Calponin Gene Research Articles

The Expression and Degradation of SM22-Alpha/Transgelin are Regulated by Mechanical Tension in the Cytoskeleton

SM22-alpha, also named transgelin, encoded by the gene TAGLN is a calponin-related protein found in smooth muscle, fibroblast and cancer cells. SM22-alpha was discovered three decades ago but its biological function remains unclear. In addition to an application as a differentiation marker for smooth muscle cells, SM22-alpha has been reported to regulate the structure and dynamics of actin cytoskeleton and cell motility in fibroblasts and cancer cells. Here we report a novel finding that the expression and degradation of SM22-alpha/transgelin are both regulated by mechanical tension. Mass spectrometry detected that SM22-alpha was significantly decreased in mouse aortic rings after incubation under low mechanical tension. Using specific monoclonal antibodies developed against chicken gizzard SM22-alpha, we found high levels of SM22-alpha in human fetal lung fibroblast cells line MRC-5 and primary neonatal mouse skin fibroblasts. Similar to that of calponin 2, the level of SM22-alpha is positively dependent on the mechanical tension in the cytoskeleton as determined by the stiffness of culture substrate. Quantitative RT-PCR demonstrated a transcriptional regulation of TAGLN gene expression by mechanical tension in the cytoskeleton. The cellular localization of SM22-alpha overlaps with that of myosin IIA and blebbistatin inhibition of myosin motor decreased the expression of SM22-alpha. The level of SM22-alpha is decreased in skin fibroblasts isolated from calponin 2 knockout mice compared to that in calponin 2-positive wild type cells, suggesting their correlated functions. With the close phylogenetic relationship between TAGLN and the calponin genes, SM22-alpha is identified as a calponin-like cytoskeleton regulatory protein. These findings laid a groundwork for understanding the physiological function of SM22-alpha in mechanoregulation of cytoskeleton and cell motility and its relationship with calponins.

NOX4-derived reactive oxygen species limit fibrosis and inhibit proliferation of vascular smooth muscle cells in diabetic atherosclerosis

Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normo- and hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe−/− mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4−/−Apoe−/−) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe−/− mice and in primary mouse SMCs through the activities of PDGF and NOX1.

Cloning and mineralization-related functions of the calponin gene in Chlamys farreri

Calponin is a widely distributed protein which is associated with the bio-mineralization process in vertebrates. Recently, a new type of calponin has been found in shellfish; the present study aimed to determine if this gene in shellfish functions in bio-mineralization, one of the most important processes in a mollusk's growth. We chose Chlamys farreri, a seashell species with great economic value, as the object of the study and obtained its full-length cDNA to study the function of calponin by gene expression analysis, shell notching experiment and RNA interference assays. Calponin in C. farreri is a basic protein that is highly conserved among mollusk species. Except for high expression in the adductor muscle and foot, which correlated with its function of regulating muscle contraction, the calponin gene was expressed more in the mantle than in other tissues. The expression of the gene was induced by shell notching and an RNA interference assay showed that inhibition of calponin expression caused the growth of irregular mineral crystals on the shell. Further analysis indicated that calponin might function by regulating the expression of other mineralization-related genes. Calponin is a mineralization-related protein in C. farreri that might influence mineral crystal growth by affecting the expressions of other proteins, such as matrix proteins and mineralization-regulating proteins.

Molecular characterization and functional analysis of a calponin gene from the pinewood nematode

SummaryIn this study, acDNAsequence corresponding to theBursaphelenchus xylophilus unc‐87homolog gene (Bx‐unc‐87) was identified inB. xylophilustranscriptome and characterized. ThiscDNAhas an 1152‐bp‐long open‐reading frame that is putatively translated into a 383 amino acids peptide. The deduced protein (Bx‐UNC‐87) contains highly conserved regions of the calponin‐like proteins and has high sequence identity to the calponin proteins of other nematodes. InCaenorhabditis elegans, the calponin protein encoded by geneunc‐87is required to maintain the structure of myofilaments in muscle cells of the body wall. The silencing ofBx‐unc‐87gene was performed byRNAi, soaking the nematodes in a solution containing dsRNAof this gene. In addition to a no dsRNAcontrol, a non‐homologous dsRNAcontrol was also included. TheBx‐unc‐87transcription was downregulated in nematodes treated withBx‐unc‐87dsRNAin comparison with the non‐dsRNA‐treated nematodes. dsRNA‐treated nematodes revealed some paralysis and uncoordinated movement in contrast to the regular and sinusoidal movement of the non‐treated nematodes. Furthermore, reproduction was reduced in dsRNA‐treated nematodes. These results suggest that this protein has a function similar to that of theC. eleganshomolog.

