Caldesmon Research Articles

CALD1 facilitates epithelial-mesenchymal transition progression in gastric cancer cells by modulating the PI3K-Akt pathway.

CALD1 has been discovered to be abnormally expressed in a variety of malignant tumors, including gastric cancer (GC), and is associated with tumor progression and immune infiltration; however, the roles and mechanisms of CALD1 in epithelial-mesenchymal transition (EMT) in GC are unknown. To investigate the role and mechanism of CALD1 in GC progression, invasion, and migration. In this study, the relationship between CALD1 and GC, as well as the possible network regulatory mechanisms of CALD1, was investigated by bioinformatics and validated by experiments. CALD1-siRNA was synthesized and used to transfect GC cells. Cell activity was measured using the CCK-8 method, cell migration and invasive ability were measured using wound healing assay and Transwell assay, and the expression levels of relevant genes and proteins in each group of cells were measured using qRT-PCR and Western blot. A GC cell xenograft model was established to verify the results of in vitro experiments. Bioinformatics results showed that CALD1 was highly expressed in GC tissues, and CALD1 was significantly higher in EMT-type GC tissues than in tissues of other types of GC. The prognosis of patients with high expression of CALD1 was worse than that of patients with low expression, and a prognostic model was constructed and evaluated. The experimental results were consistent with the results of the bioinformatics analysis. The expression level of CALD1 in GC cell lines was all higher than that in gastric epithelial cell line GES-1, with the strongest expression found in AGS and MKN45 cells. Cell activity was significantly reduced after CALD1-siRNA transfection of AGS and MKN45 cells. The ability of AGS and MKN45 cells to migrate and invade was reduced after CALD1-siRNA transfection, and the related mRNA and protein expression was altered. According to bioinformatics findings in GC samples, the CALD1 gene was significantly associated with the expression of members of the PI3K-AKT-mTOR signaling pathway as well as the EMT signaling pathway, and was closely related to the PI3K-Akt signaling pathway. Experimental validation revealed that upregulation of CALD1 increased the expression of PI3K, p-AKT, and p-mTOR, members of the PI3K-Akt pathway,while decreasing the expression of PTEN; PI3K-Akt inhibitor treatment decreased the expression of PI3K, p-AKT, and p-mTOR in cells overexpressing CALD1 (still higher than that in the normal group), but increased the expression of PTEN (still lower than that in the normal group). CCK-8 results revealed that the effect of CALD1 on tumor cell activity was decreased by the addition of the inhibitor. Scratch and Transwell experiments showed that the effect of CALD1 on tumor cell migration and invasion was weakened by the addition of the PI3K-Akt inhibitor. The mRNA and protein levels of EMT-related genes in AGS and MKN45 cells were greatly altered by the overexpression of CALD1, whereas the effect of overexpression of CALD1 was significantly weakened by the addition of the PI3K-Akt inhibitor. Animal experiments showed that tumour growth was slow after inhibition of CALD1, and the expression of some PI3K-Akt and EMT pathway proteins was altered. Increased expression of CALD1 is a key factor in the progression, invasion, and metastasis of GC, which may be associated with regulating the PI3K-Akt pathway to promote EMT.

Glucocorticoid receptor-induced non-muscle caldesmon regulates metastasis in castration-resistant prostate cancer

Lethal prostate cancer (PCa) is characterized by the presence of metastases and development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. Here, we report that in vitro downregulation of l-CaD promotes epithelial phenotype and reduces spheroid growth in 3D, which is reflected in vivo in reduced formation of metastases in zebrafish PCa xenografts. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition (EMT) transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets, and GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation.

Abstract A024: Glucocorticoid receptor-induced non-muscle caldesmon regulates growth and metastasis in castration-resistant prostate cancer

