Abstract 1058: Novel Regulation of Wnt-Inducible Secreted Protein 1 by TNF-alpha in Primary Human Cardiac Fibroblasts
Wnt1-induced secreted protein-1 (WISP-1) is a member of the CCN family of growth factors known to play a role in cellular growth, transformation, and survival. We previously demonstrated that WISP-1 is upregulated in the heart post-infarct, stimulates fibroblast proliferation and collagen expression, and is induced by the proinflammatory cytokine TNF-a. The present study was designed to investigate the molecular mechanisms involved in TNF-a-mediated WISP-1 induction in primary human cardiac fibroblasts (hCF). hCF were treated with rhTNF-a. WISP-1 expression was analyzed by Northern, Western and promoter-reporter assays. ERK1/2 and JNK activations by Western blotting using activation-specific antibodies and imunecomplex kinase assays. CREB activation by Western blotting, EMSA and reporter assay. Results demonstrate that TNF-a potently induces WISP-1 mRNA and protein expression in a time- and dose-dependent manner, effects that were blocked by anti-TNFR2, but not anti-TNFR1, neutralizing antibodies. Further investigations revealed that TNF-a stimulates WISP-1 promoter-reporter activity, an effect that was blunted when the core CREB DNA binding sequence was mutated or following the overexpression of dnCREB. TNF-a treatment induced CREB phosphorylation, in vitro and in vivo DNA binding, and reporter gene activities. TNF-a also induced ERK1/2 phosphorylation and activity, effects that were blocked by the MEK1 inhibitor U0126 and the ERK1/2 inhibitor PD98059. Furthermore, inhibition of ERK1/2 blunted CREB activation while inhibition of ERK1/2 and overexpression of dnCREB attenuated TNF-a-mediated WISP-1 promoter-reporter activity and mRNA expression. However, inhibition of JNK by SP600125 failed to modulate TNF-a-mediated WISP-1 induction. Most importantly, siRNA-mediated WISP-1 knockdown attenuated TNF-a-mediated hCF proliferation. Thus, our results demonstrate for the first time that TNF-a is a potent inducer of WISP1 expression in hCF, and TNF-a induces WISP-1 expression via TNFR2, MEK1, ERK, and CREB. Since TNF-a and WISP-1 are upregulated post-infarct, our results indicate that TNF-a/WISP-1 signaling may play a critical role in post-infarct myocardial remodeling.