Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFßR1 and ARID3a in obstructive kidney disease.

As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-ß1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-ß1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both in vivo and in vitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFßR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis in vivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFßR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.

Prostate cancer cell-derived exosomes ZNF667-AS1 reduces TGFBR1 mRNA stability to inhibit Treg expansion and DTX resistance by binding to U2AF1.

BACKGROUND: Docetaxel (DTX) resistance attenuates anti-tumor effects of DTX on prostate cancer (mCRPC) and drug resistance was related to Treg expansion in tumors. ZNF667-AS1 played a suppressing role in various tumors and tumor-derived exosomes carry lncRNAs to participate in tumor progression. Here, the effects of ZNF667-AS1 on malignant characteristics and DTX resistance in PC and the effect and its underlying molecular mechanism of tumor-derived exosomes carrying ZNF667-AS1 on Treg expansion were investigated. METHODS: The identification of exosomes were determined using TEM, NTA and western blot. The abundance of genes and proteins were evaluated using IHC, RT-qPCR, western blot and FISH. Malignant phenotypes of PC cells were evaluated by means of Edu, scratch test, transwell, CCK-8 and flow cytometry. The percentage of CD4+CD25+Foxp3+ Tregs was detected using flow cytometry. The location of ZNF667-AS1 was detected using nuclear-cytoplasmic fractionation. The co-location of ZNF667-AS1 and U2AF1 protein was detected using IF-FISH assay. The interactions among ZNF667-AS1, TGFBR1 and U2AF1 were verified using RNA pull-down, RIP and dual luciferase activity. RESULTS: ZNF667-AS1 expression in PC samples was lowered, which was negatively relative to poor prognosis and DTX resistance. ZNF667-AS1 overexpression inhibited malignant phenotypes of PC cells, tumor growth and DTX resistance. Besides, DTX resistant cell-derived exosomes expressed lower ZNF667-AS1 expression. Exosomes carrying exogenously high ZNF667-AS1 expression derived PC cells or serum of mice suppressed Treg expansion. On the mechanism, ZNF667-AS1 interacted with U2AF1 to destabilize TGFBR1 mRNA and reduce TGFBR1 expression in CD4+T cells. CONCLUSION: ZNF667-AS1 suppressed cell growth of PC cells, tumor growth of mice and DTX resistance to PC cells and exogenously high ZNF667-AS1 expression in tumor-derived exosomes destabilized TGFBR1 mRNA and reduce TGFBR1 expression through interacting with U2AF1, thus resulting in attenuated Treg expansion, which was related to DTX resistance.

Oocyte-derived growth differentiation factor 9 suppresses the expression of CYP17A1 and androgen production in human theca cells.

OBJECTIVE: To investigate the direct effect of growth differentiation factor 9 (GDF9) on androgen production in human theca cells. DESIGN: Experimental study. SETTING: Tertiary hospital-based research laboratory. PATIENT(S): Women who underwent in vitro fertilization and intracytoplasmic sperm injections at our clinic were included in this study. INTERVENTION(S): Primary cultured human theca cells from women undergoing in vitro fertilization and intracytoplasmic sperm injection treatment were treated with GDF9, an activin receptor-like kinase 5 (ALK5) inhibitor, and a SMAD4 agonist. MAIN OUTCOME MEASURE(S): The expression of androgen synthesis-related genes StAR, CYP17A1, and LHCGR, levels of androstenedione and testosterone, phosphorylation of SMAD2/3, and the interaction between bone morphogenic protein-activated type II receptor and ALK5 were evaluated using reverse transcription-quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assays, and coimmunoprecipitation assays, respectively. RESULT(S): Growth differentiation factor 9 decreased StAR, CYP17A1, and LHCGR expression levels in human theca cells, which was prevented by treatment with the ALK5 inhibitor, and suppressed production of androgen in human theca cells. Growth differentiation factor 9 increased SMAD2/3 phosphorylation, and the ALK5 inhibitor also suppressed this effect. Bone morphogenic protein-activated type II receptor and ALK5 bound to each other after GDF9 stimulation. The SMAD4 agonist kartogenin also decreased messenger RNA levels of StAR and CYP17A1 and protein levels of StAR in human theca cells. CONCLUSION(S): Growth differentiation factor 9 can activate the bone morphogenic protein-activated type II receptor-ALK5-SMAD2/3 signaling pathway, suppress CYP17A1 expression, and decrease androgen production in human theca cells.

