A screen of repurposed drugs identifies AMHR2/MISR2 agonists as potential contraceptives.

We identified the anti-Mullerian hormone (also known as Müllerian inhibiting substance or MIS) as an inhibitory hormone that induces long-term contraception in mammals. The type II receptor to this hormone, AMHR2 (also known as MISR2), represents a promising druggable target for the modulation of female reproduction with a mechanism of action distinct from steroidal contraceptives. We designed an in vitro platform to screen and validate small molecules that can activate MISR2 signaling and suppress ovarian folliculogenesis. Using a bone morphogenesis protein (BMP)­response element luciferase reporter cell­based assay, we screened 5,440 compounds from a repurposed drug library. Positive hits in this screen were tested for specificity and potency in luciferase dose­response assays, and biological activity was tested in ex vivo Mullerian duct regression bioassays. Selected candidates were further evaluated in ex vivo follicle/ovary culture assays and in vivo in mice and rats. Here, we report that SP600125, CYC-116, gandotinib, and ruxolitinib can specifically inhibit primordial follicle activation and repress folliculogenesis by stimulating the MISR2 pathway.

Identification and functional analysis of transforming growth factor-ß type I receptor (TßR1) from Scylla paramamosain: The first evidence of TßR1 involved in development and innate immunity in crustaceans.

The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24 h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Transforming growth factor ß induces bone marrow mesenchymal stem cell migration via noncanonical signals and N-cadherin.

Transforming growth factor-beta (TGF-ß) induces the migration and mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) to maintain bone homeostasis during bone remodeling and facilitate the repair of peripheral tissues. Although many studies have reported the mechanisms through which TGF-ß mediates the migration of various types of cells, including cancer cells, the intrinsic cellular mechanisms underlying cellular migration, and mobilization of BM-MSCs mediated by TGF-ß are unclear. In this study, we showed that TGF-ß activated noncanonical signaling molecules, such as Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), focal adhesion kinase (FAK), and p38, via TGF-ß type I receptor in human BM-MSCs and murine BM-MSC-like ST2 cells. Inhibition of Rac1 by NSC23766 and Src by PP2 resulted in impaired TGF-ß-mediated migration. These results suggested that the Smad-independent, noncanonical signals activated by TGF-ß were necessary for migration. We also showed that N-cadherin-dependent intercellular interactions were required for TGF-ß-mediated migration using functional inhibition of N-cadherin with EDTA treatment and a neutralizing antibody (GC-4 antibody) or siRNA-mediated knockdown of N-cadherin. However, N-cadherin knockdown did not affect the global activation of noncanonical signals in response to TGF-ß. Therefore, these results suggested that the migration of BM-MSCs in response to TGF-ß was mediated through N-cadherin and noncanonical TGF-ß signals.

Anti-Müllerian hormone reduces growth rate without altering follicular survival in isolated caprine preantral follicles cultured in vitro.

The aim of the present study was to evaluate the effect of anti-Müllerian hormone (AMH), with and without FSH, on the in vitro development of isolated caprine preantral follicles, as well as follicular steroid production and mRNA levels of AMH, hormone receptors (AMH and FSH), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP17 (cytochrome P450, family 17, subfamily A, polypeptide 1), HSD3B (3-beta-hydroxysteroid dehydrogenase) and Myc (myelocytomatosis oncogene). Isolated secondary follicles were cultured in minimum essential medium alpha (α-MEM+) alone or supplemented with 50ng mL-1 AMH and/or 100ng mL-1 FSH added sequentially on different days of culture. Follicles were cultured for a total of 18 days, with different media during the first (Days 0-9) and second (Days 10-18) halves of the culture period, resulting in six treatment groups, as follows: α-MEM+/α-MEM+, FSH/FSH, AMH/AMH, AMH+FSH/AMH+FSH, AMH/FSH, and FSH/AMH. Follicle development was evaluated on the basis of follicular growth, oocyte maturation and steroid secretion. There was a decrease in follicular growth rate in the AMH, AMH+FSH and AMH/FSH treatment groups compared with α-MEM+ and FSH treatment groups (P<0.05). However, the different culture conditions had no effect on rates of meiotic resumption and steroid secretion (P>0.05). Moreover, follicles cultured in the presence of FSH had lower levels of AMH receptor type II (AMHRII) mRNA compared with non-cultured control (freshly isolated follicles), and the AMH and AMH/FSH treatment groups. In conclusion, AMH reduces the follicular growth rate of isolated goat preantral follicles in vitro without affecting follicular survival.

