%0 Journal Article %A Yajing Ji %A Erika M. Lisabeth %A Richard R. Neubig %T TGF-β 1 Increases Expression of Contractile Genes in Human Pulmonary Arterial Smooth Muscle Cells by Potentiating Sphingosine-1-Phosphate Signaling %D 2021 %R 10.1124/molpharm.120.000019 %J Molecular Pharmacology %P MOLPHARM-AR-2020-000019 %X Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure and carries a very poor prognosis. Understanding PAH pathogenesis is needed to develop new therapeutic strategies. TGF-β drives vascular remodeling and increases vascular resistance by regulating differentiation and proliferation of smooth muscle cells (SMCs). Also, sphingosine-1-phosphate (S1P) has been implicated in PAH but the relation between these two signaling mechanisms is not well understood. Here, we characterize the signaling networks downstream of TGF-β in human pulmonary arterial smooth muscle cells (HPASMCs) which involves SMAD signaling as well as Rho GTPases. Activation of Rho GTPases regulates myocardin-related transcription factor (MRTF) and serum response factor (SRF) transcription activity and results in upregulation of contractile gene expression. Our data show that in HPASMCs, upregulation of alpha smooth muscle actin (αSMA) by TGF-β is dependent on both SMAD and Rho/MRTF-A/SRF transcriptional mechanisms. The kinetics of TGF-β-induced myosin-light chain 2 (MLC2) phosphorylation, a measure of Rho activation, is slow, as is regulation of the Rho/MRTF-A/SRF-induced αSMA expression. These results suggest that TGF-β1 activates Rho/pMLC2 through an indirect mechanism which was confirmed by sensitivity to cycloheximide treatment. As a potential mechanism for this indirect action, TGF-β1 upregulates mRNA for sphingosine kinase (SphK1), the enzyme that produces S1P, an upstream Rho activator, as well as mRNA levels of the S1P Receptor 3 (S1PR3). A SphK1 inhibitor and S1PR3 inhibitors (PF543 and TY52156/VPC23019) reduce TGF-β1-induced αSMA upregulation. Overall, we propose a model where TGF-β1 activates Rho/MRTF-A/SRF by potentiating an autocrine/paracrine S1P signaling mechanism through SphK1 and S1PR3. Significance Statement In human pulmonary arterial smooth muscle cells, TGF-β depends on S1P signaling to bridge the interaction between SMAD and Rho/MRTF signaling in regulating αSMA expression. The Rho/MRTF pathway is a signaling node in the αSMA regulatory network and is a potential therapeutic target for the treatment of PAH. %U https://molpharm.aspetjournals.org/content/molpharm/early/2021/05/22/molpharm.120.000019.full.pdf