Α-actin Gene Research Articles

Toxicological Insight from AP-1 Silencing Study on Proliferation, Migration, and Dedifferentiation of Rat Vascular Smooth Muscle Cell

There has an effective way to prevent intimal hyperplasia on vascular smooth muscle cell (VSMC) proliferation in grafted veins. The activator protein-1 (AP-1) transcription factor plays an important role in cardiovascular generation and angioplasty. Once activated, AP-1 binds its specific DNA sequence to promote the proliferation of VSMC, differentiation, and migration. The objectives of this study were to determine toxicological effects of AP-1 silencing study on proliferation, migration, and dedifferentiation of rat vascular smooth muscle cell. To suppress the expression of AP-1 gene, AP-1 siRNA was used to interfere post-transcription in rat primary VSMCs. To observe the expression of SM α-actin and downstream genes of AP-1, the activity of cell matrix metal proteinases and the migration ability of VSMC was examined by a modified Boyden chamber assay. Effects of AP-1 siRNA on proliferation and differentiation in rat VSMCs were evaluated by cell cycle analysis, DNA synthesis, MTT-test, and immunofluorescence. The results showed that the level of SM α-actin protein expression was increased. AP-1 siRNA also significantly decreased the MTT extinction value, DNA synthesis, PCNA expression, and the cell migration velocity when compared to the control group. AP-1 siRNA also clearly arrested cell cycle of VSM at the G0/G1 phase. Zymographic and Western blotting analyses showed that AP-1 siRNA suppressed serum-induced MMP-2 expression. These data suggest that the AP-1 siRNA was able to effectively inhibit the proliferation, migration, and dedifferentiation of smooth muscle cells. Thus, AP-1 siRNA provides a novel method to prevent intimal hyperplasia in blood vessel angioplasty.

Notch Induces Myofibroblast Differentiation of Alveolar Epithelial Cells via Transforming Growth Factor- -Smad3 Pathway

Notch is an ancient cell-signaling system that regulates the specification of cell fate. This study examined the role of Notch in the epithelial-mesenchymal transition (EMT) and myofibroblast differentiation of cultured RLE-6TN cells (i.e., rat alveolar epithelial cells). The activation of Notch, either by ectopic expression of the Notch intracellular domain or by the co-culture of RLE-6TN cells with L-Jagged1 cells, induces the expression of smooth muscle α-actin (SMA) and other mesenchymal marker genes (collagen I and vimentin), and reduces the expression of epithelial marker genes (E-cadherin, occludin, and zonula occludens-1). The pharmacologic inhibition of the endogenous Notch signal significantly inhibited the transforming growth factor-β (TGF-β)-induced expression of SMA. Cell migratory capacity was increased by Notch. Luciferase assays revealed that the CC(A/T)(6)GG (CArG) box and the TGF-β control element (TCE) are required for Notch-induced SMA gene transcription. DNA microarray analysis revealed that members of the TGF-β family as well as Jagged1 were induced in RLE-6TN cells by Notch. Western blot analysis showed that Notch induced the phosphorylation of Smad3, and the TGF-β receptor type I/activin receptor-like kinase 5 (ALK5) kinase inhibitor SB431542 markedly reduced the Notch-induced expression of SMA. Enzyme-linked immunosorbent assays confirmed the production of TGF-β1 from RLE-6TN cells by Notch. Immunohistochemistry of a bleomycin-induced model of pulmonary fibrosis and lung specimens from patients with idiopathic interstitial pneumonias showed that Notch was strongly expressed in myofibroblasts, identified as SMA-positive cells. These data indicate that Notch induces myofibroblast differentiation through a TGF-β-Smad3 pathway that activates SMA gene transcription in a CArG-dependent and TCE-dependent manner in alveolar epithelial cells. Our data also imply that Notch induces the EMT phenotype, with increased migratory behavior in pulmonary fibrosis.

