Expression Of Tropomyosin Research Articles

Sarcomeric tropomyosin expression during human iPSC differentiation into cardiomyocytes.

Tropomyosin (TPM) is an essential sarcomeric component, stabilizing the thin filament and facilitating actin's interaction with myosin. In mammals, including humans, there are four TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which generates a multitude of TPM isoforms via alternative splicing and using different promoters. In this study, we have examined the expression of transcripts as well as proteins of various sarcomeric TPM isoforms during human inducible pluripotent stem cell differentiation into cardiomyocytes. During the differentiation time course, we harvested cells on Days 0, 5, 10, 15, and 20 to analyze for various sarcomeric TPM transcripts by qRT-PCR and for sarcomeric TPM proteins using two-dimensional Western blot with sarcomeric TPM-specific CH1 monoclonal antibody followed by mass spectra analyses. Our results show increasing levels of total TPM transcripts and proteins during the period of differentiation, but varying levels of specific TPM isoforms during the same period. By Day 20, the rank order of TPM transcripts was TPM1α > TPM1κ > TPM2α > TPM1μ > TPM3α > TPM4α. TPM1α was the dominant protein produced with some TPM2 and much less TPM1κ and μ. Interestingly, small amounts of two lower molecular weight TPM3 isoforms were detected on Day 15. To the best of our knowledge this is the first demonstration of TPM1μ non-muscle isoform protein expression before and during cardiac differentiation.

Silencing of Tropomyosin 1 suppresses the proliferation, invasion and metastasis of oral squamous cell carcinoma in vitro

We focused on the expression of Tropomyosin 1 (TPM1) in oral squamous cell carcinoma (OSCC) on the basis of proteomic differential display analysis data. TPM1 is an actin binding and microfilament-associated protein, which is thought to affect the tumor behavior of various malignancies. In this study, we examined the role of TPM1 in proliferation, invasion and metastasis of OSCC cells in vitro as we tried to understand its usefulness as a potential biomarker for OSCC. Human OSCC cell lines were transfected by TPM1-siRNA. MTT assay, cell migration assay and wound healing assay were carried out to understand the characteristics of the TPM1-siRNA-transfected cells. Our data showed that suppression of TPM1 increased the proliferative ability, wound healing ability, and migratory ability of OSCC cells. These findings suggest that low expression of TPM1 could contribute to cancer cell invasion and metastasis in OSCC patients, and that TPM1 may have potential as a useful biomarker for OSCC. Availability of data and materialsAll data generated or analyzed during the present study are included in this published article.

Tropomyosin Is Potential Markers for the Diagnosis and Prognosis of Bladder Cancer.

Objective To investigate the correlation between tropomyosin (TM) and clinical characteristics of bladder cancer. In addition, the relationship between TM and immune cell infiltration in bladder cancer was further analyzed. Methods Based on The Cancer Genome Atlas (TCGA) database, the relationship between TM expression and clinicopathological features in bladder cancer was analyzed. Receiver operating characteristic (ROC) curve was used to evaluate the value of TM as a diagnostic marker for bladder cancer. Univariate and multivariate Cox regression was used to analyze the independent factors affecting the prognosis of patients with bladder cancer. The relationship between TM and immune cell infiltration was analyzed. Results ROC curve showed that TPM1, TPM2, and TPM3 had significant diagnostic ability (AUC was 0.845, 0.848, and 0.873, respectively). The high expression of TPM1 and TPM2 is associated with poor overall and disease-specific survival in patients with bladder cancer (P < 0.05). Multivariate Cox analysis showed that age and TPM1 were independent prognostic factors. The expression levels of TPM1, TPM2, TPM3, and TPM4 in low grade bladder cancer were lower than those in high grade bladder cancer (P < 0.05). TPM1 and TPM2 are positively correlated with the infiltration of macrophages and NK cells in bladder cancer. TPM3 is positively associated with Th2. TPM4 is positively correlated with Th1 cells, macrophages, and neutrophils (P < 0.05). Conclusions TPM1 and TPM2 are effective markers for the diagnosis of bladder cancer. TPM1 is an independent prognostic factor for bladder cancer. TM is also associated with the infiltration of various immune cells in bladder cancer. TM may have influenced the development of bladder cancer through immune inhibition.

