Calponin and SM22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development

Abstract. Calponin and SM 22 are two proteins related in sequence that are particularly abundant in smooth muscle cells. Here, the distribution patterns of calponin and SM 22 were compared with that of other smooth muscle contractile and cytoskeletal components in the avian embryo using immunofluorescence microscopy and immunoblotting. Like myosin-light-chain kinase and heavy caldesmon, both calponin and SM 22 were more or less exclusively found in smooth muscle cells, during embryonic development and in the adult. Labelling of other cell types including striated muscle was not observed. In contrast, tropomyosin, smooth muscle α-actin, filamin and desmin could also be detected in many other cell types in addition to smooth muscles, at least during part of embryonic life. Calponin and SM 22 appeared almost synchronously during the differentiation of all smooth muscle cell populations, though with a slight time difference in the case of the aorta. The appearance of calponin, SM22 and heavy caldesmon was generally delayed in relation to desmin, tropomyosin, smooth muscle α-actin, myosin-light-chain kinase and filamin and a marked increase in abundance of these proteins was observed in the late embryo and in the adult. From these observations we can conclude that both calponin and SM 22 belong to a group of late differentiation determinants in smooth muscle and may constitute convenient and reliable markers to follow the differentiation of most, if not all, smooth muscle cell populations.     

Cyclic AMP-dependent phosphorylation of filamin in mammalian smooth muscle.

Filamin is a high molecular weight actin-binding protein found in large quantities in smooth muscle and other non-muscle cells. We have studied the phosphorylation of filamin in a mammalian smooth muscle, the guinea pig vas deferens. Intact vas deferens incorporated [32P]orthophosphate into filamin. Incubation of particulate fractions of vas deferens with [gamma-32P]ATP resulted in 32P-labeling of filamin. Cyclic AMP stimulated this phosphorylation, whereas cyclic GMP and Ca2+ had no effect. Purified vas deferens filamin can be phosphorylated by purified cyclic AMP-dependent protein kinase. We have compared cyclic AMP and cyclic GMP effects on phosphorylation in smooth muscle. Cyclic GMP stimulated phosphorylation of two particulate proteins, G-I (Mr = 130,000) a protein previously described by Casnellie, J. E., and Greengard, P. (1974) Proc. Natl. Acad, Sci. U.S.A. 71, 1891-1895 and G-III (Mr = 240,000). Both proteins and the kinase responsible for their phosphorylation appear to be membrane-bound. Phosphorylation of both proteins is stimulated by cyclic GMP (Ka = 3 x 10(-8) M), cyclic AMP (Ka = 3 x 10(-7) M), and to a lesser degree by Ca2+. In contrast, filamin phosphorylation is due to a soluble kinase stimulated only by cyclic AMP (Ka = 3 x 10(-7) M) and not by cyclic GMP or Ca2+.     

SM22alpha promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo

BACKGROUND: Gene transfer into vascular smooth muscle cells (vsmcs) holds promise for studying the pathogenesis of arterial disorders. However, a potential limitation of vectors with heterologous promoters is organ toxicity resulting from unrestricted transgene expression. Vascular smooth muscle cell-specific gene expression could increase the safety of vectors for vascular diseases. MATERIALS AND METHODS: To develop vectors that target gene expression to vsmcs, we constructed vectors encoding human placental alkaline phosphatase (hpAP) and chloramphenicol transferase (CAT) driven by a 441-bp region of the murine SM22alpha promoter (AdSM22alpha-hpAP). RESULTS: Transfection of AdSM22alpha-hpAP into vascular and nonvascular cells resulted in the expression of alkaline phosphatase (AP) in primary arterial and venous smcs, but not in primary endothelial cells or National Institutes of Health (NIH) 3T3 cells. Expression of AP was observed on 32.5 +/- 1.4% of primary pig vsmcs-infected AdSM22alpha-hpAP at a multiplicity of infection (MOI) of 500; whereas, infection with AdCMV-hpAP resulted in 100 +/- 0.0% expression at a MOI of 250. In vitro, expression from the heterologous cytomegalovirus (CMV) promoter was approximately 10(3)-fold higher in vsmcs, compared with the SM22alpha promoter. Following introduction of AdSM22alpha-hpAP vectors into balloon-injured pig arteries, AP recombinant protein was detected in neointimal (2.23 +/- 1.14%) and medial (0.56 +/- 0.21%) smcs, but not in endothelial or adventitial cells. In contrast, AdCMV-hpAP vectors led to AP expression in intimal endothelial and smcs cells (39.14 +/- 10.09%) and medial smcs (2.84 +/- 1.05%). AP expression was not observed in endothelial or vsmcs following transfection with the control vector, adenoviral vector lacking E1 (AddeltaE1). CONCLUSIONS: The SM22alpha promoter programs recombinant gene expression exclusively to vascular smcs in vitro and in vivo. Although expression levels are lower than with heterologous promoters, these vectors may provide a safe and effective tool for gene therapy of vascular diseases.     

