TGF-β Signaling Molecules Research Articles

Current Perspectives in Cardiac Laterality.

The heart is the first organ to break symmetry in the developing embryo and onset of dextral looping is the first indication of this event. Looping is a complex process that progresses concomitantly to cardiac chamber differentiation and ultimately leads to the alignment of the cardiac regions in their final topology. Generation of cardiac asymmetry is crucial to ensuring proper form and consequent functionality of the heart, and therefore it is a highly regulated process. It has long been known that molecular left/right signals originate far before morphological asymmetry and therefore can direct it. The use of several animal models has led to the characterization of a complex regulatory network, which invariably converges on the Tgf-β signaling molecule Nodal and its downstream target, the homeobox transcription factor Pitx2. Here, we review current data on the cellular and molecular bases of cardiac looping and laterality, and discuss the contribution of Nodal and Pitx2 to these processes. A special emphasis will be given to the morphogenetic role of Pitx2 and to its modulation of transcriptional and functional properties, which have also linked laterality to atrial fibrillation.

An investigation of crosstalk between Wnt/β-catenin and transforming growth factor-β signaling in androgenetic alopecia

Background:Wnt and transforming growth factor-β (TGF-β) signaling pathways are known to be involved in the pathogenesis of androgenetic alopecia (AGA). However, the way that Wnt and TGF-β signaling is altered in patients with AGA and whether there exists a crosstalk between them in pathogenetic process of AGA remain unclear.Objectives:To investigate the expression of Wnt and TGF-β signaling and the crosstalk between these 2 signaling pathways in AGA.Methods:Fifteen male patients with AGA were recruited for our research. Fifteen scalp specimens of the balding were collected from frontal areas, and 9 nonbalding were collected from occipital areas. We analyzed the expression and activation of downstream Wnt and TGF-β signaling molecules in both balding and nonbalding hair follicles isolated from scalp specimens. Furthermore, we evaluated the activation of Wnt and TGF-β signaling after either of them was blocked with the inhibitor in balding and nonbalding dermal papilla (DP) cells.Results:Compared with the nonbalding counterparts, the mRNA level of Wnt10a and LEF1 was decreased. But TβRI and TβRII, and the protein expression of TGF-β1 was elevated in balding hair follicles. To investigate the crosstalk between Wnt and TGF-β signaling, we used SB431542 to inhibit the TGF-β signaling in balding DP cells and found that SB431542 significantly attenuated the phosphorylation of Smad2 and Akt. However, the mRNA level of Wnt10a, LEF1, and the nuclear translocation of β-catenin was increased. On the other hand, we suppressed the Wnt signaling by XAV939 in nonbalding DP cells, which displayed that the level of β-catenin and LEF1 was significantly inhibited; however, the level of active TGF-β1 and the phosphorylation of Smad2 and Akt were up-regulated.Conclusions:These data indicate that crosstalk between Wnt/β-catenin and TGF-β signaling pathways may exist as one of the important mechanisms contributing to AGA.

Caspase-3/7-mediated Cleavage of β2-spectrin is Required for Acetaminophen-induced Liver Damage.

The ubiquitously expressed β2-spectrin (β2SP, SPTBN1) is the most common non-erythrocytic member of the β-spectrin gene family. Loss of β2-spectrin leads to defects in liver development, and its haploinsufficiency spontaneously leads to chronic liver disease and the eventual development of hepatocellular cancer. However, the specific role of β2-spectrin in liver homeostasis remains to be elucidated. Here, we reported that β2-spectrin was cleaved by caspase-3/7 upon treatment with acetaminophen which is the main cause of acute liver injury. Blockage of β2-spectrin cleavage robustly attenuated β2-spectrin-specific functions, including regulation of the cell cycle, apoptosis, and transcription. Cleaved fragments of β2-spectrin were physiologically active, and the N- and C-terminal fragments retained discrete interaction partners and activity in transcriptional regulation and apoptosis, respectively. Cleavage of β2-spectrin facilitated the redistribution of the resulting fragments under conditions of liver damage induced by acetaminophen. In contrast, downregulation of β2-spectrin led to resistance to acetaminophen-induced cytotoxicity, and its insufficiency in the liver promoted suppression of acetaminophen-induced liver damage and enhancement of liver regeneration. Conclusions: β2-Spectrin, a TGF-β mediator and signaling molecule, is cleaved and activated by caspase-3/7, consequently enhancing apoptosis and transcriptional control to determine cell fate upon liver damage. These findings have extended our knowledge on the spectrum of β2-spectrin functions from a scaffolding protein to a target and transmitter of TGF-β in liver damage.

