A20 in hepatic stellate cells suppresses chronic hepatitis by inhibiting DCLK1-JNK pathway-dependent chemokines.

Hepatic stellate cells (HSCs) are responsible for liver fibrosis accompanied by its activation into myofibroblasts and the abundant production of extracellular matrix. However, the HSC contribution to progression of liver inflammation has been less known. We aimed to elucidate the mechanism in HSCs underlying the inflammatory response and the function of tumor necrosis factor α-related protein A20 (TNFAIP3). We established A20 conditional knockout (KO) mice crossing Twist2-Cre and A20 floxed mice. Using these mice, the effect of A20 was analyzed in mouse liver and HSCs. The human HSC line LX-2 was also used to examine the role and underlying molecular mechanism of A20. In this KO model, A20 was deficient in >80% of HSCs. Spontaneous inflammation with mild fibrosis was found in the liver of the mouse model without any exogenous agents, suggesting that A20 in HSCs suppresses chronic hepatitis. Comprehensive RNA sequence analysis revealed that A20-deficient HSCs exhibited an inflammatory phenotype and abnormally expressed chemokines. A20 suppressed JNK pathway activation in HSCs. Loss of A20 function in LX-2 cells also induced excessive chemokine expression, mimicking A20-deficient HSCs. A20 overexpression suppressed chemokine expression in LX-2. In addition, we identified DCLK1 in the genes regulated by A20. DCLK1 activated the JNK pathway and upregulates chemokine expression. DCLK1 inhibition significantly decreased chemokine induction by A20-silencing, suggesting that A20 controlled chemokine expression in HSCs via the DCLK1-JNK pathway. In conclusion, A20 suppresses chemokine induction dependent on the DCLK1-JNK signaling pathway. These findings demonstrate the therapeutic potential of A20 and the DCLK1-JNK pathway for the regulation of inflammation in chronic hepatitis.

Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses.

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, it is unclear whether it plays a role in skin or skin appendage regeneration. Here, we aimed to elucidate Twist2 expression and its role in fetal wound healing. ICR mouse fetuses were surgically wounded on embryonic day 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction. Twist2 expression was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown on E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, Twist2 hetero knockout mice (Twist + /-) formed visible scars, even on E13, when all skin structures should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

Multiple rhabdomyomatous mesenchymal hamartomas in a patient with mosaic Barber-Say syndrome.

Barber-Say syndrome (BSS) is a rare congenital ectodermal dysplasia with few cases reported in the literature. We describe a 9-year-old boy with congenital generalized hypertrichosis and multiple rhabdomyomatous mesenchymal hamartomas (RMHs) on his nose and periocular region. Next-generation sequencing, performed in DNA from a blood sample, and RMH tissue, revealed a pathogenic variant in the TWIST2 gene, which was not detected in a salivary sample of the patient, nor in his parents. Therefore, we consider this variant as de novo mosaicism. To our knowledge, this is the first case of multiple RMHs associated with BSS.

Cryptophthalmos: associated syndromes and genetic disorders.

PURPOSE: Cryptophthalmos is a rare congenital condition caused by anomalous eyelid development where the eyelid folds do not develop or fail to separate. Cryptophthalmos can be unilateral or bilateral and can occur in isolation or as part of an underlying syndrome. We aim to identify genetic syndromes associated with cryptophthalmos to facilitate genetic diagnosis. METHODS: We performed a retrospective medical record review of all patients diagnosed with cryptophthalmos followed at a single center between 2000 and 2020. The analysis included medical history, clinical examination findings, and genetic testing results. RESULTS: Thirteen patients were included, 10 (77%) males, mean age of 2.4 years. Eight (61%) had bilateral cryptophthalmos, and 4 (31%) had complete cryptophthalmos. Associated ocular abnormalities included corneal opacities (13/13, 100%), upper eyelid colobomas (12/13, 92%), and microphthalmia/clinical anophthalmia (3/13, 23%). All cases of complete cryptophthalmos had bilateral disease. An underlying clinical or molecular diagnosis was identified in 10/13 (77%) cases, including Fraser syndrome (n = 5), amniotic band syndrome (n = 1), FREM1-related disease (n = 1), Goldenhar versus Schimmelpenning syndrome (n = 1), MOTA syndrome (n = 1), and CELSR2-related disease (n = 1). CONCLUSION: This is the first report of a possible association between cryptophthalmos and biallelic CELSR2 variants. Children with cryptophthalmos, especially those with extra-ocular involvement, should be referred for comprehensive genetic evaluation.

