Oncostatin M-induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: An implication for the pathogenesis of arthritis
Journal
Arthritis and Rheumatism
Journal Volume
60
Journal Issue
5
Pages
1451-1462
Date Issued
2009
Author(s)
Abstract
Objective. To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. Methods. The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. Results. Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA-protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. Conclusion. These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis. ? 2009, American College of Rheumatology.
SDGs
Other Subjects
chloramphenicol acetyltransferase; DNA; luciferase; mitogen activated protein kinase; monocyte chemotactic protein 1; n benzyl 2 cyano 3 (3,4 dihydroxyphenyl)acrylamide; oncostatin M; phosphatidylinositol 3 kinase; protein c fos; protein kinase B; short hairpin RNA; STAT1 protein; STAT3 protein; animal experiment; animal model; arthritis; article; controlled study; disease course; disease severity; DNA protein complex; enzyme linked immunosorbent assay; gel mobility shift assay; gene silencing; human; human cell; immunohistochemistry; immunoprecipitation; male; nonhuman; Northern blotting; osteoblast; priority journal; promoter region; protein binding; protein expression; protein function; protein interaction; protein phosphorylation; rat; Western blotting; Animals; Arthritis; Blotting, Northern; Cells, Cultured; Chemokine CCL2; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Male; Oncostatin M; Osteoblasts; Phosphorylation; Phosphoserine; Proto-Oncogene Proteins c-fos; Rats; Rats, Inbred Lew; STAT1 Transcription Factor; STAT3 Transcription Factor; Transcription, Genetic; Tyrphostins
Type
journal article