Daizo Koinuma

TGF-β-induced epithelial-mesenchymal transition of A549 lung adenocarcinoma cells is enhanced by pro-inflammatory cytokines derived from RAW 264.7 macrophage cells

Cancer cells undergo epithelial-mesenchymal transition (EMT) during invasion and metastasis. Although transforming growth factor-β (TGF-β) and pro-inflammatory cytokines have been implicated in EMT, the underlying molecular mechanisms remain to be elucidated. Here, we studied the effects of proinflammatory cytokines derived from the mouse macrophage cell line RAW 264.7 on TGF-β-induced EMT in A549 lung cancer cells. Co-culture and treatment with conditioned medium of RAW 264.7 cells enhanced a subset of TGF-β-induced EMT phenotypes in A549 cells, including changes in cell morphology and induction of mesenchymal marker expression. These effects were increased by the treatment of RAW 264.7 cells with lipopolysaccharide, which also induced the expression of various proinflammatory cytokines, including TNF-α and IL-1β. The effects of conditioned medium of RAW 264.7 cells were partially inhibited by a TNF-α neutralizing antibody. Dehydroxy methyl epoxyquinomicin, a selective inhibitor of NFκB, partially inhibited the enhancement of fibronectin expression by TGF-β, TNF-α, and IL-1β, but not of N-cadherin expression. Effects of other pharmacological inhibitors also suggested complex regulatory mechanisms of the TGF-β-induced EMT phenotype by TNF-α stimulation. These findings provide direct evidence of the effects of RAW 264.7-derived TNF-α on TGF-β-induced EMT in A549 cells, which is transduced in part by NFκB signalling.

Arkadia amplifies TGF-β superfamily signalling through degradation of Smad7

Arkadia was originally identified as a protein that enhances signalling activity of Nodal and induces mammalian nodes during early embryogenesis; however, the mechanisms by which Arkadia affects transforming growth factor‐β (TGF‐β) superfamily signalling have not been determined. Here we show that Arkadia is widely expressed in mammalian tissues, and that it enhances both TGF‐β and bone morphogenetic protein (BMP) signalling. Arkadia physically interacts with inhibitory Smad, Smad7, and induces its poly‐ubiquitination and degradation. In contrast to Smurf1, which interacts with TGF‐β receptor complexes through Smad7 and degrades them, Arkadia fails to associate with TGF‐β receptors. In contrast to Smad7, expression of Arkadia is down‐regulated by TGF‐β. Silencing of the Arkadia gene resulted in repression of transcriptional activities induced by TGF‐β and BMP, and accumulation of the Smad7 protein. Arkadia may thus play an important role as an amplifier of TGF‐β superfamily signalling under both physiological and pathological conditions.

Chromatin Immunoprecipitation on Microarray Analysis of Smad2/3 Binding Sites Reveals Roles of ETS1 and TFAP2A in Transforming Growth Factor β Signaling

ABSTRACT The Smad2 and Smad3 (Smad2/3) proteins are principally involved in the transmission of transforming growth factor β (TGF-β) signaling from the plasma membrane to the nucleus. Many transcription factors have been shown to cooperate with the Smad2/3 proteins in regulating the transcription of target genes, enabling appropriate gene expression by cells. Here we identified 1,787 Smad2/3 binding sites in the promoter regions of over 25,500 genes by chromatin immunoprecipitation on microarray in HaCaT keratinocytes. Binding elements for the v-ets erythroblastosis virus E26 oncogene homolog (ETS) and transcription factor AP-2 (TFAP2) were significantly enriched in Smad2/3 binding sites, and knockdown of either ETS1 or TFAP2A resulted in overall alteration of TGF-β-induced transcription, suggesting general roles for ETS1 and TFAP2A in the transcription induced by TGF-β-Smad pathways. We identified novel Smad binding sites in the CDKN1A gene where Smad2/3 binding was regulated by ETS1 and TFAP2A. Moreover, we showed that small interfering RNAs for ETS1 and TFAP2A affected TGF-β-induced cytostasis. We also analyzed Smad2- or Smad3-specific target genes regulated by TGF-β and found that their specificity did not appear to be solely determined by the amounts of the Smad2/3 proteins bound to the promoters. These findings reveal novel regulatory mechanisms of Smad2/3-induced transcription and provide an essential resource for understanding their roles.

