Exosomal miR­194 from adipose­derived stem cells impedes hypertrophic scar formation through targeting TGF­ß1.

Hypertrophic scars, which result from aberrant fibrosis and disorganized collagen synthesis by skin fibroblasts, emerge due to disrupted wound healing processes. These scars present significant psychosocial and functional challenges to affected individuals. The current treatment limitations largely arise from an incomplete understanding of the underlying mechanisms of hypertrophic scar development. Recent studies, however, have shed light on the potential of exosomal non­coding RNAs interventions to mitigate hypertrophic scar proliferation. The present study assessed the impact of exosomes derived from adipose­derived stem cells (ADSCs­Exos) on hypertrophic scar formation using a rabbit ear model. It employed hematoxylin and eosin staining, Masson's trichrome staining and immunohistochemical staining techniques to track scar progression. The comprehensive analysis of the present study encompassed the differential expression of non­coding RNAs, enrichment analyses of functional pathways, protein­protein interaction studies and micro (mi)RNA­mRNA interaction investigations. The results revealed a marked alteration in the expression levels of long non­coding RNAs and miRNAs following ADSCs­Exos treatment, with little changes observed in circular RNAs. Notably, miRNA (miR)­194 emerged as a critical regulator within the signaling pathways that govern hypertrophic scar formation. Dual­luciferase assays indicated a significant reduction in the promoter activity of TGF­ß1 following miR­194 overexpression. Reverse transcription­quantitative PCR and immunoblotting assays further validated the decrease in TGF­ß1 expression in the treated samples. In addition, the treatment resulted in diminished levels of inflammatory markers IL­1ß, TNF­α and IL­10. In vivo evidence strongly supported the role of miR­194 in attenuating hypertrophic scar formation through the suppression of TGF­ß1. The present study endorsed the strategic use of ADSCs­Exos, particularly through miR­194 modulation, as an effective strategy for reducing scar formation and lowering pro­inflammatory and fibrotic indicators such as TGF­ß1. Therefore, the present study advocated the targeted application of ADSCs­Exos, with an emphasis on miR­194 modulation, as a promising approach to managing proliferative scarring.

miRNAs in Signal Transduction of SMAD Proteins in Breast Cancer.

The aim of this study was to identify miRNAs that could potentially influence the activity of SMAD proteins involved in TGFß signal transduction in five types of breast cancer in Polish women. Patients with five breast cancer subtypes were included in the study: luminal A (n = 130), luminal B HER2- (n = 100), luminal B HER2+ (n = 96), non-luminal HER2+ (n = 36), and TNBC (n = 43). During surgery, tumor tissue was removed along with a margin of healthy tissue (control). Molecular analysis included determination of the expression of genes related to SMAD protein signal transduction using mRNA microarrays and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Protein expression was determined using an enzyme-linked immunosorbent assay (ELISA). The miRNA profiling was performed using miRNA microarrays and the miRDB database. SMAD3 and SMAD5 were overexpressed in all types of breast cancer, which could be related to the reduced expression of miR-145, and the findings for SMAD4 and miR-155 were similar. Additionally, the level of SMAD7 was reduced, which may be due to the low activity of miR-15b and miR21b. This study determined the gene expression profiles involved in SMAD protein signal transduction across five different types of breast cancer and identified the miRNAs potentially regulating their activity. Overexpression of SMAD3, SMAD4, and SMAD5 suggests excessive activation of the TGFß pathway, potentially promoting tumor growth and development. Concurrently, a significant reduction in SMAD7 expression removes inhibitory control in the TGFß pathway, a phenomenon that is particularly evident in more aggressive breast cancer types.

Inorganic Polyphosphate Is in the Surface of Trypanosoma cruzi but Is Not Significantly Secreted.

Trypanosoma cruzi is the etiologic agent of Chagas disease, an infection that can lead to the development of cardiac fibrosis, which is characterized by the deposition of extracellular matrix (ECM) components in the interstitial region of the myocardium. The parasite itself can induce myofibroblast differentiation of cardiac fibroblast in vitro, leading to increased expression of ECM. Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that can also induce myofibroblast differentiation and deposition of ECM components and is highly abundant in T. cruzi. PolyP can modify proteins post-translationally by non-enzymatic polyphosphorylation of lysine residues of poly-acidic, serine-(S) and lysine (K)-rich (PASK) motifs. In this work, we used a bioinformatics screen and identified the presence of PASK domains in several surface proteins of T. cruzi. We also detected polyP in the external surface of its different life cycle stages and confirmed the stimulation of host cell fibrosis by trypomastigote infection. However, we were not able to detect significant secretion of the polymer or activation of transforming growth factor beta (TGF-ß), an important factor for the generation of fibrosis by inorganic polyP- or trypomastigote-conditioned medium.

