Transgelin Research Articles

Functional heterogeneity of meniscal fibrochondrocytes and microtissue models is dependent on modality of fibrochondrocyte isolation.

Collagenase digestion (d) and cellular outgrowth (og) are the current modalities of meniscus fibrochondrocytes (MFC) isolation for bioengineering and mechanobiology-related studies. However, the impact of these modalities on study outcomes is unknown. Here, we show that og- and d-isolated MFC have distinct proliferative capacities, transcriptomic profiles via RNA sequencing (RNAseq), extracellular matrix (ECM)-forming, and migratory capacities. Our data indicate that microtissue pellet models developed from og-isolated MFC display a contractile phenotype with higher expressions of alpha-smooth muscle actin (ACTA2) and transgelin (TAGLN) and are mechanically stiffer than their counterparts from d-MFC. Moreover, we introduce a novel method of MFC isolation designated digestion-after-outgrowth (dog). The transcriptomic profile of dog-MFC is distinct from d- and og-MFC, including a higher expression of mechanosensing caveolae-associated caveolin-1 (CAV1). Additionally, dog-MFC were superior chondrogenically and generated larger-size microtissue pellet models containing a higher frequency of smaller collagen fibre diameters. Thus, we demonstrate that the modalities of MFC isolation influence the downstream outcomes of bioengineering and mechanobiology-related studies.

Exclusive expression of KANK4 promotes myofibroblast mobility in keloid tissues

Keloids are characterized by abnormal wound healing with excessive accumulation of extracellular matrix. Myofibroblasts are the primary contributor to extracellular matrix secretion, playing an essential role in the wound healing process. However, the differences between myofibroblasts involved in keloid formation and normal wound healing remain unclear. To identify the specific characteristics of keloid myofibroblasts, we initially assessed the expression levels of well-established myofibroblast markers, α-smooth muscle actin (α-SMA) and transgelin (TAGLN), in scar and keloid tissues (n = 63 and 51, respectively). Although myofibroblasts were present in significant quantities in keloids and immature scars, they were absent in mature scars. Next, we conducted RNA sequencing using myofibroblast-rich areas from keloids and immature scars to investigate the difference in RNA expression profiles among myofibroblasts. Among significantly upregulated 112 genes, KN motif and ankyrin repeat domains 4 (KANK4) was identified as a specifically upregulated gene in keloids. Immunohistochemical analysis showed that KANK4 protein was expressed in myofibroblasts in keloid tissues; however, it was not expressed in any myofibroblasts in immature scar tissues. Overexpression of KANK4 enhanced cell mobility in keloid myofibroblasts. Our results suggest that the KANK4-mediated increase in myofibroblast mobility contributes to keloid pathogenesis.

Abstract 6827: Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal cancer

Abstract Background: Cancer-associated fibroblasts (CAFs) play a key role in modifying the tumor microenvironment (TME) and could be a future target for treatment. CAFs are typically categorized based on function: myofibroblastic CAFs (myCAFs) amplify extracellular matrix production that blocks therapy and immune cell infiltration while inflammatory CAFs (iCAFs) produce immuno-suppressive cytokines and chemokines. Here we sought to better understand the role and mechanisms of myCAFs in altering the TME by identifying their unique markers and potential drivers. Methods: Z-scores of ACTA2/TAGLN or ICAM1/PDPN were used to create datasets high in myCAF and iCAF markers, respectively, from TCGA data. A gene set enrichment analysis (GSEA) was performed on the resulting differential expression. Samples of patients were collected to create a cohort of colorectal cancers that were stained via immunohistochemistry (IHC) for alpha-smooth muscle actin (αSMA), transgelin (TAGLN), myocardin (MYOCD) and myocardin-related transcription factor A (MRTFA). Slides were evaluated for collagen using Masson’s trichrome stain. MYOCD and MRTFA nuclear expression was expressed as positively stained nuclei per high powered field (HPF; 400x). TAGLN, αSMA, and collagen were scored on a scale of 0-3+. Results: Of the 592 TCGA patients compiled across all disease stages, 23 (median age 63) formed the myCAF-high group (7 males, 16 females). Within this group, GSEA indicated an upregulation of myogenesis (q <0.001) with the following genes positively associated with myCAF markers: MYLK (q < 0.001, log2 fold change [FC] 1.52), DES (q < 0.001, 5.32), and MYH11 (q < 0.001, 3.57). The following were also upregulated in the myCAF high cohort: CNN1 (q < 0.001, 2.95) and MYOCD (q< 0.001, 2.27). These genes are targets of MYOCD and MRTFA transcription factors potentially driving the myCAF phenotype. To assess this potential relationship, we evaluated 306 tumor samples from 153 patients with CRC. The cohort consisted of 56 female patients and 97 male patients aged 13-90 years old across all disease stages. High amounts of nuclear MRTFA were associated with high scores for myCAF markers aSMA (average score 1.54 when MRTFA+ nuclei < 5 per HPF vs 2.06 when MRTFA + nuclei > 5 per HPF, p<0.001, Wilcoxon Rank Sum Test), TAGLN (1.37 vs 1.62, p = 0.043), and collagen (1.73 vs 2.10, p = 0.009). Likewise, for MYOCD, αSMA (1.79 vs 2.14, p = 0.001) and collagen (1.87 vs 2.15, p = 0.017) were positively correlated. Conclusions: Increased nuclear expression of MYOCD and MRTFA in stromal cells of CRC are positively correlated with myCAF gene expression, supporting our initial hypothesis of these being drivers of the phenotype. Our findings now motivate further analysis as to whether the inhibition of these transcription factors could lead to a reduction in the immune regulatory myCAF phenotype. Citation Format: Alec B. Cornelio, Katherine A. Johnson, Yousef Gadalla, Sharanya Nath, Cheri A. Pasch, Wei Zhang, Kristina A. Matkowskyj, Dustin A. Deming. Myocardin and myocardin-related transcription factor as potential drivers of the myofibroblastic cancer-associated fibroblast phenotype in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6827.