Abstract Lethal prostate cancer (PCa) is characterized by the presence of metastases and the development of resistance to therapies. Metastases form in a multi-step process enabled by dynamic cytoskeleton remodeling. An actin cytoskeleton regulating gene, CALD1, encodes a protein caldesmon (CaD). Its isoform, low-molecular-weight CaD (l-CaD), operates in non-muscle cells, supporting the function of filaments involved in force production and mechanosensing. Several factors, including glucocorticoid receptor (GR), have been identified as regulators of l-CaD in different cell types, but the regulation of l-CaD in PCa has not been defined. PCa develops resistance in response to therapeutic inhibition of androgen signaling by multiple strategies. Known strategies include androgen receptor (AR) alterations, modified steroid synthesis, and bypassing AR signaling, for example, by GR upregulation. The goal of our study was to characterize the potential role of l-CaD in PCa metastases and antiandrogen therapy resistance. In this study, we extracted co-expression data of l-CaD from the largest public PCa patient data sets and identified the common transcripts between the sets. The common co-expression hits were used to recognize biological processes associated with l-CaD in the PCa context. Next, we used in vitro techniques, including 3D culture, cell viability assays, immunofluorescence staining, and Western blotting, to study the role of l-CaD in PCa with a focus on cancer hallmarks and the recognized associated biological processes. Finally, we used in vivo zebrafish and mouse xenograft models to verify the findings observed in silico in PCa patient data and in vitro in PC3, DU145, and VCaP cell lines. Here, we report that in vitro downregulation of l-CaD promotes the epithelial phenotype and reduces the spheroid growth in 3D, which is reflected in vivo in the reduced formation of metastases in a zebrafish PCa xenograft model. In accordance, CALD1 mRNA expression correlates with epithelial-to-mesenchymal transition transcripts in PCa patients. We also show that CALD1 is highly co-expressed with GR in multiple PCa data sets and that GR activation upregulates l-CaD in vitro. Moreover, GR upregulation associates with increased l-CaD expression after the development of resistance to antiandrogen therapy in PCa xenograft mouse models. In summary, GR-regulated l-CaD plays a role in forming PCa metastases, being clinically relevant when antiandrogen resistance is attained by the means of bypassing AR signaling by GR upregulation. Citation Format: Verneri Virtanen, Kreetta Paunu, Antti Kukkula, Saana Niva, Ylva Junila, Mervi Toriseva, Terhi Jokilehto, Sari Mäkelä, Riikka Huhtaniemi, Matti Poutanen, Ilkka Paatero, Maria Sundvall. Glucocorticoid receptor-induced non-muscle caldesmon regulates growth and metastasis in castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A024.

Immunohistochemical Expression Analysis of Caldesmon Isoforms in Colorectal Carcinoma Reveals Interesting Correlations with Tumor Characteristics.

Colorectal cancer is a notorious disease, with almost half of the patients succumbing to the disease. The prevalence and incidence rates of colorectal cancer are increasing in many parts of the world, highlighting the need to discover new biomarkers for diagnosis and therapy. Caldesmon (CaD), an actin-binding protein that plays a significant role in controlling cell motility, has emerged as a promising biomarker. The CALD1 gene encodes CaD as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight (h-CaD), expressed in smooth muscle, and low-molecular-weight (l-CaD), expressed in nonsmooth muscle cells. Most studies have suggested an oncogenic role of CaD in colorectal cancer, but the exact subcellular localization of the two CaD isoforms in tumor cells and stroma have not been clarified yet. Here, we analyzed tissue samples from 262 colorectal cancer patients by immunohistochemistry analysis using specific antibodies for l-CaD and h-CaD. The results showed elevated cytoplasmic expression levels of l-Cad in 187/262 (71.4%) cases. l-Cad was expressed at low levels in the normal colon mucosa and was also consistently expressed in the cancer-associated stroma of all cases, suggesting that it could play a role in modulating the tumor microenvironment. l-CaD expression in cancer cells was associated with preinvasive stages of cancer. Survival analysis indicated that patients with high l-CaD expression in tumor cells could respond poorly to selective chemotherapeutic 5FU, but not combination chemotherapy. h-CaD was expressed in colonic and vascular smooth muscle cells as expected and to a lesser extent in the tumor-associated stroma, but it was not expressed in the cancer cells or normal colon mucosal epithelial cells. Collectively, these data clarify how the expression patterns of CaD isoforms in colorectal cancer can have applications in the management of colorectal cancer patients.

Emerging role of caldesmon in cancer: A potential biomarker for colorectal cancer and other cancers.

