Human smooth muscle alpha-actin promoter drives Cre recombinase expression in the cranial suture in addition to smooth muscle cell

Tissue-specific gene deletion by the Cre-loxp system is a powerful tool to investigate the roles of specific genes. To determine the specificity and efficiency of the Cre-mediated recombination under the control of the human smooth muscle alpha-actin promoter, we mated SMalphaA-Cre mice and R26R reporter mice. Cre-mediated recombination was observed in visceral and vascular smooth muscle cells. Partial recombination was also found in heart and musculoskeletal connective tissues. Highly efficient recombination was found in cranial sutures. Hence, we propose that SMalphaA-Cre mice are good tool for conditionally deleting gene function in the cranial suture in addition to smooth muscle cells.     

Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes

Myocardin is a serum response factor (SRF) coactivator exclusively expressed in cardiomyocytes and smooth muscle cells (SMCs). However, there is highly controversial evidence as to whether myocardin is essential for normal differentiation of these cell types, and there are no data showing whether cardiac or SMC subtypes exhibit differential myocardin requirements during development. Results of the present studies showed the virtual absence of myocardin(-/-) visceral SMCs or ventricular myocytes in chimeric myocardin knockout (KO) mice generated by injection of myocardin(-/-) embryonic stem cells (ESCs) into wild-type (WT; i.e., myocardin(+/+) ESC) blastocysts. In contrast, myocardin(-/-) ESCs readily formed vascular SMC, albeit at a reduced frequency compared with WT ESCs. In addition, myocardin(-/-) ESCs competed equally with WT ESCs in forming atrial myocytes. The ultrastructural features of myocardin(-/-) vascular SMCs and cardiomyocytes were unchanged from their WT counterparts as determined using a unique X-ray microprobe transmission electron microscopic method developed by our laboratory. Myocardin(-/-) ESC-derived SMCs also showed normal contractile properties in an in vitro embryoid body SMC differentiation model, other than impaired thromboxane A2 responsiveness. Together, these results provide novel evidence that myocardin is essential for development of visceral SMCs and ventricular myocytes but is dispensable for development of atrial myocytes and vascular SMCs in the setting of chimeric KO mice. In addition, results suggest that as yet undefined defects in development and/or maturation of ventricular cardiomyocytes may have contributed to early embryonic lethality observed in conventional myocardin KO mice and that observed deficiencies in development of vascular SMC may have been secondary to these defects.     

Identification of a CArG box-dependent enhancer within the cysteine-rich protein 1 gene that directs expression in arterial but not venous or visceral smooth muscle cells.

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.     

Identification of a CArG Box-Dependent Enhancer within the Cysteine-Rich Protein 1 Gene That Directs Expression in Arterial but Not Venous or Visceral Smooth Muscle Cells

Smooth muscle cells (SMCs) are heterogeneous with respect to their contractile, synthetic, and proliferative properties, though the regulatory factors responsible for their phenotypic diversity remain largely unknown. To further our understanding of smooth muscle gene regulation, we characterized the cis-regulatory elements of the murine cysteine-rich protein 1 gene (CRP1/Csrp1). CRP1 is expressed in all muscle cell types during embryogenesis and predominates in vascular and visceral SMCs in the adult. We identified a 5-kb enhancer within the CRP1 gene that is sufficient to drive expression in arterial but not venous or visceral SMCs in transgenic mice. This enhancer also exhibits region-specific activity in the outflow tract of the heart and the somites. Within the 5-kb CRP1 enhancer, we found a single CArG box that binds serum response factor (SRF), and by mutational analysis, demonstrate that the activity of the enhancer is dependent on this CArG element. Our findings provide further evidence for the existence of distinct regulatory programs within SMCs and suggest a role for SRF in the activation of the CRP1 gene.     

