Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

Lefty Blocks a Subset of TGFβ Signals by Antagonizing EGF-CFC Coreceptors

Members of the EGF-CFC family play essential roles in embryonic development and have been implicated in tumorigenesis. The TGFβ signals Nodal and Vg1/GDF1, but not Activin, require EGF-CFC coreceptors to activate Activin receptors. We report that the TGFβ signaling antagonist Lefty also acts through an EGF-CFC-dependent mechanism. Lefty inhibits Nodal and Vg1 signaling, but not Activin signaling. Lefty genetically interacts with EGF-CFC proteins and competes with Nodal for binding to these coreceptors. Chimeras between Activin and Nodal or Vg1 identify a 14 amino acid region that confers independence from EGF-CFC coreceptors and resistance to Lefty. These results indicate that coreceptors are targets for both TGFβ agonists and antagonists and suggest that subtle sequence variations in TGFβ signals result in greater ligand diversity.     

Activin B Antagonizes RhoA Signaling to Stimulate Mesenchymal Morphology and Invasiveness of Clear Cell Renal Cell Carcinomas

Activin B belongs to the TGFβ family of growth factors and is upregulated in clear cell renal cell carcinoma cells by hypoxia inducible factors. Expression of Activin B is required for tumor growth in vivo and tumor cell invasion in vitro. Here we show that activation of RhoA signaling counteracts Activin B mediated disassembly of actin stress fibers, mesenchymal cell morphology and invasiveness, whereas inhibition of RhoA rescues these effects in Activin B knockdown cells. Conversely, Activin B inhibits RhoA signaling suggesting that there is an antagonistic connection between both pathways. In addition we found that Rac1 plays an opposite role to RhoA, i.e. activation of Rac1 initiates loss of actin stress fibers, promotes a mesenchymal cell morphology and induces invasion in Activin B knockown cells, whereas inhibition of Rac1 abolishes these Activin B effects. Collectively, our data provide evidence that reduction of RhoA signaling by Activin B together with persistent Rac1 activity is a prerequisite for inducing an invasive phenotype in clear cell renal cell carcinoma.     

Pathogenic ACVR1R206H activation by Activin A‐induced receptor clustering and autophosphorylation

Fibrodysplasia ossificans progressiva (FOP) and diffuse intrinsic pontine glioma (DIPG) are debilitating diseases that share causal mutations in ACVR1, a TGF‐β family type I receptor. ACVR1^R206H is a frequent mutation in both diseases. Pathogenic signaling via the SMAD1/5 pathway is mediated by Activin A, but how the mutation triggers aberrant signaling is not known. We show that ACVR1 is essential for Activin A‐mediated SMAD1/5 phosphorylation and is activated by two distinct mechanisms. Wild‐type ACVR1 is activated by the Activin type I receptors, ACVR1B/C. In contrast, ACVR1^R206H activation does not require upstream kinases, but is predominantly activated via Activin A‐dependent receptor clustering, which induces its auto‐activation. We use optogenetics and live‐imaging approaches to demonstrate Activin A‐induced receptor clustering and show it requires the type II receptors ACVR2A/B. Our data provide molecular mechanistic insight into the pathogenesis of FOP and DIPG by linking the causal activating genetic mutation to disrupted signaling.     

Intra‐epithelial non‐canonical Activin A signaling safeguards prostate progenitor quiescence

The healthy prostate is a relatively quiescent tissue. Yet, prostate epithelium overgrowth is a common condition during aging, associated with urinary dysfunction and tumorigenesis. For over thirty years, TGF‐β ligands have been known to induce cytostasis in a variety of epithelia, but the intracellular pathway mediating this signal in the prostate, and its relevance for quiescence, have remained elusive. Here, using mouse prostate organoids to model epithelial progenitors, we find that intra‐epithelial non‐canonical Activin A signaling inhibits cell proliferation in a Smad‐independent manner. Mechanistically, Activin A triggers Tak1 and p38 ΜAPK activity, leading to p16 and p21 nuclear import. Spontaneous evasion from this quiescent state occurs upon prolonged culture, due to reduced Activin A secretion, a condition associated with DNA replication stress and aneuploidy. Organoids capable to escape quiescence in vitro are also able to implant with increased frequency into immunocompetent mice. This study demonstrates that non‐canonical Activin A signaling safeguards epithelial quiescence in the healthy prostate, with potential implications for the understanding of cancer initiation, and the development of therapies targeting quiescent tumor progenitors.     

