A Novel Link between YAP1 and PPP2CB/AMPK/mTORC1 Signaling in Vascular Smooth Muscle Cells

Introduction: Cardiovascular disease remains the leading cause of preventable mortality in the USA and worldwide. Vascular smooth muscle cell (VSMC) phenotypic modulation from a contractile to a synthetic state is central to the etiologies of multiple vascular wall diseases. We have previously demonstrated that Yes-associated protein 1 (YAP1), a transcriptional co-activator, plays a key role in VSMC phenotypic switching and neointima formation. However, the underlying mechanisms remain largely unknown. The mechanistic target of rapamycin complex 1 (mTORC1) is a key mediator of VSMC phenotypic switching and neointima formation. Yet, how mTORC1 is activated under these conditions remains unclear. Our preliminary data show, for the first time, that YAP1 is required and sufficient to activate mTORC1 in VSMCs. Therefore, we hypothesize that YAP1 induces VSMC phenotypic switching via the activation of mTORC1. METHODOLOGY: In vitro loss- and gain-of-function studies utilizing siRNA and adenovirus, respectively, in human coronary artery SMCs. Cell proliferation: WST-1, EdU incorporation, and CyQUANT assays. Cell signaling: Immunoblotting and qRT-PCR. Arterial injury: Left femoral artery wire injury in WT C57BL/6 mice. Results: Arterial injury in WT mice led to YAP1 upregulation, which correlated with mTORC1 activation and VSMC phenotypic switching in vivo, suggesting a key role of the YAP1/mTORC1 pathway in driving neointima formation. Importantly, mTORC1 inhibition with rapamycin markedly inhibited YAP1-induced VSMC proliferation. Next, to investigate how YAP1 regulates mTORC1, we examined the effects of YAP1 loss- and gain-of-function on potential upstream regulators of mTORC1. We found that YAP1 inhibited AMP-activated protein kinase (AMPK), a key negative upstream regulator of mTORC1, suggesting a potential intermediary role of AMPK in mediating the effects of YAP1 on mTORC1 activation, which was validated by siAMPKa1 studies. Mechanistically, bioinformatic analysis of YAP1 ChIPseq data from multiple cell lines identified the catalytic subunit (PPP2CB) and the regulatory subunit (PPP2R1B) of the protein phosphatase PP2A as potential YAP1 transcriptional targets that may mediate YAP1 effects on AMPK signaling. Consistently, we found that YAP1 induces PPP2CB and PPP2R1B at both the mRNA and protein levels. Lastly, we discovered that siPPP2CB was sufficient to rescue the effects of YAP1 on AMPK/mTORC1 signaling. Summary/Conclusion: For the first time, we identify a novel signaling pathway linking YAP1 and AMPK/mTORC1 signaling in VSMCs. We further identify PPP2CB as a direct transcriptional target of YAP1 mediating this novel link. Given the ubiquitous expression and the multitude of cellular processes regulated by YAP1, AMPK, and mTORC1, our findings will likely have greater implications beyond their described role in VSMC phenotypic switching. This work is supported by a grant to Islam Osman from the NHLBI (R00HL153896). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.