Calponin Research Articles

Puerarin blocks the aging phenotype in human dermal fibroblasts.

Dermal fibroblast aging contributes to aging-associated functional defects in the skin since dermal fibroblasts maintain skin homeostasis by interacting with the epidermis and extracellular matrix. Here, we found that puerarin, an isoflavone present in Pueraria lobata (Kudzu), can prevent the development of the aging-phenotype in human dermal fibroblasts. Normal human dermal fibroblasts (NHDFs) were subcultivated and high-passage cells were selected as senescent cells, whereas low-passage cells were selected as a young cell control. Puerarin treatment increased cell proliferation and decreased the proportion of senescence-associated beta-galactosidase-positive cells in a high-passage culture of NHDFs. Moreover, puerarin treatment reduced the number of smooth muscle actin (SMA)-positive myofibroblasts and the expression of a reticular fibroblast marker, calponin 1 (CNN1), which were induced in high-passage NHDFs. Fulvestrant, an estrogen receptor antagonist, blocked the puerarin-mediated downregulation of SMA and CNN1. Our results suggest that puerarin may be a useful functional food that alleviates aging-related functional defects in dermal fibroblasts.

Epigenomic, Transcriptomic, and Protective Effect of Carotenoid Fucoxanthin in High Glucose-Induced Oxidative Stress in Mes13 Kidney Mesangial Cells

Diabetic nephropathy (DN) is the major cause of kidney related diseases in patients induced by high glucose (HG) affecting around 40% of type 1 and 2 diabetic patients. It is characterized by excessive inflammation inducing factors, reactive oxygen species (ROS) overproduction, and potential epigenomic related changes. Fucoxanthin (FX), a carotenoid found in brown seaweed, has a structure which includes an allenic bond and a 5,6-monoepoxide in the molecule, with strong antioxidant and anti-inflammatory activity. However, understanding of the impact of FX on DN was lacking. In this study we tested the early effects of high glucose (HG) on mouse mesangial kidney Mes13 cells, a potential in vitro cell culture model of DN. Our results show that HG induced oxidative stress on kidney mesangial Mes13 cells, while FX treatment attenuates the oxidative stress by decreasing the ROS, demonstrated by flow cytometry. Next, we utilized next-generation sequencing (NGS) to profile the HG-induced early epigenomic and transcriptomic changes in this in vitro DN model and the protective effects of FX. Differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were analyzed using R software in HG and FX treated groups. Differential regulation of signaling pathways was studied using Reactome Pathway Analysis in the comparison. DEG analysis shows that novel biomarkers with specific pathways, including interleukin regulation, Toll-like receptor pathway, and PKA phosphorylation pathways, were found to be modulated by the FX treatment. TGF β 1i1 (TGFB 1i1), MAP-3-kinase-13(MAP3K13) involved in crucial cellular processes including glucose metabolism, phosphodiesterase regulation was methylated in HG, which was demethylated with FX treatment. Integrated transcriptomic and CpG methylome analysis of DEGs and DMRs revealed that genes like adenylate cyclase (Adcy7), calponin 1 (CNN1), potassium voltage-gated channel interacting protein 2 (KCNIP2), phosphatidylinositol-4-phosphate 5-kinase type 1 β (PIP5K1B), and transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1), which were modulated by FX in HG-exposed Mes13 cells, potentially modulate ion channel transport and glucose metabolism. In summary, our current study shows that novel early epigenomic and transcriptomic biomarkers were altered during the disease progression of HG-induced DN and that FX modified these alterations potentially contributing to the protective effects of mesangial cells from the HG-induced oxidative stress and damage.

Bioinformatic screening and identification of downregulated hub genes in adrenocortical carcinoma.

The molecular mechanisms of adrenocortical carcinoma (ACC) carcinogenesis and progression remain unclear. In the present study, three microarray datasets from the Gene Expression Omnibus database were screened, which identified a total of 96 differentially expressed genes (DEGs). A protein-protein interaction network (PPI) was established for these DEGs and module analysis was performed using STRING and Cytoscape. A total of eight hub genes were identified from the most significant module; namely, calponin 1 (CNN1), myosin light chain kinase (MYLK), cysteine and glycine rich protein 1 (CSRP1), myosin heavy chain 11 (MYH11), fibulin extracellular matrix protein 2 (EFEMP2), fibulin 1 (FBLN1), microfibril associated protein 4 (MFAP4) and fibulin 5 (FBLN5). The biological functions of these hub genes were analyzed using the DAVID online tool. Changes in the expression of hub genes did not affect overall survival; however, downregulated EFEMP2 decreased disease-free survival. CSRP1 and MFAP4 expression levels were associated with adverse clinicopathological features. In conclusion, although all eight hub genes were downregulated in ACC, they appeared to have important functions in ACC carcinogenesis and progression. Identification of these genes complements the genetic expression profile of ACC and provides insight for the diagnosis, treatment and prognosis of ACC.

Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification.

Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive, precluding mechanism-based therapies. Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent reactive oxygen species (ROS)-forming Nox5 (NADPH oxidase 5). In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN-1 (calponin 1), α-SMA (α-smooth muscle actin), and SM22-α (smooth muscle protein 22α) and an increase in synthetic marker S100A4 (S100 calcium binding protein A4) compared with contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca2+. Phenotypic switching was accompanied by increased levels of ROS and Ca2+-dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knockdown of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca2+-dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca2+. Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca2+ and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca2+ uptake via EVs and show that Ca2+ induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodeling and calcification in the context of mineral imbalance. Graphic Abstract: A graphic abstract is available for this article.

Biointerface topography regulates phenotypic switching and cell apoptosis in vascular smooth muscle cells

BackgroundIn-stent restenosis (ISR) is a complex disease that occurs after coronary stenting procedures. The development of quality materials and improvement of our understanding on significant factors regulating ISR are essential for enhancing prognosis. Vascular smooth muscle cells (VSMCs) are the main constituent cells of blood vessel walls, and dysfunction of VMSCs can exacerbate ISR. Accordingly, in this study, we explored the influence of wrinkled material topography on the biological functions of VSMCs. MethodsPolydimethylsiloxane with a wrinkled topography was synthesized using elastomer base and crosslinking and observed by atomic force microscopy. VSMC proliferation, apoptosis, and morphology were determined by Cell Counting Kit-8 assays, fluorescence-assisted cell sorting, and phalloidin staining. α-Smooth muscle actin (α-SMA), major histocompatibility complex (MHC), and calponin 1 (CNN-1) expression levels were measured by quantitative real-time polymerase chain reaction and western blotting. Moreover, p53 and cleaved caspase-3 expression levels were evaluated by western blotting in VSMCs to assess apoptotic induction. ResultsSurface topographies were not associated with a clear orientation or elongation of VSMCs. The number of cells was increased on wrinkled surfaces (0.7 μm in amplitude, and 3 μm in wavelength [W3]) compared with that on other surfaces, contributing to continuously increased cell proliferation. Moreover, interactions of VSMCs with the W3 surface suppressed phenotypic switching, resulting in ISR via regulation of α-SMA, calponin-1, and SM-MHC expression. The surface with an amplitude of 0.05 μm and a wavelength of 0.5 μm (W0.5) promoted apoptosis by inducing caspase 3 and p53 activities. ConclusionIntroduction of aligned topographies on biomaterial scaffolds could provide physical cues to modulate VSMC responses for engineering vascular constructs. Materials with wrinkled topographies could have applications in the development of stents to reduce ISR.

MiR-106b-5p contributes to the lung metastasis of breast cancer via targeting CNN1 and regulating Rho/ROCK1 pathway.

Objectives: Breast cancer has been the second most prevalent and fatal malignancy due to its frequent metastasis to other organs. We aim to study the effects of a key miRNA-mRNA signaling in breast cancer.Results: CNN1 was identified as the key gene in breast cancer by the bioinformatics analysis, and the downregulation of CNN1 in breast cancer tissues and cell lines was observed. Upregulating CNN1 inhibited cell survival, migration, invasion, and adhesion, but enhanced cell apoptosis. miR-106b-5p not only bound to CNN1 mRNA 3’UTR, but also promoted lung metastasis in vivo. Besides, the miR-106b-5p mimic enhanced breast cancer canceration by targeting CNN1 and activating Rho/ROCK1 signaling pathway.Conclusion: Overall, our results proved that miR-106b-5p promoted the metastasis of breast cancer by suppressing CNN1 and activating Rho/ROCK1 pathway.Methods: Bioinformatics analysis was performed to select the key gene in breast cancer. The overexpression and knockdown of Calponin 1 (CNN1) in breast cancer cell lines were performed to conduct cell viability, migrating, invasion, proliferation, adhesion, and apoptosis experiments. To identify the role of miR-106b-5p and Rho/ROCK1 in CNN1-induced breast cancer, a dual-luciferase assay, tumor lung metastasis assay, transcript half-life assay, and Rho/ROCK1 inhibition assay were performed.

