SMC Genes Research Articles

Rapid cyclic stretching induces a synthetic, proinflammatory phenotype in cultured human intestinal smooth muscle, with the potential to alter signaling to adjacent bowel cells.

Bowel smooth muscle experiences mechanical stress constantly during normal function, and pathologic mechanical stressors in disease states. We tested the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. Primary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-ε-caprolactone nano-fibrous scaffolds, were subjected to pathologic, high frequency (1 Hz) uniaxial 3% cyclic stretch (loaded) or kept unloaded in culture for 6 hours. Total RNA sequencing, qRT-PCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. Loading induced differential expression of 4537 genes in HISMCs. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle and on other cells in the bowel wall. HISMCs demonstrate remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMCs into proliferative, fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.

Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate.

Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA-arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.

Characterization of Carotid Smooth Muscle Cells during Phenotypic Transition.

Vascular smooth muscle cells (VSMCs) are central players in carotid atherosclerosis plaque development. Although the precise mechanisms involved in plaque destabilization are not completely understood, it is known that VSMC proliferation and migration participate in plaque stabilization. In this study, we analyzed expression patterns of genes involved in carotid atherosclerosis development (e.g., transcription factors of regulation of SMC genes) of VSMCs located inside or outside the plaque lesion that may give clues about changes in phenotypic plasticity during atherosclerosis. VSMCs were isolated from 39 carotid plaques extracted from symptomatic and asymptomatic patients by endarterectomy. Specific biomarker expression, related with VSMC phenotype, was analyzed by qPCR, western immunoblot, and confocal microscopy. MYH11, CNN1, SRF, MKL2, and CALD1 were significantly underexpressed in VSMCs from plaques compared with VSMCs from a macroscopically intact (MIT) region, while SPP1, KLF4, MAPLC3B, CD68, and LGALS3 were found significantly upregulated in plaque VSMCs versus MIT VSMCs. The gene expression pattern of arterial VSMCs from a healthy donor treated with 7-ketocholesterol showed high similarity with the expression pattern of carotid plaque VSMCs. Our results indicate that VSMCs isolated from plaque show a typical SMC dedifferentiated phenotype with macrophage-like features compared with VSMCs isolated from a MIT region of the carotid artery. Additionally, MYH11, KLF5, and SPP1 expression patterns were found to be associated with symptomatology of human carotid atherosclerosis.

Vascular smooth muscle cell gene regulation

Vascular smooth muscle cell (VSMC) remodeling is a hallmark of vascular diseases such as atherosclerosis, which underlie heart attack and stroke. Regional differences in atherosclerosis susceptibility in the vasculature correlate with the embryonic origin of SMC, suggesting that developmental history is disease-relevant. For example, neuroectoderm-derived cells colonise the atherogeneic aortic arch whereas VSMC in the generally disease-free descending aorta is of paraxial mesoderm origin. Embryonic origin-specific molecular and functional differences have been observed in VSMCs in vitro and in vivo, and transplantation studies demonstrated that developmental history of VSMC progenitors affect the phenotype of mature cells. Here we investigate the mechanisms underlying this developmental memory. We have established stem cell differentiation models to generate SMC of different embryonic lineages from mouse pluripotent cells. Using this model we demonstrate that upregulation of VSMC genes in response to TGFbeta/PDGF treatment depends on the developmental state of the cells. Epigenetic analysis revealed a lineage-specific correlation with chromatin reconfiguration at SMC genes and subsequent analysis of ex vivo VSMC has identified candidate chromatin modifiers that may be responsible for these changes. By increasing our understanding of how developmental history affects VSMCs this work will help elucidate the mechanisms underlying vascular disease risk.

Activation of Receptor for Advanced Glycation End Products Induces Osteogenic Differentiation of Vascular Smooth Muscle Cells