Proteasomal degradation of myocardin is required for its transcriptional activity in vascular smooth muscle cells

Myocardin is a transcriptional co-activator of serum response factor (SRF) and can be degraded through ubiquitin-proteasome system. Our preliminary studies unexpectedly revealed that accumulation of myocardin in response to proteasome inhibition by MG132 or lactacystin resulted in decrease of transcriptional activity of myocardin as indicated by reduced expression of SMC contractile marker genes (SM α-actin, SM22, and calponin) and muscle-enriched microRNAs (miR-143/145 and miR-1/133a), and reduced contractility of human vascular smooth muscle cells (SMCs) embedded in collagen gel lattices, suggesting that myocardin degradation is required for its transcriptional activity. Further studies using chromatin immunoprecipitation assay revealed that proteasome inhibition, although increased the occupancy of myocardin and SRF on the promoter of SM α-actin gene, abolished myocardin-dependent recruitment of RNA polymerase II. We further examined the degradation of myocardin in epithelioid and spindle-shaped SMCs and revealed that myocardin in more differentiated spindle-shaped SMCs was more quickly degraded and had shorter half-life than in epithelioid SMCs. In neointimal lesions, we found that stabilization of myocardin protein was companied by downregulation of transcripts of ubiquitin and proteasome subunits, further illustrating the mechanism underlying reduction of myocardin transcriptional activity. In summary, our results have suggested that proteasomal degradation of myocardin is required for its transcriptional activity.

Phylogeny and intercontinental distribution of the pneumocandin-producing anamorphic fungus Glarea lozoyensis

Glarea lozoyensis is an anamorphic ascomycete that produces pneumocandin B0, the starting molecule for the synthesis of the antifungal drug caspofungin (CANCIDAS™). Glarea lozoyensis was first isolated in 1985 from a water sample from Madrid, Spain. Until now, only the original strain was known, but we have discovered new strains from Argentina and the USA. Molecular phylogenetic reference to a 28S rDNA database of antibiotic-producing fungi quickly identified these strains as being conspecific with G. lozoyensis. Bayesian inference phylogeny of ITS, 28S rDNA and α-actin gene fragments revealed that G. lozoyensis is related to species of the genus Cyathicula (Helotiales). Glarea lozoyensis was not conspecific with any of the Cyathicula species sequenced, although it appears to share a common ancestor. Glarea lozoyensis and Cyathicula strains were fermented on nutritional microarrays in 96-well plates. Cyathicula extracts did not show antifungal activity and did not produce pneumocandins, whereas potent antifungal activity and pneumocandin A0 production were confirmed for the four G. lozoyensis isolates. Also, culture morphology differed, with G. lozoyensis strains producing a dark brown, profusely sporulating mycelium with pigmented multicellular conidia accumulating in conidial masses, while all Cyathicula species tested in culture formed hyaline to light brown mycelia and lacked conidia. The chemistry and taxonomic distribution of the echinocandin class of antifungals is comprehensively reviewed.

Mouse models of dominant ACTA1 disease recapitulate human disease and provide insight into therapies

Mutations in the skeletal muscle α-actin gene (ACTA1) cause a range of pathologically defined congenital myopathies. Most patients have dominant mutations and experience severe skeletal muscle weakness, dying within one year of birth. To determine mutant ACTA1 pathobiology, transgenic mice expressing ACTA1(D286G) were created. These Tg(ACTA1)(D286G) mice were less active than wild-type individuals. Their skeletal muscles were significantly weaker by in vitro analyses and showed various pathological lesions reminiscent of human patients, however they had a normal lifespan. Mass spectrometry revealed skeletal muscles from Tg(ACTA1)(D286G) mice contained ∼25% ACTA1(D286G) protein. Tg(ACTA1)(D286G) mice were crossed with hemizygous Acta1(+/-) knock-out mice to generate Tg(ACTA1)(D286G)(+/+).Acta1(+/-) offspring that were homozygous for the transgene and hemizygous for the endogenous skeletal muscle α-actin gene. Akin to most human patients, skeletal muscles from these offspring contained approximately equal proportions of ACTA1(D286G) and wild-type actin. Strikingly, the majority of these mice presented with severe immobility between postnatal Days 8 and 17, requiring euthanasia. Their skeletal muscles contained extensive structural abnormalities as identified in severely affected human patients, including nemaline bodies, actin accumulations and widespread sarcomeric disarray. Therefore we have created valuable mouse models, one of mild dominant ACTA1 disease [Tg(ACTA1)(D286G)], and the other of severe disease, with a dramatically shortened lifespan [Tg(ACTA1)(D286G)(+/+).Acta1(+/-)]. The correlation between mutant ACTA1 protein load and disease severity parallels effects in ACTA1 families and suggests altering this ratio in patient muscle may be a therapy for patients with dominant ACTA1 disease. Furthermore, ringbinden fibres were observed in these mouse models. The presence of such features suggests that perhaps patients with ringbinden of unknown genetic origin should be considered for ACTA1 mutation screening. This is the first experimental, as opposed to observational, evidence that mutant protein load determines the severity of ACTA1 disease.

