TGF-β Signaling Research Articles

LH promotes the proliferation of porcine primordial germ cell-like cells (pPGCLCs) by regulating the ceRNA network related to the TGF-β signaling pathway

Primordial germ cells (PGCs), as the precursors of gametes found in early embryos, provide a new direction for solving the problem of reproductive disorders. In vitro, conversion of adult stem cells (ASCs) into primordial germ cell-like cells (PGCLCs) is feasible. The means of increasing PGCLCs number in vitro has been a focus of recent stem cell research. In this study, we found that luteinizing hormone (LH) could promote porcine PGCLCs (pPGCLCs) proliferation. To investigate the proliferation regulatory network, whole transcriptome sequencing technology was employed. Results showed that the TGF-β signaling pathway played a key role. In addition, we found that TGFβR1 and SMAD4, TGF-β signaling pathway-related genes, were significantly upregulated after LH treatment. Subsequently, we predicted their target microRNAs (miRNAs) and long non-coding RNAs (lncRNAs): ssc-miR-128, ssc-miR-146b, ssc-miR-361–3p, MSTRG.11473, MSTRG.11475, MSTRG.11553, and MSTRG.11554, and constructed the competitive endogenous RNAs (ceRNA) network. Finally, to further verify the ceRNA network, the miRNA-inhibitors were transfected into cells. RT-qPCR results indicated a significant increase in the expression of MSTRG.11473, MSTRG.11475, MSTRG.11553, MSTRG.11554, TGFβR1, and SMAD4 compared to the negative control (NC) group. In conclusion, these results highlight that LH could regulate the pPGCLCs proliferation by modulating the expression of TGF-β signaling pathway-related ncRNAs.

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

The TGF-β signaling pathway plays an important role in wound healing and immune response. In this study, a TGF-β type I receptor (TGF-βRI) homolog was cloned and characterized from freshwater mussel Hyriopsis cumingii. The full-length cDNA of the TGF-β 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-βRI protein: a signal peptide, a membrane outer region, a transmembrane region, and an intracellular region. Real-time quantitative PCR results showed that the TGF-β RI gene was expressed in all tissues of healthy mussels. The transcripts of TGF-β 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-β 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-β , 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-β , MMP1/19, and TIMP2 were decreased after treatment with the inhibitor SB431542. Furthermore, the recombinant TGF-βRI proteins were expressed in vitro and existed in the form of inclusion bodies. Western blotting results showed that TGF-βRI 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-β RI is involved in the process of wound repair and immune response.

Development and in vitro evaluation of bioprinted plasma-infused biocarriers for mesenchymal stromal cell delivery in musculoskeletal disorder treatment

A meta-analysis revealed no advantage of surgical over non-surgical treatments, emphasizing the need for non-invasive methods, particularly for prevalent osteoarticular diseases like knee osteoarthritis. To enhance therapeutic efficacy, we developed a plasma-infused gelatin methacryloyl (GelMA) biocarrier loaded with bone marrow-derived mesenchymal stromal cells (BMSCs). These constructs were evaluated in vitro for their properties and paracrine interactions in non-inflamed and inflamed environments. GelMA infused with platelet-rich plasma (PRP) and platelet-poor plasma (FFP) were compared. Pristine GelMA was used as a control. Both PRP and FFP enhanced the proliferation and viability of BMSCs in biocarriers, promoting cell survival pathways while inhibiting necrotic and apoptotic events. Proteomic analysis showed no differences in BMSC behavior between PRP and FFP in the absence of inflammation (p = 0.550). However, both plasmas significantly modified cell behavior under inflammatory conditions (p = 0.001). PRP- and FFP-infused biocarriers activated ten key signaling pathways, including HIF-1a, neuroinflammation, and extracellular matrix turnover. PRP-specific pathways included IL-17, IL-6, and several growth factor signaling pathways. No significant differences in angiogenesis were linked to platelet dose (p = 0.079), but both PRP and FFP significantly enhanced angiogenesis compared to GelMA alone (p &lt; 0.001 for PRP, p = 0.002 for FFP). FFP showed stronger angiogenesis than PRP under IL-1&amp;beta; treatment (p = 0.042). Plasma-infused biocarriers altered BMSC behavior in response to inflammatory cytokines compared to GelMA (p = 0.001). PRP specifically activated TGF-b signaling under IL-1b (Z=2.308, p-value 1.02E-35), which was not seen under TNF-a exposure. These findings suggest that PRP and FFP-infused biocarriers may offer promising improvements in regenerative therapies for inflammatory osteoarticular conditions like knee osteoarthritis.