Vascular calcification is prevalent in patients with diabetes and chronic kidney disease. Receptor for advanced glycation end products (RAGE) and its multiple ligands have been implicated in the pathogenesis of accelerated atherosclerosis; however, little is known about the effects of RAGE activation on vascular calcification. Cultured rat and human aortic smooth muscle cells (HASMC) were transduced with adenovirus expressing RAGE. Expression of myocardin and the SMC-marker genes was significantly repressed in these cells. RAGE activation inhibited myocardin-induced expression of the SMC genes in mouse embryonic mesenchymal C3H10T1/2 cells. Interestingly, RAGE activation induced alkaline phosphatase (ALP) expression, calcium deposition, and Msx2 expression, a crucial transcription factor for osteogenic differentiation, in HASMC. RAGE-induced osteogenic differentiation was significantly inhibited by endogenous secretory RAGE. RAGE-induced ALP and Msx2 expression was completely abrogated by DAPT, an inhibitor of the Notch signaling pathway. PD98059 (MEK inhibitor) effectively blunted RAGE-induced Notch1 and Msx2 gene expression. Simultaneous stimulation with bone morphogenetic protein 2 (BMP2) and RAGE signaling synergistically induced expressions of Msx2 and ALP in HASMC. Immunohistochemistry revealed that the human calcifying atherosclerotic plaque expressed RAGE, Notch components and Msx2. The ALP activity induced in RAGE-overexpressing HASMCs by human serum was positively correlated with the serum creatinine level, but not with phosphate and hemoglobin A1c levels. These results indicate that activation of RAGE not only inhibits myocardin-dependent SMC gene expression, but also induces osteogenic differentiation of vascular SMC through Notch/Msx2 induction. These results provide a novel insight into the role of RAGE axis in vascular calcification.

Abstract 5549: Bone Morphogenetic Protein-2 (BMP-2) and BMP-7 Act as Instructive Factors to Specify Notch Signaling Controlling Smooth Muscle Cell Phenotype

Background: Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity including synthetic, contractive and osteogenic phenotypes in response to environmental cue. Bone morphogetic proteins (BMPs) and Notch signaling are essential determinants of cellular identity, but their mode of action to specify SMC phenotype is poorly defined. Here we investigate the cooperation between BMPs and Notch signaling in cultured VSMC. Methods and Results: We have recently shown that overexpression of Notch1 intracellular domain (N1-ICD) in human aortic SMC (HASMC) induced expression of both SMC and osteoblast marker genes. Interestingly, BMP-2, an osteogenic BMP implicating in vascular calcification, markedly enhanced N1-ICD-induced expression of osteoblast marker genes. Consistently, ALP activity as well as matrix mineralization were also synergistically induced by Notch and BMP-2 signaling. A specific silencing using siRNA confirmed that Msx-2 was responsible for the enhanced osteogenic phenotype induced by Notch and BMP-2. Experiments using site-specific mutation constructs of Msx2 promoter and ChiP assay revealed that the Msx-2 promoter contains functional RBP-Jk-binding site at −3770, and both this site and Smad-binding site are necessary for synergistic activation of the Msx-2 gene expression by Notch and BMP-2. In contrast, BMP-7 markedly augmented N1-ICD-induced expression of SMC marker genes, whereas expression of osteoblast marker genes was unchanged. Of note, BMP-7 failed to enhance N1-ICD-induced SMC marker gene expression in cells deficient of myocardin, indicating myocardin mediates the crosstalk between Notch and BMP-7 signaling. Conclusion: These results suggest that by BMP-2 and BMP-7 play an instructive role in specifying the Notch/RBP-Jk pathway which induces either differentiated phenotype or osteogenic phenotype of SMC Collectively, this study provides, at least in part, a mechanistic basis for the “context dependency” characteristic of Notch signaling.

MicroRNA-modulated targeting of vascular smooth muscle cells

VSMCs exhibit the remarkable plasticity required for development and adaptation of the cardiovascular system. The capacity of VSMCs to modulate their phenotype has evolved to facilitate angiogenesis and wound healing, but it has also been implicated in the pathogenesis of atherosclerosis, restenosis, posttransplant arteriopathy, and pulmonary hypertension. In this issue of the JCI, Boettger and colleagues report that the recently discovered Mir143/145 gene cluster promotes acquisition of the contractile phenotype of murine VSMCs (see the related article beginning on page 2634). These VSMC-restricted microRNAs, which target unique combinations of SMC genes, provide an efficient mechanism to fine-tune cardiovascular homeostasis and the response of the vessel wall to injury. This important discovery will open the door to new avenues of investigation and potentially future therapies for vascular diseases.

