Inhibition Of TGF-β Signaling Research Articles

Marine derived macrolide bryostatin 4 inhibits the TGF-β signaling pathway against acute erythroleukemia.

Acute erythroleukemia (AEL) is a rare and highly aggressive subtype of acute myeloid leukemia (AML) with an extremely poor prognosis when treated with available drugs. Therefore, new investigational agents capable of inducing remission are urgently required. Bioinformatics analysis, western blot and qRT-PCR were used to reveal the potential biological mechanism of bryostatin 4 (B4), an antineoplastic macrolide derived from the marine bryozoan Bugula neritina. Then, in vivo experiments were conducted to evaluate the role of transforming growth factor (TGF)-β signaling in the progression of AEL. Our results revealed that the proliferation of K562 cells and TF-1 cells was significantly inhibited by B4 at IC50 values of 37 nM and 52 nM, respectively. B4 inhibited TGF-β signaling and its downstream pathway targets, particularly the phosphorylation of Smad2, Smad3, Ras, C-RAF, ERK1/2, and MEK. B4 also played an important role in cell invasion and migration in K562 cells and TF-1 cells by reducing the protein levels of the mesenchymal cell marker vimentin. Moreover, Flow cytometry and western blot analyses demonstrated that B4 induced apoptosis and initiated G0/G1 phase arrest by modulating mitochondrial dysfunction and cyclin-dependent kinase (CDK) expression. These findings indicated that B4 could inhibit the proliferation, migration, invasion, and TGF-β signaling pathways of AEL cells, thus suggesting that B4 possesses therapeutic potential as a treatment for AEL.

EVs-miR-17-5p attenuates the osteogenic differentiation of vascular smooth muscle cells potentially via inhibition of TGF-β signaling under high glucose conditions

Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor β receptor type II (TGF-β RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-β RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.

High-fat and high-carbohydrate diets increase bone fragility through TGF-β-dependent control of osteocyte function.

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-β signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-β signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-β signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-β receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.

Interaction of the tumor suppressor SMAD4 and WNT signaling in progression to oral squamous cell carcinoma.

SMAD4 is a tumor suppressor mutated or silenced in multiple cancers, including oral cavity squamous cell carcinoma (OSCC). Human clinical samples and cell lines, mouse models and organoid culture were used to investigate the role that SMAD4 plays in progression from benign disease to invasive OSCC. Human OSCC lost detectable SMAD4 protein within tumor epithelium in 24% of cases, and this loss correlated with worse progression-free survival independent of other major clinical and pathological features. A mouse model engineered for KrasG12D expression in the adult oral epithelium induced benign papillomas, however the combination of KrasG12D with loss of epithelial Smad4 expression resulted in rapid development of invasive carcinoma with features of human OSCC. Examination of regulatory pathways in 3D organoid cultures of SMAD4+ and SMAD4- mouse tumors with Kras mutation found that either loss of SMAD4 or inhibition of TGFβ signaling upregulated the WNT pathway and altered the extracellular matrix. The gene signature of the mouse tumor organoids lacking SMAD4 was highly similar to the gene signature of human head and neck squamous cell carcinoma. In summary, this work has uncovered novel mechanisms by which SMAD4 acts as a tumor suppressor in OSCC. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

GVHD targets organoid-forming bile duct stem cells in a TGF-β–dependent manner

AbstractGraft-versus-host disease (GVHD) is a major life-threatening complication that occurs after allogeneic hematopoietic cell transplantation (HCT). Although adult tissue stem cells have been identified as targets of GVHD in the skin and gut, their role in hepatic GVHD is yet to be clarified. In the current study, we explored the fate of bile duct stem cells (BDSCs), capable of generating liver organoids in vitro, during hepatic GVHD after allogeneic HCT. We observed a significant expansion of biliary epithelial cells (BECs) on injury early after allogeneic HCT. Organoid-forming efficiency from the bile duct was also significantly increased early after allogeneic HCT. Subsequently, the organoid-forming efficiency from bile ducts was markedly decreased in association with the reduction of BECs and the elevation of plasma concentrations of bilirubin, suggesting that GVHD targets BDSCs and impairs the resilience of BECs. The growth of liver organoids in the presence of liver-infiltrating mononuclear cells from allogeneic recipients, but not from syngeneic recipients, was significantly reduced in a transforming growth factor-β (TGF-β)–dependent manner. Administration of SB-431542, an inhibitor of TGF-β signaling, from day 14 to day 28, protected organoid-forming BDSCs against GVHD and mitigated biliary dysfunction after allogeneic HCT, suggesting that BDSCs are a promising therapeutic target for hepatic GVHD.