LncRNA NEAT1 Promotes Proliferation, Migration And Invasion Via Regulating miR-296-5p/CNN2 Axis In Hepatocellular Carcinoma Cells.

BackgroundEmerging evidence has revealed that long noncoding RNA nuclear paraspeckle assembly transcript 1 (lncRNA NEAT1) is implicated in the development of various cancers. However, the underlying molecular mechanisms of NEAT1 in hepatocellular carcinoma (HCC) remain unclear.MethodsThe expression of NEAT1, miR-296-5p and Calponin 2 (CNN2) was detected by quantitative real-time polymerase chain reaction or Western blot, respectively. Cell proliferation and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay or flow cytometry, respectively. Transwell assay was used to determine cell migration and invasion. The interaction between miR-296-5p and NEAT1 or CNN2 was analyzed by dual-luciferase reporter assay and RIP assay. Huh7 cells transfected with sh-NEAT1 were used to establish the murine xenograft model.ResultsNEAT1 was elevated in HCC tissues and cell lines. Knockdown of NEAT1 significantly inhibited proliferation, migration and invasion of HCC cells in vitro as well as tumor growth in vivo. NEAT1 was a sponge of miR-296-5p and remarkably reduced the level of miR-296-5p in HCC cells. Furthermore, NEAT1 silence significantly decreased the expression of CNN2, which was the direct target of miR-296-5p. Besides that, the tumor suppression caused by NEAT1 silence could be rescued by CNN2 restoration or miR-296-5p inhibition in vitro. Additionally, NEAT1 indirectly regulated CNN2 expression by competing to miR-296-5p in vitro and in vivo.ConclusionLncRNA NEAT1 contributes to HCC progression by regulating miR-296-5p/CNN2 axis, providing a novel regulatory mechanism for HCC development and a promising therapeutic target for the HCC treatment.

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.

Clinicopathological features of minute meningothelial-like nodules in the lung

Objective To study the clinicopathological features of minute meningothelial-like nodule (MMLN) in the lung and its histological origin. Methods Clinicopathological data of 93 patients diagnosed with MMLN in Jiangsu Province Hospital(the First Affiliated Hospital of Nanjing Medical University) from September 2016 to September 2018 were collected and analyzed retrospectively, and the related literature was reviewed. Results Ninety-three MMLNs were identified from 5 239 patients undergoing pulmonary resection. There were 18(19.6%) males and 75(80.6%) females; age ranged 38-81 years, with mean age 58 years. The frequency of MMLNs was related to the site of origin, and single lesion most commonly (43%) arose in right lobe. There was no significant relationship with patients′ age, gender and the co-existing malignant tumors. Receiver operating characteristic (ROC) curve analysis showed that the area under curve (AUC) was 0.584 (P<0.05), and the best cut-off point was 11.5, indicating an appropriate increase in the number of slides to at least 12 would facilitate the discovery of MMLN. Histologically, MMLN cells showed bland morphology, located around the normal alveolar cavity or form nests or whorls centered on small veins. Immunohistochemistry was done in 55 cases, and all cells expressed vimentin; and a high proportion of cells expressed EMA, PR, CD56 and Calponin. They were all negative for TTF1, Syn, CgA, CKpan, SMA and CD34. The Ki-67 index was low in all cases (<2%). In addition odds ratio analysis showed that MMLN was not a risk factor for invasive carcinoma (including other malignant tumors in the lung, P=0.100). Conclusions MMLN closely resembles meningothelioma morphologically and immunophenotypically, although its origin remains uncertain. In the study, MMLN is considered as a reactive benign lesion around pulmonary pathology, functioning as an effector regulating small blood vessels caliber. MMLN probably provides nutritional support to the surrounding disease and affords a suitable environment for the development of tumor cells. Key words: Lung diseases; Solitary pulmonary nodule; Minute meningothelial-like nodule

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 &lt; 0.05) with a positive correlation with α-SMA (r=0.7219, P &lt; 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 &lt; 0.05; r=0.6438, P &lt; 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.

