNA (mRNA) and secreted type I procollagen C peptide at both temperatures, and this expression was elevated 1.6-fold to 1.8-fold at 40 degrees C. The cells expressed very low basal levels of alkaline phosphatase activity (approximately 0.02 nmol/min.mg), which was increased 2- to 5-fold in a dose-dependent manner by 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (vitamin D3) at both temperatures. Vitamin D3 also increased osteocalcin secretion in a dose-dependent manner when the clone was maintained at 34 C (approximately 6-fold), and this stimulation was enhanced > 5 fold at 40 C. In contrast to the low expression of alkaline phosphatase, the cells secreted high amounts of osteocalcin in response to vitamin D3 (approximately 15 ng/mg cell protein); this biochemical profile also resembled that of preosteocytes. Alizarin red-S histochemical staining demonstrated that these cells rapidly produced mineralized nodules at both temperatures. PTH (10 and 100 nM) had no effect on the intracellular accumulation of cAMP at 34 C but stimulated a 14- to 18-fold increase in the production of this second messenger at 40 C. In contrast, 100 nM prostaglandin E2 and 1 microM forskolin stimulated cAMP synthesis better at 34 C. Western blot analysis indicated that the cells expressed CD44, a putative osteocytic marker, at both temperatures. Finally, interleukin-1 beta and tumor necrosis factor-alpha (1-1000 pM) stimulated dose-dependent increases in the secretion of interleukin-6 and monocyte chemoattractant protein-1 at 34 C and 40C. We conclude that the HOB-01-C1 cell line has a preosteocytic phenotype. Moreover, these cells respond to calcitropic hormones and bone resorbing cytokines.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Modulator inhibits nuclear translocation of the glucocorticoid receptor and inhibits glucocorticoid-induced apoptosis in the human leukemic cell line CEM C-7.

Modulator is an endogenous low-molecular-weight regulator of both glucocorticoid and mineralocorticoid receptors as well as protein kinase C. Structural analysis of modulator purified to apparent homogeneity suggests that it is a novel ether aminophosphoglyceride. In this report, we show that modulator inhibits cytosolic human glucocorticoid receptor (GR) complex activation as measured by DNA-cellulose binding. In addition, modulator blocks glucocorticoid-induced nuclear translocation of the GR in intact human leukemic (CEM C-7) cells, as illustrated by immunocytochemical localization. Furthermore, we demonstrate that modulator, by blocking the activation and subsequent translocation of GR, inhibits glucocorticoid-mediated apoptosis, characterized by chromatin condensation, internucleosomal DNA fragmentation, and cell death in glucocorticoid-sensitive CEM C-7 cells. Modulator inhibits glucocorticoid-induced c-myc gene repression and glucocorticoid receptor gene up-regulation. These data suggest that modulator functions to regulate the GR in intact cells as well as in cytosolic preparations. In addition, the inhibition of glucocorticoid-induced programmed cell death by modulator sheds light on the cellular function of modulator as well as on the mechanism by which apoptosis occurs in CEM C-7 cells.

Amino Acid Sequence↗
PubMed1995

Regulation of c-fos expression and TGF-beta production by gonadal and adrenal androgens in normal human osteoblastic cells.

