Common Mediator Smad Research Articles

TAp73 Inhibits EMT and Cell Migration in Pancreatic Cancer Cells through Promoting SMAD4 Expression and SMAD4-Dependent Inhibition of ERK Activation.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease due to early metastatic spread, late diagnosis and the lack of efficient therapies. A major driver of cancer progression and hurdle to successful treatment is transforming growth factor (TGF)-β. Recent data from pancreatic cancer mouse models showed that transcriptionally active p73 (TAp73), a p53 family member, inhibits tumor progression through promoting tumor suppressive canonical TGF-β/Smad signaling, while preventing non-canonical TGF-β signaling through extracellular signal-regulated kinases (ERK)1/2. Here, we studied whether this mechanism also operates in human PDAC. Using the PDAC-derived tumor cell lines PANC-1, HPAFII and L3.6pl, we showed that TAp73 induces the expression of the epithelial marker and invasion suppressor E-cadherin and the common-mediator Smad, SMAD4, while at the same time suppressing expression of the EMT master regulator SNAIL and basal and TGF-β1-induced activation of ERK1 and ERK2. Using dominant-negative and RNA interference-based inhibition of SMAD4 function, we went on to show that inhibition of ERK activation by TAp73 is mediated through SMAD4. Intriguingly, both SMAD4 and the α isoform of TAp73-but not the β isoform-interfered with cell migration, as shown by xCELLigence technology. Our findings highlighted the role of TAp73-SMAD4 signaling in tumor suppression of human PDAC and identified direct inhibition of basal and TGF-β-stimulated pro-invasive ERK activation as an underlying mechanism.

SMAD7 and SMAD4 expression in colorectal cancer progression and therapy response

Inhibitory SMAD7 and common mediator SMAD4 play crucial roles in SMAD-dependent TGF-β signaling that is often disrupted in colorectal cancer (CRC). This study aimed to profile the expression of SMAD7 and SMAD4 in primary and metastatic CRC and to evaluate their significance in disease progression and therapy response. The expression of SMAD7 and SMAD4 genes was analyzed by quantitative real-time PCR in tissues from 35 primary and metastatic CRC patients and in vitro in 7 human cell lines originating from colon tissue. Expression levels of SMAD7 and SMAD4, as well as their ratio, were determined and their association with tumor characteristics and response to therapy were evaluated. SMAD4 level was significantly lower in tumors compared to non-tumor tissues in both primary (p = 0.001) and metastatic (p = 0.001) CRC patients, while tumor expression of SMAD7 was significantly lower from non-tumor tissue only in metastatic patients (p = 0.017). SMAD7/SMAD4 ratio was elevated in CRC primary tumor tissues and cell lines compared to corresponding non-tumor tissues and cell line, respectively (p = 0.003). SMAD7 expression was significantly elevated in primary tumor tissues obtained from responders to neoadjuvant chemoradiotherapy (nCRT) compared to non-responders (p = 0.014). Alterations of expression and ratio of SMAD7 and SMAD4 in CRC cell lines, primary rectal cancer, and liver metastasis emphasize the importance of these genes in different stages of disease progression. Differential expression of SMAD7 in responders versus non-responders to nCRT should be further investigated for its potential predictive value.

Visualization and Quantification of TGFβ/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay.

Transforming Growth Factor β (TGFβ)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGFβ/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGFβ/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA). Active TGFβ/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGFβ and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy. The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGFβ/BMP signaling in ECs, both quantitatively and spatially.

SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium.

Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of CDH2, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the CDH2 promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of CDH2 was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after CDH2 knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.

MicroRNA-130a Is Up-regulated in Mouse Liver by Iron Deficiency and Targets the Bone Morphogenetic Protein (BMP) Receptor ALK2 to Attenuate BMP Signaling and Hepcidin Transcription

