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Betaglycan (BG) is a transmembrane co-receptor of the transforming growth factor-β (TGF-β) family of signaling ligands. It is essential for embryonic development, tissue homeostasis and fertility in adults. It functions by enabling binding of the three TGF-β isoforms to their signaling receptors and is additionally required for inhibin A (InhA) activity. Despite its requirement for the functions of TGF-βs and InhA in vivo, structural information explaining BG ligand selectivity and its mechanism of action is lacking. Here, we determine the structure of TGF-β bound both to BG and the signaling receptors, TGFBR1 and TGFBR2. We identify key regions responsible for ligand engagement, which has revealed binding interfaces that differ from those described for the closely related co-receptor of the TGF-β family, endoglin, thus demonstrating remarkable evolutionary adaptation to enable ligand selectivity. Finally, we provide a structural explanation for the hand-off mechanism underlying TGF-β signal potentiation. Betaglycan is a co-receptor for selective TGF-β family ligands. Here, the authors solve its structure in complex with TGF-β and the signaling receptors, which explains its ligand selectivity and reveals its mechanism in potentiating TGF-β signaling.

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with the capability to act as tumour suppressor or tumour promoter depending on the cellular context. TGF-beta receptor type-2 (TGFBR2) is the ligand-binding receptor for all members of the TGF-β family. Data from mouse model experiments demonstrated that loss of Tgfbr2 expression in mammary fibroblasts was linked to tumour initiation and metastasis. Using a randomised tamoxifen trial cohort including in total 564 invasive breast carcinomas, we examined TGFBR2 expression ( n =252) and phosphorylation level of downstream target SMAD2 (pSMAD2) ( n =319) in cancer-associated fibroblasts (CAFs) and assessed links to clinicopathological markers, prognostic and treatment-predictive values. The study revealed that CAF-specific TGFBR2 expression correlated with improved recurrence-free survival. Multivariate analysis confirmed CAF-TGFBR2 to be an independent prognostic marker (multivariate Cox regression, hazard ratio: 0.534, 95% (CI): 0.360–0.793, P =0.002). CAF-specific pSMAD2 levels, however, did not associate with survival outcome. Experimentally, TGF-β signalling in fibroblasts was modulated using a TGF-β ligand and inhibitor or through lentiviral short hairpin RNA-mediated TGFBR2 -specific knockdown. To determine the role of fibroblastic TGF-β pathway on breast cancer cells, we used cell contact-dependent cell growth and clonogenicity assays, which showed that knockdown of TGFBR2 in CAFs resulted in increased cell growth, proliferation and clonogenic survival. Further, in a mouse model transfected CAFs were co-injected with MCF7 and tumour weight and proportion was monitored. We found that mouse xenograft tumours comprising TGFBR2 knockdown fibroblasts were slightly bigger and displayed increased tumour cell capacity. Overall, our data demonstrate that fibroblast-related biomarkers possess clinically relevant information and that fibroblasts confer effects on breast cancer cell growth and survival. Regulation of tumour–stromal cross-talk through fibroblastic TGF-β pathway may depend on fibroblast phenotype, emphasising the importance to characterise tumour microenvironment subtypes.

Osteoarthritis has been believed to be caused by improper mechanical function of articular joints. Xu Cao and his colleagues now show that this mechanical process leads to upregulation of transforming growth factor β1 activity in mesenchymal stem cells of subchondral bone, resulting in aberrant bone formation, further destabilization of the joint and ultimately the onset of the disease. Osteoarthritis is a highly prevalent and debilitating joint disorder. There is no effective medical therapy for the condition because of limited understanding of its pathogenesis. We show that transforming growth factor β1 (TGF-β1) is activated in subchondral bone in response to altered mechanical loading in an anterior cruciate ligament transection (ACLT) mouse model of osteoarthritis. TGF-β1 concentrations are also high in subchondral bone from humans with osteoarthritis. High concentrations of TGF-β1 induced formation of nestin-positive mesenchymal stem cell (MSC) clusters, leading to formation of marrow osteoid islets accompanied by high levels of angiogenesis. We found that transgenic expression of active TGF-β1 in osteoblastic cells induced osteoarthritis, whereas inhibition of TGF-β activity in subchondral bone attenuated the degeneration of articular cartilage. In particular, knockout of the TGF-β type II receptor (TβRII) in nestin-positive MSCs led to less development of osteoarthritis relative to wild-type mice after ACLT. Thus, high concentrations of active TGF-β1 in subchondral bone seem to initiate the pathological changes of osteoarthritis, and inhibition of this process could be a potential therapeutic approach to treating this disease.