De novo triplication at 1p36.23p36.22 further refines the dosage sensitive region of overlap in Setleis syndrome (focal facial dermal dysplasia type III).

Setleis syndrome (SS), or focal facial dermal dysplasia type III (FFDD3, MIM #227260), is an autosomal recessive condition caused by biallelic loss-of-function variants in TWIST2. It is characterized by bitemporal atrophic skin lesions and distinctive facial features. Individuals with de novo or inherited duplication or triplication of the chromosomal region 1p36.22p36.21 have also been reported to have the SS phenotype with additional neurodevelopmental challenges (rarely seen in individuals with TWIST2 mutations) and variable expressivity and penetrance. Triplication of this region is also associated with more severe manifestations compared to a duplication. We report a 2-year-old female patient with features of SS associated with a de novo 3.603 Mb triplication at 1p36.23p36.22 identified on postnatal microarray analysis. Her triplication shares a 281.263 kb overlap with gains at 1p36.22, reported by previous groups, delineating the shortest region of overlap (SRO) to date. This SRO involves 10 RefSeq and 4 OMIM morbid map genes and highlights the candidate dosage-sensitive element(s) underlying the cardinal features of SS phenotype in individuals with gains at 1p36.

Identification of underexplored mesenchymal and vascular-related cell populations in human skeletal muscle.

Skeletal muscle repair and maintenance are directly and indirectly supported by interstitial cell populations such as vascular cells and fibro-adipogenic progenitors (FAPs), a subset of which express Twist2 and possess direct myogenic potential. Furthermore, work in rodents has highlighted the potential of pericytes to act as progenitor cells, giving rise to muscle cells and transdifferentiating into endothelial cells. However, less is understood about these populations in human skeletal muscle. Here, we performed single-cell RNA sequencing (scRNAseq) on ∼2,000 cells isolated from the human semitendinosus muscle of young individuals. This demonstrated the presence of a vascular-related cell type that expressed pericyte and pan-endothelial genes that we localized to large blood vessels within skeletal muscle cross sections and termed endothelial-like pericytes (ELPCs). RNA velocity analysis indicated that ELPCs may represent a "transition state" between endothelial cells and pericytes. Analysis of published scRNAseq data sets revealed evidence for ELPCs in trunk and heart musculature, which showed transcriptional similarity. In addition, we identified a subset of FAPs expressing TWIST2 mRNA and protein. Human TWIST2-expressing cells were anatomically and transcriptionally comparable to mouse Twist2 cells as they were restricted to the myofiber interstitium, expressed fibrogenic genes but lacked satellite cell markers, and colocalized with the FAPs marker PDGFRα in human muscle cross sections. Taken together, these results highlight the complexity of stromal cells residing in human skeletal muscle and support the utility of scRNAseq for discovery and characterization of poorly described cell populations.

Twist2-expressing cells reside in human skeletal muscle and are responsive to aging and resistance exercise training.

Skeletal muscle is maintained and repaired by sub-laminar, Pax7-expressing satellite cells. However, recent mouse investigations have described a second myogenic progenitor population that resides within the myofiber interstitium and expresses the transcription factor Twist2. Twist2-expressing cells exclusively repair and maintain type IIx/b muscle fibers. Currently, it is unknown if Twist2-expressing cells are present in human skeletal muscle and if they function as myogenic progenitors. Here, we perform a combination of single-cell RNA sequencing analysis and immunofluorescence staining to demonstrate the identity and localization of Twist2-expressing cells in human skeletal muscle. Twist2-expressing cells were identified to be anatomically and transcriptionally comparable to fibro-adipogenic progenitors (FAPs) and lack expression of typical satellite cell markers such as Pax7. Comparative analysis revealed that human and mouse Twist2-expressing cells were highly transcriptionally analogous and resided within the same anatomical structures in vivo. Examination of young and aged skeletal muscle biopsy samples revealed that Twist2-positive cells are more prevalent in aged muscle and increase following 12-weeks of resistance exercise training (RET) in humans. However, the quantity of Twist2-positive cells was not correlated with indices of muscle mass or muscle fiber cross-sectional area (CSA) in young or older muscle, and their abundance was surprisingly, negatively correlated with CSA and myonuclear domain size following RET. Taken together, we have identified cells expressing Twist2 in human skeletal muscle which are responsive to aging and exercise. Further examination of their myogenic potential is warranted.