Identification of a hub gene VCL for atherosclerotic plaques and discovery of potential therapeutic targets by molecular docking

BackgroundAtherosclerosis (AS) is a pathology factor for cardiovascular diseases and instability of atherosclerotic plaques contributes to acute coronary events. This study identified a hub gene VCL for atherosclerotic plaques and discovered its potential therapeutic targets for atherosclerotic plaques.MethodsDifferential expressed genes (DEGs) were screened between unstable and stable plaques from GSE120521 dataset and then used for construction of a protein-protein interactions (PPI) network. Through topological analysis, hub genes were identified within this PPI network, followed by construction of a diagnostic model. GSE41571 dataset was utilized to validate the diagnostic model. A key hub gene was identified and its association with immune characteristics and pathways were further investigated. Molecular docking and molecular dynamics (MD) simulation were employed to discover potential therapeutic targets.ResultsAccording to the PPI network, 3 tightly connected protein clusters were found. Topological analysis identified the top 5 hub genes, Vinculin (VCL), Dystrophin (DMD), Actin alpha 2 (ACTA2), Filamin A (FLNA), and transgelin (TAGLN). Among these hub genes, VCL had the highest diagnostic value. VCL was selected for further analysis and we found that VCL was negatively correlated with immune score and AS-related inflammatory pathways. Next, we identified 408 genes that were highly correlated with VCL and determined potential drug candidates. The results from molecular docking and MD simulation showed compound DB07117 combined with VCL protein stably, the binding energy is -7.7 kcal/mol, indicating that compound DB07117 was a potential inhibitor of VCL protein.ConclusionThis study identified VCL as a key gene for atherosclerotic plaques and provides a potential therapeutic target of VCL for the treatment of atherosclerotic plaques.

Transgelin Promotes Glioblastoma Stem Cell Hypoxic Responses and Maintenance Through p53 Acetylation.

Glioblastoma (GBM) is a lethal cancer characterized by hypervascularity and necrosis associated with hypoxia. Here, it is found that hypoxia preferentially induces the actin-binding protein, Transgelin (TAGLN), in GBM stem cells (GSCs). Mechanistically, TAGLN regulates HIF1α transcription and stabilizes HDAC2 to deacetylate p53 and maintain GSC self-renewal. To translate these findings into preclinical therapeutic paradigm, it is found that sodium valproate (VPA) is a specific inhibitor of TAGLN/HDAC2 function, with augmented efficacy when combined with natural borneol (NB) in vivo. Thus, TAGLN promotes cancer stem cell survival in hypoxia and informs a novel therapeutic paradigm.

