Transforming Growth Factor Beta Receptor Research Articles

Abstract 806: TGFBR1*6A enhances breast cancer susceptibility and promote cell cycle transition

Abstract Background: TGFBR1*6A is a polymorphic variant of the Transforming Growth Factor Beta receptor 1 (TGFBR1) that has three GCG synonymous deletions in its signal peptide. It is a low penetrance breast cancer susceptibility allele (OR, 1.15; 95% CI, 1.01 to 1.31) postulated to enhance tumor formation. In this study, a novel breast cancer mouse model carrying humanized TGFBR1*6A (hTGFBR1*6A) knock-in was used to investigate TGFBR1*6A influence on breast cancer formation, and dissect its molecular pathogenicity. Methods: Humanized mouse models of the two most common TGFBR1*6A genotypes - homozygous TGFBR1*6A/6A and heterozygous TGFBR1*9A/6A, and wild-type TGFBR1 were created by replacing murine Tgfbr1 exon 1 with human TGFBR1*6A or TGFBR1 exon 1. The three genotypes were bred with MMTV.Neu mice to study breast cancer development. Between 29-35 nulliparous females from each genotype were monitored for tumor formation (≥1 tumor), multiple tumor formation (≥2 tumors) and survival. Tumor cells were isolated from the hTGFBR1 and hTGFBR1*6A mice and validated for similar Neu protein expression. They were assessed for TGF-β/FoxO1 signaling as it relates to cell cycle progression and proliferation. Results: After 548 days, the mice harboring hTGFBR1*6A developed significantly more tumors (p=0.003 for one/more tumors, and p=0.0009 for two/more tumors) than wild-type mice. About 70% hTGFBR1*6A/6A and 28.6% hTGFBR1*9A/6A variants compared to 18.5% hTGFBR1 wild-type mice developed two (2) or more tumors. The hTGFBR1*6A mice also had shorter survival when compared with the wild-type mice (Log rank X2=7.77, p=0.02). The hTGFBR1*6A/6A and hTGFBR1*9A/6A mice survived for 178-210 and 208-282 days respectively compared to 198-342 days among the wild-type. The isolated hTGFBR1*6A tumor cells formed three (3) times more colonies, and exhibited higher growth rate and G1/S cell cycle transition than wild-type tumor cells. Interestingly, the hTGFBR1*6A cells were less sensitive to TGF-β anti-proliferative effects despite showing marked increase in TGF-β/SMAD signaling. Analyses of the TGF-β/FoxO1 growth inhibition pathway showed decreased FoxO1, p21 and p27 gene and protein expression, which was not increased significantly by TGF-β treatment. This suggests that TGFBR1*6A constitutively inhibits the TGF-β/FoxO1 cell cycle tumor suppressor arm. Conclusions: Altogether, this study provides new evidence in support of TGFBR1*6A as a breast cancer susceptibility allele. It also identifies deregulation of TGF-β/FoxO1 cell cycle inhibition as a potential mechanism underlying its increased cellular proliferation. Citation Format: Kojo Agyemang, Michael Pennison, Minghui Wang, Allan Johansen, Hugo Jimenez, Carl Blackman, Ravi Singh, Ralph D'Agostino, Paul Grippo, Antonio Di Cristofano, Boris Pasche. TGFBR1*6A enhances breast cancer susceptibility and promote cell cycle transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 806.

Overexpression of TGF-β1 and SDF-1 in cervical cancer-associated fibroblasts promotes cell growth, invasion and migration

The purpose of this study was to investigatethe effect of overexpression of transforming growth factor β1 (TGF-β1) and stromal cell-derived factor 1 (SDF-1) in cervical cancer-associated fibroblasts (CAFs) on regulating cell growth, invasion and migration. CAF cells and normal fibroblast cells (NFs) were obtained from patients with cervical squamous cell carcinoma andmultiple uterine leiomyomas, respectively. Immunofluorescence assay and western blot were used to determine the expression of Vimentin andα-smooth muscle actin (α-SMA). CCK-8 assay was used to detect cell viability.Giemsa dyer was used to detect the colony formation. Flow cytometry was used to detect the growth state of cells. Transwell assayswereused to detect the migration and invasion. Vimentin and α-SMAexpressionin CAFs were significantly increased than those in NFs.In addition, TGF-β1 and SDF-1 expression were notably increased,and transforming growth factor beta receptor 2 (TβRII) expression wasmarkedly decreased in CAF cellsthan those in NFs. Similarly, TGF-β1 and SDF-1expressionin the co-culture of CAFs and Hela cells were significantly increased,and cell proliferation, migration, invasion, colony formation and cell cycle progression were also promoted, while cell apoptosis was decreased.Those phenomena were reversed in the co-culture system with neutralizing antibodies to TGF-β1 and SDF-1.Furthermore, exogenous TGF-β1and SDF-1enhanced proliferation, colony formation, cell cycle progression, migration and invasion while decreased apoptosis of cells. These phenomena were also reversed by the addition of neutralizing antibodiesto TGF-β1 and SDF-1. Overexpression ofTGF-β1and SDF-1in CAFscan promote the growth, invasion and migration of cervical cancer cells.