Colorectal cancer (CRC) is a devastating disease, mainly because of metastasis. As a result, there is a need to better understand the molecular basis of invasion and metastasis and to identify new biomarkers and therapeutic targets to aid in managing these tumors. The actin cytoskeleton and actin-binding proteins are known to play an important role in the process of cancer metastasis because they control and execute essential steps in cell motility and contractility as well as cell division. Caldesmon (CaD) is an actin-binding protein encoded by the CALD1 gene as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight CaD, expressed in smooth muscle, and low-molecular weight CaD (l-CaD), expressed in nonsmooth muscle cells. According to our comprehensive review of the literature, CaD, particularly l-CaD, plays a key role in the development, metastasis, and resistance to chemoradiotherapy in colorectal, breast, and urinary bladder cancers and gliomas, among other malignancies. CaD is involved in many aspects of the carcinogenic hallmarks, including epithelial mesenchymal transition via transforming growth factor-beta signaling, angiogenesis, resistance to hormonal therapy, and immune evasion. Recent data show that CaD is expressed in tumor cells as well as in stromal cells, such as cancer-associated fibroblasts, where it modulates the tumor microenvironment to favor the tumor. Interestingly, CaD undergoes selective tumor-specific splicing, and the resulting isoforms are generally not expressed in normal tissues, making these transcripts ideal targets for drug design. In this review, we will analyze these features of CaD with a focus on CRC and show how the currently available data qualify CaD as a potential candidate for targeted therapy in addition to its role in the diagnosis and prognosis of cancer.

Upon addition of myosin light chain phosphatase, caldesmon creates a load on actin filaments propelled by a mixture of smooth and skeletal muscle myosin

Several studies have suggested that actin-binding proteins are involved in smooth muscle force maintenance at low myosin light chain (LC20) phosphorylation levels. To better understand the role of caldesmon (CaD) in smooth muscle molecular mechanics, we injected myosin light chain phosphatase (MLCP) during in vitro motility assays performed with a mixture of 50% smooth muscle myosin and 50% skeletal muscle myosin, with and without CaD. We hypothesized that upon dephosphorylation, smooth muscle myosin would detach from actin and the velocity would increase towards that of skeletal myosin.

MIR100HG Regulates CALD1 Gene Expression by Targeting miR-142-5p to Affect the Progression of Bladder Cancer Cells in vitro, as Revealed by Transcriptome Sequencing.

Background/Aim: The role of long non-coding RNA (lncRNA) and competing endogenous RNAs (ceRNA) networks in bladder cancer, especially the function of lncRNA-miRNA-mRNA regulatory network in bladder cancer, are still relatively poorly understood. This research mainly used transcriptome sequencing to screen key lncRNAs and ceRNAs, explore their pathogenic mechanism in bladder cancer, and search for potential diagnostic and therapeutic targets. Methods: High-throughput transcriptome sequencing, combined with the limma package, Kaplan-Meier curve analysis, lncRNA-mRNA coexpression network, univariate Cox analysis, multivariate Cox analysis, protein-protein interaction (PPI), functional enrichment, weighed gene co-expression network analysis (WGCNA), ceRNA network and quantitative PCR (qPCR) analyses were performed to assess and screen differentially expressed lncRNAs and mRNAs. Then, the effects of MIR100HG on the proliferation, migration and invasion of the bladder cancer cell line 5,637 were evaluated using cell counting kit-8(CCK-8), wound-healing and transwell assays, respectively. A dual luciferase reporter assay was used to validate the MIR100HG/miR-142-5p and miR-142-5p/CALD1 targeting relationship, and the regulatory relationship among MIR100HG/miR-142-5p/CALD1 expression was explored using qPCR and western blot. Results: A total of 127 differentially expressed lncRNAs and 620 differentially expressed mRNAs were screened. Based on the survival prognosis analysis, Cox analysis, lncRNA-mRNA network, PPI network and WGCNA, we obtained 3 key lncRNAs and 13 key mRNAs, as well as the MIR100HG/miR-142-5p/CALD1 key regulatory axis. qPCR results showed that compared with the adjacent tissues, the expression of MIR100HG and CALD1 was up-regulated, and the expression of miR-142-5p was down-regulated. Moreover, MIR100HG expression was positively correlated with the tumor grade and clinical grade of patients with bladder cancer. Overexpression of MIR100HG effectively promoted the proliferation, migration and invasion of 5,637 cells, inhibited the expression of miR-142-5p, and induced the expression of CALD1 in 5,637 cells. In addition, miR-142-5p inhibited CALD1 expression in bladder cancer cells through a direct association, and reversed the proliferation and CALD1 expression in 5,637 cells overexpressing of MIR100HG. Conclusion: MIR100HG regulates CALD1 expression by targeting miR-142-5p to inhibit the proliferation, migration and invasion of bladder cancer cells. MIR100HG is an independent prognostic factor for bladder cancer, with potential as a biomarker for the diagnosis and treatment of bladder cancer.

Caldesmon ablation in mice causes umbilical herniation and alters contractility of fetal urinary bladder smooth muscle.

