Calponin-1 Expression Research Articles

Research Note: Chicken breast muscle satellite cell function: effect of expression of CNN1 and PHRF1

The Wooden Breast myopathy results in the necrosis and fibrosis of breast muscle fibers in fast-growing heavy weight meat-type broiler chickens. Myogenic satellite cells are required to repair and regenerate the damaged muscle fibers. Using Genome Wide Association, candidate genes affected with Wooden Breast have been previously reported. The effect of these genes on satellite cell proliferation, differentiation, and the synthesis of lipids by satellite cells is unknown. Satellite cells isolated from the pectoralis major muscle from commercial Ross 708 broilers and a Randombred chicken (RBch) line were used. Expression of calponin 1 (CNN1) and PHD and ring fingers domains 1 (PHRF1) were knocked down by silent interfering RNA to determine their effect on satellite cell-mediated proliferation, differentiation, and lipid accumulation. CNN1 and PHRF1 affected satellite cell activity and lipid accumulation in both lines. Proliferation was reduced in the Ross 708 and RBch lines by knocking down the expression of both genes, and differentiation was affected with a line and treatment interaction when gene expression was reduced at the beginning of proliferation. During differentiation lipid accumulation was decreased with knocking down the expression of CNN1 and PHRF1. Both CNN1 and PHRF1 have not been reported previously in skeletal muscle and further research is required to determine their effect on satellite cell-mediated growth and regeneration of the pectoralis major (breast) muscle.

Calponin 1 inhibits agonist-induced ERK activation and decreases calcium sensitization in vascular smooth muscle.

Smooth muscle cell (SMC) contraction and vascular tone are modulated by phosphorylation and multiple modifications of the thick filament, and thin filament regulation of SMC contraction has been reported to involve extracellular regulated kinase (ERK). Previous studies in ferrets suggest that the actin-binding protein, calponin 1 (CNN1), acts as a scaffold linking protein kinase C (PKC), Raf, MEK and ERK, promoting PKC-dependent ERK activation. To gain further insight into this function of CNN1 in ERK activation and the regulation of SMC contractility in mice, we generated a novel Calponin 1 knockout mouse (Cnn1 KO) by a single base substitution in an intronic CArG box that preferentially abolishes expression of CNN1 in vascular SMCs. Using this new Cnn1 KO mouse, we show that ablation of CNN1 has two effects, depending on the cytosolic free calcium level: (1) in the presence of elevated intracellular calcium caused by agonist stimulation, Cnn1 KO mice display a reduced amplitude of stress and stiffness but an increase in agonist-induced ERK activation; and (2) during intracellular calcium depletion, in the presence of an agonist, Cnn1 KO mice exhibit increased duration of SM tone maintenance. Together, these results suggest that CNN1 plays an important and complex modulatory role in SMC contractile tone amplitude and maintenance.

Potential prognostic and immunotherapeutic value of calponin 1: A pan-cancer analysis.

Background: Emerging evidence has suggested a pro-oncogenic role of calponin 1 (CNN1) in the initiation of a variety of cancers. Despite this, CNN1 remains unknown in terms of its effects and mechanisms on angiogenesis, prognosis, and immunology in cancer. Materials and Methods: The expression of CNN1 was extracted and analyzed using the TIMER, UALCAN, and GEPIA databases. Meanwhile, we analyzed the diagnostic value of CNN1 by using PrognoScan and Kaplan-Meier plots. To elucidate the value of CNN1 in immunotherapy, we used the TIMER 2.0 database, TISIDB database, and Sangerbox database. Gene set enrichment analysis (GSEA) was used to analyze the expression pattern and bio-progression of CNN1 and the vascular endothelium growth factor (VEGF) in cancer. The expressions of CNN1 and VEGF in gastric cancer were confirmed using immunohistochemistry. We used Cox regression analysis to investigate the association between pathological characteristics, clinical prognosis, and CNN1 and VEGF expressions in patients with gastric cancer. Results: CNN1 expression was higher in normal tissues than it was in tumor tissues of most types of cancers. However, the expression level rebounds during the development of tumors. High levels of CNN1 indicate a poor prognosis for 11 tumors, which include stomach adenocarcinoma (STAD). There is a relationship between CNN1 and tumor-infiltrating lymphocytes (TILs), and the marker genes NRP1 and TNFRSF14 of TILs are significantly related to CNN1 expression in gastric cancers. The GSEA results confirmed the lower expression of CNN1 in tumors when compared to normal tissues. However, CNN1 again showed an increasing trend during tumor development. In addition, the results also suggest that CNN1 is involved in angiogenesis. The immunohistochemistry results validated the GSEA result (take gastric cancer as an example). Cox analysis suggested that high CNN1 expression and high VEGF expression are closely associated with poor clinical prognosis. Conclusion: Our study has shown that CNN1 expression is aberrantly elevated in various cancers and positively correlates with angiogenesis and the immune checkpoint, contributing to cancer progression and poor prognosis. These results suggest that CNN1 could serve as a promising candidate for pan-cancer immunotherapy.

Loss of myoepithelial calponin-1 characterizes high-risk ductal carcinoma in situ cases, which are further stratified by T cell composition.