The Biological Function of TUSC7/miR-1224-3p Axis in Triple-Negative Breast Cancer.

BackgroundTriple-negative breast cancers (TNBC), comprising about 20% of breast cancers, have a poor prognosis. Currently, there is no effective target therapy for TNBC. LncRNA TUSC7 has been identified as a tumor suppressor in osteosarcoma and colorectal cancer. In this study, we investigated the clinical significance and the biological function of TUSC7 in breast cancer.MethodsWe retrospectively evaluated the expression level and clinical significance of TUSC7 in 90 paired breast cancer tissues and normal tissues. The proliferation, migration, and invasion assays were performed to investigate the biological function of TUSC7 in breast cancer. Finally, microarray, a luciferase reporter assay, and quantitative real-time polymerase chain reaction (qPCR) were used to explore the potential underlying mechanism of tumor suppressor role of TUSC7.ResultsLow TUSC7 expression was found to be an independent prognostic factor of poor overall survival (OS) in TNBC patients. Ectopic expression of TUSC7 inhibited tumor cell growth both in vitro and in vivo. TUSC7 overexpression significantly promoted the sensitivity of MDA-MB-468 cells to paclitaxel and carboplatin. In terms of the mechanism, TUSC7 might perform its biological function through binding with miR-1224-3P and regulating its expression level. Besides, genes in cell cycle pathways, such as BUB3 (budding uninhibited by benzimidazoles 3) and TGF-ß (targeting transforming growth factor β) pathways were downregulated, and genes involved in the MAPK (mitogen-activated protein kinase) (TGFBR2, transforming growth factor-beta receptor 2), PI3K-AKT (phosphoinositide 3-kinase- AKT serine/threonine kinase 1) and NF-κB (nuclear factor-kappa B subunit) pathways were upregulated in TUSC7 knockdown MDA-MB-231 cells.ConclusionThe low TUSC7 expression is an independent prognostic factor of poor OS of TNBC patients. TUSC7 might inhibit breast cancer cell growth and metastasis both in vitro and vivo through binding with miR-1224-3P and regulating MAPK, PI3K/AKT, and NF-κB signaling pathways.

Variable Expression Levels of the Hematopoietic Stem Cell Surface Antigen ESAM Depict Heterogeneity and Fluctuations in Leukemic Stem Cells