The actin-, myosin-, and calmodulin-binding protein caldesmon (CaD) is expressed in two splice isoforms: h-CaD, which is an integral part of the actomyosin domain of smooth muscle cells, and l-CaD, which is widely expressed and is involved in many cellular functions. Despite extensive research for many years, CaD's in vivo function has remained elusive. To explore the role of CaD in smooth muscle contraction in vivo, we generated a mutant allele that ablates both isoforms. Heterozygous animals were viable and had a normal life span, but homozygous mutants died perinatally, likely because of a persistent umbilical hernia. The herniation was associated with hypoplastic and dysmorphic abdominal wall muscles. We assessed mechanical parameters in isometrically mounted longitudinal strips of E18.5 urinary bladders and in ring preparations from abdominal aorta using wire myography. Ca2+ sensitivity was higher and relaxation rate was slower in Cald1-/- compared with Cald1+/+ skinned bladder strips. However, we observed no change in the content and phosphorylation of regulatory proteins of the contractile apparatus and myosin isoforms known to affect these contractile parameters. Intact fibers showed no difference in actin and myosin content, regardless of genotype, although KCl-induced force tended to be lower in homozygous and higher in heterozygous mutants than in WTs. Conversely, in skinned fibers, myosin content and maximal force were significantly lower in Cald1-/- than in WTs. In KO abdominal aortas, resting and U46619 elicited force were lower than in WTs. Our results are consistent with the notion that CaD impacts smooth muscle function dually by (1) acting as a molecular brake on contraction and (2) maintaining the structural integrity of the contractile machinery. Most importantly, CaD is essential for resolution of the physiological umbilical hernia and ventral body wall closure.

FokI-dCas9 mediates high-fidelity genome editing in pigs.

Gene editing using clustered regularly interspaced short palindromic repeats/Cas9 has great potential for improving the compatibility of porcine organs with human recipients. However, the risk of detrimental off-target mutations in gene-edited pigs remains largely undefined. We have previously generated GGTA1 knock-in pigs for xenotransplantation using FokI-dCas9, a variant of Cas9 that is reported to reduce the frequency of off-target mutagenesis. In this study, we used whole genome sequencing (WGS) and optimized bioinformatic analysis to assess the fidelity of FokI-dCas9 editing in the generation of these pigs. Genomic DNA was isolated from porcine cells before and after gene editing and sequenced by WGS. The genomic sequences were analyzed using GRIDSS variant-calling software to detect putative structural variations (SVs), which were validated by PCR of DNA from knock-in and wild-type pigs. Platypus variant-calling software was used to detect single-nucleotide variations (SNVs) and small insertions/deletions (indels). GRIDSS analysis confirmed the precise integration of one copy of the knock-in construct in the gene-edited cells. Three additional SVs were detected by GRIDSS: deletions in intergenic regions in chromosome 6 and the X chromosome and a duplication of part of the CALD1 gene on chromosome 18. These mutations were not associated with plausible off-target sites, and were not detected in a second line of knock-in pigs generated using the same pair of guide RNAs, suggesting that they were the result of background mutation rather than off-target activity. Platypus identified 1375 SNVs/indels after quality filtering, but none of these were located in proximity to potential off-target sites, indicating that they were probably also spontaneous mutations. This is the first WGS analysis of pigs generated from FokI-dCas9-edited cells. Our results demonstrate that FokI-dCas9 is capable of high-fidelity gene editing with negligible off-target or undesired on-target mutagenesis.

Caldesmon Expression in Metastatic and Non-Metastatic Oral Squamous Cell Carcinoma—A Mediator of Epithelial Mesenchymal Transition

Introduction: For the past decades long-term survival of oral squamous cell carcinoma (OSCC) patients remain unchanged which provides a wide platform for research. Caldesmon (CaD), an actin-myosin binding protein, has a contributing role in cytoskeleton modulation and cell motility. The aim of this study is to evaluate the expression of CaD in metastatic and non-metastatic OSCC and to discuss the possible role of CaD in epithelial mesenchymal transition. Materials and Methods: Archival blocks of 25 metastatic and 25 non-metastatic OSCC patients are included where CaD expression is evaluated immunohistochemically. Results: Overall expression and staining intensity of CaD were statistically significant in different grades of metastatic and non-metastatic OSCC. In summary, high expression of CaD is observed in increasing grades of OSCC but the metastatic potential of the tumour doesn’t seem to have any relation with CaD.

Neuropathy optic glaucomatosa induced by systemic hypertension through activation endothelin-1 signaling pathway in central retinal artery in rats.

