Biomedical subjects

A V Cybulsky

Publications and source records attributed to A V Cybulsky.

At least 19 recordsLinked to original sources

PubMed2000

Complement C5b-9-mediated arachidonic acid metabolism in glomerular epithelial cells : role of cyclooxygenase-1 and -2.

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG) E(2) in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A(2) was associated with a marked stimulation of PGE(2) production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE(2) production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury.

Animals↗

PubMed2000

Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA phospholipase A2 in COS-1 cells.

Phospholipases A2 (PLA2) and cyclooxygenases (COX) are important enzymes responsible for production of potent lipid mediators, including prostaglandins (PG) and thromboxane A2. We investigated coupling between PLA2 and COX isoforms by using transient transfection in COS-1 cells. Untransfected cells, incubated with or without phorbol ester + the Ca2+ ionophore ionomycin, generated trivial amounts of PGE2. In cells co-transfected with cytosolic PLA2 (cPLA2) and COX-1 or COX-2, phorbol ester + ionomycin markedly stimulated PGE2 production. There was no preferential coupling of cPLA2 to either of the COX isoforms. In contrast, group IIA secretory PLA2 (sPLA2) co-transfected with COX-1 or COX-2 did not lead to an increase in PGE2 production, despite high levels of sPLA2 enzymatic activity. Transfection of cPLA2 did not affect basal free arachidonic acid (AA) levels. Phorbol ester + ionomycin stimulated release of AA in cPLA2-transfected COS-1 cells, but not in untransfected cells, whereas sPLA2 transfection (without stimulation) led to high basal free AA. Thus, AA released by cPLA2 is accessible to both COX isoforms for metabolism to PG, whereas AA released by sPLA2 is not metabolized by COX.

Animals↗

PubMed2000

Complement-induced phospholipase A2 activation in experimental membranous nephropathy.

BACKGROUND: In the passive Heymann nephritis (PHN) model of membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. By analogy, in cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of phospholipase A2 (PLA2). This study addresses the mechanisms of PLA2 activation. METHODS: PLA2 expression was assessed with the polymerase chain reaction or immunoblotting, and activity was determined using an in vitro assay or by measurement of free AA. RESULTS: Under basal conditions, GEC in culture expressed a relatively low level of cytosolic PLA2 (cPLA2) protein, while mRNAs of groups IB, IIA and V secretory PLA2s (sPLA2) were not detectable. Incubation of GEC with sublytic C5b-9 induced 1.5- to 2.0-fold increases in free [3H]AA at 40 minutes, and three and 24 hours. C5b-9 did not increase cPLA2 protein, and did not induce group IB, IIA or V sPLA2 mRNAs. Stable overexpression of cPLA2 in GEC amplified the C5b-9-induced increases in free [3H]AA, while analogous overexpression of group IIA sPLA2 had no effect. PLA2 activity was increased in glomeruli of rats with PHN, and this enhanced activity was characterized as cPLA2. There were no differences in cPLA2 protein expression between PHN and control glomeruli. CONCLUSIONS: Release of AA by C5b-9 in GEC in culture and in vivo is mediated by cPLA2, and the mechanism is consistent with post-translational regulation of cPLA2 activity. C5b-9 does not induce expression or stimulate activity of sPLA2 isoforms in GEC.

Animals↗

PubMed2000

Growth factor pathways in proliferative glomerulonephritis.

Growth factor production, glomerular cell proliferation and glomerular extracellular matrix expansion are prominent features of mesangioproliferative and crescentic glomerulonephritides. Recent studies have provided additional insights into the mechanisms of mesangial cell proliferation and interaction with extracellular matrix, and have focused on selective interruption of relevant mediators of injury. Studies into mitogenic signalling and cell cycle regulation in glomerular epithelial cells support a pathogenetic role for growth factors and glomerular epithelial cell proliferation in focal segmental glomerulosclerosis, and possibly membranous nephropathy. A better appreciation of growth factor pathways may lead to new treatment strategies for human glomerulopathies.