Arkadia Induces Degradation of SnoN and c-Ski to Enhance Transforming Growth Factor-β Signaling

Transforming growth factor-β (TGF-β) signaling is controlled by a variety of regulators that target either signaling receptors or activated Smad complexes. Among the negative regulators, Smad7 antagonizes TGF-β signaling mainly through targeting the signaling receptors, whereas SnoN and c-Ski repress signaling at the transcriptional level through inactivation of Smad complexes. We previously found that Arkadia is a positive regulator of TGF-β signaling that induces ubiquitin-dependent degradation of Smad7 through its C-terminal RING domain. We report here that Arkadia induces degradation of SnoN and c-Ski in addition to Smad7. Arkadia interacts with SnoN and c-Ski in their free forms as well as in the forms bound to Smad proteins, and constitutively down-regulates levels of their expression. Arkadia thus appears to effectively enhance TGF-β signaling through simultaneous down-regulation of two distinct types of negative regulators, Smad7 and SnoN/c-Ski, and may play an important role in determining the intensity of TGF-β family signaling in target cells.

TGF-β drives epithelial-mesenchymal transition through δEF1-mediated downregulation of ESRP.

Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair and cancer progression in adult tissues. We have recently shown that transforming growth factor (TGF)-β-induced EMT involves isoform switching of fibroblast growth factor receptors by alternative splicing. We performed a microarray-based analysis at single exon level to elucidate changes in splicing variants generated during TGF-β-induced EMT, and found that TGF-β induces broad alteration of splicing patterns by downregulating epithelial splicing regulatory proteins (ESRPs). This was achieved by TGF-β-mediated upregulation of δEF1 family proteins, δEF1 and SIP1. δEF1 and SIP1 each remarkably repressed ESRP2 transcription through binding to the ESRP2 promoter in NMuMG cells. Silencing of both δEF1 and SIP1, but not either alone, abolished the TGF-β-induced ESRP repression. The expression profiles of ESRPs were inversely related to those of δEF1 and SIP in human breast cancer cell lines and primary tumor specimens. Further, overexpression of ESRPs in TGF-β-treated cells resulted in restoration of the epithelial splicing profiles as well as attenuation of certain phenotypes of EMT. Therefore, δEF1 family proteins repress the expression of ESRPs to regulate alternative splicing during TGF-β-induced EMT and the progression of breast cancers.

ChIP-seq reveals cell type-specific binding patterns of BMP-specific Smads and a novel binding motif

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) and pulmonary arterial smooth muscle cells (PASMCs) are implicated in human genetic disorders. Here, we generated genome-wide maps of Smad1/5 binding sites in ECs and PASMCs. Smad1/5 preferentially bound to the region outside the promoter of known genes, and the binding was associated with target gene upregulation. Cell-selective Smad1/5 binding patterns appear to be determined mostly by cell-specific differences in baseline chromatin accessibility patterns. We identified, for the first time, a Smad1/5 binding motif in mammals, and termed GC-rich Smad binding element (GC-SBE). Several sequences in the identified GC-SBE motif had relatively weak affinity for Smad binding, and were enriched in cell type-specific Smad1/5 binding regions. We also found that both GC-SBE and the canonical SBE affect binding affinity for the Smad complex. Furthermore, we characterized EC-specific Smad1/5 target genes and found that several Notch signaling pathway-related genes were induced by BMP in ECs. Among them, a Notch ligand, JAG1 was regulated directly by Smad1/5, transactivating Notch signaling in the neighboring cells. These results provide insights into the molecular mechanism of BMP signaling and the pathogenesis of vascular lesions of certain genetic disorders, including hereditary hemorrhagic telangiectasia.