Expression profiles and transcript properties of fast-twitch and slow-twitch muscles in a deep-sea highly migratory fish, Pseudocaranx dentex.

Fast-twitch and slow-twitch muscles are the two principal skeletal muscle types in teleost with obvious differences in metabolic and contractile phenotypes. The molecular mechanisms that control and maintain the different muscle types remain unclear yet. Pseudocaranx dentex is a highly mobile active pelagic fish with distinctly differentiated fast-twitch and slow-twitch muscles. Meanwhile, P. dentex has become a potential target species for deep-sea aquaculture because of its considerable economic value. To elucidate the molecular characteristics in the two muscle types of P. dentex, we generated 122 million and 130 million clean reads from fast-twitch and slow-witch muscles using RNA-Seq, respectively. Comparative transcriptome analysis revealed that 2,862 genes were differentially expressed. According to GO and KEGG analysis, the differentially expressed genes (DEGs) were mainly enriched in energy metabolism and skeletal muscle structure related pathways. Difference in the expression levels of specific genes for glycolytic and lipolysis provided molecular evidence for the differences in energy metabolic pathway between fast-twitch and slow-twitch muscles of P. dentex. Numerous genes encoding key enzymes of mitochondrial oxidative phosphorylation pathway were significantly upregulated at the mRNA expression level suggested slow-twitch muscle had a higher oxidative phosphorylation to ensure more energy supply. Meanwhile, expression patterns of the main skeletal muscle developmental genes were characterized, and the expression signatures of Sox8, Myod1, Calpain-3, Myogenin, and five insulin-like growth factors indicated that more myogenic cells of fast-twitch muscle in the differentiating state. The analysis of important skeletal muscle structural genes showed that muscle type-specific expression of myosin, troponin and tropomyosin may lead to the phenotypic structure differentiation. RT-qPCR analysis of twelve DEGs showed a good correlation with the transcriptome data and confirmed the reliability of the results presented in the study. The large-scale transcriptomic data generated in this study provided an overall insight into the thorough gene expression profiles of skeletal muscle in a highly mobile active pelagic fish, which could be valuable for further studies on molecular mechanisms responsible for the diversity and function of skeletal muscle.

The prognostic value of TPM1-4 in hepatocellular carcinoma.

BackgroundDespite advances in multiple disciplinary diagnoses and treatments, the prognosis of hepatocellular carcinoma (HCC) remains poor. Some evidence has identified that the aberrant expression of tropomyosins (TPMs) is involved with some cancers development. However, prognostic values of TPMs in HCC have not been thoroughly investigated.MethodsOriginal TPM1–4 mRNA expression of TCGA HCC data and GTEx was downloaded from UCSC XENA. Oncomine database and GSE46408 were used for verification. Clinical stages and survival analysis of TPM1–4 in HCC were performed by GEPIA2. cBioPortal was utilized to assess TPM1–4 gene alteration in HCC. TIMER2.0 was used for investigating the relevance of TPM1–4 to tumor‐infiltrating immune cells in HCC. Additionally, we constructed a TPM1–4 prognostic model to explore the value of TPM1–4 for prognostic evaluation in HCC. LinkedOmics was applied to elucidate TPM3 co‐expression networks in HCC.ResultsThis present study showed that TPM1–4 was upregulated in all HCC tissues, and TPM3 overexpression was correlated with poor survival outcomes in patients with HCC. Besides, TPM3 amplification was the main altered type in TPM1–4 genetic alteration, which affected the prognosis of HCC patients. The risk model revealed that TPM1, TPM2, and TPM3 were applied to risk assessment of HCC prognosis, among which TPM3 expression was significantly higher in the high‐risk group than that in the low‐risk group. Univariate and multivariate cox regression analyses indicated that TPM3 may be an independent prognostic factor of HCC prognosis. In addition, TPM3 co‐expression genes mainly participated in the cell cycle by maintaining microtubule cytoskeleton in HCC progression. TPM1–4 was associated with some tumor‐infiltrating immune cells in HCC.ConclusionOur study detected that the expression level of TPM1–4 was all remarkably elevated in HCC, suggesting that TPM1–4 may serve an important role in HCC development. High TPM3 expression was found to be correlated with poor overall survival, and TPM3 may be an independent prognostic factor for HCC.