Cell Cycle↗

PubMed1999

Role of extracellular matrix and Ras in regulation of glomerular epithelial cell proliferation.

Signals from extracellular matrix (ECM) to growth factor receptors regulate glomerular epithelial cell (GEC) proliferation. Epidermal growth factor (EGF), basic fibroblast growth factor, hepatocyte growth factor (HGF), or thrombin stimulated proliferation of GECs when the cells were adherent to collagen matrices, but not plastic substratum. Furthermore, EGF, HGF, or thrombin activated p42 mitogen-activated protein (MAP) kinase in collagen-adherent GECs, whereas activation was weak in GECs on plastic. To further examine the interaction of ECM with the Ras-MAP kinase cascade, GECs were stably transfected with a constitutively active Ras mutant (V12Ras). Low or moderate levels of V12Ras expression did not affect basal MAP kinase activity but, unlike parental GECs, in clones that express V12Ras, EGF was able to induce proliferation and activate MAP kinase when these cells were adherent to plastic. In parental and V12Ras-transfected GECs, MAP kinase activation was inhibited by cytochalasin D. Thus, adhesion of GECs to ECM facilitates proliferation and MAP kinase activation by mitogens acting via tyrosine kinase or non-tyrosine kinase receptors. Activation of pathway(s) downstream of V12Ras supplants signals from ECM that enable proliferation. These signals may involve the actin cytoskeleton.

Animals↗

PubMed1999

Complement C5b-9 induces receptor tyrosine kinase transactivation in glomerular epithelial cells.

In the passive Heymann nephritis (PHN) model of membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated via production of eicosanoids. Using rat GEC in culture, we demonstrated that sublytic C5b-9 induced tyrosine phosphorylation of the epidermal growth factor receptor (EGF-R), Neu, fibroblast growth factor receptor-2, and hepatocyte growth factor receptor. In addition, C5b-9 stimulated increases in tyrosine(204) phosphorylation of extracellular signal-regulated kinase-2 (ERK2), as well as free [(3)H]arachidonic acid (AA) and prostaglandin E(2) (PGE(2)). Phosphorylated EGF-R bound the adaptor protein, Grb2, and the EGF-R-selective tyrphostin, AG1478, blocked the C5b-9-induced ERK2 phosphorylation, [(3)H]AA release, and PGE(2) production by 45 to 65%, supporting a functional role for EGF-R kinase in mediating the activation of these pathways. Glomeruli isolated from rats with PHN demonstrated increases in ERK2 tyrosine(204) phosphorylation and PGE(2) production, as compared with glomeruli from control rats, and these increases were partially inhibited with AG1478. Thus, C5b-9 induces transactivation of receptor tyrosine kinases, in association with ERK2 activation, AA release, and PGE(2) production in cultured GEC and glomerulonephritis in vivo. Transactivated tyrosine kinases may serve as scaffolds for assembly and/or activation of proteins, which then lead to activation of the ERK2 cascade and AA metabolism.

Animals↗

PubMed1999

Extracellular matrix as a determinant of signaling responses in glomerular epithelial cells.

Glomerular epithelial cell proliferation occurs in renal diseases such as membranous nephropathy and crescentic glomerulonephritis. To understand the growth factors responsible for the proliferative response and inflammation of these cells, we have examined the effects of extracellular matrix on the regulation of glomerular epithelial cell proliferation and phospholipid turnover, as well as the pathophysiological consequences of this intricate process.

Animals↗

PubMed1998

Complement activates phospholipases and protein kinases in glomerular epithelial cells.

