The Four-and-a-half LIM Domain Protein 2 Regulates Vascular Smooth Muscle Phenotype and Vascular Tone

Abstract

In response to vascular injury, differentiated vascular smooth muscle cells (vSMCs) undergo a unique process known as "phenotype modulation," transitioning from a quiescent, "contractile" phenotype to a proliferative, "synthetic" state. We have demonstrated previously that the signaling pathway of bone morphogenetic proteins, members of the transforming growth factor ␤ family, play a role in the induction and maintenance of a contractile phenotype in human primary pulmonary artery smooth muscle cells. In this study, we show that a four-and-ahalf LIM domain protein 2 (FHL2) inhibits transcriptional activation of vSMC-specific genes mediated by the bone morphogenetic protein signaling pathway through the CArG box-binding proteins, such as serum response factor and members of the myocardin (Myocd) family. Interestingly, FHL2 does not affect recruitment of serum response factor or Myocd, however, it inhibits recruitment of a component of the SWI/SNF chromatin remodeling complex, Brg1, and RNA polymerase II, which are essential for the transcriptional activation. This is a novel mechanism of regulation of SMC-specific contractile genes by FHL2. Finally, aortic rings from homozygous FHL2-null mice display abnormalities in both endothelial-dependent and -independent relaxation, suggesting that FHL2 is essential for the regulation of vasomotor tone.Unlike skeletal and cardiac muscle cells, vSMCs 2 never terminally differentiate, but are capable of transitioning to a synthetic phenotype characterized by decreased vSMC marker gene expression, increased matrix deposition, and responsiveness to signals that lead to increased migration and growth (1).This plasticity is crucial for the normal development of vessels, homeostasis of blood pressure, and repair of injury, but it also contributes to the development of various vascular pathologies. Therefore, it is critical to understand the signaling pathways that modulate the phenotype of vSMCs to develop agents that can regulate aberrant vSMC responses in diseased vessels.Recently we demonstrated that the BMP signaling pathway plays an important role in the phenotype modulation of PASMCs. Activation of the BMP pathway is required not only for maintenance of the contractile phenotype in PASMCs, but also for induction of vSMC marker gene expression in nonSMCs, such as mouse mesenchymal C3H10T 1 ⁄ 2 (10T1/2) cells (2-4). BMPs are the largest group in the TGF ␤ superfamily of growth factors and play a crucial role in various physiological processes (5-7). During embryonic development, the BMP pathway participates in the induction of ventral mesoderm, cardiac myogenesis, and vasculogenesis (8). Heterozygous mutations in the gene encoding BMPRII, the type II subunit of the BMP receptor, were identified in patients with both familial and sporadic idiopathic pulmonary arterial hypertension (IPAH) (9, 10). IPAH is characterized by elevated pulmonary vascular resistance and pulmonary arterial pressure with increased muscularization of small arteries, thickening or fibrosis of the intima, ...

The Amiloride Derivative Phenamil Attenuates Pulmonary Vascular Remodeling by Activating NFAT and the Bone Morphogenetic Protein Signaling Pathway

“…It has been shown that in pulmonary vSMCs, the BMP signaling pathway is critical for maintenance of pulmonary vascular homeostasis by promoting and/or maintaining the contractile phenotype characterized by decreases in proliferation and migration and increase in contractile gene expression (5,6,11,20,28). Increased expression of Trb3 in PASMCs facilitates the induction of a contractile phenotype by BMP (6).…”

“…A previous study demonstrates that BMP increases the contractility of PASMCs through actin remodeling, visualized by a decrease in collagen lattice (28), and elevated expression of Trb3 is able to potentiate the BMP effect (5). Similar to BMP4, phenamil was able to induce contraction in the collagen lattice (Fig.…”

“…Chromatin was then incubated with either 10 g anti-NFAT2 (also known as NFATc1) antibody (MA3-024; Thermo Scientific) or rabbit nonspecific IgGs as a negative control. PCR primers specific for Trb3 phenamil response element (PRE) are available on request, while the nonspecific SMA gene promoter was described previously (28).…”

“…The chromatin immunoprecipitation (ChIP) assay was performed as described previously (28). Briefly, soluble chromatin was prepared from Ao184 cells following cross-linking reaction with 5 mM dimethyl adipimidate and 1% formaldehyde.…”

Requirement of TCF7L2 for TGF-β-dependent Transcriptional Activation of the TMEPAI Gene

“…6D, lower panel). To further confirm that TTE in the intron 1 contributes to TGF-␤-induced transcription of the TMEPAI gene, we also carried out a ChIP assay using anti-RNA polymerase II anti- body (36). The results indicated that the region including TTE was covered with RNA polymerase II (Fig.…”