Increased peritoneal TGF-β1 is associated with ascites-induced NK-cell dysfunction and reduced survival in high-grade epithelial ovarian cancer.

Natural killer (NK) cell therapy represents an attractive immunotherapy approach against recurrent epithelial ovarian cancer (EOC), as EOC is sensitive to NK cell-mediated cytotoxicity. However, NK cell antitumor activity is dampened by suppressive factors in EOC patient ascites. Here, we integrated functional assays, soluble factor analysis, high-dimensional flow cytometry cellular component data and clinical parameters of advanced EOC patients to study the mechanisms of ascites-induced inhibition of NK cells. Using a suppression assay, we found that ascites from EOC patients strongly inhibits peripheral blood-derived NK cells and CD34+ progenitor-derived NK cells, albeit the latter were more resistant. Interestingly, we found that higher ascites-induced NK cell inhibition correlated with reduced progression-free and overall survival in EOC patients. Furthermore, we identified transforming growth factor (TGF)-β1 to correlate with ascites-induced NK cell dysfunction and reduced patient survival. In functional assays, we showed that proliferation and anti-tumor reactivity of CD34+ progenitor-derived NK cells are significantly affected by TGF-β1 exposure. Moreover, inhibition of TGF-β1 signaling with galunisertib partly restored NK cell functionality in some donors. For the cellular components, we showed that the secretome is associated with a different composition of CD45+ cells between ascites of EOC and benign reference samples with higher proportions of macrophages in the EOC patient samples. Furthermore, we revealed that higher TGF-β1 levels are associated with the presence of M2-like macrophages, B cell populations and T-regulatory cells in EOC patient ascites. These findings reveal that targeting TGF-β1 signaling could increase NK cell immune responses in high-grade EOC patients.

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

TGFβ 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 TGFβ 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 TGFβ type I receptor (TβRI) to interfere with TGFβ signaling in various cancer cells in vitro. The interaction of ECM proteins with the TβRI in the migratory prostate cancer cells in response to TGFβ1 was demonstrated by several different techniques to reveal that THBS1 mediates cell migration by interacting with integrin subunit alpha V (ITGAV) and TβRI. Deletion of TβRI or THBS1 in cancer cells prevented their migration and invasion. THBS1 belongs to a group of tumorigenic ECM proteins induced via TGFβ signaling in CRPC cells, and high expression of THBS1 in human prostate cancer tissues correlated with the degree of malignancy. TGFβ-induced production of THBS1 through TβRI facilitates the invasion and metastasis of CRPC cells as shown in vivo xenograft animal experiments.

Endothelial TGF-β Signaling Regulates Endothelial-Mesenchymal Transition During Arteriovenous Fistula Remodeling in Mice With Chronic Kidney Disease.

Arteriovenous fistulae (AVFs) are the preferred vascular access for hemodialysis in patients with end-stage kidney disease. Chronic kidney disease (CKD) is associated with endothelial injury, impaired AVF maturation, and reduced patency, as well as utilization. Because CKD is characterized by multiple pathophysiological processes that induce endothelial-to-mesenchymal transition (EndMT), we hypothesized that CKD promotes EndMT during venous remodeling and that disruption of endothelial TGF (transforming growth factor)-β signaling inhibits EndMT to prevent AVF failure even in the end-stage kidney disease environment. The mouse 5/6 nephrectomy and aortocaval fistula models were used. CKD was created via 5/6 nephrectomy, with controls of no (0/6) or partial (3/6) nephrectomy in C57BL/6J mice. AVFs were created in mice with knockdown of TGF-βR1/R2 (TGF-β receptors type 1/2) in either smooth muscle cells or endothelial cells. AVF diameters and patency were measured and confirmed by serial ultrasound examination. AVF, both murine and human, were examined using Western blot, histology, and immunofluorescence. Human and mouse endothelial cells were used for in vitro experiments. CKD accelerates TGF-β activation and promotes EndMT that is associated with increased AVF wall thickness and reduced patency in mice. Inhibition of TGF-β signaling in both endothelial cells and smooth muscle cells decreased smooth muscle cell proliferation in the AVF wall, attenuated EndMT, and was associated with reduced wall thickness, increased outward remodeling, and improved AVF patency. Human AVF also showed increased TGF-β signaling and EndMT. CKD promotes EndMT and reduces AVF patency. Inhibition of TGF-β signaling, especially disruption of endothelial cell-specific TGF-β signaling, attenuates EndMT and improves AVF patency in mouse AVF. Inhibition of EndMT may be a therapeutic approach of translational significance to improve AVF patency in human patients with CKD.