Traditionally, hematopoietic stem cells (HSCs) have been regarded as a homogeneous cell population. However, many studies have shown that populations of HSCs are essentially heterogenous. Moreover, several recent studies have proposed that HSCs exhibit fluctuating biological characteristics. Although the physiological significance of this heterogeneity and fluctuation is unclear, we presume that these qualities enable HSCs to flexibly demonstrate their self-renewal or differentiation abilities dependent on physiological demands. It is, therefore, unsurprising that such heterogeneity and fluctuation is also seen in leukemic stem cells (LSCs), and that they are involved in the development of resistance to treatment.We have previously demonstrated that the endothelial-cell selective adhesion molecule (ESAM) is an important marker of HSCs in humans and mice (Blood 2009, Exp Hematol 2013), and that the expression level of this molecule indicates the activation status of HSCs (J Immunol 2012). Furthermore, we found that a subset of leukemic cells exhibit elevated levels of ESAM expression on their surfaces (Exp Hematol 2013). Approximately two-thirds of acute myelogenous leukemia cases in humans were found to be positive for ESAM expression. Since ESAM was found to be useful in monitoring the fluctuating status of HSCs after 5-FU treatment, we posited that the marker could be used to characterize LSCs.In this study, we used ESAM expression levels as an index to analyze the heterogeneity and fluctuation in LSC populations. Flow cytometric analysis of the human leukemia cell lines KG1a and CMK revealed wide-ranging heterogeneity in ESAM expression levels, with up to a 1000-fold difference in variation, even among individual cells derived from the same clone. When these cell populations were roughly fractionated into ESAM-high or ESAM-low expression groups, the ESAM-high expression group showed a higher proliferative capacity in culture. Interestingly, however, the expression levels of ESAM were not maintained indefinitely at the same level in these groups. In four weeks, both ESAM-high and ESAM-low cell fractions developed heterogeneous ESAM expression distributions similar to that in the parent cell lines. Furthermore, when single cells were sorted and cultured, each cell showed a self-renewal ability and the heterogenous nature of ESAM expression levels was reconstructed in each population derived from these single cells. These results suggest that populations of LSCs develop heterogeneity and have a fluctuating nature.When the transcriptomes of ESAM-low and ESAM-high KG1a cells were compared using RT-PCR, the expression of the transforming growth factor beta receptor type 2 (TGFBR2) gene was found to be higher in the ESAM-low cells. Reportedly, the transforming growth factor beta (TGFβ) signaling pathway is involved in various biological processes such as cell proliferation, differentiation, and migration. In tumor cells, this pathway is involved in reducing proliferation and accelerating invasion. Based on this information, it was considered likely that the TGFβ signaling pathway is involved in the heterogeneity of ESAM expression. When TGFβ1 (5 ng/ml) was added to a culture of KG1a cells (grown in RPMI 1640 medium with 10% fetal bovine serum (FBS)), the heterogeneity of ESAM expression in the population became more pronounced, mainly due to a decrease in ESAM expression in some cells.A transcriptomic analysis of cells exhibiting decreased ESAM expression after TGFβ1 stimulation showed that these cells had a higher expression of TGFBR2 than cells not exhibiting a decrease in ESAM expression. Since the presence of 10% FBS in the culture medium provides natural TGFβ1 at a concentration of ~1 ng/ml, we conducted culture experiments without FBS. When KG1a cells were cultured in serum-free medium, the ESAM-low fraction disappeared and the heterogeneity of ESAM expression could not be replicated. However, the heterogeneity of ESAM expression in KG1a cultures could be replicated in serum-free medium by the addition of 1 ng/ml TGFβ1. These results suggest that the TGFβ signaling pathway is involved in inducing heterogeneity in ESAM expression in cell populations. The fluctuation of ESAM expression levels could be caused by differences in TGF sensitivity, which in turn, may confer certain benefits to LSCs, such as drug resistance and promoting invasiveness. DisclosuresYokota:Celgene: Research Funding; MSD K.K.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Pfizer Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding. Ezoe:Taiho Pharmaceutical Co., LTD.: Research Funding. Shibayama:Novartis Pharma K.K.: Honoraria, Research Funding; Bristol-Meyer Squibb K.K.: Honoraria, Research Funding; Ono Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Jansen Pharmaceutical K.K.: Honoraria; Fujimoto Pharmaceutical Co.: Honoraria, Research Funding; Takeda Pharmaceutical Co.,LTD.: Honoraria, Research Funding; Mundipharma K.K.: Honoraria, Research Funding; Celgene K.K.: Honoraria, Research Funding. Oritani:MOCHIDA PHARMACEUTICAL: Speakers Bureau; Celgene: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma: Consultancy, Speakers Bureau. Kanakura:Kyowa Hakko Kirin: Research Funding; Chugai Pharmaceutical: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Fujimotoseiyaku: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Nippon Shinyaku: Research Funding; Toyama Chemical: Research Funding; Shionogi: Research Funding.

Long non-coding RNA CDKN2B-AS1 enhances LPS-induced apoptotic and inflammatory damages in human lung epithelial cells via regulating the miR-140-5p/TGFBR2/Smad3 signal network

BackgroundSepsis is a complicated disease with systemic inflammation or organ dysfunction, and it is the leading cause of acute lung injury (ALI). Long non-coding RNAs (lncRNAs) have played important roles in the pathogenesis of sepsis. This study was designed to explore the biological function and regulatory mechanism of cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in lipopolysaccharide (LPS)-induced lung injury.MethodsALI model was established after human lung epithelial cell line BEAS-2B was exposed to LPS. CDKN2B-AS1, microRNA-140-5p (miR-140-5p) and transforming Growth Factor Beta Receptor II (TGFBR2) levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was measured using Cell Counting Kit-8 (CCK-8). Cell apoptosis was assessed by caspase3 activity and flow cytometry. Inflammatory cytokines were examined via enzyme-linked immunosorbent assay (ELISA). Protein analysis was performed through western blot. Dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays were applied to validate the interaction between targets.ResultsCDKN2B-AS1 and TGFBR2 were abnormally upregulated in sepsis patients. Functionally, CDKN2B-AS1 or TGFBR2 knockdown promoted cell growth but inhibited cell apoptosis and inflammatory response in LPS-treated BEAS-2B cells. Moreover, the regulation of CDKN2B-AS1 in LPS-induced cell injury was achieved by increasing the TGFBR2 expression. CDKN2B-AS1 was identified as a miR-140-5p sponge and TGFBR2 was a target of miR-140-5p. Furthermore, CDKN2B-AS1 could regulate the TGFBR2/Smad3 pathway by sponging miR-140-5p.ConclusionsThese results suggested that CDKN2B-AS1 contributed to the LPS-mediated apoptosis and inflammation in BEAS-2B cells via the miR-140-5p/TGFBR2/Smad3 axis.

Inhibition of Hsp90 Counteracts the Established Experimental Dermal Fibrosis Induced by Bleomycin.

