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. 2008 Feb 29;102(4):415-22.
doi: 10.1161/CIRCRESAHA.107.157859. Epub 2007 Dec 13.

Mutation of the HEXIM1 gene results in defects during heart and vascular development partly through downregulation of vascular endothelial growth factor

Monica M Montano  1 , Yong Qui Doughman, Huayun Deng, Laura Chaplin, Jianqi Yang, Nancy Wang, Qiang Zhou, Nicole L Ward, Michiko Watanabe
Affiliations

Mutation of the HEXIM1 gene results in defects during heart and vascular development partly through downregulation of vascular endothelial growth factor

Monica M Montano et al. Circ Res. .

Abstract

Our previous studies and those of others indicated that the transcription factor Hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) is a tumor suppressor and cyclin-dependent kinase inhibitor, and that these HEXIM1 functions are mainly dependent on its C-terminal region. We provide evidence here that the HEXIM1 C-terminal region is critical for cardiovascular development. HEXIM1 protein was detected in the heart during critical time periods in cardiac growth and chamber maturation. We created mice carrying an insertional mutation in the HEXIM1 gene that disrupted its C-terminal region and found that this resulted in prenatal lethality. Heart defects in HEXIM1(1 to 312) mice included abnormal coronary patterning and thin ventricular walls. The thin myocardium can be partly attributed to increased apoptosis. Platelet endothelial cell adhesion molecular precursor-1 staining of HEXIM1(1 to 312) heart sections revealed decreased vascularization of the myocardium despite the presence of coronary vasculature in the epicardium. The expression of vascular endothelial growth factor (VEGF), known to affect angioblast invasion and myocardial proliferation and survival, was decreased in HEXIM1(1 to 312) mice compared with control littermates. We also observed decreased fibroblast growth factor 9 (FGF9) expression, suggesting that effects of HEXIM1 in the myocardium are partly mediated through epicardial FGF9 signaling. Together our results suggest that HEXIM1 plays critical roles in coronary vessel development and myocardial growth. The basis for this role of HEXIM1 is that VEGF is a direct transcriptional target of HEXIM1, and involves attenuation a repressive effects of C/EBPalpha on VEGF gene transcription.

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Figures

Figure 1
Figure 1. Genotype analyses of HEXIM1 mutant mice
(A) The upper panel shows insertion of the gene trap vector into the mouse HEXIM1 gene, resulting in the mutant HEXIM1 allele. The insertion also resulted in an in-frame fusion with the β-geo (encoding β-galactosidase and neomycin phosphotransferase) reporter gene. The lower panel shows a Southern blot wherein genomic DNA was digested with KpnI (K) restriction enzyme and analyzed by hybridization with a 3’ end probe. (B) PCR genotyping of tail DNA from E17.5 embryos. F1, F2, and R2 refer to PCR primers (see Data Supplement for primer sequences).
Figure 2
Figure 2. Expression of HEXIM1 in embryo hearts
(A) β-galactosidase staining of sections from E11.5 embryos (B) Cross sections of E17.5 embryos stained for β-galactosidase. (C) Staining of tissue sections from E13.5 WT and mutant embryos were performed using HEXIM1 antibody to the C-terminal or N-terminal region of HEXIM1.
Figure 3
Figure 3. HEXIM1 mutation resulted in features similar to dilated cardiomyopathy and increased apoptosis in the ventricular myocardium
(A) H&E stained sections of E17.5 mutant HEXIM1 embryos. (B) Higher power view (C) Increased apoptosis was evident in the wall of the left and right ventricular myocardium and intraventricular septum of HEXIM11-312 mice at E15.5. LV=left ventricle; RV=right ventricle; IVS=intraventricular septum.
Figure 4
Figure 4. Aberrant coronary vessel formation and differentiation and decreased VEGF and FGF9 expression in HEXIM1 mutant mice
(A) PECAM-1 immunostaining of E15.5 WT and HEXIM1 mutant whole-mounted embryos and heart sections. (B) The hearts of E15.5 WT and HEXIM1 mutant mice were immunostained with VEGF antibody. (C) Immunostaining of the hearts of E17.5 WT and HEXIM1 mutant mice with FGF9 antibody. (D) VEGF and FGF9 expression were also determined using western blot analyses.
Figure 5
Figure 5. Regulation of VEGF expression by HEXIM1
(A) Northern blot analyses of VEGF expression in H9C2 cells transfected with control vector or expression vectors for WT or mutant HEXM11-312. The 18S ribosomal band was used to normalize for unequal loading. The autoradiograph is representative of 3 experiments. The lower panels show Western blot analyses of WT and mutant HEXIM1 expression using the FLAG antibody. NSB = non-specific band. (B) H9C2 cells were transfected with an expression vector for HEXIM1 and luciferase reporter gene containing VEGF gene promoter. The cells were also transfected with a Renilla luciferase internal control plasmid to normalize for transfection efficiency. Fold activation over cells transfected with control expression vector is displayed as an average of three independent experiments.
Figure 6
Figure 6. VEGF is a direct transcriptional target of HEXIM1
(A) H9C2 cells were transfected with control or expression vector for HEXIM1-312 and subjected to ChIP assays. The -2079/-1252, -944/-633, and coding sequence regions of VEGF gene promoter were immunoprecipitated with HEXIM1 (B) (left) H9C2 cells were transfected with expression vector for WT HEXIM1 or HEXIM1-312 and subjected to ChIP assays. The coding sequence region of VEGF gene promoter was immunoprecipitated with serine 2 phosphorylated RNAPII (right). (B) For IP experiments (left), WT and mutant HEXIM1 were immunoprecipitated from equal concentrations of whole cell extracts using FLAG polyclonal antibody followed by Western blotting with anti-C/EBPα. Normal rabbit IgG was used as control. “Input” lane represents 10% of the total volume of lysate used in each reaction. For GST-pull down assays (right) equal amounts each of in vitro translated and [35S]methionine-labeled WT C/EBPα were incubated with equal amounts each of either glutathione-S-transferase (GST) control, WT or mutant HEXIM1. “Input” lane represents 10% of the total volume of in vitro translated product used in each reaction. (C) (left) The -2079/-1252 region of VEGF gene promoter was immunoprecipitated with C/EBPα or SP1 antibodies. The “input control” represents PCR for 2% of total chromatin used in each reaction. (right) H9C2 cells were transfected with expression vectors for WT HEXIM1, HEXIM11-312, and C/EBPα, and luciferase reporter gene containing the -2079/-1252 region of the VEGF gene promoter. (D) VEGF expression in (left) H9C2 cells transfected with control and C/EBPα siRNA using Northern blot analyses and in (right) WT (n=7) and C/EBPα (n=7) knockout mice using Western blot analyses. Densitometric quantifications of VEGF protein expression levels were expressed relative to expression levels of sarcomeric actin. NSB = non-specific band.
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