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This study provides comprehensive mechanistic evidence for the role of clusterin, a stress-response secretory chaperone protein, in the modulation of intraocular pressure (IOP) by regulating the trabecular meshwork (TM) actin cytoskeleton and the extracellular matrix (ECM). The pathological stressors on TM known to elevate IOP significantly lowered clusterin protein levels indicating stress-related clusterin function loss. Small interfering RNA-mediated clusterin loss in human TM cells in vitro induced actin polymerization and stabilization via protein kinase D1, serine/threonine-protein kinase N2 (PRK2), and LIM kinase 1 (LIMK1), and the recruitment and activation of adhesome proteins including paxillin, vinculin, and integrin αV and β5. A complete loss of clusterin as seen in clusterin knockout mice (Clu

CircRNA circ-PRDM5 (PR/SET domain 5) (circ-PRDM5) is overexpressed in age-related cataracts. Nevertheless, the biological role of circ-PRDM5 in posterior capsule opacities (PCO) (a common complication after cataract surgery) is unclear. Human lens epithelial cells SRA01/04 (LECs) were stimulated with TGF-β2 (transforming growth factor beta-2) to mimic the PCO model in vitro. Cell viability, migration, and invasion were determined by MTT, transwell, or wound-healing assays. Protein levels of EMT (epithelial-to-mesenchymal transition) markers and COL1A2 (collagen type I alpha 2 chain) were analyzed by western blotting (WB). Relative expression of circ-PRDM5, miR-92b-3p, and COL1A2 mRNA was analyzed by qRT-PCR. The targeting relationship was confirmed by dual-luciferase reporter and RIP assays. We observed that circ-PRDM5 and COL1A2 were upregulated in PCO tissues and TGF-β2-treated LECs, while miR-92b-3p was downregulated. Both circ-PRDM5 and COL1A2 knockdown impaired TGF-β2-induced LEC migration, invasion, and EMT. Also, circ-PRDM5 could adsorb miR-92b-3p to regulate COL1A2 expression. Furthermore, miR-92b-3p inhibitor offset circ-PRDM5 knockdown-mediated influence on migration, invasion, and EMT of LECs under TGF-β2 stimulation. Also, COL1A2 overexpression overturned the repressive influence of miR-92b-3p mimic on TGF-β2-induced LEC migration, invasion, and EMT. In summary, TGF-β2-induced circ-PRDM5 facilitated LEC migration, invasion, and EMT by adsorbing miR-92b-3p and increasing COL1A2 expression, offering new insights into the development of PCO.

Exosomal microRNAs (miRNAs) have been shown to be involved in the regulation of many disease progression, including proliferative vitreoretinopathy (PVR). However, the roles of exosomal miR-4488 and miR-1273 g-5p in PVR progression have not been demonstrated. Transforming growth factor β2 (TGF-β2)-induced ARPE-19 cells were used to stimulate the epithelial-mesenchymal transition (EMT) of cells. Exosomes derived from TGF-β2-induced ARPE-19 cells were identified by transmission electron microscopy and nanoparticle tracking analysis. The expression levels of miR-4488, miR-1273 g-5p and ATP-binding cassette A4 (ABCA4) were measured by quantitative real-time PCR. The promotion levels of exosomes markers, EMT markers, apoptosis markers and ABCA4 were determined by western blot analysis. The migration, invasion and apoptosis of cells were determined by transwell assay, wound healing assay and flow cytometry. Our data showed that miR-4488 and miR-1273 g-5p were lowly expressed in TGF-β2-induced ARPE-19 cells. Overexpressed exosomal miR-4488 and miR-1273 g-5p could inhibit the EMT, migration, invasion, and promote apoptosis in TGF-β2-induced ARPE-19 cells. In addition, ABCA4 was a target of miR-4488 and miR-1273 g-5p. Overexpressed ABCA4 also could reverse the negatively regulation of exosomal miR-4488 and miR-1273 g-5p on the EMT, migration, and invasion of TGF-β2-induced ARPE-19 cells. In conclusion, our data showed that exosomal miR-4488 and miR-1273 g-5p could inhibit TGF-β2-stimulated EMT in ARPE-19 cells through targeting ABCA4.

Epithelial-mesenchymal transition (EMT) occurs when polarised epithelial cells change to a mesenchymal phenotype. EMT plays a role in several chronic conditions, including ocular diseases with retinal pigment epithelium (RPE) EMT associated with retinal diseases such as diabetic retinopathy (DR). Here, EMT results in breakdown of the blood-retinal barrier (BRB) leading to sub-retinal fluid deposition and retinal detachment. Previous studies have shown that blocking connexin43 (Cx43) hemichannels can protect against RPE BRB breakdown, but the underlying mechanism is unknown. To determine whether open Cx43 hemichannels may enable EMT of RPE cells and thus result in BRB breakdown, ARPE-19 cells were either challenged with high glucose plus the inflammatory cytokines IL-1β and TNF-α (HG + Cyt) to simulate DR or treated with the Cx43 hemichannel blocker tonabersat alongside the HG + Cyt challenge. HG + Cyt induced a morphological change in RPE cells to a fibroblastic phenotype with a corresponding decrease in epithelial zonular occludens-1 and an increase in the fibroblastic marker α-SMA. The HG + Cyt challenge also induced loss of transepithelial electrical resistance while increasing dye passage between RPE cells. All of these changes were significantly reduced with tonabersat treatment, which also prevented HG + Cyt-induced transforming growth factor-β2 (TGF-β2) release. In conclusion, Cx43 hemichannel block with tonabersat attenuated both TGF-β2 release and RPE EMT under disease-mimicking conditions, offering the potential to ameliorate the progression of EMT-associated retinal diseases.

Opsin-3 (OPN3) is a potential key regulator of human melanocyte melanogenesis. How OPN3-mediated regulation of melanocyte melanogenesis is triggered is largely unclear. TGFβ can inhibit the growth of human melanocytes and reduce melanin synthesis in melanocytes. However, whether TGFβ2 can modulate pigmentation in normal human primary melanocytes through OPN3 is entirely unknown. In this study, we constructed a coculture model with human epidermal melanocytes and keratinocytes. OPN3, tyrosinase (TYR), tyrosinase-related protein (TRP)-1, and TRP-2 expression and TYR activity were detected to be higher in cocultured cells than in monocultured cells. Moreover, elevated levels of TGFβ2 were detected in the culture supernatant of melanocytes cocultured with keratinocytes. OPN3 inhibition in melanocytes decreased TYR, TRP-1, and TRP-2 expression and downregulated TYR activity. Our findings indicate that TGFβ2 upregulates TYR activity and TRP-1 and TRP-2 expression in human melanocytes through OPN3 and downstream calcium-dependent G-protein coupled signaling pathways to induce melanogenesis. Interestingly, treatment with the TGFβ2 receptor inhibitor LY2109761 (10 μM) did not inhibit TGFβ2-induced melanocyte melanogenesis though OPN3. Collectively, our data suggest that TGFβ2 upregulates TYR activity through OPN3 through a TGFβ2 receptor-independent and calcium-dependent G-protein coupled signaling pathway.

