Calponin 3 Research Articles

The modulation of lung cancer cell motility by Calponin 3 is achieved through its ability to sense and respond to changes in substrate stiffness.

The influence of extracellular matrix (ECM) stiffness on cell behavior is a well-established phenomenon. Tumor development is associated with the stiffening of the ECM. However, the understanding of the role of biomechanical behavior and mechanotransduction pathways in the oncogenesis of tumor cells remains limited. In this study, we constructed in vitro models using Polydimethylsiloxane substrates to create soft and stiff substrates. We then evaluated the migration of lung cancer cells A549 using video-microscopy and transwell assays. The mechanical properties were assessed through the utilization of atomic force microscopy, Optical Magnetic Twisting Cytometry, and traction force analysis. Additionally, the expression of Calponin 3 (CNN3) was evaluated using reverse transcription‑quantitative PCR and immunofluorescence techniques. Our observations indicate that the presence of a stiff substrate enhances A549 motility, as evidenced by increased stiffness and traction force in A549 cells on the stiff substrate. Furthermore, we observed a decrease in CNN3 expression in A549 cells on the stiff substrate. Notably, when CNN3 was overexpressed, it effectively inhibited the migration and invasion of A549 cells on the stiff substrate. The results of our study provide novel perspectives on the mechanisms underlying cancer cell migration in response to substrate mechanical properties.

Promoting effects of calponin 3 on the growth of diffuse large B‑cell lymphoma cells.

Diffuse large B‑cell lymphoma (DLBCL) is one of the most common types of lymphoma. Calponin 3 (CNN3) is a thin filament‑associated protein previously known to regulate smooth muscle contraction. Recent evidence illustrates its involvement in carcinogenesis; however, its roles in DLBCL remain unknown. CNN3 was found to be highly expressed in DLBCL specimens according to the online Gene Expression Profiling Interactive Analysis data. The aim of the present study was to investigate the roles of CNN3 in the progression of DLBCL. Invitro, the ectopic expression of CNN3 promoted the proliferation and G1/S transition of DLBCL cells, while its silencing led to opposite alterations. A similar tumor‑promoting role of CNN3 was also demonstrated by injecting nude mice with DLBCL cells over‑ or underexpressing CNN3. The results of dual‑luciferase reporter and chromatin immunoprecipitation assays revealed that forkhead box O3 (FOXO3), a known tumor suppressor in DLBCL, bound to the CNN3 promoter at ‑1955/‑1948 and ‑1190/‑1183, and suppressed the transcription of CNN3. The alterations induced by FOXO3 were partly blocked by CNN3 overexpression. On the whole, the present study demonstrates that CNN3, whose transcriptional activity is negatively regulated by FOXO3, contributes to the malignant behavior of DLBCL cells. The findings of the present study may provide novel diagnostic or therapeutic insight for DLBCL in clinical practice.

Calponin 3 Acts as a Potential Diagnostic and Prognostic Marker and Promotes Glioma Cell Proliferation, Migration, and Invasion

Calponin 3 (CNN3) is involved in the proliferation and invasion of cervical cancer and osteosarcoma cells. However, the role of CNN3 in glioma tumorigenesis remains to be elucidated. CNN3 mRNA expression in normal brain tissue and gliomas, including glioblastoma multiforme and lower-grade glioma, was analyzed using GEPIA 2 and Oncomine. CNN3 levels in glioma tissues were identified using immunohistochemical data provided by the Human Protein Atlas website. The relationship between CNN3 mRNA expression and clinical characteristics of patients with glioma was analyzed using the Oncomine database and The Cancer Genome Atlas. The diagnostic value of CNN3 expression in glioma was analyzed using receiver operating characteristic analysis according to The Cancer Genome Atlas and Genotype-Tissue Expression data. The relationship between CNN3 and prognosis was analyzed using GEPIA 2. The function of CNN3 knockdown in glioma cell lines was analyzed using cell proliferation, Transwell, and Western blot assays. Both mRNA and protein levels of CNN3 were distinctly higher in lower-grade glioma and glioblastoma multiforme tissues than those in normal brain tissues, particularly glioblastoma. A higher CNN3 mRNA level had significant relationship with higher World Health Organization grade, isocitrate dehydrogenase wild-type status, and 1p/19q noncodeletion. CNN3 mRNA expression is a highly accurate marker for the diagnosis of glioma. Patients with glioma in the high-CNN3 group had significantly lower disease-free survival and survival rates. In addition, CNN3 silencing significantly inhibited cell proliferation, migration, invasion, and the phosphorylation of P65 NF-κB. CNN3 expression is increased in glioma, particularly glioblastoma. Silencing CNN3 expression inhibited the proliferation, migration, and invasion of glioma cell lines.

