The Snail signaling branch downstream of the TGF-ß/Smad3 pathway mediates Rho activation and subsequent stress fiber formation.

Cancer cells acquire malignant phenotypes through an epithelial-mesenchymal transition, which is induced by environmental factors or extracellular signaling molecules, including transforming growth factor-ß (TGF-ß). Among epithelial-mesenchymal transition-associated cell responses, cell morphological changes and cell motility are closely associated with remodeling of the actin stress fibers. Here, we examined the TGF-ß signaling pathways leading to these cell responses. Through knockdown experiments in A549 lung adenocarcinoma cells, we found that Smad3-mediated induction of Snail, but not that of Slug, is indispensable for morphological changes, stress fiber formation, and enhanced motility in cells stimulated with TGF-ß. Ectopic expression of Snail in SMAD3-knockout cells rescued the defect in morphological changes and stress fiber formation by TGF-ß, indicating that the role of Smad3 in these responses is to upregulate Snail expression. Mechanistically, Snail is required for TGF-ß-induced upregulation of Wnt5b, which in turn activates RhoA and subsequent stress fiber formation in cooperation with phosphoinositide 3-kinase. However, ectopic expression of Snail in SMAD3-knockout cells failed to rescue the defect in cell motility enhancement by TGF-ß, indicating that activation of the Smad3/Snail/Wnt5b axis is indispensable but not sufficient for enhancing cell motility; a Smad3-dependent but Snail-independent pathway to activate Rac1 is additionally required. Therefore, the Smad3-dependent pathway leading to enhanced cell motility has two branches: a Snail-dependent branch to activate RhoA and a Snail-independent branch to activate Rac1. Coordinated activation of these branches, together with activation of non-Smad signaling pathways, mediates enhanced cell motility induced by TGF-ß.

Receptor-activated transcription factors and beyond: multiple modes of Smad2/3-dependent transmission of TGF-ß signaling.

Transforming growth factor ß (TGF-ß) is a pleiotropic cytokine that is widely distributed throughout the body. Its receptor proteins, TGF-ß type I and type II receptors, are also ubiquitously expressed. Therefore, the regulation of various signaling outputs in a context-dependent manner is a critical issue in this field. Smad proteins were originally identified as signal-activated transcription factors similar to signal transducer and activator of transcription proteins. Smads are activated by serine phosphorylation mediated by intrinsic receptor dual specificity kinases of the TGF-ß family, indicating that Smads are receptor-restricted effector molecules downstream of ligands of the TGF-ß family. Smad proteins have other functions in addition to transcriptional regulation, including post-transcriptional regulation of micro-RNA processing, pre-mRNA splicing, and m6A methylation. Recent technical advances have identified a novel landscape of Smad-dependent signal transduction, including regulation of mitochondrial function without involving regulation of gene expression. Therefore, Smad proteins are receptor-activated transcription factors and also act as intracellular signaling modulators with multiple modes of function. In this review, we discuss the role of Smad proteins as receptor-activated transcription factors and beyond. We also describe the functional differences between Smad2 and Smad3, two receptor-activated Smad proteins downstream of TGF-ß, activin, myostatin, growth and differentiation factor (GDF) 11, and Nodal.

Smad2Δexon3 and Smad3 have distinct properties in signal transmission leading to TGF-ß-induced cell motility.

In mammalian cells, Smad2 and Smad3, two receptor-regulated Smad proteins, play crucial roles in the signal transmission of transforming growth factor-ß (TGF-ß) and are involved in various cell regulatory processes, including epithelial-mesenchymal transition-associated cell responses, that is, cell morphological changes, E-cadherin downregulation, stress fiber formation, and cell motility enhancement. Smad2 contains an additional exon encoding 30 amino acid residues compared with Smad3, leading to distinct Smad2 and Smad3 functional properties. Intriguingly, Smad2 also has an alternatively spliced isoform termed Smad2Δexon3 (also known as Smad2ß) lacking the additional exon and behaving similarly to Smad3. However, Smad2Δexon3 and Smad3 signaling properties have not yet been compared in detail. In this study, we reveal that Smad2Δexon3 rescues multiple TGF-ß-induced in vitro cellular responses that would become defective upon SMAD3 KO but does not rescue cell motility enhancement. Using Smad2Δexon3/Smad3 chimeric proteins, we identified that residues Arg-104 and Asn-210 in Smad3, which are not conserved in Smad2Δexon3, are key for TGF-ß-enhanced cell motility. Moreover, we discovered that Smad2Δexon3 fails to rescue the enhanced cell motility as it does not mediate TGF-ß signals to downregulate transcription of ARHGAP24, a GTPase-activating protein that targets Rac1. This study reports for the first time distinct signaling properties of Smad2Δexon3 and Smad3.