Dally Proteoglycan Mediates the Autonomous and Nonautonomous Effects on Tissue Growth Caused by Activation of the PI3K and TOR Pathways.

How cells acquiring mutations in tumor suppressor genes outcompete neighboring wild-type cells is poorly understood. The phosphatidylinositol 3-kinase (PI3K)–phosphatase with tensin homology (PTEN) and tuberous sclerosis complex (TSC)-target of rapamycin (TOR) pathways are frequently activated in human cancer, and this activation is often causative of tumorigenesis. We utilized the Gal4-UAS system in Drosophila imaginal primordia, highly proliferative and growing tissues, to analyze the impact of restricted activation of these pathways on neighboring wild-type cell populations. Activation of these pathways leads to an autonomous induction of tissue overgrowth and to a remarkable nonautonomous reduction in growth and proliferation rates of adjacent cell populations. This nonautonomous response occurs independently of where these pathways are activated, is functional all throughout development, takes place across compartments, and is distinct from cell competition. The observed autonomous and nonautonomous effects on tissue growth rely on the up-regulation of the proteoglycan Dally, a major element involved in modulating the spreading, stability, and activity of the growth promoting Decapentaplegic (Dpp)/transforming growth factor β(TGF-β) signaling molecule. Our findings indicate that a reduction in the amount of available growth factors contributes to the outcompetition of wild-type cells by overgrowing cell populations. During normal development, the PI3K/PTEN and TSC/TOR pathways play a major role in sensing nutrient availability and modulating the final size of any developing organ. We present evidence that Dally also contributes to integrating nutrient sensing and organ scaling, the fitting of pattern to size.

Inhibitory effects of Berberine on the migratory and invasive abilities of cancer cells

The Epithelial-to-Mesenchymal Transition (EMT) is the complex program that involve in oncogenic pathways regulating in migratory and invasion of cancer cells, angiogenesis, and metastasis. As such, targeting the critical EMT inducers or EMT pathways represents an important therapeutic strategy for preventing or treating cancer cells metastasis. Berberine, an isoquinoline alkaloid traditional herbal medicine with no organ toxicity. The increasing reports indicated that berberine played anti-angiogenesis, anti-invasion, and anti-metastasis roles in different human cancer cells. However, there is still unknown about the inhibitory effect of berberine on invasive behavior and EMT of prostate cancer cells. Our study shows for the first time to indicate important role played by berberine, in repressing the metastatic process and the invasive ability of prostate cancer cells. We showed that the migratory and invasive abilities of prostate cancer cells were inhibited by berberine through inhibition of TGF-β signaling. Among the TGF-β signaling molecules were inhibited by berberine, high BMP7 and NODAL gene expressions of metastatic prostate cancer tissues were associated with shorter survival of prostate cancer patients and provide potential therapeutic interventions.

The subcellular compartmentalization of TGFβ-RII and the dynamics of endosomal formation during the signaling events: An in vivo study on rat mesothelial cells