Engineered extracellular vesicles for targeted reprogramming of cancer-associated fibroblasts to potentiate therapy of pancreatic cancer

Pancreatic cancer is one of the deadly malignancies with a significant mortality rate and there are currently few therapeutic options for it. The tumor microenvironment (TME) in pancreatic cancer, distinguished by fibrosis and the existence of cancer-associated fibroblasts (CAFs), exerts a pivotal influence on both tumor advancement and resistance to therapy. Recent advancements in the field of engineered extracellular vesicles (EVs) offer novel avenues for targeted therapy in pancreatic cancer. This study aimed to develop engineered EVs for the targeted reprogramming of CAFs and modulating the TME in pancreatic cancer. EVs obtained from bone marrow mesenchymal stem cells (BMSCs) were loaded with miR-138-5p and the anti-fibrotic agent pirfenidone (PFD) and subjected to surface modification with integrin α5-targeting peptides (named IEVs-PFD/138) to reprogram CAFs and suppress their pro-tumorigenic effects. Integrin α5-targeting peptide modification enhanced the CAF-targeting ability of EVs. miR-138-5p directly inhibited the formation of the FERMT2-TGFBR1 complex, inhibiting TGF-β signaling pathway activation. In addition, miR-138-5p inhibited proline-mediated collagen synthesis by directly targeting the FERMT2-PYCR1 complex. The combination of miR-138-5p and PFD in EVs synergistically promoted CAF reprogramming and suppressed the pro-cancer effects of CAFs. Preclinical experiments using the orthotopic stroma-rich and patient-derived xenograft mouse models yielded promising results. In particular, IEVs-PFD/138 effectively reprogrammed CAFs and remodeled TME, which resulted in decreased tumor pressure, enhanced gemcitabine perfusion, tumor hypoxia amelioration, and greater sensitivity of cancer cells to chemotherapy. Thus, the strategy developed in this study can improve chemotherapy outcomes. Utilizing IEVs-PFD/138 as a targeted therapeutic agent to modulate CAFs and the TME represents a promising therapeutic approach for pancreatic cancer.

Ultrasmall Polyphenol-NAD+ Nanoparticle-Mediated Renal Delivery for Mitochondrial Repair and Anti-Inflammatory Treatment of AKI-to-CKD Progression.

As a central metabolic molecule, nicotinamide adenine dinucleotide (NAD+) can potentially treat acute kidney injury (AKI) and chronic kidney disease (CKD); however, its bioavailability is poor due to short half-life, instability, the deficiency of targeting, and difficulties in transmembrane transport. Here a physiologically adaptive gallic acid-NAD+ nanoparticle is designed, which has ultrasmall size and pH-responsiveness, passes through the glomerular filtration membrane to reach injured renal tubules, and efficiently delivers NAD+ into the kidneys. With an effective accumulation in the kidneys, it restores renal function, immune microenvironment homeostasis, and mitochondrial homeostasis of AKI mice via the NAD+-Sirtuin-1 axis, and exerts strong antifibrotic effects on the AKI-to-CKD transition by inhibiting TGF-β signaling. It also exhibits excellent stability, biodegradable, and biocompatible properties, ensuring its long-term safety, practicality, and clinical translational feasibility. The present study shows a potential modality of mitochondrial repair and immunomodulation through nanoagents for the efficient and safe treatment of AKI and CKD.

Microbiota regulates the TET1-mediated DNA hydroxymethylation program in innate lymphoid cell differentiation

Innate lymphoid cell precursors (ILCPs) develop into distinct subsets of innate lymphoid cells (ILCs) with specific functions. The epigenetic program underlying the differentiation of ILCPs into ILC subsets remains poorly understood. Here, we reveal the genome-wide distribution and dynamics of the DNA methylation and hydroxymethylation in ILC subsets and their respective precursors. Additionally, we find that the DNA hydroxymethyltransferase TET1 suppresses ILC1 but not ILC2 or ILC3 differentiation. TET1 deficiency promotes ILC1 differentiation by inhibiting TGF-β signaling. Throughout ILCP differentiation at postnatal stage, gut microbiota contributes to the downregulation of TET1 level. Microbiota decreases the level of cholic acid in the gut, impairs TET1 expression and suppresses DNA hydroxymethylation, ultimately resulting in an expansion of ILC1s. In adult mice, TET1 suppresses the hyperactivation of ILC1s to maintain intestinal homeostasis. Our findings provide insights into the microbiota-mediated epigenetic programming of ILCs, which links microbiota-DNA methylation crosstalk to ILC differentiation.