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes‐associated protein (YAP) and the subsequent epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE‐19 cells in vitro was studied. After treated with transforming growth factor‐β2(TGF‐β2), the expressions of fibrogenic molecules, YAP activation and the TGF‐β2‐Smad signalling pathway in ARPE‐19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF‐β2–induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF‐β2–induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF‐β2–induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA‐α) and fibronectin. YAP was essential for the TGF‐β2–induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.

Background Mesenchymal stem cells (MSCs) secrete paracrine factors and extracellular matrix proteins that contribute to their ability to support tissue healing and regeneration. Both the transcriptome and the secretome of MSCs can be altered by treating the cells with cytokines, but neither have been thoroughly investigated following treatment with the specific cytokine transforming growth factor (TGF)-β2. Methods RNA-sequencing and western blotting were used to compare gene and protein expression between untreated and TGF-β2-treated equine bone marrow-derived MSCs (BM-MSCs). A co-culture system was utilized to compare equine tenocyte migration during co-culture with untreated and TGF-β2-treated BM-MSCs. Results TGF-β2 treatment significantly upregulated gene expression of collagens, extracellular matrix molecules, and growth factors. Protein expression of collagen type I and tenascin-C was also confirmed to be upregulated in TGF-β2-treated BM-MSCs compared to untreated BM-MSCs. Both untreated and TGF-β2-treated BM-MSCs increased tenocyte migration in vitro. Conclusions Treating equine BM-MSCs with TGF-β2 significantly increases production of paracrine factors and extracellular matrix molecules important for tendon healing and promotes the migration of tenocytes in vitro.

Dermal papilla cells (DPCs) tend to aggregate both in vitro and in vivo, which increases the hair inductivity of DPCs. However, the underlying mechanism of spheroid formation is unknown. We investigated whether collagen expression in human DPCs (hDPCs) is involved in the spheroid formation and hair inductivity of hDPCs and further examined the underlying molecular mechanism of collagen upregulation. The expression of diverse collagens, such as COL13A1 and COL15A1, was upregulated in three dimensional (3D)-cultured or intact DPCs, compared to 2D-cultured hDPCs. This collagen expression was a downregulated in aged hair follicle, and aged DPCs were difficult to aggregate. Blocking of COL13A1 and COL15A1 by small interfering RNA reduced aggregation, while induced senescence of hDPCs in vitro. Further, transforming growth factor-β2 (TGF-β2) expression decreases with aging, and is involved in regulating the expression of COL13A1 and COL15A1. Addition of recombinant TGF-β2 delayed cellular senescence, and recovered spheroid formation in aged hDPCs by upregulating collagen levels. On the contrary, knock-out of TGF-β2 induced the aging of DPCs, and inhibited spheroid formation. These results suggested that COL13A1 and COL15A1 expression is downregulated with aging in DPCs, and upregulation of collagen by TGF-β2 induces the spheroid formation of DPCs. Therefore, TGF-β2 supplement in DPC culture medium could enhance the maintenance and hair inductivity of DPCs.

Primary open-angle glaucoma (POAG) is tightly related with extracellular matrix (ECM) remodeling of human trabecular meshwork cells (HTMCs). Transforming growth factor-β2 (TGF-β2) can induce ECM remodeling. The aim of the study was to investigate the microRNAs (miRNAs) expression changes of extracellular vesicles (EVs) derived from HTMCs treated with TGF-β2. EVs were isolated from HTMCs supernatant cultured for 24 h with TGF-β2. The morphology of EVs pellets was examined by transmission electron microscopy. Nanoparticle tracking analysis used to demonstrate the particle size distribution. Total EVs RNAs were extracted for subsequent miRNA gene chip analysis to investigate differentially expressed miRNAs between the controls and treatment cells. Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict potential target and validate possible functions of the miRNAs. There were 23 miRNAs upregulated and 3 miRNAs downregulated and 469,102, and 94 GO terms involved in biological processes, cellular components, and molecular function for the possible functions of the 26 miRNAs. These findings indicate that TGF-β2 may alter EVs miRNAs expression to participate in the pathogenesis of POAG. They may provide significant information for potential biomarkers for POAG diagnosis and treatment.

Background aims IL-2 is a potent cytokine that activates natural killer cells and CD8+ cytotoxic T lymphocytes (CTLs) and has been approved for the treatment of metastatic renal cell carcinoma and metastatic melanoma. However, the medical use of IL-2 is restricted because of its narrow therapeutic window and potential side effects, including the expansion of regulatory T cells (Tregs). Methods In this study, the authors investigated the complementary effects of transforming growth factor-β2 (TGF-β2) anti-sense oligodeoxynucleotide (TASO) on the immunotherapeutic potential of IL-2 in a melanoma-bearing humanized mouse model. Results The authors observed that the combination of TASO and IL-2 facilitated infiltration of CTLs into the tumor, thereby potentiating the tumor killing function of CTLs associated with increased granzyme B expression. In addition, TASO attenuated the increase in Tregs by IL-2 in the peripheral blood and spleen and also inhibited infiltration of Tregs into the tumor, which was partly due to decreased CCL22. Alteration of T-cell constituents at the periphery by TGF-β2 inhibition combined with IL-2 might be associated with the synergistic augmentation of serum pro-inflammatory cytokines (such as interferon γ and tumor necrosis factor α) and decreased ratio of Tregs to CTLs in tumor tissues, which consequently results in significant inhibition of tumor growth Conclusions These results indicate that the application of TASO improves IL-2-mediated anti-tumor immunity, thus implying that blockade of TGF-β2 in combination with IL-2 may be a promising immunotherapeutic strategy for melanoma.