Role of Decorin in Posterior Capsule Opacification and Eye Lens Development.

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)β-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFβ-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFβ-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

Reference gene and tropomyosin expression in mud crab Scylla olivacea, Scylla paramamosain and Scylla tranquebarica.

Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is the most sensitive approach to produce accurate relative gene expression that has yet to be demonstrated for allergenic tropomyosin in mud crab species. This study was conducted to identify the suitable reference gene and tropomyosin expression in different body parts of local mud crabs, Scylla olivacea, Scylla paramamosain and Scylla tranquebarica. Myosin, 18S rRNA, GADPH and EF1α were selected as candidate reference genes and their expression was measured in the abdomen, walking leg and cheliped tissues of local Scylla spp. The expression stability was analyzed using the comparative delta-Ct method, BestKeeper, NormFinder and geNorm then comprehensively ranked by RefFinder algorithm. Findings showed that EF1α was the most suitable reference gene across three mud crab species. Meanwhile, the abdomen, walking leg and cheliped selected their own suitable reference gene either Myosin, 18S rRNA, EF1α or GADPH. Overall, tropomyosin was the highest in S. tranquebarica, whereas the least was in S. paramamosain. Interestingly, tropomyosin was the highest in the abdomen of all mud crab species. This is the first analysis on reference genes selection for qRT-PCR data normalization of tropomyosin expression in mud crab. These results will provide more accurate findings for further gene expression and allergen analysis in Scylla spp.

Derivation of proliferative islet1-positive cells during metamorphosis and wound response in Xenopus.

In mammalian hearts, cardiomyocytes retain a transient capacity to proliferate and regenerate following injury before birth, whereas they lose proliferative capacity immediately after birth. It has also been known that cardiac progenitor cells including islet1-positive cells do not contribute to the cardiac repair and regeneration in mammals. In contrast, hearts of zebrafish, amphibians and reptiles maintain a regenerative ability throughout life. Here, we analyzed proliferative capacity of cardiac cells during cardiac development and post-ventricular resection using Xenopus laevis, especially focusing on islet1. Immunohistochemical examination showed that islet1-positive cells were present in a wide range of the ventricle and maintained high dividing ability after metamorphosis. Interestingly, the islet1-positive cells were preserved even at 1year after metamorphosis, some of which showed tropomyosin expression. To assess the possibility of islet1-positive cells as a cellular resource, islet1 response to cardiac resection was analyzed, using adult hearts of 3months after metamorphosis. Transient gene activation of islet1 in apical region was detected within 1day after amputation. Histological analyses revealed that islet1-positive cells appeared in the vicinity of resection plane at 1day post-amputation (dpa) and increased at 3 dpa in both tropomyosin-positive and tropomyosin-negative regions. Vascular labeling analysis by biotinylated dextran amine (BDA) indicated that the islet1-positive cells in a tropomyosin-negative region were closely associated with cardiac vessels. Moreover, dividing ability at this time point was peaked. The resected region was healed with tropomyosin-positive cardiomyocytes until 3months post-amputation. These results suggest a role of islet1-positive cells as a cellular resource for vascularization and cardiogenesis in Xenopus laevis.

