Serum Response Factor Binding Research Articles

The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-κB Sites in Proliferating Cells

The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate of IE gene transcription and thus affect the outcome of HCMV infection. We have shown previously that the NF-kappaB binding sites within the MIEP enhancer and cellular NF-kappaB activity induced by HCMV infection are required for efficient MIEP activity and viral replication in quiescent cells (P. Caposio, A. Luganini, G. Hahn, S. Landolfo, and G. Gribaudo, Cell. Microbiol. 9:2040-2054, 2007). We now show that the inactivation of either the Elk-1 or serum response factor (SRF) binding site within the enhancer also reduces MIEP activation and viral replication of recombinant HCMV viruses in quiescent fibroblasts. In these cells, we show that the expression of either Elk-1 or SRF is required for optimal IE gene expression, and that the HCMV-stimulated activation of the MEK1/2-ERK1/2 signaling axis leads to Elk-1 transcriptional competency. Furthermore, the replication kinetics of recombinant viruses in which NF-kappaB, Elk-1, and SRF binding sites all are inactivated demonstrate that the higher levels of Elk-1 and SRF binding to MIEP in proliferating cells can compensate even for a lack of HCMV-induced NF-kappaB-mediated MIEP transactivation. These observations highlight the importance of the combination of different MIEP binding sites to optimize IE gene expression in cells in different physiological states.

Phosphorylation of Myocardin by Extracellular Signal-regulated Kinase

The contractile phenotype of smooth muscle (SM) cells is controlled by serum response factor (SRF), which drives the expression of SM-specific genes including SM alpha-actin, SM22, and others. Myocardin is a cardiac and SM-restricted coactivator of SRF that is necessary for SM gene transcription. Growth factors inducing proliferation of SM cells inhibit SM gene transcription, in a manner dependent on the activation of extracellular signal-regulated kinases ERK1/2. In this study, we found that ERK1/2 phosphorylates mouse myocardin (isoform B) at four sites (Ser(812), Ser(859), Ser(866), and Thr(893)), all of which are located within the transactivation domain of myocardin. The single mutation of each site either to alanine or to aspartate has no effect on the ability of myocardin to activate SRF. However, the phosphomimetic mutation of all four sites to aspartate (4xD) significantly impairs activation of SRF by myocardin, whereas the phosphodeficient mutation of all four sites to alanine (4xA) has no effect. This translates to a reduced ability of the 4xD (but not of 4xA) mutant of myocardin to stimulate expression of SM alpha-actin and SM22, as assessed by corresponding promoter, mRNA, or protein assays. Furthermore, we found that phosphorylation of myocardin at these sites impairs its interaction with acetyltransferase, cAMP response element-binding protein-binding protein, which is known to promote the transcriptional activity of myocardin. In conclusion, we describe a novel mode of modulation of SM gene transcription by ERK1/2 through a direct phosphorylation of myocardin.

Abstract 5474: Crucial Role of Nrf3 in Smooth Muscle Cell Differentiation From Stem Cells and Vessel Injury-Induced Neointima Formation