We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

Transforming growth factor-β2 (TGFβ2)-mediated epithelial to mesenchymal transition (EMT) in lens epithelial cells (LECs) has been implicated in fibrosis associated with secondary cataracts. In this study, we investigated whether the receptor for advanced glycation end products (RAGE) plays a role in TGFβ2-mediated EMT in LECs. Unlike in the LECs from wild-type mice, TGFβ2 failed to elicit an EMT response in LECs from RAGE knockout mice. The lack of RAGE also diminished TGFβ2-mediated Smad signaling. In addition, treatment with TGFβ2 increased IL-6 levels in LECs from wild-type mice but not in those from RAGE knockout mice. Treatment of human LECs with the RAGE inhibitor FPS-ZM1 reduced TGFβ2-mediated Smad signaling and the EMT response. Unlike that in wild-type lenses, the removal of fiber cell tissue in RAGE knockout lenses did not result in elevated levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and integrin β1 in capsule-adherent LECs. Taken together, these results suggest that TGFβ2 signaling is intricately linked to RAGE. Targeting RAGE could be explored as a therapeutic strategy against secondary cataracts.

Epithelial–mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a key role in proliferative retinal diseases such as age-related macular degeneration by contributing to subretinal fibrosis. To investigate the potential role of retinoic acid receptor-α (RAR-α) signaling in this process, we have now examined the effects of the RAR-α agonist Am580 on EMT induced by transforming growth factor-β2 (TGF-β2) in primary mouse RPE cells cultured in a three-dimensional type I collagen gel as well as on subretinal fibrosis in a mouse model. We found that Am580 inhibited TGF-β2-induced collagen gel contraction mediated by RPE cells. It also attenuated the TGF-β2-induced expression of the mesenchymal markers α-smooth muscle actin, fibronectin, and collagen type I; production of pro-matrix metalloproteinase 2 and interleukin-6; expression of the focal adhesion protein paxillin; and phosphorylation of SMAD2 in the cultured RPE cells. Finally, immunofluorescence analysis showed that Am580 suppressed both the TGF-β2-induced translocation of myocardin-related transcription factor-A (MRTF-A) from the cytoplasm to the nucleus of cultured RPE cells as well as subretinal fibrosis triggered by laser-induced photocoagulation in a mouse model. Our observations thus suggest that RAR-α signaling inhibits EMT in RPE cells and might attenuate the development of fibrosis associated with proliferative retinal diseases.

The main characteristic of proliferative vitreoretinopathy (PVR) is migration, adhesion, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE). Eupatilin is a naturally occurring flavone that has the potential to inhibit cell proliferation and EMT. However, its efficacy on the PVR model induced by transforming growth factor-2 (TGF-β2) is unknown. In this study, the potential effect of eupatilin on proliferation and EMT in the treatment of RPE was investigated.Serum starved human RPE cells (ARPE-19) were treated with 10 ng/ml TGF-β2 alone or co-treated with 25 μM eupatilin for 48 h. Quantitative real-time PCR and Western blot analysis were used to assess targets at the mRNA and protein expression level, respectively. Apoptosis and cell cycle progression was assessed by image-based cytometry. The effect of treatment on cell migration was evaluated by wound healing assay.Eupatilin inhibited TGF-β2-induced RPE cell proliferation via regulating the cell cycle and inducing apoptosis. TGF-β2 upregulated mRNA expression of mesenchymal markers fibronectin and vimentin was significantly downregulated by the treatment, while the epithelial markers E-cadherin and occludin expression was upregulated. The therapy significantly suppressed TGF-β2 encouraged cell migration through downregulating the expression of transcription factors Twist, Snail, and ZEB1 induced by TGF-β2. Furthermore, eupatilin significantly inhibited the expression of MMP-1, -7, and -9, and suppressed NF-κB signalling.These results suggest that eupatilin could inhibit the proliferation and transformation into fibroblast-like cells of RPE cells; thus the agent may be a potential therapeutic value in treating PVR.

Transforming growth factor beta (TGF‐β) plays an important role in the viral liver disease progression via controlling viral propagation and mediating inflammation‐associated responses. However, the antiviral activities and mechanisms of TGF‐β isoforms, including TGF‐β1, TGF‐β2 and TGF‐β3, remain unclear. Here, we demonstrated that all of the three TGF‐β isoforms were increased in Huh7.5 cells infected by hepatitis C virus (HCV), but in turn, the elevated TGF‐β isoforms could inhibit HCV propagation with different potency in infectious HCV cell culture system. TGF‐β isoforms suppressed HCV propagation through interrupting several different stages in the whole HCV life cycle, including virus entry and intracellular replication, in TGF‐β/SMAD signalling pathway–dependent and TGF‐β/SMAD signalling pathway–independent manners. TGF‐β isoforms showed additional anti‐HCV activities when combined with each other. However, the elevated TGF‐β1 and TGF‐β2, not TGF‐β3, could also induce liver fibrosis with a high expression of type I collagen alpha‐1 and α‐smooth muscle actin in LX‐2 cells. Our results showed a new insight into TGF‐β isoforms in the HCV‐related liver disease progression.

Tooth germ development involves continuous and sequential steps with reciprocal interactions between odontogenic epithelium and the adjacent mesenchyme. Several growth factors, including Wnt, are essential for tooth germ development. Molecular mechanisms underlying Wnt/β-catenin-regulated tooth germ development are poorly understood. In tooth germ rudiments culture, we recently demonstrated that Semaphorin 3A (Sema3A), an axonal guidance factor, stimulation reversed Wnt/β-catenin signaling-dependent decreased cell proliferation but did not completely rescue the morphological anomalies of tooth germ, suggesting that an uncharacterized signaling pathway may be essential in Wnt/β-catenin signaling-dependent tooth germ development. Herein, an enrichment analysis using DNA microarray data, which was obtained in our previous research, revealed that Wnt/β-catenin signaling negatively regulates YAP1 and/or TGF-β signalings. In odontogenic epithelial cells and tooth germ rudiments, Wnt/β-catenin signaling activation reduced YAP1 expression, thereby suppressing YAP1 and TGF-β signalings sequentially. Additionally, YAP1 signaling induced TGF-β2 expression to promote TGF-β signaling in the cells. Finally, Wnt/β-catenin signaling-dependent disorganized tooth germ development, in which YAP1 signaling was suppressed, was reversed by TGF-β stimulation. These results suggest that Wnt/β-catenin signaling contributes to the tooth germ development through YAP1-TGF-β signaling.