Detection of Epithelial-Mesenchymal Transition Markers in High Grade Bladder Cancer and Special Variants of Urothelial Carcinoma.

Transitional cell carcinoma is considered the most predominant type of bladder cancer. Bladder can cer can also be found as squamous cell carcinoma that accounts for 5% of the total bladder cancer due to its etiology. The biomarkers associated with grade, prognosis, and stage of the disease are not well proved and known however, many studies have pointed to the association between SNAL/SLUG and Twist2 to the overall survival in patients with bladder cancer. These biomarkers were found to have a crucial role in inhibiting cadherin mediators specifically E-cadherin which are found normally in high level to integrate cell adhesion and normal function of the bladder. This research aims to detect SNAL/SLUG and Twist2 biomarkers in specimens of patients with bladder cancer and to detect their impact on E-cadherin, a tumor suppressor mediator responsible for improving survival and prevent metastasis. MATERIALS AND METHODS: Using 150 archival tissue blocks from human bladder cancer cases to detect expression of SNAIL/SLUG and Twist2 in relation to loss of E-cadherin by immunohistochemical method. RESULTS: Our results have revealed that in squamous cell carcinoma 40 specimens showed marked Twist 2 expression, and 30 specimens showed marked snail/slug biomarkers expression while poorly differentiated cancer cases showed marked expression of Twist 2 in 60 specimens and marked expression of Snail/slug marked expression in 50 specimens. Both were associated with E-cadherin loss. Among the 100 specimens with transitional cell carcinoma, 70 specimens showed divergent differentiation with 7 subtypes each showed different medium to high expression of Snail/Slug and Twist 2 biomarkers with the loss of E-cadherin. E-cadherin was strongly associated with the inverse increase in SNAL/SLUG and Twist2 biomarkers in urothelial carcinoma. CONCLUSION: Detection of SNAIL/SLUG and Twist 2 biomarkers in urothelial cancer is an important predictor for the loss of E-cadherin, a cornerstone in urinary bladder cell adhesion and its loss in urothelial carcinoma may contribute to cancer invasion and poor prognosis. 
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Twist2 Reduced NLRP3-Induced Inflammation of Infantile Pneumonia via Regulation of Mitochondrial Permeability Transition by FOXO1.

BACKGROUND: Infantile pneumonia is an acute inflammatory lesion of the lung caused by mycoplasma pneumonia. Indeed, Twist2 signaling pathway controls inflammatory reaction, oxidative stress, and other biological reaction. However, the regulation of Twist2 on the inflammation in infantile pneumonia remains unclear. This study explained that the function and mechanism of Twist2 in infantile pneumonia. METHODS: The subjects included the serum samples of 12 patients with infantile pneumonia and normal healthy volunteers from Hunan Children's Hospital. Besides, mice were given with lipopolysaccharide (LPS) into the lung. Moreover, RAW264.7 macrophages were stimulated with LPS for 4 h and added to the culture medium. RESULTS: In present study, in serum of patients with infantile pneumonia or lung tissue of mice model with infantile pneumonia, TWIST2 expression was lessened. Apart from that, TWIST2 protein could reduce the inflammatory reaction in mice model with infantile pneumonia, resulting in an inhibition in lung injury. Conversely, over-expression of TWIST2 also decreased inflammatory reaction in macrophages model via the regulation of FOXO1/NLRP3 pathway. Downregulation of TWIST2 promoted the inflammation in macrophages model by the regulation of FOXO1/NLRP3 pathway. CONCLUSION: According to the findings, present study have identified that the TWIST2 could reduce the inflammation of infantile pneumonia by NLRP3 inflammasome through the regulation of mitochondrial permeability transition and the induction of FOXO1 expression.

Twist2-driven chromatin remodeling governs the postnatal maturation of dermal fibroblasts.

