Korean J Physiol Pharmacol.  2017 Jan;21(1):37-44. 10.4196/kjpp.2017.21.1.37.

Regulation of vascular smooth muscle phenotype by cross-regulation of krüppel-like factors

• Ha JM
• Yun SJ
• Jin SY
• Lee HS
• Kim SJ
• Shin HK
• Bae SS

• KMID: 2371066
• DOI: http://doi.org/10.4196/kjpp.2017.21.1.37

Abstract

Regulation of vascular smooth muscle cell (VSMC) phenotype plays an essential role in many cardiovascular diseases. In the present study, we provide evidence that krüppel-like factor 8 (KLF8) is essential for tumor necrosis factor α (TNFα)-induced phenotypic conversion of VSMC obtained from thoracic aorta from 4-week-old SD rats. Stimulation of the contractile phenotype of VSMCs with TNFα significantly reduced the VSMC marker gene expression and KLF8. The gene expression of KLF8 was blocked by TNFα stimulation in an ERK-dependent manner. The promoter region of KLF8 contained putative Sp1, KLF4, and NFκB binding sites. Myocardin significantly enhanced the promoter activity of KLF4 and KLF8. The ectopic expression of KLF4 strongly enhanced the promoter activity of KLF8. Moreover, silencing of Akt1 significantly attenuated the promoter activity of KLF8; conversely, the overexpression of Akt1 significantly enhanced the promoter activity of KLF8. The promoter activity of SMA, SM22α, and KLF8 was significantly elevated in the contractile phenotype of VSMCs. The ectopic expression of KLF8 markedly enhanced the expression of SMA and SM22α concomitant with morphological changes. The overexpression of KLF8 stimulated the promoter activity of SMA. Stimulation of VSMCs with TNFα enhanced the expression of KLF5, and the promoter activity of KLF5 was markedly suppressed by KLF8 ectopic expression. Finally, the overexpression of KLF5 suppressed the promoter activity of SMA and SM22α, thereby reduced the contractility in response to the stimulation of angiotensin II. These results suggest that cross-regulation of KLF family of transcription factors plays an essential role in the VSMC phenotype.

Keyword

Angiotensin; Krüppel-like factor; Phenotype; Transcription; Vascular smooth muscle cell

MeSH Terms

Angiotensin II
Angiotensins
Animals
Aorta, Thoracic
Binding Sites
Cardiovascular Diseases
Ectopic Gene Expression
Gene Expression
Humans
Muscle, Smooth, Vascular*
Phenotype*
Promoter Regions, Genetic
Rats
Transcription Factors
Tumor Necrosis Factor-alpha
Angiotensin II
Angiotensins
Transcription Factors
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 TNFα suppresses the expression of KLF8.(A) Contractile phenotype of VSMCs were stimulated with TNFα (50 ng/ml) for 4 days. Expression of contractile marker proteins were verified by the indicated antibodies. (B) Contractile cells were treated with TNFα (50 ng/ml) for the indicated times. Expression of KLF8 was verified by RT-PCR. (C) Contractile type of VSMCs were stimulated with TNFα (50 ng/ml) and PDGF (50 ng/ml), and promoter activity of KLF8 was examined as described in the “Materials and Methods” section. (D). Contractile type of VSMCs were pretreated with ERK inhibitor (PD, 10 µM) or p38 MAPK inhibitor (SB, 10 µM), and promoter activity of KLF8 was measured. Data are expressed as the mean±S.D. for three independent experiments (n=3 for each experiment). *Significantly different from values of vehicle-treated group (p<0.05).

• 

  Fig. 2 Expression of KLF8 is regulated by myocardin, KLF4 and Akt1.(A) Schematic representation of KLF8 promoter region. Cis-acting elements were indicated at upper figure and deletion mutants were denoted at lower figure. (B) Promoter activity of each deletion mutant was measured as described in the “Materials and Methods” section. (C) Promoter activity of each deletion mutant was measured in the presence of myocardin. (D) Contractile type of VSMCs was co-transfected with myocardin and promoter activity of KLF4 was measured. (E) Contractile type of VSMCs was co-transfected with KLF4 and promoter activity of KLF8 (pGL3-Δ2) was measured. (F, G) Akt1 was either silenced or overexpressed in contractile type of VSMCs, and promoter activity of KLF8 (pGL3-Δ2) was measured. Data are expressed as the mean±S.D. for three independent experiments (n=3 for each experiment). *Significantly different from values of vector group (p<0.05).

• 

  Fig. 3 Ectopic expression of KLF8 promotes VSMC differentiation.(A~C) Promoter activity of SMA, SM22a, and KLF8 was measured in either synthetic (P0) and contractile (P4) phenotype of VSMCs. (D, E) FLAG-tagged KLF8 was overexpressed in synthetic phenotype of VSMCs, and morphological changes of VMCs (bright field image) as well as expression of contractile marker proteins were examined. (F) Synthetic type of VSMCs were transfected with KLF8 and promoter activity of SMA was measured as described in the “Materials and Methods” section. Data are expressed as the mean±S.D. for three independent experiments (n=3 for each experiment). *Significantly different from values of synthetic or vector group (p<0.05).

• 

  Fig. 4 KLF8 suppresses promoter activity of KLF5.(A) Contractile type of VSMCs were stimulated with TNFα for the indicated times. Expression of KLF5 and contractile marker proteins were verified by western blot analysis. (B, C) Promoter activity of both KLF5 and KLF4 was measured in the presence of KLF8. (D, E) Promoter activity of SMA and SM22α was measured in the presence of KLF5. (F) KLF5 was overexpressed in contractile type of VSMCs, and AngII-induced contraction of VSMCs was measured as described in the “Materials and Methods” section. Data are expressed as the mean±S.D. for three independent experiments (n=3 for each experiment). *Significantly different from values of vector group (p<0.05).

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