SKI activates Hippo signalling to modulate cardiac fibroblast function and activation

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Landry, Natalie Mireille
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It has previously been established that SKI, an endogenous TGF-β1 inhibitor, halts fibroblast activation and deactivates the myofibroblast phenotype. It has also been demonstrated that conventional cell culture does not lend itself to accurate physiological studies of primary cardiac fibroblasts, in vitro. The thesis presented here establishes a method of maintaining primary cardiac fibroblasts in a resting state in two-dimensional cell culture. In addition, this improved technique was applied to test the hypothesis that SKI is a multi-functional protein that can also activate Hippo signalling, and in turn inhibit the cardiac myofibroblast phenotype. First, we developed cell culture conditions to better maintain the native phenotype of primary cells by limiting mechanical, hormonal, and nutritional input. Both rat and mouse cardiac fibroblasts could be cultured in a quiescent state for up to ten days post-isolation. Using this optimized method, we initially sought to determine the role of the nuclear effectors of the Hippo pathway, Yes-Associated Protein (YAP) and Transcriptional co-Activator with PDZ-binding motif (TAZ), in cardiac fibroblast activation. When compared to YAP signalling, TAZ demonstrated greater activation of the collagen I and III transcription. In addition, a rat model of cardiac fibrosis revealed that TAZ expression increases and translocates to the nucleus, while YAP expression was relatively unchanged in the infarcted left ventricle. Further studies examined the effects of SKI on YAP/TAZ signalling. Overexpression of SKI induced the degradation of endogenous TAZ, but not YAP, by activating LATS2 kinase. To better understand the specific SKI-TAZ link, the human cardiac fibroblast-specific SKI interactome was elucidated by using BioID2. We identified the focal adhesion-associated protein, LIMD1, as a mediator between LATS2 and SKI. Additionally, ablation of Limd1 in myofibroblasts resulted in the degradation of TAZ, but not YAP, recapitulating the TAZ-specific effects of SKI overexpression. Taken together, these data suggest that TAZ has a greater role in the pathogenesis of cardiac fibrosis, and that SKI specifically targets TAZ to inhibit fibroblast activation. The findings described here postulate that the SKI-LIMD1-TAZ signalling axis as a novel and selective therapeutic target for consideration in future treatments for cardiac fibrosis.
Cardiac fibrosis, Fibroblast, SKI, YAP, TAZ, WWTR1, Hippo Pathway, LIMD1, Myofibroblast
Landry NM, Rattan SG, Dixon IMC. "An Improved Method of Maintaining Primary Murine Cardiac Fibroblasts in Two-Dimensional Cell Culture." Sci Rep. 2019; 9 (1):12889. doi: 10.1038/s41598-019-49285-9.
Landry NM, Cohen S, Dixon IMC. "Periostin in Cardiovascular Disease and Development: A Tale of Two Distinct Roles." Basic Res Cardiol. 2017;113(1):1. doi: 10.1007/s00395-017-0659-5.