LTBP2 down-regulated FGF2 to repress vascular smooth muscle cell proliferation and vascular remodeling in a rat model of intracranial aneurysm.

This work probed into the role of latent transforming growth factor beta binding protein 2 (LTBP2) in intracranial aneurysm (IA). The rats underwent IA modeling and then stereotactic injection of short hairpin RNA against LTBP2 (shLTBP2). Hematoxylin-eosin (HE) staining was employed to assess IA model and vascular remodeling. Rat vascular smooth muscle cells (VSMCs) were transfected with shLTBP2, LTBP2 overexpression plasmid and fibroblast growth factor 2 (FGF2) overexpression plasmid. The mRNA and protein expressions of LTBP2, FGF2 and mitochondrial apoptosis-related factors (Caspase-3, Cyt-c, Mcl-1) were tested through qRT-PCR and Western blot. Cell viability, proliferation and apoptosis were examined by cell counting kit-8, EdU assay and flow cytometry. The up-regulated LTBP2 and down-regulated FGF2 were detected in IA rats. LTBP2 knockdown promoted vascular remodeling and Mcl-1 level, and restrained cell apoptosis and expressions of Caspase-3 and Cyt-c in IA model rats. Moreover, LTBP2 knockdown potentiated cell viability, proliferation and FGF2 level, and repressed apoptosis in rat VSMCs, while overexpressed LTBP2 exerted opposite effects. FGF2 overexpression promoted proliferation and Mcl-1 level, and inhibited apoptosis and expressions of Caspase-3 and Cyt-c in rat VSMCs, which also reversed the effects of overexpressed LTBP2 on these aspects. Collectively, LTBP2 down-regulates FGF2 to repress VSMCs proliferation and vascular remodeling in an IA rat model.

Association between TGF-ß/BMP signaling pathway polymorphisms and the risk of primary ovarian insufficiency in Korean women.

BACKGROUND: Primary ovarian insufficiency (POI) is one of the leading female infertility diseases in which ovarian function stops before the age of 40. Reports that POI is associated with transforming growth factor (TGF)-ß/bone morphogenetic protein (BMP) signaling pathway-associated genes (e.g., TGF-ß, and BMP15) have been continuous since publication that the TGF-ß superfamily acts as important regulators for ovary and placenta function in humans. Mechanistically, the secretion of follicle-stimulating hormone, progesterone, and estrogen is affected by the TGF-ß superfamily in granulosa cells, which are involved in the development of theca cells, oocytes, and granulosa cells. OBJECTIVE: This study aimed to identify the association between genes related to the TGF-ß/BMP signaling pathway and the risk of POI pathogenesis. METHODS: Possible associations between six gene polymorphisms and POI susceptibility were examined in 139 patients with POI and 345 control subjects. RESULTS: Allele combination of TGFBR1 rs334348 G > A and TGFBR3 rs1805110G > A exhibited association with decreased POI risk (adjusted odds ratio [AOR] = 0.165; 95% confidence interval [CI] 0.032-0.847; P = 0.031). Also, TGFBR1 rs1590 G > T and rs334348 G > A and TGFBR3 rs1805110 G > A allele combination exhibited association with decreased POI risk (OR = 0.553; 95% CI 0.374-0.816; P = 0.003). CONCLUSION: This study suggests that polymorphisms in the TGF-ß signaling pathway genes can be useful biomarkers for POI diagnosis and treatment.

TGF-ß1 overexpression in severe COVID-19 survivors and its implications for early-phase fibrotic abnormalities and long-term functional impairment.

Introduction: In post-COVID survivors, transforming growth factor-beta-1 (TGF-ß1) might mediate fibroblast activation, resulting in persistent fibrosis. Methods: In this study, 82 survivors of COVID-19-associated ARDS were examined at 6- and 24-months post-ICU discharge. At 6-months, quantitative CT analysis of lung attenuation was performed and active TGF-ß1 was measured in blood and exhaled breath condensate (EBC). Results: At 6-months of ICU-discharge, patients with reduced DmCO/alveolar volume ratio exhibited higher plasma and EBC levels of active TGF-ß1. Plasma TGF-ß1 levels were elevated in dyspneic survivors and directly related to the high-attenuation lung volume. In vitro, plasma and EBC from survivors induced profibrotic changes in human primary fibroblasts in a TGF-ß receptor-dependent manner. Finally, at 6-months, plasma and EBC active TGF-ß1 levels discriminated patients who, 24-months post-ICU-discharge, developed gas exchange impairment. Discussion: TGF-ß1 pathway plays a pivotal role in the early-phase fibrotic abnormalities in COVID-19-induced ARDS survivors, with significant implications for long-term functional impairment.

