Abstract
Abstract: :
Purpose: Stromelysin–1 (matrix metalloproteinase–3 or MMP–3) expression stimulated by TNF–α and IL–1α in trabecular meshwork (TM) cells is mediated through three MAP kinase pathways, Erk, p38 and JNK. Studies were conducted to dissect the involvement of each individual pathway and to characterize elements involved in the MMP–3 promoter. Methods: Constructs with a secreted alkaline phosphatase (SEAP) reporter driven by fragments of the MMP–3 promoter were made. A full–length 2.3 kb fragment, a fragment containing both Ets–1 and AP–1 sites, and one containing only AP–1 sites were used to characterize each of three MAP kinase pathways. Cells were co–transfected with the 2.3 kb human MMP–3 promoter construct, hMMP3p–SEAP, and one of the three dominant–negative MAP kinases. After TNF–α or IL–1α treatment, the conditioned media was assayed for SEAP activity via a chemiluminescent assay. To study the Ets–1 and AP–1 sites in the MMP–3 promoter, the cells were transfected using each of the three different promoter–reporter constructs. Results: The stimulative effect of either TNF–α or IL–1α was significantly blocked in TM cells co–transfected with Erk, p38 or JNK. The SEAP expression level in IL–1α or TNF–α treated TM cell culture was less using the MMP–3 promoter fragment containing both of Ets–1 and AP–1 sites in comparison with the full–length 2.3kb promoter. Upon using the MMP–3 promoter fragment with only the AP–1 site, the activity detected was modest. Conclusions: Each of the three MAP kinase pathways, Erk, p38 and JNK, appeared to be critical in the TNF–α or IL–1α activation of the MMP–3 promoter. However, the well–known targets of the MAP kinase pathways, Ets–1 and AP–1 sites present in 5'–deleted promoter fragment alone, are only partially effective. This study suggests participation of these and other portions of the MMP–3 promoter in regulating MMP–3 expression.
Keywords: trabecular meshwork • signal transduction • cytokines/chemokines