Expression of tropomyosin 2 in aortic dissection tissue

Objective: To investigate the expression pattern of tropomyosin 2(TPM2) in aorta of patients with aortic dissection and explore its clinical implication. Methods: Thirteen cases with acute type A aortic dissection(TAAD) diagnosed by transabdominal aortic angiography from 2015 in Tongji Hospital were included. During the operation, the aortic wall tissues of these patients were collected. Ten patients with heart transplantation were selected as control group, and normal aortic wall tissues were taken. The hematoxylin-eosin (HE) and Verhoeff's Van Gieson (EVG) staining were performed to observe the morphological changes of aorta. The mRNA expression level of TPM2 was measured by real-time fluorescent quantitative-PCR, and the protein levels of TPM2 were detected by Western blot and immunohistochemical staining. Image The J software was used to collect the optical density values of each point on the image, obtain the integrated optical density(IOD) value, and calculate the average density(%, IOD/area of the target distribution area). Results: HE and EVG staining revealed medial degeneration and broken elastic fiber in aorta of TAAD patients. The mRNA expression levels of TPM2 were significantly upregulated in aorta of TAAD patients as compared to the control group (P<0.05), so as the TPM2 protein expression levels ((9.73±1.20)% vs. (0.11±0.04)%, P<0.05). And TPM2 was mainly expressed in cytoplasm. Conclusion: The increased expression of TPM2 in TAAD patients hints that TPM2 might be involved in the pathogenesis of aortic dissection.

Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice.

Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin in skeletal, cardiac, and smooth muscle. Slow colonic transit and constipation are common in DMD patients and animal models of DMD. However, the cause of this hypocontractility and the expression of contractile proteins in smooth muscle are unknown. The aim of the study was to investigate the expression of contractile proteins in the colonic smooth muscle and the function of the colon in control and mdx mice. Muscle contraction was measured in muscle strips and isolated muscle cells. Peristaltic activity was measured in ex vivo preparations by spatiotemporal mapping, and gastrointestinal (GI) transit in vivo was measured by the distribution of fluorescent marker along the intestine and colon. mRNA expression of contractile proteins smoothelin, caldesmon, calponin, and tropomyosin was measured by qRT-PCR. Expression of mRNA for contractile proteins was decreased in colonic smooth muscle of mdx mice compared with control. Contraction in response to acetylcholine and KCl was decreased in colonic muscle strips and in isolated muscle cells of mdx mice. Distension of ex vivo colons with Krebs buffer induced peristalsis in both control and mdx mice; however, significantly fewer full peristaltic waves were recorded in the colons of mdx mice. GI transit was also inhibited in mdx mice. The data indicate that the lack of dystrophin causes decrease in colonic smooth muscle contractility, peristalsis, and GI transit and provides the basis for analysis of mechanisms involved in smooth muscle dysfunction in DMD.

Isolation and Cloning of Tropomyosin and Arginine Kinase from Tiger Prawn Penaeus monodon and Blue Swimming Crab Portunus trituberculatus