To evaluate effect of hypertension on retinal ganglion cell (RGC) apoptosis, intraocular pressure (IOP), and the activation of endothelin-1 (ET-1) signaling pathway in central retinal artery (CRA) in rats. The experimental study was performed on 20 male Sprague Dawley rats that were divided into control group, and hypertension groups. The hypertension was induced by subcutaneous deoxycorticoacetate (DOCA) 10 mg/kg twice a week and administered 0.9% NaCl solution daily for 2, 6, and 10wk. Blood pressure (BP) was measured using animal BP analyzer. IOP was measured by handheld tonometry. Retinal tissue preparations by paraffin blocks were made after enucleation. The expression of ET-1, eNOS, ET-1 receptor A (ETRA), ET-1 receptor B (ETRB), and phosphorylated myosin light chain kinase (MLCK), and caldesmon (CaD) in CRA and RGC apoptosis were evaluated through immunofluorescent staining method then observed using laser scanning confocal microscopy. BP significantly increased in all of the hypertension groups compared to control (P=0.001). Peak IOP elevation (7.78±4.14 mm Hg) and RGC apoptosis (576.15±33.28 Au) occurred on 2wk of hypertension. ET-1 expression (1238.6±55.1 Au) and eNOS expression (2814.2±70.7 Au) were found highest in 2wk of hypertension, although the ratio of ET-1/eNOS decreased since 2wk. ETRA reached peak expression in 10wk of hypertension (1219.4±6.3 Au), while ETRB significantly increased only in 2 weeks group (1069.2±9.6 Au). The highest MLCK expression (1190.09±58.32 Au), CaD (1670.28±18.36 Au) were also found in 2wk of hypertension. Hypertension effects to activation of ET-1 signaling pathway significantly in CRA, elevation of IOP, and RGC apoptosis. The highest value was achieved at 2wk, which is the development phase of hypertension.

The Influence of Caldesmon Suppression on Proliferation and Motilities of Vascular Smooth Muscle Cells

Migration of vascular smooth muscle cells (VSMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of VSMCs motility, the roles of caldesmon (CaD) were investigated previously. CaD is an actin-binding protein dynamically regulating cytoskeleton structure. In this study, the potential role of CaD in mediating proliferation and motility of VSMCs were discussed. First, structural effect of CaD on cytoskeleton integrity was analyzed with CaD knock-down; second, the proliferation of VSMCs was measured in CaD knock-down and control cells; third, the specific role of CaD on VSMCs motilities was evaluated with in vitro migration and invasion assays. We found that CaD is an integral component to maintain cytoskeleton integrity of VSMCs. Our data indicated that CaD suppression does not show significant influence on VSMCs proliferation, but negatively modulates the motilities of VSMCs, and CaD depletion would significantly facilitate migration and invasion of VSMCs.

Therapeutic Potential of Beta-Nicotinamide Adenine Dinucleotide to Reverse Endothelial Barrier Dysfunction in Sickle Cell Mouse Model

