Daeyoung D. Roh

Lysophosphatidylcholine activates mesangial cell PKC and MAP kinase by PLCγ-1 and tyrosine kinase-Ras pathways

Although lysophosphatidylcholine (LPC)-mediated cellular responses are attributed to the activation of protein kinase C (PKC), relatively little is known about the upstream signaling mechanisms that regulate the activation of PKC and downstream mitogen-activated protein (MAP) kinase. LPC activated p42 MAP kinase and PKC in mesangial cells. LPC-mediated MAP kinase activation was inhibited (but not completely) by PKC inhibition, suggesting additional signaling events. LPC stimulated protein tyrosine kinase (PTK) activity and induced Ras-GTP binding. LPC-induced MAP kinase activity was blocked by the PTK inhibitor genistein. Because LPC increased PTK activity, we examined the involvement of phospholipase Cγ-1 (PLCγ-1) as a key participant in LPC-induced PKC activation. LPC stimulated the phosphorylation of PLCγ-1. PTK inhibitors suppressed LPC-induced PKC activity, whereas the same had no effect on phorbol 12-myristate 13-acetate-mediated PKC activity. Other lysophospholipids [e.g., lysophosphatidylinositol and lysophosphatidic acid (LPA)] also induced MAP kinase activity, and only LPA-induced MAP kinase activation was sensitive to pertussis toxin. These results indicate that LPC-mediated PKC activation may be regulated by PTK-dependent activation of PLCγ-1, and both PKC and PTK-Ras pathways are involved in LPC-mediated downstream MAP kinase activation.

Effect of inhibition of cholesterol synthetic pathway on the activation of Ras and MAP kinase in mesangial cells

Intermediary metabolites of cholesterol synthetic pathway are involved in cell proliferation. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, blocks mevalonate synthesis, and has been shown to inhibit mesangial cell proliferation associated with diverse glomerular diseases. Since inhibition of farnesylation and plasma membrane anchorage of the Ras proteins is one suggested mechanism by which lovastatin prevents cellular proliferation, we investigated the effect of lovastatin and key mevalonate metabolites on the activation of mitogen-activated protein kinase (MAP kinase) and Ras in murine glomerular mesangial cells. The preincubation of mesangial cells with lovastatin inhibited the activation of MAP kinase stimulated by either FBS, PDGF, or EGF. Mevalonic acid and farnesyl-pyrophosphate, but not cholesterol or LDL, significantly prevented lovastatin-induced inhibition of agonist-stimulated MAP kinase. Lovastatin inhibited agonist-induced activation of Ras, and mevalonic acid and farnesylpyrophosphate antagonized this effect. Parallel to the MAP kinase and Ras data, lovastatin suppressed cell growth stimulated by serum, and mevalonic acid and farnesylpyrophosphate prevented lovastatin-mediated inhibition of cellular growth. These results suggest that lovastatin, by inhibiting the synthesis of farnesol, a key isoprenoid metabolite of mevalonate, modulates Ras-mediated cell signaling events associated with mesangial cell proliferation.

Oxidative Modification of Low-Density Lipoprotein Enhances Mesangial Cell Protein Synthesis and Gene Expression of Extracellular Matrix Proteins

The proliferation of intrinsic glomerular cells and the accumulation of extracellular matrix proteins are principal histopathological features seen in glomerular injury. Because of the marked similarity between the cellular and molecular events that occur in both atherosclerosis and glomerulosclerosis and the commonly accepted hypothesis that lipoproteins are implicated in the pathogenesis of glomerulosclerosis, we examined the effect of three atherogenic lipoproteins, low-density lipoprotein (LDL), oxidized (ox)-LDL, and minimally modified (mm)-LDL on the synthesis and secretion of extracellular matrix (ECM) proteins by mesangial cells. The incubation of SV-40 transformed murine mesangial cells with LDL (25–100 µg/ml) increased the synthesis and secretion of both fibronectin and laminin in a dose-dependent manner. Similarly, oxidized forms of LDL (25–100 µg/ml) increased fibronectin and laminin synthesis and secretion dose dependently. However, both oxidatively modified forms of LDL had a greater effect on increasing ECM protein synthesis than their native counterpart. Northern blot analysis showed a dose-dependent increase in mRNA transcripts for fibronectin and laminin in response to the incubation of mesangial cells with LDL, ox-LDL, and mm-LDL. Similar to the ECM protein expression data, the oxidatively modified forms of LDL had more pronounced effects on the gene expression of both fibronectin and laminin. These data show that both LDL and, perhaps more importantly, its oxidatively modified forms stimulate mesangial cells to upregulate both the gene expression and synthesis and secretion of ECM proteins, supporting a role for atherogenic lipoproteins in the pathobiology of glomerular injury.

Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: Role of protein tyrosine kinase and cyclic amp-dependent protein kinase A

Mesangial cell proliferation and extracellular matrix accumulation are fundamental in the pathogenesis of glomerulosclerosis. Platelet-derived growth factor (PDGF) is a major cytokine involved in mesangial cell proliferation, and its increased expression is seen in glomerular injury. Atherogenic lipoproteins stimulate mesangial cell proliferation and induce glomerular injury in experimental animals. We examined the effect of low-density lipoprotein (LDL) and its more atherogenic oxidized forms, minimally modified LDL (mm-LDL) and oxidized LDL (ox-LDL) on mesangial cell PDGF mRNA expression. Incubation with 2.5 to 25 microg/ml LDL or mm-LDL for 1 to 4 hours stimulated mesangial cell PDGF mRNA expression (mm-LDL 2 to 3 times greater than LDL); ox-LDL had no effect. Similarly, both LDL and mm-LDL induced mesangial cell DNA synthesis (mm-LDL 1.5 to 2 times greater). In further studies evaluating key associated intracellular signal transduction mechanisms, the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein markedly decreased basal and lipoprotein-induced PDGF mRNA expression. Both pertussis toxin and isoproterenol, cyclic AMP-generating substances, stimulated PDGF mRNA expression. Preincubation with H-8 or H-89, cyclic AMP-dependent protein kinase A (PKA) inhibitors, blocked the lipoprotein-induced PDGF message, whereas preincubation with calphostin C, a protein kinase C inhibitor, did not alter LDL- or mm-LDL-mediated PDGF mRNA expression. These data suggest that the accumulation of atherogenic lipoproteins and their endogenous oxidized forms within the glomerulus may regulate mesangial cell PDGF expression and related cellular responses. These events appear to be modulated by signal transduction pathways involving PTK and PKA.

Atherogenic Lipoproteins Enhance Murine Cortical Epithelial Cell Fibronectin Protein Synthesis and Gene Expression

Tubulointerstitial changes, characterized by the accumulation of extracellular matrix proteins (ECM) and fibrosis, are often associated with primary glomerular injury. Furthermore, these changes may be better prognostic indicators for decline in renal function than the anatomical changes seen within the glomerulus itself. Although hyperlipidemia and the increased renal accumulation of atherogenic lipoproteins are commonly seen in both human and experimental models of renal disease, the possible role that atherogenic lipoproteins may play in the cellular and molecular events associated with the development of tubulointerstitial injury remains unclear. Since atherogenic lipoproteins have been shown to be mediators of renal injury, we examined the effects of native LDL and oxidatively-modified LDL (ox-LDL, a more atherogenic form of LDL) on fibronectin protein synthesis and gene expression in proximal tubular epithelial cells (TEC). Human LDL was freshly isolated and ox-LDL prepared by incubation of LDL with 100 µM CuSO4. Incubation of TEC with LDL or ox-LDL (25–50 µg/ml) for 24 h increased the steady-state mRNA expression of fibronectin by 16–135% over control as measured by Northern blot analysis and the effect was greater with ox-LDL than native LDL. Additional studies were done to examine whether the increased fibronectin message in response to lipoprotein activation was translated into TEC protein synthesis. The activation of TEC by LDL or ox-LDL stimulated the synthesis and secretion of fibronectin (52–150%, over control) as measured by Western blot analysis. The data show that LDL and ox-LDL stimulate TEC fibronectin gene message and protein synthesis supporting a pathobiological role for these atherogenic lipoproteins in tubulointerstitial fibrosis.