Abstract 143: Role of Chronic Kidney Disease Promoted Caspase-1 Activation in Neointimal Hyperplasia

The major cause of vascular access failure is venous stenosis due to neointimal hyperplasia (NH) [62]. Vascular smooth muscle cells (VSMC) are critical for the development of NH lesions, as they have the ability to modulate their phenotype from “contractile” to a “synthetic” in the presence of uremia, through the regulation of sensor genes for uremia danger signals and VSMC-specific differentiation genes. Recent research indicates that Caspase-1 (casp-1) activation plays an essential role in sensing metabolic danger signal-associated molecular patterns[4] and initiating vascular inflammation. Therefore, the goal of this project is to examine the role of CKD-driven casp-1 activation in VSMC and CKD-related NH. We have established a CKD mouse model and published CKD-associated vascular remodeling. We exposed wild type and caspase-1 knockout mice to our CKD model, analyzed and quantified the NH lesion formed. We also examined in vitro and ex-vivo changes in VSMC-specific differentiation genes when exposed to uremic serum and cLDL, in the presence or absence of casp-1 inhibitor. We found that CKD serum induces with casp-1 activation and phenotypic changes in VSMCs from a “contractile” to a “synthetic” phenotype, which are reversed with casp-1 inhibition. In an ex-vivo model using relative quantification we found that VSMC contractile markers α -Actin, Calponin, SM-22, and Smoothelin gene expression of CKD mouse carotid VSMC were higher in casp-1 knockout mice when compared to wild-type (1.40, 1.28, 1.22, 1.41 respectively). Also using an in-vivo model, relative quantification of α-actin decreased from 1.0 to 0.329 when VSMCs were exposed to uremic serum and increased back to 0.588 when casp-1 inhibitor is added. The relative quantification of Calponin also decreased from 1.0 to 0.394 when exposed to uremic serum and increased back to 0.601 with caspa-1 inhibitor. We also found that casp-1 deficiency significantly reversed CKD-related vascular remodeling by reducing NH volume from 1,440,023.70 in wild-type mice to 71,069.97 μm2 in casp-1 knockouts (p-value 0.002). Our results provide a novel insight over the therapeutic potential of casp-1 inhibitors for CKD induced NH.

Modulation of vascular smooth muscle cell phenotype by STAT-1 and STAT-3

ObjectiveSmooth muscle cell (SMC) de-differentiation is a key step that leads to pathological narrowing of blood vessels. De-differentiation involves a reduction in the expression of the SMC contractile genes that are the hallmark of quiescent SMCs. While there is considerable evidence linking inflammation to vascular diseases, very little is known about the mechanisms by which inflammatory signals lead to SMC de-differentiation. Given that the Signal Transducers and Activators of Transcription (STAT) transcriptional factors are the key signaling molecules activated by many inflammatory cytokines and growth factors, the aim of the present study was to determine if STAT transcriptional factors play a role SMC de-differentiation. Methods and resultsUsing shRNA targeted to STAT-1 and STAT-3, we show by real time RT-PCR and Western immunoblots that STAT-1 significantly reduces SMC contractile gene expression. In contrast, STAT-3 promotes expression of SMC contractile genes. Over-expression studies of STAT-1 and STAT-3 confirmed our observation that STAT-1 down-regulates whereas STAT-3 promotes SMC contractile gene expression. Bioinformatics analysis shows that promoters of all SMC contractile genes contain STAT binding sites. Finally, using ChIP analysis, we show that both STAT-1 and STAT-3 associate with the calponin gene. ConclusionThese data indicate that the balance of STAT-1 and STAT-3 influences the differentiation status of SMCs. Increased levels of STAT-1 promote SMC de-differentiation, whereas high levels of STAT-3 drive SMC into a more mature phenotype. Thus, inhibition of STAT-1 may represent a novel target for therapeutic intervention in the control of vascular diseases such as atherosclerosis and restenosis.