Lumican/Lumikine Promotes Healing of Corneal Epithelium Debridement by Upregulation of EGFR Ligand Expression via Noncanonical Smad-Independent TGFß/TBRs Signaling.

The synthetic peptide of lumican C-terminal 13 amino acids with the cysteine replaced by an alanine, hereafter referred to as lumikine (LumC13C-A: YEALRVANEVTLN), binds to TGFß type I receptor/activin-like kinase5 (TBR1/ALK5) in the activated TGFß receptor complex to promote corneal epithelial wound healing. The present study aimed to identify the minimum essential amino acid epitope necessary to exert the effects of lumikine via ALK5 and to determine the role of the Y (tyrosine) residue for promoting corneal epithelium wound healing. This study also aimed to determine the signaling pathway(s) triggered by lumican-ALK5 binding. For such, adult Lum knockout (Lum-/-) mice (~8-12 weeks old) were subjected to corneal epithelium debridement using an Agerbrush®. The injured eyes were treated with 10 µL eye drops containing 0.3 µM synthetic peptides designed based on the C-terminal region of lumican for 5-6 h. To unveil the downstream signaling pathways involved, inhibitors of the Alk5 and EGFR signaling pathways were co-administered or not. Corneas isolated from the experimental mice were subjected to whole-mount staining and imaged under a ZEISS Observer to determine the distance of epithelium migration. The expression of EGFR ligands was determined following a scratch assay with HTCE (human telomerase-immortalized cornea epithelial cells) in the presence or not of lumikine. Results indicated that shorter LumC-terminal peptides containing EVTLN and substitution of Y with F in lumikine abolishes its capability to promote epithelium migration indicating that Y and EVTLN are essential but insufficient for Lum activity. Lumikine activity is blocked by inhibitors of Alk5, EGFR, and MAPK signaling pathways, while EGF activity is only suppressed by EGFR and MAPK inhibitors. qRT-PCR of scratched HTCE cells cultures treated with lumikine showed upregulated expression of several EGFR ligands including epiregulin (EREG). Treatment with anti-EREG antibodies abolished the effects of lumikine in corneal epithelium debridement healing. The observations suggest that Lum/lumikine binds Alk5 and promotes the noncanonical Smad-independent TGFß/TBRs signaling pathways during the healing of corneal epithelium debridement.

GPR56 facilitates hepatocellular carcinoma metastasis by promoting the TGF-ß signaling pathway.

The metastasis of hepatocellular carcinoma (HCC) poses a significant threat to the survival of patients. G protein-coupled receptor 56 (GPR56) has garnered extensive attention within malignant tumor research and plays a crucial role in cellular surface signal transmission. Nonetheless, its precise function in HCC remains ambiguous. Our investigation reveals a notable rise in GPR56 expression levels in human HCC cases, with heightened GPR56 levels correlating with unfavorable prognoses. GPR56 regulates TGF-ß pathway by interacting with TGFBR1, thereby promoting HCC metastasis. At the same time, GPR56 is subject to regulation by the canonical cascade of TGF-ß signaling, thereby establishing a positive feedback loop. Furthermore, the combination application of TGFBR1 inhibitor galunisertib (GAL) and GPR56 inhibitor Dihydromunduletone (DHM), significantly inhibits HCC metastasis. Interventions towards this signaling pathway could offer a promising therapeutic approach to effectively impede the metastasis of GPR56-mediated HCC.

Reproducible lung protective effects of a TGFßR1/ALK5 inhibitor in a bleomycin-induced and spirometry-confirmed model of IPF in male mice.

This study comprehensively validated the bleomycin (BLEO) induced mouse model of IPF for utility in preclinical drug discovery. To this end, the model was rigorously evaluated for reproducible phenotype and TGFß-directed treatment outcomes. Lung disease was profiled longitudinally in male C57BL6/JRJ mice receiving a single intratracheal instillation of BLEO (n = 10-12 per group). A TGFßR1/ALK5 inhibitor (ALK5i) was profiled in six independent studies in BLEO-IPF mice, randomized/stratified to treatment according to baseline body weight and non-invasive whole-body plethysmography. ALK5i (60 mg/kg/day) or vehicle (n = 10-16 per study) was administered orally for 21 days, starting 7 days after intratracheal BLEO installation. BLEO-IPF mice recapitulated functional, histological and biochemical hallmarks of IPF, including declining expiratory/inspiratory capacity and inflammatory and fibrotic lung injury accompanied by markedly elevated TGFß levels in bronchoalveolar lavage fluid and lung tissue. Pulmonary transcriptome signatures of inflammation and fibrosis in BLEO-IPF mice were comparable to reported data in IPF patients. ALK5i promoted reproducible and robust therapeutic outcomes on lung functional, biochemical and histological endpoints in BLEO-IPF mice. The robust lung fibrotic disease phenotype, along with the consistent and reproducible lung protective effects of ALK5i treatment, makes the spirometry-confirmed BLEO-IPF mouse model highly applicable for profiling novel drug candidates for IPF.