Calponin Isoform Expression in the Japanese Pearl Oyster, &amp;lt;i&amp;gt;Pinctada fucata&amp;lt;/i&amp;gt;

Calponin is a basic actin-binding protein found widely in invertebrate tissues including catch muscle and therefore may participate in catch contraction. There is limited information about molluscan calponin and molecular characterization to reveal its function in the regulatory system. We previously identified and partially sequenced three calponin isoforms of the Japanese pearl oyster, Pinctada fucata (Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3). In this study, the full-length nucleotide sequences of the three isoforms were determined. The primary structures revealed that Pifuc-CP-1 consists of 324 amino acids (aa) with a molecular mass (Mw) of 34.7 kDa and an isoelectric point (pI) of 9.40. Pifuc-CP-2 is 303 aa in length with a Mw of 33.3 kDa and a pI of 9.30, and Pifuc-CP-3 is 398 aa in length with a Mw of 43.8 kDa and a pI of 8.55. Domain architecture prediction showed that the three isoforms have a single calponin homology (CH) domain and multiple calponin (CN) domains. Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3 possess four, three and five CN domains, respectively. Tissue distribution analysis indicated the presence of additional calponin isoforms and these isoforms are distributed widely in muscle and non-muscle tissues. Results of cDNA cloning revealed further four calponin isoforms: Pifuc-CP-4 (402 aa, 42.8 kDa, pI = 9.10), Pifuc-CP-5 (285 aa, 30.7 kDa, pI = 9.45), Pifuc-CP-6 (286 aa, 31.1 kDa, pI = 9.60) and Pifuc-CP-7 (302 aa, 33.3 kDa, pI = 9.10). The domain architecture of these four isoforms also consists of a single CH domain and multiple CN domains. Pifuc-CP-4 possesses six CN domains, whereas Pifuc-CP-5, Pifuc-CP-6 and Pifuc-CP-7 contain three CN domains. Sequence alignment of P. fucata calponin isoforms showed that Pifuc-CP-1, Pifuc-CP-2, Pifuc-CP-3 and Pifuc-CP-4 have identical CH domain sequences, whereas Pifuc-CP-5, Pifuc-CP-6 and Pifuc-CP-7 have identical CH domain sequences. The CN repeats were not well conserved. These findings suggest that P. fucata calponin isoforms function differently in each tissue.

Deletion of calponin 2 attenuates the development of calcific aortic valve disease in ApoE−/− mice

Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular mortality and lacks non-surgical treatment. The pathogenesis of CAVD involves perturbation of valvular cells by mechanical stimuli, including shear stress, pressure load and leaflet stretch, of which the molecular mechanism requires further elucidation. We recently demonstrated that knockout (KO) of Cnn2 gene that encodes calponin isoform 2, a mechanoregulated cytoskeleton protein, attenuates atherosclerosis in ApoE KO mice. Here we report that Cnn2 KO also decreased calcification of the aortic valve in ApoE KO mice, an established model of CAVD. Although myeloid cell-specific Cnn2 KO highly effectively attenuated vascular atherosclerosis that shares many pathogenic processes with CAVD, it did not reduce aortic valve calcification in ApoE KO mice. Indicating a function in the pathogenesis of CAVD, calponin 2 participates in myofibroblast differentiation that is a leading step in the development of CAVD. The aortic valves of ApoE KO mice exhibited increased expression of calponin 2 and smooth muscle actin (SMA), a hallmark of myofibroblasts. The expression of calponin 2 increased during myofibroblast-like differentiation of primary sheep aortic valve interstitial cells and during the osteogenic differentiation of mouse myofibroblasts. Cnn2 KO attenuated TGFβ1-induced differentiation of myofibroblasts in culture as shown by the lower expression of SMA and less calcification than that of wild type (WT) cells. These findings present calponin 2 as a novel molecular target for the treatment and prevention of CAVD.

