TGF-beta Family Ligands Research Articles

Treatment of Lower Risk Myelodysplastic Syndromes

Purpose of review: Management and Optimization of therapy for lower-risk myelodysplastic syndromes will be reviewed here. Recent findings: Lower-risk MDS typically present with clinical manifestations of anemia, which is the most frequently encountered cytopenia in this setting. While therapy with erythropoietic stimulating agents (ESAs) is used in the vast majority of cases, if correctly selected, some patients do not respond, or become irresponsive to ESAs. Novel agents with very different modes of action show promising clinical results in anemic LR-MDS refractory/relapsed after ESAs. Luspatercept, a TGFbeta family ligand trap, induces nearly 50% of transfusion independence in LR MDS. Another investigational agent showing efficacy and possibly disease modifying activity is the telomerase inhibitor imetelstat. Modulation of dose and schedule of hypomethylating agents, both injectable and oral, is currently being explored, and preliminary results are positive. There is still no standard therapeutic approach for thrombocytopenic and neutropenic LR MDS, although they do represent a smaller proportion of cases. Immunosuppressive treatments, as well as TPO mimetics, could represent a good option in selected MDS cases. Summary: At present, the availability of novel active agents allows the planning of sequential therapy, especially for anemic LR MDS. Better diagnosis and prognostic stratification may allow a more precise and personalized treatment.

Temporally and spatially dynamic germ cell niches in Botryllus schlosseri revealed by expression of a TGF-beta family ligand and vasa.

BackgroundGerm cells are specified during early development and are responsible for generating gametes in the adult. After germ cells are specified, they typically migrate to a particular niche in the organism where they reside for the remainder of its lifetime. For some model organisms, the specification and migration of germ cells have been extensively studied, but how these events occur in animals that reproduce both sexually and asexually is not well understood.ResultsWe have identified a novel TGF-β family member in Botryllus schlosseri, tgfβ-f, and found that it is expressed by follicle cell progenitors and the differentiated follicle and support cells surrounding the maturing gametes. Using the expression of tgfβ-f and the germ cell marker vasa, we have found that nearly all germ cells in Botryllus are associated with tgfβ-f-expressing follicle progenitors in clusters consisting solely of those two cell types. These clusters were mostly small, consisting of ten or fewer cells, and generally contained between a 2:1 and 1:1 ratio of follicle progenitors to germ cells. Clusters of germ and follicle progenitor cells were primarily localized to niches in the primary and secondary buds, but could also be found in other locations including the vasculature. We analyzed the location of germ cell clusters throughout the asexual life cycle of Botryllus and found that at the stage when germ cells are first detected in the secondary bud niche, a dramatic change in the size and location of germ/follicle cell clusters also occurred.ConclusionsOur findings suggest that germ/follicle cell clusters have predictable migratory patterns during the weekly asexual developmental cycle in Botryllus. An increased number of small clusters and the presence of clusters in the vasculature coinciding with the appearance of clusters in the secondary bud suggest that fragmentation of clusters and the migration of smaller clusters through the vasculature may be an important aspect of Botryllus reproductive biology, ensuring the transmission of the germline to subsequent asexual generations.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-016-0047-5) contains supplementary material, which is available to authorized users.

Abstract POSTER-BIOL-1310: Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer

Abstract Tumor dormancy is an important research topic because of its potential implications in metastasis and acquired resistance to chemotherapy. Dormant tumor cells at the secondary site may persist after chemotherapy and regrow as metastatic recurrence. Seminal studies by Dr. Bissell and Dr. Hendrix have shown that the tissue microenvironment can reprogram tumor cells into dormancy. Extracellular matrix (ECM) proteins are key components of tumor microenvironment and may regulate dormancy, and disruption of ECM homeostasis may cause recurrence and metastatic growth. A major cytokine that regulates ECM remodeling is tumor growth factor-beta (TGF-beta), and it is implicated in modulating tumor cell dormancy. In the tissue microenvironment, serine protease HtrA1 was shown to inhibit TGF-beta signaling by targeting TGF-beta family ligands and receptors for proteolysis. Our lab showed that HtrA1 is down-regulated in ovarian cancer cells, and HtrA1 loss promotes resistance to anoikis and in vivo metastasis. Loss of HtrA1 is also associated with increased incidence of carcinomatosis. Finally, previous studies have shown that HtrA1 degrades ECM proteins. Therefore, we hypothesize that loss of HtrA1 in the ovarian tumor microenvironment enhances TGF-beta signaling and promotes ECM protein production by stromal cells, thereby contributing to ECM remodeling and tumor dormancy. Ovarian cancer cells with endogenous HtrA1 were transduced with HtrA1-targeting shRNA (sh1) to evaluate the effect of HtrA1 downregulation on invasion, tumor dormancy, and ECM remodeling. Cells transduced with non-targeting shRNA (NT) served as a control. Cancer cells (NT and sh1) were plated over confluent mesothelial cell layer with or without 10 nM TGF-beta for 24 hours and 48 hours. No significant difference in cell invasion was observed between those cells cultured with or without TGF-beta. In order to test which matrix proteins are critical for cell proliferation versus cell cycle arrest, MicroMatrix 36 arrays were used to culture NT and sh1 cells. Cells were cultured for 48 hours on matrix slides, then fixed and stained with anti Ki67. It was determined matrix containing combinations of fibronectin showed cells that stained positive for Ki67, when compared to those not containing fibronectin or collagen together. Changes in intracellular signaling in the presence and absence of matrix were also investigated using kinase and MAPK arrays. Mesothelial cells were cultured until confluent, and cells were removed from culture plates using 2.0 mM EDTA in PBS. Matrix produced from mesothelial cells remained on the plates. NT and sh1 cells were then plated in the presence and absence of matrix and were allowed to culture until cell attachment was observed (3 hours at 37oC, 5% CO2). Cells were lysed and lysates were cultured on arrays according to manufactures instructions. ERK and Akt pathways were observed to be activated in cells plated with matrix compared to those without matrix. Taken together the evidence thus far suggests that matrix composition is critical for cell proliferation. Currently, Stable Isotope Amino Acid labeling is being employed to investigate changes in matrix remodeling on a global scale. In addition, Imaging Mass Spectrometry will also be used to evaluate formalin fixed paraffin embedded tissue samples from patients to identify at a molecular level changes in extracellular matrix composition between tumor and normal tissues. Citation Format: Cooley, Megan K., Chien, Jeremy. Regulation of tumor dormancy by extracellular matrix remodeling as a result of increased TGF-beta signaling and loss of HtrA1 in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1310.

Abstract 323: Arginine Methylation Regulates Epicardial Cell Plasticity

Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. TGFbeta family ligands induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases following type I and type II receptor complex formation. Here we show that arginine methylation, which regulates gene expression, yet also modifies some signaling mediators, initiates TGFbeta-induced Smad signaling. TGFbeta-induced receptor complex formation promotes presentation of the methyltransferase PRMT1 to the inhibitory Smad7, resulting in Smad7 methylation at a specific arginine as revealed by mass spectrometry analysis and confirmed with a mutagenesis approach. Antibodies specific for asymmetric di-methylated arginine further confirmed Smad7 methylation and Smad7 recruitment to the cell membrane upon TGFbeta stimulation. TGFbeta-induced and PRMT1-mediated Smad7 methylation leads to Smad7 dissociation from TGFbeta type I receptors, allowing activation of effector Smads through phosphorylation. This PRMT1-mediated Smad7 methylation is required in TGFbeta-regulated epicardial plasticity, as apparent by the role of PRMT1 in TGFbeta-induced epithelial-to-mesenchymal trans-differentiation and transcriptional reprogramming in mouse epicardial cells.