We previously showed that intraperitoneal administration of Freund's adjuvant treatment resulted in acute peritonitis and TGF-β was found to be one of the main organizers of the subsequent EMT in mesothelial cells. In the present study, we investigated whether TGF-β signaling molecules are present in mesothelial cells and how their compartmentalization pattern changes with the dynamics of inflammatory events in vivo. In addition, we tried to evaluate the turnover of endosomal compartments concomitant with the internalization of signaling molecules and examine whether caveola-mediated internalization might play a role in the termination of TGF-β signaling.Using immunocytochemical approach, we could detect TβRII in EEA1 positive compartments and as the inflammation progressed, at D3, the receptor appeared in caveolin-1 positive intracellular structures as well. The latter event was accompanied by the appearance of negative regulatory protein, Smad7 in caveolae. We also found EEA1 and caveolin-1 double positive vesicular structures that were corresponded to forming MVBs affirmed by our immuno-electron microscopical results. Fine structural, morphometric and immunoblot analysis proved that Cd63 positive multivesicular body (MVB) formation was significantly increased by D3 and the IP results confirmed that TβRII as well as caveolin-1 were strongly associated with these endosomal compartments at this time. In contrast, by the termination of inflammation, by D5, caveolin-1 was found to be associated with late endosomal marker, Rab7 and entirely degraded from the system.Despite the limitations of an in vivo system, our results provide both morphological and biochemical data about the endosomal compartments involved in the internalization of TβRII upon inflammatory stimuli. Furthermore, our study implies the possible role of caveola-mediated endocytosis in the attenuation of TGF-β signaling and highlight the significance of endosomal compartments via which caveolae might meet the classical endocytic pathway under in vivo inflammatory conditions.

Expression of Airway Remodeling Proteins in Mast Cell Activated by TGF-β Released in OVA-Induced Allergic Responses and Their Inhibition by Low-Dose Irradiation or 8-oxo-dG

Allergic asthma is characterized by chronic airway remodeling, which is associated with the expression of extracellular matrix proteins (ECM) by TGF-β. However, to date there are no reports demonstrating that structural proteins are directly expressed in mast cells. This study aimed to investigate whether ECM proteins are expressed in mast cells activated with antigen/antibody reaction, and whether the resolution effects of irradiation or 8-oxo-dG may contribute to allergic asthma prevention. Bone marrow-derived mast cells (BMMCs) were activated with DNP-HSA/anti-DNP IgE antibody (act-BMMCs). C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) to induce allergic asthma. Mice were treated orally with 8-oxo-dG or exposed to whole body irradiation (using (137)Cs gamma ray at a dose of 0.5 Gy) for three consecutive days 24 h after OVA challenge. Expression of extracellular matrix (ECM) proteins, TGF-β signaling molecules and NF-κB/AP-1 was determined in the BMMCs, bronchoalveolar lavage (BAL) cells or lung tissues using Western blot, polymerase chain reaction (PCR) and electrophoretic mobility shift assay (EMSA), respectively. Act-BMMCs increased expression of ECM proteins, TGF-β/TGF-β receptor I, TGF-β signaling molecules and cytokines; and increased both NF-κB and AP-1 activity. In addition, the population of mast cells; expression of mast cell markers, TGF-β signaling molecules, ECM proteins/amounts; OVA-specific serum IgE level; numbers of goblet cells; airway hyperresponsiveness; cytokines/chemokines were increased in BAL cells and lung tissues of OVA-challenged mice. All of the above end points were reduced by irradiation or 8-oxo-dG in vitro and in vivo, respectively. The data suggest that mast cells induce expression of ECM proteins through TGF-β produced in inflammatory cells of OVA mice and that post treatment of irradiation or 8-oxo-dG after OVA-challenge may reduce airway remodeling through down-regulating mast cell re-activation by TGF-β/Smad signals.

Characterization of Dextran Sodium Sulfate-Induced Inflammation and Colonic Tumorigenesis in Smad3−/− Mice with Dysregulated TGFβ

There are few mouse models that adequately mimic large bowel cancer in humans or the gastrointestinal inflammation which frequently precedes it. Dextran sodium sulphate (DSS)-induces colitis in many animal models and has been used in combination with the carcinogen azoxymethane (AOM) to induce cancer in mice. Smad3 −/− mice are deficient in the transforming growth factor beta (TGFβ) signaling molecule, SMAD3, resulting in dysregulation of the cellular pathway most commonly affected in human colorectal cancer, and develop inflammation-associated colon cancer. Previous studies have shown a requirement for a bacterial trigger for the colitis and colon cancer phenotype in Smad3−/− mice. Studies presented here in Smad3−/− mice detail disease induction with DSS, without the use of AOM, and show a) Smad3 −/− mice develop a spectrum of lesions ranging from acute and chronic colitis, crypt herniation, repair, dysplasia, adenomatous polyps, disseminated peritoneal adenomucinosis, adenocarcinoma, mucinous adenocarcinoma (MAC) and squamous metaplasia; b) the colon lesions have variable galactin-3 (Mac2) staining c) increased DSS concentration and duration of exposure leads to increased severity of colonic lesions; d) heterozygosity of SMAD3 does not confer increased susceptibility to DSS-induced disease and e) disease is partially controlled by the presence of T and B cells as Smad3 −/− Rag2 −/− double knock out (DKO) mice develop a more severe disease phenotype. DSS-induced disease in Smad3 −/− mice may be a useful animal model to study not only inflammation-driven MAC but other human diseases such as colitis cystica profunda (CCP) and pseudomyxomatous peritonei (PMP).