Although the role of estrogens in bone formation is becoming clarified, the function of androgens in this process remains to be defined. Consequently, we have explored the mechanism of action for both gonadal and adrenal androgens in normal human osteoblastic (hOB) cells, which are responsible for the synthesis and mineralization of bone. Changes in the steady-state mRNA levels for two nuclear proto-oncogenes (c-fos and c-jun) and one cytokine (TGF-beta 1) were quantified in response to short (30 min) and long (24-48 h) treatments of these cells with physiologic concentrations of steroids. In addition, the levels of TGF-beta protein in the hOB cells conditioned-media were measured using a bioassay. The results indicated that neither 10 nM dihydrotestosterone, 10-20 nM testosterone, nor 10-100 nM androstenedione had a significant effect on the steady-state levels of c-fos, c-jun, or TGF-beta 1 mRNAs. Interestingly, 10-1000 nM dehydroepiandrosterone (DHEA) and 1-10 microM DHEA-sulfate rapidly reduced the steady-state level of c-fos mRNA by 60-80% in a dose-dependent manner within 30 min. In contrast, neither of these adrenal steroids had a significant effect on the message levels for c-jun or TGF-beta 1. Surprisingly, although TGF-beta 1 mRNA levels remained unchanged, the total amount of TGF-beta activity in the hOB cell conditioned-media increased 2-5-fold in response to 24-48 h treatments of the cells with gonadal or adrenal androgens. This increase in TGF-beta activity by DHEA-sulfate was both time- and dose-dependent, and was not blocked by cotreatment with the specific aromatase inhibitor 4-hydroxyandrostenedione (1 microM). Immunoprecipitations of the hOB cell conditioned-media with isoform-specific TGF-beta neutralizing-antibodies indicated that TGF-beta 2 was predominantly produced by the cells in response to DHEA-sulfate treatment. These results demonstrate that differences exist between the actions of estrogens and androgens on normal human osteoblasts with regard to the regulation of c-fos expression and TGF-beta production. Moreover, these data suggest that DHEA and DHEA-sulfate may play a distinct role in the regulation of human osteoblast function via the rapid repression of c-fos message levels and the slower increase in TGF-beta 2 protein levels.

Androgens↗
PubMed1995

Synthetic peptides derived from the steroid binding domain block modulator and molybdate action toward the rat glucocorticoid receptor.

Modulators are endogenous low-mol-wt inhibitors of glucocorticoid receptor activation, and are mimicked by exogenous sodium molybdate. Activation involves the dissociation of the 90-kDa heat-shock protein from the receptor. The heat-shock protein is thought to bind to a conserved 20-amino-acid region in the steroid-binding domain of the receptor (595-614 of the rat protein). Synthetic peptides corresponding to this amino acid sequence prevented the modulators and sodium molybdate from inhibiting receptor activation. These results imply that the region 595-614 of the rat glucocorticoid receptor is also a modulator/molybdate binding site.

Amino Acid Sequence↗
PubMed1995

Purification of the glucocorticoid receptor-mineralocorticoid receptor modulator-2 from rabbit liver.

Modulators-1 and -2 are endogenous low-mol-wt regulators of glucocorticoid and mineralocorticoid receptors and protein kinase C. Structural analysis of apparently purified modulators suggested that these molecules were novel ether aminophosphoglycerides. Subsequent X-ray crystallography and NMR spectroscopy indicated that the ultra-large scale modulator preparations were contaminated with glutamate and aspartate, although these amino acids lacked modulator activity. In this article, we describe the purification of modulator-2 from rabbit liver cytosol and the separation of this phosphoglyceride from these amino acids. This purification was similar to the ultra-large scale version (Bodine, P.V. and Litwack, G. [1990] J. Biol. Chem. 265, 9544-9554), but involved the chromatography of trypsinized rabbit liver cytosol on the 7-L bed volume Sephadex G-15 gel-filtration column. As before, two peaks of modulator activity (modulator-1 and -2), as well as a DNA-binding inhibitor (peak-3), eluted from the gel-filtration column. The resulting modulator-2 pool was incubated with glutamate decarboxylase and treated batch-wise with Dowex-50W cation-exchange resin and Chelex-100 resin. This enzyme/resin-treated modulator-2 preparation was then chromatographed on a Dowex-1 anion-exchange column. Finally, modulator-2 was purified by preparative silica TLC. This last purification step resulted in the separation of modulator-2 from glutamate, aspartate, and gamma-aminobutyrate. In summary, rabbit liver cytosol appears to be a reasonable source of modulator-2. In addition, treatment of the preparation with glutamate decarboxylase seems to facilitate the subsequent separation of modulator-2 from the contaminating amino acids.

Adrenalectomy↗
PubMed1994

A new synthetic ether aminophosphoglyceride exhibits partial modulator activity towards the glucocorticoid receptor.