Nuclear factor erythroid 2-related factor 3 (Nrf3), a member of the cap’n’collar family of transcription factors that binds to the DNA-antioxidant responsive elements, have been reported to be involved in muscle precursor migration during early embryo development. The present study aimed to investigate the functional role of Nrf3 in stem cell differentiation into smooth muscle cells (SMCs). When embryonic stem cells were cultivated on collagen IV-coated plates, Nrf3 was up-regulated significantly following 1–6 days of cell differentiation. Knockdown of Nrf3 resulted in down-regulation of smooth muscle specific markers including smooth muscle actin, SM22, calponin and smooth muscle myosin heavy chain, while enforced expression of Nrf3 enhanced SMC differentiation in a dose-dependent manner. SMC-specific transcription factor myocardin, but not serum response factor, was significant upregulated by Nrf3 overexpression. Strikingly, the binding of serum response factor and myocardin to the promoter of smooth muscle differentiation genes was dramatic increased by Nrf3 overexpres-sion, and Nrf3 can direct bind to the smooth muscle gene promoters as demonstrated by chromatin immunoprecipitation (CHIP) assay. Moreover, NADPH-derived ROS production during SMC differentiation was further enhanced by Nrf3 overexpression through upregualtion of NADPH oxidase (NOX4) and inhibition of anti-oxidant signalling pathway. Furthermore, overexpression of Nrf3 resulted in increase of plasma phospholipase A2 (Pla2g7) production, while knockdown Nrf3 ablated the Pla2g7 gene expression and protein activity. Additionally, both Nrf3 and Pla2g7 were up-regulated in adult SMC phenotype swiching from proliferating to contractile phenotype upon serum starvation. Importantly, in vivo knockdown of Nrf3 in the vessel wall significant increased wire-injury-induced neointima formation in ApoE-deficient mice. Our findings demonstrated for the first time that Nrf3 has a crucial role in SMC differentiation from stem cells and important protective function in vessel injury-induced neointima formation, indicating that they could be potential new therapeutic targets for intervention of SMC proliferative-related vascular diseases.

Abstract 5806: SRF Protects Brain Endothelial Cells Against Hypoxic Damage

Ischemia resulting from brain vascular disorders is the major cause of stroke. Hypoxia triggers both angiogenesis and apoptosis through the induction of HIF1 α -activated VEGF and the alteration of mitochondrial permeability, respectively. The balance between these two events is crucial to both the progression of vascular disorders and the outcome of injury healing. Our previous study showed that SRF (Serum response factor) is essential to VEGF-induced angiogenesis. This study aimed to determine the roles of SRF in hypoxia-induced brain endothelial cell angiogenesis and apoptosis. Methods : Mouse brain endothelial cells (bEND3) were transfected with either an SRF expressing plasmid, SRF shRNA, or the vehicle (control), and cultured under hypoxic condition (5% CO 2 : 94% N 2 : 1% O 2 ) for 0, 0.5, 2, 6 and 24 hours. Angiogenesis and apoptosis were assessed by 3-D in vitro angiogenesis assay and Annexin V binding assay, respectively. Results : After 30-min hypoxic treatment, SRF mRNA expression was dramatically increased by 54 fold and then gradually declined and returned to normal at 24 hours. SRF protein expression and phosphorylation were also increased by 111±24 % and 74±7 % (both P < 0.01), respectively, after 6-hour hypoxia. Hypoxia also enhanced SRF binding activity to DNA. Forced overexpression of SRF increased hypoxia-induced angiogenesis by 232±15 % (P < 0.01); and knockdown of SRF completely abolished it. After 24-hour hypoxia, 29±4 % of the cells underwent apoptosis; while overexpression of SRF reduced this percentage to 5.7±1 %; and knockdown of SRF increased it to 85±4 % (all P < 0.001). Overexpression of SRF increased Bcl-2/Bax ratio by 23±4 % at baseline and by 167±20 % after 24-hour hypoxia; knockdown of SRF decreased these ratios by 69±3 % and 75±8 %, respectively (all P < 0.01). Knockdown of SRF also increased Bax translocation to mitochondria and Cytochrome C release. Conclusions : Hypoxia triggers SRF activation. Up-regulation of SRF promotes angiogenesis. Down-regulation of SRF sensitizes cells to hypoxia-induced apoptosis. SRF protects against hypoxia-induced apoptosis likely through both direct up-regulation of Bcl-2 and indirect down-regulation of Bax. This research has received full or partial funding support from the American Heart Association, Western States Affiliate (California, Nevada & Utah).

The Smooth Muscle Cell-restricted KCNMB1 Ion Channel Subunit Is a Direct Transcriptional Target of Serum Response Factor and Myocardin

Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise alpha and beta subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.

SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin

SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.