TGF-ß-induced cell motility requires downregulation of ARHGAPs to sustain Rac1 activity.

Transforming growth factor-ß (TGF-ß) signaling promotes cancer progression. In particular, the epithelial-mesenchymal transition (EMT) induced by TGF-ß is considered crucial to the malignant phenotype of cancer cells. Here, we report that the EMT-associated cellular responses induced by TGF-ß are mediated by distinct signaling pathways that diverge at Smad3. By expressing chimeric Smad1/Smad3 proteins in SMAD3 knockout A549 cells, we found that the ß4 region in the Smad3 MH1 domain is essential for TGF-ß-induced cell motility, but is not essential for other EMT-associated responses including epithelial marker downregulation. TGF-ß was previously reported to enhance cell motility by activating Rac1 via phosphoinositide 3-kinase. Intriguingly, TGF-ß-dependent signaling mediated by Smad3's ß4 region causes the downregulation of multiple mRNAs that encode GTPase activating proteins that target Rac1 (ARHGAPs), thereby attenuating Rac1 inactivation. Therefore, two independent pathways downstream of TGF-ß type I receptor contribute cooperatively to sustained Rac1 activation, thereby leading to enhanced cell motility.

Simple digital system for the stabilization of holographic recordings of a slow grating in photorefractive materials.

We report on a digital system for the stabilization of an interference pattern of light fringes. This system uses a Raspberry Pi computer to operate a continuously stabilized setup and, because of the particular features of this stabilization setup, it is possible to record slow gratings in photorefractive materials. Our system proved to be effective, less expensive, and easy to operate, compared to the frequently employed setup with a lock-in amplifier, as it does not require specific equipment and/or specialized personnel.

Dissociation of the AhR/ARNT complex by TGF-ß/Smad signaling represses CYP1A1 gene expression and inhibits benze[a]pyrene-mediated cytotoxicity.

Cytochrome P450 1A1 (CYP1A1) catalyzes the metabolic activation of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and is transcriptionally regulated by the aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) complex upon exposure to PAHs. Accordingly, inhibition of CYP1A1 expression reduces production of carcinogens from PAHs. Although transcription of the CYP1A1 gene is known to be repressed by transforming growth factor-ß (TGF-ß), how TGF-ß signaling is involved in the suppression of CYP1A1 gene expression has yet to be clarified. In this study, using mammalian cell lines, along with shRNA-mediated gene silencing, CRISPR/Cas9-based genome editing, and reporter gene and quantitative RT-PCR assays, we found that TGF-ß signaling dissociates the B[a]P-mediated AhR/ARNT heteromeric complex. Among the examined Smads, Smad family member 3 (Smad3) strongly interacted with both AhR and ARNT via its MH2 domain. Moreover, hypoxia-inducible factor 1α (HIF-1α), which is stabilized upon TGF-ß stimulation, also inhibited AhR/ARNT complex formation in the presence of B[a]P. Thus, TGF-ß signaling negatively regulated the transcription of the CYP1A1 gene in at least two different ways. Of note, TGF-ß abrogated DNA damage in B[a]P-exposed cells. We therefore conclude that TGF-ß may protect cells against carcinogenesis because it inhibits CYP1A1-mediated metabolic activation of PAHs as part of its anti-tumorigenic activities.

ZEB1 and oncogenic Ras constitute a regulatory switch for stimulus-dependent E-cadherin downregulation.

E-cadherin, an epithelial cell-specific cell adhesion molecule, has both promoting and suppressing effects on tumor invasion and metastasis. It is often downregulated during cancer progression through gene deletion/mutation, transcriptional repression, or epigenetic silencing. We describe a novel regulatory switch to induce stimulus-dependent downregulation of mRNA encoding E-cadherin (CDH1 mRNA) in KRAS-mutated cancer cells. The regulatory switch consists of ZEB1 and oncogenic K-Ras, does not target the promoter region of CDH1, and requires an external cue to temporally downregulate E-cadherin expression. Its repressive effect is maintained as long as the external stimulus continues and is attenuated with cessation of the stimulus. Contextual external cues that turn this regulatory switch on include activation of protein kinase C or fibroblast growth factor signaling. The mode of action is distinct from that of EPCAM repression by ZEB1, which does not require an external cue. Thus, KRAS-mutated cancer cells acquire a novel mode of regulating E-cadherin expression depending on ZEB1, which could contribute to phenotypic plasticity of cancer cells during malignant progression.