Systemic iron balance is controlled by the liver peptide hormone hepcidin, which is transcriptionally regulated by the bone morphogenetic protein (BMP)-SMAD pathway. In iron deficiency, liver BMP-SMAD signaling and hepcidin are suppressed as a compensatory mechanism to increase iron availability. MicroRNAs are small regulatory RNAs that have an increasingly recognized role in many biologic processes but are only recently implicated in iron homeostasis regulation. Here, we demonstrate that liver expression of the microRNA miR-130a is up-regulated by iron deficiency in mice. We identify the BMP6-SMAD signaling pathway as a functional target of miR-130a in hepatoma-derived Hep3B cells. Although the TGF-β/BMP common mediator SMAD4 was previously reported to be an miR-130a target to inhibit TGF-β signaling, we do not confirm SMAD4 as an miR-130a target in our biologic system. Instead, we determine that the BMP type I receptor ALK2 is a novel target of miR-130a and that miR-130a binds to two specific sites in the 3'-untranslated region to reduce ALK2 mRNA stability. Moreover, we show in mice that the increased liver miR-130a during iron deficiency is associated with reduced liver Alk2 mRNA levels. Finally, we demonstrate that down-regulation of ALK2 by miR-130a has a functional effect to inhibit BMP6-induced hepcidin transcription in Hep3B cells. Our data suggest that iron deficiency increases liver miR-130a, which, by targeting ALK2, may contribute to reduce BMP-SMAD signaling, suppress hepcidin synthesis, and thereby promote iron availability.

SMAD7: a timer of tumor progression targeting TGF-β signaling.

In the context of cancer, transforming growth factor β (TGF-β) is a cell growth suppressor; however, it is also a critical inducer of invasion and metastasis. SMAD is the important mediator of TGF-β signaling pathway, which includes receptor-regulated SMADs (R-SMADs), common-mediator SMADs (co-SMADs), and inhibitory SMADs (I-SMADs). I-SMADs block the activation of R-SMADs and co-SMADs and thus play important roles especially in the SMAD-dependent signaling. SMAD7 belongs to the I-SMADs. As an inhibitor of TGF-β signaling, SMAD7 is overexpressed in numerous cancer types and its abundance is positively correlated to the malignancy. Emerging evidence has revealed the switch-in-role of SMAD7 in cancer, from a TGF-β inhibiting protein at the early stages that facilitates proliferation to an enhancer of invasion at the late stages. This role change may be accompanied or elicited by the tumor microenvironment and/or somatic mutation. Hence, current knowledge suggests a tumor-favorable timer nature of SMAD7 in cancer progression. In this review, we summarized the advances and recent findings of SMAD7 and TGF-β signaling in cancer, followed by specific discussion on the possible factors that account for the functional changes of SMAD7.

Proteomic Profiling of Smad Proteins Expression in AML: Pan Expression with High Phosphorylation Is Prognostically Adverse

