TGF-beta Superfamily Signaling Research Articles

MiR-18a Contributes to Preeclampsia by Downregulating Smad2 (Full Length) and Reducing TGF-β Signaling

The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE.

Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xeno-free Conditions.

SummaryDefects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.

SMAD3 Activation: A Converging Point of Dysregulated TGF-Beta Superfamily Signaling and Genetic Aberrations in Granulosa Cell Tumor Development?

Ovarian granulosa cell tumors (GCTs) are rare gynecologic tumors in women. Due to the rarity and limited research efforts invested, the etiology of GCTs remains poorly defined. A landmark study has discovered the mutation of forkhead box L2 (FOXL2) as a genetic hallmark of adult GCTs in the human. However, our understanding of the role of cell signaling in GCT development is far from complete. Increasing lines of evidence highlight the importance of TGF-beta (TGFB) superfamily signaling in the pathogenesis of GCTs. This review draws on findings using genetically modified mouse models and human patient specimens and cell lines to reveal SMAD3 activation as a potentially key converging point of dysregulated TGFB superfamily signaling and genetic aberrations in GCT development. It is anticipated that deciphering the role of TGFB superfamily signaling cascades in ovarian tumorigenesis will help develop new therapeutic approaches for GCTs by targeting core signaling elements essential for tumor initiation, growth, and progression.

Abstract 1932: Parallel functions of the endoplasmic reticulum chaperone protein GRP78 in tumorigenesis and the induction of pluripotency

Abstract Increasing evidence supports the concept that instances of cancer recurrence may be due to a subpopulation of cells within a tumor that behave as stem cells. Previous studies have demonstrated that pathways critical in oncogenesis parallel those necessary for the induction of pluripotency, suggesting that similar mechanisms regulate both processes. By therefore understanding the mechanisms that govern reprogramming, we may gain insight into the methods by which cancer cells acquire and exploit stem cell properties, and enable more strategic targeting of these cell populations to prevent malignant relapse. Members of the TGF-beta superfamily (e.g. activin/Nodal), which regulate many important normal cellular responses including cell growth and differentiation, have also been shown to promote tumorigenesis. Cripto, a regulator of TGF-beta superfamily ligand signaling expressed on human embryonic stem cells, is overexpressed in many types of cancer, and has also been shown to promote tumor growth and metastasis. Previous work has demonstrated that Cripto regulates TGF-beta function in tumor cell lines by forming a complex with GRP78, a protein generally restricted to the endoplasmic reticulum in normal tissues, but expressed at the cell surface in many types of tumors. Targeting GRP78 in mouse models suppresses tumor growth, and cell surface GRP78 has been shown to be a molecular target on human tumor samples. Although these studies have suggested an important role for GRP78 in promoting tumorigenesis, the mechanisms are not yet fully understood. We have discovered that GRP78 expression is induced during reprogramming, and becomes localized to the cell surface in pluripotent cells, where it co-localizes with Cripto. Overexpression of GRP78 in somatic cells induced their reprogramming efficiency. We further found that a GRP78 antibody, that disrupts cell surface GRP78/Cripto binding and Cripto-mediated TGF-beta superfamily signaling, inhibited reprogramming. Treatment of pluripotent stem cell populations with this GRP78 antibody also decreased proliferation, but did not impact pluripotency. These combined findings suggest that GRP78 may be localized to the cell surface during reprogramming where it functions to inhibit TGF-beta signaling and promote stem cell proliferation and/or survival. Interestingly, overexpression of GRP78 in human breast cancer cell lines caused an increased resistance to cisplatin. Furthermore, inhibiting cell surface function of GRP78 in these breast cancer lines resulted in a higher susceptibility to cisplatin treatment, demonstrating a specific function for cell surface GRP78 in cisplatin resistance. These combined stem cell functions of GRP78 therefore parallel many of the functions of GRP78 for cancer cells, and thus provide insight into understanding how cancer cells acquire and exploit stem cell properties. Citation Format: Athanasia D. Panopoulos, Yuriko Hishida, Min-Zu Wu, Sergio Ruiz, Erika Batchelder, Tomoaki Hishida, Jonathan A. Kelber, Peter C. Gray, Juan Carlos Izpisua Belmonte. Parallel functions of the endoplasmic reticulum chaperone protein GRP78 in tumorigenesis and the induction of pluripotency. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1932. doi:10.1158/1538-7445.AM2014-1932

Evidence Supporting a Functional Requirement of SMAD4 for Bovine Preimplantation Embryonic Development: A Potential Link to Embryotrophic Actions of Follistatin1

Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.