SM22beta encodes a lineage-restricted cytoskeletal protein with a unique developmentally regulated pattern of expression

Cytoskeletal proteins play important roles in regulating cellular morphology, cytokinesis and intracellular signaling. In this report, we describe a developmentally regulated gene encoding a novel cell lineage-restricted cytoskeletal protein, designated SM22beta. SM22beta shares high-grade sequence identity with the smooth muscle cell (SMC)-specific protein, SM22alpha, the neuron-specific protein, NP25, and the Drosophila melanogaster flight muscle-specific protein, mp20. The mouse SM22beta cDNA encodes a 199-amino acid polypeptide that contains a single conserved calponin-like repeat domain. During mouse embryonic development, the SM22beta gene is expressed in a temporally and spatially regulated pattern in the tunica media of arteries and veins, endocardium and compact layer of the myocardium, bronchial epithelium and mesenchyme of the lung, gastrointestinal epithelium and cartilaginous primordia. During postnatal development, SM22beta is co-expressed with SM22alpha in arterial and venous SMCs. In addition, SM22beta is expressed at high levels in the bronchial epithelium and lung mesenchyme, gastrointestinal epithelial cells and in the cartilagenous and periosteal layer of bones. Three-dimensional deconvolution microscopic analyses of A7r5 SMCs revealed that SM22beta co-localizes with SM22alpha to cytoskeletal actin filaments. Taken together, these data demonstrate that SM22beta is a novel actin-associated protein with a unique cell lineage-restricted pattern of expression.     

Targeted insertion of an IRES Cre into the Hnf4alpha locus: Cre-mediated recombination in the liver, kidney, and gut epithelium

The Hnf4alpha gene belongs to a family of trancriptional regulators required for liver development and function. Hnf4alpha is also expressed in other tissues, including the newly formed visceral endoderm of the early postimplantation embryo, and later in embryogenesis in the gut epithelium and the kidney. The regulatory sequences involved in controlling expression of Hnf4alpha at these diverse sites are not clearly understood. Here we used homologous recombination to introduce Cre recombinase coding sequences into the endogenous Hnf4alpha locus. Crossing Hnf4alpha(Creex2/+) mice with R26R partners allowed us to follow the pattern of Cre-mediated recombination. Our results show that recombination of the reporter allele closely follows endogenous Hnf4alpha expression, but with a slight temporal delay. Thus, the Hnf4alpha(Creex2) strain should prove useful for conditionally deleting gene activity in the liver, gut epithelium, or kidney.     

Phenotype-dependent expression of cadherin 6B in vascular and visceral smooth muscle cells

We used mRNA subtraction of differentiated and dedifferentiated smooth muscle cells (SMCs) to reveal the molecular mechanisms underlying the phenotypic modulation of SMCs. With this approach, we found that a 10 kb mRNA encoding a homotypic cell adhesion molecule, cadherin 6B, was strongly expressed in differentiated vascular and visceral SMCs, but not in the dedifferentiated SMCs derived from them. In vivo, cadherin 6B was expressed in vascular and visceral SMCs, in addition to brain, spinal cord, retina and kidney, at a late stage of chicken embryonic development. These results suggest that cadherin 6B is a novel molecular marker for vascular and visceral SMC phenotypes and is involved in the late differentiation of SMCs.     

Smooth muscle protein 22 alpha-Cre is expressed in myeloid cells in mice

Background: Experiments using Cre recombinase to study smooth muscle specific functions rely on strict specificity of Cre transgene expression. Therefore, accurate determination of Cre activity is critical to the interpretation of experiments using smooth muscle specific Cre. Methods and results: Two lines of smooth muscle protein 22 α-Cre (SM22α-Cre) mice were bred to floxed mice in order to define Cre transgene expression. Southern blotting demonstrated that SM22α-Cre was expressed not only in tissues abundant of smooth muscle, but also in spleen, which consists largely of immune cells including myeloid and lymphoid cells. PCR detected SM22α-Cre expression in peripheral blood and peritoneal macrophages. Analysis of SM22α-Cre mice crossed with a recombination detector GFP mouse revealed GFP expression, and hence recombination, in circulating neutrophils and monocytes by flow cytometry. Conclusions: SM22α-Cre mediates recombination not only in smooth muscle cells, but also in myeloid cells including neutrophils, monocytes, and macrophages. Given the known contributions of myeloid cells to cardiovascular phenotypes, caution should be taken when interpreting data using SM22α-Cre mice to investigate smooth muscle specific functions. Strategies such as bone marrow transplantation may be necessary when SM22α-Cre is used to differentiate the contribution of smooth muscle cells versus myeloid cells to observed phenotypes.     