The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.

Microglia are CNS-resident macrophages, but studying their functions in health and disease has been challenging due to a lack of specific markers. Greter and colleagues identify the transcription factor Sall1 as being uniquely associated with microglia in the CNS, where it is important for defining their fate and homeostatic function. Microglia are the resident macrophages of the central nervous system (CNS). Gene expression profiling has identified Sall1 , which encodes a transcriptional regulator, as a microglial signature gene. We found that Sall1 was expressed by microglia but not by other members of the mononuclear phagocyte system or by other CNS-resident cells. Using Sall1 for microglia-specific gene targeting, we found that the cytokine receptor CSF1R was involved in the maintenance of adult microglia and that the receptor for the cytokine TGF-β suppressed activation of microglia. We then used the microglia-specific expression of Sall1 to inducibly inactivate the murine Sall1 locus in vivo , which resulted in the conversion of microglia from resting tissue macrophages into inflammatory phagocytes, leading to altered neurogenesis and disturbed tissue homeostasis. Collectively, our results show that transcriptional regulation by Sall1 maintains microglial identity and physiological properties in the CNS and allows microglia-specific manipulation in vivo .

Multiple sclerosis (MS) is a T cell-mediated autoimmune disease of the central nervous system. Foxp3 + regulatory T (Treg) cells are reduced in frequency and dysfunctional in patients with MS, but the underlying mechanisms of this deficiency are unclear. Here, we show that induction of human IFN-γ − IL-17A − Foxp3 + CD4 + T cells is inhibited in the presence of circulating exosomes from patients with MS. The exosomal miRNA profile of patients with MS differs from that of healthy controls, and let-7i , which is markedly increased in patients with MS, suppresses induction of Treg cells by targeting insulin like growth factor 1 receptor ( IGF1R ) and transforming growth factor beta receptor 1 ( TGFBR1 ). Consistently, the expression of IGF1R and TGFBR1 on circulating naive CD4 + T cells is reduced in patients with MS. Thus, our study shows that exosomal let-7i regulates MS pathogenesis by blocking the IGF1R/TGFBR1 pathway. MiRNAs are small RNA molecules that can regulate gene expression. Here the authors show that expression of several exosomal miRNAs are altered in patients with multiple sclerosis, and that let-7i modulates regulatory T cell homeostasis to contribute to pathogenesis.

Cancer immunotherapies have shown clinical success in various types of tumors but the patient response rate is low, particularly in breast cancer. Here we report that malignant breast cancer cells can transfer active TGF-β type II receptor (TβRII) via tumor-derived extracellular vesicles (TEV) and thereby stimulate TGF-β signaling in recipient cells. Up-take of extracellular vesicle-TβRII (EV-TβRII) in low-grade tumor cells initiates epithelial-to-mesenchymal transition (EMT), thus reinforcing cancer stemness and increasing metastasis in intracardial xenograft and orthotopic transplantation models. EV-TβRII delivered as cargo to CD8 + T cells induces the activation of SMAD3 which we demonstrated to associate and cooperate with TCF1 transcription factor to impose CD8 + T cell exhaustion, resulting in failure of immunotherapy. The levels of TβRII + circulating extracellular vesicles (crEV) appears to correlate with tumor burden, metastasis and patient survival, thereby serve as a non-invasive screening tool to detect malignant breast tumor stages. Thus, our findings not only identify a possible mechanism by which breast cancer cells can promote T cell exhaustion and dampen host anti-tumor immunity, but may also identify a target for immune therapy against the most devastating breast tumors. Understanding the factors that hamper immune therapy in breast cancer may increase the range of patients who benefit. Here authors show that breast cancer cells produce and subsequently transfer active TGF-β type II receptors to CD8 + T cells to render them exhausted, thus paralyzing the anti-tumor immune response.

