Transforming Growth Factor-β Signaling Pathway Research Articles

Update of genetic susceptibility in patients with Kawasaki disease.

Kawasaki disease (KD) is an acute systemic vasculitis that predominantly affects children, and can result in coronary artery lesions (CAL). A patient with KD who is resistant to treatment with intravenous immunoglobulin (IVIG) has a higher risk of developing CAL. Incomplete KD has increased in prevalence in recent years, and is another risk factor for the development of CAL. Although the pathogenesis of KD remains unclear, there has been increasing evidence for the role of genetic susceptibility to the disease since it was discovered in 1967. We retrospectively reviewed previous genetic research for known susceptibility genes in the pathogenesis of KD, IVIG resistance, and the development of CAL. This review revealed numerous potential susceptibility genes including genetic polymorphisms of ITPKC, CASP3, the transforming growth factor-β signaling pathway, B lymphoid tyrosine kinase, FCGR2A, KCNN2, and other genes, an imbalance of Th17/Treg, and a range of suggested future treatment options. The results of genetic research may improve our understanding of the pathogenesis of KD, and aid in the discovery of new treatment modalities for high-risk patients with KD.

Cysteine-rich, angiogenic inducer, 61 expression in patients with ovarian epithelial carcinoma

Cysteine-rich, angiogenic inducer, 61 (CYR61) is a key gene in the transforming growth factor-β signalling pathway, which is involved in the development of many tumour types. This study aimed to clarify the status and clinical significance of CYR61 expression in patients with ovarian epithelial carcinoma. Tissue from patients with ovarian epithelial carcinoma or benign ovarian tumours were investigated retrospectively for CYR61 expression at mRNA and protein levels, using reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. Correlations between immunohistochemical scores and several clinicopathological parameters were investigated. In 50 patients with ovarian epithelial carcinoma and 50 patients with benign ovarian tumours, CYR61 expression on mRNA and protein levels was significantly higher in ovarian epithelial carcinoma tissue than in benign ovarian tissue. CYR61 expression was associated with regional lymph node metastases and progression of clinical disease stage. There was no difference in CYR61 expression between patients aged <50 years and ≥ 50 years. CYR61 expression was significantly upregulated in ovarian carcinoma tissue compared with benign ovarian tumour tissue samples. Protein CYR61 levels were associated with lymph node metastases and Union for International Cancer Control stage. Protein CYR61 may be useful in targeted diagnosis and therapy, for patients with ovarian epithelial carcinoma.

GATA-2 inhibits transforming growth factor-β signaling pathway through interaction with Smad4

GATA-2, a member of zinc finger GATA transcription factor family, plays key role in the hematopoietic stem cells self-renewal and differentiation. The transforming growth factor-β (TGFβ) signaling pathway is a major signaling network that controls cell proliferation, differentiation and tumor suppression. Here we found that GATA-2 negatively regulated TGF-β signaling pathway in Smad4-dependent manner. GATA-2 specifically interacts with Smad4 with its N-terminal while the zinc finger domain of GATA-2 is essential for negative regulation of TGFβ. Although GATA-2 did not affect the phosphorylation of Smad2/3 and the complex Smad2/3/4 formation in response to TGFβ, the DNA binding activity of Smad4 was decreased significantly by GATA-2 overexpression. Overexpression of GATA-2 in K562 cells led to reduced TGFβ-induced erythroid differentiation while knockdown of GATA-2 enhanced TGFβ-induced erythroid differentiation. All these results suggest that GATA-2 is a novel negative regulator of TGFβ signal pathway.

Synthesis and Biological Evaluation of Novel Tetrahydro-β-carboline Derivatives as Antitumor Growth and Metastasis Agents through Inhibiting the Transforming Growth Factor-β Signaling Pathway

The transforming growth factor beta (TGFβ) signaling cascade is considered as one of the pivotal oncogenic pathways in most advanced cancers. Inhibition of the TGFβ signaling pathway by specific antagonists, neutralizing antibodies, or small molecules is considered as an effective strategy for the treatment of tumor growth and metastasis. Here we demonstrated the identification of a series of tetrahydro-β-carboline derivatives from virtual screening which potentially inhibit the TGFβ signaling pathway. Optimization of the initial hit compound 2-benzoyl-1,3,4,9-tetrahydro-β-carboline (8a) through substitution at different positions to define the structure-activity relationship resulted in the discovery of potent inhibitors of the TGFβ signaling pathway. Among them, compound 8d, one of the tested compounds, not only showed potent inhibition of lung cancer cell proliferation and migration in vitro but also strongly suppressed growth of lung cancer and breast cancer in vivo.

