Osteopontin in rat renal fibroblasts: functional properties and transcriptional regulation by aldosterone.
Osteopontin (OPN), a proinflammatory cytokine, plays an important role in renal fibrosis. We reported that plasma OPN levels were higher in patients with primary aldosteronism than with essential hypertension. However, the regulatory mechanism of OPN by aldosterone remains unclear. Here, we report the transcriptional regulation of OPN expression by aldosterone and the functional effects of aldosterone-mediated OPN transcription in renal fibroblasts. Aldosterone induced OPN expression in a dose-dependent manner with significant responses at 10 nmol/L (1.6+/-0.2-fold of controls, P<0.05, n=5) and elicited maximal effects at 10 micromol/L (3.5+/-0.4-fold of controls, P<0.01, n=5). Aldosterone increased OPN expression in a time-dependent manner with a maximal effect after 48 hours (2.7+/-0.3-fold of controls, P<0.01, n=5). This effect was abolished by the mineralocorticoid receptor (MR) antagonist spironolactone. Luciferase promoter deletion assays identified a novel cis regulatory element (-2153 to -1758) in the OPN promoter that is responsive to aldosterone. This element contains an activator protein-1 (AP-1) and nuclear factor kappa B (NF kappaB) site. Electrophoretic mobility shift assays, supershift assays, and chromatin immunoprecipitation assays identified both AP-1 and NF kappaB as the DNA binding proteins induced by aldosterone with spironolactone inhibiting aldosterone-induced AP-1 or NF kappaB activity. OPN-siRNA inhibited completely the induction of cell proliferation, type I, III, and IV collagen synthesis by aldosterone. These results indicate that aldosterone induced MR-mediated OPN expression through AP-1 and NF kappaB activation and suggest that aldosterone plays an important role in renal fibrosis through the induction of OPN.
D000450:Aldosterone / Q000008:administration & dosage / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000502:physiology*; D017354:Point Mutation / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000502:physiology; D016760:Proto-Oncogene Proteins c-fos / Q000502:physiology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D018161:Receptors, Mineralocorticoid / Q000502:physiology; D050436:Regulatory Elements, Transcriptional / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology*
D016328:NF-kappa B; D016760:Proto-Oncogene Proteins c-fos; D034741:RNA, Small Interfering; D018161:Receptors, Mineralocorticoid; D018808:Transcription Factor AP-1; D053495:Osteopontin; D000450:Aldosterone; D003094:Collagen; D048031:JNK Mitogen-Activated Protein Kinases