Berberine suppressed the epithelial-mesenchymal transition (EMT) of colon epithelial cells through the TGF-ß1/Smad and NF-κB pathways associated with miRNA-1269a.

Objective: To explore the mechanisms of the TGF-ß1/Smad and NF-κB pathways in the effect of berberine (BBR) on colon cancer epithelial-mesenchymal transition (EMT) and their regulatory relationships with microRNAs (miRNAs). Methods: TGF-ß1 was used to induce EMT in normal colon epithelial HCoEpiC cells and colon cancer HT29 cells in vitro. After BBR intervention, the expression of EMT-related markers and the major molecules involved in the TGF-ß1/Smad and NF-κB pathways were detected via western blotting. Cell migration was detected via wound healing assays. SMAD2 and NF-κB p65 were overexpressed and transfected into cells, and the inhibitors SB431542 and BAY 11-7082 were added to block the TGF-ß1/Smad and NF-κB pathways, respectively. The mRNA expression levels of related microRNA genes were detected by using RT‒PCR. Results: Treatment with 10 ng/ml TGF-ß1 for 72 h significantly induced EMT in HCoEpiC and HT29 cells, which was repressed by BBR. BBR significantly inhibited the TGF-ß1-induced migration of HCoEpiC and HT29 cells and the TGF-ß1-promoted expression of p-Smad2/3, NF-κB p65, and p-IκBα. Compared to those in the group treated with TGF-ß1, the expression of NF-κB p65 and p-Smad2 in the group treated with NF-κB pathway inhibitor BAY 11-7082 was decreased (P < 0.05), and TGF-ß1 signalling inhibitor SB431542 significantly reduced the expression of NF-κB p65 (P < 0.05). Overexpression of NF-κB p65 and SMAD2 in HT29 cells decreased the expression of E-cadherin and caused a relative increase in N-cadherin. BBR mediated the expression profile of microRNAs in TGF-ß1-induced HCoEpiC cells, but this pattern differed from that in HT29 cells. SB431542 and BAY 11-7082 significantly reduced the mRNA level of miR-1269a in HCoEpiC and HT29 cells (P < 0.05). Overexpressed NF-κB p65 and SMAD2 increased the mRNA level of miR-1269a in both cell lines; however, this increase was significantly lower than that in the TGF-ß1 treatment group (P < 0.05). Conclusion: BBR can significantly inhibit TGF-ß1-induced EMT in normal and cancerous colon epithelial cells through the inhibition of the TGF-ß1/Smad and NF-κB p65 pathways. TGF-ß1/Smads can promote the NF-κB p65 pathway, which is a common target of miR-1269a, and can partially regulate the expression of miR-1269a.

Correlation of Immunomodulatory Cytokines with Tumor Volume and Cerebrospinal Fluid in Vestibular Schwannoma Patients.

Sporadic vestibular schwannomas (VSs) often exhibit slow or negligible growth. Nevertheless, some VSs increase significantly in volume within a few months or grow continuously. Recent evidence indicates a role of inflammation in promoting VS growth. Therefore, our study aimed to identify cytokines, which are associated with larger VSs. The expression of different cytokines in VS tumor samples and VS primary cultures was investigated. Additionally, the concentration of cytokines in cell culture supernatants of VS primary cultures and cerebrospinal fluid (CSF) of VS patients and healthy controls were determined. Correlation analysis of cytokine levels with tumor volume, growth rate, Koos grade, age, and hearing was examined with Spearman's-rank test. The mRNA expression of CC-chemokine ligand (CCL) 18, growth differentiation factor (GDF) 15, and interferon regulatory factor 4 correlated positively with tumor volume. Moreover, the amount of GDF15 in the cell culture supernatant of primary cells correlated positively with tumor volume. The concentrations of the cytokines CCL2, CCL5, and CCL18 and transforming growth factor beta (TGFB) 1 in the CSF of the patients were significantly different from those in the CSF controls. Inhibition of immune cell infiltration could be a putative approach to prevent and control VS growth.

