Four isoforms of serum response factor that increase or inhibit smooth-muscle-specific promoter activity.
Ontology highlight
ABSTRACT:
SUBMITTER: Kemp PR
PROVIDER: S-EPMC1220776 | biostudies-other | 2000 Feb
REPOSITORIES: biostudies-other
Similar Datasets
Project description: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.
| S-EPMC2783385 | biostudies-literature
Project description:IFNγ exerts multiple biological effects on effector cells by regulating many downstream genes, including smooth muscle-specific genes. However, the molecular mechanisms underlying IFNγ-induced inhibition of smooth muscle-specific gene expression remain unclear. In this study, we have shown that serum response factor (SRF), a common transcriptional factor important in cell proliferation, migration, and differentiation, is targeted by IFNγ in a STAT1-dependent manner. We show that the molecular mechanism by which IFNγ regulates SRF is via activation of the 2-5A-RNase L system, which triggers SRF mRNA decay and reduced SRF expression. As a result, decreased SRF expression reduces expression of SRF target genes such as smooth muscle α-actin and smooth muscle myosin heavy chain. Additionally, IFNγ reduced p300 and acetylated histone-3 binding in both smooth muscle α-actin and SRF promoters, epigenetically decreasing smooth muscle α-actin and SRF transcriptional activation. Our data reveal that SRF is a novel IFNγ-regulated gene and further elucidate the molecular pathway between IFNγ, IFNγ-regulated genes, and SRF and its target genes.
| S-EPMC2952243 | biostudies-literature
Project description:We have previously shown that the transcription-promoting activity of serum response factor (SRF) is partially regulated by its extranuclear redistribution. In this study, we examined the cellular mechanisms that facilitate SRF nuclear entry in canine tracheal smooth muscle cells. We used in vitro pull-down assays to determine which karyopherin proteins bound SRF and found that SRF binds KPNA1 and KPNB1 through its nuclear localization sequence. Immunoprecipitation studies also demonstrated direct SRF-KPNA1 interaction in HEK293 cells. Import assays demonstrated that KPNA1 and KPNB1 together were sufficient to mediate rapid nuclear import of SRF-GFP. Our studies also suggest that SRF is able to gain nuclear entry through an auxiliary, nuclear localization sequence-independent mechanism.
| S-EPMC3175573 | biostudies-literature
Project description:BACKGROUND & AIMS:Smooth muscle cells (SMCs) change phenotypes under various pathophysiological conditions. These changes are largely controlled by the serum response factor (SRF), a transcription factor that binds to CC (A/T)6 GG (CArG) boxes in SM contractile genes. MicroRNAs (miRNA) regulate transitions among SMC phenotypes. The SMC miRNA transcriptome (SMC miRNAome) and its regulation by SRF have not been determined. METHODS:We performed massively parallel sequencing to identify gastrointestinal (GI) SMC miRNA transcriptomes in mice and humans. SMC miRNA transcriptomes were mapped to identify all CArG boxes, which were confirmed by SRF knockdown and microarrays. Quantitative polymerase chain reaction was used to identify SMC-phenotypic miRNAs in differentiated and proliferating SMCs. Bioinformatics and target validation analysis showed regulation of SMC phenotype by SRF-dependent, SMC-phenotype miRNAs. RESULTS:We cloned and identified GI miRNA transcriptomes using genome-wide analyses of mouse and human cells. The SM miRNAome consisted of hundreds of unique miRNAs that were highly conserved among both species. We mapped miRNAs CArG boxes and found that many had an SRF-dependent signature in the SM miRNAome. The SM miRNAs CArG boxes had several distinct features. We also identified approximately 100 SMC-phenotypic miRNAs that were induced in differentiated or proliferative SMC phenotypes. We showed that SRF-dependent, SMC-phenotypic miRNAs bind and regulate Srf and its cofactors, myocadin (Myocd) and member of ETS oncogene family Elk1. CONCLUSIONS:The GI SMC phenotypes are controlled by SRF-dependent, SMC-phenotypic miRNAs that regulate expression of SRF, MYOCD, and ELK1.
| S-EPMC3129374 | biostudies-literature
Project description:ObjectiveDrug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia.Approach and resultsWe found that germ-line-specific or smooth muscle-specific deletion of Akt2 resulted in more severe intimal hyperplasia compared with control mice after arterial denudation injury. Conversely, smooth muscle-specific Akt1 knockout prevented intimal hyperplasia, whereas germ-line Akt1 deletion caused severe thrombosis. Notably, rapamycin prevented intimal hyperplasia in wild-type mice but had no therapeutic benefit in Akt2 knockouts. We identified opposing roles for AKT1 and AKT2 isoforms in smooth muscle cell proliferation, migration, differentiation, and rapamycin response in vitro. Mechanistically, rapamycin induced MYOCD (myocardin) mRNA expression. This was mediated by AKT2 phosphorylation and nuclear exclusion of FOXO4 (forkhead box O4), inhibiting its binding to the MYOCD promoter.ConclusionsOur data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin's therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.
