Date Published: April 11, 2014
Publisher: Public Library of Science
Author(s): Kuldeep Kumawat, Mark H. Menzen, Ralph M. Slegtenhorst, Andrew J. Halayko, Martina Schmidt, Reinoud Gosens, Wenhui Hu.
WNT-5A, a key player in embryonic development and post-natal homeostasis, has been associated with a myriad of pathological conditions including malignant, fibroproliferative and inflammatory disorders. Previously, we have identified WNT-5A as a transcriptional target of TGF-β in airway smooth muscle cells and demonstrated its function as a mediator of airway remodeling. Here, we investigated the molecular mechanisms underlying TGF-β-induced WNT-5A expression. We show that TGF-β-activated kinase 1 (TAK1) is a critical mediator of WNT-5A expression as its pharmacological inhibition or siRNA-mediated silencing reduced TGF-β induction of WNT-5A. Furthermore, we show that TAK1 engages p38 and c-Jun N-terminal kinase (JNK) signaling which redundantly participates in WNT-5A induction as only simultaneous, but not individual, inhibition of p38 and JNK suppressed TGF-β-induced WNT-5A expression. Remarkably, we demonstrate a central role of β-catenin in TGF-β-induced WNT-5A expression. Regulated by TAK1, β-catenin is required for WNT-5A induction as its silencing repressed WNT-5A expression whereas a constitutively active mutant augmented basal WNT-5A abundance. Furthermore, we identify Sp1 as the transcription factor for WNT-5A and demonstrate its interaction with β-catenin. We discover that Sp1 is recruited to the WNT-5A promoter in a TGF-β-induced and TAK1-regulated manner. Collectively, our findings describe a TAK1-dependent, β-catenin- and Sp1-mediated signaling cascade activated downstream of TGF-β which regulates WNT-5A induction.
WNT-5A is a member of the Wingless/integrase 1 (WNT) family of secreted glycoproteins. There are 19 WNT ligands known in humans that act through 10 Frizzled (FZD) receptors, low-density lipoprotein receptor-related protein (LRP) 5/6 co-receptors and many non-FZD receptors, including ROR1, ROR2, RYK . WNT signaling is broadly subdivided into two main streams- canonical (β-catenin-dependent) and non-canonical (β-catenin-independent) WNT signaling. In the canonical signaling, binding of a WNT ligand to a FZD receptor and LRP5/6 co-receptors activates signaling mechanisms resulting in stabilization of the transcriptional co-activator β-catenin, leading to its accumulation in the cytosol. Stabilized β-catenin translocates to the nucleus where it partners with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and activates target gene transcription. Non-canonical WNT signaling functions exclusive of β-catenin and LRP5/6 and involves a multitude of pathways regulating gene transcription, cytoskeletal reorganization, cell polarity and cell movements. WNT/Ca2+ and WNT/planar cell polarity (PCP) are the best characterized non-canonical WNT signaling pathways among others. In the WNT/Ca2+ signaling, binding of WNT ligands to FZD or non-FZD receptors activates calcium-dependent signaling molecules, including protein kinase C (PKC), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor of activated T-cell (NFAT), whereas the WNT/PCP pathway involves activation of the RhoA signaling or c-Jun N-terminal Kinases (JNKs) via small Rho-GTPases .
In the present study, we have delineated the signaling mechanisms driving TGF-β-induced WNT-5A expression in airway smooth muscle cells. To the best of our knowledge, this is the first report describing a signaling cascade consisting of TAK1, β-catenin and Sp1 that regulates WNT-5A expression. We demonstrate that TAK1 activity is required for WNT-5A expression in response to TGF-β stimulation and provide evidence for the involvement of β-catenin in this process which, in turn, is regulated by TAK1 signaling. We further identify Sp1 as transcription factor for WNT-5A and demonstrate its interaction with β-catenin in airway smooth muscle cells. We provide evidence that Sp1 is recruited to the WNT-5A promoter in response to TGF-β, a phenomenon regulated by TAK1 activity. Collectively, our study identifies a novel pathway involved in WNT-5A regulation, thus, providing an understanding of mechanisms governing WNT-5A homeostasis.