Loss of Smad4 in Sertoli and Leydig Cells Leads to Testicular Dysgenesis and Hemorrhagic Tumor Formation in Mice1

As the central component of canonical TGFbeta superfamily signaling, SMAD4 is a critical regulator of organ development, patterning, tumorigenesis, and many other biological processes. Because numerous TGFbeta superfamily ligands are expressed in developing testes, there may exist specific requirements for SMAD4 in individual testicular cell types. Previously, we reported that expansion of the fetal testis cords requires expression of SMAD4 by the Sertoli cell lineage. To further uncover the role of Smad4 in murine testes, we produced conditional knockout mice lacking Smad4 in either Leydig cells or in both Sertoli and Leydig cells simultaneously. Loss of Smad4 concomitantly in Sertoli and Leydig cells led to underdevelopment of the testis cords during fetal life and mild testicular dysgenesis in young adulthood (decreased testis size, partially dysgenic seminiferous tubules, and low sperm production). When the Sertoli/Leydig cell Smad4 conditional knockout mice aged (56- to 62-wk old), the testis phenotypes became exacerbated with the appearance of hemorrhagic tumors, Leydig cell adenomas, and a complete loss of spermatogenesis. In contrast, loss of Smad4 in Leydig cells alone did not appreciably alter fetal and adult testis development. Our findings support a cell type-specific requirement of Smad4 in testis development and suppression of testicular tumors.

The TGFbeta Superfamily Signaling Pathway

The transforming growth factor (TGF)beta superfamily of secreted factors is comprised of over 30 members including Activins, Nodals, Bone Morphogenetic Proteins (BMPs), and Growth and Differentiation Factors (GDFs). Members of the family, which are found in both vertebrates and invertebrates, are ubiquitously expressed in diverse tissues and function during the earliest stages of development and throughout the lifetime of animals. Indeed, key roles in embryonic stem cell self-renewal, gastrulation, differentiation, organ morphogenesis, and adult tissue homeostasis have been delineated. Consistent with this ubiquitous activity, aberrant TGFbeta superfamily signaling is associated with a wide range of human pathologies including autoimmune, cardiovascular and fibrotic diseases, as well as cancer. TGFbeta superfamily ligands signal through cell-surface serine/threonine kinase receptors to the intracellular Smad proteins, which in turn accumulate in the nucleus to regulate gene expression. In addition to this universal cascade, Smad-independent pathways are also employed in a cell-specific manner to transduce TGFbeta signals. Ligand access to the signaling receptors is regulated by numerous secreted agonists and antagonists and by membrane-associated coreceptors that act in a context-dependent manner. Given the fundamental role of the TGFbeta superfamily in metazoans and the diversity of biological responses, it is not surprising that the signaling pathway is subject to tight and complex regulation at levels both outside and inside the cell. WIREs Dev Biol 2013, 2:47-63. doi: 10.1002/wdev.86 For further resources related to this article, please visit the WIREs website.

Sensory Regulation of the C. elegans Germline through TGF-β-Dependent Signaling in the Niche

The proliferation/differentiation balance of stem and progenitor cell populations must respond to the physiological needs of the organism [1, 2]. Mechanisms underlying this plasticity are not well understood. The C. elegans germline provides a tractable system to study the influence of the environment on progenitor cells (stem cells and their proliferative progeny). Germline progenitors accumulate during larval stages to form an adult pool from which gametes are produced. Notch pathway signaling from the distal tip cell (DTC) niche to the germline maintains the progenitor pool [3-5], and the larval germline cell cycle is boosted by insulin/IGF-like receptor signaling [6]. Here we show that, independent of its role in the dauer decision, TGF-β regulates the balance of proliferation versus differentiation in the C. elegans germline in response to sensory cues that report population density and food abundance. Ciliated ASI sensory neurons are required for TGF-β-mediated expansion of the larval germline progenitor pool, and the TGF-β receptor pathway acts in the germline stem cell niche. TGF-β signaling thereby couples germline development to the quality of the environment, providing a novel cellular and molecular mechanism linking sensory experience of the environment to reproduction.

Alterations in the Smad pathway in human cancers.

Members of the TGF-beta superfamily exhibit various biological activities, and perturbations of their signaling are linked to certain clinical disorders including cancer. The role of TGF-beta signaling as a tumor suppressor pathway is best illustrated by the presence of inactivating mutations in genes encoding TGF-beta receptors and Smads in human carcinomas. This perspective is further supported by studies of tumor development in mouse models after modulation of receptors and Smads. TGF-beta also controls processes such as cell invasion, immune regulation, and microenvironment alterations that cancer cells may exploit to their advantage for their progression. Consequently, the output of a TGF-beta response is highly situation dependent, across different tissues, and also in cancer in general. Understanding the mechanisms of TGF-beta superfamily signaling is thus important for the development of new ways to treat various types of cancer. This review focuses on recent advances in understanding the Smad dependent TGF-beta pathway as it relates to human carcinogenesis.