CNN3 in glioma: The prognostic factor and a potential immunotherapeutic target.

Background:Gliomas are the most intrinsic type of primary intracranial tumors. The protein encoded by The calponin 3 (CNN3) has been proven to be a member of the calponin family. Its relationships with cervical cancer, colorectal cancer, gastric cancer, and colon cancer have been emphasized by several studies. Our research aims to explore the prognosis value and immunotherapeutic targetability of CNN3 in glioma patients using bioinformatics approach.Methods:CNN3 expression in glioma was analyzed based on GEO and TCGA datasets. Gene expression profiling with clinical information was employed to investigate the correlation between clinicopathological features of glioma patients and relative CNN3 expression levels. Survival analysis was conducted using Kaplan-Meier analysis and the Cox proportional-hazards regression model. Gene set enrichment analysis was conducted to select the pathways significantly enriched for CNN3 associations. Correlations between inflammatory activities, immune checkpoint molecules and CNN3 were probed by gene set variation analysis, correlograms, and correlation analysis.Results:CNN3 was enriched in gliomas, and high expression of CNN3 correlated with worse clinicopathological features and prognosis. Associations between CNN3 and several immune-related pathways were confirmed using a bioinformatics approach. Correlation analysis revealed that CNN3 was associated with inflammatory and immune activities, tumor microenvironment, and immune checkpoint molecules.Conclusion:Our results indicate that high CNN3 expression levels predict poor prognosis, and CNN3 may be a promising immunotherapy target.

Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway.

BackgroundMyogenesis is a complex process that requires optimal outside–in substrate–cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12.MethodsMyoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA).ResultsDuring proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines.ConclusionCNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

Calponin 3 suppresses proliferation, migration and invasion of non-small cell lung cancer cells.

Calponin 3 (CNN3) is known to serve a role in certain types of cancer, such as gastric cancer and colorectal cancer. The present study investigated the clinical significance of CNN3 in non-small cell lung cancer (NSCLC) by evaluating its expression profile and relationship with disease prognosis using the Gene Expression Omnibus repository, Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Kaplan-Meier plotter analysis. CNN3 mRNA expression was measured using reverse transcription-quantitative PCR, while the protein expression level was measured using western blot analysis. Cell proliferation, cell cycle and apoptosis, and migration and invasion were analyzed using MTS assay, flow cytometry and Transwell assays, respectively. These results revealed that CNN3 mRNA expression was downregulated in NSCLC tissues compared with that in normal tissues. Additionally, CNN3 expression had a high diagnostic value based on the GSE2514 dataset and the data from The Cancer Genome Atlas and the Genotype Tissue Expression database, whereas it had a low diagnostic value based on the GSE10072 dataset. Furthermore, CNN3 expression was associated with survival in patients with lung adenocarcinoma (LUAD), whereas it was not associated with survival in patients with lung squamous cell carcinoma (LUSC) according to the Kaplan-Meier plotter results. According to the data from GEPIA2, and the GSE72094, GSE41271 and GSE31210 datasets, CNN3 expression was not associated with the prognosis of patients with LUAD and LUSC. The mRNA and protein expression levels of CNN3 were lower in two NSCLC cell lines (A549 and SK-MES-1) than in a human bronchial epithelial cell line (BEAS-2B). CNN3 overexpression suppressed cell proliferation, migration and invasion, induced G1-phase arrest, promoted apoptosis and suppressed PI3K/AKT signaling pathway activation in the NSCLC cell lines, whereas CNN3 overexpression had no effect on cell morphology. In conclusion, CNN3 suppressed the proliferation and metastasis of NSCLC cells by downregulating the PI3K/AKT signaling pathway, making it a potential therapeutic target in this disease.