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

LP340, a novel histone deacetylase inhibitor, decreases liver injury and fibrosis in mice: role of oxidative stress and microRNA-23a.

Effective therapy for liver fibrosis is lacking. Here, we examined whether LP340, the lead candidate of a new-generation of hydrazide-based HDAC1,2,3 inhibitors (HDACi), decreases liver fibrosis. Liver fibrosis was induced by CCl4 treatment and bile duct ligation (BDL) in mice. At 6 weeks after CCl4, serum alanine aminotransferase increased, and necrotic cell death and leukocyte infiltration occurred in the liver. Tumor necrosis factor-α and myeloperoxidase markedly increased, indicating inflammation. After 6 weeks, α-smooth muscle actin (αSMA) and collagen-1 expression increased by 80% and 575%, respectively, indicating hepatic stellate cell (HSC) activation and fibrogenesis. Fibrosis detected by trichrome and Sirius-red staining occurred primarily in pericentral regions with some bridging fibrosis in liver sections. 4-Hydroxynonenal adducts (indicator of oxidative stress), profibrotic cytokine transforming growth factor-β (TGFβ), and TGFβ downstream signaling molecules phospho-Smad2/3 also markedly increased. LP340 attenuated indices of liver injury, inflammation, and fibrosis markedly. Moreover, Ski-related novel protein-N (SnoN), an endogenous inhibitor of TGFβ signaling, decreased, whereas SnoN expression suppressor microRNA-23a (miR23a) increased markedly. LP340 (0.05mg/kg, ig., daily during the last 2weeks of CCl4 treatment) decreased 4-hydroxynonenal adducts and miR23a production, blunted SnoN decreases, and inhibited the TGFβ/Smad signaling. By contrast, LP340 had no effect on matrix metalloproteinase-9 expression. LP340 increased histone-3 acetylation but not tubulin acetylation, indicating that LP340 inhibited Class-I but not Class-II HDAC in vivo. After BDL, focal necrosis, inflammation, ductular reactions, and portal and bridging fibrosis occurred at 2 weeks, and αSMA and collagen-1 expression increased by 256% and 560%, respectively. LP340 attenuated liver injury, ductular reactions, inflammation, and liver fibrosis. LP340 also decreased 4-hydroxynonenal adducts and miR23a production, prevented SnoN decreases, and inhibited the TGFβ/Smad signaling after BDL. In vitro, LP340 inhibited immortal human hepatic stellate cells (hTERT-HSC) activation in culture (αSMA and collagen-1 expression) as well as miR23a production, demonstrating its direct inhibitory effects on HSC. In conclusions, LP340 is a promising therapy for both portal and pericentral liver fibrosis, and it works by inhibiting oxidative stress and decreasing miR23a.

Suppression of the TGF-β signaling exacerbates degeneration of auditory neurons in kanamycin-induced ototoxicity in mice

Transforming growth factor-β (TGF-β) signaling plays a significant role in multiple biological processes, including inflammation, immunity, and cell death. However, its specific impact on the cochlea remains unclear. In this study, we aimed to investigate the effects of TGF-β signaling suppression on auditory function and cochlear pathology in mice with kanamycin-induced ototoxicity. Kanamycin and furosemide (KM-FS) were systemically administered to 8-week-old C57/BL6 mice, followed by immediate topical application of a TGF-β receptor inhibitor (TGF-βRI) onto the round window membrane. Results showed significant TGF-β receptor upregulation in spiral ganglion neurons (SGNs) after KM-FA ototoxicity, whereas expression levels in the TGF-βRI treated group remained unchanged. Interestingly, despite no significant change in cochlear TGF-β expression after KM-FS ototoxicity, TGF-βRI treatment resulted in a significant decrease in TGF-β signaling. Regarding auditory function, TGF-βRI treatment offered no therapeutic effects on hearing thresholds and hair cell survival following KM-FS ototoxicity. However, SGN loss and macrophage infiltration were significantly increased with TGF-βRI treatment. These results imply that inhibition of TGF-β signaling after KM-FS ototoxicity promotes cochlear inflammation and SGN degeneration.