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-β signaling pathways and cell cycle. Among the ligand factors of TGF-β 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.

MiRNAs in Signal Transduction of SMAD Proteins in Breast Cancer.

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

Legumain is a paracrine regulator of osteoblast differentiation and mediates the inhibitory effect of TGF-β1 on osteoblast maturation.

Transforming growth factor-beta 1 (TGF-β1) is a critical regulator of skeletal homeostasis and has diverse effects on osteoblastogenesis. To date, the mechanisms behind the intriguing inhibitory effect of TGF-β1 on osteoblast maturation are not fully understood. Here, we demonstrate a novel mechanism by which TGF-β1 modulates osteoblast maturation through the lysosomal protease legumain. We observed that addition of TGF-β1 to osteogenic cultures of human bone marrow derived mesenchymal stromal (stem) cells enhanced legumain activity and secretion, in-spite of decreased legumain mRNA expression, suggesting post-transcriptional regulation. We further showed that osteogenic cells internalize and activate prolegumain, associated with inhibited osteoblast maturation, revealing legumain as a paracrine regulator of osteoblast maturation. Interestingly, TGF-β1 treatment exacerbated legumain internalization and activity, and showed an additive effect on legumain-induced inhibition of osteoblast maturation. Importantly, pharmacological inhibition of legumain abolished the inhibitory effect of TGF-β1 on osteoblast maturation. Our findings reveal that TGF-β1 inhibits osteoblast maturation by stimulating secretion and activity of endogenous legumain, as well as enhancing internalization and activation of extracellular prolegumain. Therefore, our study provides a deeper understanding of the complex regulation of osteoblastogenesis and unveils a novel TGF-β1-legumain axis in regulation of osteoblast maturation, offering novel insights for possible therapeutic interventions related to bone diseases associated with aberrant TGF-β1 signaling.

Ursodeoxycholic Acid Platinum(IV) Conjugates as Antiproliferative and Antimetastatic Agents: Remodel the Tumor Microenvironment through Suppressing JAK2/STAT3 Signaling.

Tumor microenvironment (TME) is a pivotal factor driving the tumor metastasis and leading to the failure of tumor therapy. Here, a series of ursodeoxycholic acid platinum(IV) conjugates with potency in remodeling the TME through suppressing JAK2/STAT3 signaling was developed. A candidate was screened out, which displayed potent antiproliferative and antimetastatic performance both in vitro and in vivo. It displayed superior pharmacokinetic properties compared to cisplatin. Serious DNA injury was induced, and then mitochondria-mediated apoptosis was initiated through the Bcl-2/Bax/Caspase3 pathway. The JAK2/STAT3 and TGF-β1 signaling pathways were remarkably inhibited, and pro-death autophagy was subsequently promoted. The inflammatory and hypoxic TME was suppressed by downregulating COX-2, MMP9, and HIF-1α, which resulted in inhibited angiogenesis in tumors by inhibiting the HIF-1α/VEGFA axis. Additionally, the immunosuppressive TME was reversed by blocking the immune checkpoint PD-L1, further improving the density of CD3+ and CD8+ tumor-infiltrating lymphocytes, and promoting macrophage polarization from M2- to M1-type.

A Paradigm Shift in Tumor Immunology: Th17 Cells and TGF-β in Intestinal Cancer Initiation.