Amino acid sequence of chicken gizzard smooth muscle SM22 alpha

The complete amino acid sequence of SM22 alpha, a novel and abundant 22-kDa protein from chicken gizzard smooth muscle, was determined by a combination of automated and manual Edman degradation methods on fragments produced by suitable chemical and proteolytic cleavages. The protein consists of a single polypeptide chain of 197 residues, has a Mr of 21, 978, and a net charge of +4.5 at neutral pH. The pattern of alternating hydrophilic and hydrophobic regions throughout the length of SM23 alpha is typical of a globular protein. The overall secondary structural analysis, using several algorithms based on the sequence, predicts approximately 31% alpha-helix, 24% beta-sheet, 18% beta-turn, and 27% random coil. A search against the National Biomedical Research Foundation Protein Sequence Databank (Washington) and GenBank (Los Alamos) failed to demonstrate significant similarity with any other protein of known sequence.     

Absence of calponin phosphorylation in contracting or resting arterial smooth muscle

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 265, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 32P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [32P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.     

Chemical energy usage and myosin light chain phosphorylation in mammalian smooth muscle

Experiments have been done to determine the relationships among active force output, average rate of high-energy phosphate utilization, and the degree of phosphorylation of the 20,000-dalton myosin light chain in the rabbit tenia coli at 18 C. During an isometric tetanus at l0 the degree of light chain phosphorylation increases to a maximum of 30-40% before maximum force is developed, and then phosphorylation slowly decreases while active force is maintained. During the period when there is a small decrease in degree of phosphorylation, the average rate of chemical energy usage falls by fourfold. In contrast, when the calcium concentration of the bathing medium is lowered from 1.9 to 1.0 mM a very large decrease in degree of phosphorylation is associated with only a small decrease in both energy usage and active force. At lower calcium levels both force and chemical energy usage decrease proportionately with little further decrease in degree of phosphorylation. We conclude that under isometric conditions there is no consistent relationship between degree of myosin light chain phosphorylation and the rate of cross-bridge cycling as measured by the rate of high-energy phosphate usage in this mammalian smooth muscle.     

绵羊钙调蛋白调节基因Calponin h1 的分子克隆

在一项研究中我们发现雌激素体在胚胎发育后期对绵羊子宫平滑肌Calponin (CaP) 基因的活动有明显上调作用,而CaP一直被作为观察其他基因表达水平变化的基准参照基因（Reference Gene）。迄今为止, 绵羊CaP尚未完整克隆,为进一步了解其结构和功能,根据人、小鼠和家猪的同源保守区序列设计锚定寡核苷酸引物,通过5′-RACE及3′-RACE方法克隆了绵羊子宫平滑肌组织全长CaP h1 cDNA (GenBank登录号： AY327118), 在cDNA序列的基础上, 又通过PCR-SSP方法获得了CaP h1基因除内含子1、2之外的其余4个内含子全部序列 （GenBank登录号分别为：AY771807,AY771808, AY771809, AY771810） 。DNA序列测定和分析表明,绵羊子宫平滑肌CaP h1 cDNA全长1499bp, 编码297个氨基酸,5′-UTR及3′-UTR分别为79bp和529bp。CaP h1基因组DNA的克隆和序列分析表明,绵羊CaP全长约8kb,由 7个外显子和6个内含子组成。 同源序列比较发现,该基因外显子在不同物种间相对保守;与人类、野猪、小鼠、大鼠和鸡Calponin mRNA同源性分别为88％、92％、81％、79％和81％,但不同物种间内含子存在较大差异(>50%)。本研究填补了绵羊CaP基因分子克隆的空白,为进一步研究该基因的功能及子宫平滑肌收缩的调节机理奠定了基础。     

cDNA cloning and mRNA expression of calponin and SM22 in rat aorta smooth muscle cells