Our previous study demonstrated that heat shock protein 90 (Hsp90) is overexpressed in the involved skin of patients with systemic sclerosis (SSc) and in experimental dermal fibrosis. Pharmacological inhibition of Hsp90 prevented the stimulatory effects of transforming growth factor-beta on collagen synthesis and the development of dermal fibrosis in three preclinical models of SSc. In the next step of the preclinical analysis, herein, we aimed to evaluate the efficacy of an Hsp90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in the treatment of established experimental dermal fibrosis induced by bleomycin. Treatment with 17-DMAG demonstrated potent antifibrotic and anti-inflammatory properties: it decreased dermal thickening, collagen content, myofibroblast count, expression of transforming growth factor beta receptors, and pSmad3-positive cell counts, as well as leukocyte infiltration and systemic levels of crucial cytokines/chemokines involved in the pathogenesis of SSc, compared to vehicle-treated mice. 17-DMAG effectively prevented further progression and may induce regression of established bleomycin-induced dermal fibrosis to an extent comparable to nintedanib. These findings provide further evidence of the vital role of Hsp90 in the pathophysiology of SSc and characterize it as a potential target for the treatment of fibrosis with translational implications due to the availability of several Hsp90 inhibitors in clinical trials for other indications.

Identification of TGFBR2 mutation as a negative predictor of immunotherapy in NSCLC.

e21002 Abstract Background: Immune adaptive resistance is one of the reasons contributing to the resistance of immune checkpoint inhibitors (ICIs). Previous studies have demonstrated the role of TGFβ-SMAD signaling in the dysfunction of immune response. We hypothesized that transforming growth factor beta receptor 2 (TGFBR2) mutation in non-small cell lung cancer (NSCLC) might be a predictor of immunotherapeutic resistance. Methods: Genomic and transcriptomic data of NSCLC patients from The Cancer Genome Atlas (TCGA) were analyzed by the in silico Pathway Activation Network Decomposition Analysis (iPNADA) to study the association between TGFBR2 mutation and intracellular signaling pathway activation. Clinical and genomic data from public cohorts with patients treated with ICIs including POPLAR/OAK, MSKCC and Van Allen cohorts were obtained and analyzed. An independent China cohort was used to demonstrate the association between TGFBR2 mutation and immunotherapeutic efficacy and we further demonstrated a case with mutated TGFBR2 treated with ICI. Results: TGF-β signaling was decreased and JAK-STAT signaling was increased in patients with mutated TGFBR2 (P < 0.05). The immune checkpoints including CD274, LAG3, TIGHT, CTLA-4, PDCD1 and PDCD1LG2 were significantly increased in patients with mutated TGFRB2 (P < 0.05). The immune dysfunction score tended to be increased in patients with mutated TGFBR2 though no significant difference was observed (P = 0.23). In the POPLAR/OAK cohort, patients with mutated TGFBR2 had shorter progression-free survival (P = 0.004; HR, 2.83; 95% CI, 1.34-6.00) and overall survival (OS) (P = 0.0006; HR, 3.46; 95% CI, 1.63-7.35) than patients with wild-type TGFBR2 when treated with ICIs. While in patients treated with chemotherapy, there was no difference in PFS (P = 0.69; HR, 0.86; 95% CI, 0.41-1.82) and OS (P = 0.61; HR, 1.21; 95% CI, 0.57-2.57) between patients with mutated and wild-type TGFBR2. In the merged MSKCC and Van Allen cohorts, similar result was observed that the OS was shorter in patients with mutated TGFBR2 (P = 0.007; HR, 2.53; 95% CI, 1.25-5.12). At last, we demonstrated a 39-year-old case with TGFBR2 mutation who was treated with 3 mg/kg nivolumab as second-line treatment. The patient progressed after three cycles of nivolumab and the PFS was only 1.3 months. Conclusions: We identified TGFBR2 mutation as a negative predictor for ICIs in NSCLC and the clinical use of ICIs needs to be cautious in those patients, highlighting the importance of genomic profiling in the treatment of ICIs. Combination ICIs and TGF-β signaling inhibitors may overcome the resistance for those patients harboring TGFBR2 mutation and need to be developed in the future.

Association of TGFBR2 mutations with shorter survival in non-small cell lung cancer treated with immune checkpoint inhibitors.