A hallmark of ductal carcinoma in situ (DCIS) progression is a loss of the surrounding ductal myoepithelium. However, whether compromise in myoepithelial differentiation, rather than overt cellular loss, can be used to predict the risk of DCIS progression is unknown. Here we address this question utilizing pure and mixed DCIS cases (N = 30) as surrogates for DCIS at low and high risk for progression, respectively. We used multiplex immunohistochemical staining to evaluate the relationship between myoepithelial cell differentiation and lymphoid immune cell types associated with poor prognostic DCIS. Our results show that myoepithelial calponin‐1 discriminates between pure and mixed DCIS lesions better than histological subtype, presence of necrosis, or nuclear grade. Additionally, focal loss of myoepithelial cells associated with increased PD‐1+CD8+ T cells, which suggests a link between the myoepithelium and immune surveillance. To identify associations between calponin‐1 expression and immune response, we performed unsupervised hierarchical clustering of myoepithelial and immune cell biomarkers on 219 DCIS lesions from 30 cases. Notably, the majority of pure (low‐risk) DCIS lesions clustered in a high calponin‐1, T cell low group, whereas the majority of mixed (high‐risk) DCIS lesions clustered in a low calponin‐1, T cell high group, specifically with CD8+ and PD‐1+CD8+ T cells. However, a subset of pure DCIS lesions had a similar calponin‐1 and immune signature as the majority of mixed DCIS lesions, which have low calponin‐1 and T cell enrichment—raising the possibility that these pure DCIS lesions might be at a high risk for progression.

Matrix stiffness regulates SMC functions via TGF-β signaling pathway

The stiffness change of the vessel wall is involved in many pathological processes of the blood vessel. However, how stiffness changes regulate vascular cell phenotype is not well understood. In this study, we investigated the effects of matrix stiffness on the phenotype and functions of vascular smooth muscle cells (SMCs). SMCs were cultured on the matrices with the stiffness between 1 and 100 kPa. The expression of contractile markers calponin-1 (CNN1) and smoothelin (SMTN) increased with stiffness; in contrast, the expression of synthetic markers osteopontin (OPN) and epiregulin (EREG) were the highest on the soft surface (1 kPa). In addition, matrix metalloproteinase 2 (MMP-2) was significantly upregulated on 1-kPa surface. Consistently, the stiffness of atherosclerotic lesions in human arteries decreased by up to 10 folds compared to normal area (>40 kPa), which was accompanied by a decrease of CNN1 expression and collagen content and an increase of OPN and MMP-2 in the area of lipid deposition. Furthermore, the phosphorylation of Smad2/3 increased with matrix stiffness; when TGF-β signaling pathway was inhibited, the stiffness effects on the SMCs were reversed. Our findings suggest that matrix stiffness regulates SMC phenotype and matrix remodeling through TGF-β signal pathway. This study unravels a mechanobiological mechanism in vascular remodeling, and will help us develop strategies for vascular tissue engineering, disease modeling and therapies.

Expression of calponin-1 and its pathogenic role in systemic sclerosis

To investigate the expression of calponin-1 (CNN1) in systemic sclerosis (SSc) and its pathogenic role in fibrosis. Skin biopsy samples were collected from 19 patients with SSc and 21 healthy subjects. Real-time PCR was used to detect the expression of CNN1 and α-SMA mRNAs in the samples, and the protein expression of CNN1 was detected using immunohistochemistry. In cultured primary human dermal fibroblasts, CNN1 expression was knocked down via RNA interference, and the mRNA expression levels of CNN1 and the fibrosis-related genes α-SMA, CTGF, COL1A1, COL1A2, and COL3A1 were detected using real-time PCR; the proliferation of the cells was assessed using a real-time cell proliferation detection system. Compared with that in samples from normal subjects, the expression of CNN1 mRNA was significantly increased in the skin tissue of patients with SSc (P < 0.05) with a positive correlation with α-SMA (r=0.7219, P < 0.0001); the protein expression of CNN1 was also significantly increased in the skin tissue of patients with SSc. In cultured primary skin fibroblasts, the expression of CNN1 mRNA was positively correlated with α-SMA and COL1A1 mRNA expressions (r=0.6547, P < 0.05; r=0.6438, P < 0.05). CNN1 knockdown in the fibroblasts significantly inhibited the cell proliferation, obviously lowered the expressions of fibrosis-related genes, and reduced the protein expression of collagen. The expression of CNN1 is increased in the skin tissues of patients with SSc, and CNN1 knockdown can reduce the activity of dermal fibroblasts, suggesting the close correlation of CNN1 with fibrosis in SSc.

Novel noninvasive quantification of penile corpus cavernosum lesions in hyperlipidemia-induced erectile dysfunction in rabbits by two-dimensional shear-wave elastography.