Transforming Growth Factor–β–Induced Differentiation of Airway Smooth Muscle Cells Is Inhibited by Fibroblast Growth Factor–2

In asthma, basic fibroblast growth factor (FGF-2) plays an important (patho)physiological role. This study examines the effects of FGF-2 on the transforming growth factor-β (TGF-β)-stimulated differentiation of airway smooth muscle (ASM) cells in vitro. The differentiation of human ASM cells after incubation with TGF-β (100 pM) and/or FGF-2 (300 pM) for 48 hours was assessed by increases in contractile protein expression, actin-cytoskeleton reorganization, enhancements in cell stiffness, and collagen remodeling. FGF-2 inhibited TGF-β-stimulated increases in transgelin (SM22) and calponin gene expression (n = 15, P < 0.01) in an extracellular signal-regulated kinase 1/2 (ERK1/2) signal transduction-dependent manner. The abundance of ordered α-smooth muscle actin (α-SMA) filaments formed in the presence of TGF-β were also reduced by FGF-2, as was the ratio of F-actin to G-actin (n = 8, P < 0.01). Furthermore, FGF-2 attenuated TGF-β-stimulated increases in ASM cell stiffness and the ASM-mediated contraction of lattices, composed of collagen fibrils (n = 5, P < 0.01). However, the TGF-β-stimulated production of IL-6 was not influenced by FGF-2 (n = 4, P > 0.05), suggesting that FGF-2 antagonism is selective for the regulation of ASM cell contractile protein expression, organization, and function. Another mitogen, thrombin (0.3 U ml(-1)), exerted no effect on TGF-β-regulated contractile protein expression (n = 8, P > 0.05), α-SMA organization, or the ratio of F-actin to G-actin (n = 4, P > 0.05), suggesting that the inhibitory effect of FGF-2 is dissociated from its mitogenic actions. The addition of FGF-2, 24 hours after TGF-β treatment, still reduced contractile protein expression, even when the TGF-β-receptor kinase inhibitor, SB431542 (10 μM), was added 1 hour before FGF-2. We conclude that the ASM cell differentiation promoted by TGF-β is antagonized by FGF-2. A better understanding of the mechanism of action for FGF-2 is necessary to develop a strategy for therapeutic exploitation in the treatment of asthma.

Calponin gene expression in Chironomus riparius exposed to di(2‐ethylhexyl) phthalate

Di(2-ethylhexyl) phthalate, which is a ubiquitous environmental contaminant because of its extensive use as a plasticizer, is a potential nongenotoxic carcinogen. To assess the effects of DEHP exposure on the cytoskeleton of Chironomus, we characterized full-length cDNA sequences of the calponin gene from Chironomus riparius. The expression of the calponin gene was analyzed during different life-history stages and under various DEHP concentrations for short and long periods. A phylogenetic investigation was then conducted to compare different orders of insects using sequence database analysis. The complete cDNA sequence of the calponin gene was found to be 555 bp in length. The results of phylogenetic analysis revealed that C. riparius calponin is most closely related to that of beetles. The basal level of calponin mRNA was highly expressed during different life-history stages. In addition, calponin gene expression decreased within 1 h of short-term exposure to DEHP, regardless of the concentration. We also investigated expression of the calponin gene following long-term exposure (10 days). Calponin gene expression was found to decrease significantly in C. riparius that were exposed to a low dose of DEHP, and this response was found to occur in a dose-dependent manner. Taken together, these results suggest that DEHP affects the functions of Ca(2+) binding muscle proteins such as calponin in Chironomus species.

Integrin-linked kinase regulates smooth muscle differentiation marker gene expression in airway tissue

Phenotypic changes in airway smooth muscle occur with airway inflammation and asthma. These changes may be induced by alterations in the extracellular matrix that initiate signaling pathways mediated by integrin receptors. We hypothesized that integrin-linked kinase (ILK), a multidomain protein kinase that binds to the cytoplasmic tail of beta-integrins, may be an important mediator of signaling pathways that regulate the growth and differentiation state of airway smooth muscle. We disrupted signaling pathways mediated by ILK in intact differentiated tracheal muscle tissues by depleting ILK protein using ILK antisense. The depletion of ILK protein increased the expression of the smooth muscle differentiation marker genes myosin heavy chain (SmMHC), SM22alpha, and calponin and increased the expression of SmMHC protein. Conversely, the overexpression of ILK protein reduced the mRNA levels of SmMHC, SM22alpha, and calponin and SmMHC protein. Analysis by chromatin immunoprecipitation showed that the binding of the transcriptional regulator serum response factor (SRF) to the promoters of SmMHC, SM22alpha, and calponin genes was increased in ILK-depleted tissues and decreased in tissues overexpressing ILK. ILK depletion also increased the amount of SRF that localized within the nucleus. ILK depletion and overexpression, respectively, decreased and increased the activation of its downstream substrate protein kinase B (PKB/Akt). The pharmacological inhibition of Akt activity also increased SRF binding to the promoters of smooth muscle-specific genes and increased expression of smooth muscle proteins, suggesting that ILK may exert its effects by regulating the activity of Akt. We conclude that ILK is a critical regulator of airway smooth muscle differentiation. ILK may mediate signals from integrin receptors that control airway smooth muscle differentiation in response to alterations in the extracellular matrix.