Effects of 4-Nonylphenol and Bisphenol A on Stimulation of Cell Growth via Disruption of the Transforming Growth Factor-β Signaling Pathway in Ovarian Cancer Models

Transforming growth factor β (TGF-β) signaling pathway is a major pathway in cellular processes such as cell growth, apoptosis, and cellular homeostasis. The signaling pathway activated by 17β-estadiol (E2) appeared to inhibit the TGF-β signaling pathway by cross-talk with the TGF-β components in estrogen receptor (ER) positive cells. In this study, we examined the inhibitory effects of endocrine disrupting chemicals (EDCs), including 4-nonylphenol (NP), 4-otylphenol (OP), bisphenol A (BPA), and benzophenon-1 (BP-1), in the TGF-β signaling pathway in BG-1 ovarian cancer cells expressing estrogen receptors (ERs). The transcriptional and translational levels of TGF-β related genes were examined by reverse transcription-PCR (RT-PCR), Western blot analysis, and xenograft mouse models of ovarian cancer cells. As a result, treatment with NP, OP, and BPA induced the expressions of SnoN, a TGF-β pathway inhibitor, and c-Fos, a TGF-β target transcription factor. Treatment with NP, BPA, and BP-1 resulted in decreased phosphorylation of Smad3, a downstream target of TGF-β. These results indicate that NP and BPA may stimulate the proliferation of BG-1 cells via inhibition of the TGF-β signaling pathway. In a xenograft mouse model, transplanted BG-1 ovarian cancer cells showed significantly decreased phosphorylation of Smad3 and increased expression of SnoN in the ovarian tumor masses following treatment with E2, NP, or BPA. In parallel with an in vitro model, the expressions of these TGF-β signaling pathway were similarly regulated by NP or BPA in a xenograft mouse model. These results support the fact that the existence of an unproven relationship between EDCs/ER-α and TGF-β signaling pathway and a further study are required in order to verify more profound and distinct mechanism(s) for the disturbance of the TGF-β signaling pathway by diverse EDCs.

Antimicrobial effects of coprisin on wounds infected with Staphylococcus aureus in rats

Antimicrobial peptides (AMPs) are naturally produced antibiotics that play important roles in host defense mechanisms. These proteins are found in variety of animal and plant species. The antibiotic effects of AMPs are gaining attention for use in human medicine. In this study, the antimicrobial effects of coprisin, a novel AMP isolated from the dung beetle (Copris tripartitus), were evaluated. The peptide was used to treat rats with wounds infected with Staphylococcus aureus. Coprisin accelerated wound closure both grossly and microscopically compared with the untreated group. Additionally, treatment with this peptide decreased phosphorylated-Smad2/3 (p-Smad2/3) levels, a downstream factor of the transforming growth factor-β signaling pathway which is believed to inhibit reepithelization, in the nucleus and cytoplasm of regenerating cells. Moreover, increased cell populations and angiogenesis were observed in lesions treated with coprisin, suggesting that this peptide promotes wound healing via its antimicrobial activity against S. aureus. Our results demonstrated that coprisin is a potential therapeutic agent that can possibly replace traditional antibiotics and overcome microbial resistance.

Pulmonary arterial hypertension in familial hemiplegic migraine with ATP1A2 channelopathy

