Exogenous S1P via S1P receptor 2 induces CTGF expression through Src-RhoA-ROCK-YAP pathway in hepatic stellate cells

Mol Biol Rep. 2024 Sep 2;51(1):950. doi: 10.1007/s11033-024-09868-w.

Abstract

Background: Hepatic fibrosis, a prevalent chronic liver condition, involves excessive extracellular matrix production associated with aberrant wound healing. Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis, activated by inflammatory factors such as sphingosine 1-phosphate (S1P). Despite S1P's involvement in fibrosis, its specific role and downstream pathway in HSCs remain controversial.

Methods: In this study, we investigated the regulatory role of S1P/S1P receptor (S1PR) in Hippo-YAP activation in both LX-2 cell lines and primary HSCs. Real-time PCR, western blot, pharmacological inhibitors, siRNAs, and Rho activity assays were adopted to address the molecular mechanisms of S1P mediated YAP activation.

Results: Serum and exogenous S1P significantly increased the expression of YAP target genes in HSCs. Pharmacologic inhibitors and siRNA-mediated knockdowns of S1P receptors showed S1P receptor 2 (S1PR2) as the primary mediator for S1P-induced CTGF expression in HSCs. Results using siRNA-mediated knockdown, Verteporfin, and Phospho-Tag immunoblots showed that S1P-S1PR2 signaling effectively suppressed the Hippo kinases cascade, thereby activating YAP. Furthermore, S1P increased RhoA activities in cells and ROCK inhibitors effectively blocked CTGF induction. Cytoskeletal-perturbing reagents were shown to greatly modulate CTGF induction, suggesting the important role of actin cytoskeleton in S1P-induced YAP activation. Exogeneous S1P treatment was enough to increase the expression of COL1A1 and α-SMA, that were blocked by YAP specific inhibitor.

Conclusions: Our data demonstrate that S1P/S1PR2-Src-RhoA-ROCK axis leads to Hippo-YAP activation, resulting in the up-regulation of CTGF, COL1A1 and α-SMA expression in HSCs. Therefore, S1PR2 may represent a potential therapeutic target for hepatic fibrosis.

Keywords: Cytoskeletal rearrangement; Hepatic fibrosis; Hepatic stellate cell; Hippo-YAP signaling pathway; Sphingosine 1-phosphate (S1P); Sphingosine 1-phosphate receptor (S1PR).

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Cell Line
  • Collagen Type I / genetics
  • Collagen Type I / metabolism
  • Connective Tissue Growth Factor* / genetics
  • Connective Tissue Growth Factor* / metabolism
  • Hepatic Stellate Cells* / drug effects
  • Hepatic Stellate Cells* / metabolism
  • Hippo Signaling Pathway
  • Humans
  • Liver Cirrhosis / genetics
  • Liver Cirrhosis / metabolism
  • Liver Cirrhosis / pathology
  • Lysophospholipids* / metabolism
  • Lysophospholipids* / pharmacology
  • Receptors, Lysosphingolipid / genetics
  • Receptors, Lysosphingolipid / metabolism
  • Signal Transduction*
  • Sphingosine* / analogs & derivatives
  • Sphingosine* / metabolism
  • Sphingosine-1-Phosphate Receptors / genetics
  • Sphingosine-1-Phosphate Receptors / metabolism
  • Transcription Factors* / genetics
  • Transcription Factors* / metabolism
  • YAP-Signaling Proteins* / metabolism
  • rho-Associated Kinases* / genetics
  • rho-Associated Kinases* / metabolism
  • rhoA GTP-Binding Protein* / metabolism
  • src-Family Kinases / metabolism

Substances

  • Connective Tissue Growth Factor
  • Lysophospholipids
  • sphingosine 1-phosphate
  • rho-Associated Kinases
  • Sphingosine
  • YAP-Signaling Proteins
  • rhoA GTP-Binding Protein
  • CCN2 protein, human
  • Transcription Factors
  • Sphingosine-1-Phosphate Receptors
  • S1PR2 protein, human
  • src-Family Kinases
  • Adaptor Proteins, Signal Transducing
  • Receptors, Lysosphingolipid
  • YAP1 protein, human
  • RHOA protein, human
  • Collagen Type I