Sunday, 18 July 2010

Purpose: MMP-9 is the prominent isozyme of matrix metalloproteinases (MMP) known to be highly expressed in human atherosclerotic plaques. One potent inhibitor of MMP-9 production is the transforming growth factor beta (TGF-β). Similar to this, we also observed inhibitory effects with sphingosine 1-phosphate (S1P), accumulation in high density lipoprotein (HDL), which presumably suppresses MMP-9 production. The aim of this study was to reveal the inhibitory influence of HDL on MMP-9 production and the molecular mechanisms behind these processes. Methods: Rat vascular smooth muscle cells (VSMC) were stimulated with interleukin-1β (IL-1β). The following MMP-9 expression was quantified by real-time PCR and activity by zymography. For receptor knockdown studies, cells were transfected with siRNA against S1P3 receptor. Results: TGF-β significantly down-regulated IL-1β stimulated MMP-9 expression (78.6% ± 5.4 decrease; p < 0.05, n = 10). This inhibition could be significantly blocked with the TGF-β receptor inhibitor R1 (36.7% ± 2.9 vs. 78.6% ± 5.4 decrease; p < 0.05; n = 9). Like S1P, HDL also significantly suppressed the IL-1β stimulated MMP-9 expression (S1P: 42.2% ± 8.3 decrease n = 5; HDL: 44.3% ± 9.8 decrease; n = 10; p < 0.05). To reveal if there is any cross-communication between the TGF-β and S1P receptors, we also tested the TGF-β receptor inhibitor in the IL-1β/HDL and IL-1β/S1P costimulation, which significantly reversed the effect of HDL (12.2 % ± 4.6 vs. 44.3 % ± 2.4 decrease; p < 0.05; n = 7) and S1P (42.2% ± 3.5 vs. 72.3% ± 8.3 decrease; p < 0.05; n = 7). Investigation of the MMP-9 activity via zymography showed corresponding results after TGF-β and S1P treatment in the absence and presence of the inhibitor. The specific downregulation of the S1P3 receptor by siRNA abolished the HDL/S1P induced TGF-β signaling. Conclusions: HDL, S1P and TGF-β all down-regulate MMP-9 expression. The HDL and S1P mediated inhibition of MMP-9 expression could be diminished by a TGF-β receptor inhibitor and by S1P3 receptor knockdown. Therefore, HDL and its component S1P could cross activate the TGF-β signaltransduction pathway via the S1P3 receptor.