Smad4-Irf6 genetic interaction and TGFβ-mediated IRF6 signaling cascade are crucial for palatal fusion in mice

Cleft palate is one of the most common human birth defects and is associated with multiple genetic and environmental risk factors. Although mutations in the genes encoding transforming growth factor beta (TGFβ) signaling molecules and interferon regulatory factor 6 (Irf6) have been identified as genetic risk factors for cleft palate, little is known about the relationship between TGFβ signaling and IRF6 activity during palate formation. Here, we show that TGFβ signaling regulates expression of Irf6 and the fate of the medial edge epithelium (MEE) during palatal fusion in mice. Haploinsufficiency of Irf6 in mice with basal epithelial-specific deletion of the TGFβ signaling mediator Smad4 (Smad4(fl/fl);K14-Cre;Irf6(+/R84C)) results in compromised p21 expression and MEE persistence, similar to observations in Tgfbr2(fl/fl);K14-Cre mice, although the secondary palate of Irf6(+/R84C) and Smad4(fl/fl);K14-Cre mice form normally. Furthermore, Smad4(fl/fl);K14-Cre;Irf6(+/R84C) mice show extra digits that are consistent with abnormal toe and nail phenotypes in individuals with Van der Woude and popliteal pterygium syndromes, suggesting that the TGFβ/SMAD4/IRF6 signaling cascade might be a well-conserved mechanism in regulating multiple organogenesis. Strikingly, overexpression of Irf6 rescued p21 expression and MEE degeneration in Tgfbr2(fl/fl);K14-Cre mice. Thus, IRF6 and SMAD4 synergistically regulate the fate of the MEE, and TGFβ-mediated Irf6 activity is responsible for MEE degeneration during palatal fusion in mice.

Muscle growth in teleost fish is regulated by factors utilizing the activin II B receptor

The activin type IIB receptor (Acvr2b) is the cell surface receptor for multiple transforming growth factor β (TGF-β) superfamily ligands, several of which regulate muscle growth in mammals. To investigate the role of the Acvr2b signaling pathway in the growth and development of skeletal muscle in teleost fish, transgenic rainbow trout (RBT; Oncorhynchus mykiss) expressing a truncated form of the acvr2b-2a (acvr2b(▵)) in muscle tissue were produced. High levels of acvr2b(▵) expression were detected in the majority of P1 transgenic fish. Transgenic P1 trout developed enhanced, localized musculature in both the epaxial and hypaxial regions (dubbed 'six pack'). The F1 transgenic offspring did not exhibit localized muscle growth, but rather developed a uniform body morphology with greater girth, condition factor and increased muscle fiber hypertrophy. There was a high degree of variation in the mass of both P1 and F1 transgenic fish, with several fish of each generation exhibiting enhanced growth compared with other transgenic and control siblings. The 'six pack' phenotype observed in P1 transgenic RBT overexpressing acvr2b(▵) and the presence of F1 individuals with altered muscle morphology provides compelling evidence for the importance of TGF-β signaling molecules in regulating muscle growth in teleost fish.