Hyaluronic acid modified extracellular vesicles targeting hepatic stellate cells to attenuate hepatic fibrosis

RationaleTransforming growth factor-beta1 (TGF-β1) plays a pivotal role in promoting hepatic fibrosis, pirfenidone (PFD) could inhibit TGF-β1 signaling pathway to alleviate hepatic stellate cells (HSC) activation mediated hepatic fibrosis. The targeting delivery strategy of PFD to hepatic stellate cells is a challenge. Extracellular vesicles (EVs), cell-derived membranous particles are intraluminal nano-vesicles that play a vital role in intercellular communication, they also be considered as an ideal nano-carrier. MethodsIn this study, we developed a target strategy to deliver PFD to HSC with CD44 over-expression by EVs, hyaluronic acid (HA) modified DSPE-PEG2000 endows the active targeting ability of activated HSCs to PFD-loaded EVs. ResultsIn both rat hepatic stellate cell line HSC-T6 and rat hepatocyte cell line BRL, HA@EVs-PFD demonstrated the capacity to down-regulate the expression of collagen-synthesis-related proteins and showed superior inhibition efficacy of HSC-T6 activation compared to free PFD. In hepatic fibrosis model, 4 weeks of HA@EVs-PFD treatment resulted in a reduction in liver collagen fibers, significant improvement in hepatic cell morphology, and amelioration of hepatic fibrosis. ConclusionsHA@EVs-PFD, as a drug delivery system that effectively targets and inhibits activated HSCs to treat hepatic fibrosis, holds promise as a potential therapeutic agent against hepatic fibrosis.

Abstract 1071: TGFβ Signaling Negatively Regulates Cardiac Lymphangiogenesis Following Myocardial Ischemia

Cardiovascular disease is the leading cause of death worldwide. Despite high prevalence, standard treatments have made little progress, and developing novel therapeutic treatments to improve cardiac diseases is the top priority of the field. Lymphatics are essential in maintaining tissue-fluid homeostasis, clearing cell debris, and trafficking immune cells. Over recent years, cardiac lymphatics have emerged as a potential target for treatment of cardiovascular diseases, as therapeutic lymphangiogenesis is shown to improve cardiac function following myocardial infarction (MI). However, pathologic lymphangiogenesis induced by MI is reported to be dysfunctional, suggesting cellular or molecular changes occurring in MI directly impact the structure and function of cardiac lymphatics. However, the underlying mechanisms leading to these changes have not been fully explored. In this study we propose TGFβ, a profibrotic signaling molecule increased in ischemic heart diseases, negatively regulates cardiac lymphangiogenesis and lymphatic function following MI. We generated mice with conditional deletion of TGFβ receptor in Lymphatic endothelial cells (LECs) and found that these conditional null (LEC-DKO) mice showed improved post-MI cardiac function and increased lymphangiogenesis compared to controls. Mechanistically, we found that LECs with TGFβ treatment showed increased Endothelial to Mesenchymal Transition (EndMT), and impaired junction integrity. Bulk RNAseq results confirmed these findings as genes involved in EndMT were highly upregulated, and LEC marker related genes were significantly downregulated in LECs in the presence of TGFβ. More importantly, GSEA revealed metabolic reprogramming in LECs treated with TGFβ, with significantly increased expression in glycolysis related genes, but significant downregulation of genes related to fatty acid oxidation. These data provide a possible mechanism by which TGFβ signaling induces LEC metabolic switch to impede lymphangiogenesis and promote cardiac dysfunction following MI, and inhibition of TGFβ signaling in LECs could provide a novel therapeutic strategy to improve lymphatic vascular integrity and lymphangiogenesis, thereby improving cardiac function following MI.

MAD1 deficiency accelerates hepatocellular proliferation via suppressing TGF-β signaling

Numerous researches have reported on the regulatory network of liver regeneration induced by partial hepatectomy (PH). However, information on key molecules and/or signaling pathways regulating the termination stage of liver regeneration remains limited. In this study, we identify hepatic mitotic arrest deficient 1 (MAD1) as a crucial regulator of transforming growth factor β (TGF-β) in the hepatocyte to repress liver regeneration. MAD1 has a low expression level at the rapid proliferation phase but significantly increases at the termination phase of liver regeneration. We show that MAD1 deficiency accelerates hepatocyte proliferation and enhances mitochondrial biogenesis and respiratory. Mechanistically, MAD1 deficiency in hepatocytes enhances mitochondrial function and promotes hepatocyte proliferation by suppressing TGF-β signaling. Our study reveals MAD1 as a novel suppressor of hepatocyte proliferation, which may provide a new therapeutic target for the recovery of liver function after liver transplant and partial hepatectomy.