Relative expression of genes encoding SMAD signal transduction factors in human granulosa cells is correlated with oocyte quality

To determine the expression of SMAD transcripts in human granulosa cells. Luteinized mural granulosa cells were harvested from forty women undergoing oocyte retrieval, and RNAs were isolated. SMAD expression levels were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (q-RTPCR). SMAD1-7 and 9 are expressed in human granulosa cells, with SMAD2, 3 and 4 showing the highest expression levels. Peak estradiol (E2) levels correlated with the number of oocytes retrieved during IVF. Oocyte number showed no correlation with SMAD expression levels or ratios. Fertilization rates also did not correlate with the expression levels of individual SMADs, but did correlate with higher SMAD4:SMAD3 ratios (p = 0.0062) and trended with SMAD4:SMAD2 (p = 0.0698). SMAD transcripts are differently expressed in human granulosa cells, where they may mediate TGF-beta superfamily signaling during folliculogenesis and ovulation. Further, the relative expression ratios of SMAD2, 3 and 4 may differentially affect fertilization rate.

Klf10 and Klf11 as mediators of TGF-beta superfamily signaling

Klf10 and Klf11 belong to the family of Sp1/Krüppel-like zinc finger transcription factors that play important roles in a variety of cell types and tissues. Although Klf10 and Klf11 were initially introduced as transforming growth factor-beta (TGF-beta)-inducible genes, several studies have described their upregulation by a plethora of growth factors, cytokines and hormones. Here, we review the current knowledge of the inductive cues for Klf10 and Klf11 and focus on their transcriptional regulation by members of the TGF-beta superfamily. We further summarize their involvement in the regulation of the TGF-beta signaling pathway and discuss their possible role as molecules mediating crosstalk between various signaling pathways. Finally, we provide an overview of the pro-apoptotic and anti-proliferative functions of Klf10 and Klf11.

The role of Smad signaling in vascular and hematopoietic development revealed by studies using genetic mouse models

Smads are intracellular mediators of transforming growth factor beta (TGF-beta) superfamily signaling. In this review, we focus on the genetic mouse models for Smad pathways, which have provided functional evidence regarding the complex circuitry in angiogenesis and hematopoiesis during development. In the early stages of vascular development, TGF-beta signaling is a contributing factor in angiogenesis and vascular maturation. Whereas in the later embryogenesis, selected molecules of Smad pathways, such as TGF-beta type II receptor (TbRII), ALK5, and Smad5, seem to be dispensable for vessel morphogenesis and integrity. TGF-beta signaling is not required in the induction of hematopoietic precursors from mesoderm, but inhibits the subsequent expansion of committed hematopoietic precursors. By contrast, bone morphogenetic protein 4 (BMP4) has long been acknowledged pivotal in mesoderm induction and hematopoietic commitment during development. However, recent genetic evidence shows the BMP4-ALK3 axis is not crucial for the formation of hematopoietic cells from FLK1(+) mesoderm. Because of the highly redundant mechanisms within the Smad pathways, the precise role of the Smad signaling involved in vascular and hematopoietic development remains nebulous. The generation of novel cell lineage restricted Cre transgenes would shed new light on the future relevant investigations.

ALK5 phosphorylation of the endoglin cytoplasmic domain regulates Smad1/5/8 signaling and endothelial cell migration

Endoglin, an endothelial cell-specific transforming growth factor-beta (TGF-beta) superfamily coreceptor, has an essential role in angiogenesis. Endoglin-null mice have an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin result in the vascular disease hereditary hemorrhagic telangiectasia type I. Increased endoglin expression in the proliferating endothelium of tumors has been correlated with metastasis, tumor grade and decreased survival. Although endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta-responsive pathways, the activin receptor-like kinase 5 (ALK5)/Smad2/3 pathway and the activin receptor-like kinase 1 (ALK1)/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Here, we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by ALK5 in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to TGF-beta and endothelial cell migration, whereas loss of phosphorylation at both serines 646 and 649 resulted in a loss of endoglin-mediated inhibition of Smad1/5/8 signaling in response to bone morphogenetic protein-9. Taken together, these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.

Prdm16 is required for normal palatogenesis in mice

Transcriptional cofactors are essential to the regulation of transforming growth factor beta (TGFbeta) superfamily signaling and play critical and widespread roles during embryonic development, including craniofacial development. We describe the cleft secondary palate 1 (csp1) N-ethyl-N-nitrosourea-induced mouse model of non-syndromic cleft palate (NSCP) that is caused by an intronic Prdm16 splicing mutation. Prdm16 encodes a transcriptional cofactor that regulates TGFbeta signaling, and its expression pattern is consistent with a role in palate and craniofacial development. The cleft palate (CP) appears to be the result of micrognathia and failed palate shelf elevation due to physical obstruction by the tongue, resembling human Pierre Robin sequence (PRS)-like cleft secondary palate. PRDM16 should be considered a candidate for mutation in human clefting disorders, especially NSCP and PRS-like CP.