Background: Thepulmonary endothelium is a metabolically active monolayer on the luminal surface of the lung vasculature. It regulates a variety of functions including the passage of macromolecules and fluid between the blood and lung interstitial tissue. Inflammatory agents such as bacterial endotoxin lipopolysaccharide (LPS) cause lung endothelial cells (EC) dysfunction leading to pulmonary edema predisposing to acute chest syndrome, a leading cause of death in sickle cell disease (SCD); therefore, a drug which repairs the damaged EC barrier function might be of therapeutic potential. Recently we demonstrated that β-nicotinamide adenine dinucleotide (β-NAD) reverses LPS-induced EC barrier dysfunction in a model of acute lung injury (Exp Lung Res 2012, 38, 223-32). In this study, we investigated the protective effect of β-NAD against the damaging effects of LPS using primary ECs from human and sickle cell mouse lungs as well as survival studies using the preclinical sickle cell mouse model.Methods: Early passaged human lung microvascular EC (HLMVEC) were obtained from Lonza and mouse lung microvascular EC (MLMVEC) were isolated from the Townes knock-in homozygous sickle cell (SS) and heterozygous sickle trait (AS) transgenic mice. The EC barrier function was measured using electrical cell-substrate impedance sensing (ECIS) technique. Western blot was done to measure phosphorylation of proteins involved in the regulation of EC barrier function. Survival studies were performed in SS mice treated with intraperitoneal (IP) LPS (0.05 mg/kg) alone or combined with β-NAD (11 mg/kg); normal saline served as a control. Lungs were harvested at 10-12 hrs from the LPS group and at 24 hrs from saline or LPS/β-NAD groups and stained with Hematoxylin and Eosin for to assess interstitial edema and myeloperoxidase as a marker of neutrophil infiltration.Results: Barrier function measurements using ECIS of HLMVEC indicate that β-NAD (100 µM) enhanced basal EC barrier function by 50-60%. In addition, β-NAD attenuated LPS-induced EC gap formation and actin stress fibers. In human lung endothelium, myosin light chain phosphatase (MLCP) plays a crucial role in the maintenance of the EC barrier integrity. Furthermore, LPS mediates MLCP inactivation by phosphorylating the MLCP regulatory subunit MYPT1 and cytoskeletal targets ERM (Ezrin, Radixin, and Moesin) and CaD (Caldesmon). Western blot analysis demonstrated that β-NAD reversed LPS-induced phosphorylation of MYPT1, ERM and CaD proteins. To determine a functional role, we performed studies using 10 nM MYPT1 siRNA treatment followed by β-NAD (100 μM) for 5 hrs. We observed a 50% attenuation of β-NAD enhanced barrier function suggesting MYPT1 is involved in the mechanism of action for β-NAD. Subsequent studies with SS-MLMVEC demonstrated β-NAD mediated a 40% improvement in barrier function which was comparable to HLMVEC. Further, LPS treatment for 16 hrs produced a 25% decrease in barrier function in SS-MLMVEC compared to a 7% decrease in AS-MLMVEC suggesting impaired EC barrier function in SS lungs. Finally we performed survival studies to determine the ability of β-NAD to protect against LPS-induced mortality in SS mice. In dose optimization studies of LPS (0.1-1.0 mg/kg/dose), SS mice survived for 10-12 hrs with the 0.05 mg/kg LPS dose. By contrary, all LPS doses tested had no effect on AS mice survival. Based on this result, SS mice were given IP injections of saline (3 mice), LPS (4 mice) or LPS/β-NAD (4 mice) and survival rates were monitored. The saline or LPS/β-NAD treated mice survived for 24 hrs (100%) compared with LPS mice which died by 10-12 hrs (p=0.01) supporting a protective effect of β-NAD. Histological assessment of the lung tissue showed increased interstitial edema by Hematoxylin and Eosin stain and neutrophil infiltration by myeloperoxidase in the LPS-treated lungs; these changes were much less prominent in the lungs from LPS/β-NAD treated SS mice.Conclusions: Our data support a role of MLCP and its cytoskeletal targets in the protective effects of β-NAD against LPS-induced HLMVEC barrier dysfunction. Studies using SS-MLMVEC suggest that SS mice have impaired barrier function and greater susceptibility to LPS toxicity. However, β-NAD treatment improved survival of SS mice supporting the therapeutic potential of this agent to treat the complication of acute chest syndrome in SCD. DisclosuresMakala:Georgia Regents University: Employment.

Primary culture and characteristics of colorectal cancer-associated fibroblasts

To compare the biological characteristics of colorectal cancer associated fibroblasts (CAFs) with normal fibroblasts (NFs). CAFs and NFs were isolated from fresh specimens of colorectal cancer and their paired normal colon tissue and cultured by tissue explant method. Light microscopy, quantitative polymerase chain reaction (qPCR), Western blot, immunofluorescence microscopy, electron microscopy and flow cytometry were used to identify isolated fibroblasts and to explore their characteristics of activation and growth. Primary colorectal CAFs and NFs were isolated and cultured successfully. NFs showed spindled morphology and were arranged in interlacing or spiral bundles. CAFs were polygonal or spindle, but were fatter than NFs. They were distributed randomly and arranged irregularly, and had obvious actin expression. CAFs and NFs both expressed fibronectin, but not E-cadherin, CD31 and caldesmon. qPCR showed that CAFs expressed more fibroblast activation protein (FAP) and less fibroblast specific protein 1 (FSP1) than that of NFs. There was no difference in the expression of α-SMA between NFs and CAFs by Western blot. α-SMA was bundled in parallel to the long axis of the cell by immunofluorescence. By electron microscopy, CAFs but not NFs showed dense myofilament that was arranged regularly. Flow cytometry showed that the percentage of S- and G2-phase in CAFs were significantly lower than that in NFs. mRNA expression of transforming growth factor β1, stromal derived factor 1 (SDF-1) and platelet derived growth factor (PDGF)-D in CAFs were lower while that for PDGFC was higher than that in NFs. That indicated the proliferation of CAFs was inhibited and the secretion of some cytokines was different when compared with NFs. CAFs show differences with NFs in morphology, characteristics of activation and secretion of some cytokines. The proliferation of CAFs is down regulated as compared with NFs.