CircRNA CDR1as Promotes Pulmonary Artery Smooth Muscle Cell Calcification by Upregulating CAMK2D and CNN3 via Sponging miR-7-5p

Emerging evidence has suggested that circular RNAs (circRNAs) are involved in multiple physiological processes and participate in a variety of human diseases. However, the underlying biological function of circRNAs in pulmonary hypertension (PH) is still ambiguous. Herein, we investigated the implication and regulatory effect of a typical circRNA, CDR1as, in the pathological process of vascular calcification in PH. Human pulmonary artery smooth muscle cell (HPASMC) calcification was analyzed by western blotting, immunofluorescence, alizarin red S staining, alkaline phosphatase activity analysis, and calcium deposition quantification. CDR1as targets were identified by bioinformatics analysis and validated by dual-luciferase reporter and RNA antisense purification assays. We identified that CDR1as was upregulated in hypoxic conditions and promoted a phenotypic switch of HPASMCs from a contractile to an osteogenic phenotype. Moreover, microRNA (miR)-7-5p was shown to be a target of CDR1as, and calcium/calmodulin-dependent kinase II-delta (CAMK2D) and calponin 3 (CNN3) were suggested to be the putative target genes and regulated by CDR1as/miR-7-5p. The results showed that the CDR1as/miR-7-5p/CNN3 and CAMK2D regulatory axis mediates HPASMC osteoblastic differentiation and calcification induced by hypoxia. This evidence reveals an approach to the treatment of PH.

Calponin 3 is associated with poor prognosis and regulates proliferation and metastasis in osteosarcoma.

Osteosarcoma is a malignant, life-threatening tumor that affects children and adolescents. In this study, we identified high levels of calponin 3 (CNN3) protein in osteosarcoma tissues and cell lines. The receiver operating characteristic curve analysis revealed that CNN3 has diagnostic value for patients with osteosarcoma. We also found that high CNN3 expression was associated with tumor size, tumor stage, and lymph node and distant metastases. Moreover, high levels of CNN3 mRNA were associated with a poor overall survival rate and a shorter disease-free survival period. CNN3 silencing inhibited cell proliferation, induced apoptosis and cell cycle arrest at the G1 stage, and inhibited cell migration and invasion in vitro. Furthermore, CNN3 silencing also inhibited subcutaneous tumor growth and lung metastasis in vivo. Western blotting revealed that silencing of CNN3 resulted in downregulated expression of MMP9, VEGF, and vimentin, and upregulation of E-cadherin. CNN3 silencing also resulted in downregulation of the ERK1/2 and p38 signaling pathways. In conclusion, high CNN3 expression was found to help in the diagnosis of osteosarcoma, and was found to be associated with poor prognosis in patients. Therefore, CNN3 may play an oncogenic role during the progression of osteosarcoma by activating the ERK1/2 and p38 pathways.

CNN3 acts as a potential oncogene in cervical cancer by affecting RPLP1 mRNA expression

The prognosis of advanced stage cervical cancer is poorer due to cancer invasion and metastasis. Exploring new factors and signalling pathways associated with invasiveness and metastasis would help to identify new therapeutic targets for advanced cervical cancer. We searched the cancer microarray database, Oncomine, and found elevated calponin 3 (CNN3) mRNA expression in cervical cancer tissues. QRT-PCR verified the increased CNN3 expression in cervical cancer compared to para-cancer tissues. Proliferation, migration and invasion assays showed that overexpressed CNN3 promoted the viability and motility of cervical cancer cells, the opposite was observed in CNN3-knockdown cells. In addition, xenografted tumours, established from SiHa cells with CNN3 knockdown, displayed decreased growth and metastasis in vivo. Furthermore, RNA-sequencing showed that ribosomal protein lateral stalk subunit P1 (RPLP1) was a potential downstream gene. Gene function experiments revealed that RPLP1 had the same biological effects as CNN3 did. Rescue experiments demonstrated that the phenotypes inhibited by CNN3 silencing were partly or completely reversed by RPLP1 overexpression. In conclusion, we verified that CNN3 acts as an oncogene to promote the viability and motility of cervical cancer cells in vitro and accelerate the growth and metastasis of xenografted tumours in vivo, by affecting RPLP1 expression.