Mutations in TGFBR2, a component of the transforming growth factor (TGF)-β signaling pathway, occur in high-frequency microsatellite instability (MSI-H) colorectal cancer (CRC). In mouse models, Tgfbr2 inactivation in the intestinal epithelium accelerates the development of malignant intestinal tumors in combination with disruption of the Wnt-β-catenin pathway. However, no studies have further identified the genes influenced by TGFBR2 inactivation following disruption of the Wnt-β-catenin pathway. We previously described CDX2P-G19Cre;Apcflox/flox mice, which is stochastically null for Apc in the colon epithelium. In this study, we generated CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice, with simultaneous loss of Apc and Tgfbr2. These mice developed tumors, including adenocarcinoma in the proximal colon. We compared gene expression profiles between tumors of the two types of mice using microarray analysis. Our results showed that the expression of the murine homolog of GSDMC was significantly upregulated by 9.25-fold in tumors of CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice compared with those of CDX2P-G19Cre;Apcflox/flox mice. We then investigated the role of GSDMC in regulating CRC tumorigenesis. The silencing of GSDMC led to a significant reduction in the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GSDMC enhanced cell proliferation. These results suggested that GSDMC functioned as an oncogene, promoting cell proliferation in colorectal carcinogenesis. In conclusion, combined inactivation of both Apc and Tgfbr2 in the colon epithelium of a CRC mouse model promoted development of adenocarcinoma in the proximal colon. Moreover, GSDMC was upregulated by TGFBR2 mutation in CRC and promoted tumor cell proliferation in CRC carcinogenesis, suggesting that GSDMC may be a promising therapeutic target.

Transforming growth factor-β (TGF-β) mimetic peptides offer significant therapeutic potential due to their superior pharmacological properties over the native cytokine. Our previous work identified two such peptides, TB1 and TB2, which bind to the type II TGF-β receptor (TβRII) yet elicit distinct cellular responses. To uncover the mechanistic basis for the functional divergence, we employed integrated molecular dynamics (MD) simulations with the AlphaFold3-predicted structures. Our analytical results indicated that TB2 stabilizes a dynamic complex with TβRII and is predicted to facilitate type I receptor (TβRI) engagement possibly involving a critical hydrogen bond between TB2-Gly11 and TβRI-Phe60. The resulting trimeric assembly (TB2–TβRII–TβRI) exhibits a higher relative binding affinity (−67.76 ± 7.70 kcal/mol) and structural stability. In contrast, the TB1–TβRII complex fails to productively engage TβRI. These computational results were experimentally validated. Western blot analysis confirmed that TB2, but not TB1, activates the canonical TGF-β/Smad pathway by enhancing the expression and phosphorylation of Smad3. This study will elucidate the dynamic structural basis for the activity of TGF-β mimetic peptides and suggest TB2 as a promising lead candidate for the rational design of tissue-regenerative therapeutics.

A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1). Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8 + and T H 1 cells. To address this therapeutic challenge, we invent bifunctional antibody–ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFβ receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFβ in the target cell microenvironment ( a -CTLA4-TGFβRII ecd and a -PDL1-TGFβRII ecd ). a -CTLA4-TGFβRII ecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab). Likewise, a -PDL1-TGFβRII ecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab). Our data demonstrate that Y-traps counteract TGFβ-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors. Antitumor T cells can be inhibited by a TGFβ rich tumor microenvironment. The authors develop bifunctional proteins comprising CTLA-4 or PD-L1 immune checkpoint-targeted antibodies fused to a “TGFβ trap” and show that they counteract tumor immune tolerance and enhance the efficacy of these antibodies.