e21166 Background: Transforming growth factor beta receptor 2 (TGFBR2) is a transmembrane receptor that plays an important role in both cellular functions and immune reaction, including cell proliferation, cell differentiation and extracellular matrix production. It forms a heterodimeric complex with TGF-beta receptor type-1 and binds TGF-beta. The association between TGFBR2 gene mutations and efficiency of immune checkpoint inhibitors (ICIs) has not been investigated in non-small cell lung cancer (NSCLC). Methods: Two published cohorts of NSCLC patients treat with ICIs were used to explore the immunotherapeutic predictive role of TGFBR2 gene mutations, including a discovery cohort (Gandara et al., Nature Genetics, 2018) and a validation cohort(Samstein et al. Nature Genetics, 2019). The Cancer Genome Altas (TCGA) Pan -Lung Cancer dataset was used for prognostic analysis. Results: TGFBR2 mutations were identified in 1.6% (7/429) and 2.3% (8/344) patients with NSCLC in the discovery and validation cohort, respectively. In the discovery cohort, patients with TGFBR2 mutations had shorter progression-free survival (PFS, median, 1.3 months vs 2.7 months; HR = 2.83; 95% CI, 1.33–6; p = 0.0046) and dramatically shorter overall survival (OS, median, 2.4 months vs 11.1 months; HR = 3.46; 95% CI, 1.63–7.35; p = 0.00058) than those with wild-type TGFBR2. Consistent with this, TGFBR2 mutations were significantly correlated with shorter OS (3 months vs 10 months; HR = 3.46; 95% CI, 1.63–7.35; p = 0.00058) in the validation cohort. In addition, no significant difference was found in OS between TGFBR2-mutant and TGFBR2-wild-type NSCLC patients with standard treatment in TCGA cohort (p = 0.821). Conclusions: TGFBR2 gene mutations are associated with shorter survival in NSCLC patients treated with ICIs, suggesting that TGFBR2 mutations be used as a potential biomarker of predicting the efficiency of immunotherapy and guiding NSCLC systemic treatment.

Genes associated with amyloid-beta-induced inflammasome-mediated neuronal death identified using functional gene trap mutagenesis approach.

Alzheimer's disease is an irreversible neurodegenerative disease, which accounts for most dementia cases. Neuroinflammation is increasingly recognised for its roles in Alzheimer's disease pathogenesis which, in part, links amyloid-beta to neuronal death. Neuroinflammatory signalling can be exhibited by neurons themselves, potentially leading to widespread neuronal cell death, although neuroinflammation is commonly associated with glial cells. The presence of the inflammasomes such as nucleotide-binding leucine-rich repeat receptors protein 1 in neurons accelerates amyloid-beta -induced neuroinflammation and has been shown to trigger neuronal pyroptosis in murine Alzheimer's disease models. However, the pathways involved in amyloid-beta activation of inflammasomes have yet to be elucidated. In this study, a gene trap mutagenesis approach was utilised to resolve the genes functionally involved in inflammasome signalling within neurons, and the mechanism behind amyloid-beta-induced neuronal death. The results indicate that amyloid-beta significantly accelerated neuroinflammatory cell death in the presence of a primed inflammasome (the NLR family pyrin domain-containing 1). The mutagenesis screen discovered the atypical mitochondrial Ras homolog family member T1 as a significant contributor to amyloid-beta-induced inflammasome -mediated neuronal death. The mutagenesis screen also identified two genes involved in transforming growth factor beta signalling, namely Transforming Growth Factor Beta Receptor 1 and SNW domain containing 1. Additionally, a gene associated with cytoskeletal reorganisation, SLIT-ROBO Rho GTPase Activating Protein 3 was found to be neuroprotective. In conclusion, these genes could play important roles in inflammasome signalling in neurons, which makes them promising therapeutic targets for future drug development against neuroinflammation in Alzheimer's disease.

Sustained Delivery of SB-431542, a Type I Transforming Growth Factor Beta-1 Receptor Inhibitor, to Prevent Arthrofibrosis.

Fibrosis of the knee is a common disorder resulting from an aberrant wound healing response and is characterized by extracellular matrix deposition, joint contraction, and scar tissue formation. The principal regulator of the fibrotic cascade is transforming growth factor beta-1 (TGF-β1), a factor that induces rapid proliferation and differentiation of resident fibroblasts. In this study, we demonstrate successful inhibition of TGF-β1-driven myofibroblastic differentiation in human fibroblast-like synoviocytes using a small molecule TGF-β1 receptor inhibitor, SB-431542. We also demonstrate successful encapsulation of SB-431542 in poly(D,L-lactide-co-glycolide) (PLGA) as a potential prophylactic treatment for arthrofibrosis and characterize drug release and bioactivity in a three-dimensional collagen gel contraction assay. We assessed the effects of TGF-β1 and SB-431542 on cell proliferation and viability in monolayer cultures. Opposing dose-dependent trends were observed in cell proliferation, which increased in TGF-β1-treated cultures and decreased in SB-431542-treated cultures relative to control (p < 0.05). SB-431542 was not cytotoxic at the concentrations studied (0-50 μM) and inhibited TGF-β1-induced collagen gel contraction in a dose-dependent manner. Specifically, TGF-β1-treated gels contracted to 18% ± 1% of their initial surface area, while gels treated with TGF-β1 and ≥10 μM SB-431542 showed no evidence of contraction (p < 0.0001). Upon removal of the compound, all gels contracted to control levels after 44 h in culture, necessitating sustained delivery for prolonged inhibition. To this end, SB-431542 was encapsulated in PLGA microspheres (SBMS) that had an average diameter of 87.5 ± 24 μm and a loading capacity of 4.3 μg SB-431542 per milligram of SBMS. Functional assessment of SBMS revealed sustained inhibition of TGF-β1-induced gel contraction as well as hallmark features of myofibroblastic differentiation, including α-smooth muscle actin expression and connective tissue growth factor production. These results suggest that SB-431542 may be used to counter TGF-β1-driven events in the fibrotic cascade in the knee cartilage. Impact statement Arthrofibrosis is the most prevalent comorbidity resulting from orthopedic procedures such as total knee arthroplasty that is characterized by excess deposition and accumulation of extracellular matrix. Despite its prevalence, treatments are generally palliative, and there is no effective prophylactic therapy. We report that the small molecule transforming growth factor beta-1 (TGF-β1) receptor inhibitor, SB-431542, can inhibit the TGF-β1-driven myofibroblastic differentiation of fibroblast-like synoviocytes. To provide sustained inhibition, we explored the use of SB-laden microspheres as a prophylactic therapy in a three-dimensional contraction model of fibrosis and propose that such therapies will have the potential to improve the standard of care for arthrofibrosis.