Cancer remains one of the most complex challenges in modern medicine, with intricate relationships between immune responses and tumor development. This article examines a groundbreaking study by Fesneau, Thevin and colleagues, published in Nature Immunology. This elegant body of work explores the link between chronic inflammation and cancer, particularly focusing on Th17 cells involved in intestinal cancer initiation. Th17 cells, known for their dual roles in immunity, can promote or inhibit tumor growth depending on their environment. This study reveals that a specific subset of Th17 cells, derived from IL-17-producing cells, can transition to a tumorigenic state when TGF-β signaling is impaired. Surprisingly, TGF-β acts as a crucial regulatory factor, maintaining the balance between immune tolerance and tumorigenesis by preventing Th17 cells from becoming tumorigenic. This research highlights the potential for therapeutic interventions targeting TGF-β signaling to prevent cancer initiation in chronic inflammatory conditions. The findings have clinical implications for improving cancer immunotherapies, including immune checkpoint inhibitors and adoptive T cell therapies, by enhancing the efficacy of treatments and mitigating the risk of tumorigenic transformations. Overall, this study provides insights into the mechanisms linking inflammation and cancer, paving the way for innovative strategies to harness the immunity in cancer treatment.

The Biological Effects and Mechanisms of Fisetin on Hepatotoxicity, Liver Injury, and Liver Fibrosis: A Systematic Review

Background: Liver disease is a common cause of death worldwide. Objectives: This study aims to investigate the effects and mechanisms of Fisetin on hepatotoxicity, liver injury, and liver fibrosis. Methods: We adhered to the PRISMA 2020 guidelines in this systematic review. Our search used MeSH keywords encompassed Embase, PubMed, Web of Science, Scopus, and Cochrane Library for articles published before March 2, 2024. Relevant data was extracted from the publications, meticulously recorded in a standard form, and subsequently reviewed for outcomes and mechanisms. Results: Fisetin protects hepatocytes from oxidative stress by neutralizing free radicals (O2 −and H2O2), reduces oxidative stress, prevents lipid peroxidation, and increases endogenous antioxidants. It also reduces inflammation via lowering the production of tumor necrosis factor α (TNF-α), interleukins (IL)1α, IL-6, IL-18, IL-1β suppressing nuclear factor kappa B (NF-κB) activation, and cyclooxygenase- 2 (COX-2), inducible nitric oxide synthase (iNOS) inhibition, reducing monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), NLR family pyrin domain-containing 3 (NLRP3) inflammasome and interferon-gamma (IFN‐γ). Moreover, it inhibited apoptosis-modulated enzyme activity and detoxification enzymes via modulating the activity of cytochrome P450 and Phase II detoxification enzymes. Fisetin prevented fibrosis by inhibiting the activation of hepatic stellate cells (HSCs), attenuating extracellular matrix (ECM) remodeling-associated genes, and suppressing transforming growth factor-β (TGF-β) signaling pathway and attenuating collagen production. It decreased lipid accumulation and liver function tests. Conclusion: In vivo and in vitro studies indicated that Fisetin can enhance detoxification, attenuate liver injury, and reduce fibrosis, which helps maintain liver health.

PDCD10 promotes the tumor-supporting functions of TGF-β in pancreatic cancer.

The progression of pancreatic ductal adenocarcinoma (PDAC) is significantly affected by transforming growth factor (TGF)-β but targeting TGF-β can also compromize physiological effects in patients. Our study examined the functions of the ubiquitously expressed protein, PDCD10, as a modulator of TGF-β signaling in PDAC. Using in silico analyses we found that in patient samples, PDCD10 is significantly higher expressed in PDAC tumor tissue compared with normal pancreas and it is highly correlated with reduced survival. We created stable KO's of PDCD10 in two PDAC lines, PaTu 8902 (SMAD4 +/+) and PaTu 8988t (SMAD4 -/-), and found that KO lines are more sensitive to 5-FU and Gemcitabine treatment than their wild-type counterparts. Performing viability and wound closure assays we further found that PDCD10 promotes cell survival and proliferation by enhancing specifically the mitogenic functions of TGF-β. The molecular mechanism underlying this effect was further investigated using Western blots and with primary organoid lines derived from patient PDAC tissue samples. The data imply that PDCD10 mediates an increase in p-ERK through a non-SMAD4 pathway, leading to EMT promotion. Furthermore, PDCD10 facilitates deactivation of RB via a SMAD4-dependent pathway, thereby counter-acting the anti-proliferative actions of TGF-β. By performing proximity ligation assays (PLA) we found that PDCD10 associates with the kinase MST4, translocates it intracellularly and thereby facilitates phosphorylations of RB and ERK1/2. Our study indicates that PDCD10 promotes the proliferative function and EMT induction of TGF-β in pancreatic cancer cells. Therefore, targeting PDCD10 in PDAC patients could represent a promising new strategy to optimize TGF-β targeted therapies.

