Effect of hypoxia on monocyte chemotactic protein-1 (MCP-1) gene expression induced by Interleukin-1beta in human synovial fibroblasts.
OBJECTIVE
Rheumatoid arthritis (RA) synovial membrane is characterized by leucocyte infiltration and secretion of chemotactic and proinflammatory factors. Since hypoxia is an important pathogenic factor in inflamed synovium, we examined the effects of hypoxia on monocyte chemotactic protein-1 (MCP-1) expression in human rheumatoid arthritis synovial fibroblasts (RASF) under IL-1beta-stimulated and -unstimulated conditions.


METHODS
Synovial fibroblasts were isolated from RA, osteoarthritis (OA) and healthy knee joints and subjected to hypoxia or/and IL-1beta treatment. MCP-1 expression and protein secretion were measured by real-time PCR and ELISA, respectively.


RESULTS
Hypoxia reduces MCP-1 expression and protein secretion in RASF. The same response to hypoxia was found in OA and healthy SF cultures. Treatment with actinomycin D showed that hypoxic down-regulation of MCP-1 expression was due to a decrease in transcription, since the half-life of MCP-1 mRNA was unchanged. A cycloheximide study demonstrated that de novo protein synthesis was not required for the hypoxic effect. The decrease in MCP-1 expression by hypoxia was mimicked by cobalt chloride in unstimulated RASF with no effect on IL-1beta-activated MCP-1, suggesting differences in the signaling mechanisms. The analysis of IkappaB degradation and NF-kappaB translocation revealed that hypoxia did not affect IL-1beta activation of NF-kappaB.


CONCLUSIONS
Hypoxia regulates MCP-1 expression under both basal and cytokine-stimulated conditions, suggesting that reduced oxygen supply is an important factor that mediates chemotaxis of monocytes to the area of inflammation.
Safronova
O
O
Department of Cellular Physiological Chemistry, 1-5-45, Yushima, Bunkyo-ku, 113-8549, Tokyo, Japan.
Nakahama
K
K
Onodera
M
M
Muneta
T
T
Morita
I
I
D001172:Arthritis, Rheumatoid / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis*; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D000860:Hypoxia; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism / Q000502:physiology*; D007717:Knee / Q000473:pathology; D010003:Osteoarthritis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013583:Synovial Membrane / Q000378:metabolism*; D013997:Time Factors
D018932:Chemokine CCL2; D007375:Interleukin-1; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D003609:Dactinomycin; D003035:Cobalt; D003513:Cycloheximide; C018021:cobaltous chloride