Picroside â ¡ alleviates renal fibrosis through YY1-dependent transcriptional inhibition of TGFß1.

Diabetic Nephropathy (DN) has become the leading cause of end-stage renal disease worldwide. Studies have indicated that Transforming Growth Factor beta1 (TGFß1) is the most potent factor contributing to renal fibrosis, and understanding the exact pathogenic mechanism of renal fibrosis is crucial for alleviating the condition. Previous research has identified Yin Yang 1 (YY1) as an effective inhibitor of TGF-ß1. Our study, through dual-luciferase reporter gene assays and Western blot experiments, screened and obtained the small molecule compound PdⅡ. Subsequently, validation in a high-glucose-induced renal mesangial cell injury model showed that PdⅡ treatment significantly increased the expression of YY1 protein and mRNA, while correspondingly reducing the expression of TGFß1 protein and mRNA. Dual-luciferase reporter gene assay results revealed that, compared to the control group, the luciferase transcription activity of YY1 molecules increased in the PdⅡ treatment group, and the luciferase transcription activity of TGFß1 decreased. By further designing mutations in the binding sites between TGFß1 and YY1 on the promoter, transfecting fluorescent enzyme reporter gene plasmids with TGFß1 mutant promoter into mesangial cells damaged by high glucose, and then treating the cells with PdⅡ, it was observed that the luciferase transcription activity of TGFß1 did not decrease. Therefore, these results suggest that PdⅡ may inhibit TGFß1 transcriptional activity by activating YY1, thereby slowing down the progression of diabetic nephropathy.

Unraveling the role of MiR-181 in skin fibrosis pathogenesis by targeting NUDT21.

Systemic sclerosis (SSc) is a life-threatening autoimmune disease characterized by widespread fibrosis in the skin and several internal organs. Nudix Hydrolase 21 (NUDT2 or CFIm25) downregulation in fibroblasts is known to play detrimental roles in both skin and lung fibrosis. This study aims to investigate the upstream mechanisms that lead to NUDT21 repression in skin fibrosis. We identified transforming growth factor ß (TGFß1) as the primary cytokine that downregulated NUDT21 in normal skin fibroblasts. In the bleomycin-induced dermal fibrosis model, consistent with the peak activation of TGFß1 at the late fibrotic stage, NUDT21 was downregulated at this stage, and delayed NUDT21 knockdown during this fibrotic phase led to enhanced fibrotic response to bleomycin. Further investigation suggested TGFß downregulated NUDT21 through microRNA (miRNA) 181a and 181b induction. Both miR-181a and miR-181b were elevated in bleomycin-induced skin fibrosis in mice and primary fibroblasts isolated from SSc patients, and they directly targeted NUDT21 and led to its downregulation in skin fibroblasts. Functional studies demonstrated that miR-181a and miR-181b inhibitors attenuated bleomycin-induced skin fibrosis in mice in association with decreased NUDT21 expression, while miR-181a and miR-181b mimics promoted bleomycin-induced fibrosis. Overall, these findings suggest a novel role for miR-181a/b in SSc pathogenesis by repressing NUDT21 expression.

Overexpression of DUSP26 gene suppressed the proliferation, migration, and invasion of human prostate cancer cells.

Prostate cancer (PCa) is threatening the health of millions of people, the pathological mechanism of prostate cancer has not been fully elaborated, and needs to be further explored. Here, we found that the expression of DUSP26 is dramatically suppressed, and a positive connection of its expression with PCa prognosis was also observed. In vitro, overexpression of DUSP26 significantly inhibited the proliferative, migrative, and invasive capacities of PC3 cells, DUSP26 silencing presented opposite results. Tumor formation experiments in subcutaneous nude mice demonstrated that DUSP26 overexpression could significantly suppress PC3 growth in vivo. Moreover, the mechanism of DUSP26 gene and PCa was discovered by RNA-Seq analysis. We found that DUSP26 significantly inhibited MAPK signaling pathway activation, and further experiments displayed that DUSP26 could impair TAK1, p38, and JNK phosphorylation. Interestingly, treatment with the TAK1 inhibitor (iTAK1) attenuated the effect of DUSP26 on PC3 cells. Together, these results suggested that DUSP26 may serve as a novel therapeutic target for PC3 cell type PCa, the underlying mechanism may be through TAK1-JNK/p38 signaling.

Hyperactivation of ATF4/TGF-ß1 signaling contributes to the progressive cardiac fibrosis in Arrhythmogenic cardiomyopathy caused by DSG2 Variant.

BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized with progressive cardiac fibrosis and heart failure. However, the exact mechanism driving the progression of cardiac fibrosis and heart failure in ACM remains elusive. This study aims to investigate the underlying mechanisms of progressive cardiac fibrosis in ACM caused by newly identified Desmoglein-2 (DSG2) variation. METHODS: We identified homozygous DSG2F531C variant in a family with 8 ACM patients using whole-exome sequencing and generated Dsg2F536C knock-in mice. Neonatal and adult mouse ventricular myocytes isolated from Dsg2F536C knock-in mice were used. We performed functional, transcriptomic and mass spectrometry analyses to evaluate the mechanisms of ACM caused by DSG2F531C variant. RESULTS: All eight patients with ACM were homozygous for DSG2F531C variant. Dsg2F536C/F536C mice displayed cardiac enlargement, dysfunction, and progressive cardiac fibrosis in both ventricles. Mechanistic investigations revealed that the variant DSG2-F536C protein underwent misfolding, leading to its recognition by BiP within the endoplasmic reticulum, which triggered endoplasmic reticulum stress, activated the PERK-ATF4 signaling pathway and increased ATF4 levels in cardiomyocytes. Increased ATF4 facilitated the expression of TGF-ß1 in cardiomyocytes, thereby activating cardiac fibroblasts through paracrine signaling and ultimately promoting cardiac fibrosis in Dsg2F536C/F536C mice. Notably, inhibition of the PERK-ATF4 signaling attenuated progressive cardiac fibrosis and cardiac systolic dysfunction in Dsg2F536C/F536C mice. CONCLUSIONS: Hyperactivation of the ATF4/TGF-ß1 signaling in cardiomyocytes emerges as a novel mechanism underlying progressive cardiac fibrosis in ACM. Targeting the ATF4/TGF-ß1 signaling may be a novel therapeutic target for managing ACM.

Blocking TSP1 Ameliorates Diabetes Mellitus-Induced Erectile Dysfunction by Inhibiting the TGF-ß/SMAD Pathway.

PURPOSE: To examine the role and mechanism of thrombospondin-1 (TSP1) in the development of fibrosis in diabetes mellitus-induced erectile dysfunction (DMED). MATERIALS AND METHODS: DMED was induced by intraperitoneal streptozotocin injection. All rats were categorized into three groups: control group (n=8), DMED group (n=8) and DMED+Leu-Ser-Lys-Leu (LSKL) group (n=8). After eight weeks following the induction of diabetes mellitus, the DMED+LSKL group was subjected to intraperitoneal injections of LSKL twice weekly for four weeks. To measure intracavernous pressure (ICP), a 25-gauge needle connected to a PE tube containing heparin was inserted into the corpus cavernosum (CC). Additionally, a needle was inserted into the carotid artery to measure mean arterial pressure (MAP). Sirius red staining and Masson trichrome staining were utilized to assess CC fibrosis. Moreover, high glucose (HG)-induced CC smooth muscle cells (CCSMCs) and CC fibroblasts (CCFs) were treated with or without LSKL. Western blotting and immunofluorescence were utilized to assess the phosphorylation and expression of related proteins. RESULTS: Compared with those in the control group, the ratio of the maximum ICP to the MAP markedly decreased in the DMED group, as did the ratio of smooth muscle to collagen and the ratio of collagen I to collagen III. These ratios were greater in the DMED+LSKL group than in the DMED group. TSP1 was highly expressed in the CC of DMED rats. In vitro experiments indicated that TSP1 expression significantly increased in the medium of CCSMCs and CCFs cultured in HG media and that the TGF-ß pathway was activated in CCSMCs. Collagen IV was overexpressed in CCSMCs, indicating severe fibrosis was severe. Adding LSKL or knocking TSP1 down can prevent the activation of TGF-ß signaling, as well as the overexpression of collagen IV in CCSMCs promoted by TSP1 secreted from CCSMCs itself or CCFs. CONCLUSIONS: TSP1 expression is increased in the CC of DMED rats. HG-induced TSP1 secretion via autocrine signaling from CCSMCs and/or paracrine signaling from CCFs to accelerate penile fibrosis. LSKL, an antagonist of TSP1, could improve erectile dysfunction by inhibiting the TGF-ß/SMAD pathway.

ΔNp63 bookmarks and creates an accessible epigenetic environment for TGFß-induced cancer cell stemness and invasiveness.