Induced pluripotent stem cell-derived vascular smooth muscle cells as an in vitro model of coronary spastic angina

Abstract Background Coronary spastic angina (CSA), also known as Prinzmetal’s variant angina, is a form of angina caused by coronary artery spasm. CSA is characterized by enhanced basal tone of coronary artery smooth muscle and hyperreactivity to vasoconstrictor stimuli. The vascular smooth muscle hyperreactivity is likely due to a post-receptorial cellular abnormality in the regulation of myofibril contraction, suggesting the possibility of multiple stimuli acting on various cellular receptors. However, its detailed mechanism is still unclear. To investigate it, various experiments using coronary artery smooth muscle cells obtained from the CSA patients is preferable, but it is impossible to do it clinically and ethically. Purpose The purpose of this study was to establish induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (VSMCs) and to characterize this VSMC model for CSA research. Methods Three CSA patients with ST-segment elevation, which is a characteristic of variant angina, were recruited for this study. The isolated skin fibroblasts or peripheral blood mononuclear cells were reprogrammed into iPSCs through electroporation of episomal plasmids encoding the reprogramming genes OCT3/4, SOX2, KLF4, L-MYC, LIN28, and mouse p53DD. We differentiated mesodermal using Glycogen synthase kinase 3 inhibition and Bone morphogenetic protein 4 treatment from iPSCs and resulted in the differentiation of VSMCs by platelet-derived growth factor-BB and Activin A. The differentiation of VSMCs was confirmed by VSMC-specific gene expression in qRT–PCR and fluorescence-activated cell sorting (FACS). We examined biological features and intracellular calcium response to acetylcholine (ACh) in VSMCs obtained from 3 CSA patients and 3 healthy controls. Intracellular free Ca2+ concentration ([Ca2+]i; nM) was measured by fura-2. Results CSA iPSCs were confirmed to have normal karyotypes and pluripotency to differentiate. FACS showed that VSMCs differentiated from iPSCs exhibited smooth muscle cell marker expressions including alpha-smooth muscle actin and SM22-alpha. qRT–PCR showed that VSMCs from iPSCs had expressions of ACTA2 and TAGLN gene similar to the human coronary artery smooth muscle cells. Differentiation induction rate of VSMCs differentiated from iPSCs between CSA and control did not show significant difference. [Ca2+]i at baseline did not differ significantly between them (143 nM in CSA versus 120 nM in control, p=0.63, n=3), but peak increase in [Ca2+]i from baseline after ACh stimulation was significantly increased in CSA group compared with control (372 nM versus 177 nM, p<0.05, n=3). Conclusions iPSC-derived VSMCs obtained from CSA patients showed an enhanced intracellular calcium response to ACh, which is likely responsible for CSA phenotype. Our iPSC-derived VSMC model can be useful for elucidating the mechanism of coronary spasm and developing therapeutic strategy such as drug efficacy.

Prognostic Value of Tumor Size in Colon Cancer-Smaller is Better?

The prognostic value of tumor size in colon cancer remains controversial. This study aimed to reveal the correlation between tumor size and prognosis of colon cancer. A total of 491 patients with colon cancer were included in this study. The correlation of tumor size with prognosis, mismatch repair status, and other clinicopathological characteristics as well as tumor microenvironment was analyzed. For stage IIA microsatellite stable (MSS) colon cancer, tumors sized <3.5cm and ≥5cm were associated with a poorer disease free survival (DFS) compared with tumors sized between 3.5 and 5cm (P = .002). Small tumor size (HR = 5.098, P = .001) and large tumor size (HR = 2.749, P = .029) were found to be independent prognostic factors for stage IIA MSS colon cancer. Moreover, high expression of transgelin (TAGLN), a marker of cancer-associated fibroblasts (CAFs), was found to be an independent prognostic factor for poorer DFS (HR = 9.651, P = .009), which was also associated with smaller tumor size (P = .027). Small (<3.5cm) and large (≥5cm) tumor sizes are associated with decreased DFS in stage IIA MSS colon cancer. Enrichment of TAGLN+ CAFs is associated with decreased DFS and small tumor size.

Transgelin promotes ferroptosis to inhibit the malignant progression of esophageal squamous cell carcinoma.