| S-EPMC5699966 | biostudies-literature
Project description:A large number of tropomyosin (Tm) isoforms function as gatekeepers of the actin filament, controlling the spatiotemporal access of actin-binding proteins to specialized actin networks. Residues ∼40-80 vary significantly among Tm isoforms, but the impact of sequence variation on Tm structure and interactions with actin is poorly understood, because structural studies have focused on skeletal muscle Tmα. We describe structures of N-terminal fragments of smooth muscle Tmα and Tmβ (sm-Tmα and sm-Tmβ). The 2.0-Å structure of sm-Tmα81 (81-aa) resembles that of skeletal Tmα, displaying a similar super-helical twist matching the contours of the actin filament. The 1.8-Å structure of sm-Tmα98 (98-aa) unexpectedly reveals an antiparallel coiled coil, with the two chains staggered by only 4 amino acids and displaying hydrophobic core interactions similar to those of the parallel dimer. In contrast, the 2.5-Å structure of sm-Tmβ98, containing Gly-Ala-Ser at the N terminus to mimic acetylation, reveals a parallel coiled coil. None of the structures contains coiled-coil stabilizing elements, favoring the formation of head-to-tail overlap complexes in four of five crystallographically independent parallel dimers. These complexes show similarly arranged 4-helix bundles stabilized by hydrophobic interactions, but the extent of the overlap varies between sm-Tmβ98 and sm-Tmα81 from 2 to 3 helical turns. The formation of overlap complexes thus appears to be an intrinsic property of the Tm coiled coil, with the specific nature of hydrophobic contacts determining the extent of the overlap. Overall, the results suggest that sequence variation among Tm isoforms has a limited effect on actin binding but could determine its gatekeeper function.
| S-EPMC3270971 | biostudies-literature
Project description:We have determined the specificity of sera elicited by glutaraldehyde-stabilized bovine aortic actin. This modification induces a high titre of antibodies directed against the N-terminal (residues 1-39) and the C-terminal region of smooth-muscle actins. The crude antisera were purified on peptide (corresponding to the 1-9 or 1-8 N-terminal sequences of smooth-muscle isoactins)-polyacrylic-resin columns. By fractionating the antisera we obtained oligoclonal antibody populations specific for each isoactin.
| S-EPMC1147662 | biostudies-other
Project description:Serum response factor (SRF) is a transcription factor known to mediate phenotypic plasticity in smooth muscle cells (SMCs). Despite the critical role of this protein in mediating intestinal injury response, little is known about the mechanism through which SRF alters SMC behavior. Here, we provide compelling evidence for the involvement of SRF-dependent microRNAs (miRNAs) in the regulation of SMC apoptosis. We generated SMC-restricted Srf inducible knockout (KO) mice and observed both severe degeneration of SMCs and a significant decrease in the expression of apoptosis-associated miRNAs. The absence of these miRNAs was associated with overexpression of apoptotic proteins, and we observed a high level of SMC death and myopathy in the intestinal muscle layers. These data provide a compelling new model that implicates SMC degeneration via anti-apoptotic miRNA deficiency caused by lack of SRF in gastrointestinal motility disorders.
| S-EPMC4720888 | biostudies-literature
Project description:Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells.
| S-EPMC2785029 | biostudies-literature
Project description:Actin filaments propelled in vitro by groups of skeletal muscle myosin motors exhibit distinct phases of active sliding or arrest, whose occurrence depends on actin length (L) within a range of up to 1.0 ?m. Smooth muscle myosin filaments are exponentially distributed with ?150 nm average length in vivo--suggesting relevance of the L-dependence of myosin group kinetics. Here, we found L-dependent actin arrest and sliding in in vitro motility assays of smooth muscle myosin. We perturbed individual myosin kinetics with varying, physiological concentrations of phosphate (Pi, release associated with main power stroke) and adenosine diphosphate (ADP, release associated with minor mechanical step). Adenosine triphosphate was kept constant at physiological concentration. Increasing [Pi] lowered the fraction of time for which actin was actively sliding, reflected in reduced average sliding velocity (?) and motile fraction (fmot, fraction of time that filaments are moving); increasing [ADP] increased the fraction of time actively sliding and reduced the velocity while sliding, reflected in reduced ? and increased fmot. We introduced specific Pi and ADP effects on individual myosin kinetics into our recently developed mathematical model of actin propulsion by myosin groups. Simulations matched our experimental observations and described the inhibition of myosin group kinetics. At low [Pi] and [ADP], actin arrest and sliding were reflected by two distinct chemical states of the myosin group. Upon [Pi] increase, the probability of the active state decreased; upon [ADP] increase, the probability of the active state increased, but the active state became increasingly similar to the arrested state.
| S-EPMC4317549 | biostudies-literature