Abstract 716: Generation of γ-secretase Inhibitor-loaded PLGA-Fe 3 O 4 - Magnetic Nanoparticles

Cardiovascular disease (CVD) is the number one killer in Ireland and the wider EU. A hallmark of the disease is the obstruction to blood flow due to the build-up of vascular smooth muscle (SMCs)-like cells within the vessel wall. While polymer-coated DES have significantly reduced the incident of in-stent restenosis, current DESs lack the fundamental capacity for (i) adjustment of the drug dose and release kinetics and the (ii) ability to replenish the stent with a new drug on depletion. This limitation can be overcome by a strategy combining magnetic targeting via a uniform field-induced magnetization effect and a biocompatible magnetic nanoparticle (MNP) formulation designed for efficient entrapment and delivery of specific drugs that target the resident vascular stem cell source of the SMC. Magnetic nanoparticles (MNP’s) containing magnetite (Fe3O4) were fabricated, polymer coated with poly (DL-lactide-co-glycolide) polyvinyl alcohol [PLGA-PVA] and loaded with a γ-secretase inhibitor (GSI) of Notch signalling, DAPT using an oil in water emulsification technique. The free GSI’s and GSI-loaded MNP’s were assessed for drug release, the efficacy at controlling mesenchymal stem cell (MSC) growth (proliferation and apoptosis) and inhibiting myogenic differentiation under magnetic and non-magnetic conditions. The DAPT-loaded MNPs had an average hydrodynamic diameter of 351 d.nm Up to 40% of drug was released from MNPs within 48 h rising to 65% after 1 week under magnetic conditions. The Notch ligand, Jagged1 increased Hey1 mRNA levels and promoted myogenic differentiation of MSCs in vitro by increasing SMC differentiation markers, myosin heavy chain 11 (Myh11) and calponin1 (CNN1) expression, respectively. This effect was significantly attenuated following treatment of cells with MNP’s loaded with DAPT when compared to unloaded MNP’s. Notch GSI -loaded magnetic nanoparticles are functional at targeting vascular stem cells in vitro.

Carcinosarcoma of Stomach Confined to the Mucosa.

Carcinosarcoma is a malignant tumor composed of both epithelial and mesenchymal malignant tumor components. A 78-year-old man was transferred to our hospital because of hematemesis and tarry stool. An emergency gastrointestinal endoscopic examination revealed active bleeding from an ulcerative lesion on the posterior wall of the gastric body; endoscopic hemostasis was successfully performed. A gastrointestinal endoscopy performed two months later showed a polypoid lesion at the same place where the ulcer had been. The biopsy specimen was histologically diagnosed as well to moderately differentiated tubular adenocarcinoma. The patient underwent a laparoscopic distal gastrectomy with D2 lymph node dissection under a diagnosis of gastric adenocarcinoma. A 28 × 15 mm polypoid tumor was resected from the gastric body, and was found on microscopic examination to consist of both carcinoma and sarcoma components, showing atypical spindle cells, which were positive for α-smooth muscular actin, calponin, and h-caldesmon, but negative for CD34, CD117 (c-kit), desmin, and dog 1. These findings led to a diagnosis of gastric carcinosarcoma. The tumor was confined to the mucous membrane. Lymph node metastasis was found in one node and contained only the carcinoma component. The postoperative course was uneventful. The patient lived without recurrence for 2 years. Carcinosarcoma of the stomach is a rare tumor with high malignant potential and poor prognosis. Careful follow up is required for early detection of any recurrence.

Lentivirus-mediated shRNA Targeting CNN2 Inhibits Hepatocarcinoma in Vitro and in Vivo.

Objective: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with a high rate of mortality. Our previous study shows the expression of calponin 2 (CNN2) is up-regulated in hepatocellular carcinoma tissues, especially in metastatic ones. To better understand the role of CNN2 in HCC, RNA interference (RNAi) was used to explore its role in tumor growth and metastasis.Methods: Lentivirus-mediated CNN2-shRNA was transfected into SK-hep-1 cells, and the efficacy of CNN2 expression, cell migration, invasion, proliferation and cell cycles were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB), Transwell assay, methyl thiazol tetrazolium assay and flow cytometry, respectively. SK-hep-1 cells transfected with Lentivirus-CNN2 shRNA were xenografted in Balb/C nude mice to explore the effect of CNN2-shRNA in tumor growth. Xenograft tumor tissues were examined for their histopathology, cell apoptosis, the expression of total protein and their corresponding phosphorylated protein of MEK1/2, ERK1/2, AKT, by hematoxylin and eosin stain (H & E staining), TUNEL assay, immunohistochemical technique, respectively.Results: Our research shows it is evident that CNN2 shRNA can effectively down-regulate the expressions of CNN2 mRNA and protein, inhibit cell proliferations, arrest cell cycles at the S phase and reduce cell migration and invasion. SK-hep-1 cells with CNN2 down-regulation have markedly attenuated tumor growth in nude mice. Xenograft tumor tissues have displayed typical tumor characteristics and no apoptosis is detected in shRNA group or in control group. No metastatic tumor was found in any group of nude mice. With CNN2 protein down-regulation, the protein of pMEK1/2 and pERK1/2 are effectively down-regulated, except pAKT, AKT, MEK1/2 and ERK1/2.Conclusions: CNN2 plays an important role in tumor growth and metastasis, possibly through MEK1/2-ERK1/2 signaling pathway. Our study illustrate that CNN2 might be a potential target in HCC molecular target therapy.