The aim of the present study was to examine the function of transgelin (TAGLN) and its underlying mechanism in the ferroptosis of esophageal squamous cell carcinoma (ESCC) cells. To meet this aim, the association between TAGLN expression and the prognosis of patients with ESCC was determined using tissue samples and clinical data. Gene Expression Omnibus databank and Gene Set Enrichment Analysis data were used to examine which genes were co‑expressed with TAGLN, as well as the influence of TAGLN on ESCC. Subsequently, Transwell chamber, wound healing, Cell Counting Kit‑8 viability and colony formation assays were performed to observe the effects of TAGLN on the migration, invasion, viability and proliferation of Eca‑109 and KYSE‑150 cells. The interaction between TAGLN and p53 in the regulation of ferroptosis was detected using reverse transcription‑quantitative PCR, co‑immunoprecipitation and fluorescence co‑localization assays, and a xenograft tumor model was established to examine the effect of TAGLN on tumor growth. The level of TAGLN expression in patients with ESCC was found to be low, compared with normal esophageal tissue, and a positive association was identified between the prognosis of ESCC and TAGLN expression. The expression of the ferroptosis marker protein, glutathione peroxidase 4, was found to be high, whereas that of acyl‑CoA synthetase long‑chain family member 4 was lower in patients with ESCC compared with expression levels in healthy patients. The overexpression of TAGLN resulted in a significant decrease in the invasive and proliferative capabilities of Eca‑109 and KYSE‑150 cells invitro compared with the control group; invivo, TAGLN overexpression was found to significantly decrease tumor size, volume and weight after one month of growth. In addition, the proliferation, migration and invasion of Eca‑109 cells invivo was stimulated by the knockdown of TAGLN. The results of the transcriptome analysis further demonstrated that TAGLN was able to induce ferroptosis‑associated cell functions and pathways. Finally, TAGLN overexpression was found to promote ferroptosis in ESCC through its interaction with p53.Taken together, the findings of the present study suggested that the malignant development of ESCC may be inhibited by TAGLN through the manifestation of ferroptosis.

Interaction of TAGLN and USP1 promotes ZEB1 ubiquitination degradation in UV-induced skin photoaging

BackgroundUltraviolet A (UVA) irradiation can lead to skin damage and premature skin aging known as photoaging. This work found that UVA irradiation caused an imbalance between dermal matrix synthesis and degradation through the aberrant upregulation of transgelin (TAGLN) and studied the underlying molecular mechanism.ResultsCo-immunoprecipitation and proximal ligation assay results showed that TAGLN can interact with USP1. USP1 can be retained in the cytoplasm by TAGLN in UVA-induced cells, which inhibits the interaction between USP1/zinc finger E-box binding homeobox 1 (ZEB1), promote the ubiquitination degradation of ZEB1, and lead to photoaging. TAGLN knockdown can release USP1 retention and help human skin fibroblasts (HSFs) resist UVA-induced damage. The interactive interface inhibitors of TAGLN/USP1 were screened via virtual docking to search for small molecules that inhibit photoaging. Zerumbone (Zer), a natural product isolated from Zingiber zerumbet (L.) Smith, was screened out. Zer can competitively bind TAGLN to reduce the retention of USP1 in the cytoplasm and the degradation of ZEB1 ubiquitination in UV-induced HSFs. The poor solubility and permeability of Zer can be improved by preparing it as a nanoemulsion, which can effectively prevent skin photoaging caused by UVA in wild-type (WT) mice. Zer cannot effectively resist the photoaging caused by UVA in Tagln−/− mice because of target loss.ConclusionsThe present results showed that the interaction of TAGLN and USP1 can promote ZEB1 ubiquitination degradation in UV-induced skin photoaging, and Zer can be used as an interactive interface inhibitor of TAGLN/USP1 to prevent photoaging.

Abstract 674: Transgelin Deficiency Is Associated With Elevated Low-density Lipoprotein Cholesterol And Atherogenesis In Mice.

Genetic hyperlipidemias are a major risk factor for atherosclerotic cardiovascular disease due to life-long exposure to elevated plasma lipids. However, the causality of hyperlipidemia is not known in a large proportion of patients, suggesting that unknown genes remain to be discovered. Applying a CRISPR/Cas9 screen, we recently identified transgelin (TAGLN), an actin binding protein, as a new gene involved in LDL metabolism. TAGLN supports LDL endocytosis presumably by interacting with actin filaments during LDL endocytosis; however, if TAGLN impacts on plasma lipid levels and atherogenesis in vivo is unknown. Aim: To investigate TAGLN’s impact on lipid profile and atherosclerosis development in a Tagln -knockout (KO) mouse model. Methods: Tagln -KO (n=18) and wild-type (WT) (n=18) mice were fed a high-fat diet (42% Cal from fat) for 12 weeks. We collected plasma for lipoprotein analysis, liver specimens for histopathology and lipid extraction/quantification, and aortas for quantifying atherosclerotic plaque area. Results: Tagln -KO mice showed higher total cholesterol than WT mice (20% increase, mean±SD: 299±51 vs. 251±59 mg/dL, p=0.014), without changes in triglyceride levels (p=0.36). Lipoprotein subfraction analysis by FPLC revealed an increase in the lipids associated with the LDL fraction of Tagln -KO mice. Although no differences were observed in hepatic histology or triglyceride content, Tagln -KO animals showed an increase in the hepatic content of total cholesterol (mean±SD: 376±65 vs. 317±70 μg/100 mg, p=0.012). Finally, Tagln -KO mice developed about twice more atherosclerotic lesion area than WT mice (Median(IQR): 0.997(0.347-1.451) vs 0.428(0.336-0.859), p=0.015). Conclusions: In line with a reduction in LDL uptake in hepatocytes, shown by our previous in vitro data, TAGLN deficiency was associated with hypercholesterolemia and high LDL cholesterol in mice, likely due to decreased hepatic LDL clearance. Despite the modest hypercholesterolemia, Tagln -KO mice showed disproportionally increased atherosclerosis, suggesting that TAGLN may regulate other atherogenic mechanisms, beyond LDL cholesterol.