Mechanistic insights into lethal hyper progressive disease induced by PD-L1 inhibitor in metastatic urothelial carcinoma

Hyper progressive disease (HPD) is a paradoxical phenomenon characterized by accelerated tumor growth following treatment with immune checkpoint inhibitors. However, the pathogenic causality and its predictor remain unknown. We herein report a fatal case of HPD in a 50-year-old man with metastatic bladder cancer. He had achieved a complete response (CR) through chemoradiation therapy followed by twelve cycles of chemotherapy, maintaining CR for 24 months. Three weeks after initiating maintenance use of a PD-L1 inhibitor, avelumab, a massive amount of metastases developed, leading to the patient’s demise. Omics analysis, utilizing metastatic tissues obtained from an immediate autopsy, implied the contribution of M2 macrophages, TGF-β signaling, and interleukin-8 to HPD pathogenesis.

Carbon Fiber-Mediated Electrospinning Scaffolds Can Conduct Electricity for Repairing Defective Tendon.

Partial or complete rupture of the tendon can damage the collagen structure, resulting in the disruption of the electrical signal pathway. It is a great challenge to reconstruct the original electrical signal pathway of the tendon and promote the regeneration and functional recovery of defective tendon. In this study, carbon fiber-mediated electrospinning scaffolds were fabricated by wrapping conductive, high-strength, loose single-bundle carbon fibers with nanofiber membranes. Due to the presence of nanofiber membranes, the maximum tensile force of the scaffolds was 2.4 times higher than that of carbon fibers, while providing excellent temporal and spatial prerequisites for tenocytes to adapt to electrical stimulation to accelerate proliferation and expression. The diameter of the carbon fiber monofilaments used in this study was 5.07 ± 1.20 μm, which matched the diameter of tendon collagen, allowing for quickly establishing the connection between the tendon tissue and the scaffold, and better promoting the recovery of the electrical signal pathway. In a rabbit Achilles tendon defect repair model, the carbon fiber-mediated electrospinning scaffold was almost filled with collagen fibers compared to a nonconductive polyethylene glycol terephthalate scaffold. Transcriptome sequencing revealed that fibromodulin and tenomodulin expression were upregulated, and their related proteoglycans and glycosaminoglycan binding proteins pathways were enhanced, which could regulate the TGF-β signaling pathway and optimize the extracellular matrix assembly, thus promoting tendon repair. Therefore, the scaffold in this study makes up for the shortage of conductive scaffolds for repairing tendon defects, revealing the potential impact of conductivity on the signaling pathway of tendon repair and providing a new approach for future clinical studies.

Biochanin A suppresses Klf6-mediated Smad3 transcription to attenuate renal fibrosis in UUO mice

BackgroundRenal fibrosis is a hallmark of chronic kidney disease (CKD). Smad3 serves as the principal transcription factor mediating the pro-fibrosis effects of TGF-β signaling in renal fibrosis. Biochanin A (BCA), a natural isoflavone, has been shown to attenuate renal fibrosis by inhibiting TGF-β signaling but the detailed mechanisms remain unresolved. This study aimed to elucidate the specific mechanisms by which BCA modulates TGF-β signaling. MethodsRenal fibrosis models were established both in vitro, using TGF-β1-stimulated mouse renal tubular TCMK1 cells, and in vivo, employing mice with unilateral ureter obstruction (UUO). RNA-seq was conducted to identify BCA-regulated genes. The AnimalTFDB4.0 database was utilized to predict transcription factors with potential binding to Smad3 promoter. The activities of TGF-β signaling and the cloned mouse Smad3 promoter were assessed using luciferase reporter assays. Plasmid transfection was performed using polyethylenimine in TCMK1 cells or ultrasound microbubbles in UUO kidneys. Gene expression was analyzed by RT-PCR, western blot, and immunohistochemistry assays. ResultsBCA significantly inhibited TGF-β signaling activity and suppressed TGF-β1-induced fibrotic gene expression in TCMK1 cells. RNA-seq and in silico analyses identified Smad3 as the key gene downregulated by BCA, while leaving Smad2 unaffected. This selective transcriptional suppression of Smad3 by BCA was validated by luciferase reporter assays using the cloned Smad3 promoter. Furthermore, transcription factor binding prediction identified that Klf6, a transcription factor downregulated by BCA, has binding potential to the Smad3 promoter and promotes Smad3 transcription. Klf6 expression was induced in TGF-β1-stimulated TCMK1 cells and UUO kidneys, but this induction was abolished upon BCA treatment. Importantly, Klf6 overexpression restored Smad3 expression and counteracted the anti-fibrosis effects of BCA in both TGF-β1-stimulated TCMK1 cells and UUO kidneys. ConclusionTGF-β-responsive Klf6 transcriptionally transactivates Smad3 expression. BCA exerts anti-renal fibrosis effects by inhibiting the Klf6-Smad3 signaling axis, underscoring its therapeutic potential in the treatment of CKD.

