Relaxin-induced matrix metalloproteinase-9 expression is associated with activation of the NF-kappaB pathway in human THP-1 cells.
Matrix metalloproteinases (MMPs) and relaxin (RLX) are reported to play an important role in tissue remodeling and wound repair. When macrophages populate wound sites, they secrete biologically active substances, including MMPs. The transcription factor NF-kappaB is important in MMP gene regulation in macrophage cells. Thus, a monocyte/macrophage cell line, THP-1, was used to study the molecular mechanism of RLX action on MMP-2 and MMP-9 expression. After 24 h incubation with porcine RLX (100 ng/ml), conditioned media (CM) and THP-1 cells were collected. Gelatin zymography demonstrated an increase in pro-MMP-9 activity in response to RLX in CM, and no significant change in pro-MMP-2 expression was observed. Immunoblot analysis also revealed an increase in pro-MMP-9 in CM from RLX-treated THP-1 cells. Gel EMSA showed that NF-kappaB DNA-binding activity was elevated in THP-1 cells treated with RLX for 10 min and reached a peak at 30 min. The NF-kappaB DNA complex was supershifted using antibodies against NF-kappaB subunits p50 and p65. Increased expression of the p50 and p65 NF-kappaB subunits was also detected in THP-1 cells after RLX treatment. Incubation with RLX (90 min) reduced THP-1 expression of the NF-kappaB inhibitor protein, IkappaB-alpha. Using a specific NF-kappaB inhibitor, pyrrolidine dithiocarmate (PDTC) inhibited nuclear binding of NF-kappaB. Pre-exposure to PDTC suppressed pro-MMP-9 activity and protein levels in RLX-treated THP-1 cells. In conclusion, these data suggest that RLX-induced tissue remodeling through increasing MMP-9 expression is dependent on NF-kappaB activation.
Ho
Teh-Yuan
TY
Department of Animal Sciences, Rutgers University, New Brunswick, NJ 08901-8525, USA.
Yan
Wenbo
W
Bagnell
Carol A
CA
D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000276:immunology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020672:I-kappa B Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis* / Q000187:drug effects* / Q000276:immunology; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000187:drug effects* / Q000276:immunology*; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D012065:Relaxin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects* / Q000276:immunology; D013329:Structure-Activity Relationship; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D013859:Thiocarbamates; C066229:prolinedithiocarbamate; D000072000:NF-KappaB Inhibitor alpha; D012065:Relaxin; D004247:DNA; D011392:Proline; D020780:Matrix Metalloproteinase 9