Thrombin induces epidermal growth factor receptor transactivation and CCL2 expression in human osteoblasts.
OBJECTIVE
Thrombin is a key factor involved in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of rheumatoid arthritis (RA). The aim of this study was to investigate the intracellular signaling pathways involved in thrombin-induced CCL2 expression in human osteoblasts.


METHODS
Thrombin-mediated CCL2 expression was assessed by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The mechanisms of action of thrombin in different signaling pathways were studied using Western blotting. Knockdown of protease-activated receptor (PAR) protein was achieved by small interfering RNA (siRNA) transfection. Chromatin immunoprecipitation assays were used to study in vivo binding of c-Jun to the CCL2 promoter. Transient transfection was used to examine activator protein 1 (AP-1) activity.


RESULTS
Stimulation of human primary osteoblasts and MG-63 cells with thrombin induced CCL2 expression. PAR-1-specific siRNA (but not other PAR siRNA) was involved in thrombin-mediated up-regulation of CCL2. Thrombin-mediated CCL2 production was attenuated by the thrombin inhibitor PPACK, the protein kinase Cδ (PKCδ) inhibitor rottlerin, the c-Src inhibitor PP2, epidermal growth factor receptor (EGFR) inhibitor AG-1478, MEK inhibitors PD98059 and U0126, or AP-1 inhibitors curcumin and tanshinone IIA. Stimulation of cells with thrombin increased PKCδ, c-Src, EGFR, MEK, and ERK activation. Treatment of osteoblasts with thrombin also increased c-Jun phosphorylation, AP-1 luciferase activity, and c-Jun binding to the AP-1 element on the CCL2 promoter.


CONCLUSIONS
Our results suggest that the interaction between thrombin and PAR-1 increases CCL2 expression in human osteoblasts via the PKCδ/c-Src/ EGFR transactivation/MEK/ERK/c-Jun/AP-1 pathway.
China Medical University and China Medical University Hospital, Taichung, Taiwan.
Chun-Yin
CY
Huang
Shi-Yann
SY
Chen
Hsiao-Chi
HC
Tsai
Horng-Chaung
HC
Hsu
Chih-Hsin
CH
Tang
D000098:Acetophenones / Q000494:pharmacology; D000328:Adult; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D044463:Receptor, PAR-1 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology; D013917:Thrombin / Q000494:pharmacology*; D015533:Transcriptional Activation / Q000187:drug effects; D020032:Tyrphostins / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C412373:AG 1879; D000098:Acetophenones; D000590:Amino Acid Chloromethyl Ketones; D001578:Benzopyrans; D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; D044463:Receptor, PAR-1; D020032:Tyrphostins; C101044:RTKI cpd; C085746:rottlerin; D066246:ErbB Receptors; D019061:src-Family Kinases; D011493:Protein Kinase C; D013917:Thrombin; C050815:phenylalanyl-prolyl-arginine chloromethyl ketone-thrombin