To the Editor: Pulmonary arterial hypertension (PAH) has been the focus of major research in recent years [1]. Involvement of mutations in genes encoding for members of the transforming growth factor-β signalling pathway ( BMPR2 , ACVRL1 , ENG and SMAD8 ) has been demonstrated in the development of heritable PAH, allowing novel experimental and clinical approaches [2–4]. However, ∼30% of familial forms of PAH remain without any identification of genetic mutations. Recently, mutations of the KCNK3 gene (encoding K+ channel subfamily K member 3) have been reported in patients with familial and sporadic PAH [5]. KCNK3 belongs to a family of mammalian K+ channels, and are involved in the regulation of resting membrane potential, pulmonary vascular tone and in vascular remodelling. This result paves the way to the involvement of novel signalling pathways in the development of heritable PAH. Herein, we describe a novel association of PAH and a channelopathy due to mutation in ATP1A2 (encoding the α2-subunit of the Na+/K+-ATPase), a mutation known to cause familial hemiplegic migraine (FHM), a rare autosomal dominant disease [6]. A 24-year-old male was referred with a 1-year history of progressive exertional dyspnoea. Since the age of 8 years, he has reported recurrent episodes of hemiplegic migraine associated with muscle weakness and pain. The proband’s mother (II4) (fig. 1) and two of his brothers (III6 and III7) had recurrent hemiplegic migraine with aura. There was no familial history of PAH. On admission, the patient was in New York Heart Association (NYHA) functional class III. His 6-min …

Novel mutations in ADAMTSL2 gene underlying geleophysic dysplasia in families from United Arab Emirates

Geleophysic dysplasia (GD) is an autosomal recessive disorder characterized by short stature, brachydactyly, stiff joints, thick skin, and cardiac valvular abnormalities that are often responsible for early death. Mutations in ADAMTSL2 and FBN1 genes have been shown to cause GD due to the dysregulation of transforming growth factor-β signaling pathways. Small numbers of mutations in ADAMTSL2 have been reported so far in patients with GD type 1 (GD1). In this study, we clinically evaluated two children from two consanguineous Arab families living in the United Arab Emirates with GD1. In addition we have sequenced all the coding exons of ADAMTSL2 gene using Sanger sequencing. The two patients exhibited most of the typical features of this rare bone dysplasia. Molecular analysis of the ADAMTSL2 gene revealed two novel homozygous missense mutations (c.938T>C, p.M313T and c.499G>A, p.D167N). The mutations segregated well in the studied families with the parents being heterozygous. In addition, bioinformatics analyses showed that these mutations are affecting conserved amino acids residues and thus strongly support their pathogenicity. We describe the clinical phenotypes of two patients with GD1 that are caused by two novel homozygous missense mutations in the ADAMTSL2 gene.

Genetics of Thoracic Aortic Aneurysm

Aortic aneurysm, including both abdominal aortic aneurysm and thoracic aortic aneurysm, is the cause of death of 1% to 2% of the Western population. This review focuses only on thoracic aortic aneurysms and dissections. During the past decade, the genetic contribution to the pathogenesis of thoracic aortic aneurysms and dissections has revealed perturbed extracellular matrix signaling cascade interactions and deficient intracellular components of the smooth muscle contractile apparatus as the key mechanisms. Based on the study of different Marfan mouse models and the discovery of several novel thoracic aortic aneurysm genes, the involvement of the transforming growth factor-β signaling pathway has opened unexpected new avenues. Overall, these discoveries have 3 important consequences. First, the pathogenesis of thoracic aortic aneurysms and dissections is better understood, although some controversy still exists. Second, the management strategies for the medical and surgical treatment of thoracic aortic aneurysms and dissections are becoming increasingly gene-tailored. Third, the pathogenetic insights have delivered new treatment options that are currently being investigated in large clinical trials.

Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EnMT) is a cell transformation process involved in both morphogenesis and pathogenesis. EnMT of corneal endothelial cells happens after endothelial injury and during ex vivo culture. Previous studies have shown that the transforming growth factor-β signaling pathway is involved in this transition. In this study, we found that rat corneal endothelial cells could spontaneously undergo EnMT during ex vivo culture. This change in rat corneal endothelial cells was associated with Notch signaling pathway activation after the first passage, which was blocked by the Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). This inhibitor also prevented transforming growth factor β1-, β2-, and β3-induced EnMT and reversed transformed rat corneal endothelial cells to a normal phenotype. Furthermore, DAPT treatment blocked retrocorneal membrane formation in a rat corneal endothelium damage model. Our study indicates that the Notch signaling pathway is involved in the corneal EnMT process, which may be a novel therapeutic target for treating corneal endothelial fibrogenic disorders.