PKA-Dependent Phosphorylation of Serum Response Factor Inhibits Smooth Muscle–Specific Gene Expression

Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRF's involvement in smooth muscle cell (SMC)-specific and early response gene expression. By screening phosphorylation-deficient and mimetic mutations in SRF(-/-) embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG. Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.

Interaction of Hsp70 with p49/STRAP, a serum response factor binding protein

Members of the Hsp70 protein family must work with other co-chaperones to exert their function. Herein, we identified a new Hsp70 co-chaperone, p49/STRAP, previously shown to interact with serum response factor. We demonstrated that a fraction of p49/STRAP was cytosolic, and that it interacted with the β-sandwich domain of Hsp70. Although p49/STRAP had little effect on the intrinsic ATPase activity of Hsp70, it reduced the ATP-hydrolytic activity of Hsp70 stimulated by Hsp40, and inhibited the refolding activity of the Hsp70/Hsp40 system. Thus, p49/STRAP can be considered a bona fide co-chaperone of Hsp70.

Negative Regulation of the EGFR-MAPK Cascade by Actin-MAL-Mediated Mig6/Errfi-1 Induction

We analyzed the G-actin-regulated transcriptome by gene expression analysis using previously characterized actin-binding drugs. We found many known MAL/MRTF-dependent target genes of serum response factor (SRF), as well as additional directly regulated genes. Surprisingly, several putative antiproliferative target genes were identified, including mig6/errfi-1, a negative regulator of the EGFR family. Mig6 induction occurred through actin-MAL-SRF signaling, and MAL was inducibly recruited to and activated a mig6 promoter element. Upregulation of Mig6 by lipid agonists such as LPA and S1P or actin drugs involved MAL and correlated with decreased activation of EGFR, MAPK/Erk, and c-fos. Mig6 depletion restored EGFR signaling and provided a proliferative advantage. Overexpression of MAL exhibited strong antiproliferative effects requiring the domains for SRF binding and transactivation, which supports antagonistic functions of MAL on growth-promoting signals. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signaling pathways toward SRF.

Enhancer of Polycomb1 Acts on Serum Response Factor to Regulate Skeletal Muscle Differentiation

Skeletal muscle differentiation is well regulated by a series of transcription factors. We reported previously that enhancer of polycomb1 (Epc1), a chromatin protein, can modulate skeletal muscle differentiation, although the mechanisms of this action have yet to be defined. Here we report that Epc1 recruits both serum response factor (SRF) and p300 to induce skeletal muscle differentiation. Epc1 interacted physically with SRF. Transfection of Epc1 to myoblast cells potentiated the SRF-induced expression of skeletal muscle-specific genes as well as multinucleation. Proximal CArG box in the skeletal alpha-actin promoter was responsible for the synergistic activation of the promoter-luciferase. Epc1 knockdown caused a decrease in the acetylation of histones associated with serum response element (SRE) of the skeletal alpha-actin promoter. The Epc1.SRF complex bound to the SRE, and the knockdown of Epc1 resulted in a decrease in SRF binding to the skeletal alpha-actin promoter. Epc1 recruited histone acetyltransferase activity, which was potentiated by cotransfection with p300 but abolished by si-p300. Epc1 directly bound to p300 in myoblast cells. Epc1+/- mice showed distortion of skeletal alpha-actin, and the isolated myoblasts from the mice had impaired muscle differentiation. These results suggest that Epc1 is required for skeletal muscle differentiation by recruiting both SRF and p300 to the SRE of muscle-specific gene promoters.

The SWI/SNF Chromatin Remodeling Complex Regulates Myocardin-Induced Smooth Muscle–Specific Gene Expression

Regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle-specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle-specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle-specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, which lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle-specific genes. Whereas, reconstitution of wild-type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle-specific genes. Brg1 and Brm directly bind to the N terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. These data demonstrate that the ability of myocardin to induce smooth muscle-specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes.

