[Role of testosterone in tumor necrosis factor-alpha and monocyte chemotactic protein-1 expression in mouse macrophage RAW264.7 and its molecular mechanism].
OBJECTIVE
To investigate the role of androgen in TNF-alpha and MCP-1 expression in RAW264.7 macrophage and its molecular mechanism.


METHODS
(1) RAW264.7 macrophage was treated with 10(-9) mol/L or 10(-7) mol/L testosterone (T) and then subjected to the measurement of: 1) the cellular expression of TNF-alpha and MCP-1 mRNA by RT-PCR; 2) the expression of TNF-alpha and MCP-1 in cell culture supernatant by ELISA; (2) The expression of phospho-NF-kappaB after treatment with T was measured by Western blot. (3) Cells were pre-incubated with 10(-4) mol/L PDTC (an inhibitor of NF-kappaB) for 1 hour, followed by T treatment. Expression of mRNA and supernatant levels of TNF-alpha and MCP-1 were measured by RT-PCR and ELISA.


RESULTS
(1) 1) After a 6 h treatment with 10(-9) mol/L or 10(-7) mol/L T, the expression of TNF-alpha mRNA increased by 1.78 and 1.87 folds, MCP-1 by 1.58 and 1.66 folds respectively (all P < 0.05). 2) Incubated with both concentration of T for 6 h showed no significant changes of supernatant levels of TNF-alpha and MCP-1. After a 24 h treatment, the levels of TNF-alpha and MCP-1 increased significantly (all P < 0.05) while more significant increase was found in 10(-7) mol/L T group (P < 0.05). (2) The expression of phospho-NF-kappaB (ser276) increased significantly after cells were treated with 10(-7) mol/L T for 30 min (P < 0.05) and peaked at 60 min. (3) With 1 h PDTC pre-incubation, T (10(-9) mol/L or 10(-7) mol/L) treatment for 6 h led to a lower mRNA expression and 24 h led to lower supernatant levels of TNF-alpha and MCP-1 than those without (P < 0.05). However, both cellular and supernatant expression of TNF-alpha and MCP-1 with PDTC pre-incubation were still higher than those of blank controls (all P < 0.05, except for TNF-alpha in 10(-9) mol/L T treatment).


CONCLUSIONS
Testosterone can increase TNF-alpha and MCP-1 expression in RAW264.7 macrophage in vitro. Activation of cellular NF-kappaB by testosterone may be one of underlying molecular mechanisms.
Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China.
Jie-ping
JP
Zhu
Chun-lin
CL
Su
Min
M
Chen
Jin-fang
JF
Lin
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D013739:Testosterone / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D013739:Testosterone