Circular RNA Plek promotes fibrogenic activation by regulating the miR-135b-5p/TGF-βR1 axis after spinal cord injury.

Objectives: The spinal cord rarely repairs itself when damaged; however, methods for encouraging nerves to regrow are on the horizon. Although circular RNAs (circRNAs) contribute to various biological processes, including neuronal processes, their functions in the subacute phase of spinal cord injury (SCI) have not been elucidated. In this study, we identified a novel circRNA, named CircPlek, with increased expression in spinal tissues after SCI.Materials and Methods: We predicted a regulatory relationship between CircPlek and miR-135b-5p, which showed the most obvious decrease in post-SCI expression. We established the CircPlek/miR-135b-5p/transforming growth factor-beta receptor type I (TGF-βR1) axis using a bioinformatics approach and further evaluated the potential function of the interaction network in vitro.Results: We confirmed that in TGF-β1-induced fibroblasts, the overexpression of miR-135b-5p or/and inhibition of CircPlek inhibited fibrosis activation via the Smad pathway. Inhibitors of miR-135b-5p had antagonistic effects on CircPlek.Conclusions: the CircPlek/miR-135b-5p/TGF-βR1 axis may exert important functions in SCI and is a potential therapeutic target.

Adipose-Derived Mesenchymal Stem Cells-Derived Exosomes Carry MicroRNA-671 to Alleviate Myocardial Infarction Through Inactivating the TGFBR2/Smad2 Axis

Mesenchymal stem cells (MSCs) and their derived extracellular vesicles have been reported as promising tools for the management of heart disease. The aim of this study was to explore the function of adipose-derived MSCs (adMSCs)-derived exosomes (Exo) in the progression of myocardial infarction (MI) and the molecules involved. Mouse cardiomyocytes were treated with oxygen-glucose deprivation (OGD) to mimic an MI condition in vitro. The adMSCs-derived Exo were identified and administrated into the OGD-treated cardiomyocytes, and then the viability and apoptosis of cells, and the secretion of fibrosis- and inflammation-related cytokines in cells were determined. Differentially expressed microRNAs (miRNAs) in cells after Exo treatment were screened using a microarray analysis. The downstream molecules regulated by miR-671 were explored through bioinformatic analysis. Involvements of miR-671 and transforming growth factor beta receptor 2 (TGFBR2) in the exosome-mediated events were confirmed by rescue experiments. A murine model with MI was induced and treated with Exo for functional experiments in vivo. Compared to phosphate-buffered saline treatment, the Exo treatment significantly enhanced viability while reduced apoptosis of cardiomyocytes, and in reduced myocardial fibrosis and inflammation both in vitro and in vivo. miR-671 was significantly upregulated in cells after Exo treatment. Downregulation of miR-671 blocked the protective functions of Exo. miR-671 targeted TGFBR2 and suppressed phosphorylation of Smad2. Artificial downregulation of TGFBR2 enhanced viability of the OGD-treated cardiomyocytes. This study suggested that adMSC-derived exosomal miR-671 directly targets TGFBR2 and reduces Smad2 phosphorylation to alleviate MI-like symptoms both in vivo and in vitro.

FGD5-AS1 Inhibits Osteoarthritis Development by Modulating miR-302d-3p/TGFBR2 Axis.