The role of SWI/SNF in regulating smooth muscle differentiation

The myocardin related transcription factors (MRTFs) play a critical role in regulating serum response factor's (SRF) ability to drive smooth muscle differentiation. MRTFs can induce the transcription of smooth muscle‐specific genes in nonmuscle cells through a mechanism involving the induction of SRF binding to the promoters of these genes, within intact chromatin. The goal of our studies is to understand how MRTFs can facilitate SRF binding within an otherwise closed chromatin landscape. We found that MRTFs can recruit SWI/SNF ATP‐dependent chromatin remodeling complexes, to the promoters of smooth muscle‐specific genes thereby permitting high affinity binding of SRF. Expression of dominant negative Brg1 or knockdown of Brg1 attenuated expression of SRF dependent smooth muscle‐specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and Brg1 directly binds MRTFs in vitro. Dominant negative Brg1 significantly attenuated the ability of MRTFs to increase SRF binding to the promoters of smooth muscle‐specific genes. Mice harboring a smooth muscle‐specific knockout of Brg1 were generated and preliminary analysis of these mice revealed defects in GI development, including a significantly shorter gut with attenuated contractility in Brg1 knockout mice. Supported by DK61130, an AHA fellowship to MZ and an AHA SDG to LZ.

Gene regulation and deregulation by MAL/MRTF coactivators

Proteins of the MRTF family play a key role in regulated gene expression downstream of Rho family GTPases. MAL/MKL1/MRTF-A is a transcriptional coactivator of serum response factor (SRF) and is bound and inhibited by G-actin in the non-induced state. In acute megakaryocytic leukaemia in infants, a recurrent translocation t(1;22) results in the OTT-MAL/RBM15-MKL1 fusion oncoprotein. How it contributes to the malignancy is unknown. We tested by biochemical analysis whether OTT-MAL is functionally deregulated. We showed that OTT-MAL is a constitutive activator of SRF and target gene expression. This requires the SRF binding motif and the MAL-derived transactivation domain. OTT-MAL localises to the nucleus and is not regulated by upstream signalling. OTT-MAL deregulation reflects its independence from control by G-actin, which fails to interact with OTT-MAL in co-immunoprecipitation experiments. OTT-MAL also caused a delayed induction of the MAL-independent, TCF-dependent target genes c-fos and egr-1, and the MAPK/Erk pathway. In addition, RBPJ/CBF-1 regulated gene expression was activated by OTT-MAL but not by MAL. Our data suggest that the deregulated activation of MAL-dependent and independent promoters results in tissue-specific functions of OTT-MAL. When tested in heterologous tissue culture systems, however, we observed strong anti-proliferative effects of OTT-MAL. Similarly, overexpression of MAL exhibited antiproliferative and pro-apoptotic effects requiring transcription through SRF. To gain insight into the molecular mechanisms involved, we performed gene expression analysis. By using a combination of actin binding drugs, which specifically interfere with the actin-MAL complex, we identified on a genome wide basis 210 genes primarily regulated by G-actin. We found many known MAL-dependent SRF target genes, as well as novel directly regulated genes. Several putative antiproliferative target genes were newly identified. We showed that a group of MAL regulated genes negatively interferes with the EGFR-MAPK pathway, thereby reducing proliferative signalling. Our results show the existence of negatively acting transcriptional networks between pro- and antiproliferative signalling pathways towards subsets of SRF target genes.