Modulator is an endogenous low-molecular weight regulator of both glucocorticoid and mineralocorticoid receptors, as well as protein kinase C. Analogs of the putative modulator structure have been synthesized. These compounds include 1-O-(3'-carboxypropyl) or (5'-carboxypentyl)-L-glycero-3-phospho-L-serine or L-threonine, and the D-glycerol stereoisomers. These compounds were tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. One of the ether phosphoglycerides, 1-O-(5'-carboxypentyl)-L-glycero-3-phospho-L-threonine (H-GPT-1), partially inhibited steroid-receptor complex activation in a dose-dependent manner. However, none of the other compounds exhibited any modulator activity towards the glucocorticoid-receptor complex. Like modulator, H-GPT-1 did not inhibit activated glucocorticoid-receptor complex binding to DNA-cellulose. Surprisingly, in contrast to modulator, H-GPT-1 partially inhibited unoccupied receptor steroid-binding in a dose-dependent manner. These results suggest that although modulator is not exactly mimicked by this compound, H-GPT-1 is the first synthetic organic molecule to exhibit some modulator activity towards the glucocorticoid receptor.

Adrenalectomy↗
PubMed1992

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone↗
PubMed1991

Endogenous modulators of glucocorticoid receptor function also regulate purified protein kinase C.

Modulator-1 and -2, proposed to be novel ether-linked aminophosphoglycerides, were originally identified as regulators of glucocorticoid receptor function (Bodine, P. V., and Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554). We now demonstrate that these modulators are also potent new stimulators of protein kinase C activity in vitro. These endogenous biomolecules regulate purified protein kinase C activity in a biphasic and dose-dependent pattern, as determined by histone phosphorylation. Modulators, at concentrations within their apparent cellular range, stimulate protein kinase C-catalyzed histone phosphorylation 2-4-fold when added separately, or 10-12-fold when added together. This enhancement of kinase activity apparently is specific for protein kinase C, since neither protein kinase M, nor cAMP-dependent protein kinase A are stimulated by the modulators. The stimulation of purified protein kinase C occurs only when the enzyme has been initially activated by calcium, phosphatidylserine, and diacylglycerol, indicating that the modulators do not simply substitute for one of the enzyme cofactors. In addition, the modulators appear to interact directly with protein kinase C, perhaps with the regulatory domain of the enzyme, since these biomolecules inhibit the binding of phorbol ester to purified protein kinase C. Finally, time-course studies of protein kinase C-catalyzed histone phosphorylation indicate that the velocity of the enzyme reaction is increased by the modulators. Taken together, these results suggest that the modulators are a new class of regulators of protein kinase C.

Animals↗
PubMed1991

Evaluation of synthetic novel ether aminophosphoglycerides for glucocorticoid-receptor complex modulator activity.

Modulator is an endogenous low-mol wt regulator of the glucocorticoid-receptor complex. Structural analysis of purified modulator suggested that it was a novel ether aminophosphoglyceride (Bodine and Litwack 1988b). Analogs of the putative modulator structure have now been synthesized. The synthetic compounds are 1-O-(6-carboxylhexyl)-glycero-3-phosphoserine and the sn-2-methoxy and sn-1-ethylester derivatives. Like modulator, these novel synthetic compounds are water soluble. However, thin-layer chromatography and spectroscopic analysis of these phosphoglycerides indicated significant structural differences between modulator and the synthetic analogs. In particular, the chromatographic behavior of the compounds suggests that modulator is more highly charged than the synthetic derivatives. The synthetic compounds, as well as lysophosphatidylserine, were also tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. None of the analogs exhibited modulator activity in these assays. However, the synthetic compounds were generally less detrimental to receptor steroid-binding than lysophosphatidylserine. From this work, we conclude that although modulator is not mimicked by one of these synthetic phosphoglycerides, a starting point for future structure-function studies has nonetheless been established.

Amines↗
PubMed1990

Purification and characterization of two novel phosphoglycerides that modulate the glucocorticoid-receptor complex. Evidence for two modulator binding sites in the occupied/unactivated steroid hormone receptor.

