The Tyrosine Kinase Src is a Critical Mediator of High Glucose-induced EGF Receptor (EGFR)Transactivation, MAPK Phosphorylation and Collagen Acc umulation in Mesangial Cells

Abstract

In high glucose glomerular mesangial cells (MC) are stimulated and produce excess extracellular matrix (ECM) proteins such as collagen IV which accumulate to cause glomerulosclerosis. The cell signaling mechanisms of these changes, e,g, the MAPK family of Ser/Thr kinases and the role of Tyr kinases is not clear. To study these, primary rat MC were exposed to 5.6 mM (NG) or 25 mM (HG) glucose for up to 48 h. HG stimulated phosphorylation of MAPK (ERK1/2), p38, and EGFRTyr845 (Src phosphorylation site) and Tyr1173 (autophosphorylation site) with a maximum at 48 h. Phosphorylation of SrcTyr416 (activation site) was also induced by HG and reached maximum by 18 h. Two Src kinase inhibitors, PP2 or S U6656, inhibited MAPK and EGFR phosphorylation, while the specific EGFR inhibitor AG1478 inhibited ERK1/2, p38 and EGFR phosphorylation but not Src, placing Src upstream of EGFR. Src has been implicated in EGFR transactivation via cleavage of membrane bound HB-EGF by TACE (TNF-α converting enzyme, ADAM17). The HB-EGF inhibitor (CRM197) and TACE inhibitor (TAPI-2) both blocked HG-stimulated EGFR, ERK1/2 and p38 phosphorylation. To document that ECM accumulation is dependent on this signaling pathway, collagen IV content was determined by immunofluorescence confocal imaging. PP2, AG1478 and TAPI-2, all blocked HG-stimulated collagen IV accumulation. To confirm that Src is a key mediator in vivo, streptozotocin -induced diabetic DBA/2 J mouse renal cortical tissue was examined. Immunoblotting revealed that diabetes stimulated phosphorylation of SrcTyr416 and ERK1/2 which were blocked by administration of PP2. These data indicate that in renal MC, HG activates a Src-TACE mediated EGFR transactivation which is necessary to stimulate the MAPK enzymes and collagen