Over, and Underexpression of Endothelin 1 and TGF-Beta Family Ligands and Receptors in Lung Tissue of Broilers with Pulmonary Hypertension

Transforming growth factor beta (TGFβ) is a family of genes that play a key role in mediating tissue remodeling in various forms of acute and chronic lung disease. In order to assess their role on pulmonary hypertension in broilers, we determined mRNA expression of genes of the TGFβ family and endothelin 1 in lung samples from 4-week-old chickens raised either under normal or cold temperature conditions. Both in control and cold-treated groups of broilers, endothelin 1 mRNA expression levels in lungs from ascitic chickens were higher than levels from healthy birds (P < 0.05), whereas levels in animals with cardiac failure were intermediate. Conversely, TGFβ2 and TGFβ3 gene expression in lungs were higher in healthy animals than in ascitic animals in both groups (P < 0.05). TGFβ1, TβRI, and TβRII mRNA gene expression among healthy, ascitic, and chickens with cardiac failure showed no differences (P > 0.05). BAMBI mRNA gene expression was lowest in birds with ascites only in the control group as compared with the values from healthy birds (P < 0.05).

TGF-beta Family Signaling in Embryonic Stem Cells

Ligands of transforming growth factor beta (TGF-β) family members have been implicated in the development and patho-physiological process of various organs. Embryonic stem cells (ESCs) are characterized by their ability to proliferate indefinitely and differentiated into all three germ layer cells, which are termed as pluripotency and self-renewal,respectively. For successful therapeutic application of ESCs, it is essential to understand the mechanisms underlying self-renewal and pluripotency, which involve complex networks among key factors including transcription factors, epigenetic control, microRNAs and signaling pathways. In this review, we discuss recent progress on the function of TGF beta family ligands and their canonical SMAD signaling in the maintenance of ESC' s identity.

Abstract 4922: A single-cell TGFbetaR-JUND dichotomy and its role in basal-like breast cancer

Abstract Basal-like carcinoma is a subtype of breast cancer with poor prognosis that is characterized by pronounced cell-to-cell non-uniformities. Intratumor heterogeneity could involve sporadic single-cell activation of key pathways implicated in cancer progression, but it has not been possible to identify such pathways in an unbiased way. Our lab has developed a new approach for globally identifying coregulated, heterogeneously expressed genes among single cells (Nat Methods 7:311 [2010]). In a 3D in vitro culture model of a basal-like breast epithelial cell line (MCF10A), we stochastically profiled 4557 transcripts for heterogeneity and identified 547 that were predicted to be strongly dichotomous among extracellular matrix (ECM)-attached cells in the developing acinus. Hierarchical clustering of the heterogeneous transcripts revealed a group of ∼15 biosynthetic genes that contained the transcription factor, JUND, and the basal-cell cytokeratin, KRT5. In the same dataset, we identified a second group of genes-containing a transforming growth factor-beta (TGF-beta) family ligand (GDF11), a TGF-beta receptor (TGFBR3), and a TGF-beta marker protein (TGFBI)-with strong sampling fluctuations that were opposite to those of JUND cluster. Here, we show that perturbation of JUND or TGFBR3 expression profoundly disrupts 3D morphogenesis. Constitutive JUND expression causes stable cribiform-like bridges across the acinar lumen, whereas TGFBR3 knockdown induces a ductal-branching phenotype. These perturbations further revealed that JUND and TGFBR3 are reciprocally coupled by interlinked positive and negative feedback loops that could give rise to asynchronous oscillations during morphogenesis. Live-cell imaging with a fluorescently tagged Smad2 revealed sporadic pulses of TGF-betaR signaling in matrix-attached cells during morphogenesis. Last, in clinical specimens of breast cancer with dichotomous basal cytokeratin expression, we find that junD and Krt5 proteins are correlated in ECM-attached cells. Interestingly, junD and Krt5 are anticorrelated in cells not directly in contact with stromal ECM, and this regulation is mimicked in 3D culture of MCF10A cells. Together, our results raise the possibility that heterogeneous basal-like breast cancers switch between two states that depend critically on the ECM context. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4922. doi:10.1158/1538-7445.AM2011-4922