Human transforming growth factor ß type I receptor in complex with kinase inhibitor SB505124.

The crystal structure of the intracellular domain of transforming growth factor ß type I receptor (TßR1) in complex with the competitive inhibitor SB505124 is presented. The study provides insights into the structure and function of TßR1 in complex with SB505124, and as such offers molecular-level understanding of the inhibition of this critical signalling pathway. The potential of SB505124 as an avenue for therapy in cancer treatment is discussed on basis of the results.

Cloning and Expression Analysis of TGF-ß Type I Receptor Gene in Hyriopsis cumingii.

The TGF-ß signaling pathway plays an important role in wound healing and immune response. In this study, a TGF-ß type I receptor (TGF-ßRI) homolog was cloned and characterized from freshwater mussel Hyriopsis cumingii. The full-length cDNA of the TGF-ß RI gene was 2017 bp, with a 1554 bp open reading frame (ORF), and encoded 517 amino acids. The predictive analysis further identified distinct regions within the TGF-ßRI protein: a signal peptide, a membrane outer region, a transmembrane region, and an intracellular region. Real-time quantitative PCR results showed that the TGF-ß RI gene was expressed in all tissues of healthy mussels. The transcripts of TGF-ß RI in hemocytes and hepatopancreas were significantly up-regulated at different periods after stimulation with Aeromonas hydrophila and peptidoglycan (PGN) (P < 0.05). The mRNA expression of TGF-ß RI progressively increased from day 1 to day 10 after trauma (P < 0.05), and it returned to the initial level by day 15. The expression levels of TGF-ß , Smad5, MMP1/19, and TIMP1/2, but not Smad3/4, were significantly up-regulated at different time points after trauma. However, the expression levels of TGF-ß , MMP1/19, and TIMP2 were decreased after treatment with the inhibitor SB431542. Furthermore, the recombinant TGF-ßRI proteins were expressed in vitro and existed in the form of inclusion bodies. Western blotting results showed that TGF-ßRI proteins were expressed constitutively in various tissues of mussels, and their expression was up-regulated after trauma, which was consistent with the mRNA expression trend. These results indicate that TGF-ß RI is involved in the process of wound repair and immune response.

Protective Effects of Vitamin D on Proteoglycans of Human Articular Chondrocytes through TGF-ß1 Signaling.

The extracellular matrix of cartilage primarily constitutes of collagen and aggrecan. Cartilage degradation starts with aggrecan loss in osteoarthritis (OA). Vitamin D (VD) plays an essential role in several inflammation-related diseases and can protect the collagen in cartilage during OA. The present study focused on the role of VD in aggrecan turnover of human articular chondrocytes treated with tumor necrosis factor α (TNF-α) and the possible mechanism. Treatment with different doses of VD and different periods of intervention with TNF-α and TGF-ß1 receptor (TGFßR1) inhibitor SB525334 were investigated. The viability of human chondrocytes and extracellular secretion of TGF-ß1 were measured. The expression of intracellular TGFßR1 and VD receptor was examined. Transcriptional and translational levels of aggrecan and the related metabolic factors were analyzed. The results showed that TNF-α markedly reduced the viability, TGFßR1 expressions and aggrecan levels of human chondrocytes, and increased disintegrin and metalloproteinase with thrombospondin motifs. The alterations were partially inhibited by VD treatment. Furthermore, the effects of VD were blocked by the TGFßR1 inhibitor SB525334 in TNF-α-treated cells. VD may prevent proteoglycan loss due to TNF-α via TGF-ß1 signaling in human chondrocytes.

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

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

Discovery of a urea-based hit compound as a novel inhibitor of transforming growth factor-ß type 1 receptor: in silico and in vitro studies.