Abstract 985: BAC Transgenic Analysis of the Smooth Muscle Calponin Gene

Regulatory elements controlling gene expression are often found in separate, sometimes remote, regions around gene loci. Artificial chromosomes offer a means to capture all regulatory elements for study of gene regulation in a near-correct genomic context. The smooth muscle calponin gene (CNN1) encodes for a multifunctional protein involved in signaling, contractile force generation, and growth regulation. While CNN1’s physiology has been studied extensively, its transcriptional regulation has proven to be intractable to conventional in vivo assays. Four evolutionarily-conserved serum response factor (SRF) binding sites (called CArG boxes) are present in the first intron of CNN1 and appear to be required for full transcriptional competence of CNN1 in cultured smooth muscle cells (SMC). To assess the functionality of CNN1 CArG elements in vivo , we exploited a 103-kb bacterial artificial chromosome (BAC) containing the human CNN1 locus shown previously to completely recapitulate endogenous mouse CNN1 expression in vivo . We now show similar SMC-restricted expression of human CNN1 from only 38-kb of BAC sequence. BAC recombineering studies in which the first intron (containing four CArG elements) of CNN1 is replaced with a bacterial selection gene ( galK ) reveal complete loss of human CNN1 expression in vascular SMC, with residual expression in some visceral SMC-containing tissues such as bladder. Transgenic mice carrying the 38-kb BAC with point mutations in all four intronic CArG elements show similar findings suggesting that SRF directly mediates in vivo expression of CNN1 primarily through intronic CArG elements. Bioinformatic, luciferase, and ChIP assays reveal the presence of distal, conserved CArG elements, including a previously unrecognized one located more than 3-kb upstream of the CNN1 transcription start site. The latter CArG-like element is responsive to the SRF coactivator myocardin and is essential for full CNN1 promoter activity in vitro . Moreover, this distal CArG-like element is embedded in a cis regulatory module with conserved binding sites to other distinct transcription factors. We propose that full in vivo CNN1 gene expression requires multiple elements, including five CArG boxes, spread over 14-kb of genomic sequence.

Direct association and translocation of PKC-alpha with calponin.

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-alpha and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-alpha, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-alpha in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.

Characterization of a mammalian smooth muscle cell line that has retained transcriptional and posttranscriptional potencies.

Unlike skeletal and cardiac muscle cells that differentiate irreversibly, smooth muscle cells (SMCs) retain a high degree of plasticity. During the so-called phenotypic modulation, SMCs can undergo transition between a contractile phenotype and a highly proliferative synthetic phenotype, as apparent from the extinction of numerous smooth muscle (SM) markers when they are passaged in culture. It would be very useful to have an SMC line that can be indefinitely propagated for the cellular and molecular analysis of the mechanisms that underlie the control of SM differentiation. This report describes an immortalized rabbit aorta SMC-derived cell line (U8A4) that has conserved differentiated properties through multiple subcultures. U8A4 cells can grow in the absence of serum and express the SMC markers studied, including SM alpha-actin, SM calponin, SM22alpha, SM alpha-tropomyosin (alpha-TM), SM myosin heavy chain (SM-MHC), and myocardin. U8A4 cells can activate SMC-restricted promoters like those of SM22alpha, SM calponin, and SM-MHC genes as efficiently as described previously for rat SMC lines (PAC1, A7r5, and A10). These cells can also process exogenous alpha-TM transcripts according to an SM-specific pattern. These results demonstrate that the U8A4 cell line constitutes a good alternative model to existing SMC lines that could facilitate the study of the transcriptional and posttranscriptional regulatory mechanisms underlying SMC differentiation.