Structural alterations in fibroelastic components of the penile corpus cavernousum (CC) may impair its compliance, resulting in venous leakage and erectile dysfunction (ED). Our study evaluated the effectiveness of noninvasive two-dimensional shear-wave elastography (2-D SWE) in quantifying penile CC lesions in rabbits with hyperlipidemia-induced ED. A total of 12 New Zealand white rabbits were randomly divided into two groups. Six were fed a high-cholesterol diet containing 2% cholesterol and 8.5% lard for 10 weeks and the other six were fed normal diet as controls. We measured the shear-wave elastic quantitative (SWQ) value of penile CC by 2-D SWE. Erectile function was investigated by intracavernous injection of papaverine, and immunohistochemical (IHC) staining and the western blot analysis to determine the penile CC lesions. After 10 weeks, the SWQ values obtained from penile CC were remarkably higher in the high-cholesterol-fed compared with the control group, and the ΔICP (ICP plateau minus ICP baseline)/MAP (ICP: intracavernous pressure, MAP: mean arterial pressure) was markedly decreased. The IHC staining and western blot revealed extracellular matrix (ECM) accumulation in penile cavernous tissues, and the smooth muscle cell (SMC) phenotypic transition was affected, as indicated by reduced alpha-smooth muscle actin and calponin-1 expression and increased phospho-myosin light chain20 (p-MLC20)/MLC20 and osteopontin expression. Hyperlipidemia resulted in ECM accumulation accompanied with SMC phenotypic transition in penile CC and impaired the erectile function eventually. These might, in turn, lead to variations in the SWQ values. It suggests that 2-D SWE may be a novel, noninvasive and effective approach that distinguishes penile CC lesions secondary to hyperlipidemia from normal.

Matrix Metalloproteinase-2 Activity is Associated with Divergent Regulation of Calponin-1 in Conductance and Resistance Arteries in Hypertension-induced Early Vascular Dysfunction and Remodelling.

Matrix metalloproteinase (MMP)-2 participates in hypertension-induced maladaptive vascular remodelling by degrading extra- and intracellular proteins. The consequent extracellular matrix rearrangement and phenotype switch of vascular smooth muscle cells (VSMCs) lead to increased cellular migration and proliferation. As calponin-1 degradation by MMP-2 may lead to VSMC proliferation during hypertension, the hypothesis of this study is that increased MMP-2 activity contributes to early hypertension-induced maladaptive remodelling in conductance and resistance arteries via regulation of calponin-1. The main objective was to analyse whether MMP-2 exerts similar effects on the structure and function of the resistance and conductance arteries during early hypertension. Two-kidney, one-clip (2K-1C) hypertensive male rats and corresponding controls were treated with doxycycline (30 mg/kg/day) or water until reaching one week of hypertension. Systolic blood pressure was increased in 2K-1C rats, and doxycycline did not reduce it. Aortas and mesenteric arteries were analysed. MMP-2 activity and expression were increased in both arteries, and doxycycline reduced it. Significant hypertrophic remodelling and VSMC proliferation were observed in aortas but not in mesenteric arteries of 2K-1C rats. The contractility of mesenteric arteries to phenylephrine was increased in 2K-1C rats, and doxycycline prevented this alteration. The potency of phenylephrine to contract aortas of 2K-1C rats was increased, and doxycycline decreased it. Whereas calponin-1 expression was increased in 2K-1C mesenteric arteries, calponin-1 was reduced in aortas. Doxycycline treatment reverted changes in calponin-1 expression. MMP-2 contributes to hypertrophic remodelling in aortas by decreasing calponin-1 levels, which may result in VSMC proliferation. On the other hand, MMP-2-dependent increased calponin-1 in mesenteric arteries may contribute to vascular hypercontractility in 2K-1C rats. Divergent regulation of calponin-1 by MMP-2 may be an important mechanism that leads to maladaptive vascular effects in hypertension.

Transforming Growth Factor-β and Notch Signaling Mediate Stem Cell Differentiation into Smooth Muscle Cells

The differentiation of stem cells into smooth muscle cells (SMCs) plays an important role in vascular development and remodeling. In addition, stem cells represent a potential source of SMCs for regenerative medicine applications such as constructing vascular grafts. Previous studies have suggested that various biochemical factors, including transforming growth factor-beta (TGF-beta) and the Notch pathway, may play important roles in vascular differentiation. However, the interactions of these two signaling pathways in the differentiation of bone marrow mesenchymal stem cells (MSCs) have not been clearly defined. In this study, we profiled the gene expression in MSCs in response to TGF-beta, and showed that TGF-beta induced Notch ligand Jagged 1 (JAG1) and SMC markers, including smooth muscle alpha-actin (ACTA2), calponin 1 (CNN1), and myocardin (MYOCD), which were dependent on the activation of SMAD3 and Rho kinase. In addition, knocking down JAG1 expression partially blocked ACTA2 and CNN1 expression and completely blocked MYOCD expression, suggesting that JAG1 plays an important role in TGF-beta-induced expression of SMC markers. On the other hand, the activation of Notch signaling induced the expression of SMC markers in MSCs and human embryonic stem cells (hESCs). Notch activation in hESCs also resulted in an increase of neural markers and a decrease of endothelial markers. These results suggest that Notch signaling mediates TGF-beta regulation of MSC differentiation and that Notch signaling induces the differentiation of MSCs and hESCs into SMCs, which represents a novel mechanism involved in stem cell differentiation.