Transforming growth factor ß type 1 receptor (TGFßR1), a crucial serine-threonine kinase, is central to the TGFß/Smad signaling pathway, governing cellular processes like growth, differentiation, apoptosis, and immune response. This pathway is closely linked to the epithelial-mesenchymal transition (EMT) process, which plays an important role in the metastasis of hepatocellular carcinoma (HCC). To date, only limited inhibitors targeting TGFßR1 have entered clinical trials, yet they encounter challenges, notably high toxicity, in clinical applications. Herein, an efficient virtual screening pipeline was developed. Eighty compounds were screened from a pool of over 17 million molecules based on docking scores and binding free energy. Four compounds were manually selected with the assistance of enhanced sampling method BPMD (binding pose metadynamics). The binding stability of these four compounds complexed with TGFßR1 was subsequently studied through long-timescale conventional molecular dynamics simulations. The three most promising compounds were subjected to in vitro bioactivity assays. Cpd272 demonstrated moderate inhibitory activity against TGFßR1, with an IC50 value of 1.57 ± 0.33 µM. Moreover, it exhibited cytotoxic effects on human hepatocellular carcinoma cell line Bel-7402. By shedding light on the binding mode of the receptor-ligand complexes, Cpd272 was identified as a hit compound featuring a novel urea-based scaffold capable of effectively inhibiting TGFßR1.

Sulforaphane Promotes Proliferation of Porcine Granulosa Cells via the H3K27ac-Mediated GDF8-ALK5-ERK Pathway.

Follicle development, a crucial process in reproductive biology, hinges upon the dynamic proliferation of granulosa cells (GCs). Growth differentiation factor-8 (GDF8) is well-known as myostatin for inhibiting skeletal muscle growth, and it also exists in ovarian GCs and follicle fluid. However, the relationship between GCs proliferation and GDF8 remains elusive. Sulforaphane (SFN) is a potent bioactive compound, which in our study has been demonstrated to induce the expression of GDF8 in GCs. Meanwhile, we discover a novel role of SFN in promoting the proliferation of porcine GCs. Specifically, SFN enhances GCs proliferation by accelerating the progression of the cell cycle through the G1 phase to the S phase. By performing gene expression profiling, we showed that the promoting proliferative effects of SFN are highly correlated with the TGF-ß signaling pathways and cell cycle. Among the ligand factors of TGF-ß signaling, we identify GDF8 as a critical downstream effector of SFN, which acts through ALK5 to mediate SFN-induced proliferation and G1/S transition. In addition, we identify a noncanonical downstream pathway by which GDF8 induces the activation of MAPK/ERK to facilitate the cell cycle progression in GCs. Moreover, we reveal that the expression of GDF8 is regulated by SFN through epigenetic modifications of H3K27 acetylation. These findings not only provide mechanistic insights into the regulation of GCs proliferation but also establish a previously unrecognized role of GDF8 in follicle development, which have significant implications for developing strategies to improve female fertility.

Cancer-associated fibroblasts barrier breaking via TGF-ß blockade paved way for docetaxel micelles delivery to treat pancreatic cancer.

TGF-ß is a crucial regulator in tumor microenvironment (TME), especially for myofibroblastic cancer-associated fibroblasts (myCAFs). The myCAFs can be motivated by TGF-ß signaling to erect pro-tumor TME, meanwhile, myCAFs overexpress TGF-ß to mediate the crosstalk between tumor and stromal cells. The blockade of TGF-ß can break cancer-associated fibroblasts barrier, consequently opening the access for drugs into tumor. The TGF-ß is a promising target in anti-tumor therapy. Herein, we introduced a two-stage combination therapy (TC-Therapy), including TGF-ß receptor I inhibitor SB525334 (SB) and cytotoxicity agent docetaxel micelle (DTX-M). We found that SB and DTX-M synergistically inhibited myCAFs proliferation and elevated p53 protein expression in BxPC-3/3T3 mixed cells. Gene and protein tests demonstrated that SB cut off TGF-ß signaling via receptor blockade and it did not arouse TGF-ß legend compensated internal autocrine. On the contrary, two agents combined decreased TGF-ß secretion and inhibited myCAFs viability marked by α-SMA and FAPα. TC-Therapy was applied in BxPc-3/3T3 mixed tumor-bearing mice model. After TC-Therapy, the α-SMA+/ FAPα+ myCAFs faded increasingly and collagenous fibers mainly secreted by myCAFs decreased dramatically as well. More than that, the myCAFs barrier breaking helped to normalize micro-vessels and paved way for micelle penetration. The TGF-ß protein level of TC-Therapy in TME was much lower than that of simplex DTX-M, which might account for TME restoration. In conclusion, TGF-ß inhibitor acted as the pioneer before nano chemotherapeutic agents. The TC-Therapy of TGF-ß signaling inhibition and anti-tumor agent DTX-M is a promising regimen without arising metastasis risk to treat pancreatic cancer. The therapeutic regimen focused on TGF-ß related myCAFs reminds clinicians to have a comprehensive understanding of pancreatic cancer.