Cumulus expansion is necessary for the release of a fertilizable oocyte from the ovary, which is critical for the normal fertilization of mammals. Cumulus expansion requires cooperation between epidermal growth factor (EGF)-like growth factors and oocyte paracrine factors. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are well-known paracrine factors secreted by oocytes. In addition, transforming growth factor-β2 (TGFB2) was primarily expressed in oocytes and its membrane receptors type 1 receptor (TGFBR1) and type 2 receptor (TGFBR2) were located in cumulus cells. In our present study, TGFB2 induced expansion of oocytectomized (OOX) complexes and increased the expression of expansion-related genes in the presence of EGF, suggesting that TGFB2 enables cumulus expansion. Inhibition of TGF-β signaling with SD208 blocked TGFB2-promoted cumulus expansion. Furthermore, in the culture of OOX complexes from mice of Tgfbr2-specific depletion in granulosa cells, TGFB2-promoted cumulus expansion and the expression of expansion-related genes were impaired. These results suggest that TGFB2 could induce cumulus expansion through TGFBR-SMAD2/3 signaling. Tgfb2-specific depletion in oocytes using Zp3-Cre mice had no effect on cumulus expansion in vivo, possibly due to the compensatory effect of other cumulus expansion-enabling factors. Taken together, TGFB2 is involved in expansion-related gene expression and consequent cumulus expansion.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein-protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)-AP3D1-CTGF- TGFβ2. We find that TGFβ2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFβ2 through an AP-3 (adaptor protein 3)-dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat-cargo interaction. Moreover, we find that TGFβ2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.

Radioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

The COVID-19 pandemic is an ongoing threat to public health. Since the identification of COVID-19, the disease caused by SARS-CoV-2, no drugs have been developed to specifically target SARS-CoV-2. To develop effective and safe treatment options, a better understanding of cellular mechanisms underlying SARS-CoV-2 infection is required. To fill this knowledge gap, researchers require reliable experimental systems that express the host factor proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, angiotensin-converting enzyme 2 (ACE2), and the proteases, transmembrane serine protease 2 (TMPRSS2) and furin. A number of studies have reported cell-type-specific expression of the genes encoding these molecules. However, less is known about the protein expression of these molecules. We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in an air-liquid interface (ALI) as an experimental system for studying SARS-CoV-2 infection in vitro. During cellular differentiation, we measured the expression of ACE2, TMPRSS2, and furin over progressive ALI days by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-β on the expression of these proteins in well-differentiated HBE cells. Like ACE2, TMPRSS2 and furin proteins are localized in differentiated ciliated cells, as confirmed by immunofluorescence staining. These data suggest that well-differentiated HBE cells maintained in ALI are a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that the profibrotic mediators, TGF-β1 and TGF-β2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.

Endothelial-to-mesenchymal transition (EndMT) and fibroblast-to-myofibroblast (FibroMF) differentiation are frequently reported in organ fibrosis. Stromelysin1, a matrix metalloprotease-3 (MMP3) has been indicated in vascular pathologies and organ injuries that often lead to fibrosis. In the current study, we investigated the role of stromelysin1 in EndMT and FibroMF differentiation, which is currently unknown. In our results, whereas TGFβ2 treatment of endothelial cells (ECs) induced EndMT associated with increased expression of stromelysin1 and mesenchymal markers such as α-smooth muscle actin (αSMA), N-cadherin, and activin linked kinase-5 (ALK5), inhibition of stromelysin1 blunted TGFβ2-induced EndMT. In contrast, treatment of NIH-3T3 fibroblasts with TGFβ1 promoted FibroMF differentiation accompanied by increased expression of αSMA, N-cadherin, and ALK5. Intriguingly, stromelysin1 inhibition in TGFβ1-stimulated myofibroblasts further exacerbated fibroproliferation with increased FibroMF marker expression. Gene Expression Omnibus (GEO) data analysis indicated increased stromelysin1 expression associated with EndMT and decreased stromelysin1 expression in human pulmonary fibrosis fibroblasts. In conclusion, our study has identified that EndMT and FibroMF differentiation are reciprocally regulated by stromelysin1.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes’ expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß‐2) was used to induce epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co‐cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT‐promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P

Aberrant remodeling of trabecular meshwork (TM) extracellular matrix (ECM) may induce ocular hypertensive phenotypes in human TM (hTM) cells to cause ocular hypertension, via a yet unknown mechanism. Here, we show that, in the absence of exogenous transforming growth factor-beta2 (TGFβ2), compared with control matrices (VehMs), glucocorticoid-induced cell-derived matrices (GIMs) trigger non-Smad TGFβ2 signaling in hTM cells, correlated with overexpression/activity of structural ECM genes (fibronectin, collagen IV, collagen VI, myocilin), matricellular genes (connective tissue growth factor [CTGF], secreted protein, acidic and rich in cysteine), crosslinking genes/enzymes (lysyl oxidase, lysyl oxidase-like 2–4, tissue transglutaminase-2), and ECM turnover genes/enzymes (matrix metalloproteinases-MMP2,14 and their inhibitors-TIMP2). However, in the presence of exogenous TGFβ2, VehMs and GIMs activate Smad and non-Smad TGFβ2 signaling in hTM cells, associated with overexpression of α-smooth muscle actin (α-SMA), and differential upregulation of aforementioned ECM genes/proteins with new ones emerging (collagen-I, thrombospondin-I, plasminogen activator inhibitor, MMP1, 9, ADAMTS4, TIMP1); with GIM-TGFβ2-induced changes being mostly more pronounced. This suggests dual glaucomatous insults potentiate profibrotic signaling/phenotypes. Lastly, we demonstrate type I TGFβ receptor kinase inhibition abrogates VehM-/GIM- and/or TGFβ2-induced upregulation of α-SMA and CTGF. Collectively, pathological TM microenvironments are sufficient to elicit adverse cellular responses that may be ameliorated by targeting TGFβ2 pathway.

Evidence indicates that long noncoding RNAs (lncRNAs) participate in the regulation of various physiological and pathological processes including organ fibrosis and eye-related diseases. The important pathological manifestations of open-angle glaucoma (OAG) are human trabecular meshwork cells (HTMCs) apoptosis and excessive deposition of extracellular matrix (ECM) components in TM, which can cause pathological changes in the outflow pathway. To investigate the role and regulation mechanism of lncRNA in HTMCs under oxidative stress, we established an oxidative stress model in HTMCs using hydrogen peroxide (H2O2) followed by RNA sequencing and found that subsets of lncRNAs and mRNAs that closely associate with TGF-β signaling are differentially regulated in these cells. We then constructed a network with the TGF-β2 -colocalized and -coexpressed lncRNAs, to investigate the effects and regulatory mechanisms of the potential lncRNAs on ECM deposition in HTMCs. The gain-of-function and loss-of-function experiments demonstrated that lnc-TGFβ2-AS1 promotes ECM production via TGF-β2 in HTMC, suggesting that lnc-TGFβ2-AS1 may be a potential glaucoma treatment target.

