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.

Phosphatidylcholine-directed phospholipase C: activation by complement C5b-9.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria. In cultured rat GEC, C5b-9 stimulates a phosphoinositide-directed phospholipase (PL) C and products of PLC downregulate C5b-9-mediated GEC injury. We now report that C5b-9-induced hydrolysis of phosphatidylcholine (PC) provides an additional source of 1,2-diacylglycerol (DAG). PC was labeled in intact GEC by brief incubation with 1-O-[alkyl-3H]2-lyso-PC. Assembly of C5b-9 stimulated an increase in PC-derived [3H]DAG (173 +/- 18% control), which was reduced in GEC depleted of protein kinase C (PKC) by prolonged preincubation with phorbol 12-myristate 13-acetate (PMA). Similar to C5b-9, [3H]DAG was released from PC after brief incubation of GEC with Ca2+ ionophore A23187 plus PMA. The increases in [3H]DAG induced by C5b-9 and A23187 plus PMA were paralleled by increases in DAG mass. C5b-9 also increased [3H]phosphatidic acid (PA; 182 +/- 37% control), but there was no significant interconversion of DAG and PA. Thus DAG probably originated via PLC. PC-directed PLC activity was also studied in GEC homogenates by release of [14C]DAG from exogenous 1-palmitoyl-2-[arachidonoyl-14C]PC. PLC activity was present at physiological Ca2+ concentration (200-1,200 nM), and PMA stimulated PLC activity in cell homogenates (in presence of ATP). These results demonstrate directly that PMA stimulates release of DAG from PC and are in keeping with the effect of PMA in [3H]lyso-PC-labeled GEC. Thus GEC contain a PC-directed PLC, whose activity is physiologically regulated and is present at nanomolar Ca2+ concentration. C5b-9 stimulates PC-directed PLC, leading to production of DAG. This DAG might trigger a mechanism for limiting injury during complement attack.

Extracellular matrix-stimulated phospholipase activation is mediated by beta 1-integrin.

beta 1-Integrins are major mediators of interactions between cells and extracellular matrix (ECM). Adhesion of rat glomerular epithelial cells (GEC) to collagen stimulated phospholipase C. As a result, 1,2-diacylglycerol (DAG) was increased, and inositol phospholipids were decreased in collagen-adherent cells, as compared with GEC adherent to plastic substrata. Adhesion to collagen also stimulated production of free arachidonic acid (the precursor for eicosanoids) due to metabolism of DAG through the DAG lipase pathway and due to phospholipase A2-induced hydrolysis of phospholipids. Phospholipase A2 appeared to be stimulated as a result of protein kinase C (PKC) activation, probably secondary to increased DAG. The collagen-induced increases in DAG and free arachidonic acid, as well as the decrease in inositol phospholipids, were partially inhibited by lowering extracellular Ca2+ concentration to 200 nM or less and by anti-beta 1-integrin antibody Fab. In contrast, anti-beta 1-integrin immunoglobulin G (IgG) enhanced collagen-mediated increases in DAG and arachidonic acid. Proliferation of GEC adherent to collagen was reduced in the presence of anti-beta 1-integrin IgG. The antiproliferative effect of anti-beta 1-IgG appeared to be mediated through PKC, since it was absent in PKC-depleted GEC. Immunoprecipitation with integrin subunit-specific antibodies demonstrated alpha 2 beta 1- and alpha 3 beta 1-integrins in GEC. Thus, in GEC, ECM induces activation of phospholipases C and A2, which is mediated, at least in part, by beta 1-integrins. Products of integrin-mediated phospholipase activation may modulate GEC proliferation.

Adhesion of rat glomerular epithelial cells to extracellular matrices: role of beta 1 integrins.

Glomerular epithelial cells (GEC) maintain glomerular permselectivity and are a target of immunological glomerular injury, which may lead to proliferation or detachment from extracellular matrix (ECM). We studied adhesion mechanisms in rat GEC in culture, focusing on adhesion molecules of the beta 1 integrin family. At early time points (1 hr after plating of cells into culture wells that had been pre-incubated with purified ECM proteins), adhesion of GEC to collagen I, collagen IV, laminin, and fibronectin was inhibited with anti-beta 1 integrin antibody. The peptide RGDS inhibited adhesion to fibronectin and laminin. Immunoprecipitation studies demonstrated the presence of alpha 2, alpha 3, and beta 1 integrins; the alpha 1, alpha 4, alpha 5, alpha 6, alpha v, and beta 3 subunits were undetectable. Adhesion to all ECM proteins was dependent on divalent cations, but the effects of individual cations varied among substrata. In rat GEC, alpha 2 beta 1 and/or alpha 3 beta 1 integrins appear to mediate adhesion to collagen I, collagen IV, and laminin. The alpha 3 beta 1 integrin is also the likely receptor for fibronectin, interacting through an RGD binding site. Furthermore, single integrins or combinations of integrins appear to have distinct ligand-binding functions that are differentially regulated by divalent cations. Characterization of GEC adhesion molecules may facilitate the understanding of mechanisms of glomerular development, and cell detachment or proliferation in immune glomerular injury.

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells.

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.