Calponin 3 promotes invasion and drug resistance of colon cancer cells.

BACKGROUNDCalponin 3 (CNN3) is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells. It is required for cytoskeletal rearrangement and wound healing.AIMTo dissect the role of CNN3 in carcinogenesis with a focus on colon cancer.METHODSA total of 20 cancer cell lines (8 breast, 11 colon, and HeLa cervical cancer cell as a positive control for mesenchymal phenotype) and 57 formalin-fixed, paraffin-embedded sections from archived sporadic colorectal carcinomas were included in this study. CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses. The CNN3 gene was silenced by specific small interfering RNA (commonly known as siRNA), followed by confirmation of the silencing efficiency by western blotting. Then, the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers, invasion, and response to 5-fluoruracil treatment. We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins.RESULTSCNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells. Interestingly, the colorectal adenocarcinoma line SW480 was negative, while the cell line developed from its matching lymph node metastasis (SW620) was positive for CNN3. CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others. We selected SW620 for subsequent functional analyses. CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers. CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil. Phospho-kinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase, β-Catenin, mutant p53, c-Jun, and heat shock protein 60 activities but increased that of checkpoint kinase 2. CNN3 was expressed in 20/57 (35%) colon cancer cases as shown by immunohistochemistry. CNN3 was associated with a decrease in overall survival in colon cancer in silico.CONCLUSIONThese results show the involvement of CNN3 in lymph node metastasis and resistance to chemotherapy in colon cancer and suggest that significant oncogenic pathways are involved in these CNN3-related actions.

Calponin 3 Regulates Cell Invasion and Doxorubicin Resistance in Gastric Cancer.

Calponin 3 (CNN3) is an F-actin-binding protein that regulates actin cytoskeletal rearrangement. However, the role of CNN3 in cancer cell invasion and resistance to chemotherapeutic agents has not yet been investigated. The present study was undertaken to investigate whether CNN3 influences cancer-related phenotypes in gastric cancer. We demonstrate that CNN3 contributes to cell invasion and resistance to doxorubicin in gastric cancer. CNN3 expression was markedly elevated in highly invasive cancer cell lines compared to less invasive or noninvasive cancer cell lines. Depletion of CNN3 protein suppressed the invasive ability of gastric cancer cells. The highly invasive MKN-28 gastric cancer cells were more resistant to doxorubicin than the noninvasive MKN-45 cells; however, knockdown of CNN3 expression in MKN-28 cells resensitized them to doxorubicin treatment. Taken together, our results suggest that CNN3 plays a key role in invasiveness and doxorubicin resistance in gastric cancer cells.

PO-206 Calponin 3 (CNN3) promotes epithelial to mesenchymal transition and drug resistance of colon cancer cells