Structural basis for inhibitory effects of Smad7 on TGF-ß family signaling.

Smad6 and Smad7 are classified as inhibitory Smads (I-Smads). They are crucial in the fine-tuning of signals by cytokines of the transforming growth factor-ß (TGF-ß) family. They are negative feedback regulators and principally target the activated type I receptors as well as the activated Smad complexes, but with distinct specificities. Smad7 inhibits Smad signaling from all seven type I receptors of the TGF-ß family, whereas Smad6 preferentially inhibits Smad signaling from the bone morphogenetic protein (BMP) type I receptors, BMPR1A and BMPR1B. The target specificities are attributed to the C-terminal MH2 domain. Notably, Smad7 utilizes two alternative molecular surfaces for its inhibitory function against type I receptors. One is a basic groove composed of the first α-helix and the L3 loop, a structure that is shared with Smad6 and receptor-regulated Smads (R-Smads). The other is a three-finger-like structure (consisting of residues 331-361, 379-387, and the L3 loop) that is unique to Smad7. The underlying structural basis remains to be elucidated in detail. Here, we report the crystal structure of the MH2 domain of mouse Smad7 at 1.9 Å resolution. The three-finger-like structure is stabilized by a network of hydrogen bonds between residues 331-361 and 379-387, thus forming a molecular surface unique to Smad7. Furthermore, we discuss how Smad7 antagonizes the activated Smad complexes composed of R-Smad and Smad4, a common partner Smad.

A comparative analysis of Smad-responsive motifs identifies multiple regulatory inputs for TGF-ß transcriptional activation.

Smad proteins are transcriptional regulators activated by TGF-ß. They are known to bind to two distinct Smad-responsive motifs, namely the Smad-binding element (SBE) (5'-GTCTAGAC-3') and CAGA motifs (5'-AGCCAGACA-3' or 5'-TGTCTGGCT-3'). However, the mechanisms by which these motifs promote Smad activity are not fully elucidated. In this study, we performed DNA CASTing, binding assays, ChIP sequencing, and quantitative RT-PCR to dissect the details of Smad binding and function of the SBE and CAGA motifs. We observed a preference for Smad3 to bind CAGA motifs and Smad4 to bind SBE, and that either one SBE or a triple-CAGA motif forms a cis-acting functional half-unit for Smad-dependent transcription activation; combining two half-units allows efficient activation. Unexpectedly, the extent of Smad binding did not directly correlate with the abilities of Smad-binding sequences to induce gene expression. We found that Smad proteins are more tolerant of single bp mutations in the context of the CAGA motifs, with any mutation in the SBE disrupting function. CAGA and CAGA-like motifs but not SBE are widely distributed among stimulus-dependent Smad2/3-binding sites in normal murine mammary gland epithelial cells, and the number of CAGA and CAGA-like motifs correlates with fold-induction of target gene expression by TGF-ß. These data, demonstrating Smad responsiveness can be tuned by both sequence and number of repeats, provide a compelling explanation for why CAGA motifs are predominantly used for Smad-dependent transcription activation in vivo.

Lipidome-based rapid diagnosis with machine learning for detection of TGF-ß signalling activated area in head and neck cancer.

BACKGROUND: Several pro-oncogenic signals, including transforming growth factor beta (TGF-ß) signalling from tumour microenvironment, generate intratumoural phenotypic heterogeneity and result in tumour progression and treatment failure. However, the precise diagnosis for tumour areas containing subclones with cytokine-induced malignant properties remains clinically challenging. METHODS: We established a rapid diagnostic system based on the combination of probe electrospray ionisation-mass spectrometry (PESI-MS) and machine learning without the aid of immunohistological and biochemical procedures to identify tumour areas with heterogeneous TGF-ß signalling status in head and neck squamous cell carcinoma (HNSCC). A total of 240 and 90 mass spectra were obtained from TGF-ß-unstimulated and -stimulated HNSCC cells, respectively, by PESI-MS and were used for the construction of a diagnostic system based on lipidome. RESULTS: This discriminant algorithm achieved 98.79% accuracy in discrimination of TGF-ß1-stimulated cells from untreated cells. In clinical human HNSCC tissues, this approach achieved determination of tumour areas with activated TGF-ß signalling as efficiently as a conventional histopathological assessment using phosphorylated-SMAD2 staining. Furthermore, several altered peaks on mass spectra were identified as phosphatidylcholine species in TGF-ß-stimulated HNSCC cells. CONCLUSIONS: This diagnostic system combined with PESI-MS and machine learning encourages us to clinically diagnose intratumoural phenotypic heterogeneity induced by TGF-ß.