UNC-129 regulates the balance between UNC-40 dependent and independent UNC-5 signaling pathways

SummaryThe UNC-5 receptor mediates axon repulsion from UNC-6/netrin through UNC-40 dependent (‘UNC-5+UNC-40’) and independent (‘UNC-5-alone’) signaling pathways. A requirement for UNC-40 dependent signaling has been shown in long-range repulsion from UNC-6/netrin, however, the mechanisms used to regulate distinct UNC-5 signaling pathways are poorly understood. Here we demonstrate that the C. elegans TGF-β family ligand UNC-129, graded opposite to UNC-6/netrin, functions independent of the canonical TGF-β receptors to regulate UNC-5 cellular responses. We provide evidence that UNC-129 facilitates long-range repulsive guidance of UNC-6 by enhancing ‘UNC-5+UNC-40’ signaling at the expense of ‘UNC-5-alone’ signaling through interaction with the UNC-5 receptor. This increases the set point sensitivity of growth cones to UNC-6/netrin as they simultaneously migrate up the UNC-129 gradient and down the UNC-6 gradient. Similar regulatory interactions between oppositely graded extracellular cues may be a common theme in guided cell and axon migrations.

Differential response to exogenous and endogenous myostatin in myoblasts suggests that myostatin acts as an autocrine factor in vivo.

Myostatin is a member of the TGF-beta superfamily that is essential for proper regulation of skeletal muscle growth. As do other TGF-beta superfamily members, myostatin signals into the cell via a receptor complex that consists of two distinct transmembrane proteins, known as the type I and type II receptors. Vertebrates have seven distinct type I receptors, each of which can mix and match with one of five type I receptors to mediate signals for all the TGF-beta family ligands. Accumulating evidence indicates that myostatin shares its pair of receptors with activin, and therefore, the question arises about how specificity in signaling is achieved. Our hypothesis is that a mechanism has to exist to restrict myostatin actions to the muscle cells. To investigate this possibility, we compared the effect of endogenous myostatin (myostatin overexpressed by myoblasts) and exogenous myostatin (recombinant myostatin added to the culture medium) in cultured myoblasts. As opposed to exogenous myostatin, endogenous myostatin induced the transcription of a reporter vector in cultured myoblasts. Notably, the myostatin concentrations that failed to induce a response in myoblasts were effective in MCF-7 cells (human mammary carcinoma) and in HepG2 cells (human hepatic carcinoma). Based on our observations, we propose that a mechanism exists that differentially regulates the bioavailability of endogenous and exogenous myostatin to muscle cells. This is consistent with a model in which myostatin actions are exerted in vivo in an autocrine fashion.

The 26S proteasome system in the signaling pathways of TGF-beta superfamily.

Recent studies of the Smad family proteins, which are the key signal transducers of the TGF-beta family ligands, have revealed the ability of Smads to interact with various components of the 26S proteasome system. Such interactions are now known to contribute to the regulation of Smad protein levels before and after Smad activation. Most importantly, such interactions are also shown to be an integral part of the signaling functions of Smads. Through a physical interaction with different ubiquitin E3 ligases (HECT family, SCF and APC complex), the TGF-beta/activin responsive Smad3 exhibits the novel ability to regulate the ubiquitination of several key regulators, such as the oncoprotein SnoN and the multi-domain docking protein HEF1. The proteasomal degradation of these two proteins links TGF-beta signaling to multiple signaling pathways involving SnoN and HEF1. Through the interaction with proteasome beta subunit HsN3 and the substrate marker protein ornithine decarboxylase antizyme (AZ), the BMP responsive Smad1 regulates the proteasomal targeting events that contribute to the degradation of Smad1 and its interacting proteins, one of which is SNIP1, a repressor of the transcriptional co-activator CBP/p300. Thus, the novel physical link between Smads and components in the 26S proteasome system allow the intracellular events triggered by the TGF-beta family ligands to connect with those induced by many other extracellular regulators, thereby forming an extremely complex signaling network to regulate a wide range of biological activities.