A phospholipase C 1-activated pathway regulates transcription in human vascular smooth muscle cells

Growth factor-induced repression of smooth muscle (SM) cell marker genes is an integral part of vascular SM (VSM) cell proliferation. This is partly regulated via translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) to the nucleus which activates the transcription factor Elk-1. The mediators involved in ERK1/2 nuclear translocation in VSM cells are unknown. The aim of this study is to examine the mechanisms which regulate growth factor-induced nuclear translocation of ERK1/2 and gene expression in VSM cells. In cultured human VSM cells, phospholipase C (PLC)γ1 expression was required for platelet-derived growth factor (PDGF)-induced ERK1/2 nuclear translocation, Elk-1 phosphorylation, and subsequent repression of SM α-actin gene expression. The mechanisms of a role for PLCγ1 in ERK1/2 nuclear localization were further examined by investigating interacting proteins. The ERK1/2-binding phosphoprotein, protein enriched in astrocytes-15 (PEA-15), was phosphorylated by PDGF and this phosphorylation required activation of PLCγ1. In cells pre-treated with PEA-15 siRNA, ERK1/2 distribution significantly increased in the nucleus and resulted in decreased SM α-actin expression and increased VSM cell proliferation. Overexpression of PEA-15 increased ERK1/2 localization in the cytoplasm. The regulatory role of PEA-15 phosphorylation was assessed. In VSM cells overexpressing a non-phosphorylatable form of PEA-15, PDGF-induced ERK1/2 nuclear localization was inhibited. These results suggest that PEA-15 phosphorylation by PLCγ1 is required for PDGF-induced ERK1/2 nuclear translocation. This represents an important level of phenotypic control by directly affecting Elk-1-dependent transcription and ultimately SM cell marker protein expression in VSM cells.

A novel ACTA1 mutation resulting in a severe congenital myopathy with nemaline bodies, intranuclear rods and type I fibre predominance

We describe a severe congenital myopathy patient of Xhosa native African origin with a novel de novo p.Gly152Ala skeletal muscle α-actin gene ( ACTA1) mutation, who died at 6 months of age. The muscle pathology demonstrated abundant cytoplasmic and intranuclear rods, core-like areas and the unusual feature of larger type I than type II fibres. Our results further expand the phenotypes associated with ACTA1 mutations and provide support for the hypothesis that the structural abnormalities seen are a pathological continuum dependent on the precise mutation and biopsy location. Our results also demonstrate the likely world-wide distribution of de novo mutations in this gene.

Isolation and characterization of the core single-stranded DNA-binding domain of purine-rich element binding protein B (Purβ)

Purβ is a single-stranded nucleic acid-binding protein implicated in the injury-induced repression of genes encoding certain muscle-restricted isoforms of actin and myosin expressed in the heart, skeletal muscle, and vasculature. To better understand how the modular arrangement of the primary sequence of Purβ affects the higher order structure and function of the protein, purified recombinant Purβ was subjected to partial proteolysis in an attempt to identify a well-folded truncation protein that retained purine-rich single-stranded DNA-binding activity. Limited tryptic digestion of Purβ liberated a core ∼30 kDa fragment corresponding to residues 29–305 as determined by epitope mapping and mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to self-associate while circular dichroism analysis confirmed that the Purβ core domain is stably folded in the absence of glycine-rich N- and C-terminal sequences. Comparative DNA-binding assays revealed that the isolated core domain interacts with purine-rich cis-elements from the smooth muscle α-actin gene with similar specificity but increased affinity compared to full-length Purβ. These findings suggest that the highly conserved modular repeats of Purβ fold to form a core functional domain, which mediates the specific and high affinity binding of the protein to single-stranded DNA.