Nonclinical Profile of PF-06952229 (MDV6058), a Novel TGFßRI/Activin Like Kinase 5 Inhibitor Supports Clinical Evaluation in Cancer.

The development of transforming growth factor ßreceptor inhibitors (TGFßRi) as new medicines has been affected by cardiac valvulopathy and arteriopathy toxicity findings in nonclinical toxicology studies. PF-06952229 (MDV6058) selected using rational drug design is a potent and selective TGFßRI inhibitor with a relatively clean off-target selectivity profile and good pharmacokinetic properties across species. PF-06952229 inhibited clinically translatable phospho-SMAD2 biomarker (≥60%) in human and cynomolgus monkey peripheral blood mononuclear cells, as well as in mouse and rat splenocytes. Using an optimized, intermittent dosing schedule (7-day on/7-day off/cycle; 5 cycles), PF-06952229 demonstrated efficacy in a 63-day syngeneic MC38 colon carcinoma mouse model. In the pivotal repeat-dose toxicity studies (rat and cynomolgus monkey), PF-06952229 on an intermittent dosing schedule (5-day on/5-day off cycle; 5 cycles, 28 doses) showed no cardiac-related adverse findings. However, new toxicity findings related to PF-06952229 included reversible hepatocellular (hepatocyte necrosis with corresponding clinically monitorable transaminase increases) and lung (hemorrhage with mixed cell inflammation) findings at ≥ targeted projected clinical efficacious exposures. Furthermore, partially reversible cartilage hypertrophy (trachea and femur in rat; femur in monkey) and partially to fully reversible, clinically monitorable decreases in serum phosphorus and urinary phosphate at ≥ projected clinically efficacious exposures were observed. Given the integral role of TGFß in endochondral bone formation, cartilage findings in toxicity studies have been observed with other TGFßRi classes of compounds. The favorable cumulative profile of PF-06952229 in biochemical, pharmacodynamic, pharmacokinetic, and nonclinical studies allowed for its evaluation in cancer patients using the intermittent dosing schedule (7-day on/7-day off) and careful protocol-defined monitoring. SIGNIFICANCE STATEMENT: Only a few TGFßRi have progressed for clinical evaluation due to adverse cardiac findings in pivotal nonclinical toxicity studies. The potential translations of such findings in patients are of major concern. Using a carefully optimized intermittent dosing schedule, PF-06952229 has demonstrated impressive pharmacological efficacy in the syngeneic MC38 colon carcinoma mouse model. Additionally, a nonclinical toxicology package without cardiovascular liabilities and generally monitorable toxicity profile has been completed. The compound presents an acceptable International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use S9-compliant profile for the intended-to-treat cancer patients.

LMTK2 switches on canonical TGF-ß1 signaling in human bronchial epithelial cells.