Modulator is a novel ether aminophosphoglyceride that is commonly known as the low-molecular weight inhibitor of glucocorticoid-receptor complex activation. An ultra-large scale purification of modulator has been performed from 1000 rat livers. This purification was similar to our previous one (Bodine, P. V., and Litwack, G. (1988) J. Biol. Chem. 263, 3501-3512), but involved the chromatography of heated rat liver cytosol on a 7-liter bed volume Sephadex G-15 gel filtration column. Two peaks of modulator activity eluted from the giant gel-filtration column, and these two modulators (peak-1 and peak-2) were chromatographed separately on Dowex-1 anion-exchange columns. Both modulators were determined to be homogeneous after this step by analytical high-performance thin-layer chromatography, analytical high-performance liquid chromatography, and nuclear magnetic resonance spectroscopy. Furthermore, although peak-1 and peak-2 differed in molecular weight, the two modulators co-chromatographed by anion-exchange, high-performance thin-layer, and high-performance liquid chromatography. These results suggest that the two modulators have similar structures and therefore appear to be isoforms of each other. In addition, both of the modulators are organic molecules that are devoid of molybdenum and 62 other metals. Activity assays indicated that the larger peak-1 modulator was five times more potent than the smaller peak-2 modulator at inhibiting receptor activation and at stabilizing the steroid-binding ability of the occupied and unoccupied receptors. Mixing experiments indicated that the activities of the two modulators were synergistic for both receptor activation inhibition and for occupied receptor steroid-binding stabilization. However, the effects of peak-1 and peak-2 modulator on unoccupied receptor steroid-binding stabilization were additive. Thus, although the two modulators have similar chemical structures, the biological potencies of the two compounds are different. Moreover, these results suggest that although the unoccupied/unactivated receptor has only one modulator binding site, the occupied/unactivated receptor has two modulator binding sites, one site for each of the isoforms.

Animals↗
PubMed1988

Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride.

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.

Animals↗
PubMed1988

Evidence that the modulator of the glucocorticoid-receptor complex is the endogenous molybdate factor.

We have recently purified the modulator of the glucocorticoid-receptor complex from rat liver. Purified modulator inhibits glucocorticoid-receptor complex activation and stabilizes the steroid-binding ability of the unoccupied glucocorticoid receptor. Since these activities are shared by exogenous sodium molybdate, modulator appears to be the endogenous factor that sodium molybdate mimics. In this report, we present additional evidence for the mechanism of action of purified modulator. (i) Molybdate and modulator inhibit receptor activation as measured by DNA-cellulose binding, DEAE-cellulose chromatography, and Sepharose 4B gel filtration. (ii) The ability of molybdate and modulator to inhibit receptor activation and stabilize the unoccupied receptor appears to be additive. (iii) Scatchard analysis of heat-destabilized unoccupied receptors indicates that the number of steroid-binding sites is reduced during destabilization, whereas the steroid dissociation constant remains unchanged. Molybdate and modulator stabilize the receptor by maintaining the number of steroid-binding sites. (iv) Molybdate and modulator do not inhibit alkaline phosphatase-induced destabilization of the unoccupied receptor. However, alkaline phosphatase-induced destabilization is reversed by the addition of dithiothreitol in the presence, but not in the absence, of molybdate or modulator. These results suggest that the mechanism of action for modulator is identical to that of sodium molybdate, and we propose that modulator is the endogenous molybdate factor for the glucocorticoid receptor.

DNA↗
PubMed1984

Calmodulin antagonists decrease the binding of epidermal growth factor to transformed, but not to normal, human fibroblasts.

Four psychoactive agents which inhibit calmodulin activity were used to study their effect on the binding of epidermal growth factor (EGF) to normal and simian-virus-40-transformed human fibroblasts (WI38). These calmodulin antagonists decreased the binding of 125I-labelled EGF to the transformed, but not to the normal, cell in a dose-dependent manner. The mechanism of this effect appears to be due to a decrease in the apparent affinity of the plasma-membrane EGF receptor for the EGF molecule.

Calmodulin↗
PubMed1984

Evidence that pH induced activation of the rat hepatic glucocorticoid-receptor complex is irreversible.