Osteoarthritis (OA) is a common joint disorder, accompanied by extracellular matrix (ECM) degradation. Reportedly, long noncoding RNAs (lncRNAs) are involved in OA pathogenesis. However, the role of lncRNA FYVE, RhoGEF, and PH domain containing 5 antisense RNA 1 (FGD5-AS1) in OA development is still not fully clarified. This study was aimed to clarify the role of FGD5-AS1 in OA. FGD5-AS1 and miR-302d-3p expression levels were determined in cartilage tissues and chondrocytes by quantitative real-time polymerase chain reaction (qRT-PCR). Chondrocytes (C20/A4 cells) were stimulated with interleukin 1β (IL-1β) to mimic the inflammatory environment of OA. Cell viability was detected by cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays. Cell apoptosis was measured by the caspase-3 activity assay and flow cytometry. Transforming growth factor beta receptors II (TGFBR2), matrix metalloproteinase 13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 5 expression levels were examined by qRT-PCR or Western blot. The regulatory relationships among FGD5-AS1, miR-302d-3p, and TGFBR2 were predicted by the StarBase v2.0, miRanda, miRDB, and TargetScan databases, and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. FGD5-AS1 and TGFBR2 expression levels were downregulated while miR-302d-3p expression was increased in cartilage tissues of OA patients. Knocking down FGD5-AS1 inhibited the viability of C20/A4 cells but induced apoptosis and ECM degradation, while FGD5-AS1 overexpression exerted opposite effects. MiR-302d-3p was identified as a target of FGD5-AS1, and TGFBR2 was identified as a target of miR-302d-3p. FGD5-AS1 positively regulated TGFBR2 expression by repressing miR-302d-3p expression, and miR-302d-3p inhibition or TGFBR2 restoration reversed the changes of cell viability, apoptosis, and ECM degradation induced by FGD5-AS1 knockdown. FGD5-AS1 can probably inhibit OA progression by regulating miR-302d-3p/TGFBR2 axis.

Creatine promotes cancer metastasis through activation of Smad2/3

As one of the most popular nutrient supplements, creatine has been highly used to increase muscle mass and improve exercise performance. Here, we report an adverse effect of creatine using orthotopic mouse models, showing that creatine promotes colorectal and breast cancer metastasis and shortens mouse survival. We show that glycine amidinotransferase (GATM), the rate-limiting enzyme for creatine synthesis, is upregulated in liver metastases. Dietary uptake, or GATM-mediated de novo synthesis of creatine, enhances cancer metastasis and shortens mouse survival by upregulation of Snail and Slug expression via monopolar spindle 1 (MPS1)-activated Smad2 and Smad3 phosphorylation. GATM knockdown or MPS1 inhibition suppresses cancer metastasis and benefits mouse survival by downregulating Snail and Slug. Our findings call for using caution when considering dietary creatine to improve muscle mass or treat diseases and suggest that targeting GATM or MPS1 prevents cancer metastasis, especially metastasis of transforming growth factor beta receptor mutant colorectal cancers.

Chi-miR-370-3p regulates hair follicle morphogenesisof Inner Mongolian cashmere goats.

MicroRNAs (miRNAs), a class of 22 nucleotide (nt) noncoding RNAs, negatively regulate mRNA posttranscriptional modification in various biological processes. Morphogenesis of skin hair follicles in cashmere goats is a dynamic process involving many key signaling molecules, but the associated cellular biological mechanisms induced by these key signaling molecules have not been reported. In this study, differential expression, bioinformatics, and Gene Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed on miRNA expression profiles of Inner Mongolian cashmere goats at 45, 55, and 65 days during the fetal period, and chi-miR-370-3p was identified and investigated further. Real-time fluorescence quantification (qRT-PCR), dual luciferase reporting, and Western blotting results showed that transforming growth factor beta receptor 2 (TGF-βR2) and fibroblast growth factor receptor 2 (FGFR2) were the target genes of chi-miR-370-3p. Chi-miR-370-3p also regulated the expression of TGF-βR2 and FGFR2 at mRNA and protein levels in epithelial cells and dermal fibroblasts. DNA staining, Cell Counting Kit-8, and fluorescein-labelled Annexin V results showed that chi-miR-370-3p inhibited the proliferation of epithelial cells and fibroblasts but had no effect on apoptosis. Cell scratch test results showed that chi-miR-370-3p promoted the migration of epithelial cells and fibroblasts. Chi-miR-370-3p inhibits the proliferation of epithelial cells and fibroblasts by targeting TGF-βR2 and FGFR2, thereby improving cell migration ability and ultimately regulating the fate of epithelial cells and dermal fibroblasts to develop the placode and dermal condensate, inducing hair follicle morphogenesis.

MicroRNA-665 Regulates Cell Proliferation and Apoptosis of Vascular Smooth Muscle Cells by Targeting TGFBR1.