TGFβ2 is an essential regulator of immune cell functionality, but the mechanisms whereby it drives immune infiltration in gastric cancer remain uncertain. The Oncomine and Tumor Immunoassay Resource (TIMER) databases were used for assessing the expression of TGFβ2, after which TIMER was used to explore the relationship between TGFβ2 and tumour immune infiltration. Finally, we assessed how TGFβ2 expression correlated with the expression of a set of marker genes associated with immune infiltration using TIMER and GEPIA. We determined TGFβ2 expression to be significantly correlated with outcome in multiple types of cancer in the Cancer Genome Atlas (TCGA), with the effect being particularly pronounced in gastric cancer. Furthermore, elevated TGFβ2 expression was found to be significantly correlated with gastric cancer N staging, and with the expression of a variety of immune markers associated with particular immune cell subsets. These results indicate that TGFΒ2 is associated with patient outcome and tumour immune cell infiltration in multiple cancer types. This suggests that TGFβ2 is a key factor which governs immune cell recruitment to gastric cancer tumours, potentially playing a vital role in governing immune cell infiltration and thus representing a valuable prognostic biomarker in gastric cancer patients.

Bronchopulmonary dysplasia (BPD) is a major cause of mortality and morbidity in premature infants, characterized by alveolar simplification, surfactant deficiency, and respiratory distress. In the present study, we have investigated the functional roles of sumoylated CCAAT/enhancer binding protein alpha (C/EBPα) in the BPD rat model. A significant increase in small ubiquitin‐like modifier 1 (SUMO1) and sumoylated C/EBPα protein levels were observed in BPD rats, and the levels of the sumoylated C/EBPα were associated with the pulmonary surfactant proteins (SPs). In order to confirm the role of sumoylated C/EBPα in BPD rats, SUMO1 was knocked down by lentiviral transfection of neonatal rat lungs with SUMO1‐RNAi‐LV. We found that the expression of C/EBPα and surfactant proteins increased following SUMO1 knockdown. Furthermore, the relatively low decrease in the levels of C/EBPα sumoylation was correlated with reduced glycogen consumption. Besides, co‐immunoprecipitation assays revealed that sumoylation is involved in the regulation of the interaction between C/EBPα and TGFβ2 in the lung. In conclusion, our findings indicate that sumoylation may act as a negative regulator of the C/EBPα‐mediated transactivation in BPD rats.

Building a left-right (L-R) asymmetric organ requires asymmetric information. This comes from various sources, including asymmetries in embryo-scale genetic cascades (including the left-sided Nodal cascade), organ-intrinsic mechanical forces, and cell-level chirality, but the relative influence of these sources and how they collaborate to drive asymmetric morphogenesis is not understood. During zebrafish heart development, the linear heart tube extends to the left of the midline in a process known as jogging. The jogged heart then undergoes dextral (i.e. rightward) looping to correctly position the heart chambers relative to one another. Left lateralized jogging is governed by the left-sided expression of Nodal in mesoderm tissue, while looping laterality is mainly controlled by heart-intrinsic cell-level asymmetries in the actomyosin cytoskeleton. The purpose of lateralized jogging is not known. Moreover, after jogging, the heart tube returns to an almost midline position and so it is not clear whether or how jogging may impact the dextral loop. Here, we characterize a novel loss-of-function mutant in the zebrafish Nodal homolog southpaw (spaw) that appears to be a true null. We then assess the relationship between jogging and looping laterality in embryos lacking asymmetric Spaw signals. We found that the probability of a dextral loop occurring, does not depend on asymmetric Spaw signals per se, but does depend on the laterality of jogging. Thus, we conclude that the role of leftward jogging is to spatially position the heart tube in a manner that promotes robust dextral looping. When jogging laterality is abnormal, the robustness of dextral looping decreases. This establishes a cooperation between embryo-scale Nodal-dependent L-R asymmetries and organ-intrinsic cellular chirality in the control of asymmetric heart morphogenesis and shows that the transient laterality of the early heart tube has consequences for later heart morphogenetic events.

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open-angle glaucoma patients compared to non-glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti-apoptotic and anti-TGF-β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF-β2-induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride-induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF-α-induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF-β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.

To explore the effects of transforming growth factor-β2 (TGF-β2) and TGF-β1 on the odontogenic and osteogenic differentiation of mesenchymal stem cells (MSCs).We used lentiviral transduction to knock down TGF-β1 or TGF-β2 in stem cells from dental apical papilla (SCAPs), and to generate bone marrow mesenchymal stem cells (BMSCs) with overexpression of TGF-β1 or TGF-β2. We investigated the odontogenic and osteogenic differentiation abilities of these transductants in vitro and in vivo.In vitro, TGF-β2 knockdown in SCAPs reduced the expression of odontoblast-related markers DSPP and DMP-1, and increased the expression of osteoblast-related markers OCN and RUNX-2. Conversely, TGF-β1 knockdown had the opposite effects. TGF-β2 overexpression promoted expression of odontoblast-related markers in BMSCs at early differentiation, but inhibited the expression of odontoblast-related markers at later stages. TGF-β2 overexpression attenuated expression of osteogenic-related markers in BMSCs, while TGF-β1 overexpression enhanced odontoblast-related and osteoblast-related markers. SCAP or BMSC transductants were transplanted underneath kidneys in vivo. Masson staining showed that knockdown of TGF-β1, but not TGF-β2 promoted the expression of type I collagen in SCAPs. Immunohistochemical staining showed that TGF-β2 knockdown inhibited DSPP expression in SCAPs, but TGF-β1 knockdown had no obvious effect on DSPP expression. In vivo, TGF-β1 overexpression and TGF-β2 overexpression had no effect on the expression of type I collagen and DSPP in BMSCs.TGF-β2 promotes odontogenic differentiation of SCAPs and attenuates osteogenic differentiation of SCAPs and BMSCs. TGF-β1 promotes osteogenic differentiation of BMSCs and plays a complex role in regulating odontogenic differentiation of MSCs.