Shellfish is an important source of food and plays a significant role in human nutrition and health. However, shellfish allergy is a long-lasting disorder which mostly persists throughout life and is often associated with severe reactions [1]. Among the various consumed shellfish, prawns and crabs are the most widely consumed and can lead to the most severe reactions. At present, allergies to shellfish are diagnosed similarly to other food allergies. The diagnosis relies upon careful evaluation of history, the presence of appropriate clinical signs and confirmation with in vivo or in vitro tests to demonstrate the presence of allergen-specific immunoglobulin E (IgE) [2]. However, both in vivo or in vitro diagnostic approaches are mainly based on the use of crude allergen extracts. Crude allergen extracts are obtained from biological sources and consist of mixture of allergenic components with high amounts of undesirable products that can interfere with diagnosis. In many cases, only few of the several proteins found in crude allergen extracts act as the essential allergens in the majority of patients that are allergic to the substance. The most important ones are called major allergens. Problems associated with using crude allergen extracts for allergy diagnosis may be overcome with recombinant allergens. Recombinant allergens with high purity can be produced by using controlled production procedures that yield defined molecules with known molecular, immunologic and biological characteristics [1]. Tiger prawn Penaeus monodon and blue swimming crab Portunus trituberculatus, are among the widely consumed shellfish in Malaysia. Our earlier study involving 131 atopic patients in Allergy Clinic, Kuala Lumpur Hospital demonstrated that patients in Malaysia suffering from allergic responses to shellfish including tiger prawn Penaeus monodon and blue swimming crab Portunus trituberculatus. Amongst the shellfish extracts tested, prawn elicited the highest frequency of positive reactivity in 39% of the patients. Further, crab was the second most common shellfish to elicit a positive reaction in 24% of the patients [3]. Our first phase study has successfully identified tropomyosin and arginine kinase as the major allergens in both species of shellfish. However, more information about the individual allergenic species-specific components is needed. Therefore, we continued our study to isolate and clone the tropomyosin and arginine kinase from these two species of shellfish, tiger prawn Penaeus monodon and blue swimming crab Portunus trituberculatus. Tropomyosin and arginine kinase were isolated from the total RNA (Ribonucleic Acid) obtained from both prawn and crab muscles followed by RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). The RT-PCR products were then cloned into the cloning vector, pJET 1.2 and transformed into Escherichia coli host. Transformants were screened for positive clones by PCR (Polymerase Chain Reaction) colony and sequenced. The 855 bp tropomyosins have been isolated and sequenced from both prawn and crab. Arginine kinases isolated and sequenced from prawn and crab were 1071 bp and 1074 bp, respectively (Figure 1). The GenBank BLAST search for the sequences showed high homology to the targeted proteins as shown in Table 1. Tropomyosin is a 34 to 38 kDa heat-stable protein that belongs to a highly conserved family of actin filament binding proteins, which plays a functional role in contractile activities in muscle cells [4]. Arginine kinase is a 40 to 42 kDa heat-labile protein that plays an important role in regenerating adenosine triphosphate (ATP) during bursts of cellular activity [5]. Tropomyosin and arginine kinase from the prawn and crab have been isolated and the full-length sequences were obtained. Current ongoing study focuses on sub-cloning and full-length expression of tropomyosin and arginine kinase in order to produce respective recombinant proteins, and subsequently investigate their physicochemical and allergenic characteristics.

Tanshinone IIA attenuates high glucose induced human VSMC proliferation and migration through miR-21-5p-mediated tropomyosin 1 downregulation

The proliferation and migration of vascular smooth muscle cells (VSMCs) play important roles in the development and progression of diabetes-related vascular complications. Recently, microRNAs (miRNAs) have been suggested to be involved in the pathogenesis of vascular diseases. This study was designed to investigate the influences of tanshinone IIA, an active compound extracted from Chinese herb Salvia miltiorrhiza, on the proliferation and migration of human aortic VSMCs (HASMCs).cultured in a high glucose medium and the underlying mechanisms related miRNAs. Using a miRNA microarray method, we profiled the miRNA expression signature in human aortic VSMCs (HASMCs) exposed to normal glucose, high glucose with and without Tanshinone IIA. Cell proliferation was measured with 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. Cell migration was evaluated using transwell migration assay and wound scratch assay. Western blot was used to examine the expression of tropomyosin 1 (TPM1) and miRNA level was quantified by real-time PCR. The results showed that several miRNAs that were highly expressed in the high glucose group were significantly decreased in the high glucose with Tanshinone IIA group compared with the normal glucose group (P < 0.05). Among these miRNAs, miR-21–5p was significantly upregulated in the high glucose group and downregulated after Tanshinone IIA treatment (P < 0.05). The depletion of miR-21–5p in HASMCs resulted in decreased cell proliferation and migration (P < 0.05). Moreover, we found that Tanshinone IIA inhibited proliferation and migration partly through miR-21-5p-mediated TPM1 downregulation (P < 0.05). In conclusion, the present study demonstrates that Tanshinone IIA is able to protect HASMCs from high glucose-induced proliferation and migration through regulating expression of miRNAs.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments.