Ca 2+ /Calmodulin-Dependent Kinase IIδ Causes Heart Failure by Accumulation of p53 in Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM), characterized by dilatation and dysfunction of the left ventricle, is an important cause of heart failure. Many mutations in various genes, including cytoskeletal protein genes and contractile protein genes, have been identified in DCM patients, but the mechanisms of how such mutations lead to DCM remain unknown. We established the mouse model of DCM by expressing a mutated cardiac alpha-actin gene, which has been reported in patients with DCM, in the heart (mActin-Tg). mActin-Tg mice showed gradual dilatation and dysfunction of the left ventricle, resulting in death by heart failure. The number of apoptotic cardiomyocytes and protein levels of p53 were increased in the hearts of mActin-Tg mice. Overexpression of Bcl-2 or downregulation of p53 decreased the number of apoptotic cardiomyocytes and improved cardiac function. This mouse model showed a decrease in myofilament calcium sensitivity and activation of calcium/calmodulin-dependent kinase IIdelta (CaMKIIdelta). The inhibition of CaMKIIdelta prevented the increase in p53 and apoptotic cardiomyocytes and ameliorated cardiac function. CaMKIIdelta plays a critical role in the development of heart failure in part by accumulation of p53 and induction of cardiomyocyte apoptosis in the DCM mouse model.

GH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model

The aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish (Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the α-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.

Repression of Smooth Muscle Differentiation by a Novel High Mobility Group Box-containing Protein, HMG2L1

The molecular mechanisms regulating smooth muscle-specific gene expression during smooth muscle development are poorly understood. Myocardin is an extraordinarily powerful cofactor of serum response factor (SRF) that stimulates expression of smooth muscle-specific genes. In an effort to search for proteins that regulate myocardin function, we identified a novel HMG box-containing protein HMG2L1 (high mobility group 2 like 1). We found that HMG2L1 expression is correlated with the smooth muscle cell (SMC) synthetic phenotype. Overexpression of HMG2L1 in SMCs down-regulated smooth muscle marker expression. Conversely, depletion of endogenous HMG2L1 in SMCs increases smooth muscle-specific gene expression. Furthermore, we found HMG2L1 specifically abrogates myocardin-induced activation of smooth muscle-specific genes. By GST pulldown assays, the interaction domains between HMG2L1 and myocardin were mapped to the N termini of each of the proteins. Finally, we demonstrated that HMG2L1 abrogates myocardin function through disrupting its binding to SRF and abolishing SRF-myocardin complex binding to the promoters of smooth muscle-specific genes. This study provides the first evidence of this novel HMG2L1 molecule playing an important role in attenuating smooth muscle differentiation.

Characterization of bacterial artificial chromosome transgenic mice expressing mCherry fluorescent protein substituted for the murine smooth muscle α‐actin gene

Smooth muscle alpha actin (SMA) is a cytoskeletal protein expressed by mesenchymal and smooth muscle cell types, including mural cells (vascular smooth muscle cells and pericytes). Using Bacterial Artificial Chromosome (BAC) recombineering technology, we generated transgenic reporter mice that express a membrane localized cherry red fluorescent protein (mCherry), driven by the full-length SMA promoter and intronic sequences. We determined that the founders and F1 progeny of five independent lines contain 1-3 copies of the mCherry-substituted BAC vector. Furthermore, we characterized the expression of SMA-mCherry in relation to endogenous SMA in the embryo and in adult tissues, and found that the transgenic reporter in each line recapitulated endogenous SMA expression at all time points. We were also able to isolate SMA expressing cells from embryonic tissues using fluorescence-activated cell sorting (FACS). We demonstrated that this marker can be combined with other vital fluorescent reporters and it can be used for live imaging of embryonic cardiodynamics. Therefore, these transgenic mice will be useful for isolating live SMA-expressing cells via FACS and for studying the emergence, behavior, and regulation of SMA-expressing cells, including vascular smooth muscle cells and pericytes throughout embryonic and postnatal development.