The possible reversibility of pH induced activation of the glucocorticoid-receptor complex was studied. Generally, this was accomplished by activating rat liver cytosol at pH 8.5 (15 degrees C, 30 min), and then returning it to pH 6.5 for a second incubation (15 degrees C, 30 min). Activation was quantitated by measuring the binding of [3H]triamcinolone acetonide [( 3H]TA)-receptor complexes to DNA-cellulose. When cytosol was incubated at pH 6.5, only 4.1% of the [3H]TA-receptor complexes bound to DNA-cellulose. However, 39.2% of the complexes bound when the cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 47.0% of the steroid-receptor complexes bound. Thus, according to the DNA-cellulose binding assay, pH induced activation was irreversible. In order to visualize both activated and unactivated [3H]TA-receptor complexes during this process, diethylaminoethyl (DEAE)-cellulose chromatography was performed. When cytosol was incubated at pH 6.5, only 19.6% of the [3H]TA-receptor complexes were eluted in the activated form from DEAE-cellulose. However, 67.5% of the complexes were eluted in the activated form when cytosol was pH activated. When pH activation was followed by a second incubation at pH 6.5, 74.9% of the steroid-receptor complexes were eluted in the activated form. Thus, DEAE-cellulose chromatography also showed that pH induced activation was irreversible. This is the first known report that the combination of DNA-cellulose binding and DEAE-cellulose chromatography have been used to study pH induced activation of the glucocorticoid-receptor complex. By these criteria, we conclude that in vitro pH induced activation is irreversible.

Adrenalectomy↗
PubMed1983

Calcium effects on epidermal growth factor receptor-mediated endocytosis in normal and SV40-transformed human fibroblasts.

Lowering of extracellular Ca2+ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus40(SV40)-transformed WI38 cells do not exhibit this Ca2+-dependent arrest. One possibility for this difference in Ca2+ requirement is that extracellular or surface membrane-bound Ca2+ may be required for growth factor receptor-mediated endocytosis and this Ca2+ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca2+ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV40WI38 cell. The binding of [125I]EGF to the cell surface is not significantly altered by lowering of Ca2+ to 10(5)-M levels in either the normal or transformed cell. At this Ca2+ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca2+ deprivation. Degradation of the EGF-receptor complex is also sensitive to Ca2+. A twofold reduction in the rate of release of acid-soluble 125I occurs in the normal but not the transformed cell under conditions of lowered medium Ca2+. In contrast, 2-chloro-10-3-aminopropyl phenothiazine (CP), an inhibitor of the Ca2+-dependent regulator protein calmodulin, causes an inhibition of [125I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [125I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.

Calcium↗
PubMed1982

Membrane transport properties differ following return of serum-deprived versus Ca++-deprived human fibroblasts to a proliferative state.

Human lung fibroblasts (W138) can be brought to a quiescent state by removal of serum from the medium or by lowering of the extracellular Ca++. Upon return of Ca++ or serum, the cells enter the G1 phase and progress to S within 15-18 hours. Since multiple G1 phase blocks have been demonstrated, we wished to determine whether the Ca++ and serum block were equivalent since previous data suggested that these two medium components may act at a common point in the initiation of proliferation. We have evaluated the membrane transport of 86Rb, 3-O-methylglucose, AIB, and cycloleucine following stimulation of quiescent cells by Ca++ or serum. Serum stimulation results in large increases in the influx of all the substances tested. These increases are prevented if Ca++ is absent upon serum stimulation or they are rapidly diminished following Ca++ removal. In contrast, Ca++ stimulation of Ca++-deprived cells causes little or no enhancement of any of the transport system, yet the cells progress to S phase in a manner similar to serum-stimulated cells. These results indicate that the Ca++ and serum G0 and G1 block are not equivalent and that the serum-induced change in transport of these components does not appear necessary for successful G1 phase progression. Furthermore, the data suggest that the sequence in which Ca++ or serum are presented to the cells alters the ability of Ca++ to modulate the transport systems. Quiescent cells which are exposed to Ca++ prior to serum possess a Ca++ modulation of several transport systems. Cells which are exposed to Ca++ subsequent to serum do not appear to possess this Ca++ regulation.

Biological Transport↗