IntroductionCalponin 3 (CNN3) is one of the three isoforms in the Calponin family of actin-binding proteins. It is expressed in smooth muscle cells and non-smooth muscle cells and is required for cytoskeletal rearrangement and wound healing. Epithelial to Mesenchymal Transition (EMT) is important step in the development of epithelial cancers during which the cancer cell exploit the preexisting wound healing program. Therefore, it is expected that CNN3 plays a role in EMT. The few available data on the role of CNN3 in cancer shows that CNN3 is expressed in mammary cells in response to ErbB2-overexpression and in Duke’s stage C of colon cancer, but there is no comprehensive analysis of its role in cancer which is the aim of this study.Material and methodsTotal of 21 cell lines were included in this study (8 from breast cancer, 12 from colon cancer, and a cervical cancer cell (HeLa) as a positive control for EMT). CNN3 expression was examined by Western blotting, followed by siRNA silencing in the metastatic cell line SW620. The influence of CNN3 silencing on EMT was detected by EMT markers expression by Western blot and collagen invasion assay. Cell viability was performed after exposure to 5 Fluorouracil (5FU) using Sulforhodamine B (SRB) assay. Furthermore CNN3 expression was examined by immunohistochemistry in 56 formalin-fixed paraffin-embedded (FFPE) colon cancer tissue samples.Results and discussionsCNN3 showed positive expression in 6/8 breast lines and 7/12 colon lines. The primary line SW480 was negative while its metastasis from the same patient (SW620) was positive, suggesting that CNN3 was associated with metastasis. The CNN3 silencing was >98% efficient in the metastatic cell line SW620. Silenced cells were less invasive compared to the control and showed loss of mesenchymal markers and acquisition of epithelial markers. CNN3 partially restored the colon cancer cell sensitivity to 5FU. CNN3 was expressed in 20/56 (39%) of FFPE colon cancer by immunohistochemistry and it was not related to p53 stabilisation consistent with our cell line findings.ConclusionThe results suggested involvement of CNN3 in EMT and hence metastasis and also in resistance to standard chemotherapy in colon cancer. These data deserves further exploration in vivo and in clinical studies to validate the potential clinical applications of CNN3 in cancer treatment and prognosis.

Proteomic Screen of Brain Glycoproteome Reveals Prion Specific Marker of Pathogenesis.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by the presence of an infectious prion protein. The primary site of pathology is the brain characterized by neuroinflammation, astrogliosis, prion fibrils, and vacuolation. The events preceding the observed pathology remain in question. We sought to identify biomarkers in the brain of TSE-infected and aged-matched control mice using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Since the brain proteome is too complex to resolve all proteins using 2D-DIGE, protein samples are initially filtered through either concanavalin A (ConA) or wheat-germ agglutinin (WGA) columns. Four differentially abundant proteins are identified through screening of the two different glycoproteomes: Neuronal growth regulator 1 (NEGR1), calponin-3 (CNN3), peroxiredoxin-6 (Prdx6), and glial fibrillary acidic protein (GFAP). Confirmatory Western blots are performed with samples from TSE-infected and comparative Alzheimer's disease (AD) affected brains and their respective controls from time points throughout the disease courses. The abundance of three of the four proteins increases significantly during later stages of prion disease whereas NEGR1 decreases in abundance. Comparatively, no significant changes are observed in later stages of AD. Our lab is the first to associate the glycosylated NEGR1 protein with prion disease pathology.

Overexpressed Calponin3 by Subsonic Vibration Induces Neural Differentiation of hUC-MSCs by Regulating the Ionotropic Glutamate Receptor.

In this study, we used proteomics to investigate the effects of sonic vibration (SV) on mesenchymal stem cells derived from human umbilical cords (hUC-MSCs) during neural differentiation to understand how SV enhances neural differentiation of hUC-MSCs. We investigated the levels of gene and protein related to neural differentiation after 3 or 5 days in a group treated with 40-Hz SV. In addition, protein expression patterns were compared between the control and the 40-Hz SV-treated hUC-MSC groups via a proteomic approach. Among these proteins, calponin3 (CNN3) was confirmed to have 299 % higher expression in the 40-Hz SV stimulated hUC-MSCs group than that in the control by Western blotting. Notably, overexpression of CNN3-GFP in Chinese hamster ovary (CHO)-K1 cells had positive effects on the stability and reorganization of F-actin compared with that in GFP-transfected cells. Moreover, CNN3 changed the morphology of the cells by making a neurite-like form. After being subjected to SV, messenger RNA (mRNA) levels of glutamate receptors such as PSD95, GluR1, and NR1 as well as intracellular calcium levels were upregulated. These results suggest that the activity of glutamate receptors increased because of CNN3 characteristics. Taken together, these results demonstrate that overexpressed CNN3 during SV increases expression of glutamate receptors and promotes functional neural differentiation of hUC-MSCs.

