Low-density lipoprotein stimulation of mesangial cell fibronectin synthesis: role of protein kinase C and transforming growth factor-beta.
Low-density lipoprotein (LDL) cholesterol has been implicated in the pathogenesis of glomerulosclerosis in diabetes and other forms of glomerular injury. In the present study we evaluated the effect of LDL on fibronectin synthesis in cultured rat mesangial cells (MCs) and the roles of protein kinase C (PKC) and transforming growth factor-beta (TGF-beta) in mediating this LDL action. In MCs, 25 micrograms to 100 micrograms/ml LDL increased PKC activity within 15 minutes, as reflected by enhanced in situ phosphorylation of the 80 kd myristoylated alanine-rich C kinase substrate protein, a specific endogenous substrate of PKC in MC. The same concentrations of LDL subsequently (18 to 72 hours) enhanced fibronectin synthesis, as reflected by increased incorporation of labeled methionine into fibronectin. GF 109203X, a selective inhibitor of PKC, blocked increases in both PKC activity and fibronectin synthesis induced by LDL in MCs. Furthermore, prior downregulation of PKC to less than 1% of basal activity by exposure of MCs to 0.5 mumol/L phorbol myristate acetate (PMA) also prevented LDL stimulation of fibronectin synthesis. The activation of PKC by LDL seen after 15 minutes of exposure was transient and was not observed after 4 or 48 hours of exposure of MCs to LDL. However, exposure to LDL for 48 hours, but not for 15 minutes or 4 hours, increased both maximal PKC responses to phorbol dibutyrate (PDBu) and tritiated PDBu binding to MCs by 30%. These findings suggest that chronic exposure to LDL increases the total PKC content in MCs and thereby might modulate responses to other PKC agonists. Neither the cyclooxygenase inhibitor piroxicam nor the thromboxane/prostaglandin endoperoxide receptor blocker Sq-29548 altered LDL stimulation of fibronectin synthesis in MCs, suggesting that this action of LDL was not mediated by changes in MC eicosanoid generation. By contrast, antibody to TGF-beta blocked LDL stimulation of fibronectin synthesis in MCs. TGF-beta bioactivity, determined with the mink lung epithelial cell assay, was two to three times higher in the medium of MCs cultured with LDL for 24 to 48 hours as compared with corresponding control values. Total TGF-beta bioactivity examined after heat activation of latent TGF-beta was also two times higher in the medium of MCs exposed to LDL as compared with that of controls. Prior down-regulation of PKC by exposure of MCs to PMA blocked the increases in TGF-beta bioactivity induced by LDL.(ABSTRACT TRUNCATED AT 400 WORDS)
Studer
R K
RK
Veterans Affairs Medical Center, Pittsburgh, PA 15240.
Craven
P A
PA
DeRubertis
F R
FR
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002478:Cells, Cultured; D005231:Fatty Acids, Unsaturated; D005260:Female; D005353:Fibronectins / Q000096:biosynthesis*; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D006834:Hydrazines / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010766:Phosphorylation; D010894:Piroxicam / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013928:Thromboxane A2 / Q000037:antagonists & inhibitors; D016212:Transforming Growth Factor beta / Q000502:physiology*
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D005353:Fibronectins; D006834:Hydrazines; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL; D008301:Maleimides; D008565:Membrane Proteins; D011506:Proteins; D016212:Transforming Growth Factor beta; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010894:Piroxicam; D015240:Phorbol 12,13-Dibutyrate; D013928:Thromboxane A2; C045749:SQ 29548; D011493:Protein Kinase C; C070515:bisindolylmaleimide I