Transgelin promotes lung cancer progression via activation of cancer-associated fibroblasts with enhanced IL-6 release

Cancer-associated fibroblasts (CAFs), the principal constituent of the heterogenous tumor microenvironment, have been shown to promote tumor progression; however, the underlying mechanism is still less clear. Here, we find that transgelin (TAGLN) protein levels increased in primary CAFs isolated from human lung cancer, compared with those in paired normal fibroblasts. Tumor microarrays (TMAs) revealed that increased stromal TAGLN levels correlates with more lymphatic metastasis of tumor cells. In a subcutaneous tumor transplantation model, overexpression of Tagln in fibroblasts also increased tumor cell spread in mice. Further experiments show that Tagln overexpression promoted fibroblast activation and mobility in vitro. And TAGLN facilitates p-p65 entry into the nucleus, thereby activating the NF-κB signaling pathway in fibroblasts. Activated fibroblasts promote lung cancer progression via enhancing the release of pro-inflammatory cytokines, especially interleukine-6 (IL-6). Our study revealed that the high levels of stromal TAGLN is a predictive risk factor for patients with lung cancer. Targeting stromal TAGLN may present an alternative therapeutic strategy against lung cancer progression.

Differentially expressed genes related to lymph node metastasis in advanced laryngeal squamous cell cancers.

Understanding the molecular mechanisms and gene expression in laryngeal squamous cell carcinoma (LSCC) may explain its aggressive biological behavior and regional metastasis pathways. In the present study, patients with locally advanced LSCC tumors were examined for differential gene expression in the normal mucosa (non-tumoral mucosa), tumors and lymph node tissues. The aim was to identify possible predictive genes for lymph node metastasis. A total of 16 patients who had undergone total laryngectomy with neck dissection for advanced LSCC were randomly selected from a hospital database: Eight of the patients had lymph node metastasis (Group 1) and the other eight patients did not have metastasis (Group 2). Overall survival (OS), disease-free survival (DFS) and disease-specific survival (DSS) were analyzed. For each patient, paraffin-embedded tissue samples were collected from non-tumoral mucosa, tumoral lesions and lymph node tissues. RNA was extracted from the tissue samples and used for complementary DNA synthesis, and microarray analysis was subsequently performed on each sample. Gene expression levels were determined in each specimen, and Groups 1 and 2 were compared and statistically analyzed. The microarray results for lymph node metastasis-positive and -negative groups, indicated the differential expression of 312 genes in the lymph nodes, 691 genes in the normal mucosal tissue and 93 genes in the tumor tissue. Transgelin (TAGLN) and cofilin 1 (CFL1) were identified as possible target genes and validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR results for TAGLN and CFL1 supported the microarray data. OS, DFS and DSS times were longer in Group 2 than in Group 1 (P=0.002, 0.015 and 0.009, respectively). In addition, TAGLN and CFL1 were associated with DFS and DSS. On the basis of these results, it is suggested that TAGLN and CFL1 expression may play an important role in the pathogenesis of regional metastasis and poor prognosis in advanced LSCC.