BACKGROUND: In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by eicosanoids. In cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of cytosolic phospholipase A2 (cPLA2). In this study, we address the role of protein kinases in cPLA2 activation. METHODS: GEC were stably transfected with cDNAs of wild-type (wt) cPLA2, and serine505-->alanine mutant (cPLA2-SA505), which lacks the mitogen-activated protein kinase (MAPK) phosphorylation site. RESULTS: Complement stimulated protein kinase C (PKC) activity in GEC, and activated p42 (but not p38) MAPK. Overexpression of either cPLA2-wt or cPLA2-SA505 markedly amplified the release of [3H]AA by C5b-9. Depletion of PKC blocked the complement-dependent activation of cPLA2-wt or cPLA2-SA505, but inhibition of the p42 MAPK pathway had no effect. Epidermal growth factor was a strong activator of p42 MAPK, but stimulated PKC activity weakly. Unlike complement, activation of cPLA2-wt by epidermal growth factor was dependent on PKC, and was augmented significantly by p42 MAPK. Stable overexpression of phospholipase C-gamma 1 in GEC amplified C5b-9-induced production of [3H]inositol phosphates and [3H]diacylglycerol, an endogenous activator of PKC, and complement stimulated tyrosine phosphorylation of phospholipase C-gamma 1. CONCLUSIONS: C5b-9 induces activation of cPLA2 that is dependent on the diacylglycerol-PKC pathway. The role of p42 MAPK in cPLA2 activation becomes redundant in the presence of relatively potent PKC activation.

Animals↗

PubMed1997

Activation of phospholipase A2 by complement C5b-9 in glomerular epithelial cells.

In rat membranous nephropathy, C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. In cultured rat GEC, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids, due to activation of phospholipase A2 (PLA2). To address mechanisms of PLA2 activation, GEC were stably transfected with cDNAs of wild-type cytosolic PLA2 (cPLA2-wt), or group II secretory PLA2, producing overexpression of PLA2 activity. Sublytic C5b-9 markedly increased free [3H]AA in cPLA2-wt-transfected GEC, but only trivial increases were evident in secretory PLA2-transfected, or neo (control) GEC. In cPLA2-wt-transfected GEC, reduction of extracellular free Ca2+ or down-regulation of protein kinase C inhibited [3H]AA release. To further address the regulation of cPLA2, we stably expressed a mutant cPLA2 in which the Ca2+-dependent lipid binding domain was deleted (deltaCaLB). In GEC that express cPLA2-deltaCaLB, the C5b-9-induced increase in free [3H]AA was comparable with neo, despite expression of cPLA2-deltaCaLB at levels similar to cPLA2-wt. We then stably expressed another cPLA2 mutant (cPLA2-srcmyr) in which the CaLB domain was replaced by the N-terminal myristoylation domain of c-Src. cPLA2-srcmyr is permanently membrane associated. At low extracellular free Ca2+, C5b-9 increased free [3H]AA significantly in GEC that express cPLA2-srcmyr, while in neo GEC, the change was negligible. Thus, C5b-9 activates the cPLA2 isoform. Activation is dependent on the CaLB domain, and is mediated by phosphorylation, Ca2+ influx, and membrane association.

Animals↗

PubMed1997

Extracellular matrix is required for MAP kinase activation and proliferation of rat glomerular epithelial cells.

This study examined the role of extracellular matrix (ECM) in the regulation of glomerular epithelial cell (GEC) proliferation. Epidermal growth factor (EGF) stimulated proliferation of GEC when the cells were adherent to collagen matrices, but not plastic substratum. Significant and prolonged EGF receptor (R) tyrosine autophosphorylation (which reflects EGF-R kinase activation) was induced by EGF only in GEC adherent to collagen. In addition, EGF stimulated the activity and tyrosine phosphorylation of p42 mitogen-activated protein (MAP) kinase (ERK2) in collagen-adherent GEC, but not in cells on plastic. An inhibitor of the p-42 MAP kinase pathway, PD98059, blocked EGF-induced MAP kinase activity and proliferation. Thus, adhesion to ECM enables EGF to induce proliferation of GEC, by facilitating activation of EGF-R and the p42 MAP kinase pathway. Signals from ECM to growth factor receptor tyrosine kinases may regulate cell turnover in the glomerulus under normal conditions and during immune glomerular injury.