Impaired Myofibroblast Proliferation is a Central Feature of Pathologic Post-Natal Alveolar Simplification.

Premature infants with bronchopulmonary dysplasia (BPD) have impaired alveolar gas exchange due to alveolar simplification and dysmorphic pulmonary vasculature. Advances in clinical care have improved survival for infants with BPD, but the overall incidence of BPD remains unchanged because we lack specific therapies to prevent this disease. Recent work has suggested a role for increased transforming growth factor-beta (TGFβ) signaling and myofibroblast populations in BPD pathogenesis, but the functional significance of each remains unclear. Here, we utilize multiple murine models of alveolar simplification and comparative single-cell RNA sequencing to identify shared mechanisms that could contribute to BPD pathogenesis. Single-cell RNA sequencing reveals a profound loss of myofibroblasts in two models of BPD and identifies gene expression signatures of increased TGFβ signaling, cell cycle arrest, and impaired proliferation in myofibroblasts. Using pharmacologic and genetic approaches, we find no evidence that increased TGFβ signaling in the lung mesenchyme contributes to alveolar simplification. In contrast, this is likely a failed compensatory response, since none of our approaches to inhibit TGFb signaling protect mice from alveolar simplification due to hyperoxia while several make simplification worse. In contrast, we find that impaired myofibroblast proliferation is a central feature in several murine models of BPD, and we show that inhibiting myofibroblast proliferation is sufficient to cause pathologic alveolar simplification. Our results underscore the importance of impaired myofibroblast proliferation as a central feature of alveolar simplification and suggest that efforts to reverse this process could have therapeutic value in BPD.

A regulatory loop involving the cytochrome P450-soluble epoxide hydrolase axis and TGF-β signaling

Fatty acid metabolites, produced by cytochrome P450 enzymes and soluble epoxide hydrolase (sEH), regulate inflammation. Here, we report that the transforming growth factor β (TGF-β)-induced polarization of macrophages to a pro-resolving phenotype requires Alk5 and Smad2 activation to increase sEH expression and activity. Macrophages lacking sEH showed impaired repolarization, reduced phagocytosis, and maintained a pro-inflammatory gene expression profile. 11,12-Epoxyeicosatrienoic acid (EET) was one altered metabolite in sEH-/- macrophages and mimicked the effect of sEH deletion on gene expression. Notably, 11,12-EET also reduced Alk5 expression, inhibiting TGF-β-induced Smad2 phosphorylation by triggering the cytosolic translocation of the E3 ligase Smurf2. These findings suggest that sEH expression is controlled by TGF-β and that sEH activity, which lowers 11,12-EET levels and promotes TGF-β signaling by metabolizing 11,12-EET to prevent Alk5 degradation. Thus, an autocrine loop between sEH/11,12-EET and TGF-β1 regulates macrophage function.

Triphenyl Phosphate Alters Methyltransferase Expression and Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

SMURF1/2 are novel regulators of WNK1 stability.

Angiogenesis is essential for remodeling and repairing existing vessels, and this process requires signaling pathways including those controlled by transforming growth factor beta (TGF-β). We have previously reported crosstalk between TGF-β and the protein kinase With No lysine (K) 1 (WNK1). Homozygous disruption of the gene encoding WNK1 results in lethality in mice near embryonic day E12 due to impaired angiogenesis and this defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase Oxidative Stress-Responsive 1 (OSR1). However, molecular processes regulated via a collaboration between TGF-β and WNK1/OSR1 are not well understood. Here we show that WNK1 interacts with the E3 ubiquitin ligases SMURF1/2. In addition, we discovered that WNK1 regulates SMURF1/2 protein stability and vice versa. We also demonstrate that WNK1 activity regulates TGF-β receptor levels, in turn, controlling TGF-β signaling.