Ex vivo generation of mature and functional human smooth muscle cells differentiated from skeletal myoblasts

We described the ex vivo production of mature and functional human smooth muscle cells (SMCs) derived from skeletal myoblasts. Initially, myoblasts expressed all myogenic cell-related markers such as Myf5, MyoD and Myogenin and differentiate into myotubes. After culture in a medium containing vascular endothelial growth factor (VEGF), these cells were shown to have adopted a differentiated SMC identity as demonstrated by alphaSMA, SM22alpha, calponin and smooth muscle-myosin heavy chain expression. Moreover, the cells cultured in the presence of VEGF did not express MyoD anymore and were unable to fuse in multinucleated myotubes. We demonstrated that myoblasts-derived SMCs (MDSMCs) interacted with endothelial cells to form, in vitro, a capillary-like network in three-dimensional collagen culture and, in vivo, a functional vascular structure in a Matrigel implant in nonobese diabetic-severe combined immunodeficient mice. Based on the easily available tissue source and their differentiation into functional SMCs, these data argue that skeletal myoblasts might represent an important tool for SMCs-based cell therapy.     

SM22alpha is required for agonist-induced regulation of contractility: evidence from SM22alpha knockout mice

The present study was undertaken to determine whether SM22alpha participates in the regulation of vascular smooth muscle contractility using SM22alpha knockout mice and, if so, to investigate the mechanisms involved. Aortic ring preparations were mounted and equilibrated in organ baths for 60 min before observing contractile responses to 50 mM KCl, and then exposed to contractile agents such as phenylephrine and phorbol ester. Measurement of isometric contractions using a computerized data acquisition system was combined with molecular or cellular experiments. Interestingly, the aortas from SM22alpha-deficient mice (SM22(-/-LacZ)) displayed an almost three-fold increase in the level of SM22beta protein compared to wild-type mice, but no change in the levels of caldesmon, actin, desmin or calponin. Ca2+-independent contraction in response to phenylephrine or phorbol ester was significantly decreased in the SM22alpha-deficient mice, whereas in the presence of Ca2+ neither contraction nor subcellular translocation of myosin light chain kinase (MLCK) in response to phenylephrine or 50 mM KCl was significantly affected. A decrease in phosphorylation of extracellular signal regulated kinase (ERK) 1/2 was observed in the SM22alpha-deficient mice and this may be related to the decreased vascular contractility. Taken together, this study provides evidence for a pivotal role of SM22alpha in the regulation of Ca2+-independent vascular contractility.     

Generation and characterization of Csrp1 enhancer-driven tissue-restricted Cre-recombinase mice

Cell type-specific genetic modification using the LoxP/Cre system is a powerful tool for genetic analysis of distinct cell lineages. Because of the unique arterial smooth muscle-restricted expression of a 5.0 kb cysteine-rich protein (Csrp1) enhancer (Lilly et al.,2001, Dev Biol 240:531-547), we hypothesized that a transgenic Cre line would prove useful for the smooth muscle lineage-specific genetic manipulation. Here we describe a transgenic mouse line, ECsrp1(Cre), where Cre is initially specifically expressed in arterial smooth muscle cells. Use of the ROSA26R reporter allele confirmed that Cre-mediated recombination in vascular smooth muscle cells began at approximately E10.0 and was highly proficient. Subsequently, Cre is expressed in restricted skeletal and nonvascular smooth muscle lineages. This lineage tracing data is important for future conditional knockout studies to understand where and when Cre-mediated deletion occurs and where Cre-expressing daughter cells finally localize. Additionally, we crossed the ECsrp1(Cre) mice to the ROSA26(-eGFP-DTA) diphtheria toxin A-expressing mice to genetically ablate ECsrp1(Cre) expressing cells. This ECsrp1(Cre) transgenic line should thus prove useful for genetic analysis of diverse aspects of cardiovascular morphogenesis and as a general smooth muscle lineage deletor line.