Dermal fibroblasts lose stem cell potency after birth, which prevents regenerative healing. However, the underlying intracellular mechanisms are largely unknown. We uncover the postnatal maturation of papillary fibroblasts (PFs) driven by the extensive Twist2-mediated remodeling of chromatin accessibility. A loss of the regenerative ability of postnatal PFs occurs with decreased H3K27ac levels. Single-cell transcriptomics, assay for transposase-accessible chromatin sequencing (ATAC-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) reveal the postnatal maturation trajectory associated with the loss of the regenerative trajectory in PFs, which is characterized by a marked decrease in chromatin accessibility and H3K27ac modifications. Histone deacetylase inhibition delays spontaneous chromatin remodeling, thus maintaining the regenerative ability of postnatal PFs. Genomic analysis identifies Twist2 as a major regulator within chromatin regions with decreased accessibility during the postnatal period. When Twist2 is genetically deleted in dermal fibroblasts, the intracellular cascade of postnatal maturation is significantly delayed. Our findings reveal the comprehensive intracellular mechanisms underlying intrinsic postnatal changes in dermal fibroblasts.

Notch1 signaling in keratocytes maintains corneal transparency by suppressing VEGF expression.

The cornea fends off chemicals, dirt, and infectious particles and provides most of the eye's focusing power. Corneal transparency is of paramount importance to normal vision, yet how it is established and maintained remains unclear. Here, we ablated Notch1 in keratocytes using Twist2-Cre mice and found that Twist2-Cre; Notch1f/f mice developed stroma expansion and neovascularization, followed by hyperproliferation and metaplasia of corneal epithelial progenitor cells and plaque formation at central cornea, leading to loss of transparency. Development of these phenotypes does not involve bacteria-caused inflammation; instead, Notch1 deletion upregulates Vegfa and Vegfc via Hif1α in keratocytes. Vascular endothelial growth factor (VEGF) receptor inhibitor axitinib prevented development of these anomalies in Twist2-Cre; Notch1f/f mice, suggesting that VEGFs secreted by keratocytes promote not only neovascularization but also proliferation and metaplasia of epithelial progenitor cells at central cornea. This study uncovers a Notch1-Hif1α-VEGF pathway in keratocytes that maintains corneal transparency and represents a potential target for treatment of related corneal disorders.

TWIST2 inhibits EMT and induces oxidative stress in lung cancer cells by regulating the FGF21-mediated AMPK/mTOR pathway.

Twist related protein 2 (TWIST2) plays an important role in bone development, tumorigenesis, tumour progression and epithelial mesenchymal transition (EMT). At present, there are few reports about the role of TWIST2 in lung cancer, which need to be further explored. Therefore, the purpose of this study is to explore the role and molecular mechanism of TWIST2 in the occurrence and development of lung cancer. The expression of TWIST2 in tissues of patients and cell lines was measured using RT-qPCR and western blotting. MTT and CCK8 assays were used to detect cell proliferation and viability. Western blotting was used to measure the expression of EMT-related proteins, including E-cadherin, N-cadherin, Vimentin and Slug. The results revealed that TWIST2 is lowly expressed in the tissues of lung cancer patients and cell lines. Further studies found that overexpression of TWIST2 significantly induced apoptosis and promoted the expression of E-cadherin, as well as inhibiting the expression of N-cadherin, Vimentin and Slug. More importantly, TWIST2 induced oxidative stress in lung cancer cells. In addition, TWIST2 regulated the FGF21 and AMPK/mTOR signalling pathway, which is involved in the molecular mechanism of the gene in lung cancer cells. We suggest that the mechanism of TWIST2 inhibition of the progression of lung cancer is by regulating the FGF21-mediated AMPK/mTOR signalling pathway.

Zebrafish twist2/dermo1 regulates scale shape and scale organization during skin development and regeneration.

Scales are skin appendages in fishes that evolutionarily predate feathers in birds and hair in mammals. Zebrafish scales are dermal in origin and develop during metamorphosis. Understanding regulation of scale development in zebrafish offers an exciting possibility of unraveling how the mechanisms of skin appendage formation evolved in lower vertebrates and whether these mechanisms remained conserved in birds and mammals. Here we have investigated the expression and function of twist 2/dermo1 gene - known for its function in feather and hair formation - in scale development and regeneration. We show that of the four zebrafish twist paralogues, twist2/dermo1 and twist3 are expressed in the scale forming cells during scale development. Their expression is also upregulated during scale regeneration. Our knockout analysis reveals that twist2/dermo1 gene functions in the maintenance of the scale shape and organization during development as well as regeneration. We further show that the expression of twist2/dermo1 and twist3 is regulated by Wnt signaling. Our results demonstrate that the function of twist2/dermo1 in skin appendage formation, presumably under regulation of Wnt signaling, originated during evolution of basal vertebrates.