Sclerostin is involved in osteogenic transdifferentiation of vascular smooth muscle cells in chronic kidney disease-associated vascular calcification with non-canonical Wnt signaling

Vascular calcification is prominent in patients with chronic kidney disease (CKD) and is a strong predictor of cardiovascular mortality in the CKD population. However, the mechanism underlying CKD-associated vascular calcification remains unclear. To identify potential therapeutic targets, a 5/6 nephrectomy rat model was established by feeding of a high-phosphorous diet as the CKD group and compared with sham group rats at 4 and 16 weeks. Sequencing analyses of the rat aorta revealed 643 upregulated and 1023 downregulated genes at 4 weeks, as well as 899 upregulated and 1185 downregulated genes at 16 weeks in the CKD group compared to the sham group. Bioinformatics analyses suggested that SOST (which encodes sclerostin) and Wnt signaling are involved in CKD-associated vascular calcification. Furthermore, protein-protein interactions analysis revealed interactions between SOST, WNT5A, and WNT5B, that involved runt-related transcription factor 2 (RUNX2) and transgelin (TAGLN). SOST was increased in CKD-associated vascular calcification following reduction of the Wnt signaling, including WNT5A and WNT5B, both in vivo and in vitro. TargetScan was used to predict the microRNAs (miRNAs) targeting WNT5A and WNT5B. The expression levels of miR-542-3p, miR-298-3p, miR-376b-5p, and miR-3568 were significantly reduced, whereas that of miR-742-3p was significantly increased in calcified rat aortic vascular smooth muscle cells (VSMCs). In CKD rat aortas, the expression of miR-542-3p, miR-298-3p, miR-376b-5p, miR-3568, miR-742-3p, and miR-22-5p were significantly reduced at both 4 and 16 weeks. Altogether, owing to several assessments, potentially diagnostic and prognostic biomarkers for improving common CKD diagnostic tools were identified in this study. Abbreviations: BUN: blood urea nitrogen; CKD: chronic kidney disease; CKD-MBD: chronic kidney disease-mineral bone disorder; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GO: the Gene Ontology; HE: hematoxylin-eosin; HRP: horseradish peroxidase; KEGG: Kyoto Encyclopedia of Genes and Genomes; MiRNAs: microRNAs; PAS: periodic acid-Schiff; RUNX2: runt-related transcription factor 2; SCr: serum creatinine; STRING: the Search Tool for the Retrieval of Interacting Genes/Proteins; TAGLN: transgelin; VSMC: vascular smooth muscle cell.

The quantitative proteomic analysis reveals schisantherin a prevents liver fibrosis through regulating extracellular matrix organization

The activation of hepatic stellate cell (HSC) induced by transforming growth factor-β1 (TGF-β1) is the key event in the pathogenesis of liver fibrosis. Schisantherin A (SCA), a main active ingredient of Schisandra chinensis, has anti-tumor and anti-inflammatory activities. Here, we reported that SCA inhibited the activation, proliferation, and cell cycle of human HSC cell line LX2. Then, by performing the tandem mass tag (TMT)-based quantitative proteomic analysis on LX2, we identified a total of 6045 proteins across the control, TGF-β1-activated and SCA treated LX2 groups, of which 544 proteins were significantly changed among the three groups. All the differentially expressed proteins (DEPs) were assigned to 4 clusters by fuzzy c-means (FCM) clustering analysis. The changed expression of DEPs in cluster 3 and 4 was reversed by SCA treatment. Bioinformatic analysis revealed that SCA regulated the expression of several DEPs involved in extracellular matrix organization, such as thrombospondin 1 (THBS1), transgelin (TAGLN) and tissue inhibitor of metalloproteinase 3 (TIMP3). The Western blot and RT-qPCR analysis confirmed these proteins increased in TGF-β1 group and decreased by the SCA treatment. In summary, we found that SCA may exert anti-fibrotic effect on HSCs by regulating the process of extracellular matrix organization in HSCs. Keywords: liver fibrosis, schisantherin A, proteomics, extracellular matrix organization.

GRASP55 regulates the unconventional secretion and aggregation of mutant huntingtin

Recent studies demonstrated that the Golgi reassembly stacking proteins (GRASPs), especially GRASP55, regulate Golgi-independent unconventional secretion of certain cytosolic and transmembrane cargoes; however, the underlying mechanism remains unknown. Here, we surveyed several neurodegenerative disease–related proteins, including mutant huntingtin (Htt-Q74), superoxide dismutase 1 (SOD1), tau, and TAR DNA–binding protein 43 (TDP-43), for unconventional secretion; our results show that Htt-Q74 is most robustly secreted in a GRASP55-dependent manner. Using Htt-Q74 as a model system, we demonstrate that unconventional secretion of Htt is GRASP55 and autophagy dependent and is enhanced under stress conditions such as starvation and endoplasmic reticulum stress. Mechanistically, we show that GRASP55 facilitates Htt secretion by tethering autophagosomes to lysosomes to promote autophagosome maturation and subsequent lysosome secretion and by stabilizing p23/TMED10, a channel for translocation of cytoplasmic proteins into the lumen of the endoplasmic reticulum–Golgi intermediate compartment. Moreover, we found that GRASP55 levels are upregulated by various stresses to facilitate unconventional secretion, whereas inhibition of Htt-Q74 secretion by GRASP55 KO enhances Htt aggregation and toxicity. Finally, comprehensive secretomic analysis identified novel cytosolic cargoes secreted by the same unconventional pathway, including transgelin (TAGLN), multifunctional protein ADE2 (PAICS), and peroxiredoxin-1 (PRDX1). In conclusion, this study defines the pathway of GRASP55-mediated unconventional protein secretion and provides important insights into the progression of Huntington’s disease.