Coronary artery disease (CAD) is one of the heavy health burdens worldwide. Aberrant proliferation of vascular smooth muscle cells (VSMCs) contributes to the occurrence and development of CAD. This study aimed at exploring differentially expressed microRNAs (miRNAs) and their regulatory mechanisms in the development of CAD.The miRNA expression profile of GSE28858 was obtained from the Gene Expression Omnibus database. Differentially expressed miRNAs (DEmiRNAs) between CAD and healthy control samples were analyzed using limma package in R. Target genes of DEmiRNAs were predicted, and a miRNA-target gene network was constructed. The relationship between miR-665 and transforming growth factor beta receptor 1 (TGFBR1) was selected for further analysis. The interaction between miR-665 and TGFBR1 was confirmed by dual luciferase reporter assay. Effects of miR-665 on cell viability and apoptosis of VSMCs were evaluated by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. Besides, western blot assays for BCL2L11 and caspase 3 were also conducted.A total of 38 upregulated miRNAs and 28 downregulated miRNAs were identified. The expression level of miR-665 was significantly downregulated in patients with CAD. TGFBR1 was proved to be a target gene of miR-665. Besides, ectopic expression of miR-665 obviously inhibited VSMC growth and promoted VSMC apoptosis. TGFBR1 overexpression in VSMCs transfected with miR-665 mimic could restore the effect of miR-665 on the proliferation and apoptosis of VSMCs.MiR-665 might participate in the proliferation and apoptosis of VSMCs by targeting TGFBR1.

Extracellular Vesicles Derived From Adult and Fetal Bone Marrow Mesenchymal Stromal Cells Differentially Promote ex vivo Expansion of Hematopoietic Stem and Progenitor Cells.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Global Transcriptomic Analyses Reveal Genes Involved in Conceptus Development During the Implantation Stages in Pigs.

Prenatal mortality remains a significant concern to the pig farming industry around the world. Spontaneous fetal loss ranging from 20 to 45% by term occur after fertilization, with most of the loss happening during the implantation period. Since the factors regulating the high mortality rates of early conceptus during implantation phases are poorly understood, we sought to analyze the overall gene expression changes during this period, and identify the molecular mechanisms involved in conceptus development. This work employed Illumina’s next-generation sequencing (RNA-Seq) and quantitative real-time PCR to analyze differentially expressed genes (DEGs). Soft clustering was subsequently used for the cluster analysis of gene expression. We identified 8236 DEGs in porcine conceptus at day 9, 12, and 15 of pregnancy. Annotation analysis of these genes revealed rRNA processing (GO:0006364), cell adhesion (GO:1904874), and heart development (GO:0007507), as the most significantly enriched biological processes at day 9, 12, and 15 of pregnancy, respectively. In addition, we found various genes, such as T-complex 1, RuvB-like AAA ATPase 2, connective tissue growth factor, integrins, interferon gamma, SLA-1, chemokine ligand 9, PAG-2, transforming growth factor beta receptor 1, and Annexin A2, that play essential roles in conceptus morphological development and implantation in pigs. Furthermore, we investigated the function of PAG-2 in vitro and found that PAG-2 can inhibit trophoblast cell proliferation and migration. Our analysis provides a valuable resource for understanding the mechanisms of conceptus development and implantation in pigs.

Structures of HCMV Trimer reveal the basis for receptor recognition and cell entry

Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFβR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFβR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFβR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.

MiR-128-3p inhibits apoptosis and inflammation in LPS-induced sepsis by targeting TGFBR2.

BackgroundSepsis is a systemic inflammatory response that can lead to the dysfunction of many organs. The aberrant expression of miRNAs is associated with the pathogenesis of sepsis. However, the biological functions of miR-128-3p in sepsis remain largely unknown, and its mechanism should be further investigated. This study aimed to determine the regulatory network of miR-128-3p and TGFBR2 in lipopolysaccharide (LPS)-induced sepsis.MethodsThe expression levels of miR-128-3p and transforming growth factor beta receptors II (TGFBR2) were detected by quantitative polymerase chain reaction (qPCR). The protein levels of TGFBR2, Bcl-2, Bax, cleaved caspase 3, Smad2, and Smad3 were measured by western blot. Cell apoptosis was analyzed by flow cytometry. Cytokine production was detected by enzyme-linked immunosorbent assay (ELISA). The binding sites of miR-128-3p and TGFBR2 were predicted by Targetscan online software and confirmed by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay.ResultsThe level of miR-128-3p was decreased, and TGFBR2 expression was increased in serum samples of sepsis patients and LPS-induced HK2 cells. Overexpression of miR-128-3p or knockdown of TGFBR2 ameliorated LPS-induced inflammation and apoptosis. Moreover, TGFBR2 was a direct target of miR-128-3p, and its overexpression reversed the inhibitory effects of miR-128-3p overexpression on inflammation and apoptosis in LPS-induced HK2 cells. Besides, overexpression of miR-128-3p downregulated TGFBR2 to suppress the activation of the Smad signaling pathway.ConclusionmiR-128-3p could inhibit apoptosis and inflammation by targeting TGFBR2 in LPS-induced HK2 cells, which might provide therapeutic strategy for the treatment of sepsis.