Requirement of heat shock protein 90 in mesangial cell mitogenesis

Abstract

Background. Hyperplasia of mesangial cells (MCs) is a frequent finding in glomerulonephritis. Heat shock protein 90 (HSP90) is a major cellular chaperone that assists protein folding under physiological and stress conditions. Methods. To identify genes that are potentially involved in the pathogenesis of glomerulonephritis, we analyzed glomerular gene expression in mesangioproliferative rat anti-Thy1.1 nephritis by representational difference analysis (RDA). Expression of HSP90β in anti-Thy1.1 nephritis was studied by Northern and Western blot analyses and immunohistochemistry. In cultured rat MCs, the requirement of HSP90 for mitogenic signaling steps and MC replication was studied by incubation with the specific HSP90 inhibitor geldanamycin. Results. By RDA, a cDNA fragment homologous to HSP90β was identified. Glomerular mRNA and protein expression of HSP90β was markedly and transiently up-regulated during the course of anti-Thy1.1 nephritis, with a maximum at day 6, coinciding with the peak of MC proliferation. By immunohistochemistry, HSP90β expression in normal glomeruli was detected in podocytes. However, in anti-Thy1.1 nephritis, glomerular HSP90β protein expression was strongly and transiently increased in mesangial localization. In vitro, mitogenic stimulation of rat MCs led to the induction of HSP90β mRNA and protein. Incubation of MCs with geldanamycin dose-dependently inhibited DNA synthesis and replication. Moreover, geldanamycin interfered with mitogen-induced phosphorylation of extracellular signal-regulated kinase and transcription of c-fos and Egr-1, but not with transactivation of STAT1 transcription factor. Cell cycle analysis of serum-stimulated MCs revealed that geldanamycin inhibited kinase activity of cyclin D1/CDK4 complexes and blocked progression in the G0/G1 phase and at the S/G2 phase transition. Conclusions. The up-regulation of HSP90β in anti-Thy1.1 nephritis may reflect its functional involvement in phenotypical alterations of MCs in mesangioproliferative glomerulonephritis. Our in vitro studies indicate that HSP90 governs the capacity of MCs to respond to proliferative stimuli by regulating critical mitogenic signaling steps necessary for G1 entry and S-phase progression.