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.2012 Nov;53(5):707-14.
doi: 10.1016/j.yjmcc.2012.08.012. Epub 2012 Aug 23.

5-HT(2B) antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

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5-HT(2B) antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

Joshua D Hutcheson et al. J Mol Cell Cardiol.2012 Nov.

Abstract

Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT(2B)) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT(2B) opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT(2B) antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT(2B) antagonism. Interestingly, 5-HT(2B) antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT(2B) antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT(2B) antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT(2B) antagonism as a novel therapeutic approach for CAVD that merits further investigation.

Copyright © 2012 Elsevier Ltd. All rights reserved.

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Figures

Fig. 1
Fig. 1. 5-HT2B antagonism prevents TGF-β1-induced myofibroblast activation and calcific nodule morphogenesis in AVICs
A, Treating AVICs with 1 ng/ml TGF-β1 for 24 h leads to a significant increase in markers for myofibroblast activation, αSMA expression and SM22α promoter activity. Both of these myofibroblast activation markers are reduced to basal levels by pretreating AVICs with either of two 5-HT2B antagonists, SB204741 or SB228357, or an inhibitor of Alk5 (n ≥ 3).B, Adding 15% strain to TGF-β1 treated AVICs leads to calcific nodule morphogenesis that is decreased in a dose dependent manner by treatment with SB228357 and SB204741 (p < 0.005, n = 3).C, Representative images from samples treated with TGF-β1 and increasing dose of SB228357 demonstrate calcific nodules identified using Alizarin Red.D, Calcific nodules were found to be dystrophic with an apoptotic ring (green) of AVICs surrounding a necrotic core (red); bright field (top) and fluorescence (bottom) of a single calcific nodule.E, Neither 5-HT2B antagonist affects cell viability.F, The 5-HT2B antagonists do not affect AVIC proliferation over 24 h. All error bars indicate standard error of the mean. * indicates significant difference (p < 0.005) versus control. Scale bar = 250 µm.
Fig. 2
Fig. 2. 5-HT2B antagonism prevents non-canonical p38 MAPK but not canonical Smad3 signaling
A, TGF-β1 treatment leads to increased Smad3 phosphorylation (pSmad3) after 15 min and p38 phosphorylation (pp38) after 1 h. 5-HT2B antagonism inhibits TGF-β1-induced pp38 but not pSmad3.B, 24 h of TGF-β1 treatment leads to a five-fold increase in canonical PAI-1 promoter activity that is not inhibited by either of the 5-HT2B antagonists (n ≥ 3).C, Average densitometry (n = 5) reveals a two-fold increase in pp38 after TGF-β1 treatment that is completely inhibited by 5-HT2B antagonism.D, p38 inhibitor blocks TGF-β1-induced αSMA expression and SM22α promoter activity but not PAI-1 promoter activity.e, p38 inhibition blocks TGF-β1-induced calcific nodule morphogenesis but Smad3 inhibitor does not.d, Representative images indicate the presence of calcific nodules in AVICs treated with TGF-β1 alone or in combination with Smad3 or p38 inhibitors. All error bars indicate standard error of the mean. * indicates significant difference (p < 0.005) versus control. Scale bar = 250 µm.
Fig. 3
Fig. 3. 5-HT2B antagonism inhibits Src function
A, TGF-β1 induces Src phosphorylation (pSrc) after 15 min of incubation that is inhibited by an Alk5 inhibitor.B, Neither 5-HT2B antagonist inhibit TGF-β1-induced pSrc; however, pCas is reduced with antagonist treatment in the presence of TGF-β1.C, Average densitometry (n = 6) reveal a 2.5-fold increase in pSrc by TGF-β1 at 15 min that return to near basal levels after 1 h. pSrc levels remain elevated in AVICs treated with TGF-β1 and either 5-HT2B antagonist after 1 h.D, Average densitometry (n = 4) reveals that TGF-β1 treatment leads to a trending increase in Cas phosphorylation (pCas); however, 5-HT2B antagonists reduce pCas to sub-basal levels.E, Increasing doses of SB204741 lead to decreasing αSMA but increasing PAI promoter activity in AVICs treated with TGF-β1 (n = 3).F, SYF−/− MEFs exhibit significantly less SM22α promoter activity compared to wild-type MEFs following 24 h of TGF-β1 treatment.G, SYF−/− genetically modified MEFs exhibit a 45-fold increase in PAI-1 promoter activity due to TGF-β1, whereas wild-type MEFs exhibit a four-fold increase in PAI-1 promoter activity.H, AVICs transfected to express constitutively active Src (caSrc) exhibit significantly greater SM22α promoter activity independent of TGF-β1, and SB228357 abrogates this increase. * indicates significant difference (p < 0.005) versus control.
Fig. 4
Fig. 4. 5-HT2B antagonism arrests Src motility within AVICs
A, AVICs transfected with mCherry-N1-Src and treated with DMSO vehicle for 1 h were imaged by time-lapse microscopy. Images were taken every 15 s for a total of 15 min.B, A subsection of a DMSO vehicle treated AVIC allows for clear identification of Src movement at t = 0, 1, and 1.5 min.C, Eulerian based analysis of pixel intensity changes within the image over the course of 15 min represents Src kinematics. Red indicates a high degree of movement, and blue indicates little movement within a pixel.D, E, AVICs treated with 1 µM SB228357 for 1 h exhibit little to no Src movement at t = 0, 1, and 1.5 min.F, 5-HT2B antagonism arrests Src movement over the 15 min imaging time.
Fig. 5
Fig. 5. Proposed mechanism of 5-HT2B antagonist inhibition of TGF-β1 signaling
A, TGF-β1 ligand binding to TβRII leads to Alk5-dependent canonical signaling through Smad phosphorylation (pSmad) and non-canonical signaling through Src phosphorylation (pSrc) and subsequent p38 phosphorylation (pp38).B, Treatment with a 5-HT2B antagonist physically sequesters pSrc preventing non-canonical TGF-β1 signaling by restricting Src’s ability to phosphorylate TβRII and Cas (indicated with × on arrows), but enhancing canonical signaling by suppressing Cas phosphorylation (pCas), which prevents pCas inhibition of pSmad (shown by double hatched arrow).
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References

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