Smad3-STAT3 crosstalk in pathophysiological contexts.

Smad3 and STAT3 are intracellular molecules that transmit signals from plasma membrane receptors to the nucleus. Smad3 operates downstream of growth/differentiation factors that utilize activin receptor-like kinase (ALK)-4, 5, or 7, such as transforming growth factor-ß (TGF-ß), activin, and myostatin. STAT3 principally functions downstream of cytokines that exert their effects via gp130 and Janus family kinases, including interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and oncostatin M. Accumulating evidence indicates that Smad3 and STAT3 engage in crosstalk in a highly context-dependent fashion, cooperating in some conditions while acting antagonistically each other in others. Here, we review the crosstalk between Smad3 and STAT3 in various biological contexts, including early tumorigenesis, epithelial-mesenchymal transition, fibrosis, and T cell differentiation.

Ets1 and ESE1 reciprocally regulate expression of ZEB1/ZEB2, dependent on ERK1/2 activity, in breast cancer cells.

The epithelial-mesenchymal transition (EMT) is a crucial morphological event that occurs during progression of epithelial tumors. We reported previously that levels of the δ-crystallin/E2-box factor 1 (δEF1) family proteins (Zinc finger E-box binding homeobox 1 [ZEB1]/δEF1 and ZEB2/ Smad-interacting protein 1), key regulators of the EMT, are positively correlated with EMT phenotypes and aggressiveness of breast cancer. Here, we show that Ets1 induces ZEB expression and activates the ZEB1 promoter, independently of its threonine 38 phosphorylation status. In the basal-like subtype of breast cancer cells, siRNAs targeting Ets1 repressed expression of ZEBs and partially restored their epithelial phenotypes and sensitivity to antitumor drugs. Epithelium-specific ETS transcription factor 1 (ESE1), a member of the Ets transcription factor family, was originally characterized as being expressed in an epithelial-restricted pattern, placing it within the epithelium-specific ETS subfamily. ESE1, highly expressed in the luminal subtype of breast cancer cells, was repressed by activation of the MEK-ERK pathway, resulting in induction of ZEBs through Ets1 upregulation. Conversely, Ets1, highly expressed in the basal-like subtype, was repressed by inactivation of MEK-ERK pathway, resulting in reduction of ZEBs through ESE1 upregulation. These findings suggest that ESE1 and Ets1, whose expressions are reciprocally regulated by the MEK-ERK pathway, define the EMT phenotype through controlling expression of ZEBs in each subtype of breast cancer cells.

Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma.

Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-ß (TGF-ß) signaling pathways play crucial regulatory roles in cancer-associated inflammation. However, mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-ß signaling, c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827 cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance as repressing transcription of Smad3, whereas TGF-ß enhanced gefitinib sensitivity as activating transcription of Smad3 in HCC827 cells with gefitinib-sensitizing EGFR mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4-induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our results suggest that derepression of Smad3 can be a therapeutic strategy to prevent gefitinib-resistance in NSCLCs with gefitinib-sensitizing EGFR mutation.

Dual Roles for Epithelial Splicing Regulatory Proteins 1 (ESRP1) and 2 (ESRP2) in Cancer Progression.

Epithelial splicing regulatory protein 1 (ESRP1) and 2 (ESRP2) are members of the hnRNP family of RNA binding proteins that regulate alternative splicing events associated with epithelial phenotypes. These proteins play crucial roles during organogenesis, including craniofacial and epidermal development as well as branching morphogenesis in the lungs and salivary glands. Recent reports have also addressed their roles during cancer progression. Expression of ESRP proteins is low in normal epithelium but upregulated in carcinoma in situ and advanced carcinomas. Intriguingly, they are downregulated in invasive fronts. The plastic nature of ESRP expression suggests dual roles for them in cancer progression. Consistently, it has been shown that ESRPs suppress motility and anchorage-independent growth of cancer cells while supporting cell survival by enhancing resistance to reactive oxygen species. Regulatory circuits that fine-tune ESRP gene expression have recently emerged. Here, we summarize recent findings on the molecular mechanisms by which ESRPs exert positive as well as negative effects on cancer progression.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition.

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Impaired NFKBIE gene function decreases cellular uptake of methotrexate by down-regulating SLC19A1 expression in a human rheumatoid arthritis cell line.