BACKGROUND: p63 is a transcription factor with intrinsic pioneer factor activity and pleiotropic functions. Transforming growth factor ß (TGFß) signaling via activation and cooperative action of canonical, SMAD, and non-canonical, MAP-kinase (MAPK) pathways, elicits both anti- and pro-tumorigenic properties, including cell stemness and invasiveness. TGFß activates the ΔNp63 transcriptional program in cancer cells; however, the link between TGFß and p63 in unmasking the epigenetic landscape during tumor progression allowing chromatin accessibility and gene transcription, is not yet reported. METHODS: Small molecule inhibitors, including protein kinase inhibitors and RNA-silencing, provided loss of function analyses. Sphere formation assays in cancer cells, chromatin immunoprecipitation and mRNA expression assays were utilized in order to gain mechanistic evidence. Mass spectrometry analysis coupled to co-immunoprecipitation assays revealed novel p63 interactors and their involvement in p63-dependent transcription. RESULTS: The sphere-forming capacity of breast cancer cells was enhanced upon TGFß stimulation and significantly decreased upon ΔNp63 depletion. Activation of TGFß signaling via p38 MAPK signaling induced ΔNp63 phosphorylation at Ser 66/68 resulting in stabilized ΔNp63 protein with enhanced DNA binding properties. TGFß stimulation altered the ratio of H3K27ac and H3K27me3 histone modification marks, pointing towards higher H3K27ac and increased p300 acetyltransferase recruitment to chromatin. By silencing the expression of ΔNp63, the TGFß effect on chromatin remodeling was abrogated. Inhibition of H3K27me3, revealed the important role of TGFß as the upstream signal for guiding ΔNp63 to the TGFß/SMAD gene loci, as well as the indispensable role of ΔNp63 in recruiting histone modifying enzymes, such as p300, to these genomic regions, regulating chromatin accessibility and gene transcription. Mechanistically, TGFß through SMAD activation induced dissociation of ΔNp63 from NURD or NCOR/SMRT histone deacetylation complexes, while promoted the assembly of ΔNp63-p300 complexes, affecting the levels of histone acetylation and the outcome of ΔNp63-dependent transcription. CONCLUSIONS: ΔNp63, phosphorylated and recruited by TGFß to the TGFß/SMAD/ΔNp63 gene loci, promotes chromatin accessibility and transcription of target genes related to stemness and cell invasion.

NOR1 promotes the osteoblastic differentiation of human periodontal ligament stem cells via TGF-ß signaling pathway.

Alveolar bone loss is a main manifestation of periodontitis. Human periodontal ligament stem cells (PDLSCs) are considered as optimal seed cells for alveolar bone regeneration due to its mesenchymal stem cell like properties. Osteogenic potential is the premise for PDLSCs to repair alveolar bone loss. However, the mechanism regulating osteogenic differentiation of PDLSCs remain elusive. In this study, we identified Neuron-derived orphan receptor 1 (NOR1), was particularly expressed in PDL tissue in vivo and gradually increased during osteogenic differentiation of PDLSCs in vitro. Knockdown of NOR1 in hPDLSCs inhibited their osteogenic potential while NOR1 overexpression reversed this effect. In order to elucidate the downstream regulatory network of NOR1, RNA-sequencing was used. We found that downregulated genes were mainly enriched in TGF-ß, Hippo, Wnt signaling pathway. Further, by western blot analysis, we verified that the expression level of phosphorylated-SMAD2/3 and phosphorylated-SMAD4 were all decreased after NOR1 knockdown. Additionally, ChIP-qPCR and dual luciferase reporter assay indicated that NOR1 could bind to the promoter of TGFBR1 and regulate its activity. Moreover, overexpression of TGFBR1 in PDLSCs could rescue the damaged osteogenic potential after NOR1 knockdown. Taken together, our results demonstrated that NOR1 could activate TGF-ß/SMAD signaling pathway and positively regulates the commitment of osteoblast lineages of PDLSCs by targeting TGFBR1 directly.

Androgens and androgen receptor directly induce the thickening, folding, and vascularization of the seahorse abdominal dermal layer into a placenta-like structure responsible for male pregnancy via multiple signaling pathways.