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase and myocardin

We have previously reported that in ovine fetal pulmonary venous smooth muscle cells (FPVSMC), decreased expression of cGMP-dependent protein kinase (PKG) by hypoxia could explain hypoxia-induced SMC phenotype modulation. In this study, we investigated the role of myocardin, a possible downstream effector of PKG, in SMC phenotype modulation induced by 1 and 24 h of hypoxia. Hypoxia for 1 h induced the phosphorylation of E-26-like protein 1 (Elk-1), indicating a quick activation of Elk-1 after hypoxia. Either hypoxia (1 h) or treatment with DT-3, a PKG inhibitor, increased associations of Elk-1 with myosin heavy chain (MHC) gene and serum response factor (SRF), which was paralleled by a decrease in association of myocardin with MHC gene and SRF. Exposure to hypoxia of FPVSMC for 24 h significantly decreased the promoter activity of multiple SMC marker genes, downregulated protein and mRNA expression of myocardin, and upregulated mRNA expression of Elk-1, but had no significant effects on the phosphorylation of Elk-1. Inhibition of myocardin by siRNA transfection downregulated the expression of SMC marker proteins, while overexpression of myocardin prevented the hypoxia-induced decrease in expression of SMC marker proteins. Inhibition of PKG by siRNA transfection downregulated the expression of myocardin, but upregulated that of Elk-1. Overexpression of PKG prevented hypoxia-induced effects on protein expression of myocardin and Elk-1. These data suggest that PKG induces displacement of myocardin from SRF and upregulates myocardin expression, thus activating the SMC genes transcription. The inhibitory effects of hypoxia on PKG may explain hypoxia-induced SMC phenotype modulation by decreasing the effects of PKG on myocardin.

New Insights to Vascular Smooth Muscle Cell and Pericyte Differentiation of Mouse Embryonic Stem Cells In Vitro

The molecular mechanisms that regulate pericyte differentiation are not well understood, partly because of the lack of well-characterized in vitro systems that model this process. In this article, we develop a mouse embryonic stem (ES) cell-based angiogenesis/vasculogenesis assay and characterize the system for vascular smooth muscle cell (VSMC) and pericyte differentiation. ES cells that were cultured for 5 days on OP9 stroma cells upregulated their transcription of VSMC and pericyte selective genes. Other SMC marker genes were induced at a later time point, which suggests that vascular SMC/pericyte genes are regulated by a separate mechanism. Moreover, sequence analysis failed to identify any conserved CArG elements in the vascular SMC and pericyte gene promoters, which indicates that serum response factor is not involved in their regulation. Gleevec, a tyrosine kinase inhibitor that blocks platelet-derived growth factor (PDGF) spell-receptor signaling, and a neutralizing antibody against transforming growth factor (TGF) beta1, beta2, and beta3 failed to inhibit the induction of vascular SMC/pericyte genes. Finally, ES-derived vascular sprouts recruited cocultured MEF cells to pericyte-typical locations. The recruited cells activated expression of a VSMC- and pericyte-specific reporter gene. We conclude that OP9 stroma cells induce pericyte differentiation of cocultured mouse ES cells. The induction of pericyte marker genes is temporally separated from the induction of SMC genes and does not require platelet-derived growth factor B or TGFbeta1 signaling.

Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.

Mental Retardation and Abnormal Skeletal Development (Dyggve-Melchior-Clausen Dysplasia) Due to Mutations in a Novel, Evolutionarily Conserved Gene

Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC) are similar, rare autosomal recessive osteochondrodysplasias. The radiographic features and cartilage histology in DMC and SMC are identical. However, patients with DMC exhibit significant developmental delay and mental retardation, the major features that distinguish the two conditions. Linkage studies localized the SMC and DMC disease genes to chromosome 18q12-21.1, providing evidence suggesting that they are allelic disorders. Sequence analysis of the coding exons of the FLJ90130 gene, a highly evolutionarily conserved gene within the recombination interval defined in the linkage study, identified mutations in SMC and DMC patients. The affected individuals in two consanguinous DMC families were homozygous for a stop codon mutation and a frameshift mutation, respectively, demonstrating that DMC represents the FLJ90130-null phenotype. The data confirm the hypothesis that SMC and DMC are allelic disorders and identify a gene necessary for normal skeletal development and brain function.

Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB.

Structural maintenance of chromosomes (SMC) proteins are present in all eukaryotes and in many prokaryotes. Eukaryotic SMC proteins form complexes with various non-SMC subunits, which affect their function, whereas the prokaryotic homologues had no known non-SMC partners and were thought to act as simple homodimers. Here we describe two novel families of proteins, widespread in archaea and (Gram-positive) bacteria, which we denote 'segregation and condensation proteins' (Scps). ScpA genes are localized next to smc genes in nearly all SMC- containing archaea, suggesting that they belong to the same operon and are thus involved in a common process in the cell. The function of ScpA was studied in Bacillus subtilis, which also harbours a well characterized smc gene. Here we show that scpA mutants display characteristic phenotypes nearly identical to those of smc mutants, including temperature- sensitive growth, production of anucleate cells, formation of aberrant nucleoids, and chromosome splitting by the so-called guillotine effect. Thus, both SMC and ScpA are required for chromosome segregation and condensation. Interestingly, mutants of another B. subtilis gene, scpB, which is localized downstream from scpA, display the same phenotypes, which indicate that ScpB is also involved in these functions. ScpB is generally present in species that also encode ScpA. The physical interaction of ScpA and SMC was proven (i) by the use of the yeast two-hybrid system and (ii) by the isolation of a complex containing both proteins from cell extracts of B. subtilis. By extension, we speculate that interaction of orthologues of the two proteins is important for chromosome segregation in many archaea and bacteria, and propose that SMC proteins generally have non-SMC protein partners that affect their function not only in eukaryotes but also in prokaryotes.

Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein

Disruption of ypuG and ypuH open reading frames in Bacillus subtilis leads to temperature-sensitive slow growth, a defect in chromosome structure and formation of anucleate cells. The genes, which were named scpA and scpB, were found to be epistatic to the smc gene. Fusions of ScpA and ScpB to the fluorescent proteins YFP or CFP showed that both proteins co-localize to two or four discrete foci that were present at mid-cell in young cells, and within both cell halves, generally adjacent to chromosomal origin regions, in older cells. ScpA and ScpB foci are associated with DNA and depend on the presence of SMC and both Scps. ScpA and ScpB are associated with each other and with SMC in vivo, as determined using the FRET technique and immunoprecipitation assays. Genes similar to scpA and scpB are present in many bacteria and archaea, which suggests that their gene products form a condensation complex with SMC in most prokaryotes. The observed foci could constitute condensation factories that pull DNA away from mid-cell into both cell halves.

Prokaryotic structural maintenance of chromosomes (SMC) proteins: distribution, phylogeny, and comparison with MukBs and additional prokaryotic and eukaryotic coiled-coil proteins

Structural maintenance of chromosomes (SMC) proteins are known to be essential for chromosome segregation in some prokaryotes and in eukaryotes. A systematic search for the distribution of SMC proteins in prokaryotes with fully or partially sequenced genomes showed that they form a larger family than previously anticipated and raised the number of known prokaryotic homologs to 54. Secondary structure predictions revealed that the length of the globular N-terminal and C-terminal domains is extremely well conserved in contrast to the hinge domain and coiled-coil domains which are considerably shorter in several bacterial species. SMC proteins are present in all gram-positive bacteria and in nearly all archaea while they were found in less than half of the gram-negative bacteria. Phylogenetic analyses indicate that the SMC tree roughly resembles the 16S rRNA tree, but that cyanobacteria and Aquifex aeolicus obtained smc genes by lateral transfer from archaea. Fourteen out of 22 smc genes located in fully sequenced genomes seem to be co-transcribed with a second gene out of six different gene families, indicating that the deduced gene products might be involved in similar functions. The SMC proteins were compared with other prokaryotic proteins with long coiled-coil domains. The lengths of different protein domains and signature sequences allowed to differentiate SMCs, MukBs, which were found to be confined to gamma proteobacteria, and two subfamilies of COG 0419 including the SbcC nuclease from E. coli. A phylogenetic analysis was performed including the prokaryotic coiled-coil proteins as well as SMCs and Rad18 proteins from selected eukaryotes.

Analysis of mutation rates in the SMCY/SMCX genes shows that mammalian evolution is male driven.

Mammalian evolution is believed to be male driven because the greater number of germ cell divisions per generation in males increases the opportunity for errors in DNA replication. Since the Y Chromosome (Chr) replicates exclusively in males, its genes should also evolve faster than X or autosomal genes. In addition, estimating the overall male-to-female mutation ratio (alpha m) is of great importance as a large alpha m implies that replication-independent mutagenic events play a relatively small role in evolution. A small alpha m suggests that the impact of these factors may, in fact, be significant. In order to address this problem, we have analyzed the rates of evolution in the homologous X-Y common SMCX/SMCY genes from three different species--mouse, human, and horse. The SMC genes were chosen because the X and Y copies are highly homologous, well conserved in evolution, and in all probability functionally interchangeable. Sequence comparisons and analysis of synonymous substitutions in approximately 1kb of the 5' coding region of the SMC genes reveal that the Y-linked copies are evolving approximately 1.8 times faster than their X homologs. The male-to-female mutation ratio alpha m was estimated to be 3. These data support the hypothesis that mammalian evolution is male driven. However, the ratio value is far smaller than suggested in earlier works, implying significance of replication-independent mutagenic events in evolution.