ZNF451 stabilizes TWIST2 through SUMOylation and promotes epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is the process by which epithelial cells lose their tightly packed polarized characteristics and acquire a migratory mesenchymal phenotype. EMT plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. The basic helix-loop-helix (bHLH) transcription factors TWIST1/2 are key EMT-inducing transcription factors that govern transcription of EMT-associated genes. Although regulation of TWIST1 activity and stability has been well studied, little is known about how TWIST2 is post-translationally regulated. Here we have identified ZNF451, a SUMO2/3 specific E3 ligase, as a novel regulator of TWIST2 in promoting its stability. ZNF451 directly binds to and SUMOylates TWIST2 at K129 residue, and consequently blocks ubiquitination and proteasome-dependent degradation of TWIST2. Ectopic expression of ZNF451 increases the protein level of TWIST2 in mammary epithelial cells, leading to increased expression of mesenchymal markers, whereas depletion of ZNF451 suppresses mesenchymal phenotypes. Collectively, our findings demonstrate that ZNF451 plays a vital role in EMT through SUMOylation-dependent stabilization of TWIST2.

TWIST2 and the PPAR signaling pathway are important in the progression of nonalcoholic steatohepatitis.

BACKGROUND: To investigate the roles of the transcription factors twist family bHLH transcription factor 1 (TWIST1), twist family bHLH transcription factor 2 (TWIST2), and peroxisome proliferator activated receptor gamma (PPARγ) in the progression of nonalcoholic steatohepatitis. METHODS: The protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of nonalcoholic fatty liver disease (NAFLD) patients and healthy controls by enzyme-linked immunosorbent assay (ELISA). An in vivo model for fatty liver was established by feeding C57BL/6 J mice a high-fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on untreated and OA-treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis. RESULTS: The TWIST2 serum protein levels decreased significantly in all fatty liver groups (P < 0.05), while TWIST1 varied. TWIST2 tended to be lower in mice fed an HFD and was significantly lower at 3 months. Similarly, in the in vitro model, the TWIST2 protein level was downregulated significantly at 48 and 72 h after OA treatment. RNA sequencing of LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with no obvious change in TWIST1 and PPARγ. The PPAR signaling pathway was enriched, with 4 genes upregulated in OA-treated cells (P = 0.0018). The interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways were enriched in OA-treated cells. CONCLUSIONS: The results provide evidence that the TWIST2 and PPAR signaling pathways are important in NAFLD and shed light on a potential mechanism of steatosis.

Expression Profiling Identifies TWIST2 Target Genes in Setleis Syndrome Patient Fibroblast and Lymphoblast Cells.

Background: Setleis syndrome (SS) is a focal facial dermal dysplasia presenting with bilateral temporal skin lesions, eyelash abnormalities and absent meibomian glands. SS is a rare autosomal recessive disorder caused by mutations in the TWIST2 gene, which codes for a transcription factor of the bHLH family known to be involved in skin and facial development. Methods: We obtained gene expression profiles by microarray analyses from control and SS patient primary skin fibroblast and lymphoblastoid cell lines. Results: Out of 983 differentially regulated genes in fibroblasts (fold change ≥ 2.0), 479 were down-regulated and 509 were up-regulated, while in lymphoblasts, 1248 genes were down-regulated and 73 up-regulated. RT-PCR reactions confirmed altered expression of selected genes. Conclusions: TWIST2 is described as a repressor, but expression profiling suggests an important role in gene activation as well, as evidenced by the number of genes that are down-regulated, with a much higher proportion of down-regulated genes found in lymphoblastoid cells from an SS patient. As expected, both types of cell types showed dysregulation of cytokine genes. These results identify potential TWIST2 target genes in two important cell types relevant to rare disorders caused by mutations in this bHLH gene.

LINC01235-TWIST2 feedback loop facilitates epithelial-mesenchymal transition in gastric cancer by inhibiting THBS2.