Abstract 891The Transforming growth factor β (TGF-β) signaling pathway has been previously known to play a tumor suppressor role in hematologic malignancies. Smad proteins and their phosphorylation play a vital role in TGF-β signaling pathway. There are three class of Smads; Receptor-regulated Smads (1, 2, 3, 5, 8), Common mediator Smad (4) and Inhibitory Smads (6, 7). However, little is known about the expression and activation of Smad proteins in AML and nothing has been reported about correlation with clinical features or outcomes. Interestingly, Tabe et.al, (ASH 2012) recently identified pro-survival effect of TGF-β in leukemia cells via upregulation of MMP-1 and that the anti-apoptotic effect of TGF-β was associated with G0/G1 cell cycle arrestWe performed proteomic profiling of Smad expression, measuring the level of total Smad 1, 2, 3, 4, 5, 6, and phosphorylated Smad 2 (p245, p465) and 5 (p463) using reverse phase protein array (RPPA) technology. All antibodies were strictly validated. Analysis was performed on a cohort of 511 newly diagnosed AML (non APL) cases randomly divided into training and test sets. Normal bone marrow derived CD34+ cells (n=11) served as expression controls. Hierarchical clustering with Wald linkage rules and Euclidean distance matrix were used to define signatures. Cox model and long rank test were used to assess the survival outcome with different sample signatures.When comparing expression of individual Smad proteins with control CD34+ cells, most cases had expression within the normal CD34+ cell range, but levels of Smad 2, 2p465 and 4 had higher percentages of cases with expression below normal, while levels of Smad 3, 5, 5 (p463) and 6 were more frequently expressed at levels above normal. There were no major differences in expression between bone marrow and blood, and diagnosis and relapse samples. When unbiased hierarchical clustering was performed on the training set, four distinct Smad protein expression signatures were identified (Figure 1). Group 1 is characterized by pan-low Smad expression; Group 2 by high expression in Smad 2, 5, 5 (p463); Group 3 by high expression of phosphorylated Smad 2 (p245, p465) and 5 (p463); and Group 4, by pan-high Smad expression. Group 2 was associated with Ras mutation (28% vs. 9% for the other 3, p=0.03) and FLT3 ITD (p=0.009) mutation frequency was significantly lower in group 1. Smad group was not associated with FAB classification, demographics, prior treatment history, cytogenetics, and other molecular mutations. Higher Smad expression was statistically significantly correlated with higher counts of WBC (p=0.04), bone marrow and peripheral blast % (p=0.009, 0.004), CD33 and 34 counts (p=0.006, 0.002). Intriguingly, among 210 other proteins assayed in RPPA, expression of Integrin/Adhesion proteins IGFBP2, CD49B, CD11A and Fibronectin were inversely correlated with Smad expression consistent with the above observation. Pan Smad expression was strongly correlated with AKT pathway activation and high expression of several proliferation promoting proteins. Pan-high Smad expression (group 4) was associated with inferior overall survival (OS) (Figure 2) and event free survival (EFS) whereas the OS and EFS of Group 1, 2, and 3 were similar (log-rank test OS p=0.017; EFS p=0.03). Median OS, EFS were 36.3 and 19.3 weeks in Group 4 versus 56.1 and 26.9 weeks in other groups, respectively. Patients in group 4 had a lower remission rate (51% vs. 66%). When validated with the test set, similar results were observed and group 4 again had inferior survival (median 26.7 vs. 58 weeks, p = 0.0047) compared to the other groups.In conclusion, we observed that Smad expression in AML segregates into four distinct heterogeneous expression and activation patterns. Pan-high Smad expression was linked with significantly worse OS, EFS, and trends for inferior CR rates. The clinical features (high WBC and % PB and BM blasts) and inferior clinical outcome associated with pan-high Smad expression suggest that dominant TGF-β signaling is adverse in AML and that these patients may benefit from TGF-β blockade. Our finding suggest a tumor promoting, rather than tumor suppressing role, for TGF- β in AML, possibly mediated via MMP-1 activation. Further studies are required to investigate the mechanism of TGF-β pathway activation possibly inducing chemotherapy resistance leading to poor survival. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Spatial Restriction of Bone Morphogenetic Protein Signaling in Mouse Gastrula through the mVam2-Dependent Endocytic Pathway

The embryonic body plan is established through positive and negative control of various signaling cascades. Late endosomes and lysosomes are thought to terminate signal transduction by compartmentalizing the signaling molecules; however, their roles in embryogenesis remain poorly understood. We showed here that the endocytic pathway participates in the developmental program by regulating the signaling activity. We modified the mouse Vam2 (mVam2) locus encoding a regulator of membrane trafficking. mVam2-deficient cells exhibited abnormally fragmented late endosomal compartments. The mutant cells could terminate signaling after theremoval of the growth factors including TGF-β and EGF, except BMP-Smad1/Smad5 signaling. mVam2-deficient embryos exhibited ectopic activation of BMP signaling and disorganization of embryo patterning. We found that mVam2, which interacts with BMP type I receptor, is required for the spatiotemporal modulation of BMP signaling, via sequestration of the receptor complex in the late stages of the endocytic pathway.

Negative Regulation of TGFβ Signaling by the Kinase LKB1 and the Scaffolding Protein LIP1

Signal transduction by the Smad pathway elicits critical biological responses to many extracellular polypeptide factors, including TGFβ and bone morphogenetic protein. Regulation of Smad signaling imparts several cytoplasmic and nuclear mechanisms, some of which entail protein phosphorylation. Previous work established a protein complex between Smad4 and the scaffolding protein LKB1-interacting protein 1 (LIP1). LKB1 is a well studied tumor suppressor kinase that regulates cell growth and polarity. Here, we analyzed the LKB1-LIP1 and the Smad4-LIP1 protein complexes and found that LIP1 can self-oligomerize. We further demonstrate that LKB1 is capable of phosphorylating Smad4 on Thr77 of its DNA-binding domain. LKB1 inhibits Smad4 from binding to either TGFβ- or bone morphogenetic protein-specific promoter sequences, which correlates with the negative regulatory effect LKB1 exerts on Smad4-dependent transcription. Accordingly, LKB1 negatively regulates TGFβ gene responses and epithelial-mesenchymal transition. Thus, LKB1 and LIP1 provide negative control of TGFβ signaling.