Expression of smooth muscle calponin in tumor vessels of human hepatocellular carcinoma and its possible association with prognosis.

Hepatocellular carcinoma (HCC) is a vascular-rich tumor. The tumor vessels in HCC were demonstrated to have alpha-smooth muscle actin positive smooth muscle cells (SMCs). However, it is unclear whether the SMCs in the wall of the tumor vessels are differentiated or undifferentiated. Basic calponin is an actin-, tropomyosin-, and calmodulin-binding protein, and expression of the calponin gene in SMCs has been recognized as one of the late stage differentiation markers of SMCs. The authors investigated the differentiation state of SMCs in tumor vessels by immunohistochemical examination of calponin in patients with HCC, and whether it is associated with the patients' prognosis. Tumor and nontumor tissues were obtained from 75 patients with HCC who underwent radical hepatic resection. The differentiation state of the smooth muscle cells were evaluated based on the expression level of calponin, an actin-binding protein, using immunohistochemistry and reverse transcription-polymerase chain reaction analysis. The disease free survival (DFS) rates were estimated according to the Kaplan-Meier method comparing groups of patients with calponin positive and negative tumor vessels. A multivariate analysis based on the Cox proportional hazards regression model was performed to estimate whether the expression of calponin is an independent prognostic factor. In the 75 patients with HCC examined, 36 patients (48%) possessed calponin positive SMCs, and the remaining 39 (52%) did not. There were no significant differences in either clinical or pathologic factors between the two groups of patients. The 5- and 8-year DFS rate of the patients with calponin positive vessels were 37% and 26%, respectively. These values were significantly higher (11% and 5%) than those of patients with calponin negative vessels. Gender, TNM classification, perioperative transfusion, and calponin expression were found to be independent prognostic factors for DFS. Immunohistochemical examination of the calponin expression in the tumor vessels is a new and useful means to predict the prognosis of HCC patients after hepatic resection.

Smooth-muscle calponin in mesangial cells: regulation of expression and a role in suppressing glomerulonephritis.

The basic or h1 calponin gene, which encodes an actin-binding protein involved in the regulation of smooth-muscle shortening velocity, is known to be a smooth-muscle differentiation-specific gene. It was found that basic calponin was expressed by cultured mesangial cells and localized along the actin filaments. Among the growth factors involved in the mesangial cell pathophysiology, including platelet-derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1), TNF-alpha potently downregulates basic calponin expression in both the mRNA and protein levels, whereas TGF-beta1 upregulates the calponin expression. PDGF-BB also reduced its mRNA expression. The half-life of basic calponin mRNA was determined to be similar between TNF-alpha-treated and -untreated mesangial cells, whereas cell transfection assays that used a luciferase reporter gene construct containing the functional basic calponin promoter showed that TNF-alpha and PDGF-BB reduced the transcriptional activity. Because stimulation with TNF-alpha and PDGF-BB was associated with mesangial cell proliferation, basic calponin may play a role in the suppression of mesangial cell proliferation. Treatment with anti-glomerular basement membrane antibody in calponin knockout mice induced more severe nephritis than in wild type mice, as judged from an increase in the urinary protein excretion, glomerular cellularity, and number of proliferating cell nuclear antigen-positive cells in glomerulus. These results suggest that basic calponin expression may serve as one of the intrinsic regulators of glomerular nephritis. Elucidation of the molecular mechanisms for regulation of the basic calponin expression in mesangial cells may improve the understanding of the molecular basis and pathogenesis of the glomerular response to injury.

Angiotensin II modulates calponin gene expression in rat vascular smooth muscle cells in vivo.

