Abstract 5872: 17-beta-Estradiol Inhibits Matrix Metalloproteinase-2 Transcriptional Activity via MAP-Kinase Pathway in Fibroblasts
Background and aims: Matrix metalloproteinase-2 (MMP-2) plays an important role in myocardial extracellular matrix (ECM) remodeling. 17β-Estradiol (E2) modulates ECM composition and function. Here we analyze the effect of E2 on MMP-2 gene expression in cardiac fibroblasts. Moreover, we study the 5′-flanking region of the human (h) MMP-2 gene and the molecular mechanisms involved in the E2-dependent regulation of its promoter in a fibroblast cell line.
Methods: Isolated cardiac fibroblasts from male and female rats were treated with E2. A series of hMMP-2 promoter-luciferase reporter constructs were co-transfected with human estrogen receptor alpha (ERα) expression-vector into human fibrosarcoma cell line (HT1080). After treatment with E2 (10 – 8M) or vehicle and/or pre-treatment with ICI182, 780 (10 –5M) or PD98059 (10μM), luciferase reporter assays were carried out. Electrophoretic mobility shift assays (EMSA)/supershift assays were used to identify the cis-and transacting elements, important for E2/ER-mediated hMMP-2 gene expression in HT1080 cells.
Results: E2 significantly decreased MMP-2 gene expression in rat cardiac fibroblasts of both sexes in an ER-dependent manner. Co-transfection of the hMMP-2 expression-constructs and hERa expression-vector in human fibroblast cells showed a significant reduction of promoter activity after E2-treatment. The inhibiting E2-effect is mediated through a defined region between −324bp to −259bp of the hMMP-2 promoter. EMSA and supershift analysis demonstrated the binding of the transcription factor Elk-1 within this regulatory region. Elk-1 is phosphorylated by E2 via activation of MAP-Kinase signalling-pathway. Pre-treatment of HT1080 cells with ER antagonist ICI182, 780 and MAP-Kinase inhibitor PD98059 significantly abolished the phosphorylation of Elk-1 and the inhibiting E2-effect on the hMMP-2 promoter activity.
Conclusion: We showed a down-regulation of MMP-2 gene expression in cardiac fibroblasts and the inhibition of the hMMP-2 promoter activity by E2 via activation of ERα and MAP-Kinase pathway in fibroblast cells. These findings suggest that a deficiency or excess of E2 may cause a dysregulation of the ECM turnover in the human heart by the modulation of hMMP-2 gene expression.