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.
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.


CONCLUSIONS
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.
Kok
Sang-Heng
SH
School fo Dentistry, College of Medicine, National Taiwan University, and National Taiwan University Hospital, Taipei, Taiwan.
Hong
Chi-Yuan
CY
Kuo
Mark Yen-Ping
MY
Wang
Chih-Chiang
CC
Hou
Kuo-Liang
KL
Lin
Yi-Ting
YT
Galson
Deborah L
DL
Lin
Sze-Kwan
SK
D000818:Animals; D001168:Arthritis / Q000209:etiology*; D015152:Blotting, Northern; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis*; D024202:Electrophoretic Mobility Shift Assay; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D008297:Male; D053683:Oncostatin M / Q000502:physiology*; D010006:Osteoblasts / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000502:physiology; D051381:Rats; D011917:Rats, Inbred Lew; D050794:STAT1 Transcription Factor / Q000502:physiology*; D050796:STAT3 Transcription Factor / Q000502:physiology*; D014158:Transcription, Genetic; D020032:Tyrphostins / Q000494:pharmacology
D018932:Chemokine CCL2; D047908:Intracellular Signaling Peptides and Proteins; D016760:Proto-Oncogene Proteins c-fos; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D053683:Oncostatin M; D010768:Phosphoserine