Structural and functional characterization of the core single‐stranded DNA‐binding domain of purine‐rich element binding protein B (Purβ)

Purβ is a sequence‐specific single‐stranded DNA (ssDNA)‐binding protein that represses smooth muscle α‐actin gene transcription in stress‐activated fibroblasts and smooth muscle cells via cooperative interaction with two sites in the purine‐rich strand of a 5′ enhancer element (dubbed PE32‐F). To better understand how the modular structure of Purβ dictates its specific ssDNA‐binding function, we evaluated the interaction of purified recombinant Purβ with PE32‐F using spectroscopic and biochemical approaches. Results of intrinsic protein fluorescence and circular dichroism analyses pointed to a ssDNA‐induced change in protein conformation. Consistent with this interpretation, Purβ was protected from proteolysis when complexed with PE32‐F. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment spanning residues 29‐305 as determined by mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to dimerize. Quantitative ssDNA‐binding assays revealed that the core domain binds to PE32‐F with similar specificity but increased affinity compared to full‐length Purβ and a truncated 37‐263 mutant absent a putative amphipathic helix‐forming module. These findings provide new insight into the structural basis for the specific and high affinity binding of Purβ to ssDNA. NIH R01 HL054281, T32 HL007594, P20 RR16462.

SHP2 mediates gp130-dependent cardiomyocyte hypertrophy via negative regulation of skeletal alpha-actin gene

Morphological and biochemical phenotypes of cardiomyocyte hypertrophy are determined by neurohumoral factors. Stimulation of G protein-coupled receptor (GPCR) results in uniform cell enlargement in all directions with an increase in skeletal α-actin (α-SKA) gene expression, while stimulation of gp130 receptor by interleukin-6 (IL-6)-related cytokines induces longitudinal elongation with no increase in α-SKA gene expression. Thus, α-SKA is a discriminating marker for hypertrophic phenotypes; however, regulatory mechanisms of α-SKA gene expression remain unknown. Here, we clarified the role of SH2-containing protein tyrosine phosphatase 2 (SHP2) in α-SKA gene expression. In neonatal rat cardiomyocytes, endothelin-1 (ET-1), a GPCR agonist, but not leukemia inhibitory factor (LIF), an IL-6-related cytokine, induced RhoA activation and promotes α-SKA gene expression via RhoA. In contrast, LIF, but not ET-1, induced activation of SHP2 in cardiomyocytes, suggesting that SHP2 might negatively regulate α-SKA gene expression downstream of gp130. Therefore, we examined the effect of adenovirus-mediated overexpression of wild-type SHP2 (SHP2WT), dominant-negative SHP2 (SHP2C/S), or β-galactosidase (β-gal), on α-SKA gene expression. LIF did not upregulate α-SKA mRNA in cardiomyocytes overexpressing either β-gal or SHP2WT. In cardiomyocytes overexpressing SHP2C/S, LIF induced upregulation of α-SKA mRNA, which was abrogated by concomitant overexpression of either C3-toxin or dominant-negative RhoA. RhoA was activated after LIF stimulation in the cardiomyocytes overexpressing SHP2C/S, but not in myocytes overexpressing β-gal. Furthermore, SHP2 mediates LIF-induced longitudinal elongation of cardiomyocytes via ERK5 activation. Collectively, these findings indicate that SHP2 negatively regulates α-SKA expression via RhoA inactivation and suggest that SHP2 implicates ERK5 in cardiomyocyte elongation downstream of gp130.

G.P.12.09 Actin myopathy with myofibrillar dysgenesis and abnormal ossification

Skeletal muscle α-actin (ACTA1) gene mutations result in a myopathy with histopathological characteristics that include cytoplasmic nemaline rods, intranuclear rods and abnormal actin aggregates. The severity of muscle weakness and cardiomyopathy show clinical variability with onset age from neonatal period to adulthood, but extramuscular manifestations have been rarely described in patients with actinopathy.