Overexpression of caldesmon is associated with tumor progression in patients with primary non-muscle-invasive bladder cancer.

The expression and function of caldesmon (CAD) in urothelial bladder carcinoma (BC) have not been reported. Here, we investigated the expression, prognostic value, and potential functional mechanism of CAD in primary non-muscle-invasive bladder cancer (NMIBC). Protein profiling of tissue samples using antibody microarrays showed significantly higher CAD expression in muscle-invasive BC tissues compared with NMIBC tissues. We then validated the CAD expression in BC cells by immunohistochemistry analysis using paraffin-embedded tissue blocks and western blots using BC cell lines. In addition, we examined the expression of CAD variants by reverse transcription-polymerase chain reaction, and confirmed the expression of low-molecular-weight isoforms (L-CAD), specifically encoded by WI-38 L-CAD II (transcript variant 2), in BC cells. Survival analysis in an independent primary NMIBC cohort comprising 132 patients showed that positive CAD expression was significantly associated with poorer prognosis than no CAD expression with regard to recurrence- and progression-free survival (p = 0.001 and 0.014, respectively). Multivariate analyses further indicated that positive CAD expression was an independent predictor of progression-free survival (p = 0.032; HR = 5.983). Data obtained from in vitro silencing and overexpression studies indicated that L-CAD promotes migration and invasiveness of BC cells. Immunofluorescence assays showed dramatic structural changes in the actin cytoskeleton of BC cells after L-CAD overexpression. Our findings collectively suggest that L-CAD overexpression in primary NMIBC is significantly associated with tumor progression and that a possible mechanism for L-CAD's activity is implicated in increased cell motility and invasive characteristics through morphological changes in BC cells.

The Effect of Hypertension on Intraocular Pressure and Apoptosis of Retinal Ganglion Cell Through ET‐1 Signaling Pathway Activation in Trabecular Meshwork of Hypertension Rat Model

PurposeTo evaluate the effect of Deoxycorticoacetate (DOCA)‐salt Hypertension model on IOP,retinal ganglion cell (RGC) apoptosis, ratio of endothelin (ET‐1)/ endothelial Nitric Oxide Synthase (eNOS), ETA and ETB Receptor (ETRA and ETRB), Myosin Light Chain Kinase (MLCK), and Caldesmon (CaD) in endothelial cells of Trabecular Meshwork (TM).MethodsExperimental study was performed on 20 male Spraque Dawley rats divided into control group(1), hypertension group (2–4): DOCA subcutaneous 10 mg/kg BW twice a week+ NaCl 0.9% daily for 2, 6, and 10 weeks respectively. Blood pressure were measured by BP analyzer with animal tail‐cuff method and IOP measured by handheld tonometry before study and before enucleation. ET‐1 signaling pathway and RGC apoptosis were evaluated by immunofluorescent staining, then observed by laser scanning confocal microscopy. Data were analyzed by one way Anova.ResultsPeak of IOP elevation occurred on 2 weeks of hypertension (7.78 ± 4.14 mmHg). The average ratio of ET‐1/eNOS was highest on 2 weeks (1.31 ± 0.025 au). The ETRA were significantly increased in 2 and 6 weeks (1476.3 ± 20.9 au and 1209.7 ± 6.1 au), while ETRB only in 2 weeks (1160.5 ± 18.2 au).The highest average of MLCK (1916.68 ± 6.41 au), CaD (1676.37 ± 7.72 au), and RGC apoptosis (576.15 ± 33.28 au) were found in 2 weeks hypertension.ConclusionsHypertension induced by DOCA‐salt stimulated significant activation of ET‐1 signaling pathway on TM, elevation of IOP, and RGC apoptosis. The peak of activation was achieved at 2 weeks of hypertension.