The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

Downregulated Expression of Tropomyosin 1 in Intrahepatic Cholangiocarcinoma: A Predictor of Recurrence and Prognosis.

BackgroundThe downregulation of tropomyosin 1 (TPM1) has been observed in various tumors, but few studies have focused on the clinical significance of TPM1 in intrahepatic cholangiocarcinoma (ICC). In the present study, we investigated the prognostic significance of TPM1 in ICC.Material/MethodsA total of 124 patients with ICC were enrolled in this study. Quantitative real-time polymerase chain reaction (qRT-RCR) was performed to examine the mRNA levels of TPM1 in ICC tissue samples and adjacent noncancerous tissue specimens, while the protein level of TPM1 in tissue specimens were investigated using immunohistochemistry assay. The correlation of TPM1 with clinicopathological features of ICC was analyzed by chi-square test. Survival analysis was performed with Kaplan-Meier method. The Cox proportional hazards model was used to evaluate the prognostic value of TPM1 in patients with ICC.ResultsTPM1 expression was significantly downregulated in ICC tissues at mRNA and protein levels (P<0.001 for both). Downregulated TPM1 mRNA was negatively associated with tumor size (P=0.001) and TNM stage (P=0.007). Moreover, survival analysis demonstrated that patients with low TPM1 expression had a shorter overall survival (OS) (P<0.001) and recurrence-free survival (RFS) (P<0.001) than those with high TPM1 expression. Additionally, multivariate analysis showed that TPM1 could be a potential biomarker for predicting the recurrence (HR=4.632, 95% CI: 3.832–10.368, P<0.001) and survival outcome (HR=5.320, 95% CI: 2.627–11.776, P<0.001) of ICC.ConclusionsTPM1 may serve as a useful biomarker for predicting tumor recurrence and prognosis in patients with ICC.

Rapamycin ameliorates psoriasis by regulating the expression and methylation levels of tropomyosin via ERK1/2 and mTOR pathways in vitro and in vivo.

Psoriasis is a chronic inflammatory disease, affecting more than millions of people in the world. Recently, the mTOR inhibitor rapamycin (RAPA) was reported to be involved in the pathogenesis of psoriasis. However, the underlying mechanism remains unclear. Haematoxylin and eosin staining was used to examine the effects of RAPA on inflammatory level of lesional tissues from patients with psoriasis and animal models. Quantitative real-time PCR, immunohistochemistry and western blot assay were performed to assess the effects of RAPA on tropomyosins (TPMs) expression in patients with psoriasis, cell models and animal models. Phalloidin staining was used to assess the RAPA effects on cell skeleton. The effects of RAPA on cell proliferation and cell cycle were detected by CCK-8 assay, EdU staining and flow cytometry. Methylation status of TPMs was analysed by methylation-specific PCR. The expression of TPM1 and TPM2 was significantly downregulated, while their methylation level was obviously higher in the lesional tissues, cell models and animal models of psoriasis. After treated with RAPA, the expression and methylation levels of TPMs were all restored in the cell models and animal models of psoriasis. RAPA inhibited cell proliferation and decreased the ratio of S phase cell in Hacat or human epidermal keratinocytes cell models of psoriasis. Finally, the activated ERK1/2 and mTOR pathways in the cell model and animal model of psoriasis were suppressed by the treatment of RAPA. RAPA could be used as an effective agent for the treatment of psoriasis by decreasing the methylation level of TPM1 and TPM2 via inhibiting the ERK1/2 and mTOR signalling pathways.