EGR-1 is an active transcription factor in TGF-ß2-mediated small intestinal cell differentiation.

Human milk contains growth factors that maintain intestinal mucosal homeostasis, but the molecular mechanisms behind how these growth factors regulate gene transcription are largely unknown. In this study, IEC-6 (rat intestinal epithelial cells) cells were used as a model to study cell differentiation mediated by transforming growth factor-ß2 (TGF-ß2), the most abundant growth factor in human milk. We focused on the transcription factor early growth response-1 (EGR-1), as we found a robust and rapid response in our initial transcription factor screen. Immunoblotting and immunofluorescent assays confirmed the phenotype change upon TGF-ß2 treatment and EGR-1 stimulation in the nucleus, with maximum expression occurring at 1 h. Chromatin immunoprecipitation sequencing was performed to map genome-wide EGR-1 binding sites on more than 1800 genes, widely involved in processes such as gene expression, transcription, membrane invagination and metabolism. In particular, more than 15 Wnt signaling pathway genes have EGR-1 binding sites; among them, Axin1 was the limiting factor, ensuring proper ß-catenin accumulation in the cytoplasm. We further used chromatin immunoprecipitation quantitative PCR to validate that EGR-1 binds to the region of -636/-454 bp and -454/-200 bp of the Axin1 promoter and functionally activates gene expression. The effect of TGF-ß2 on maintaining small intestinal cell homeostasis was partially explained by Axin1 activation through EGR-1.

Differential effect of hypoxia on early endothelial-mesenchymal transition response to transforming growth beta isoforms 1 and 2.

Angiogenesis is essential for mammalian development and tissue homeostasis, and is involved in several pathological processes, including tumor growth and dissemination. Many factors within the tissue microenvironment are known to modulate angiogenesis, including cytokines, such as transforming growth factor beta (TGFß), and oxygen level. TGFß exists in three different isoforms (1, 2 and 3), all of which (albeit in different contexts) might mediate angiogenesis and are able to induce endothelial-mesenchymal transition (EndoMT), a process involved in heart development, pathologic fibrosis and, as recently reported, in angiogenesis. Low oxygen level, referred to as hypoxia, has been independently shown to induce angiogenesis, modulate TGFß signalling and promote EndoMT. However, how these phenomena might be interconnected to drive angiogenesis is rather unexplored. To begin addressing the potential contribution of TGFß-induced EndoMT to angiogenesis, and to explore how microenvironmental hypoxia might influence these processes, we investigated the effect of TGFß isoforms 1 and 2 on early EndoMT response in cultured adult endothelium under standard (21 %) and hypoxic (1 %) culture conditions. Our data indicates that EndoMT-like changes, such as an increase in expression and nuclear translocation of Snail, Slug and Zeb1, and reduction of VE-cadherin expression, occur in response to TGFß1 and/or TGFß2 as early as 6h after stimulation and might be enhanced by hypoxia in an isoform-specific manner. Further, hypoxia enhances canonical TGFß signalling, and appears to be a key determinant of Snail's differential involvement in endothelial cell responses to TGFß1 versus TGFß2.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

Inhibition of TGF-ß Signaling Promotes Human Pancreatic ß-Cell Replication.