Astrocytes within the optic nerve head undergo actin cytoskeletal rearrangement early in glaucoma, which coincides with astrocyte reactivity and extracellular matrix (ECM) deposition. Elevated transforming growth factor beta 2 (TGFβ2) levels within astrocytes have been described in glaucoma, and TGFβ signaling induces actin cytoskeletal remodeling and ECM deposition in many tissues. A key mechanism by which astrocytes sense and respond to external stimuli is via mechanosensitive ion channels. Here, we tested the hypothesis that inhibition of mechanosensitive channels will attenuate TGFβ2-mediated optic nerve head astrocyte actin cytoskeletal remodeling, reactivity, and ECM deposition. Primary optic nerve head astrocytes were isolated from C57BL/6J mice and cell purity was confirmed by immunostaining. Astrocytes were treated with vehicle control, TGFβ2 (5 ng/ml), GsMTx4 (a mechanosensitive channel inhibitor; 500 nM), or TGFβ2 (5 ng/ml) + GsMTx4 (500 nM) for 48 h. FITC-phalloidin staining was used to assess the formation of f-actin stress fibers and to quantify the presence of crosslinked actin networks (CLANs). Cell reactivity was determined by immunostaining and immunoblotting for GFAP. Levels of fibronectin and collagen IV deposition were also quantified. Primary optic nerve head astrocytes were positive for the astrocyte marker GFAP and negative for markers for microglia (F4/80) and oligodendrocytes (OSP1). Significantly increased %CLAN-positive cells were observed after 48-h treatment with TGFβ2 vs. control in a dose-dependent manner. Co-treatment with GsMTx4 significantly decreased %CLAN-positive cells vs. TGFβ2 treatment and the presence of f-actin stress fibers. TGFβ2 treatment significantly increased GFAP, fibronectin, and collagen IV levels, and GsMTx4 co-treatment ameliorated GFAP immunoreactivity. Our data suggest inhibition of mechanosensitive channel activity as a potential therapeutic strategy to modulate actin cytoskeletal remodeling within the optic nerve head in glaucoma.

To establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

Transforming growth factor‑β2 (TGF‑β2) has been implicated in the pathogenesis of proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), due to its ability to stimulate the overproduction of pro‑angiogenic factors, such as vascular endothelial growth factor (VEGF), and remodeling of the extracellular matrix (ECM). Although intravitreal triamcinolone acetonide (TA) is clinically useful in the treatment of PVR and PDR, its molecular mechanism has yet to be fully elucidated. The present study investigated whether TA treatment altered TGF‑β2‑driven biological effects on the behavior of cultured human retinal pigment epithelial (RPE) cells, in order to determine which signaling pathway may be essential for the pharmacological action of TA. The R‑50 human RPE cell line was treated with TA in the presence of TGF‑β2, followed by analyses of cell viability and contraction using cell viability and collagen gel contraction assays. VEGF mRNA expression and protein production were measured using reverse transcription‑quantitative PCR and ELISA, respectively. The phosphorylation status of signaling mediators and the protein expression of type I collagen (COL1A1), α‑smooth muscle actin (α‑SMA), and ECM‑remodeling enzymes, including MMP‑2 and MMP‑9, were analyzed using western blotting. The gelatinolytic activity of MMPs was detected using gelatin zymography. TA treatment exhibited no prominent cytotoxicity but markedly antagonized TGF‑β2‑induced cytostatic effects on RPE cell viability and TGF‑β2‑enhanced contractility in collagen gels. In the context of TGF‑β2‑related signaling, TA significantly attenuated TGF‑β2‑elicited Smad2, extracellular‑regulated kinase (ERK)1/2 and p38 mitogen‑activated protein kinase (MAPK) phosphorylation. Moreover, TA markedly mitigated TGF‑β2‑induced VEGF upregulation through ablation of p38 signaling activity. TA also partially attenuated TGF‑β2‑elicted expression of COL1A1, α‑SMA, MMP‑2, and MMP‑9, but only suppressed TGF‑β2‑induced MMP‑9 gelatinolytic activity. Mechanistically, the MEK/ERK signaling pathway may have a critical role in the TGF‑β2‑induced upregulation of COL1A1, α‑SMA and MMP‑9. In conclusion, TA may be considered a useful therapeutic agent for treating TGF‑β2‑associated intraocular angiogenesis and tissue remodeling, the underlying mechanism of which may involve the ERK and p38 MAPK signaling pathways.

Osteonecrosis of the femoral head (ONFH) is a pathological process that initially occurs in the weight-bearing field of the femoral head. Due to the unknown pathogenesis, this study was for the investigation of the effect of microRNA-141 (miR-141) targeting transforming growth factor-β2 (TGF-β2) on regulating osteoblast activity and osteoclast activity in steroid-induced ONFH. Tissues of ONFH and normal femoral head were collected for detecting the expression of miR-141 and TGF-β2. A rat model of ONFH was constructed by injection of hormones, and transfected with miR-141 inhibitors and overexpressed TGF-β2. The apoptosis of bone cells was detected by TUNEL staining. The expression of osteoprotegerin (OPG), osteoprotegerin ligand (OPGL), Bcl-2, Bax, Runx2, BMP2 and RANK were detected. Highly expressed miR-141 and lowly expressed TGF-β2 existed in femoral head tissues in ONFH. Inhibited miR-141 resulted in elevated TGF-β2 in femoral head tissues in ONFH of rats. Depressed miR-141 or overexpressed TGF-β2 inhibited the apoptosis of bone cells of rats with ONFH and induced elevated OPG, Bcl-2, BMP2, Runx2 and declined OPGL, Bax and RANK expression in the femoral head tissues of rats with ONFH. Altogether, we find that down-regulated miR-141 promotes osteoblast activity and inhibits osteoclast activity to ameliorate ONFH via up-regulated TGF-β2 expression.

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF‐β2–induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co‐localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p‐KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF‐β2–induced EMT process, we found that p‐KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p‐KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p‐KRT8 in the prevention and treatment of PVR.

Hair regenerative medicine, a promising strategy for the treatment of hair loss, will likely involve the transplantation of autologous hair follicular stem cells (HFSCs) and dermal papilla cells (DPCs) into regions of hair loss. Cyclic hair regeneration results from the periodic partial activation of HFSCs. However, previous studies have not successfully achieved large-scale HFSC expansion in vitro without the use of feeder cells, with a lack of research focused on regulating HFSC fate for hair follicular (HF) regeneration. Hence, an emerging focus in regenerative medicine is the reconstruction of natural extracellular matrix (ECM) regulatory characteristics using biomaterials to generate cellular microenvironments for expanding stem cells and directing their fate for tissue regeneration. Methods: HFSCs were coated with gelatin and alginate using layer-by-layer (LbL) self-assembly technology to construct biomimetic ECM for HFSCs; after which transforming growth factor (TGF)-β2 was loaded into the coating layer, which served as a sustained-release signal molecule to regulate the fate of HFSCs both in vitro and in vivo. In vitro experiments (cell culture and siRNA) were employed to investigate the molecular mechanisms involved and in vivo implantation was carried out to evaluate hair induction efficiency. Results: Nanoscale biomimetic ECM was constructed for individual HFSCs, which allowed for the stable amplification of HFSCs and maintenance of their stem cell properties. TGF-β2 loading into the coating layer induced transformation of CD34+ stem cells into highly proliferating Lgr5+ stem cells, similar to the partial activation of HFSCs in HF regeneration. Thus, LbL coating and TGF-β2 loading partially reconstructed the quiescent and activated states, respectively, of stem cells during HF regeneration, thereby mimicking the microenvironment that regulates stem cell fate for tissue regeneration during HF cycling. Improved HF regeneration was achieved when the two HFSC states were co-transplanted with neonatal mouse dermal cells into nude mice. Conclusion: This study provides novel methods for the construction of stem cell microenvironments and experimental models of HF regeneration for the treatment of hair loss.