OBJECTIVE: A non-synonymous single nucleotide polymorphism (nsSNP, rs2233434, Val194Ala) in the NFKBIE (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon) gene is known to be a rheumatoid arthritis (RA) susceptibility polymorphism in the Japanese RA population and could be closely associated with nuclear factor kappaB (NF-κB) activity. Inflammation caused by RA is sometimes associated with changes in expression levels of MTX (methotrexate) pathway-related genes. It is of interest to examine whether the NFKBIE gene had any influences on the mode of MTX action. METHODS: Both knockdown of NFKBIE gene expression and overexpression of wild-type NFKBIE and Val194Ala mutation were performed. A transfected human RA synovial cell line was cultured and then gene expressions in the MTX pathway were measured. In addition, we measured the uptake and efflux of MTX derivatives under the NFKBIE knockdown condition. RESULTS: Knockdown of NFKBIE reduced the mRNA for SLC19A1, a main MTX membrane transporter, and the intracellular accumulations of MTX derivatives. Moreover, our experiments also confirmed that overexpression of Val194Ala mutant NFKBIE decreased the SLC19A1 mRNA when compared to that of wild-type NFKBIE. CONCLUSIONS: We suggest that the impairment of NFKBIE gene function can reduce the uptake of MTX into cells, suggesting that the gene is an important factor for the RA outcome.

Epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) suppress cancer cell motility via different mechanisms.

ESRP1 (epithelial splicing regulatory protein 1) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells, and both are down-regulated during the epithelial-mesenchymal transition. However, little is known about their expression and functions during carcinogenesis. In this study, we found that expression of both ESRP1 and ESRP2 is plastic: during oral squamous cell carcinogenesis, these proteins are up-regulated relative to their levels in normal epithelium but down-regulated in invasive fronts. Importantly, ESRP1 and ESRP2 are re-expressed in the lymph nodes, where carcinoma cells metastasize and colonize. In head and neck carcinoma cell lines, ESRP1 and ESRP2 suppress cancer cell motility through distinct mechanisms: knockdown of ESRP1 affects the dynamics of the actin cytoskeleton through induction of Rac1b, whereas knockdown of ESRP2 attenuates cell-cell adhesion through increased expression of epithelial-mesenchymal transition-associated transcription factors. Down-regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells.

TGF-ß regulates isoform switching of FGF receptors and epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair, and cancer progression in adult tissues. Here, we demonstrate that transforming growth factor (TGF)-ß induced EMT and that long-term exposure to TGF-ß elicited the epithelial-myofibroblastic transition (EMyoT) by inactivating the MEK-Erk pathway. During the EMT process, TGF-ß induced isoform switching of fibroblast growth factor (FGF) receptors, causing the cells to become sensitive to FGF-2. Addition of FGF-2 to TGF-ß-treated cells perturbed EMyoT by reactivating the MEK-Erk pathway and subsequently enhanced EMT through the formation of MEK-Erk-dependent complexes of the transcription factor δEF1/ZEB1 with the transcriptional corepressor CtBP1. Consequently, normal epithelial cells that have undergone EMT as a result of combined TGF-ß and FGF-2 stimulation promoted the invasion of cancer cells. Thus, TGF-ß and FGF-2 may cooperate with each other and may regulate EMT of various kinds of cells in cancer microenvironment during cancer progression.

Oligodendrocyte transcription factor 1 (Olig1) is a Smad cofactor involved in cell motility induced by transforming growth factor-ß.

Transforming growth factor (TGF)-ß plays crucial roles in embryonic development and adult tissue homeostasis by eliciting various cellular responses in target cells. TGF-ß signaling is principally mediated through receptor-activated Smad proteins, which regulate expression of target genes in cooperation with other DNA-binding transcription factors (Smad cofactors). In this study, we found that the basic helix-loop-helix transcription factor Olig1 is a Smad cofactor involved in TGF-ß-induced cell motility. Knockdown of Olig1 attenuated TGF-ß-induced cell motility in chamber migration and wound healing assays. In contrast, Olig1 knockdown had no effect on bone morphogenetic protein-induced cell motility, TGF-ß-induced cytostasis, or epithelial-mesenchymal transition. Furthermore, we observed that cooperation of Smad2/3 with Olig1 is regulated by a peptidyl-prolyl cis/trans-isomerase, Pin1. TGF-ß-induced cell motility, induction of Olig1-regulated genes, and physical interaction between Smad2/3 and Olig1 were all inhibited after knockdown of Pin1, indicating a novel mode of regulation of Smad signaling. We also found that Olig1 interacts with the L3 loop of Smad3. Using a synthetic peptide corresponding to the L3 loop of Smad3, we succeeded in selectively inhibiting TGF-ß-induced cell motility. These findings may lead to a new strategy for selective regulation of TGF-ß-induced cellular responses.

Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.