A Rare Human Sequence Variant Reveals Myocardin Autoinhibition

Myocardin (MYOCD) is a transcriptional co-activator that promotes cardiac or smooth muscle gene programs through its interaction with myocyte-enhancing factor (MEF2) or serum-response factor (SRF). Isoforms of MYOCD with a truncated amino terminus show increased activity when compared with those with the full-length amino terminus, but how this is achieved remains unknown. We identified a rare human sequence variation in MYOCD in a patient with congenital heart disease that resulted in a missense mutation at codon 259 (K259R). This variation created a hypomorphic cardiac isoform with impaired SRF binding and transactivation capacity but did not impair the smooth muscle isoform of MYOCD, which lacks the amino terminus. Consistent with differential effects of the amino terminus on the K259R mutation, we found that the cardiac-specific amino terminus acted in an autoinhibitory fashion to bind MYOCD via specific negatively charged residues and thereby repressed SRF-dependent MYOCD activity. This effect was exaggerated in the MYOCD-K259R mutant. The amino terminus was sufficient to impair MYOCD-dependent fibroblast conversion into smooth muscle cells as well as cardiomyocyte hypertrophy. These findings identify a novel mechanism that regulates levels of MYOCD-dependent activation of the SRF genetic program differentially in cardiac and smooth muscle.

Thymine DNA Glycosylase Represses Myocardin-induced Smooth Muscle Cell Differentiation by Competing with Serum Response Factor for Myocardin Binding

Myocardin is a serum response factor (SRF) co-activator that regulates transcription of many smooth muscle-specific genes and is essential for development of vascular smooth muscle. We used a yeast two-hybrid screen, with myocardin as bait in a search for factors that regulate myocardin transcriptional activity. From this screen, thymine DNA glycosylase (TDG) was identified as a myocardin-associated protein. TDG was originally identified as an enzyme involved in base excision repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. However, TDG has also been shown to act as a transcriptional co-activator or co-repressor. The interaction between TDG and myocardin was confirmed in vitro by glutathione S-transferase pull down and in vivo by co-immunoprecipitation assays. We found that TDG abrogates myocardin induced expression of smooth muscle-specific genes and represses the trans-activation of the promoters of myocardin of these genes. Overexpression of TDG in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous TDG in SMCs increased smooth muscle-specific myosin heavy chain (SM MHC) and Telokin gene expression. Glutathione S-transferase pull-down assays demonstrated that TDG binds to a region of myocardin that includes the SRF binding domain. Furthermore, TDG was found to compete with SRF for binding to myocardin in vitro and in vivo, suggesting that TDG can inhibit expression of smooth muscle-specific genes, at least in part, through disrupting SRF/myocardin interactions. Finally, we demonstrated that the glycosylase activity of TDG is not required for its inhibitory effects on myocardin function. This study reveals a previously unsuspected role for the repair enzyme TDG as a repressor of smooth muscle differentiation via competing with SRF for binding to myocardin.

“FRNKly, Smooth Muscle, I Don’t Give a CArG!”

Studies spanning the last two decades have begun to uncover the molecular mechanisms underlying smooth muscle cell (SMC) differentiation, specifically the critical role of the ubiquitously expressed serum response factor (SRF) transcription factor. SRF selectively binds to conserved cis-regulatory “CArG box” elements, CC(A/T)6GG consensus sequence, in the promoters or first introns of numerous smooth muscle-selective genes. The SMC contractile marker genes smooth muscle alpha actin (SMαA), smooth muscle myosin heavy chain (SM-MHC), and smooth muscle 22 alpha (SM22α) all require multiple CArG boxes for proper transcriptional activation. On SRF binding, the coactivator myocardin is recruited to the promoter through direct interaction with SRF and is required for expression of CArG containing SMC-selective genes.1,2 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Sayers et al detail the in vivo regulation of a SMC-selective CArG-independent gene, focal adhesion kinase related nonkinase (FRNK), that plays a critical role in CArG-dependent contractile gene expression during SMC differentiation and after vascular injury.3 See accompanying article on page 2115 Unlike other muscle cell types, adult SMCs exist in a highly plastic nonterminally differentiated state. SMCs can assume a proliferative and migratory synthetic phenotype, allowing for both complete investment of the endothelial tube during vascular development and repair of the vessel wall after arterial injury. Various environmental cues direct SMCs to phenotypically modulate into a highly …

Integrin-linked kinase regulates smooth muscle differentiation marker gene expression in airway tissue

Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of beta-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22alpha, and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22alpha, and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22alpha, and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix.