Diabetes is associated with loss of functional pancreatic ß-cells, and restoration of ß-cells is a major goal for regenerative therapies. Endogenous regeneration of ß-cells via ß-cell replication has the potential to restore cellular mass; however, pharmacological agents that promote regeneration or expansion of endogenous ß-cells have been elusive. The regenerative capacity of ß-cells declines rapidly with age, due to accumulation of p16(INK4a), resulting in limited capacity for adult endocrine pancreas regeneration. Here, we show that transforming growth factor-ß (TGF-ß) signaling via Smad3 integrates with the trithorax complex to activate and maintain Ink4a expression to prevent ß-cell replication. Importantly, inhibition of TGF-ß signaling can result in repression of the Ink4a/Arf locus, resulting in increased ß-cell replication in adult mice. Furthermore, small molecule inhibitors of the TGF-ß pathway promote ß-cell replication in human islets transplanted into NOD-scid IL-2Rg(null) mice. These data reveal a novel role for TGF-ß signaling in the regulation of the Ink4a/Arf locus and highlight the potential of using small molecule inhibitors of TGF-ß signaling to promote human ß-cell replication.

Ubiquitination and regulation of Smad7 in the TGF-ß1/Smad signaling of aristolochic acid nephropathy.

Aristolochic acid I (AAI) affects TGF-ß1/Smad signaling, which causes AA nephropathy (AAN), but the mechanisms are not fully understood. We aimed to clarify whether Arkadia and UCH37 participate in TGF-ß1/Smad signaling via Smad7, and the regulatory mechanisms of Smad7. One side, mice and cultured mouse renal tubular epithelial cells (RTECs) were treated with various AAI doses and concentrations, respectively; on the other side, RTECs were transfected with small interfering RNA (siRNA) expression vectors against Arkadia and UCH37 and then treated with 10 µg/ml AAI. And then detect the mRNA and protein levels of Smad7, UCH37, Arkadia and any other relative factors by RT-PCR and Western blotting. In kidney tissues and RTECs, the mRNA and protein levels of Smad7 decreased with increasing AAI doses concentrations by real-time PCR and Western blotting, whereas those of Arkadia, UCH37, Smad2, Smad3 and TßRI increased. Cells transfected with the Arkadia siRNA expression vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3 and TßRI after AAI treatment, while those of CK18 and Smad7 increased compared with those of untransfected RTECs. Conversely, cells transfected with the UCH37 siRNA expression vector showed the opposite effect on analyzed signaling molecules after AAI treatment. Arkadia and UCH37 participate in TGF-ß1/Smad signaling-mediated renal fibrosis, and Smad7 blocks TGF-ß1 signaling by inhibiting Smad2/Smad3 phosphorylation and enhancing the degradation of TßRI.

The Immune Adaptor ADAP Regulates Reciprocal TGF-ß1-Integrin Crosstalk to Protect from Influenza Virus Infection.

Highly pathogenic avian influenza virus (HPAI, such as H5N1) infection causes severe cytokine storm and fatal respiratory immunopathogenesis in human and animal. Although TGF-ß1 and the integrin CD103 in CD8+ T cells play protective roles in H5N1 virus infection, it is not fully understood which key signaling proteins control the TGF-ß1-integrin crosstalk in CD8+ T cells to protect from H5N1 virus infection. This study showed that ADAP (Adhesion and Degranulation-promoting Adapter Protein) formed a complex with TRAF6 and TAK1 in CD8+ T cells, and activated SMAD3 to increase autocrine TGF-ß1 production. Further, TGF-ß1 induced CD103 expression via an ADAP-, TRAF6- and SMAD3-dependent manner. In response to influenza virus infection (i.e. H5N1 or H1N1), lung infiltrating ADAP-/- CD8+ T cells significantly reduced the expression levels of TGF-ß1, CD103 and VLA-1. ADAP-/- mice as well as Rag1-/- mice receiving ADAP-/- T cells enhanced mortality with significant higher levels of inflammatory cytokines and chemokines in lungs. Together, we have demonstrated that ADAP regulates the positive feedback loop of TGF-ß1 production and TGF-ß1-induced CD103 expression in CD8+ T cells via the TßRI-TRAF6-TAK1-SMAD3 pathway and protects from influenza virus infection. It is critical to further explore whether the SNP polymorphisms located in human ADAP gene are associated with disease susceptibility in response to influenza virus infection.

Transforming Growth Factor ß Signaling in Colorectal Cancer Cells With Microsatellite Instability Despite Biallelic Mutations in TGFBR2.