Nemaline (actin) myopathy with myofibrillar dysgenesis and abnormal ossification

We report a 2-year-old boy who presented with marked hypotonia and was dependent on artificial ventilation since birth. He was diagnosed with nemaline (actin) myopathy, based on the cytoplasmic accumulation of thin filament aggregates and marked myofibrillar dysgenesis. Intranuclear rods and dispersed tiny nemaline bodies were also observed. The patient was shown to be heterozygous for a de novo mutation, c.430C>T (p.Leu144Phe), in the α-actin ( ACTA1) gene. He also showed orbital osteosclerosis, longitudinal striations of the iliac bones, hepatomegaly, undescended testis, a unilateral vesico-ureteric stenosis, severe failure to thrive, and dilatation of the lateral cerebral ventricles. Besides the severe muscle involvement, these clinical findings further broaden the clinical spectrum of actinopathy phenotypes.

AP-1 Inhibits Expression of MMP-2/9 and Its Effects on Rat Smooth Muscle Cells

We designed AP-1 small interfering RNA (siRNA) to study the relationship between AP-1 expression and matrix metalloproteinase (MMP)-2 activation. To suppress the expression of AP-1 gene, we used RNA interference to silence the AP-1 gene post-transcriptionally in rat smooth muscle cells. Effects of AP-1 siRNA on mRNA expression of AP-1 were examined using reverse transcriptase polymerase chain reaction (RT-PCR). Phosphorylation levels of c-Jun, c-Fos, and the activity of AP-1 were determined by Western blotting and AP-1 DNA-binding activity assay. To observe the expression of SM alpha-actin and downstream genes of AP-1, we simultaneously examined the activity of cell matrix metal proteinases and the migration ability of smooth muscle cells using a modified Boyden-chamber assay. Effects of AP-1 siRNA on rat vascular smooth muscle cells (VSMC) in vitro were evaluated using cell cycle analysis, MTT-tests, BrdU-ELISA and immunofluorescence. AP-1 siRNA decreased not only AP-1 mRNA and AP-1 binding activity, but also c-Jun and c-Fos phosphorylation levels and the expression levels of urokinase-type plasminogen (u-PA), MMPs, TGF-beta1 and bFGF in VSMCs. The AP-1 siRNA also significantly inhibited PDGF/IL-1-induced MMP-2 and MMP-9 gelatinolytic activity in VSMCs. The invasion and migration of VSMC were also induced greatly. AP-1 inhibited expression of MMP-2/9 and AP-1 siRNA was able to effectively inhibit the proliferation, migration and de-differentiation of rat smooth muscle cells.

Transforming Growth Factor β1-mediated Activation of the Smooth Muscle α-Actin Gene in Human Pulmonary Myofibroblasts Is Inhibited by Tumor Necrosis Factor-α via Mitogen-activated Protein Kinase Kinase 1-dependent Induction of the Egr-1 Transcriptional Repressor

Transforming growth factor (TGF) beta1 is a mediator of myofibroblast differentiation in healing wounds in which it activates transcription of the smooth muscle alpha-actin (SMalphaA) gene via dynamic interplay of nuclear activators and repressors. Targeting components of TGFbeta1 signaling may be an effective strategy for controlling myofibroblasts in chronic fibrotic diseases. We examined the ability of proinflammatory tumor necrosis factor (TNF)-alpha to antagonize TGFbeta1-mediated human pulmonary myofibroblast differentiation. TNF-alpha abrogated TGFbeta1-induced SMalphaA gene expression at the level of transcription without disrupting phosphorylation of regulatory Smads. Intact mitogen-activated protein kinase kinase (Mek)-extracellular signal-regulated kinase (Erk) kinase signaling was required for myofibroblast repression by TNF-alpha via induction of the early growth response factor-1 (Egr-1) DNA-binding protein. Egr-1 bound to the GC-rich SPUR activation element in the SMalphaA promoter and potently suppressed Smad3- and TGFbeta1-mediated transcription. Reduction in Smad binding to the SMalphaA promoter in TNF-alpha-treated myofibroblasts was accompanied by an increase in Egr-1 and YB-1 repressor binding, suggesting that the molecular mechanism underlying repression may involve competitive interplay between Egr-1, YB-1, and Smads. The ability of TNF-alpha to attenuate myofibroblast differentiation via modulation of a Mek1/Erk/Egr-1 regulatory axis may be useful in designing new therapeutic targets to offset destructive tissue remodeling in chronic fibrotic disease.