Role for TGF-β superfamily signaling in telencephalic GABAergic neuron development

Signaling mechanisms mediated by the Transforming Growth Factor-β (TGF-β) superfamily regulate a variety of developmental processes. Here we show that components of both bone morphogenetic protein/growth differentiation factor and TGF-β/activin/Nodal branches of TGF-β superfamily signaling are expressed in the developing subpallium. Furthermore, Smad proteins, transcriptional effectors of TGF-β signaling, are co-expressed and physically interact in the basal ganglia with Dlx homeodomain transcription factors, which are critical regulators of the differentiation, migration and survival of telencephalic GABAergic neurons. We also show that Dlx and Smad proteins localize to promoters/enhancers of a number of common telencephalic genes in vivo and that Smad proteins co-activate transcription with Dlx family members, except with certain mutated human DLX proteins identified in autistic individuals. In agreement with these observations, expression of dominant-negative Smads in the developing basal ganglia phenocopies the cell migration defects observed in Dlx1/2-deficient mice. Together, these results suggest that TGF-β superfamily signaling plays a role in telencephalic GABAergic neuron development through functional interactions with Dlx transcription factors.Electronic supplementary materialThe online version of this article (doi:10.1007/s11689-009-9035-6) contains supplementary material, which is available to authorized users.

Mutations in LTBP4 Cause a Syndrome of Impaired Pulmonary, Gastrointestinal, Genitourinary, Musculoskeletal, and Dermal Development

We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

Oocytes from Pre-Antral and Antral Follicles Are Targets of TGF-Beta Superfamily Signaling.

Oocytes from Pre-Antral and Antral Follicles Are Targets of TGF-Beta Superfamily Signaling.

Regulated production of SnoN2 is a feature of testicular differentiation

Transforming growth factor betas (TGF beta s) and activins are key regulators of male fertility, affecting somatic and germ cell proliferation and differentiation in the developing and adult testis. Several studies have shown that these ligands influence discrete developmental stages, suggesting that temporal expression of modifying factors may determine their specific signaling outcomes. Upon binding to cell surface receptors, TGFbeta and activin signals are transduced intracellularly by the phosphorylation and nuclear accumulation of SMAD2 and SMAD3 transcription factors. The objective of this study was to determine the cellular localization of phosphorylated SMAD2/3 and the transcriptional repressor SnoN (Ski-like), a modifier of SMAD2/3 transcriptional activity, in mouse testes. Western blot established that only the smaller SnoN isoform, SnoN2, is produced in the testis. By immunohistochemistry, widespread phospho-SMAD2/3 distribution was observed in somatic and germ cells at all ages. In contrast, SnoN2 production was highly regulated, being detected only in gonocytes and interstitial cells at birth and in pachytene spermatocytes at puberty. In the adult, SnoN2 expression differed to that during the first wave, being ubiquitously expressed but exhibiting regulated nuclear localization. In another model of spermatogenic differentiation, the irradiated rat testis, widespread phospho-SMAD2/3 contrasted with restricted SnoN2 expression. SnoN2 was limited to interstitial cells, with reduced staining intensity observed associated with the timing of spermatogenesis resumption. We conclude that somatic and germ cells at all differentiation stages are actively transducing TGFbeta superfamily signals but that responses to these ligands may be selectively modulated by controlled production and nuclear localization of SnoN2.

New insights into the aetiology of colorectal cancer from genome-wide association studies

Genome-wide association studies have recently identified ten common genetic variants associated with colorectal cancer susceptibility, several suggesting the involvement of components of the transforming growth factor beta (TGFbeta) superfamily signalling pathway. To date, no causal sequence variants have been identified, and risk seems to be mediated through effects on gene regulation. Several markers are located close to poorly characterized genes or in gene deserts, raising challenges for elucidating mechanisms of susceptibility. Disease-associated common genetic variation offers the potential to refine risk stratification within populations and enable more targeted disease prevention strategies.

Negative Smad Expression and Regulation in the Developing Chick Limb

The inhibitory or negative Smads, Smad6 and Smad7, block TGFβ superfamily signals of both the BMP and TGFβ classes by antagonizing the intracellular signal transduction machinery. We report the cloning of one Smad6 and two Smad7 (Smad7a and Smad7b) chick homologs and their expression and regulation in the developing limb. Smad6 and Smad7a are expressed in dynamic patterns reflecting the domains of BMP gene expression in the limb. Activation and inhibition of the BMP signaling pathway in limb mesenchyme indicates that negative Smad gene expression is regulated, at least in part, by BMP family signals.