BACKGROUND & AIMS: Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor ß receptor II (TGFBR2). TGFß signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFß. We investigated how TGFß signaling might continue in MSI-H CRC cells. METHODS: We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGFß signaling was detected by SMAD2 phosphorylation and through use of a TGFß-responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGFß, and their activities were measured. RESULTS: SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2­even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein. CONCLUSION: TGFß signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGFß signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated.

TGFß and BMP signaling in skeletal muscle: potential significance for muscle-related disease.

The transforming growth factor beta (TGFß) superfamily comprises a large number of secreted proteins that regulate various fundamental biological processes underlying embryonic development and the postnatal regulation of many cell types and organs. Sequence similarities define two ligand subfamilies: the TGFß/activin subfamily and the bone morphogenetic protein (BMP) subfamily. The discovery that myostatin, a member of the TGFß/activin subfamily, negatively controls muscle mass attracted attention to this pathway. However, recent findings of a positive role for BMP-mediated signaling in muscle have challenged the model of how the TGFß network regulates skeletal muscle phenotype. This review illustrates how this complex network integrates crosstalk among members of the TGFß superfamily and downstream signaling elements to regulate muscle in health and disease.

Targeting TGFß superfamily ligand accessory proteins as novel therapeutics for chronic lung disorders.

Dysregulation of the transforming growth factor ß (TGFß) pathway has been implicated to underlie a number of disease indications including chronic lung disorders such as asthma, chronic obstructive pulmonary disease (COPD), interstitial pneumonias, and pulmonary arterial hypertension (PAH). Consequently, the pharmaceutical industry has devoted significant resources in the pursuit of TGFß pathway inhibitors that target the cognate type I and II receptors and respective ligands. The progress of these approaches has been painfully slow, due in part to dose-limiting safety issues that result from the antagonism of a pathway that is responsible for regulating many fundamental biological processes including immune surveillance and cardiovascular responses. These disappointments have led many in the field to conclude that modulating the TGFß pathway for chronic indications with a sufficient safety window using conventional approaches may be extremely difficult to achieve. Here we review the rationale and limitations of the use of TGFß pathway inhibitors in chronic lung disorders and the possibility of targeting TGFß superfamily ligand accessory proteins to allow rheostatic regulation of signaling to achieve efficacy while maintaining a sufficient therapeutic index.

A forward chemical screen using zebrafish embryos with novel 2-substituted 2H-chromene derivatives.

We synthesized 2-substituted 2H-chromene derivatives from salicylaldehyde using potassium vinylic borates in the presence of secondary amines. Our goal was to generate novel compounds that might modulate transforming growth factor-beta signaling, based on limited rational design. Potassium vinyl trifluoroborates react with salicylaldehydes at 80 degrees C in the presence of a secondary amine and produce 2-substituted 2H-chromene derivatives with a 70-90% yield. A small library of these compounds, predicted to potentially interact with transforming growth factor-beta receptors, was screened for bioactivity in living zebrafish embryos. We found that the related compounds differentially affect development, and demonstrate one compound that produces severe body axis alterations in early embryogenesis and at lower doses affects specifically cardiovascular development. This compound modulates specifically a Smad-independent transforming growth factor-beta-regulated mitogen-activated protein kinase pathway, namely p-SAPK/JNK. These compounds, as suggested by our biological assays, may prove useful to manipulate developmental programs and develop therapeutic tools.

TGF beta2-induced changes in LRP-1/T beta R-V and the impact on lysosomal A beta uptake and neurotoxicity.

Numerous studies suggest a central role for the low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V in Alzheimer's Disease. We continue our investigation of a ligand for this receptor, transforming growth factor beta2, which is also implicated in Alzheimer Disease pathogenesis, but whose mechanism(s) remain elusive. Confocal imaging reveals that transforming growth factor beta2 rapidly targets amyloid beta peptide to the lysosomal compartment in cortical neurons and induces cell death. Low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V is known as an endocytic receptor, delivering proteins to the lysosomal compartment for degradation. Transforming growth factor beta2 may alter this pathway resulting in increased uptake, intracellular accumulation and toxicity of amyloid beta peptide. RT-PCR and Western blot analysis of transforming growth factor beta2-treated cells demonstrate that transforming growth factor beta2 modestly increases the mRNA and protein levels of low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V as well as increases the uptake activity. Furthermore, transforming growth factor beta2 alters the morphology and numbers of lysosomes in neurons. Lucifer Yellow and lysosomal hydrolase analysis show that transforming growth factor beta2 makes lysosomal membranes unstable and leaky and this effect is exacerbated with the addition of amyloid beta protein. Our data support a key role for low-density lipoprotein receptor-related protein/transforming growth factor beta receptor V in mediating transforming growth factor beta2 enhancement of amyloid beta peptide uptake and neurotoxicity.