Intrinsically disordered caldesmon binds calmodulin via the “buttons on a string” mechanism

We show here that chicken gizzard caldesmon (CaD) and its C-terminal domain (residues 636–771, CaD136) are intrinsically disordered proteins. The computational and experimental analyses of the wild type CaD136 and series of its single tryptophan mutants (W674A, W707A, and W737A) and a double tryptophan mutant (W674A/W707A) suggested that although the interaction of CaD136 with calmodulin (CaM) can be driven by the non-specific electrostatic attraction between these oppositely charged molecules, the specificity of CaD136-CaM binding is likely to be determined by the specific packing of important CaD136 tryptophan residues at the CaD136-CaM interface. It is suggested that this interaction can be described as the “buttons on a charged string” model, where the electrostatic attraction between the intrinsically disordered CaD136 and the CaM is solidified in a “snapping buttons” manner by specific packing of the CaD136 “pliable buttons” (which are the short segments of fluctuating local structure condensed around the tryptophan residues) at the CaD136-CaM interface. Our data also show that all three “buttons” are important for binding, since mutation of any of the tryptophans affects CaD136-CaM binding and since CaD136 remains CaM-buttoned even when two of the three tryptophans are mutated to alanines.

KAT5-mediated SOX4 acetylation orchestrates chromatin remodeling during myoblast differentiation.

Transcription factor SOX4 has been implicated in skeletal myoblast differentiation through the regulation of Cald1 gene expression; however, the detailed molecular mechanism underlying this process is largely unknown. Here, we demonstrate that SOX4 acetylation at lysine 95 by KAT5 (also known as Tip60) is essential for Cald1 promoter activity at the onset of C2C12 myoblast differentiation. KAT5 chromodomain was found to facilitate SOX4 recruitment to the Cald1 promoter, which is involved in chromatin remodeling at the promoter. Chromatin occupancy analysis of SOX4, KAT5, and HDAC1 indicated that the expression of putative SOX4 target genes during C2C12 myoblast differentiation is specifically regulated by the molecular switching of the co-activator KAT5 and the co-repressor HDAC1 on SOX4 transcriptional activation.

Leupaxin stimulates adhesion and migration of prostate cancer cells through modulation of the phosphorylation status of the actin-binding protein caldesmon.

The focal adhesion protein leupaxin (LPXN) is overexpressed in a subset of prostate cancers (PCa) and is involved in the progression of PCa. In the present study, we analyzed the LPXN-mediated adhesive and cytoskeletal changes during PCa progression. We identified an interaction between the actin-binding protein caldesmon (CaD) and LPXN and this interaction is increased during PCa cell migration. Furthermore, knockdown of LPXN did not affect CaD expression but reduced CaD phosphorylation. This is known to destabilize the affinity of CaD to F-actin, leading to dynamic cell structures that enable cell motility. Thus, downregulation of CaD increased migration and invasion of PCa cells. To identify the kinase responsible for the LPXN-mediated phosphorylation of CaD, we used data from an antibody array, which showed decreased expression of TGF-beta-activated kinase 1 (TAK1) after LPXN knockdown in PC-3 PCa cells. Subsequent analyses of the downstream kinases revealed the extracellular signal-regulated kinase (ERK) as an interaction partner of LPXN that facilitates CaD phosphorylation during LPXN-mediated PCa cell migration. In conclusion, we demonstrate that LPXN directly influences cytoskeletal dynamics via interaction with the actin-binding protein CaD and regulates CaD phosphorylation by recruiting ERK to highly dynamic structures within PCa cells.

Targeted Deletion of all Caldesmon Splice Variants in Mice Results in Abdominal Wall Closure Defects

Ample evidence obtained in vitro indicates that the thin filament linked protein, caldesmon (CaD) is involved in regulation of smooth muscle contraction. Homozygous h‐CaD deficient mice were reported to be viable and exhibit a but small slowing of relaxation (Guo et al., 2013). However, the results are confounded by upregulation of non‐muscle l‐CaD in smooth muscle. Thus, the in vivo function of CaD is still not clear. Here we report that deletion of all splice variants of Cald1 results in perinatal lethality whereas the life span of heterozygotes (hets) is normal. Gene targeting was achieved by deleting exons 2‐13 in the murine Cald1 gene. Homologous recombination was confirmed on DNA level by Southern blots and PCR, and on protein level by Western Blotting. Homozygous offspring followed Mendelian rules at E15.5 but was abrogated at E19.5. The bodyweight of the ‐/‐ fetuses at E18.5 was reduced by ~20%. The most prominent feature of the ‐/‐ fetuses is the persistence of the physiological umbilical hernia whereby the size of the abdominal wall defect is variable. Bladder contractility of adult hets was not significantly affected. In conclusion, our results indicate that deletion of CaD does not result in early embryonic lethality but it may be essential for organ maturation. Moreover, lack of h‐CaD but not lack of all splice variants including non‐muscle l‐CaD is compatible with life.