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.

The Transforming Growth Factor Beta 1/SMAD Signaling Pathway Involved in Human Chronic Myeloid Leukemia

Transforming growth factor beta 1 (TGF-beta1) is the prototypic member of a large family of structurally related pleiotropic-secreted cytokines. The TGF-beta1/SMAD signaling pathway usually participates in a wide range of cellular processes such as growth, proliferation, differentiation and apoptosis. Upon binding on TGF-beta1, the dimerized TGF-beta type II receptors recruit and phosphorylate the TGF-beta type I receptors, which phosphorylate the receptor-regulated SMAD (SMAD2 and SMAD3) presented by the SMAD anchor for receptor activation. The phosphorylated receptor-regulated SMAD form heterologous complexes with the common-mediator SMAD (SMAD4) and subsequently translocate into the nucleus, where they interact with other transcription factors to regulate the expression of target genes. This multi-functional signaling pathway modulated by various elements with complex mechanisms at different levels is also inevitably involved in cancer. We herein present data on the role of the TGF-beta1/SMAD signaling pathway in human chronic myeloid leukemia and explain the potent biological effects of TGF-beta1 on leukemia cells. The paper is based on a review of articles selected from Cancerline and Medline data bases. The constitutively active tyrosine kinase produced by the specific Bcr-Abl fusion gene on the Philadelphia chromosome can enhance the resistance of malignant cells to TGF-beta1-induced growth inhibition and apoptosis, which contributes to enhancement of proteasomal degradation of p27. However, overexpression of the EVI1 gene, which is also caused by Bcr-Abl, can recruit the C-terminal binding protein and histone deacetylase to prevent the MH2 domain on SMAD3. The later is essential for transcription activation on target genes and leads to blockage of the TGF-beta1/SMAD signaling pathway. Some studies have indicated that certain therapeutic agents applied in clinical treatment can inhibit proliferation and promote differentiation of leukemia cells by way of modulation of the TGF-beta1/SMAD signal pathway. For example, arsenic trioxide can promote specific degradation of the AML1/MDS1/EVI1 oncoprotein and inhibit the proliferation of leukemia cells. However, specific histone deacetylase inhibitors can interrupt the effect of histone deacetylase to alleviate EVI1-mediated suppression of TGF-beta1/SMAD signaling. The tyrosine kinase inhibitor in the target therapy of chronic myeloid leukemia can effectively inhibit the tyrosine kinase activity of Bcr-Abl and induce suppression on the TGF-beta1/SMAD signaling pathway. The TGF-beta1/SMAD signaling pathway plays an important role in chronic myeloid leukemia cells and leads the leukemia cells to growth inhibition, differentiation and apoptosis. The positive influence of the TGF-beta1/SMAD signaling pathway in chronic myeloid leukemia is fairly significant, and its potential effects in clinical treatment will bring about definite benefits. Since it is a complex signaling pathway widely involved in many aspects of cellular activities, further study and comprehensive analysis of the TGF-beta1/SMAD signaling pathway are imperative and will have a guiding significance in research and clinical applications. It is an exciting area for future research.

Role of TGF-β1, its receptor TGFβRII, and Smad proteins in the progression of colorectal cancer

In the current study, we investigated the expression of TGF-beta1, its receptor TGFbetaRII, and the signaling proteins Smad4 and Smad7 in colorectal cancer tissue in relation to infiltration with antigen-presenting cells and some clinical and pathologic parameters of disease progression in patients with colorectal cancer (CRC). The immunohistochemical expression of TGF-beta1, TGFbetaRII, Smad4, Smad7, HLA-DR antigen, CD1a, CD83, and CD68 was evaluated in 142 patients (50 females and 92 males) with CRC, followed-up for 6-8 years period. In our study, 127 (89.4%) out of 142 colorectal cancers displayed cytoplasmic TGF-beta1 immunoreactivity. Common-mediator Smad4 was detected in the tumor cytoplasm in 124 cancers (79.5%) and inhibitory Smad7 immunostaining was observed in 110 (77.4%) tumor specimens. TGFbetaRII was expressed on tumor cell membranes in 119 (76.3%) of the cancers. The increased TGF-beta1 expression in tumor cytoplasm was related to low CD68(+)- and CD83(+)-cell infiltration in tumor tissues. Patients with TGF-beta1 overexpression had worse prognosis after surgical therapy compared to those with low expression of TGF-beta1. The observed association was more pronounced for the patients in T1-T2 stage (p = 0.0015). The expression of TGF-beta1, its receptor TGFbetaRII, and signaling proteins Smad4 and Smad7 was observed in the majority of colorectal cancer specimens. Our results suggest that TGF-beta1 production by tumor cells may affect the tumor environment via suppression of tumor-infiltrating immune cells and probably contributes to tumor cells aggressiveness through autocrine activation of Smad signaling.