MiR-181a promotes osteoblastic differentiation through repression of TGF-β signaling molecules

Osteoblastic differentiation is controlled by complex interplay of several signaling pathways and associated key transcription factors, as well as by microRNAs (miRNAs). In our current study, we found miR-181a to be highly upregulated during BMP induced osteoblastic differentiation of C2C12 and MC3T3 cells. Overexpression of miR-181a led to upregulation of key markers of osteoblastic differentiation as well as enhanced ALP levels and Alizarin red staining, indicating the importance of this miRNA for osteoblastic differentiation. Further, we show that miR-181 isoforms (181a, 181b, 181c) are expressed during different stages of mouse calvarial and tibial development, implying their role in both endochondral and intramembranous ossification. We found several direct and indirect targets of miR-181a to be downregulated by global mRNA expression profiling. Our results demonstrate that miR-181a promotes osteoblastic differentiation via repression of TGF-β signaling molecules by targeting the negative regulator of osteoblastic differentiation Tgfbi (Tgf-beta induced) and TβR-I/Alk5 (TGF-β type I receptor). Furthermore, our findings suggest that Rgs4 and Gata6 are direct targets of miR-181a. Taken together, we provide evidence for a crucial functional link between a specific miRNA, miR-181a and osteoblastic differentiation.

Regulation of mesenchymal stem cell chondrogenesis by glucose through protein kinase C/transforming growth factor signaling

Effective induction of human mesenchymal stem cell (hMSC) differentiation for regenerative medicine applications remains a great challenge. While much research has studied hMSC activity during differentiation, it is unclear whether pre-differentiation culture can modulate differentiation capacity. We investigate the effect of glucose concentration in pre-differentiation/expansion culture on modulating chondrogenic capacity of hMSCs, and explore the underlying molecular mechanism. The extent of chondrogenesis of hMSCs previously cultured with different concentrations of glucose was evaluated. Transforming growth factor-beta (TGF-β) signaling molecules and protein kinase C (PKC) were analyzed to identify the role of these molecules in the regulation of glucose on chondrogenesis. In addition, hMSCs in high-glucose expansion culture were treated with the PKC inhibitor to modulate the activity of PKC and TGF-β signaling molecules. High-glucose maintained hMSCs were less chondrogenic than low-glucose maintained cells upon receiving differentiation signals. Interestingly, we found that high-glucose culture increased the phosphorylation of PKC and expression of type II TGF-β receptor (TGFβRII) in pre-differentiation hMSCs. However, low-glucose maintained hMSCs became more responsive to chondrogenic induction with increased PKC activation and TGFβRII expression than high-glucose maintained hMSCs during differentiation. Inhibiting the PKC activity of high-glucose maintained hMSCs during expansion culture upregulated the TGFβRII expression of chondrogenic cell pellets, and enhanced chondrogenesis. Our findings demonstrate the effect of glucose concentration on regulating the chondrogenic capability of pre-differentiation hMSCs, and provide insight into the mechanism of how glucose concentration regulates PKC and TGF-β signaling molecules to prime pre-differentiation hMSCs for subsequent chondrogenesis.

Role of TGF-β in Melanoma

Human malignant melanoma is highly resistant to chemotherapy and current immunotherapeutic approaches induce long term remission only in the minority of patients. The transforming growth factor-β (TGF-β) has attracted much attention as a therapeutic target because it plays an important and pleiotropic role in melanoma progression. TGF-β is a multifunctional cytokine involved in the regulation of many cellular processes including cell proliferation, differentiation and survival. Resistance to the growth inhibitory effects of TGF-β without alterations of TGF-β signaling molecules is characteristic of cutaneous melanoma. Melanoma produces increasing amounts of TGF-β with disease progression, inhibiting immune responses and providing an optimal microenvironment for undisturbed tumor growth. In addition, TGF-β exerts its tumor promoting functions via direct effects on tumor cell motility and invasiveness and indirectly by modulating tumor stroma and extracellular matrix, supporting angiogenesis and inhibiting immune surveillance. TGF-β acts through multiple intracellular signaling pathways and the outcome of TGF-β signaling is context-dependent. Defining the impact of the different TGF-β signaling pathways on melanoma progression will help to identify suitable therapeutic targets. Here we review the current knowledge of TGF-β in melanoma and discuss recent therapeutic approaches targeting the TGF-β pathway.

New glimpses of caveolin-1 functions in embryonic development and human diseases.