Transforming growth factor (TGF-ß1) is a critical profibrotic mediator in chronic lung disease, and there are no specific strategies to mitigate its adverse effects. Activation of TGF-ß1 signaling is a multipart process involving ligands, transmembrane receptors, and transcription factors. In addition, an intricate network of adaptor proteins fine-tunes the signaling strength, duration, and activity. Namely, Smad7 recruits growth arrest and DNA damage (GADD34) protein that then interacts with the catalytic subunit of phosphoprotein phosphatase 1 (PP1c) to inactivate TGF-ß receptor (TßR)-I and downregulate TGF-ß1 signaling. Little is known about how TGF-ß1 releases TßR-I from the GADD34-PP1c inhibition to activate its signaling. Transmembrane lemur tyrosine kinase 2 (LMTK2) is a PP1c inhibitor, and our published data showed that TGF-ß1 recruits LMTK2 to the cell surface. Here, we tested the hypothesis that TGF-ß1 recruits LMTK2 to inhibit PP1c, allowing activation of TßR-I. First, LMTK2 interacted with the TGF-ß1 pathway in the human bronchial epithelium at multiple checkpoints. Second, TGF-ß1 inhibited PP1c by an LMTK2-dependent mechanism. Third, TGF-ß1 used LMTK2 to activate canonical Smad3-mediated signaling. We propose a model whereby the LMTK2-PP1c and Smad7-GADD34-PP1c complexes serve as on-and-off switches in the TGF-ß1 signaling in human bronchial epithelium.NEW & NOTEWORTHY Activation of the transforming growth factor (TGF)-ß1 signaling pathway is complex, involving many ligands, transmembrane receptors, transcription factors, and modulating proteins. The mechanisms of TGF-ß1 signaling activation/inactivation are not fully understood. We propose for the first time a model by which transmembrane lemur tyrosine kinase 2 (LMTK2) forms a complex with phosphoprotein phosphatase 1 (PP1c) to activate TGF-ß1 signaling and Smad7, growth arrest and DNA damage (GADD34), and PP1C form a complex to inactivate TGF-ß1 signaling in human bronchial epithelium.

The TßRI promotes migration and metastasis through thrombospondin 1 and ITGAV in prostate cancer cells.

TGFß potently modifies the extracellular matrix (ECM), which is thought to favor tumor cell invasion. However, the mechanism whereby the cancer cells employ the ECM proteins to facilitate their motility is largely unknown. In this study we used RNA-seq and proteomic analysis to examine the proteins secreted by castration-resistant prostate cancer (CRPC) cells upon TGFß treatment and found that thrombospondin 1 (THBS1) was observed to be one of the predominant proteins. The CRISPR Cas9, or siRNA techniques was used to downregulate TGFß type I receptor (TßRI) to interfere with TGFß signaling in various cancer cells in vitro. The interaction of ECM proteins with the TßRI in the migratory prostate cancer cells in response to TGFß1 was demonstrated by several different techniques to reveal that THBS1 mediates cell migration by interacting with integrin subunit alpha V (ITGAV) and TßRI. Deletion of TßRI or THBS1 in cancer cells prevented their migration and invasion. THBS1 belongs to a group of tumorigenic ECM proteins induced via TGFß signaling in CRPC cells, and high expression of THBS1 in human prostate cancer tissues correlated with the degree of malignancy. TGFß-induced production of THBS1 through TßRI facilitates the invasion and metastasis of CRPC cells as shown in vivo xenograft animal experiments.

Loss of TGFß-Mediated Repression of Angiopoietin-2 in Pericytes Underlies Germinal Matrix Hemorrhage Pathogenesis.

BACKGROUND: TGF (transforming growth factor)-ß pathway is central to blood-brain barrier development as it regulates cross talk between pericytes and endothelial cells. Murine embryos lacking TGFß receptor Alk5 (activin receptor-like kinase 5) in brain pericytes (mutants) display endothelial cell hyperproliferation, abnormal vessel morphology, and gross germinal matrix hemorrhage-intraventricular hemorrhage (GMH-IVH), leading to perinatal lethality. Mechanisms underlying how ALK5 signaling in pericytes noncell autonomously regulates endothelial cell behavior remain elusive. METHODS: Transcriptomic analysis of human brain pericytes with ALK5 silencing identified differential gene expression. Brain vascular cells isolated from mutant embryonic mice with GMH-IVH and preterm human IVH brain samples were utilized for target validation. Finally, pharmacological and genetic inhibition was used to study the therapeutic effects on GMH-IVH pathology. RESULTS: Herein, we establish that the TGFß/ALK5 pathway robustly represses ANGPT2 (angiopoietin-2) in pericytes via epigenetic remodeling. TGFß-driven SMAD (suppressor of mothers against decapentaplegic) 3/4 associates with TGIF1 (TGFß-induced factor homeobox 1) and HDAC (histone deacetylase) 5 to form a corepressor complex at the Angpt2 promoter, resulting in promoter deacetylation and gene repression. Moreover, murine and human germinal matrix vessels display increased ANGPT2 expression during GMH-IVH. Isolation of vascular cells from murine germinal matrix identifies pericytes as a cellular source of excessive ANGPT2. In addition, mutant endothelial cells exhibit higher phosphorylated TIE2 (tyrosine protein kinase receptor). Pharmacological or genetic inhibition of ANGPT2 in mutants improves germinal matrix vessel morphology and attenuates GMH pathogenesis. Importantly, genetic ablation of Angpt2 in mutant pericytes prevents perinatal lethality, prolonging survival. CONCLUSIONS: This study demonstrates that TGFß-mediated ANGPT2 repression in pericytes is critical for maintaining blood-brain barrier integrity and identifies pericyte-derived ANGPT2 as an important pathological target for GMH-IVH.