Effect of nifedipine on renal microvascular cholesterol accumulation and prostacyclin biosynthesis in cholesterol-fed rabbits

Studies, performed in rabbits, examined the effect of feeding a high cholesterol diet and/or a calcium antagonist, nifedipine, on renal microvascular prostacyclin biosynthesis and cholesterol accumulation. After 30 days, cholesterol-fed rabbits had elevated serum and tissue cholesterol levels associated with decreased microvascular prostacyclin biosynthesis and histologic evidence of microvascular and glomerular lipid accumulation. Nifedipine reduced tissue cholesterol levels, enhanced prostacyclin biosynthesis, and reduced the histologic evidence for lipid accumulation in renal microvessels and glomeruli. These studies suggest that calcium antagonists may have a beneficial effect in preventing the tissue cholesterol accumulation associated with a high-cholesterol diet and further suggest that these agents may have beneficial effects in the treatment of renal diseases associated with microvascular or glomerular lipid accumulation.

Comparative studies on acid cholesterol esterase in renal blood vessels and aorta of control and hypercholesterolemic rabbits

Decreased acid cholesterol esterase has been linked to cholesteryl ester accumulation and may be fundamental in the development of atherosclerosis. The present study compared cholesterol esterase activity with the accumulation of cholesterol and its esters in aorta, renal artery and renal preglomerular microvessels. Tissue was obtained from white New Zealand rabbits fed either a control or 2%-cholesterol diet for 1 month. Cholesterol esterase was increased in microvessels from cholesterol-fed animals when compared to aorta and renal artery. Cholesterol feeding generally produced an increase in cholesterol and cholesteryl ester accumulation in all vascular tissues. The percent distribution of esterified/total cholesterol in renal microvessels was decreased consistent with the concomitant increase in cholesterol esterase. In contrast, aorta and renal artery exhibited an increase in cholesterol and cholesteryl ester accumulation and an increase in the percent of esterified cholesterol consistent with a decrease in acid cholesterol esterase after cholesterol feeding. The data suggest that renal microvessels, when compared to aorta and renal artery, may be relatively protected from developing atherosclerotic microvascular lesions through an organ-specific increase in acid cholesterol esterase activity.

Effect of Dietary Cholesterol on Rat Glomerular Cholesterol Esterase

The accumulation of tissue cholesterol and cholesteryl esters is commonly seen during the development of both atherosclerosis and glomerulosclerosis. The intracellular cholesterol content is regulated, in part, by the hydrolysis of cholesteryl esters to cholesterol, a reaction catalyzed by cholesterol esterase. Decreased cholesterol esterase has been linked to cholesteryl ester accumulation in vascular cells and has been postulated to be an important factor in the progression of atherosclerosis and, possibly, glomerulosclerosis. In order to determine whether cholesterol esterase regulates glomerular cholesterol accumulation, the effect of cholesterol feeding on the cholesterol content and the activity of cholesterol esterase was examined in rat glomeruli. Cholesterol esterase was measured using a cholesteryl[1-14C]oleate-lecithin liposome substrate. Total and free glomerular cholesterol was measured spectrofluorometrically. Feeding rats 4% cholesterol for 2 months decreased total glomerular (acid plus neutral) cholesterol esterase activity when compared to glomeruli from similar rats fed a normal chow (1.8 +/- 0.1 versus 1.48 +/- 0.2 nmol/mg protein/h, p < 0.05). Total, free and esterified cholesterol concentrations were higher in glomeruli from cholesterol-fed rats than from controls, consistent with decreased cholesterol esterase activity. Thus, glomerular cholesterol accumulation appears to be regulated by cholesterol esterase. This finding is similar to that in other vascular tissues which have been investigated and which are prone to accumulate cholesterol during the development of atherosclerosis.