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. 2012 Apr 13;287(16):13084-93.
doi: 10.1074/jbc.M111.288944. Epub 2012 Feb 3.

Cardiac lineage protein-1 (CLP-1) regulates cardiac remodeling via transcriptional modulation of diverse hypertrophic and fibrotic responses and angiotensin II-transforming growth factor β (TGF-β1) signaling axis

Eduardo Mascareno  1 , Josephine Galatioto, Inna Rozenberg, Louis Salciccioli, Haroon Kamran, Jason M Lazar, Fang Liu, Thierry Pedrazzini, M A Q Siddiqui
Affiliations

Cardiac lineage protein-1 (CLP-1) regulates cardiac remodeling via transcriptional modulation of diverse hypertrophic and fibrotic responses and angiotensin II-transforming growth factor β (TGF-β1) signaling axis

Eduardo Mascareno et al. J Biol Chem. .

Abstract

It is well known that the renin-angiotensin system contributes to left ventricular hypertrophy and fibrosis, a major determinant of myocardial stiffness. TGF-β1 and renin-angiotensin system signaling alters the fibroblast phenotype by promoting its differentiation into morphologically distinct pathological myofibroblasts, which potentiates collagen synthesis and fibrosis and causes enhanced extracellular matrix deposition. However, the atrial natriuretic peptide, which is induced during left ventricular hypertrophy, plays an anti-fibrogenic and anti-hypertrophic role by blocking, among others, the TGF-β-induced nuclear localization of Smads. It is not clear how the hypertrophic and fibrotic responses are transcriptionally regulated. CLP-1, the mouse homolog of human hexamethylene bis-acetamide inducible-1 (HEXIM-1), regulates the pTEFb activity via direct association with pTEFb causing inhibition of the Cdk9-mediated serine 2 phosphorylation in the carboxyl-terminal domain of RNA polymerase II. It was recently reported that the serine kinase activity of Cdk9 not only targets RNA polymerase II but also the conserved serine residues of the polylinker region in Smad3, suggesting that CLP-1-mediated changes in pTEFb activity may trigger Cdk9-dependent Smad3 signaling that can modulate collagen expression and fibrosis. In this study, we evaluated the role of CLP-1 in vivo in induction of left ventricular hypertrophy in angiotensinogen-overexpressing transgenic mice harboring CLP-1 heterozygosity. We observed that introduction of CLP-1 haplodeficiency in the transgenic α-myosin heavy chain-angiotensinogen mice causes prominent changes in hypertrophic and fibrotic responses accompanied by augmentation of Smad3/Stat3 signaling. Together, our findings underscore the critical role of CLP-1 in remodeling of the genetic response during hypertrophy and fibrosis.