Animals↗

PubMed1997

Receptor protein tyrosine kinases in perinatal developing rat kidney.

We have identified receptor protein tyrosine kinases (PTKs) that are expressed and/or activated during kidney development. mRNA from fetal rat kidneys in late gestation (embryonic day 21), was used to prepare a cDNA template for polymerase chain reaction amplification with primers based on conserved regions of PTKs, and products were subcloned and sequenced. Among 346 clones, we identified epidermal growth factor receptor (EGF-R), Tie-2, platelet-derived growth factor receptor (PDGF-R)-alpha, PDGF-R beta, Flk-1, Flt-4, fibroblast growth factor receptor (FGF-R)-1, FGF-R3, FGF-R4, Met, and RYK/Nbtk-1. PTK expression was studied by immunoprecipitation and immunoblotting of kidney membrane proteins with specific antibodies. EGF-R, PDGF-R alpha, FGF-R1, FGF-R3, Met, and in some cases Tie-2 protein expression was greater in fetal kidneys, as compared with kidneys from 12-week-old adult rats (controls). Flk-1, PDGF-R beta, and FGF-R4 proteins were expressed comparably, however, Flt-4 was not detected. As a reflection of receptor PTK activity, we assessed endogenous tyrosine phosphorylation, and in vitro autophosphorylation. EGF-R and PDGF-R alpha displayed activity in fetal, but not adult kidneys. FGF-R3 and Flk-1 were active in some fetal kidneys, and the other PTKs were not active. Thus, in late gestational rat kidney, there are distinct patterns of receptor PTK expression and activity. EGF-R, PDGF-R alpha, FGF-R3 and Flk-1 are among the PTKs that are activated, and they may mediate perinatal development of renal epithelial, interstitial, or vascular structures.

Amino Acid Sequence↗

PubMed1997

Glomerular epithelial cell products stimulate mesangial cell proliferation in culture.

Glomerular epithelial cells (GEC) and mesangial cells (MC) are both involved in glomerular diseases. To elucidate potential interactions between these glomerular cell types, we examined whether products of GEC affect the proliferative activity of MC. We found that cultured rat GEC secrete soluble factors into the supernate (GEC-CM) that induce proliferation of quiescent rat MC. The mitogenic activity was trypsin sensitive and partially heat-labile. Biochemical analysis of GEC-CM by gel filtration HPLC, reverse phase HPLC, and isoelectric focusing revealed at least three mitogenic fractions as well as inhibitory activity present in GEC-CM. Competitive binding assays with 125I-labeled PDGF did not show significant amounts of PDGF in GEC-CM. The biochemical features of the GEC-derived MC growth factors are distinct from IL-6, PDGF, bFGF, and endothelin, previously described GEC-derived MC growth factors. Additionally, significant contributions of known growth factors such as IL-1, IL-2, IL-3, IL-4, IL-5, TNF alpha, TGF beta, and GM-CSF are unlikely. The results indicate that GEC produce several biochemically-distinct MC growth regulators. While these epithelial cell-derived mitogens for MC require further characterization, they may play an important role in the regulation of MC replication, such as during embryogenesis and glomerular disease.

Animals↗

PubMed1996

Extracellular matrix stimulates production and breakdown of inositol phospholipids.

