Activation of protein kinase C by phorbol esters induces DNA synthesis and protein phosphorylations in glomerular mesangial cells

Abstract

The tumor‐promoting phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) is shown to be mitogenic for quiescent glomerular mesangial cells cultured in serum‐free conditions. TPA induces DNA synthesis measured by PH thymidine incorporation in a dose‐dependent manner with an ED10 of 7 ng/ml and an optimal response for 50 ng/ml. The phorbol ester action is potentiated by insulin with an increase of the maximal effect from 232 ± 15% of TPA alone to 393 ± 96% for TPA plus insulin. Down‐regulation of protein kinase C by prolonged exposure to TPA completely abolishes the mitogenic effect of the phorbol ester. Using a highly resolutive 2D electrophoresis, we have shown that TPA is able to stimulate the phosphorylation of 2 major proteins of M 1 80000, pI 4.5 (termed 80K) and M 1 28000, pI 5.7–5.9 (termed 28K). The 80K protein phosphorylation is time‐ and dose‐dependent with an ED10 of 8 ng/ml TPA. Exposure or mesangial cells to heat‐shock induces synthesis of a 28K protein among a set of other proteins suggesting that the 28K protein kinase C substrate belongs to the family of low molecular mass stress proteins. Mitogenic concentrations of TPA and phorbol 12,13‐dibutyrate inhibit [125I]epidermal growth factor binding and stimulate the 80K protein phosphorylation with the same order of potency. The inactive tumor‐promoter 4x‐phorbol was found to be ineffective both on these 2 parameters and on DNA synthesis. These results suggest a positive role of protein kinase C on mesangial cell proliferation and indicate the existence in this cell line of 2 major protein kinase C substrates.

Basic Fibroblast Growth Factor Stimulates Glomerular Mesangial Cell Proliferation Through a Protein Kinase C-Independent Pathway

“…For experiments using staurosporine, 5 /g/ml of insulin were added to the incubation medium. As previously reported, insulin alone has no effect by itself but potentiates the effect of TPA and Des-Arg'bradykinin (Issandou and Darbon, 1991). By contrast, we found that insulin did not increase the mitogenic response observed with optimal bFGF concentrations (data not shown).…”

“…Prolonged exposure of cells with phorbol esters is known to down-regulate protein kinase C in mesangial cells (Pfeilshifter, 1989), as well as in other cell lines (Darbon et al, 1987;Rozengurt, 1989,1990). After such a pretreatment, we have previously shown that TPA was unable to trigger DNA synthesis while serum was still effective (Issandou and Darbon, 1991). As shown in Figure 2 (closed circles), a 2-day pretreatment with 320nM TPA did not prevent bFGF from stimulating DNA synthesis.…”

“…The Mr=28,000/pI 5.7-5.9 protein (termed 28K) has been identified as a protein kinase C substrate in mesangial cells and characterized as a stress protein (Issandou and Darbon, 1991). The fact that bFGF induced 28 K phosphorylation in normal cells as well as in protein kinase Cdepleted cells suggests that this protein may be also a substrate for other types of protein kinases putatively involved in the cascade of phosphorylation events leading to mitogenesis.…”

“…Protein kinase C has been recently shown to be implicated in the proliferation of glomerular mesangial cells. Activation of the enzyme leads to a variety of early molecular events in growth-arrested mesangial cells including an increase in the phosphorylation of a M,= 80,00O/pI 4.5 protein (termed 80 K) (Issandou and Darbon, 1991), described as a major and specific protein kinase C substrate in Swiss 3T3 cells (Rozengurt et al, 1983) and other cultured fibroblasts (Blackshear et al, 19861, a s well as a Mr=28,000/pI 5.7-5.9 protein which has been shown as a heat-shock protein (Issandou and Darbon, 1991).…”

Des-Arg10-kallidin Engagement of the B1 Receptor Stimulates Type I Collagen Synthesis via Stabilization of Connective Tissue Growth Factor mRNA

“…For example, both the B1 and B2 receptors mediate pain perception (45); both receptors activate phospholipase C in mesangial cells (46); and both receptors stimulate mitogenesis in fibroblasts (36). In addition, the structural similarities of the B1 and B2 receptor agonists suggest that the kinin receptors may be isoforms.…”