Osteoarthritis (OA) is a progressive joint disorder that is primarily characterized by the degeneration and destruction of the articular cartilage. Cartilage matrix degradation, production of proinflammatory mediators, chondrocyte apoptosis and activation of macrophages in the synovial are involved in OA pathogenesis. Current non-surgical therapies for OA mainly aim at relieving pain but can barely alleviate the progression of OA. Quercetin, a naturally occurring flavonoid has shown potent anti-inflammatory effects, however, its effects and underlying mechanisms on OA have seldom been systematically illuminated. In this study, we explored the protective effects of quercetin on repairing OA-induced cartilage injuries and its possible mechanisms. In vitro, quercetin remarkably suppressed the expression of matrix degrading proteases and inflammatory mediators, meantime promoted the production of cartilage anabolic factors in interleukin-1β-induced (IL-1β) rat chondrocytes. In addition, quercetin exhibited anti-apoptotic effects by decreasing intracellular reactive oxygen species (ROS), restoring mitochondrial membrane potential (MMP) and inhibiting the Caspase-3 pathway in apoptotic rat chondrocytes. Moreover, quercetin induced M2 polarization of macrophages and upregulated the expression of transforming growth factor β (TGF-β) and insulin-like growth factor (IGF), which in turn created a pro-chondrogenic microenvironment for chondrocytes and promoted the synthesis of glycosaminoglycan (GAG) in chondrocytes. In vivo, intra-articular injection of quercetin alleviated the degradation of the cartilage and the apoptosis of chondrocytes in a rat OA model. Moreover, the expression of TGF-β1 and TGF-β2 in the synovial fluid and the ratio of M2 macrophages in the synovial membrane were elevated. In summary, our study proves that quercetin exerts chondroprotective effects by inhibiting inflammation and apoptosis of chondrocytes, modulating synovial macrophages polarization to M2 macrophages and creating a pro-chondrogenic environment for chondrocytes to enhance cartilage repair under OA environment. It is suggested that quercetin may serve as a potential drug for OA treatment.

Macular fibrosis is a vital obstacle of vision acuity improvement of age‐related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL‐2) on epithelial‐mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF‐β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL‐2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α‐smooth muscle actin (α‐SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL‐1), TGF‐β2, and the activation of the JAK/STAT3 and NF‐κB signaling pathway. Furthermore, JAK/STAT3 and NF‐κB signaling pathways were specifically blocked by WP1066 or BAY11‐7082, respectively, and the expression of α‐SMA, COL‐1, Fn and TGF‐β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL‐2 with or without WP1066 or BAY11‐7082. After induction of IL‐2, the expressions of Fn, COL‐1, TGF‐β2 protein were significantly increased, and this effect was correlated with IL‐2 treatment duration, while α‐SMA protein expression did not change significantly. Both WP1066 and BAY11‐7082 could effectively downregulate the expression of Fn, COL‐1 and TGF‐β2 induced by IL‐2. What's more, both NF‐κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF‐κB inhibitor BAY 11‐7082 could obviously decrease RPE cells migration capability induced by IL‐2. IL‐2 promotes cell migration, ECM synthesis and TGF‐β2 expression in RPE cells via JAK/STAT3 and NF‐κB signaling pathways, which may play an important role in proliferative vitreoretinopathy., The study presented the non‐canonical effects of IL‐2 on the migration, mechanisms of extracellular matrix synthesis as well as the effects of TGF‐β2 expression in RPE cells via NF‐κB and JAK/STAT3 signaling pathway.

Transforming growth factor beta is a key cytokine involved in the pathogenesis of fibrosis in many organs, whereas interleukin-6 plays an important role in the regulation of inflammation. They are both potent angiogenesis inducers with opposite effects on cell survival and apoptosis. TGF-β2 induces apoptosis; in contrast, IL-6 protects cells from apoptosis. The possible interaction between these two cytokines is indicated in various disease states. In this study, we have assessed the effect of TGF-β2 on IL-6 signaling and found that TGF-β2 could strongly inhibit IL-6-induced STAT3 activation and synergy with IL-6 resulting in enhanced SOCS3 expression. Interestingly, IL-6 also slows down the decay of TGF-β2 mRNA. Consistent with this mechanism, we found that TGF-β2 could antagonize IL-6 effect on cell survival in both γ-irradiation and UV light-induced apoptosis. Taken together, the finding shows that TGF-β2 serves as a negative regulator of IL-6 signaling and antagonizes the anti-apoptosis effect of IL-6.

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-β family that selectively binds transforming growth factor-β (TGF-β) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo. Here, using NMR titrations of methyl-labeled TGF-β2 with BG's C-terminal binding domain, BG(ZP-C), and surface plasmon resonance binding measurements with TGF-β2 variants, we found that the BG(ZP-C)–binding site on TGF-β2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-β isoforms and the InhA α-subunit, but they are unconserved in other TGF-β family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-β2 variants with the corresponding residues from BMP-2 bound BG(ZP-C) more weakly than corresponding alanine variants. The BG(ZP-C)–binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-β2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-β2 and InhA bind BG(ZP-C) through a site on the inside of their extended finger regions. Overall, these results identify the BG(ZP-C)–binding site on TGF-β2 and shed light on the specificity of BG for select TGF-β–type GFs and the mechanisms by which BG influences their signaling.