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Figures

FIGURE 1.
FIGURE 1.
Enhanced left ventricular hypertrophy in αMHC-ANG/CLP-1+/− mice. A, representative cross-section at the level of the left ventricle of adult mouse hearts of wild type heart, transgenic wild type hearts, nontransgenic heterozygous heart, and transgenic heterozygous heart. B, panel shows collagen deposition in a section of the left ventricle of αMHC-ANG/CLP-1+/+ and αMHC-ANG/CLP-1+/− mice obtained by Masson Trichrome stain in magnification of ×200. C, bar graph shows the distribution of collagen deposition between αMHC-ANG/CLP-1+/+ and αMHC-ANG/CLP-1+/− hearts sections (n = 3 per group). *, p < 0.05 between αMHC-ANG/CLP-1+/− versus αMHC-ANG/CLP-1+/+. D, heart/body weight ratios for nontransgenic wild type heart, nontransgenic heterozygous heart, and transgenic heterozygous heart. *, p values < 0.05. For wild type versus αMHC-ANG, the values are means ± S.E.; **, p values are <0.01 αMHC-ANG/CLP-1+/− versus αMHC-ANG. E, bar graph shows the quantification of cross-sectional area. Values are mean ± S.E.; *, p < 0.05 αMHC-ANG versus wild type; **, p values are <0.01 αMHC-ANG/CLP-1+/− versus αMHC-ANG.
FIGURE 2.
FIGURE 2.
A, two upper panels represent the CLP-1 and αMHC-ANG genotypes of each genetic background Western-blotted with specific antibodies. GAPDH was used as loading control (n = 4). Statistical analysis on the expression levels of CLP-1 and cyclin T1 is depicted in the graphs. CLP-1 expression, *, p < 0.05 CLP-1+/+ versus αMHC-ANG/CLP-1+/+; **, p < 0.05 CLP-1+/− versus αMHC-ANG/CLP-1+/−. p value was not significant between CLP-1+/+ versus αMHC-ANG/CLP-1+/−. CyclinT1 expression, *, p < 0.05 CLP-1+/− versus αMHC-ANG/CLP-1+/−. B, representative Western blot showing CLP-1/CyclinT1 interaction by immunoprecipitation (IP) of total protein heart extracts from mice of each genetic background with anti-CLP-1 followed by blotting with anti-CyclinT1 antibody. Loading control was performed using a chicken anti-CLP-1 antibody (bottom panel). C, AngII triggers activation of the Jak/Stat pathway. The upper panel is a Western blot showing tyrosine phosphorylation of Jak2. Total heart tissue extracts were immunoprecipitated with polyclonal anti-Jak2 antibody and probed with anti-phosphotyrosine-Jak2 antibody. A direct Western blot using anti-Jak2 antibody serves as loading control. D, lower panel shows the tyrosine phosphorylation of Stat3. As above, total heart tissue extracts were immunoprecipitated with rabbit anti-Stat3 followed by Western blotting with anti-phosphotyrosine Stat3 antibodies. Data are expressed as means ± S.E. of four independent experiments. *, p < 0.05 control mice versus transgenic mice. **, p < 0.05 αMHC-ANG/CLP-1+/− versus αMHC-ANG.
FIGURE 3.
FIGURE 3.
Enhanced expression of smooth muscle α-actin in CLP-1+/− fibroblasts. A, Western blots show the expression of smooth muscle α-actin in wild type (SM-α-actin+/+) and CLP-1+/− (SM-α-actin+/+) fibroblasts. GAPDH expression was used as loading control. Data are expressed as means ± S.E. of four independent experiments. SM-α-actin, *, p < 0.05 wild type versus wild type treated with agonist; *, p < 0.05 control untreated versus treated with TGF-β, and **, p < 0.05 CLP-1+/− versus wild type at 24 h treated with agonists. B, Western blot of wild type (CLP-1+/+) and heterozygous (CLP-1+/−) cardiac fibroblasts treated for 24 h with TGF-β (10 ng/ml) and/or AngII peptide (10−7 m). The Western blot shows that the expression of Nox4 paralleled the increase expression of smooth muscle α-actin in CLP-1+/− fibroblast. Data are expressed as means ± S.E. of four independent experiments. Nox4, *, p < 0.05 wild type versus CLP-1+/− treated with agonists. C, immunofluorescence assay performed in mouse embryonic in wild type (+/+), heterozygous (CLP-1+/−) and null (CLP-1−/−) fibroblasts, showing the AngII or TGF-β1 induction of nuclear translocation of serine-phosphorylated Smad3 (red) and expression of smooth muscle α-actin (green). Nuclear staining was obtained with DAPI (blue).
FIGURE 4.
FIGURE 4.
CLP-1 gene dosage modulates serine-Smad3 transcriptional activity. A, transient transfections using the Smad3-luciferase reporter (pT3×Luc), the FLAG-Smad3, or the mutant FLAG-MSmad3 were examined in mouse embryonic fibroblasts wild type, heterozygous, and mice null for the CLP-1 gene. In the absence of the ligands AngII or TGF-β1, there was a significant increase in the luciferase reporter activity inversely proportional to the CLP-1 gene dosage. However, substitution mutations of the conserved serine residues in the polylinker region of Smad3 (MSmad3) completely abolished luciferase activity. B, AngII mediates Smad3 transcriptional activity in mouse embryonic fibroblasts, and a similar response as above using AngII as inducer of Smad activity was observed. C, even a more significant transcriptional induction was observed with TGF-β1 treatment that was reversed by co-transfection with pcDNA CLP-1 (Null + C (CLP-1) + TGF). Data are expressed as means ± S.E. of four independent experiments. *, p < 0.05 control versus TGF-β treatment; **, p < 0.05 Null TGF-β-treated versus Null TGF-β treated and CLP-1 added. D, upper panels shows a representative Western blot with specific antibodies against MMP3, MMP9, and CCN1. GAPDH was used as loading control (n = 4). E, bar graphs represent the quantitative determination of atrial natriuretic peptide and brain natriuretic peptide by qPCR. Data are expressed as means ± S.E. of three independent experiments. ANP and BNP expression shows a *, p < 0.05 αMHC-ANG/CLP-1+/+ versus αMHC-ANG/CLP-1+/−. F, Western blots show the expression of serine 208-Smad3 in heart protein extracts from wild type (WT), heterozygous (CLP-1+/−), transgenic wild type-αMHC-ANG (TG+/+), and transgenic heterozygous-αMHC-ANG (TG+/−) mice hearts. GAPDH expression was used as loading control. Data are expressed as means ± S.E. of three independent experiments. Serine 203-Smad3 expression shows the following: *, p < 0.05 αMHC-ANG/CLP-1+/− versus αMHC-ANG/CLP-1+/+.
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