Caveolin-1 (Cav-1) isoforms, including Cav-1α and Cav-1β, were identified as integral membrane proteins and the major components of caveolae. Cav-1 proteins are highly conserved during evolution from {itCaenorhabditis elegans} to human and are capable of interacting with many signaling molecules through their caveolin scaffolding domains to regulate the activities of multiple signaling pathways. Thus, Cav-1 plays crucial roles in the regulation of cellular proliferation, differentiation and apoptosis in a cell-specific and contextual manner. In addition, Cav-1 is essential for embryonic development of vertebrates owing to its regulation of BMP, Wnt, TGF-β and other key signaling molecules. Moreover, Cav-1 is mainly expressed in terminally differentiated cells and its abnormal expression is often associated with human diseases, such as tumor progression, cardiovascular diseases, fibrosis, lung regeneration, and diseases related to virus. In this review, we will further discuss the potential of Cav-1 as a target for disease therapy and multiple drug resistance.

β-Catenin is essential for Müllerian duct regression during male sexual differentiation

During male sexual differentiation, the transforming growth factor-β (TGF-β) signaling molecule anti-Müllerian hormone (AMH; also known as Müllerian inhibiting substance, MIS) is secreted by the fetal testes and induces regression of the Müllerian ducts, the primordia of the female reproductive tract organs. Currently, the molecular identity of downstream events regulated by the AMH signaling pathway remains unclear. We found that male-specific Wnt4 expression in mouse Müllerian duct mesenchyme depends upon AMH signaling, implicating the WNT pathway as a downstream mediator of Müllerian duct regression. Inactivation of β-catenin, a mediator of the canonical WNT pathway, did not affect AMH signaling activation in the Müllerian duct mesenchyme, but did block Müllerian duct regression. These data suggest that β-catenin mediates AMH signaling for Müllerian duct regression during male sexual differentiation.

Transforming Growth Factor-β (TGFβ) Receptors I/II Differentially Regulate TGFβ1 and IGF-Binding Protein-3 Mitogenic Effects in the Human Placenta

Maternal IGFs regulate cytotrophoblast proliferation and, thereby, placental growth and function. IGF bioavailability is controlled by IGF-binding proteins (IGFBPs); in placenta, IGFBP-3 is particularly abundant. In other systems, IGFBP-3 can regulate cellular events independently of IGFs; these effects are thought to be mediated by TGFbeta receptors (TbetaR). We have examined IGFBP-3 regulation of IGF-dependent and -independent cytotrophoblast proliferation in first-trimester placental explants and the role of TbetaRII in mediating these effects. In the presence of IGFBP-3 (50 nm), IGF-induced (10 nm) proliferation (monitored by immunohistochemical analysis of Ki67 expression and bromodeoxyuridine incorporation) was significantly reduced (P < 0.05). IGFBP-3 also reduced basal proliferation independently of IGF receptor signaling. Immunohistochemical analysis demonstrated that TGFbeta signaling molecules [TGFbeta receptor I (TbetaRI), TbetaRII, TbetaRV, Smad-2, and ERK] are expressed in syncytium and/or cytotrophoblast. TGFbeta1 (10 ng/ml) enhanced cytotrophoblast proliferation and activated both Smad-2 and ERK-1/2, whereas IGFBP-3 activated only Smad-2. The function of both TGFbeta1 and IGFBP-3 was attenuated by a TbetaRII function-blocking antibody and by small interfering RNA-mediated knockdown of TbetaRII (P < 0.05); this was accompanied by a reduction in Smad-2 activation. This study demonstrates that both TGFbeta1 and IGFBP-3 signal through TbetaRI/II to influence human cytotrophoblast proliferation. However, downstream pathways are distinct, because IGFBP-3 acts only through Smad-2, whereas TGFbeta1 also phosphorylates ERK, resulting in opposite effects on cytotrophoblast proliferation. The effects of maternal growth signals on placental growth and function therefore depend on the balance of ligands, receptors, and signaling molecules at the syncytiotrophoblast surface. Therapeutic manipulation of this balance might offer a strategy to optimize placental development and pregnancy outcome.