Regulation of SRF/CArG‐dependent gene transcription during chronic partial obstruction of murine small intestine

Intestinal obstructions lead to a variety of motility disorders. Small intestine smooth muscles undergo dramatic phenotypic changes in response to obstruction, but the underlying molecular mechanisms are unknown. Using RT-PCR, ChIP, Re-ChIP, and Western blots, we examined the effect of small bowel mechanical obstruction on smooth muscle gene expression. Obstruction caused a transient hyperplasia, followed by a prolonged hypertrophic response of small intestine smooth muscle cells. Smooth muscle myosin heavy chain (MHC), alpha-actin, and gamma-actin expression decreased initially, and then increased as hypertrophy developed. Myocardin expression decreased initially and then increased, while kruppel-like factors (KLF)4 and KLF5 expression increased initially, and then decreased. Serum response factor (SRF) expression decreased initially, and then recovered to sham-operated levels as hypertrophy developed. SRF binding to smooth muscle MHC and alpha-actin promoters decreased initially, but then increased above sham-operated levels as hypertrophy developed. Elk-1 binding to smooth muscle myosin heavy chain and alpha-actin promoters increased initially, and then decreased to sham-operated levels as hypertrophy developed. c-fos expression increased initially, which was associated with increased SRF/Elk-1 binding to the c-fos promoter. The Elk-1 phosphorylation inhibitor U-0126 inhibited the increase in c-fos expression. These findings indicate a dynamic response of small intestine smooth muscles to bowel obstruction involving switching between differentiated, proliferative, and hypertrophic phenotypes. These results suggest that changes in the expression and interactions between SRF, myocardin, Elk-1, and c-fos play key roles in the phenotypic switching of small intestine smooth muscles in response to mechanical obstruction.

Regulation of myocardin factor protein stability by the LIM-only protein FHL2

Extensive evidence indicates that serum response factor (SRF) regulates muscle-specific gene expression and that myocardin family SRF cofactors are critical for smooth muscle cell differentiation. In a yeast two hybrid screen for novel SRF binding partners expressed in aortic SMC, we identified four and a half LIM domain protein 2 (FHL2) and confirmed this interaction by GST pull-down and coimmunoprecipitation assays. FHL2 also interacted with all three myocardin factors and enhanced myocardin and myocardin-related transcription factor (MRTF)-A-dependent transactivation of smooth muscle alpha-actin, SM22, and cardiac atrial natriuretic factor promoters in 10T1/2 cells. The expression of FHL2 increased myocardin and MRTF-A protein levels, and, importantly, this effect was due to an increase in protein stability not due to an increase in myocardin factor mRNA expression. Treatment of cells with proteasome inhibitors MG-132 and lactacystin strongly upregulated endogenous MRTF-A protein levels and resulted in a substantial increase in ubiquitin immunoreactivity in MRTF-A immunoprecipitants. Interestingly, the expression of FHL2 attenuated the effects of RhoA and MRTF-B on promoter activity, perhaps through decreased MRTF-B nuclear localization or decreased SRF-CArG binding. Taken together, these data indicate that myocardin factors are regulated by proteasome-mediated degradation and that FHL2 regulates SRF-dependent transcription by multiple mechanisms, including stabilization of myocardin and MRTF-A.

MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

Transforming growth factor-beta1 (TGF-beta1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates alpha-smooth muscle actin (alpha-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-beta1. Herein, we study the effect of MKL1 expression on alpha-SMA in these cells. We demonstrate that TGF-beta1 stimulation of alpha-SMA transcription is mediated through CC(A/T)(6)-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates alpha-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces alpha-SMA expression regardless of treatment with TGF-beta1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce alpha-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-beta1 stimulation of alpha-SMA expression. Therefore, MKL1 is also absolutely required for TGF-beta1 stimulation of alpha-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-beta1 induces binding of endogenous SRF and MKL1 to the alpha-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating alpha-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.