Zili Inhibits Transforming Growth Factor-β Signaling by Interacting with Smad4

Piwi proteins are required for germ cell proliferation, differentiation, and germ line stem cell maintenance. In normal tissues, human and mouse Piwil2 are primarily expressed in testis but widely expressed in tumors. However, the underlying mechanism remains largely unknown. In vertebrates, transforming growth factor (TGF)-β signaling plays an important role in patterning embryo and control of cell growth and differentiation. A previous study has shown a role for Zili, a Piwil2 gene in zebrafish, in germ cells in zebrafish. Here we report that zili functions in patterning the early embryo and inhibits TGF-β signaling. Whole mount expression analysis shows that zili expresses not only in PGCs but also in axis. Ectopic expression of zili causes fusion of the eyes and reduction of mesodermal marker genes expression, suggesting that zili functions to inhibit Nodal signaling and mesoderm formation. Genetic interaction shows that zili inhibits Nodal and bone morphogenetic protein signaling. The results of protein interaction assays identify that Zili binds to Smad4 via its N-terminal domain and prevents the formation of Smad2/3/4 and Smad1/5/9/4 complexes to antagonize TGF-β signaling. This work shows that zili plays a role in early embryogenesis beyond germ line as a novel negative regulator of TGF-β signaling, extending the function of Piwi proteins in vertebrates.

VE-cadherin is a critical endothelial regulator of TGF-β signalling

VE-cadherin is an endothelial-specific transmembrane protein concentrated at cell-to-cell adherens junctions. Besides promoting cell adhesion and controlling vascular permeability, VE-cadherin transfers intracellular signals that contribute to vascular stabilization. However, the molecular mechanism by which VE-cadherin regulates vascular homoeostasis is still poorly understood. Here, we report that VE-cadherin expression and junctional clustering are required for optimal transforming growth factor-beta (TGF-beta) signalling in endothelial cells (ECs). TGF-beta antiproliferative and antimigratory responses are increased in the presence of VE-cadherin. ECs lacking VE-cadherin are less responsive to TGF-beta/ALK1- and TGF-beta/ALK5-induced Smad phosphorylation and target gene transcription. VE-cadherin coimmunoprecipitates with all the components of the TGF-beta receptor complex, TbetaRII, ALK1, ALK5 and endoglin. Clustered VE-cadherin recruits TbetaRII and may promote TGF-beta signalling by enhancing TbetaRII/TbetaRI assembly into an active receptor complex. Taken together, our data indicate that VE-cadherin is a positive and EC-specific regulator of TGF-beta signalling. This suggests that reduction or inactivation of VE-cadherin may contribute to progression of diseases where TGF-beta signalling is impaired.

Molecular characterisation of a second structurally unusual AR-Smad without an MH1 domain and a Smad4 orthologue from Echinococcus multilocularis

Members of the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP) family of cytokines play crucial roles in animal development and are candidate molecules for host–parasite cross-communication in helminth diseases. TGF-β/BMP-signalling involves binding of the cytokines to receptor kinases which subsequently activate intracellular transcription factors of the Smad family. We have previously characterized two members of the receptor-regulated Smad (R-Smad) family, EmSmadA and EmSmadB, from the human parasitic cestode Echinococcus multilocularis and now present evidence for two additional Smads that are expressed by the larval stages of the parasite. The full-length cDNAs coding for a third R-Smad, EmSmadC, and a common mediator Smad (Co-Smad), EmSmadD, were characterized. While EmSmadD displayed a typical Co-Smad structure, EmSmadC lacked the N-terminal MH1 domain which is typically found in Smads. In yeast two-hybrid analyses, EmSmadC and EmSmadD were capable of homo- and heterodimer formation with other Echinococcus Smads. Furthermore, EmSmadC displayed autonomous transcription activation activity and interacted with EmSkip, a member of the SNW/SKIP family of transcriptional regulators. In a heterologous expression system, EmSmadC was specifically phosphorylated by mammalian TGF-β receptors, indicating that it is a member of the AR-Smad sub-family. Finally, in activity assays, the parasite’s Erk-like kinase EmMPK1 phosphorylated EmSmadD, indicating cross-regulation between mitogen-activated protein kinase cascade- and TGF-β/BMP-signalling in Echinococcus. The data presented herein significantly broaden our knowledge of Smad-signalling factors in E. multilocularis and will facilitate studies on TGF-β/BMP-regulated genes in the parasite as well as TGF-β/BMP mediated host–parasite cross-interaction during alveolar echinococcosis.

