Requirement of heat shock protein 90 in mesangial cell mitogenesis.
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 HSP90beta 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 HSP90beta was identified. Glomerular mRNA and protein expression of HSP90beta 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, HSP90beta expression in normal glomeruli was detected in podocytes. However, in anti-Thy1.1 nephritis, glomerular HSP90beta protein expression was strongly and transiently increased in mesangial localization. In vitro, mitogenic stimulation of rat MCs led to the induction of HSP90beta 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 HSP90beta 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.
Pieper
M
M
Medizinische Klinik IV, Universität Erlangen-Nürnberg, Erlangen, Germany, and Department of Pathology, University of Leiden, Leiden, The Netherlands.
Rupprecht
H D
HD
Bruch
K M
KM
De Heer
E
E
Schöcklmann
H O
HO
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D016227:Benzoquinones; D001798:Blood Proteins / Q000494:pharmacology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D002921:Cicatrix; D019938:Cyclin D1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D016195:G2 Phase / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D005920:Glomerular Mesangium / Q000201:enzymology / Q000473:pathology*; D005921:Glomerulonephritis / Q000139:chemically induced / Q000235:genetics / Q000473:pathology*; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D006965:Hyperplasia; D047029:Lactams, Macrocyclic; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D017510:Protein Folding; D011518:Proto-Oncogene Proteins; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D016192:Resting Phase, Cell Cycle / Q000187:drug effects / Q000502:physiology; D016196:S Phase / Q000187:drug effects / Q000502:physiology; D050794:STAT1 Transcription Factor; D018800:Thy-1 Antigens / Q000276:immunology; D015534:Trans-Activators / Q000378:metabolism
D000906:Antibodies; D016227:Benzoquinones; D001798:Blood Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D011518:Proto-Oncogene Proteins; D011809:Quinones; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494081:Stat1 protein, rat; D018800:Thy-1 Antigens; D015534:Trans-Activators; D019938:Cyclin D1; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D020928:Mitogen-Activated Protein Kinases; C001277:geldanamycin