Blocking transforming growth factor-β signaling pathway augments antitumor effect of adoptive NK-92 cell therapy

Natural killer (NK) cells hold great potential for improving the immunotherapy of cancer. However, existing data indicate that tumor cells can effectively escape NK cell-mediated apoptosis through immunosuppressive effect in the tumor microenvironment. Transforming growth factor-β (TGF-β) is a potent immunosuppressant. The present study was intended to develop a treatment strategy through adoptive transfer of TGF-β insensitive NK-92 cells. To block TGF-β signaling pathway, NK-92 cells were genetically modified with dominant negative TGF-β type II receptor (DNTβRII) by optimizing electroporation using the Amaxa Nucleofector system. These genetically modified NK-92 cells were insensitive to TGF-β and able to resist the suppressive effect of TGF-β on Calu-6 lung cancer cells in vitro. To determine the antitumor activity in vivo, recipient mice were challenged with a single subcutaneous injection of Calu-6 cells. Adoptive transfer of TGF-β insensitive NK-92 cells decreased tumor proliferation, reduced lung metastasis, produced more IFN-γ, and increased the survival rate of nude mice bearing established Calu-6 cells. Hence, we have demonstrated that blocking transforming growth factor-β signaling pathway in NK cells provides a novel therapeutic strategy and warrants further investigation.

Secretory/releasing proteome-based identification of plasma biomarkers in HBV-associated hepatocellular carcinoma

For successful therapy, hepatocellular carcinoma (HCC) must be detected at an early stage. Herein, we used a proteomic approach to analyze the secretory/releasing proteome of HCC tissues to identify plasma biomarkers. Serum-free conditioned media (CM) were collected from primary cultures of cancerous tissues and surrounding noncancerous tissues. Proteomic analysis of the CM proteins permitted the identification of 1365 proteins. The enriched molecular functions and biological processes of the CM proteins, such as hydrolase activity and catabolic processes, were consistent with the liver being the most important metabolic organ. Moreover, 19% of the proteins were characterized as extracellular or membrane-bound. For validation, secretory proteins involved in transforming growth factor-β signaling pathways were validated in plasma samples. Alphafetoprotein (AFP), metalloproteinase (MMP)1, osteopontin (OPN), and pregnancy-specific beta-1-glycoprotein (PSG)9 were significantly increased in HCC patients. The overall performance of MMP1 and OPN in the diagnosis of HCC remained greater than that of AFP. In addition, this study represents the first report of MMP1 as a biomarker with a higher sensitivity and specificity than AFP. Thus, this study provides a valuable resource of the HCC secretome with the potential to investigate serological biomarkers. MMP1 and OPN could be used as novel biomarkers for the early detection of HCC and to improve the sensitivity of biomarkers compared with AFP.

Analysis of altered microRNA expression profiles in peripheral blood mononuclear cells from patients with primary biliary cirrhosis

MicroRNA, as an important regulator of gene expression, has been found to be associated with several diseases. MicroRNA expression profiles have been identified in several autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. However, the expression profile in peripheral blood mononuclear cells (PBMCs) from primary biliary cirrhosis (PBC) patients and the role of microRNA in PBC remained unclear. The present study aimed to explore abnormal microRNA regulation in PBC. MicroRNA array was performed in PBMCs obtained from PBC patients versus healthy controls. Then, six of the 17 differentially expressed microRNAs were confirmed using quantitative real-time polymerase chain reaction. Based on bioinformatics analysis, we identified the potential biological processes and significant signaling pathways affected by these microRNAs, and generated the microRNA-gene network. According to microRNA array, a total of 17 microRNAs were found to be differentially expressed. Six microRNAs have been validated using quantitative real-time polymerase chain reaction, and the results were consistent with microRNA array analysis. The bioinformatics analysis showed that the potential target genes of these microRNAs were involved in cell proliferation, cell differentiation, apoptosis, and signal transduction. Similarly, these microRNAs also affected endocytosis, mitogen-activated protein kinase signaling pathway, transforming growth factor-β signaling pathway, Wnt signaling pathway, calcium signaling pathway, etc. In the present study, 17 microRNAs were identified to be differentially expressed in PBMCs from PBC patients. Functional bioinformatics analysis demonstrated that prediction genes targeted by these microRNAs were involved in multiple biological processes and signaling pathways. The present study offers intriguing new perspectives on the involvement of microRNA in PBC, but the precise mechanisms need to be validated further.