Knockdown of calponin 2 suppressed cell growth in gastric cancer cells.

Calponin family members are actin filament-associated regulatory proteins with distinct expression patterns. Previous studies on CNN2 (calponin 2) have demonstrated that CNN2 is expressed in a broad range of tissues and cell types, exhibiting potential regulatory roles in a number of cellular activities, including cell proliferation, cell migration, and platelet adhesion. In this work, we found that both messenger RNA and protein expression levels of CNN2 were remarkably upregulated in 60%-70% of gastric cancer tissues by comparison with those of neighboring non-tumorous mucosa. By utilizing specific shCNN2 (small hairpin RNA targeting CNN2), the potential role of CNN2 in regulating AGS gastric cancer cell growth was then further investigated. AGS cells infected with shCNN2 exhibited significantly decreased cell growth ability by comparison with control cells in vitro. Moreover, while there was no obvious difference in cell cycle distribution between two groups, enhanced cell apoptosis was detected in cells with reduced CNN2 expression. Consistently, caspase 3/7 activity was also remarkably activated upon shCNN2 lentivirus infection. Taken together, our results demonstrated that knockdown of endogenous CNN2 in AGS cells could significantly activate cell apoptosis pathway and therefore suppress cell growth in vitro. The deletion of CNN2 might be a potential therapeutic approach to inhibit aggressive growth of gastric cancer.

Actin cytoskeleton mediates BMP2-Smad signaling via calponin 1 in preosteoblast under simulated microgravity

Microgravity influences the activity of osteoblast, induces actin microfilament disruption and leads to bone loss during spaceflight. Mechanical stress such as gravity, regulates cell function, response and differentiation through dynamic cytoskeleton changes, but the mechanotransduction mechanism remains to be fully elucidated. Previous, we demonstrated actin microfilament mediated osteoblast Cbfa1 responsiveness to BMP2 under simulated microgravity (SMG). Here, we explored a potential molecular and its detailed mechanism of actin cytoskeleton functioning on BMP2-Smad signaling in MC3T3-E1 under SMG. Results showed that the actin microfilament-disrupting agent, cytochalasin B (CB), reduced BMP2-induced activation, translocation of Smad1/5/8 and Runx2 expression. SMG also inhibited BMP2-Smad signaling, which was rescued by actin cytoskeleton stabilizing agent, Jasplakinolide (JAS). Furthermore, we found that siRNA mediated knockdown of calponin 1 (CNN1), an actin binding protein, markedly promoted BMP2-Smad signaling and abolished both inhibition of CB, SMG on BMP2-Smad signaling and the rescue action of JAS. Overexpression of CNN1 inhibited the p-Smad induced by BMP2. Bidirectional Co-IP experiments demonstrated CNN1 could interacted with Smad or p-Smad protein. Furthermore, CB or SMG decreased the phosphorylated CNN1 and increased its interaction with Smad or p-Smad. Combined with the phosphorylation of CNN1 inhibites its actin binding activity, these results indicate that actin cytoskeleton depolymerization inhibites BMP2 signaling via blocking of Smad by dephosphorylated CNN1 in osteoblast cells. Thus, we provide new important insights into the mechanism of mechanotransduction under SMG condition, which probably contribute to bone formation decrease induced by SMG.

Aortic dissection is associated with reduced polycystin-1 expression, an abnormality that leads to increased ERK phosphorylation in vascular smooth muscle cells.