Although the anomalous expression of long non-coding RNAs (lncRNAs) has been extensively investigated in numerous carcinomas including gastric cancer (GC), their function remains unclear. The aim of our study was to explore the role of LINC01235 in GC. We used real-time quantitative PCR (RT-qPCR) to measure the expression of LINC01235 and twist family bHLH transcription factor 2 (TWIST2) in GC tissues. Scratch and transwell assays were performed to evaluate cellular capacity for migration and invasion. Gene relationships were explored by Weighted Gene Co-Expression Network Analysis (WGCNA). We measured TWIST2, thrombospondin 2 (THBS2) and epithelial-mesenchymal transition (EMT)-related proteins with western blot. We also used Pearson correlation analysis and the Kaplan-Meier method to detect associations among genes and overall survival. We found that LINC01235 was upregulated in GC tissues and cells. LINC01235 down-regulation restricted migration and invasion. Interestingly, we found the LINC01235-TWIST2-THBS2 axis induced EMT. Additionally, TWIST2 upregulated LINC01235 transcription in luciferase and chromatin immunoprecipitation (ChIP) assays. Bioinformatics analysis showed that microRNA (miR)-6852-5p might be a key gene involved in the regulation of TWIST2 by LINC01235. The LINC01235-TWIST2 positive feedback loop mainly affected migration and invasion of GC cells, which suggests it may serve as a potential therapeutic target in gastric cancer.

Periostin induces epithelial­to­mesenchymal transition via the integrin α5ß1/TWIST­2 axis in cholangiocarcinoma.

Periostin (PN) (also known as osteoblast­specific factor OSF­2) is a protein that in humans is encoded by the POSTN gene and has been correlated with a reduced survival of cholangiocarcinoma (CCA) patients, with the well­known effect of inducing epithelial­to­mesenchymal transition (EMT). The present study investigated the effect of PN, through integrin (ITG)α5ß1, in EMT­mediated CCA aggressiveness. The alterations in EMT­related gene and protein expression were investigated by real­time PCR, western blot analysis and zymogram. The effects of PN on migration and the level of TWIST­2 were assessed in CCA cells with and without siITGα5 transfection. PN was found to induce CCA cell migration and EMT features, including increments in Twist­related protein 2 (TWIST­2), zinc finger protein SNAI1 (SNAIL­1), α-smooth muscle actin (ASMA), vimentin (VIM) and matrix metallopeptidase 9 (MMP­9), and a reduction in cytokeratin 19 (CK­19) together with cytoplasmic translocation of E-cadherin (CDH­1). Additionally, PN markedly induced MMP­9 activity. TWIST­2 was significantly induced in PN­treated CCA cells; this effect was attenuated in the ITGα5ß1­knockdown cells and corresponded to reduced migration of the cancer cells. These results indicated that PN induced CCA migration through ITGα5ß1/TWIST-2­mediated EMT. Moreover, clinical samples from CCA patients showed that higher levels of TWIST­2 were significantly correlated with shorter survival time. In conclusion, the ITGα5ß1­mediated TWIST­2 signaling pathway regulates PN­induced EMT in CCA progression, and TWIST­2 is a prognostic marker of poor survival in CCA patients.

The renoprotective effects of soy protein in the aging rat kidney.

Aging is a risk factor for chronic kidney disease (CKD) and is itself associated with alterations in renal structure and function. There are no specific interventions to attenuate age-dependent renal dysfunction and the mechanism(s) responsible for these deficits have not been fully elucidated. In this study, male Fischer 344 rats, which develop age-dependent nephropathy, were feed a casein- or soy protein diet beginning at 16 mon (late life intervention) and renal structure and function was assessed at 20 mon. The soy diet did not significantly affect body weight, but was renoprotective as assessed by decreased proteinuria, increased glomerular filtration rate (GFR) and decreased urinary kidney injury molecule-1 (Kim-1). Renal fibrosis, as assessed by hydroxyproline content, was decreased by the soy diet, as were several indicators of inflammation. RNA sequencing identified several candidates for the renoprotective effects of soy, including decreased expression of Twist2, a basic helix-loop-helix transcription factor that network analysis suggest may regulate the expression of several genes associated with renal dysfunction. Twist2 expression is upregulated in the aging kidney and the unilateral ureteral obstruction of fibrosis; the expression is limited to distal tubules of mice. Taken together, these data demonstrate the renoprotective potential of soy protein, putatively by reducing inflammation and fibrosis, and identify Twist2 as a novel mediator of renal dysfunction that is targeted by soy.

Sema3a-Nrp1 Signaling Mediates Fast-Twitch Myofiber Specificity of Tw2+ Cells.

We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.