The TGFß type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed/refractory multiple myeloma: a phase 1b trial.

Functional blockade of the transforming growth factor-beta (TGFß) signalling pathway improves the efficacy of cytotoxic and immunotherapies. Here, we conducted a phase 1b study (ClinicalTrials.gov., NCT03143985) to determine the primary endpoints of safety, tolerability, and maximal tolerated dose (200 mg twice daily) for the orally-available TGFß type I receptor kinase inhibitor vactosertib in combination with pomalidomide in relapsed and/or refractory multiple myeloma (RRMM) patients who had received ≥2 lines of chemoimmunotherapy. Secondary endpoints demonstrated sustained clinical responses, favorable pharmacokinetic parameters and a 6-month progression-free survival of 82%. Vactosertib combined with pomalidomide was well-tolerated at all dose levels and displayed a manageable adverse event profile. Exploratory analysis indicated that vactosertib co-treatment with pomalidomide also reduced TGFß levels in patient bone marrow as well as the level of CD8+ T-cells that expressed the immunoinhibitory marker PD-1. In vitro experiments indicated that vactosertib+pomalidomide co-treatment decreased the viability of MM cell lines and patient tumor cells, and increased CD8+ T-cell cytotoxic activity. Vactosertib is a safe therapeutic that demonstrates tumor-intrinsic activity and can overcome immunosuppressive challenges within the tumor microenvironment to reinvigorate T-cell fitness. Vactosertib offers promise to improve immunotherapeutic responses in heavily-pretreated MM patients refractory to conventional agents.

LncRNA H19 accelerates renal fibrosis by negatively regulating the let-7b-5p/TGF-ßR1/COL1A1 axis.

BACKGROUND: Transforming growth factor-beta1 (TGF-ß1)-mediated renal fibrosis is a critical pathological process of chronic kidney disease worsening to end-stage renal disease. Recent studies have shown that long noncoding RNA H19 (lncRNA H19) is widely involved in the formation and progression of fibrosis in multiple organs. However, its molecular events in renal fibrosis remain to be elucidated. METHODS: Rats were treated with adenine intragastrically and HK-2 cells were induced by TGF-ß1 to construct renal fibrosis models in vivo and in vitro, respectively. Renal histopathological examination was performed using HE and Masson staining. Gene expression levels of interleukin-1beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), TGF-ß1, fibronectin (Fn), alpha-smooth muscle actin (α-SMA), H19, let-7b-5p, TGF-ß receptor 1 (TGF-ßR1), and type I collagen (COL1A1) were detected by qRT-PCR. Immunohistochemistry, immunofluorescence, and western blot analysis were used to evaluate the expression of renal fibrosis biomarkers. Dual-luciferase reporter assay was used to verify the presence of binding sites between H19 and let-7b-5p, and between let-7b-5p and TGF-ßR1 and COL1A1. RESULTS: H19 was overexpressed in both in vivo and in vitro renal fibrosis models. H19 knockdown significantly reversed TGF-ß1-induced upregulation of fibronectin, COL1A1, and α-SMA and downregulation of E-cadherin in HK-2 cells, accompanied by an increase in let-7b-5p. Let-7b-5p was bound to H19 in HK-2 cells, and its overexpression inhibited TGF-ß1-induced HK-2 cell fibrosis. Further experiments determined that let-7b-5p directly targets TGF-ßR1 and COL1A1 in HK-2 cells. In addition, inhibition of let-7b-5p reversed the reduction in HK-2 cell fibrosis induced by H19 knockdown. Finally, knockdown of H19 alleviated renal fibrosis in vivo and was associated with regulation of the let-7b-5p/TGF-ßR1/COL1A1 axis. CONCLUSION: Our results indicate that knockdown of H19 inhibits renal tubular epithelial fibrosis by negatively regulating the let-7b-5p/TGF-ßR1/COL1A1 axis, which may provide new mechanistic insights into CRF progression.

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

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