The vascular smooth muscle cell (VSMC) phenotypic switch is a key pathophysiological change in various cardiovascular diseases, such as aortic dissection (AD), with a high morbidity. Polycystin-1 (PC1) is significantly downregulated in the VSMCs of AD patients. PC1 is an integral membrane glycoprotein and kinase that regulates different biological processes, including cell proliferation, apoptosis, and cell polarity. However, the role of PC1 in intracellular signaling pathways remains poorly understood. In this study, PC1 downregulation in VSMCs promoted the expression of SM22, ACTA2 and calponin 1 (CNN1) proteins. Furthermore, PC1 downregulation in VSMCs upregulated phospho-MEK, phospho-ERK and myc, but did not change phospho-JNK and phospho-p38. These findings suggest that the MEK/ERK/myc signaling pathway is involved in PC1-mediated human VSMC phenotypic switch. Opposite results were observed when an ERK inhibitor was used in VSMCs downregulated by PC1. When the C-terminal domain of PC1 (PC1 C-tail) was overexpressed in VSMCs, the expression levels of phosphor-ERK, myc, SM22, ACTA2 and CNN1 proteins were downregulated. The group with the overexpressed mutant protein (S4166A) in the PC1 C-tail showed similar results to the group with the downregulated PC1 in VSMCs. These results suggest that the Ser at the 4166 site in PC1 is crucial in the PC1 mediated MEK/ERK/myc signaling pathway, which might be the key pathophysiological cause of AD.

Deletion of calponin 2 in macrophages attenuates the severity of inflammatory arthritis in mice.

Calponin is an actin cytoskeleton-associated protein that regulates motility-based cellular functions. Three isoforms of calponin are present in vertebrates, among which calponin 2 encoded by the Cnn2 gene is expressed in multiple types of cells, including blood cells from the myeloid lineage. Our previous studies demonstrated that macrophages from Cnn2 knockout (KO) mice exhibit increased migration and phagocytosis. Intrigued by an observation that monocytes and macrophages from patients with rheumatoid arthritis had increased calponin 2, we investigated anti-glucose-6-phosphate isomerase serum-induced arthritis in Cnn2-KO mice for the effect of calponin 2 deletion on the pathogenesis and pathology of inflammatory arthritis. The results showed that the development of arthritis was attenuated in systemic Cnn2-KO mice with significantly reduced inflammation and bone erosion than that in age- and stain background-matched C57BL/6 wild-type mice. In vitro differentiation of calponin 2-null mouse bone marrow cells produced fewer osteoclasts with decreased bone resorption. The attenuation of inflammatory arthritis was confirmed in conditional myeloid cell-specific Cnn2-KO mice. The increased phagocytotic activity of calponin 2-null macrophages may facilitate the clearance of autoimmune complexes and the resolution of inflammation, whereas the decreased substrate adhesion may reduce osteoclastogenesis and bone resorption. The data suggest that calponin 2 regulation of cytoskeleton function plays a novel role in the pathogenesis of inflammatory arthritis, implicating a potentially therapeutic target.

The clinical diagnostic value of SYT-SSX gene detection combined with immunohistochemistry in synovial sarcoma

Objective To analyse the clinicopathological characteristics of synovial sarcoma (SS) from histomorphology, immunohistochemical (IHC) and fluorescence in situ hybridization (FISH), and to investigate the related factors influencing the prognosis of SS patients. Methods 94 cases were collected, including 60 SS cases and 34 in dined to SS cases. The expressions of common antibodies related to the diagnosis of SS Vimentin, CD99, AE1/AE3, EMA, CD34, Calponin, Ki-67 were detected by IHC, and the SYT-SSX fusion gene was detected by FISH. The clinical pathologic factors that affecting the prognosis of patients were analyzed through the statistical method. Results The positive rates of Vimentin, CD99, AE1/AE3, EMA, CD34, Calponin, Ki-67 were 100.00 %, 74.47 %, 36.17 %, 28.72 %, 17.02 %, 90.43 % and 60.64 % respectively. FISH results showed that 81 patients were tested with SYT gene translocation, including 54 cases who were considered as SS, and 27 cases who were suspected/inclined to SS, and the number of positive cells surpassed 80 %. The gene translocation was not related with the patient's age, sex, tumor position, histological type, differentiation of the cancer (all P > 0.05). Single factor survival analysis showed that the degree of differentiation, tumor diameter, the recurrence and metastasis had certain influences on patients'survival time (all P 1, P < 0.05). Conclusions IHC combined with FISH SYT-SSX fusion gene detection have the vital significance in the diagnosis of SS. The degree of differentiation and tumor size are the risk factors influencing the prognosis of SS. Key words: Sarcoma, synovial; Genes; Diagnosis; Immunohistochemistry; Fluorescence in situ hybridization; Prognosis