Effects of Bulleyaconitine A on Extracellular Matrix Secretion and Expression of Related Proteins in Acetaldehyde-Activated Hepatic Stellate Cells.

Activated hepatic stellate cells differentiate into myofibroblasts, which synthesize and secrete extracellular matrix (ECM) leading to liver fibrosis. It was previously demonstrated that bulleyaconitine A (BLA), an alkaloid from Aconitum bulleyanum, inhibits proliferation and promotes apoptosis of human hepatic Lieming Xu-2 (LX-2) cells. In this study, we analyzed the effect of BLA on the production of ECM and related proteins by LX-2 cells activated with acetaldehyde (AA). The cells were randomized into the control group, AA group (cells activated with 400 μM AA), and BLA+AA group (cells cultured in the presence of 400 μM AA and 18.75 μg/ml BLA). In the BLA+AA group, the contents of collagens I and III and the expression of α-smooth muscle actin and transforming growth factor-β1 (TGF-β1) were statistically significantly higher than in the control, but lower than in the AA group. Expression of MMP-1 in the BLA+AA group was also significantly higher than in the AA group, but lower than in the control. Expression of TIMP-1 in the BLA+AA group was significantly higher than in the control, but lower than in the AA group. Thus, BLA suppressed activation and proliferation of LX-2 cells by inhibiting TGF-β1 signaling pathway and decreasing the content of collagens I and III by reducing the MMP-1/TIMP-1 ratio.

Cyclic strain alters the transcriptional and migratory response of scleral fibroblasts to TGFβ

In glaucoma, scleral fibroblasts are exposed to IOP-associated mechanical strain and elevated TGFβ levels. These stimuli, in turn, lead to scleral remodeling. Here, we examine the scleral fibroblast migratory and transcriptional response to these stimuli to better understand mechanisms of glaucomatous scleral remodeling. Human peripapillary scleral (PPS) fibroblasts were cultured on parallel grooves, treated with TGFβ (2 ng/ml) in the presence of vehicle or TGFβ signaling inhibitors, and exposed to uniaxial strain (1 Hz, 5%, 12–24 h). Axis of cellular orientation was determined at baseline, immediately following strain, and 24 h after strain cessation with 0° being completely aligned with grooves and 90° being perpendicular. Fibroblasts migration in-line and across grooves was assessed using a scratch assay. Transcriptional profiling of TGFβ-treated fibroblasts with or without strain was performed by RT-qPCR and pERK, pSMAD2, and pSMAD3 levels were measured by immunoblot. Pre-strain alignment of TGFβ-treated cells with grooves (6.2 ± 1.5°) was reduced after strain (21.7 ± 5.3°, p < 0.0001) and restored 24 h after strain cessation (9.5 ± 2.6°). ERK, FAK, and ALK5 inhibition prevented this reduction; however, ROCK, YAP, or SMAD3 inhibition did not. TGFβ-induced myofibroblast markers were reduced by strain (αSMA, POSTN, ASPN, MLCK1). While TGFβ-induced phosphorylation of ERK and SMAD2 was unaffected by cyclic strain, SMAD3 phosphorylation was reduced (p = 0.0004). Wound healing across grooves was enhanced by ROCK and SMAD3 inhibition but not ERK or ALK5 inhibition. These results provide insight into the mechanisms by which mechanical strain alters the cellular response to TGFβ and the potential signaling pathways that underlie scleral remodeling.

Inhibition of MERTK reduces organ fibrosis in mouse models of fibrotic disease.

Transforming growth factor-β (TGFβ) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFβ signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFβ-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFβ expression and drove TGFβ signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFβ signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.

BRCC3 Regulation of ALK2 in Vascular Smooth Muscle Cells: Implication in Pulmonary Hypertension.