Localization of TGF-β Signaling Intermediates Smad2, 3, 4, and 7 in Developing and Mature Human and Mouse Kidney

Smad proteins are signaling intermediates of the TGF-beta superfamily and are involved in a range of biological activities including development and immune responses. We studied the expression of TGF-beta-receptor activated Smads (Smad2 and Smad3), the common partner Smad (Smad4), an inhibitory Smad (Smad7), and the activated (phosphorylated) Smad2 (pSmad2) in developing and adult kidneys of humans and mice. These studies demonstrate associated expression of these Smads in multiple renal cell types in all developmental stages and in mature non-diseased kidneys. Smad expression is in general most widespread at the earliest stages of nephron development and diminishes as components of the nephrons become more differentiated. Paucity of Smad expression in mesangial cells in contrast to widespread expression of these Smads in glomerular visceral epithelial cells in both developing and mature kidneys was remarkable. Divergent and less extensive expression of Smad4, compared with other Smad proteins, was also demonstrated in tubules of human kidneys. Based on the observed expression patterns, these findings demonstrate, for the first time, expression of the TGF-beta-receptor-activated Smad2 and Smad3, the common mediator Smad4, and the inhibitory Smad7 in the developing human fetal kidney, extending observations previously made in rodent systems to humans.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

A New Twist in Smad Signaling

Signaling by members of the TGFbeta family is much dependent on the common-mediator Smad4, which forms transcriptionally active complexes with all receptor-activated Smads (R-Smads). New findings demonstrate that transcriptional intermediary factor 1gamma (TIF1gamma) also can bind to R-Smads, as an alternative to Smad4, and mediate different transcriptional effects.

Structural basis for the inhibition of activin signalling by follistatin

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatin's domain structure and shown that the minimal activin-inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 A resolution. The complex structure shows how Fs12 molecules wrap around the back of the 'wings' of activin, blocking the type II receptor-binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activin's fingers. Complex formation imposes a novel orientation for the EGF- and Kazal-like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF-beta family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF-beta family proteins.

The Tumor Suppressor Smad4 Is Required for Transforming Growth Factor β–Induced Epithelial to Mesenchymal Transition and Bone Metastasis of Breast Cancer Cells

Transforming growth factor beta (TGF-beta) can act as suppressor and promoter of cancer progression. Intracellular Smad proteins (i.e., receptor regulated Smads and common mediator Smad4) play a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-beta, but their function in TGF-beta-induced invasion and metastasis is unclear. Here, we have investigated the role of Smad4 in a cellular and mouse model for TGF-beta-induced breast cancer progression. Consistent with its tumor suppressor function, specific silencing of Smad4 in NMuMG mammary gland epithelial cells using small hairpin RNA (shRNA)-expressing RNAi vectors strongly mitigated TGF-beta-induced growth inhibition and apoptosis. Smad4 knockdown also potently inhibited TGF-beta-induced epithelial to mesenchymal transition of NMuMG cells as measured by morphologic transformation from epithelial to fibroblast-like cells, formation of stress fibers, inhibition of E-cadherin expression, and gain of expression of various mesenchymal markers. Furthermore, we show that knockdown of Smad4 in MDA-MB-231 breast cancer cells strongly inhibited the frequency of bone metastasis in nude mice by 75% and significantly increased metastasis-free survival. Communication of MDA-MB-231 cells with the bone microenvironment, which is needed for optimal tumor cell growth and metastasis, may be affected in Smad4 knockdown cells as TGF-beta-induced expression of interleukin 11 was attenuated on Smad4 knockdown. Taken together, our results show that Smad4 plays an important role in both tumor suppression and progression of breast cancer cells.