Krüppel-like factor 4, Elk-1, and histone deacetylases cooperatively suppress smooth muscle cell differentiation markers in response to oxidized phospholipids

Phenotypic switching of vascular smooth muscle cells (SMCs), such as increased proliferation, enhanced migration, and downregulation of SMC differentiation marker genes, is known to play a key role in the development of atherosclerosis. However, the factors and mechanisms controlling this process are not fully understood. We recently showed that oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)- sn-glycero-3-phosphocholine (POVPC), which accumulate in atherosclerotic lesions, are potent repressors of expression of SMC differentiation marker genes in cultured SMCs as well as in rat carotid arteries in vivo. Here, we examined the molecular mechanisms whereby POVPC induces suppression of SMC differentiation marker genes in cultured SMCs. Results showed that POVPC induced phosphorylation of ERK1/2 and Elk-1. The MEK inhibitors U-0126 and PD-98059 attenuated POVPC-induced suppression of smooth muscle ( SM) α-actin and SM-myosin heavy chain. POVPC also induced expression of Krüppel-like factor 4 (Klf4). Chromatin immunoprecipitation assays revealed that POVPC caused simultaneous binding of Elk-1 and Klf4 to the promoter region of the SM α-actin gene. Moreover, coimmunoprecipitation assays showed a physical interaction between Elk-1 and Klf4. Results in Klf4-null SMCs showed that blockade of both Klf4 induction and Elk-1 phosphorylation completely abolished POVPC-induced suppression of SMC differentiation marker genes. POVPC-induced suppression of SMC differentiation marker genes was also accompanied by hypoacetylation of histone H4 at the SM α-actin promoter, which was mediated by the recruitment of histone deacetylases (HDACs), HDAC2 and HDAC5. Coimmunoprecipitation assays showed that Klf4 interacted with HDAC5. Results provide evidence that Klf4, Elk-1, and HDACs coordinately mediate POVPC-induced suppression of SMC differentiation marker genes.

PIAS1 Mediates TGFβ-Induced SM α-Actin Gene Expression Through Inhibition of KLF4 Function-Expression by Protein Sumoylation

TGFbeta and proliferation/phenotypic switching of smooth muscle cells (SMCs) play a pivotal role in pathogenesis of atherosclerotic and restenotic lesions after angioplasty. We have previously shown that the protein inhibitor of activated STAT (PIAS)1 activates expression of SMC differentiation marker genes including smooth muscle (SM) alpha-actin by interacting with serum response factor (SRF) and class I bHLH proteins. Here, we tested the hypothesis that TGFbeta activates SM alpha-actin through PIAS1. An siRNA specific for PIAS1 and ubc9, an E2-ligase for sumoylation, inhibited TGFbeta-induced expression of SM alpha-actin in cultured SMCs as determined by real-time RT-PCR. Overexpression of PIAS1 increased SM alpha-actin promoter activity in a TGFbeta control element (TCE)-dependent manner. Because the TCE within the SM alpha-actin promoter could mediate repression through interaction with KLF4, we tested whether PIAS1 regulates the function of KLF4 for SMC gene expression. PIAS1 interacted with KLF4 in mammalian two-hybrid and coimmunoprecipitation assays, and overexpression of PIAS1 inhibited KLF4-repression of SM alpha-actin promoter activity. Moreover, PIAS1 promoted degradation of KLF4 through sumoylation. These results provide evidence that PIAS1 promotes TGFbeta-induced activation of SM alpha-actin gene expression at least in part by promoting sumoylation and degradation of the TCE repressor protein, KLF4.