An imbalance of antiproliferative BMP (bone morphogenetic protein) signaling and proliferative TGF-β (transforming growth factor-β) signaling is implicated in the development of pulmonary arterial hypertension (PAH). The posttranslational modification (eg, phosphorylation and ubiquitination) of TGF-β family receptors, including BMPR2 (bone morphogenetic protein type 2 receptor)/ALK2 (activin receptor-like kinase-2) and TGF-βR2/R1, and receptor-regulated Smads significantly affects their activity and thus regulates the target cell fate. BRCC3 modifies the activity and stability of its substrate proteins through K63-dependent deubiquitination. By modulating the posttranslational modifications of the BMP/TGF-β-PPARγ pathway, BRCC3 may play a role in pulmonary vascular remodeling, hence the pathogenesis of PAH. Bioinformatic analyses were used to explore the mechanism by which BRCC3 deubiquitinates ALK2. Cultured pulmonary artery smooth muscle cells (PASMCs), mouse models, and specimens from patients with idiopathic PAH were used to investigate the rebalance between BMP and TGF-β signaling in regulating ALK2 phosphorylation and ubiquitination in the context of pulmonary hypertension. BRCC3 was significantly downregulated in PASMCs from patients with PAH and animals with experimental pulmonary hypertension. BRCC3, by de-ubiquitinating ALK2 at Lys-472 and Lys-475, activated receptor-regulated Smad1/5/9, which resulted in transcriptional activation of BMP-regulated PPARγ, p53, and Id1. Overexpression of BRCC3 also attenuated TGF-β signaling by downregulating TGF-β expression and inhibiting phosphorylation of Smad3. Experiments in vitro indicated that overexpression of BRCC3 or the de-ubiquitin-mimetic ALK2-K472/475R attenuated PASMC proliferation and migration and enhanced PASMC apoptosis. In SM22α-BRCC3-Tg mice, pulmonary hypertension was ameliorated because of activation of the ALK2-Smad1/5-PPARγ axis in PASMCs. In contrast, Brcc3-/- mice showed increased susceptibility of experimental pulmonary hypertension because of inhibition of the ALK2-Smad1/5 signaling. These results suggest a pivotal role of BRCC3 in sustaining pulmonary vascular homeostasis by maintaining the integrity of the BMP signaling (ie, the ALK2-Smad1/5-PPARγ axis) while suppressing TGF-β signaling in PASMCs. Such rebalance of BMP/TGF-β pathways is translationally important for PAH alleviation.

Protein Disulfide Isomerase 4 Is an Essential Regulator of Endothelial Function and Survival.

Endothelial autophagy plays an important role in the regulation of endothelial function. The inhibition of endothelial autophagy is associated with the reduced expression of protein disulfide isomerase 4 (PDIA-4); however, its role in endothelial cells is not known. Here, we report that endothelial cell-specific loss of PDIA-4 leads to impaired autophagic flux accompanied by loss of endothelial function and apoptosis. Endothelial cell-specific loss of PDIA-4 also induced marked changes in endothelial cell architecture, accompanied by the loss of endothelial markers and the gain of mesenchymal markers consistent with endothelial-to-mesenchymal transition (EndMT). The loss of PDIA-4 activated TGFβ-signaling, and inhibition of TGFβ-signaling suppressed EndMT in PDIA-4-silenced endothelial cells in vitro. Our findings help elucidate the role of PDIA-4 in endothelial autophagy and endothelial function and provide a potential target to modulate endothelial function and/or limit autophagy and EndMT in (patho-)physiological conditions.

Abstract 728: Oral TGF-beta receptor1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity

Abstract Osteosarcoma (OS) is an aggressive malignant bone cancer, with the lung as the most frequent site of metastasis. Unresectable pulmonary metastasis remains a significant challenge with a survival rate of less than 20%. Identification of novel therapeutic strategies are desperately needed. Transforming growth factor-β1 (TGF-β) is a potent immune suppressive cytokine in OS tumor microenvironment (TME). TGF-β1 expression is increased in the sera and tumor tissues of OS patients and this increase is associated with high-grade OS and lung metastases. Therefore, blocking TGF-β1 signaling may be a novel therapy for OS treatment. In this study, we show that blocking TGF-β1 signaling using the orally bioavailable small molecule TGF-βR1 inhibitor, Vactosertib, significantly inhibited OS proliferation in vitro and in vivo. Notably, Vactosertib inhibits c-Myc expression in the OS cells and oral administration of Vactosertib significantly reduces OS growth in vivo. Vactosertib increased immune effectors (e.g., IFNγ+CD8+ cells and NK cells) and inhibited immune suppressors (e.g., M2-like TAM, MDSC) in the OS TME. Our results suggest that inhibition of TGF-β1 signaling is an effective therapeutic strategy against OS through a multi-pronged approach that targets tumor intrinsic and extrinsic factors to achieve optimal immune-effector functions and maximal clinical response. Citation Format: Sung Hee Choi, Jay Myers, Suzanne Tomchuck, Melissa Bonner, Saada Eid, Daniel Kingsley, Kristen VanHeyst, Seong-Jin Kim, Byung-Gyu Kim, Alex Y. Huang. Oral TGF-beta receptor1 inhibitor vactosertib promotes osteosarcoma regression by targeting tumor proliferation and enhancing anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 728.