Mechanosensitive TRPV4 Channel is Involved in Fibrotic Transdifferentiation of Retinal Pigment Epithelial Cells

Sustained presence of myofibroblasts during wound healing can cause fibrosis. Within the eye, transdifferentiation of retinal pigment epithelial (RPE) cells to myofibroblasts have been implicated as a key event leading to fibrotic scarring of the retina. While TGF‐beta signaling has been shown to play key roles in this process, inhibition of this pathway can result in undesirable side‐effects by preventing physiological function of this multifaceted cytokine. Therefore, identification of other pathways/molecules that could potentially be targeted to prevent fibrotic scarring is warranted. In this study, roles of mechanosensitive channel TRPV4, which has been implicated to play roles in several fibrotic diseases, and its downstream effector MLCK in myofibroblast transdifferentiation of RPE cells as well as matrix contraction, a key phenomenon of retinal scarring, were examined.Primary cultured porcine RPE cells were used to assess matrix contraction. Cells were cultured with media containing TGF‐beta on collagen matrices in the presence or absence of TRPV4 inhibitor HC‐067047 or MLCK inhibitor ML‐7. Collagen matrices with attached cells were released from 24 well plates and contraction was assessed as change in size of matrices. Cells on these matrices were then lysed to examine the expression of myofibroblast marker protein tropomyosin 1 (TPM1) as well as TRPV4 by western blot analyses.TGF‐beta treatment significantly increased collagen matrix contraction by cultured RPE cells compared to untreated controls. TGF‐beta treatment did not significantly affect TRPV4 expression. Inhibitors for TRPV4 and MLCK significantly reduced matrix contraction as well as TPM1 expression. These data implicate that TRPV4 signaling pathway, while not directly regulated by TGF‐beta, is involved in myofibroblast transdifferentiation of RPE cells and subsequent fibrotic contraction of the matrix.Support or Funding InformationResearch to Prevent Blindness Unrestricted Grant, DoD Award No. W81XWH‐15‐1‐0298This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Myocardin‐Related Transcription Factor Plays a Role in Myofibroblast Transdifferentiation of Retinal Pigment Epithelial Cells

Retinal pigmented epithelial (RPE) cells have been implicated in fibrotic scarring of the retina. Past studies have shown transforming growth factor (TGF)‐beta plays a critical role in this process by inducing myofibroblast transdifferentiation. Myocardin‐related transcription factor (MRTF) has been implicated to play a key role in TGF‐beta initiated signaling in several fibrotic diseases, and thus, we examined its role in an in vitro model using small molecule inhibitors.Primary cultured porcine RPE cells were cultured for 3 days in media containing TGF‐beta2 (10ng/ml) in the presence or absence of the following inhibitors: TGF‐beta receptor inhibitor SB431542 (10 micro M), MRTF signaling inhibitors CCG100602 and CCG203971 (25 microM), and CCG1423 (10 microM). Staining of actin cytoskeleton and alpha‐smooth muscle actin (SMA) were performed using phalloidin and anti‐alpha‐SMA antibody, respectively. For the collagen matrix contraction assay, an in vitro model for fibrosis, cells were cultured three days on type 1 collagen gels, which were subsequently released from 24 well plates and photographed to evaluate contraction by measuring gel size. These same gels with attached cells were then lysed by RIPA buffer and used for Western blot analysis of alpha‐SMA, and tropomyosin1.TGF‐beta treatment induced myofibroblast transdifferentiation as evidenced by expression of alpha‐SMA incorporated prominent stress fibers and the associated significant increase in contractility. This phenotypic change was completely blocked by the TGF‐beta receptor inhibitor SB431542. Similarly, MRTF inhibitors CCG100602, CCG203671, and CCG1423 prevented cytoskeletal changes as well as the enhanced collagen matrix contraction induced by TGF‐beta. Interestingly, MRTF inhibitors did not significantly alter alpha‐SMA expression, but reduced expression of tropomyosin 1, a MRTF target that has been implicated to play a role in alpha‐SMA incorporation into stress fiber.These data strongly implicate that MRTF plays a key role in myofibroblast transdifferentiation of RPE cells downstream of TGF‐beta receptor activation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of starvation on expression of troponin complex genes and ER stress associated genes in skeletal muscles