TGF-beta activates ERK5 in human renal epithelial cells.

The role of the MAP kinase, extracellular signal-regulated kinase 5 (ERK5) remains unknown, however it is involved in cell differentiation and survival as highlighted by the embryonic lethality of the ERK5 knockout. ERK5 can be activated by growth factors and other extracellular signals. TGF-beta, a powerful controller of epithelial cell phenotype, is known to activate the MAP kinase, ERK1/2 however its effect on ERK5 remains unknown. This study demonstrates, fort the first time, ERK5 activation by TGF-beta, observed in both transformed and primary adult human PTEC; activation required ALK-5 receptor activity. In addition this work demonstrates expression of myocyte enhancer factor-2 (MEF2C) by PTEC and that TGF-beta increased the association of MEK5 with phospho-ERK5 and MEF2C. ERK5 activation by either TGF-beta or epidermal growth factor (EGF) was also inhibited by the p38 MAP kinase inhibitor, SB-202190.

TGF-beta: a fibrotic factor in wound scarring and a potential target for anti-scarring gene therapy.

Hypertrophic scar and keloid are common and difficult to treat diseases in plastic surgery. Results of wound healing research over the past decades have demonstrated that transforming growth factor-beta (TGF-beta) plays an essential role in cutaneous scar formation. In contrast, fetal wounds, which heal without scarring, contain a lower level of TGF-beta than adult wounds. How to translate the discovery of basic scientific research into the clinical treatment of wound scarring has become an important issue to both clinicians and basic researchers. The development of gene therapy techniques offers the potential to genetically modify adult wound healing to a healing process similar to fetal wounds, and thus reduces wound scarring. This article intends to review the roles of TGF-beta in the formation of wound scarring, the possible strategies of antagonizing wound TGF-beta, and our preliminary results of scar gene therapy, which show that wound scarring can be significantly reduced by targeting wound TGF-beta.

Transforming growth factor beta receptor family ligands inhibit hepatocyte growth factor synthesis and secretion from astrocytoma cells.

Transforming growth factor beta (TGFbeta) and hepatocyte growth factor (HGF) promote glioma progression. Using U87human astrocytoma cells, which express TGFbeta receptors (TbetaRs), we show (1) mRNA expression of Smads (2, 3, 4), bone morphogenetic protein (BMP)- and activin-A receptors; (2) TGFbeta1 inhibits and HGF induces proliferation; (3) TGFbeta1 and activin-A equipotently inhibit HGF secretion more than BMP-2, but none alters c-Met expression. Because interfering with TbetaR signaling might nullify the beneficial inhibition of HGF secretion, activin-A should instead be considered for combination glioma therapy.

Expression of recombinant extracellular domain of the type II transforming growth factor-beta receptor: utilization in a modified enzyme-linked immunoabsorbent assay to screen TGF-beta agonists and antagonists.

TGF-beta is a ubiquitous protein that exhibits a broad spectrum of biological activity. The prokaryotic expression and purification of the extracellular domain of the type II TGF-beta receptor (T beta R-II-ED), without the need for fusion protein cleavage and refolding, is described. The recombinant T beta R-II-ED fusion protein bound commercially available TGF-beta 1 and displayed an affinity of 11.1 nM. In a modified ELISA, receptor binding to TGF-beta1 was inhibited by TGF-beta 3. The technique lends itself to high-throughput screening of combinatorial libraries for the identification of TGF-beta agonists and antagonists and this, in turn, may have important therapeutic implications.