Halofuginone mediated protection against radiation-induced leg contracture

Fibrosis of normal tissues often accompanies radiation treatment of cancer. Activation of the transforming growth factor-beta (TGF-beta) signaling pathway is thought to play a major role in radiation-induced fibrosis and has prompted the development and assessment of low molecular weight inhibitors of the pathway. Previous studies with halofuginone have shown it to inhibit TGF-beta signaling in vitro and protect mice from radiation-induced leg contraction (a model for soft tissue fibrosis). The current study confirms these findings for HaCaT cells stimulated with exogenous TGF-beta treatment. Reducing the halifuginone treatment from 7 days/week (used previously) to 5 days/week post-radiation exposure provided significant protection against radiation-induced leg contraction in mice 3 and 4 months post-radiation treatment. Halofuginone treatment was shown to attenuate TGF-beta signaling molecules taken from irradiated skin including TGF-betaRII, pSmad3, Smad7, and TSP1. The latter, TSP1, a co-activator of TGF-beta may serve as a suitable biomarker for monitoring the efficacy of halofuginone should it be evaluated in a clinical setting for protection against radiation-induced fibrosis.

Bacterial Infection of Smad3/Rag2 Double-Null Mice with Transforming Growth Factor-β Dysregulation as a Model for Studying Inflammation-Associated Colon Cancer

Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.

Snail and Slug Promote Epithelial-Mesenchymal Transition through β-Catenin–T-Cell Factor-4-dependent Expression of Transforming Growth Factor-β3

Members of the Snail family of transcription factors have been shown to induce epithelial-mesenchymal transition (EMT), a fundamental mechanism of embryogenesis and progressive disease. Here, we show that Snail and Slug promote formation of beta-catenin-T-cell factor (TCF)-4 transcription complexes that bind to the promoter of the TGF-beta3 gene to increase its transcription. Subsequent transforming growth factor (TGF)-beta3 signaling increases LEF-1 gene expression causing formation of beta-catenin-lymphoid enhancer factor (LEF)-1 complexes that initiate EMT. TGF-beta1 or TGF-beta2 stimulates this signaling mechanism by up-regulating synthesis of Snail and Slug. TGF-beta1- and TGF-beta2-induced EMT were found to be TGF-beta3 dependent, establishing essential roles for multiple TGF-beta isoforms. Finally, we determined that beta-catenin-LEF-1 complexes can promote EMT without upstream signaling pathways. These findings provide evidence for a unified signaling mechanism driven by convergence of multiple TGF-beta and TCF signaling molecules that confers loss of cell-cell adhesion and acquisition of the mesenchymal phenotype.

Thrombospondin-1 Is an Endogenous Activator of TGF-β in Experimental Diabetic Nephropathy In Vivo

Transforming growth factor-beta (TGF-beta), the central cytokine responsible for the development of diabetic nephropathy, is usually secreted as a latent procytokine complex that has to be activated before it can bind to its receptors. Recent studies by our group demonstrated that thrombospondin-1 (TSP-1) is the major activator of latent TGF-beta in experimental glomerulonephritis in the rat, but its role in diabetic nephropathy in vivo is unknown. Type 1 diabetes was induced in wild-type (n = 27) and TSP-1-deficient mice (n = 36) via streptozotocin injection, and diabetic nephropathy was investigated after 7, 9.5, and 20 weeks. Renal histology, TGF-beta activation, matrix accumulation, and inflammation were assessed by immunohistology. Expression of fibronectin and TGF-beta was evaluated using real-time PCR. Furthermore, functional parameters were examined. In TSP-1-deficient compared with wild-type mice, the amount of active TGF-beta within glomeruli was significantly lower, as indicated by staining with specific antibodies against active TGF-beta or the TGF-beta signaling molecule phospho-smad2/3 or the typical TGF-beta target gene product plasminogen activator inhibitor-1. In contrast, the amount of glomerular total TGF-beta remained unchanged. The development of diabetic nephropathy was attenuated in TSP-1-deficient mice as demonstrated by a significant reduction of glomerulosclerosis, glomerular matrix accumulation, podocyte injury, renal infiltration with inflammatory cells, and renal functional parameters. We conclude that TSP-1 is an important activator of TGF-beta in diabetic nephropathy in vivo. TSP-1-blocking therapies may be considered a promising future treatment option for diabetic nephropathy.