Seahorses exhibit the unique characteristic of male pregnancy, which incubates numerous embryos in a brood pouch that plays an essential role in enhancing offspring survivability. The pot-belly seahorse (Hippocampus abdominalis) possesses the largest body size among seahorses and is a significant species in Chinese aquaculture. In this study, we revealed the cytological and morphological characteristics, as well as regulatory mechanisms, throughout the entire brood pouch development in H. abdominalis. The brood pouch originated from the abdominal dermis, extending towards the ventral midline. As the dermal layers thicken, the inner epithelium folds, the stroma loosens, and vascularization occurs, culminating in the formation of the brood pouch. Furthermore, through transcriptomic analysis of brood pouches at various developmental stages, 8 key genes (tgfb3, fgf2, wnt7a, pgf, mycn, tln2, jund, ccn4) closely related to the development of brood pouch were identified in the MAPK, Rap1, TGF-ß, and Wnt signaling pathways. These genes were highly expressed in the pseudoplacenta and dermal layers at the newly formed stage as examined by in situ hybridization (ISH). The angiogenesis, densification of collagen fibers, and proliferation of fibroblasts and endothelial cells in seahorse brood pouch formation may be regulated by these genes and pathways. Additionally, the expression of the androgen receptor gene (ar) was significantly upregulated during the formation of the brood pouch, and ISH confirmed the expression of the ar gene in the dermis and pseudoplacenta of the brood pouch, highlighting its role in the developmental process. Androgen and flutamide (androgen receptor antagonist) treatments significantly accelerated the formation of the brood pouch and completely inhibited its occurrence respectively, concomitant to the upregulated expression of differentially expressed genes involved above signaling pathways. These findings demonstrated that formation of the brood pouch is determined by androgen and the androgen receptor activates the above signaling pathways in the brood pouch through the regulation of fgf2, tgfb3, pgf, and wnt7a. Interestingly, androgen even induced the formation of the brood pouch in females. We firstly elucidated the formation of the seahorse brood pouch, demonstrating that androgens and their receptors directly induce the thickening, folding, and vascularization of the abdominal dermal layer into a placenta-like structure through multiple signaling pathways. These findings provide foundational insights to further exploring the evolution of male pregnancy and adaptive convergence in viviparity across vertebrates.

RNA-seq transcriptomic profiling of TGF-ß2-exposed human trabecular meshwork explants: Advancing insights beyond conventional cell culture models.

Primary open-angle glaucoma (POAG), a leading cause of irreversible vision loss, is closely linked to increased intraocular pressure (IOP), with the trabecular meshwork (TM) playing a critical role in its regulation. The TM, located at the iridocorneal angle, acts as a sieve, filtering the aqueous humor from the eye into the collecting ducts, thus maintaining proper IOP levels. The transforming growth factor-beta 2 (TGF-ß2) signaling pathway has been implicated in the pathophysiology of primary open-angle glaucoma POAG particularly, in the dysfunction of the TM. This study utilizes human TM explants to closely mimic in vivo conditions, thereby minimizing transcriptional changes that could arise from cell culture enabling an exploration of the transcriptomic impacts of TGF-ß2. Through bulk RNA sequencing and immunohistological analysis, we identified distinct gene expression patterns and morphological changes induced by TGF-ß2 exposure (5 ng/ml for 48 h). Bulk RNA sequencing identified significant upregulation in genes linked to extracellular matrix (ECM) regulation and fibrotic signaling. Immunohistological analysis further elucidated the morphological alterations, including cytoskeletal rearrangements and ECM deposition, providing a visual confirmation of the transcriptomic data. Notably, the enrichment analysis unveils TGF-ß2's influence on both bone morphogenic protein (BMP) and Wnt signaling pathways, suggesting a complex interplay of molecular mechanisms contributing to TM dysfunction in glaucoma. This characterization of the transcriptomic modifications on an explant model of TM obtained under the effect of this profibrotic cytokine involved in glaucoma is crucial in order to develop and test new molecules that can block their signaling pathways.