A role for age-related changes in TGFβ signaling in aberrant chondrocyte differentiation and osteoarthritis

Transforming growth factor beta (TGFβ) is a growth factor with many faces. In our osteoarthritis (OA) research we have found that TGFβ can be protective as well as deleterious for articular cartilage. We postulate that the dual effects of TGFβ on chondrocytes can be explained by the fact that TGFβ can signal via different receptors and related Smad signaling routes. On chondrocytes, TGFβ not only signals via the canonical type I receptor ALK5 but also via the ALK1 receptor. Notably, signaling via ALK5 (Smad2/3 route) results in markedly different chondrocyte responses than ALK1 signaling (Smad1/5/8), and we postulate that the balance between ALK5 and ALK1 expression on chondrocytes will determine the overall effect of TGFβ on these cells. Importantly, signaling via ALK1, but not ALK5, stimulates MMP-13 expression by chondrocytes. In cartilage of ageing mice and in experimental OA models we have found that the ALK1/ALK5 ratio is significantly increased, favoring TGFβ signaling via the Smad1/5/8 route, changes in chondrocyte differentiation and MMP-13 expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. In this paper we summarize concepts in OA, its link with ageing and disturbed growth factor responses, and a potential role of TGFβ signaling in OA development.     

Hepsin regulates TGFβ signaling via fibronectin proteolysis

Transforming growth factor‐beta (TGFβ) is a multifunctional cytokine with a well‐established role in mammary gland development and both oncogenic and tumor‐suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFβ activity by acting as a storage compartment of latent‐TGFβ, but how TGFβ is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFβ signaling through the release of latent‐TGFβ from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFβ signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent‐TGFβ1, while overexpression of hepsin in mammary tumors increased TGFβ signaling. Cell‐free and cell‐based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent‐TGFβ and, importantly, that the ability of hepsin to activate TGFβ signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFβ pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.     

Distinct functions of transforming growth factor-β signaling in c-MYC driven hepatocellular carcinoma initiation and progression

Dysregulation of transforming growth factor-beta (TGFβ) signaling has been implicated in liver carcinogenesis with both tumor promoting and inhibiting activities. Activation of the c-MYC protooncogene is another critical genetic event in hepatocellular carcinoma (HCC). However, the precise functional crosstalk between c-MYC and TGFβ signaling pathways remains unclear. In the present investigation, we investigated the expression of TGFβ signaling in c-MYC amplified human HCC samples as well as the mechanisms whereby TGFβ modulates c-Myc driven hepatocarcinogenesis during initiation and progression. We found that several TGFβ target genes are overexpressed in human HCCs with c-MYC amplification. In vivo, activation of TGFβ1 impaired c-Myc murine HCC initiation, whereas inhibition of TGFβ pathway accelerated this process. In contrast, overexpression of TGFβ1 enhanced c-Myc HCC progression by promoting tumor cell metastasis. Mechanistically, activation of TGFβ promoted tumor microenvironment reprogramming rather than inducing epithelial-to-mesenchymal transition during HCC progression. Moreover, we identified PMEPA1 as a potential TGFβ1 target. Altogether, our data underline the divergent roles of TGFβ signaling during c-MYC induced HCC initiation and progression.Subject terms: Cancer microenvironment, Liver cancer, Apoptosis, Growth factor signalling     

干扰TGFβRⅠ增加乳腺癌细胞MDA-MB-231对化疗药物的敏感性

转化生长因子β(transforming growth factorβ,TGFβ)与其受体(transforming growth factorβreceptor,TGFβR)结合激活下游信号通路,在肿瘤的发生发展和转移中具有重要意义,且已有迹象表明该通路可能介导肿瘤的化疗耐受.本研究构建了干扰转化生长因子βⅠ型受体(transforming growth factor receptor typeⅠ,TGFβRⅠ)的重组质粒pRNAT-U6.1/TGFβRⅠ-sh1,发现其可在mRNA和蛋白质水平均显著下调TGFβRⅠ的表达,P0.01.后将该干扰质粒转染乳腺癌细胞MCF-7/5Fu、MCF-7、MDA-MB-231、MDA-MB-453,建立了稳定的乳腺癌TGFβRⅠ干扰模型;MTS法检测上述4种细胞模型对5-氟尿嘧啶(5-fluorouracil,5-FU)和紫杉醇(taxol,TAX)的敏感性.结果显示,MCF-7、MCF-7/5Fu和MDA-MB-453细胞在干扰TGFβRⅠ之后对5-FU和TAX敏感性并未发生改变;但是间质表型的MDA-MB-231细胞在干扰TGFβRⅠ之后对5-FU和TAX的敏感性显著增加.由此可见,干扰TGFβRⅠ的表达阻断TGFβ信号通路,进而显著增加间质型乳腺癌细胞对化疗药物的敏感性,这为临床乳腺癌治疗提供了一定的理论依据.     