The cause of posterior capsular opacification (PCO) is the dysfunction of lens epithelial cells (LECs). Circular RNA (circRNA) was found to regulate cell biological functions, including LECs. However, the role of circ-GGA3 in PCO formation is unclear. Quantitative real-time PCR was used to measure the expression of circ-GGA3, miR-497-5p and SMAD4. Cell proliferation, invasion and migration were determined via MTT assay, EdU staining, transwell assay and wound healing assay. The protein expression of epithelial-mesenchymal transition (EMT) markers, fibrosis markers, TGF-β/SMAD pathway markers and SMAD4 were determined by western blot assay. The interaction between miR-497-5p and circ-GGA3 or SMAD4 was confirmed using dual-luciferase reporter assay. Circ-GGA3 was highly expressed in PCO patients, and its silencing inhibited the proliferation, invasion, migration, EMT process and fibrosis of TGF-β2-induced LECs. Circ-GGA3 could sponge miR-497-5p to regulate SMAD4. Further experiments revealed that miR-497-5p inhibitor recovered the negative regulation of circ-GGA3 knockdown on the biological functions of TGF-β2-induced LECs, and SMAD4 overexpression also abolished the suppressive effect of miR-497-5p. In addition, circ-GGA3/miR-497-5p/SMAD4 axis could activate the TGF-β/SMAD pathway. Our results indicated that circ-GGA3 could enhance the biological functions of LECs, suggesting that circ-GGA3 might be a potential target for PCO therapy.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

A pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFβ2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFβ2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFβ2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFβ2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFβ2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFβ2. Those were significantly and differently modulated by Rip or KD025.

Glaucoma is a group of optic neuropathies that leads to irreversible vision loss. The optic nerve head (ONH) is the site of initial optic nerve damage in glaucoma. ONH-derived lamina cribrosa (LC) cells synthesize extracellular matrix (ECM) proteins, however, these cells are adversely affected in glaucoma and cause detrimental changes to the ONH. LC cells respond to mechanical strain by increasing the profibrotic cytokine transforming growth factor-beta 2 (TGFβ2) and ECM proteins. Moreover, microRNAs (miRNAs or miR) regulate ECM gene expression in different fibrotic diseases, including glaucoma. A delicate homeostatic balance between profibrotic and anti-fibrotic miRNAs may contribute to the remodeling of ONH. This study aimed to determine whether modulation of miRNAs alters the expression of ECM in human LC cells. Primary human normal and glaucoma LC cells were grown to confluency and treated with or without TGFβ2 for 24 h. Differences in expression of miRNAs were analyzed using miRNA qPCR arrays. miRNA PCR arrays showed that the miR-29 family was significantly decreased in glaucomatous LC cell strains compared to age-matched controls. TGFβ2 treatment downregulated the expression of multiple miRNAs, including miR-29c-3p, compared to controls in LC cells. LC cells transfected with miR-29c-3p mimics or inhibitors modulated collagen expression.

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

Purpose The multifunctional profibrotic cytokine TGF-β2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-β2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-β2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-β2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-β2 profibrotic responses in cultured primary human TM cells. Methods Semiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-β2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining. Results Quiescent primary human TM cells cultured in the presence of TGF-β2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-β2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-β2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-β2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation. Conclusions TGF-β2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-β2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-β2 elicited profibrotic responses within the TM.

The trabecular meshwork (TM) is composed of TM cells and beams of the extracellular matrix, together contributing to aqueous humor (AH) outflow resistance. Herein, we validated that our culture system on 2D Matrigel expressed putative TM markers and myocilin, of which the latter was upregulated by dexamethasone. Continuous passage of these cells on 2D Matrigel resulted in a gradual loss of expression of these markers. However, such a loss was restored by seeding cells in 3D Matrigel where expression of TM markers was further upregulated upon continuous passage. In contrast, TM cells seeded on fibronectin, collagen I/IV, or laminin lost expression of these markers and turned into myofibroblasts with expression of αSMA, which were dose-dependently upregulated by TGF-β1/TGF-β2. TM cells in 3D Matrigel also expressed TGF-β1/TGF-β3 despite challenge of TGF-β1. The maintenance of TM phenotype by 3D Matrigel was linked to inhibition of canonical TGF-β signaling and activation of pFAK-pSrc-pP190RhoGAP-P120RasGAP signaling. These findings indicate that basement membrane matrix with low rigidity plays an active role in maintaining TM phenotype in the presence of TGF-β1 and shed light on its physiological role. Furthermore, abnormal matrices may perpetuate the pathological TM phenotype when the level of TGF-β2 is elevated in glaucoma patients.

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.

Chronic obstructive pulmonary disease (COPD) is a common, complex disease and a major cause of morbidity and mortality. Although multiple genetic determinants of COPD have been implicated by genome-wide association studies (GWASs), the pathophysiological significance of these associations remains largely unknown. From a COPD protein–protein interaction network module, we selected a network path between two COPD GWAS genes for validation studies: FAM13A (family with sequence similarity 13 member A)–AP3D1–CTGF– TGFβ2. We find that TGFβ2, FAM13A, and AP3D1 (but not CTGF) form a cellular protein complex. Functional characterization suggests that this complex mediates the secretion of TGFβ2 through an AP-3 (adaptor protein 3)–dependent pathway, with FAM13A acting as a negative regulator by targeting a late stage of this transport that involves the dissociation of coat–cargo interaction. Moreover, we find that TGFβ2 is a transmembrane protein that engages the AP-3 complex for delivery to the late endosomal compartments for subsequent secretion through exosomes. These results identify a pathophysiological context that unifies the biological network role of two COPD GWAS proteins and reveal novel mechanisms of cargo transport through an intracellular pathway.

Abundant regulatory genes and complex circuits involving non-coding RNAs (ncRNAs) monitor the formation and development of hepatic fibrosis (HF). Circular RNAs (circRNAs) are a class of RNAs generated from protein coding genes by back-splicing, playing crucial roles in various pathological processes, including HF. However, little is known about mechanisms of action of circRNAs, let alone in HF. In this study, we found circUbe2k enhanced in CCl4 -induced HF mice and LX-2 cells stimulated with TGF-β1, regulating the development of HF. Restraining the expression of circUbe2k inhibited α-SMA and Col1α1 expression in CCl4 -induced HF mice and in LX-2 cells stimulated with TGF-β1. Furthermore, inhibiting circUbe2k expression reduced hepatic stellate cells (HSCs) activation and proliferation in vivo and in vitro. Mechanistically, we demonstrated a direct interaction between circUbe2k and miR-149-5p, which results in the modulation of TGF-β2 expressions. Together, circUbe2k may act as a "catalyst" of HSCs activation and HF through the circUbe2k/miR-149-5p/TGF-β2 axis. Our results provide unprecedented evidence for a significant role for circUbe2k to serve as a potential biomarker for HF therapy.