Engineering bioactive nanoparticles to rejuvenate vascular progenitor cells

Fetal exposure to gestational diabetes mellitus (GDM) predisposes children to future health complications including type-2 diabetes mellitus, hypertension, and cardiovascular disease. A key mechanism by which these complications occur is through stress-induced dysfunction of endothelial progenitor cells (EPCs), including endothelial colony-forming cells (ECFCs). Although several approaches have been previously explored to restore endothelial function, their widespread adoption remains tampered by systemic side effects of adjuvant drugs and unintended immune response of gene therapies. Here, we report a strategy to rejuvenate circulating vascular progenitor cells by conjugation of drug-loaded liposomal nanoparticles directly to the surface of GDM-exposed ECFCs (GDM-ECFCs). Bioactive nanoparticles can be robustly conjugated to the surface of ECFCs without altering cell viability and key progenitor phenotypes. Moreover, controlled delivery of therapeutic drugs to GDM-ECFCs is able to normalize transgelin (TAGLN) expression and improve cell migration, which is a critical key step in establishing functional vascular networks. More importantly, sustained pseudo-autocrine stimulation with bioactive nanoparticles is able to improve in vitro and in vivo vasculogenesis of GDM-ECFCs. Collectively, these findings highlight a simple, yet promising strategy to rejuvenate GDM-ECFCs and improve their therapeutic potential. Promising results from this study warrant future investigations on the prospect of the proposed strategy to improve dysfunctional vascular progenitor cells in the context of other chronic diseases, which has broad implications for addressing various cardiovascular complications, as well as advancing tissue repair and regenerative medicine.

A narrative review of research advances in the study of molecular markers of airway smooth muscle cells.

Background and ObjectiveAirway smooth muscle cells (ASMCs) are an important component of the airway. Their thickening and proliferation are important in pathological situations, such as airway remodeling in asthma, but their origin remains unclear. Therefore, characterizing molecular markers of ASMCs were sought to identify the source of increased ASMCs in asthmatic airway remodeling.MethodsArticles for this review were derived from a review of the literature related to surface markers and biological properties of ASMCs and smooth muscle cells (SMCs) using PubMed, Google Scholar, and Web of Science.Key Content and FindingsThis review discusses several SMC molecular markers, describes the different developmental stages of SMCs that express different molecular markers, and summarizes several classical SMC molecular markers. However, the establishment of a specific molecular marker detection system for ASMCs still faces great challenges.ConclusionsAlthough there is no recognized molecular marker detection system for ASMCs, and the study of the properties and sources of increased ASMCs in asthma airway remodeling is still in a state of exploration, the future is promising. Among the SMC markers described in this review, Myosin heavy chain 11 (MYH11) is a molecular marker for mature SMCs and Transgelin (TAGLN) is an early marker for SMC differentiation, and different molecular markers or combinations of molecular markers can be selected for the identification of the properties and sources of increased ASMCs in asthma airway remodeling according to the differentiation period and research needs.

Transgelin: a new gene involved in LDL endocytosis identified by a genome-wide CRISPR-Cas9 screen