Little is known about the mechanism of gene expression changes in skeletal muscle during starvation. This study investigated the influence of starvation on gene expression of troponin complex (TnC, TnI and TnT), tropomyosin (Tpm), and endoplasmic reticulum (ER) stress associated proteins (BiP, calreticulin, PDI, and ATF6) in three kinds of skeletal muscles of Gryllus bimaculatus. Dorsal ventral flight muscle (DVM), dorsal longitudinal, and dorsal wing flight muscles (DWM) were identified as typical skeletal muscles by H&E staining. Obvious gene expression was detected in the DVM, but not in the DLM or DWM. In addition to gene expression in DVM, gene expression of decorin, a type of myokine, was also significantly increased by starvation. This is the first study that reports the effect of starvation on expression of troponin complex genes and ER stress associated genes in three types of skeletal muscles.

Developmental Profiling of Tropomyosin Expression in Mouse Brain Reveals Tpm4.2 as the Major Post-synaptic Tropomyosin in the Mature Brain.

Nerve cell connections, formed in the developing brain of mammals, undergo a well-programmed process of maturation with changes in their molecular composition over time. The major structural element at the post-synaptic specialization is the actin cytoskeleton, which is composed of different populations of functionally distinct actin filaments. Previous studies, using ultrastructural and light imaging techniques have established the presence of different actin filament populations at the post-synaptic site. However, it remains unknown, how these different actin filament populations are defined and how their molecular composition changes over time. In the present study, we have characterized changes in a core component of actin filaments, the tropomyosin (Tpm) family of actin-associated proteins from embryonal stage to the adult stage. Using biochemical fractionation of mouse brain tissue, we identified the tropomyosin Tpm4.2 as the major post-synaptic Tpm. Furthermore, we found age-related differences in the composition of Tpms at the post-synaptic compartment. Our findings will help to guide future studies that aim to define the functional properties of actin filaments at different developmental stages in the mammalian brain.

Tumor suppressor tropomyosin Tpm2.1 regulates sensitivity to apoptosis beyond anoikis characterized by changes in the levels of intrinsic apoptosis proteins.

The actin cytoskeleton is a polymer system that acts both as a sensor and mediator of apoptosis. Tropomyosins (Tpm) are a family of actin binding proteins that form co-polymers with actin and diversify actin filament function. Previous studies have shown that elevated expression of the tropomyosin isoform Tpm2.1 sensitized cells to apoptosis induced by cell detachment (anoikis) via an unknown mechanism. It is not yet known whether Tpm2.1 or other tropomyosin isoforms regulate sensitivity to apoptosis beyond anoikis. In this study, rat neuroepithelial cells overexpressing specific tropomyosin isoforms (Tpm1.7, Tpm2.1, Tpm3.1, and Tpm4.2) were screened for sensitivity to different classes of apoptotic stimuli, including both cytoskeletal and non-cytoskeletal targeting compounds. Results showed that elevated expression of tropomyosins in general inhibited apoptosis sensitivity to different stimuli. However, Tpm2.1 overexpression consistently enhanced sensitivity to anoikis as well as apoptosis induced by the actin targeting drug jasplakinolide (JASP). In contrast the cancer-associated isoform Tpm3.1 inhibited the induction of apoptosis by a range of agents. Treatment of Tpm2.1 overexpressing cells with JASP was accompanied by enhanced sensitivity to mitochondrial depolarization, a hallmark of intrinsic apoptosis. Moreover, Tpm2.1 overexpressing cells showed elevated levels of the apoptosis proteins Bak (proapoptotic), Mcl-1 (prosurvival), Bcl-2 (prosurvival) and phosphorylated p53 (Ser392). Finally, JASP treatment of Tpm2.1 cells caused significantly reduced Mcl-1, Bcl-2 and p53 (Ser392) levels relative to control cells. We therefore propose that Tpm2.1 regulates sensitivity to apoptosis beyond the scope of anoikis by modulating the expression of key intrinsic apoptosis proteins which primes the cell for death.