Sphingosine 1-phosphate elicits RhoA-dependent proliferation and MRTF-A mediated gene induction in CPCs

Abstract

Although c-kit(+) cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. G-protein coupled receptors (GPCRs) play an important role in regulating these processes in mammalian cell types thus we assessed GPCR mRNA expression in c-kit(+) cells isolated from adult mouse hearts. Our data provide the first comprehensive overview of the distribution of this fundamental class of cardiac receptors in CPCs and reveal notable distinctions from that of adult cardiomyocytes. We focused on GPCRs that couple to RhoA activation in particular those for sphingosine-1-phosphate (S1P). The S1P2 and S1P3 receptors are the most abundant S1P receptor subtypes in mouse and human CPCs while cardiomyocytes express predominantly S1P1 receptors. Treatment of CPCs with S1P, as with thrombin and serum, increased proliferation through a pathway requiring RhoA signaling, as evidenced by significant attenuation when Rho was inhibited by treatment with C3 toxin. Further analysis demonstrated that both S1P- and serum-induced proliferation are regulated through the S1P2 and S1P3 receptor subtypes which couple to Gα12/13 to elicit RhoA activation. The transcriptional co-activator MRTF-A was activated by S1P as assessed by its nuclear accumulation and induction of a RhoA/MRTF-A luciferase reporter. In addition S1P treatment increased expression of cardiac lineage markers Mef2C and GATA4 and the smooth muscle marker GATA6 through activation of MRTF-A. In conclusion, we delineate an S1P-regulated signaling pathway in CPCs that introduces the possibility of targeting S1P2/3 receptors, Gα12/13 or RhoA to influence the proliferation and commitment of c-kit(+) CPCs and improve the response of the myocardium following injury.

Keywords: Cardiac progenitor cells; GPCR; MRTF-A; RhoA; Sphingosine-1-phosphate.

Figure 1. Expression of S1 PR subtypes in adult cardiomyocytes, and in mouse and human cardiac progenitor cells

S1P receptor mRNA levels were analyzed by qPCR and expressed as fold versus GAPDH. (A) Cardiomyocytes (CMs) were isolated from adult mouse heart as described [28] and cardiac progenitor cells (CPCs) (B) mouse and (C) human isolated as described in Methods. S1PR₁ was the highest receptor in CMs while S1PR₂ and S1PR₃ were the highest in mouse and human CPCs respectively. n=4 in A, n = 5 in B, n=1 in C.

Figure 2. S1P induced proliferation in CPCs is RhoA dependent and mediated through S1PR₂ and S1PR₃

(A) Serum starved mouse CPCs were stimulated with S1P (3µM), thrombin (0.5U/ml) or 20% serum for 24 hours and proliferation was measured using the CyQUANT assay. Pre-treatment with C3 toxin (1µg/ml) blocked proliferation induced by S1P (3µM), thrombin and serum. #p≤0.05 vs. control, *p≤0.05 vs. treated by one-way ANOVA; n=15, 7 independent experiments (B) Serum starved CPCs isolated from WT, S1PR₂ KO and S1PR₃ KO mice stimulated with 20% serum. Proliferation was significantly lower in S1PR₂ or S1PR₃ KO CPCs than in WT. *p≤0.05, **p≤0.01, ***p≤0.005 vs. WT by two-ways ANOVA; n=6, 3 independent experiments (C) CPCs were transfected with control, S1P₁, S1P₂ or S1P₃ receptor siRNA for 48 hours prior to addition of S1P (3µM) for 24 hours. Knockdown of S1P receptor mRNA assessed by qPCR was ~80–90% for all three receptor subtypes (data not shown). *p≤0.05 vs. S1P treated Control (siCTL) by one-way ANOVA; n=4, 2 independent experiments.

Figure 3. MRTF-A nuclear accumulation is diminished in S1PR KO CPCs

Serum starved WT, S1PR₂ and S1PR₃ KO mouse CPCs were stimulated with S1P (3µM) for 30 minutes. MRTF-A was localized by immunofluorescence; z-stack confocal images were compressed for analysis. The percentage of cells with nuclear MRTF-A was quantified by counting 10 fields in 3 independent experiments (n=50 cells). (A) Representative immunofluorescence of MRTF-A localization Green- MRTF-A; (B) Quantification of MRTF-A nuclear accumulation. *p≤0.05 vs. non-treated (NT) by unpaired t-test.

Figure 4. S1P induces MRTF-A accumulation in the nucleus in a Rho dependent manner

Serum starved mouse CPCs were stimulated with S1P (3µM) or serum (20%) with or without pre-treatment with C3 toxin (1ug/ml). MRTF-A was localized by immunofluorescence; z-stack confocal images were compressed for analysis. The percentage of cells with nuclear MRTF-A was quantified by counting 10 fields in 3 independent experiments (n=50 cells). (A) Representative immunofluorescence of MRTF-A localization Green- MRTF-A (B) Quantification of MRTF-A nuclear accumulation. *p≤0.05 **p≤0.01, and *** p≤0.005 vs. non-treated (NT) by unpaired t-test

Figure 5. S1P induces RhoA mediated gene expression through Gα_12/13

Mouse CPCs were transfected with an SRE.L luciferase reporter construct (0.5 µg) for 48 hrs (A) Cells were stimulated with S1P (µM) or 20% serum for 6 hours with or without pre-treatment with C3 toxin (1ug/ml). #p≤0.05 vs. non-treated (NT); *p≤0.05 vs. agonist alone by one-way ANOVA; n=12, 4 independent experiments (B) Cells were transfected with siControl or siRNA targeting various G protein α subunits. Knockdown of G protein mRNAs assessed by qPCR was ~80% for Gα₁₂, ~100% for Gα₁₃, ~90% for Gα_q and Gα_i . SRE.L luciferase activity was normalized to Renilla and expressed as percentage of maximal S1P induction with control siRNA. ***p≤0.005 vs. Max S1P response (100%) by unpaired t-test; n=4 independent experiments.

Figure 6. S1P induces cardiac lineage gene expression in a MRTF-A dependent manner

Starved mouse CPCs were treated with S1P (3µM) for 3 days and Mef2C, GATA4 and GATA6 expression levels were measured by qPCR. Fold changes over control at time 0 are graphed. Pharmacological inhibition (10µM, CCG-293971) (A) or silencing (B) of MRTF-A attenuate the effect of S1P on cardiac gene expression. *p≤0.05, **p≤0.01, ***p≤0.005 vs. S1P treated by unpaired t-test; n=8, 3 independent experiments. (C) CPCs were transfected with a plasmid encoding MRTF-A (0.5 µg). Overexpression of MRTF-A for 48 hours induced cardiac gene expression in CPCs. n=12, 4 independent experiments. *p≤0.05 and ***p≤0.005 vs. control at time 0 by unpaired T-test. (D) qPCR analysis of gene expression in S1PR KO CPCs following 3 days of S1P stimulation *p≤0.05 **p≤0.01 vs. WT S1P treated by unpaired t-test; n = 9, 3 independent experiments