A significant proportion of patients with elevated LDL and a clinical presentation of familial hypercholesterolemia do not carry known genetic mutations associated with hypercholesterolemia, such as defects in the LDL receptor. To identify new genes involved in the cellular uptake of LDL, we developed a novel whole-genome clustered regularly interspaced short palindromic repeat-Cas9 KO screen in HepG2 cells. We identified transgelin (TAGLN), an actin-binding protein, as a potentially new gene involved in LDL endocytosis. In silico validation demonstrated that genetically predicted differences in expression of TAGLN in human populations were significantly associated with elevated plasma lipids (triglycerides, total cholesterol, and LDL-C) in the Global Lipids Genetics Consortium and lipid-related phenotypes in the UK Biobank. In biochemical studies, TAGLN-KO HepG2 cells showed a reduction in cellular LDL uptake, as measured by flow cytometry. In confocal microscopy imaging, TAGLN-KO cells had disrupted actin filaments as well as an accumulation of LDL receptor on their surface because of decreased receptor internalization. Furthermore, TAGLN-KO cells exhibited a reduction in total and free cholesterol content, activation of SREBP2, and a compensatory increase in cholesterol biosynthesis. TAGLN deficiency also disrupted the uptake of VLDL and transferrin, other known cargoes for receptors that depend upon clathrin-mediated endocytosis. Our data suggest that TAGLN is a novel factor involved in the actin-dependent phase of clathrin-mediated endocytosis of LDL. The identification of novel genes involved in the endocytic uptake of LDL may improve the diagnosis of hypercholesterolemia and provide future therapeutic targets for the prevention of cardiovascular disease.

Bioinformatics analysis reveals the roles of cytoskeleton protein transgelin in occurrence and development of proteinuria.

BackgroundProteinuria is a sensitive hallmark for progressive renal dysfunction. Transgelin (TAGLN) has been demonstrated to participate in etiology of proteinuria and dynamics of podocyte foot process; however, the mechanism of TAGLN involvement in proteinuria is unknown. The present study aimed to explore the roles of TAGLN in the development of proteinuria.MethodsDifferentially expressed genes (DEGs) were detected from microarray expression profiling datasets from Gene Expression Omnibus, and analyzed by the short time series expression miner to cluster the DEGs in proteinuria progression. Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to determine the top 20 enriched pathways, and construct a gene interaction network.ResultsIn total, 2,409 DEGs for nephropathy and 10,612 DEGs for podocyte foot process and proteinuria were detected. Additionally, 76 common DEGs (25 upregulated and 41 downregulated) between nephropathy and podocyte foot process were primarily involved in innate immunity, positive regulation of transcription-DNA−templated, immunity and negative regulation of cell proliferation, enriched in cytokine-cytokine receptor interaction signaling pathway, Ras signaling pathway, axon guidance, tumor necrosis factor (TNF) signaling pathway and apoptosis.ConclusionsWe discovered a TAGLN-mediated regulatory network involved in proteinuria progression. These findings provide novel insight to understand the molecular mechanisms underlying the pathogenesis of proteinuria.

Transgelin Inhibits the Malignant Progression of Esophageal Squamous Cell Carcinomas by Regulating Epithelial-Mesenchymal Transition.

ObjectiveThis article investigates the role of Transgelin (TAGLN) in the epithelial–mesenchymal transition (EMT) of esophageal squamous cell carcinomas (ESCC) and its possible mechanism of inhibiting the invasion of these cancers.MethodsTissue specimens and clinical information of patients with ESCC were collected to analyze the relationship between Transgelin expression level and prognosis of patients with ESCC. Transgelin siRNA was used to knock down Transgelin expression. The expression of Transgelin in Eca-109 and KYSE-150 cells was overexpressed by Transgelin-overexpressing plasmid. The effects of Transgelin overexpression and knockdown on the proliferation of Eca-109 and KYSE-150 cells were examined by Transwell chamber, scratch assay, and CCK-8 cell activity assay. RT-PCR and Western blot were used to detect the effect of Transgelin overexpression or knockdown on the mRNA and protein expressions of E-cadherin and Vimentin. TCGA data were used to analyze Transgelin co-expressed genes and further study the GO and KEGG enrichment analysis results under the influence of Transgelin.ResultsThe expression of Transgelin was low in ESCC, and its expression level was positively correlated with the prognosis of patients with ESCC. The targeted Transgelin siRNA and Transgelin-overexpressing plasmid can effectively regulate the expression of Transgelin mRNA and protein in Eca-109 and KYSE-150 cells. After overexpression of Transgelin, the invasion and proliferation abilities of Eca-109 and KYSE-150 cells were significantly decreased compared with those of the control group (P < 0.05). However, Transgelin knockdown could promote the proliferation, migration, and invasion of ESCC cells. The overexpression of Transgelin inhibits EMT in ESCC. With the increase of Transgelin expression in Eca-109 and KYSE-150 cells, the expression of E-cadherin increased, while the expression of Vimentin decreased, and the difference was statistically significant (P < 0.05).ConclusionTransgelin can inhibit the malignant progression of ESCC by inhibiting the occurrence of EMT.