Suppression TGF-β Signaling by Phospholipase D

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-β signals that block cell cycle progression in G₁ are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-β signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467 – sites on Smads that get phosphorylated by the TGF-β receptor and positively regulate TGF-β signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-β. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255 – sites that are phosphorylated by MAP kinase and negatively regulate TGF-β signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-β. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-β signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-β signals that promote G₁ arrest.     

Activin/TGFbeta and BMP crosstalk determines digit chondrogenesis

The progress zone (PZ) is a specialized area at the distal margin of the developing limb where mesodermal cells are kept in proliferation and undifferentiated, allowing limb outgrowth. At stages of digit morphogenesis the PZ cells can undergo two possible fates, either aggregate initiating chondrogenic differentiation to configure the digit blastemas, or to die by apoptosis if they are incorporated in the interdigital mesenchyme. While both processes are controlled by bone morphogenetic proteins (BMPs) the molecular basis for such contrasting differential behavior of the autopodial mesoderm remains unknown. Here we show that a well-defined crescent domain of high BMP activity located at the tip of the forming digits, which we termed the digit crescent (DC), directs incorporation and differentiation of the PZ mesenchymal cells into the digit aggregates. The presence of this domain does not correlate with an exclusive expression domain of BMP receptors and its abrogation by surgical approaches or by local application of BMP antagonists is followed by digit truncation and cell death. We further show that establishment of the DC is directed by Activin/TGFβ signaling, which inhibits Smad 6 and Bambi, two specific BMP antagonists expressed in the interdigits and progress zone mesoderm. The interaction between Activin/TGFβ and BMP pathways at the level of DC promotes the expression of the chondrogenic factor SOX9 accompanied by a local decrease in cell proliferation. Characteristically, the DC domain is asymmetric, it being extended towards the posterior interdigit. The presence of the DC is transitorily dependent of the adjacent posterior interdigit and its maintenance requires also the integrity of the AER.     

Tumor Necrosis Factor-Receptor–associated Factor-4 Is a Positive Regulator of Transforming Growth Factor-β Signaling That Affects Neural Crest Formation

The transforming growth factor (TGF)-β superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFβ signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFβ. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor–associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.     

<Emphasis Type="Italic">C. elegans</Emphasis> serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation

Background  

In C. elegans there are two well-defined TGFβ-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFβ signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29.     

Expression cloning of Xenopus Os4, an evolutionarily conserved gene, which induces mesoderm and dorsal axis.

Multiple factors, including members of the FGF, TGF beta, and Wnt family of proteins, are important mediators in the regulation of dorsal-ventral pattern formation during vertebrate development. By using an expression cloning approach to identify novel factors that could regulate dorsal-ventral patterning in the Xenopus embryo, we isolated the Xenopus homologue of the human Os4 gene by virtue of its ability to induce a secondary dorsal axis. While Os4 homologues have been identified in a variety of species, and human Os4 is overexpressed in human tumors, the biological function of Os4 is unknown. To explore the mechanism by which Xenopus Os4 (XOs4) induces a secondary dorsal axis, we used Xenopus explant and whole-embryo assays. The secondary axis induced by XOs4 is distinct from that induced by activation of Wnt or FGF pathways but similar to that induced by inhibition of BMP signaling or activation of an Activin pathway. However, XOs4 did not inhibit BMP signaling in dissociated animal cap explants, indicating that XOs4 does not inhibit BMP signaling. Similar to activation of an Activin-like pathway, expression of XOs4 induces molecular markers for mesoderm in animal cap explants, although expression of gastrula-stage mesodermal markers was very weak and substantially delayed. Yet, XOs4 does not require activity of the Activin signal-transduction pathway for mesoderm induction as dominant-negative components of the Activin/Nodal/Vg1 pathway did not prevent XOs4-mediated induction of mesodermal derivatives. Finally, like Activin/Nodal/Vg1 pathways, XOs4 requires FGF signaling for expression of mesoderm markers. Results presented in this study demonstrate that XOs4 can induce mesoderm and dorsalize ventral mesoderm resulting in ectopic dorsal axis formation, suggesting a role for this large evolutionarily conserved gene family in early development.