Talabostat, fibroblast activation protein inhibitor, attenuates inflammation and fibrosis in systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disorder characterized by excessive fibrosis, where activated fibroblasts play a pivotal role in disease progression. This study aimed to investigate the potential of Talabostat, a small molecule inhibitor of dipeptidyl peptidases, in alleviating fibrosis and inflammation associated with SSc pathogenesis. METHODS: Dermal fibroblasts were obtained from skin biopsies of ten diffuse cutaneous SSc patients and healthy controls. These fibroblasts were subjected to treatment with either TGF-ß alone or in combination with Talabostat. Immunofluorescence staining was conducted to evaluate FAPα and α-SMA protein levels. The expression of activated fibroblast markers (FAPα and ACAT2), pro-fibrotic (COL1A1 and COL1A2), anti-fibrotic (MMP1, MMP2, and MMP9), and inflammatory (IL-6 and TGFß1) related genes was measured by quantitative real-time PCR. Talabostat-treated fibroblasts were assessed for their migratory capacity using a scratch assay and for their viability through MTT assay and Annexin V staining. RESULTS: The basal expression of COL1A1 and TGFß1 was notably higher in healthy subjects, while MMP1 expression showed a significant increase in SSc patients. Furthermore, TGF-ß stimulation led to upregulation of activated fibroblast markers, pro-fibrotic, and inflammatory-related genes in SSc-derived fibroblasts, which were attenuated upon Talabostat treatment. Interestingly, Talabostat treatment resulted in an upregulation of MMP9 expression. Moreover, Talabostat exhibited a concentration-dependent inhibition of activated fibroblast viability in both healthy and SSc fibroblasts, and suppressed fibroblast migration specifically in SSc patients. CONCLUSION: In summary, Talabostat modulates fibrotic genes in SSc, thereby inhibiting myofibroblast differentiation, activation, and migration. These findings suggest promising therapeutic avenues for targeting fibrosis in SSc.

ASPP2 Upregulation as a Novel Approach to TGF-ß2-Induced Proliferative Vitreoretinopathy In Vivo and In Vitro.

PURPOSE: Proliferative vitreoretinopathy (PVR) can cause blindness and the pathogenesis is unclear. Transforming growth factor (TGF)-ß-induced epithelial-mesenchymal transition (EMT) of RPE cells is vital. P53 protein 2 (ASPP2) was previously reported to inhibit EMT in PVR rats, but the specific mechanism is unveiled. METHODS: TGF-ß was used to induce EMT in ARPE-19 cells, and evaluated by immunofluorescence and western blot. ARPE-19 cells were transfected with scrambled/ASPP2-lentivirus, followed by TGF-ß treatment. After that, alterations of EMT and autophagy were measured by western blot and transmission electron microscopy. Moreover, TGF-ß and ARPE-19 cells treated with scrambled/ASPP2-lentivirus were employed to establish the PVR model via intravitreal injection to SD rats, and retinal changes as well as EMT and autophagy activity were evaluated accordingly. RESULTS: ASPP2 expression was decreased during TGF-ß-induced EMT in ARPE-19 cells. In vitro, EMT and autophagy was activated by TGF-ß, which could be partly reversed by ASPP2 upregulation. In vivo, ASPP2 upregulation protected against structural and functional changes in PVR retinas. Additionally, expressions of EMT and autophagy markers in retinas were inhibited by ASPP2 upregulation. CONCLUSIONS: ASPP2 upregulation inhibited the EMT and autophagy process caused by TGF-ß in ARPE-19 cells. Correspondingly, upregulation of ASPP2 alleviated intraocular fibrosis and protected visual function in PVR rats.

Silencing SPP1 in M2 macrophages inhibits the progression of castration-resistant prostate cancer via the MMP9/TGFß1 axis.

Background: M2 macrophages can promote the progression of castration-resistant prostate cancer (CRPC), but the specific mechanism is still unclear. Therefore, we are preliminarily exploring the molecular mechanism by which M2 macrophages regulate the progression of CRPC. Methods: The genes positively correlated with CRPC and with the most significant differences in the GEO32269 dataset were obtained. Database and immunofluorescence experiments were used to validate the localization of secreted phosphoprotein 1 (SPP1) in localized prostate cancer (PCa), hormone-sensitive prostate cancer (HSPC), and CRPC tumor tissues. The function of SPP1 in M2 macrophages was verified through cell scratch, Transwell, and an orthotopic PCa model. PCa database and Western blot were used to verify the relationship between SPP1 and matrix metallopeptidase 9 (MMP9), as well as the ability of MMP9 in M2 macrophages to promote epithelial-mesenchymal transition (EMT) in PCa cells. Results: The primary localization of SPP1 in prostate and CRPC tissues is in macrophages. Silencing SPP1 expression in M2 macrophages promotes their polarization towards the M1 phenotype and significantly inhibits the malignant progression of PCa in vitro and in vivo. SPP1 promotes the expression of MMP9 through the PI3K/AKT signaling pathway in M2 macrophages. Furthermore, MMP9 enhances the EMT and migratory capabilities of PC3 cells by activating the TGFß signaling pathway. Conclusions: We have found that the high expression of SPP1 in M2 macrophages promotes the progression of CRPC through cell-cell interactions. These findings can contribute to the development of novel therapeutic approaches for combating this deadly disease.