CNN3 Is Regulated by microRNA-1 during Muscle Development in Pigs

The calponin 3 (CNN3) gene has important functions involved in skeletal muscle development. MicroRNAs (miRNAs) play critical role in myogenesis by influencing the mRNA stability or protein translation of target gene. Based on paired microRNA and mRNA profiling in the prenatal skeletal muscle of pigs, our previous study suggested that CNN3 was differentially expressed and a potential target for miR-1. To further understand the biological function and regulation mechanism of CNN3, we performed co-expression analysis of CNN3 and miR-1 in developmental skeletal muscle tissues (16 stages) from Tongcheng (a Chinese domestic breed, obese-type) and Landrace (a Western, lean-type) pigs, respectively. Subsequently, dual luciferase and western blot assays were carried out. During skeletal muscle development, we observe a significantly negative expression correlation between the miR-1 and CNN3 at mRNA level. Our dual luciferase and western blot results suggested that the CNN3 gene was regulated by miR-1. We identified four single nucleotide polymorphisms (SNPs) contained within the CNN3 gene. Association analysis indicated that these CNN3 SNPs are significantly associated with birth weight (BW) and the 21-day weaning weight of the piglets examined. These facts indicate that CNN3 is a candidate gene associated with growth traits and regulated by miR-1 during skeletal muscle development in pigs.

Calponin 3 regulates stress fiber formation in dermal fibroblasts during wound healing

Skin wound healing is an intricate process involving various cell types and molecules. In granulation tissue, fibroblasts proliferate and differentiate into myofibroblasts and generate mechanical tension for wound closure and contraction. Actin stress fibers formed in these cells, especially those containing α-smooth muscle actin (α-SMA), are the central machinery for contractile force generation. In the present study, calponin 3 (CNN3), which has a phosphorylation-dependent actin-binding property, was identified in the molecular mechanism underlying stress fiber formation. CNN3 was expressed by fibroblasts/myofibroblasts in the proliferation phase of wound healing, and was associated with α-SMA in stress fibers formed by cultured dermal fibroblasts. CNN3 expression was post-transcriptionally regulated by tension, as demonstrated by disruption of actin filament organization under floating culture or blebbistatin treatment. CNN3 knockdown in primary fibroblasts impaired stress fiber formation, resulting in a phenotype of decreased cellular dynamics such as cell motility and contractile ability. These findings indicate that CNN3 participates in actin stress fiber remodeling, which is required for cell motility and contraction of dermal fibroblasts in the wound healing process.

Rock-dependent calponin 3 phosphorylation regulates myoblast fusion

Myogenesis occurs during embryonic development as well as regeneration following postnatal muscle fiber damage. Herein, we show that acidic calponin or calponin 3 (CNN3) regulates both myoblast cell fusion and muscle-specific gene expressions. Overexpression of CNN3 impaired C2C12 cell fusion, whereas CNN3 gene knockdown promoted skeletal myosin expression and fusion. CNN3 was phosphorylated at Ser293/296 in the C-terminal region. The basal inhibitory property of CNN3 against myoblast differentiation was enhanced by Ser293/296Ala mutation or deletion of the C-terminal region, and this inhibition was reversed by Ser293/296Asp mutation. Ser293/296 phosphorylation was required for CNN3 to bind actin and was dependent on Rho-associated kinases 1/2 (ROCK 1/2). Gene knockdown of ROCK1/2 suppressed CNN3 phosphorylation and impaired myoblast fusion, and these effects were partially attenuated by additional CNN3 overexpression of Ser293/296Asp CNN3. These findings indicated that CNN3 phosphorylation by ROCK blunts CNN3's inhibitory effects on muscle cell differentiation and fusion. In muscle tissues, satellite cells, but not mature myofibrils, expressed CNN3. CNN3 was also expressed and phosphorylated during myotube induction in isolated muscle satellite cells. Taken together, these results indicate that CNN3 is a downstream regulator of the ROCK signaling pathway for myogenesis.

Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

Cell-cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.