We cloned and sequenced cDNAs encoding calponin (Calp) and SM22 (smooth muscle-specific 22-kDa protein) from rat aorta (RaA) smooth muscle (Smu) cells. The 1504-bp calp cDNA contains a single open reading frame (ORF) which encodes 297 amino acids (aa) (M_r 33 342). The 1186-bp SM22 cDNA contains a single ORF which encodes 201 aa (M_r 22 601). There were 43% identical aa in a 181-aa overlap between RaA Calp and SM22. Especially for the C-terminal region of SM22 and for the first repeat motif of Calp, 70% identity was observed. Northern blot analysis revealed that the calp and SM22 mRNAs were expressed in RaA Smu, but not in rat cardiac and skeletal muscles. SM22 mRNA was much more abundant than calp mRNA in RaA (3- to 4-fold). The expression levels of the calp and SM22 mRNAs in RaA showed a significant increase for 5 to 15 week old rats (1.5- to 3-fold) with vascular development and blood pressure elevation. No significant differences were observed in the expression of the RaA calp and SM22 mRNAs between normotensive (Wistar Kyoto) and spontaneously hypertensive rats (SHR).     

Regulation of SM22 alpha expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM alpha-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22 alpha appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22 alpha expression by AVP and PDGF in cultured VSMC. Levels of SM22 alpha mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22 alpha promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22 alpha promoter. Expression of constitutively active Ras produced similar suppressions on SM22 alpha promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.     

Purified desmin from adult mammalian skeletal muscle: a peptide mapping comparison with desmins from adult mammalian and avian smooth muscle.

Comparative one-dimensional peptide maps were prepared by the electrophoresis of digests derived from treatment of desmins with Ca²⁺-activated muscle protease, trypsin, $\text{Staphylococcus}\text{aureus}$ V8 protease, and cyanogen bromide. Desmins from adult mammalian skeletal and smooth muscles were very similar. Avian smooth muscle desmin, although homologous with respect to many peptides, was different from the mammalian smooth and skeletal desmins. The amino acid compositions of the three desmins were quite similar.     

Distribution of vimentin and desmin filaments in smooth muscle tissue of mammalian and avian aorta

The presence of intermediate filament proteins in vascular tissue cells has been examined by immunofluorescence microscopy on frozen sections of the aortic wall of diverse vertebrates (rat, cow, human and chicken) and by gel electrophoresis of cytoskeletal proteins from whole aortic tissue or from stripped tunica media of cow and man. Most cells of the aortic wall in these species contain vimentin filaments, including smoooth muscle cells of the tunica media. In addition, we have observed aortic cells that are positively stained by antibodies to desmin. The presence of desmin in aortic tissue has also been demonstrated by gel electrophoresis for rat, cow and chicken. In aortic tissue some smooth muscle cells contain both types of intermediate filament proteins, vimentin and desmin. Bovine aorta contains, besides cells in which vimentin and desmin seem to co-exist, distinct bundles of smooth muscle cells, located in outer regions of the tunica media, which contain only desmin. The results suggest that (i) intermediate-sized filaments of both kinds, desmin and vimentin, can occur in vascular smooth muscle in situ and (ii) smooth muscle cells of the vascular system are heterogeneous and can be distinguished by their intermediate filament proteins. The finding of different vascular smooth muscle cells is discussed in relation to development and differentiation of the vascular system.     

Isolation and characterization of an abundant and novel 22-kDa protein (SM22) from chicken gizzard smooth muscle

Chicken gizzard smooth muscle contains a highly abundant protein (SM22) with an apparent Mr on sodium dodecyl sulfate-polyacrylamide electrophoretic gels of 23,000. The ratio of actin:SM22:tropomyosin in this tissue is estimated to be 6.5(+/- 0.8):2.0(+/- 0.2):1.0. At least three isoelectric isoforms are present in ratios of alpha:beta:gamma of 14:5:1 with alpha the most basic and gamma the most acidic. A method for the purification of SM22 and partial separation of its isoforms is described. Amino acid analyses of purified alpha and beta demonstrate the presence of 1 and 2 half-cystines, respectively, and a lower content of basic amino acids in beta. A value of 22,000 for the Mr of alpha estimated by sedimentation equilibrium indicated its presence as a monomer at physiological ionic strengths. Estimates of the translational frictional coefficient (f/fmin) of alpha calculated from its Stokes radius (25.5 A) and Mr were consistent with its existence as a moderately asymmetric globular protein. Calculations based on its far-ultraviolet CD spectrum provided values of 37% alpha-helix, 31% beta-sheet, 5% beta-turn, and 27% random coil. SM22 was shown not to share functional properties with several proteins of similar Mr and isoelectric point such as myokinase, brain 23-kDa protein, and troponin I. We conclude that it is a novel protein not previously isolated or characterized from any tissue.     

Filamin isoforms in molluscan smooth muscle

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.