Stage-specific expression of Smad2 and Smad3 during folliculogenesis.

Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development.

Identification of new signaling components in the Drosophila genome sequence

The availability of the complete sequence of the Drosophila genome and the assignment of putative reading frames, provides an opportunity to search for new members in families of proteins generating signaling cascades. The six major pathways that dictate patterning were examined: receptor tyrosine kinases, transforming growth factor beta (TGF beta), Wnt, Toll, Hedgehog and Notch. Several new components were identified for the first four pathways, including ligands, receptors, cytoplasmic components and transcription factors. Most notable is the identification of a vascular endothelial growth factor (VEGF) receptor tyrosine kinase, two insulin/insulin growth factor I (IGF I) receptors without cytoplasmic protein kinase domains, and a family of proteins similar to Rhomboid (a protein involved in cleavage of TGF alpha-like ligands). A new TGF beta family ligand, two new Wnts and a Frizzled receptor were also identified. Finally, for the Toll pathway, two new potential Spatzle-like ligands and two new receptors were identified.

Can't get no SMADisfaction: Smad proteins as positive and negative regulators of TGF-beta family signals.

The identification of Smad proteins as molecular components of the transforming growth factor-beta (TGF-beta) signaling cascade has enhanced our understanding of how ligand-mediated activation of TGF-beta receptors leads to modulation of target gene transcription. Recent studies have identified a distinct, structurally related class of Smads which inhibits, rather than transduces, TGF-beta family signals. The molecular mechanism of action and the exact signaling pathways that are targeted by antagonistic Smads are not completely understood. These proteins appear to participate in autoregulatory negative feedback loops in which signaling initiated by specific TGF-beta family ligands induces the expression of an inhibitory Smad that then functions to modulate the amplitude or duration of signaling. Negative feedback circuits such as these play important roles in fine-tuning the activity of multifunctional signaling molecules during embryonic patterning and in response to pathologic stimuli in adults.

Xenopus Smad8 acts downstream of BMP-4 to modulate its activity during vertebrate embryonic patterning.

Bone morphogenetic proteins (BMPs) participate in the development of nearly all organs and tissues. BMP signaling is mediated by specific Smad proteins, Smad1 and/or Smad5, which undergo serine phosphorylation in response to BMP-receptor activation and are then translocated to the nucleus where they modulate transcription of target genes. We have identified a distantly related member of the Xenopus Smad family, Smad8, which lacks the C-terminal SSXS phosphorylation motif present in other Smads, and which appears to function in the BMP signaling pathway. During embryonic development, the spatial pattern of expression of Smad8 mirrors that of BMP-4. We show that an intact BMP signaling pathway is required for its expression. Overexpression of Smad8 in Xenopus embryos phenocopies the effect of blocking BMP-4 signaling, leading to induction of a secondary axis on the ventral side of intact embryos and to direct neural induction in ectodermal explants. Furthermore, Smad8 can block BMP-4-mediated induction of ventral mesoderm-specific gene expression in ectodermal explants. Overexpression of Smad8 within dorsal cells, however, causes patterning defects that are distinct from those reported in BMP-4-deficient embryos, suggesting that Smad8 may interact with additional signaling pathways. Indeed, overexpression of Smad8 blocks expression of Xbra in whole animals, and partially blocks activin signaling in animal caps. In addition, Smad8 inhibits involution of mesodermal cells during gastrulation, a phenotype that is not observed following blockade of activin or BMPs in Xenopus. Together, these results are consistent with the hypothesis that Smad8 participates in a negative feedback loop in which BMP signaling induces the expression of Smad8, which then functions to negatively modulate the amplitude or duration of signaling downstream of BMPs and, possibly, downstream of other transforming growth factor-beta (TGF-beta) family ligands.