Adhesion of rat glomerular epithelial cells (GEC) to collagen stimulates production of D-myo-inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. This process is mediated via beta 1-integrins, and it modulates GEC proliferation. In this study, we address the changes in inositol-lipid turnover induced by GEC adhesion to extracellular matrix (ECM). The masses of both phosphatidylinositol 4,5-bisphosphate (PIP2) and IP3, as well as [3H]inositol phosphates, were increased in GEC adherent to collagen, compared with plastic substratum. Phosphatidylinositol-4-phosphate (PIP) 5-kinase activity was predominantly membrane associated and was enhanced in GEC on collagen. Phospholipase C (PLC) activity and PLC-gamma 1 protein were increased in membrane fractions of GEC adherent to collagen, compared with plastic. Stable overexpression of PLC-gamma 1 in GEC amplified the effect of ECM on the production of [3H]inositol phosphates. In addition, the PLC-gamma 1 that was membrane associated in collagen-adherent GEC was tyrosine phosphorylated. Thus production of IP3 in GEC adherent to ECM is associated with increased production of PIP2. Moreover, adhesion to ECM increases tyrosine phosphorylation and membrane association of PLC-gamma 1, which may facilitate PIP2 hydrolysis by increasing the catalytic activity of PLC-gamma 1 and the proximity of PLC-gamma 1 and its substrate. Understanding the process of ECM-induced inositol lipid production and breakdown in GEC may provide insights into the regulation of GEC proliferation and differentiated functions in normal conditions and during glomerular injury.

Animals↗

PubMed1995

Phospholipase A2 activity is increased in guinea pig uterine cervix in late pregnancy and at parturition.

Release of arachidonic acid from phospholipids by phospholipases A2 (PLA2) is the rate-limiting step in prostaglandin (PG) synthesis. PGE2 and PGF2 alpha are essential intermediates in interstitial collagenase-mediated degradation of type I collagen, a key step in cervical dilatation at parturition. We demonstrate that PLA2 is present in cytosolic fractions of guinea pig cervix and PLA2 activity is increased during cervical dilatation at parturition. In the cervix of nonpregnant animals, PLA2 activity against phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was 13 +/- 6 and 49 +/- 27 pmol.min-1.mg protein-1, respectively. Levels were similar at day 25 of pregnancy. At day 50, PLA2-PC increased to 190 +/- 54 pmol.min-1.mg-1, and PLA2-PE rose to 592 +/- 127 pmol.min-1.mg-1. At parturition (68 +/- 2 days) there were further increases of 49-67%. PLA2 activity declined toward basal levels 2 days postpartum. Almost 50% of the enhanced cytosolic PLA2 (cPLA2) activity at day 50 or at parturition was of high molecular mass and was identified as the "85-kDa cPLA2." Increases in cPLA2 activity at these time points were not, however, associated with increases in cPLA2 protein or phosphorylation of cPLA2, compared with nonpregnant animals. This study suggests that multiple PLA2 enzymes, including cPLA2, are involved in cervical dilatation at parturition.

Animals↗

PubMed1995

Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.

Animals↗

PubMed1995

Insulin-like growth factor-1 enhances epidermal growth factor receptor activation and renal tubular cell regeneration in postischemic acute renal failure.

Growth factors such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and hepatocyte growth factor have been shown to accelerate the recovery from postischemic acute renal failure (ARF) with a concomitant increase in DNA synthesis. Interactions between growth factors have been demonstrated in a number of in vitro studies. This study examined the effect of exogenous IGF-1 on the DNA synthesis and EGF receptor (EGF-R) activation in postischemic rat kidneys. Thirty minutes after the relief of 30-minute total occlusion of the left renal artery in anesthetized 225 to 300 gm Sprague-Dawley rats, either IGF-1 (75 micrograms/kg) or normal saline solution (NS, 0.2 ml) was given by intravenous bolus, followed by twice daily subcutaneous injections of IGF-1 (50 micrograms/kg) or 0.2 ml NS for 4 days, respectively, in IGF-1-Tx) and NS treated (NS-Tx) groups (n = 8 each). On the day after the completion of treatment, inulin clearance (ml/kg/min) of the postischemic kidneys in the IGF-1-Tx group was significantly higher (p < 0.01) than inulin clearance of kidneys in the NS-Tx group. This was associated with improved kidney morphology. IGF-1 treatment also enhanced the labeling index of 5-bromo-2'-deoxyuridine (percent of stained tubule cells), a marker for active DNA synthesis, in the outer medulla of postischemic kidneys at 1 day and 2 days after the injury. EGF-R tyrosine phosphorylation (which reflects receptor activation) increased in postischemic kidneys in both NS-Tx (n = 5) and IGF-1-Tx (n = 3) groups 1 day after the injury as compared with nonischemic contralateral kidneys. In the IGF-1-Tx group there was also increased iodine 125-labeled EGF binding and EGF-R protein. Our results demonstrate a beneficial effect of IGF-1 on postischemic ARF. Furthermore, they suggest that EGF-R activation is involved in tubular regeneration and that IGF-1 may enhance EGF-R activation by increasing EGF-R expression.