Pin1 Down-regulates Transforming Growth Factor-β (TGF-β) Signaling by Inducing Degradation of Smad Proteins

Transforming growth factor-β (TGF-β) is crucial in numerous cellular processes, such as proliferation, differentiation, migration, and apoptosis. TGF-β signaling is transduced by intracellular Smad proteins that are regulated by the ubiquitin-proteasome system. Smad ubiquitin regulatory factor 2 (Smurf2) prevents TGF-β and bone morphogenetic protein signaling by interacting with Smads and inducing their ubiquitin-mediated degradation. Here we identified Pin1, a peptidylprolyl cis-trans isomerase, as a novel protein binding Smads. Pin1 interacted with Smad2 and Smad3 but not Smad4; this interaction was enhanced by the phosphorylation of (S/T)P motifs in the Smad linker region. (S/T)P motif phosphorylation also enhanced the interaction of Smad2/3 with Smurf2. Pin1 reduced Smad2/3 protein levels in a manner dependent on its peptidyl-prolyl cis-trans isomerase activity. Knockdown of Pin1 increased the protein levels of endogenous Smad2/3. In addition, Pin1 both enhanced the interaction of Smurf2 with Smads and enhanced Smad ubiquitination. Pin1 inhibited TGF-β-induced transcription and gene expression, suggesting that Pin1 negatively regulates TGF-β signaling by down-regulating Smad2/3 protein levels via induction of Smurf2-mediated ubiquitin-proteasomal degradation.

Specific interactions between Smad proteins and AP-1 components determine TGFβ-induced breast cancer cell invasion

Deregulation of the transforming growth factor β (TGFβ) signal transduction cascade is functionally linked to cancer. In early phases, TGFβ acts as a tumor suppressor by inhibiting tumor cell proliferation, whereas in late phases, it can act as a tumor promoter by stimulating tumor cell invasion and metastasis. Smad transcriptional effectors mediate TGFβ responses, but relatively little is known about the Smad-containing complexes that are important for epithelial–mesenchymal transition and invasion. In this study, we have tested the hypothesis that specific members of the AP-1 transcription factor family determine TGFβ signaling specificity in breast cancer cell invasion. Using a 3D model of collagen-embedded spheroids of MCF10A-MII premalignant human breast cancer cells, we identified the AP-1 transcription factor components c-Jun, JunB, c-Fos and Fra1 as essential factors for TGFβ-induced invasion and found that various mesenchymal and invasion-associated TGFβ-induced genes are co-regulated by these proteins. In situ proximity ligation assays showed that TGFβ signaling not only induces complexes between Smad3 and Smad4 in the nucleus but also complexes between Smad2/3 and Fra1, whereas complexes between Smad3, c-Jun and JunB could already be detected before TGFβ stimulation. Finally, chromatin immunoprecipitations showed that c-Jun, JunB and Fra1, but not c-Fos, are required for TGFβ-induced binding of Smad2/3 to the mmp-10 and pai-1 promoters. Together these results suggest that in particular formation of Smad2/3-Fra1 complexes may reflect activation of the Smad/AP-1-dependent TGFβ-induced invasion program.

Transforming growth factor-β decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-β (TGF-β) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-β repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-β, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-β negatively contributes to maintain the CICs within the cancer.

Tumor-promoting functions of transforming growth factor-β in progression of cancer

Abstract Transforming growth factor-β (TGF-β) elicits both tumor-suppressive and tumor-promoting functions during cancer progression. Here, we describe the tumor-promoting functions of TGF-β and how these functions play a role in cancer progression. Normal epithelial cells undergo epithelial-mesenchymal transition (EMT) through the action of TGF-β, while treatment with TGF-β and fibroblast growth factor (FGF)-2 results in transdifferentiation into activated fibroblastic cells that are highly migratory, thereby facilitating cancer invasion and metastasis. TGF-β also induces EMT in tumor cells, which can be regulated by oncogenic and anti-oncogenic signals. In addition to EMT promotion, invasion and metastasis of cancer are facilitated by TGF-β through other mechanisms, such as regulation of cell survival, angiogenesis, and vascular integrity, and interaction with the tumor microenvironment. TGF-β also plays a critical role in regulating the cancer-initiating properties of certain types of cells, including glioma-initiating cells. These findings thus may be useful for establishing treatment strategies for advanced cancer by inhibiting TGF-β signaling.