It has been shown that angiotensin II (Ang II) induces the expression of calponin, a 34 kD actin-binding protein, in vascular smooth muscle cells in vitro. The aim of this study was to investigate whether Ang II can modulate calponin gene expression in rat aorta in vivo. Aortic calponin gene expression was studied after chronic exogenous Ang II administration and in Goldblatt hypertension. To investigate the effect of Ang II administration, Sprague Dawley rats were treated for 6 days with a continuous infusion of Ang II (200 ng/kg per min) or saline by osmotic minipumps. The effect of endogenous Ang II on aortic calponin mRNA expression was studied in Goldblatt hypertensive rats with (2K1C model), or without (1K1C model) activation of the renin-angiotensin system. In particular, calponin gene expression in 2K1C rats was studied both at 1 week (2K1C-HR, high renin) and 4 weeks after the onset of hypertension, when plasma renin activity (PRA) was returned to normal values (2K1C-NR, normal renin). Systolic blood pressure (SBP) was measured twice a week. At the end of the experimental period, PRA was measured by radioimmunoassay, and aortic calponin gene expression was measured by Northern hybridization. SBP was significantly higher (P < 0.01), whereas PRA was suppressed (P < 0.01), in Ang II versus saline-treated rats. Northern hybridization showed that the aortic calponin gene expression significantly increased (2.5-fold) in Ang II-treated rats (P = 0.01). In Goldblatt hypertensive rats, SBP was significantly higher in 2K1C-HR (P < 0.01), 2K1C-NR (P < 0.01) and 1K1C (P < 0.01) rats compared with the corresponding sham-treated rats. Activation of the renin-angiotensin system was present only in 2K1C-HR rats (P < 0.01), and Northern analysis showed that aortic calponin mRNA expression was significantly increased (2.2-fold) in this group of rats only (P < 0.01). Our data demonstrate that both exogenous and endogenous Ang II increase calponin gene expression in aortic smooth muscle cells, independently of the hemodynamic effect of Ang II.

G-protein-coupled-receptor activation of the smooth muscle calponin gene.

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [alpha-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

G-protein-coupled-receptor activation of the smooth muscle calponin gene

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

Expression and chromosomal mapping of the mouse smooth muscle calponin gene.

Smooth muscle calponin (Cnn1) is a multifunctional protein whose expression is tightly restricted to differentiated smooth muscle cell (SMC) lineages during embryonic and post-natal life. As such, Cnn1 represents an ideal locus from which to dissect out regulatory elements that control its expression and hence the mature SMC phenotype. Previous work has focused on the expression and chromosomal mapping of the rat and human Cnn1 orthologs. In this report, we describe a unique pattern of Cnn1 expression during the growth and differentiation of BC3H1 cells, a mouse cell line that has transcriptional characteristics of both smooth and skeletal muscle lineages. Actively growing BC3H1 cells exhibit Cnn1 mRNA expression, which is extinguished when these cells are induced to differentiate upon serum withdrawal. Replating differentiated BC3H1 cells restores steady-state Cnn1 mRNA levels. The down-regulation of Cnn1 mRNA during BC3H1 differentiation coincides with the induction of myogenin, a skeletal muscle transcription factor that is not present in SMC lineages. Results from cycloheximide and actinomycin D studies suggest the existence of a labile repressor protein(s) that destabilizes the pool of Cnn1 mRNA and/or silences transcription of the Cnn1 locus. Mapping of the mouse Cnn1 locus to Chr 9, which is homologous to human Cnn1 on 19p13.2 and rat Cnn1 on 8q, suggests no gross rearrangement of this locus in the BC3H1 cell line. These results are the first to show reversible expression of Cnn1 and demonstrate the utility of the BC3H1 muscle cell line as a model system for the further characterization of Cnn1 gene regulation.

CDNA cloning and expression analysis of a calponin gene from the plant-parasitic nematode Meloidogyne incognita

cDNA cloning and expression analysis of a calponin gene from the plant-parasitic nematode Meloidogyne incognita

Angiotensin II Increases Calponin Expression in Cultured Rat Vascular Smooth Muscle Cells

Angiotensin II (Ang II) action on vascular smooth muscle cells is not limited to contraction, but includes long term effects such as hypertrophy and hyperplasia. This implies a complex pattern of gene modulation, which remains largely unknown. We used the mRNA differential display method to screen rat aortic smooth muscle cells cultured with or without Ang II. We demonstrated that Ang II induces the expression of calponin, a 34-kD protein, which has been shown to regulate smooth muscle cell contraction and to be a marker of smooth muscle cell differentiation. We demonstrated this induction both at gene and protein level in vascular smooth muscle cells. Calponin mRNA was dose-dependently induced by Ang II, with an effect still evident at 5 × 10−9M, and it did not require active protein synthesis, since cycloheximide treatment did not suppress this induction. Calponin gene expression was maximal at 3 h, while protein expression was maximal at 8 h. Calponin expression was completely abolished by the AT1 receptor antagonist, losartan, at 1 × 10−6M. Our data demonstrate that Ang II increases calponin gene expression and protein level in rat aortic smooth muscle cells in vitro.