Acute Kidney Injury↗

PubMed1994

Epidermal growth factor receptor activation in developing rat kidney.

Epidermal growth factor (EGF) binding increases in late-gestational rat kidney and then falls toward basal adult levels postnatally during the 1st wk. We report that the increase in EGF binding is accompanied by an increase in EGF receptor (EGFR) protein and activation of EGFR tyrosine kinase. Multiple proteins were endogenously tyrosine phosphorylated in kidney membranes from fetal rats, and the phosphorylation pattern was similar in rats ranging from 16 to 21 days of gestation. Tyrosine phosphorylation was, however, almost undetectable in 12-wk adult rat kidneys (controls). Among the phosphoproteins in fetal kidney, a prominent 170-kDa protein was identified as EGFR. Endogenous tyrosine phosphorylation of EGFR (reflecting receptor activation) was 30-fold higher in fetal kidney membranes than in adult (3- to 7-fold higher when adjusted for differences in EGF binding or EGFR protein content). The EGFR substrate, phospholipase C-gamma 1, was tyrosine phosphorylated in fetal kidneys but not adult, and a greater proportion was membrane-associated in fetal kidneys, consistent with activation of phospholipase C-gamma 1. Thus EGFR tyrosine kinase activity is increased in late-gestational rat kidney. Induction and activation of EGFR may mediate perinatal renal cell growth and development.

Aging↗

PubMed1994

Extracellular matrix modulates epidermal growth factor receptor activation in rat glomerular epithelial cells.

To understand how glomerular epithelial cell (GEC) proliferation may be regulated in health and disease, we studied the effects of type I collagen extracellular matrices (ECM) on EGF receptor (EGF-R) activation in cultured rat GEC. EGF stimulated proliferation of GEC adherent to ECM, but not of GEC on a plastic substratum. Significant and prolonged EGF-R tyrosine autophosphorylation (which reflects receptor kinase activation) was induced by EGF in GEC adherent to collagen, but EGF did not stimulate EGF-R autophosphorylation in GEC on plastic (at 37 degrees C). However, EGF-R autophosphorylation increased significantly in plastic-adherent GEC that were stimulated with EGF at 4 degrees C or in the presence of vanadate, an inhibitor of phosphotyrosine phosphatases. Furthermore, dephosphorylation of EGF-R was enhanced in GEC on plastic as compared with collagen. At 4 degrees C, [125I]EGF binding was not different between substrata, and there was negligible accumulation of intracellular [125I]EGF (which reflects EGF-R internalization). At 37 degrees C, EGF-R internalization was reduced significantly in collagen-adherent GEC as compared with GEC on plastic. Thus, contact with ECM facilitates proliferation and EGF-R activation in GEC. The enhanced activity of EGF-R tyrosine kinase may be due to ECM-induced reduction in EGF-R internalization and dephosphorylation by phosphotyrosine phosphatase(s). Signals from ECM to growth factor receptors may regulate cell turnover in the glomerulus under normal conditions and during immune glomerular injury.

Animals↗