Disparate profiles of dys-regulated miRNAs in activated hepatic stellate cells

We read with great interest the article by Lakner et al. in a recent issue of HEPATOLOGY.1 In this study, the authors found a novel regulator of hepatic stellate cells (HSCs), miR-19b, which acted via the transforming growth factor-β signaling pathway. miR-19b was expressed differently between quiescent and activated HSCs, using comparative analysis of microRNA (miRNA) expression. As is well known, comparative analysis is the gold standard approach for detecting dys-regulated miRNAs. This same approach has been used on HSC in 4 other studies related to the topic.2-5 The profiles of dys-regulated miRNAs in activated HSCs are summarized in Table 1. The same protocol was executed with the following steps in these studies: step 1, quiescent HSCs were isolated from normal rat liver; step 2, activated HSCs were acquired by culturing quiescent HSCs in vitro for 10 or 14 days until activated; step 3, the different miRNA expression patterns of activated and quiescent HSCs were analyzed by comparative analysis. However, there was an interesting phenomenon shown in Table 1, which was that the profiles of dys-expressed miRNAs in activated HSCs varied greatly across the studies. The issue remains why the same protocol for detecting dys-regulated miRNAs in activated HSCs resulted in such different miRNA profiles. Shao-Long Chen M.D.*, Ming-Hua Zheng M.D.*, Tao Yang M.D.*, Mei Song M.D.*, Yong-Ping Chen M.D.*, * Department of Infection and Liver Diseases, Liver Research Center,The First Affiliated Hospital of Wenzhou Medical College, Wenzhou, China.

Biota orientalis induces cell proliferation through growth factor receptor expression in human dermal fibroblasts

The dried seeds of Biota orientalis are considered to be moistening and brightening one’s complexion. Ancient Chinese and Korean herbal textbook also reference it as a treatment of skin disorders like alopecia, scabies and tinea. However, no study has looked at the possible mechanism and molecular rationale of healing potential of B. orientalis. This study investigated the effects of a water extract of seeds from B. orientalis on human dermal fibroblasts (Hs68) and its mechanism related to wound healing. The effect of B. orientalis seed extract on Hs68 cell viability was investigated using the MTT assay. Gene expression analysis was performed using a microarray from Hs68 cells. The identities of the genes with altered expression after B. orientalis extract treatment were verified by RT-PCR. Western blot was used to explore the functional mechanism of cell proliferation. B. orientalis seed extract promoted Hs68 cell proliferation in a dose-dependent manner, to a level of 138 % of control at a concentration of 100 μg/mL. Microarray analysis indicated that B. orientalis seed extract upregulated the expression of genes related to cell proliferation, especially those involved in the mitogen-activated protein kinase and transforming growth factor-β signaling pathways, including transforming growth factor-β receptor 1 and fibroblast growth factor receptor 1. B. orientalis also caused the upregulation of TGFBR1, FGFR1 protein expression as shown by Western blotting assay. B. orientalis seed extract effectively stimulated cell proliferation in human dermal cells and may be useful for promoting wound healing.

Insights into the Transforming Growth Factor-β Signaling Pathway in Cutaneous Melanoma.

Transforming growth factor-β (TGF-β) is a pleiotropic growth factor with broad tissue distribution that plays critical roles during embryonic development, normal tissue homeostasis, and cancer. While its cytostatic activity on normal epithelial cells initially defined TGF-β signaling as a tumor suppressor pathway, there is ample evidence indicating that TGF-β is a potent pro-tumorigenic agent, acting via autocrine and paracrine mechanisms to promote peri-tumoral angiogenesis, together with tumor cell migration, immune escape, and dissemination to metastatic sites. This review summarizes the current knowledge on the implication of TGF-β signaling in melanoma.

Raloxifene and Bevacizumab for severe complications of hereditary haemorrhagic telangiectasia in a haemodialysis patient.

Hereditary haemorrhagic telangiectasia (HHT), or Osler–Weber–Rendu syndrome, is inherited in an autosomal-dominant fashion. There are two different forms of HHT: HHT type 1, caused by mutations in the endoglin gene, located in chromosome 9 (which encodes endoglin), and HHT type 2, caused by mutations in the gene encoding activin A receptor type II-like 1 (ALK1) on chromosome 12. Mucocutaneous telangiectasias, arteriovenous malformations, epistaxis, gastrointestinal bleeding and iron-deficiency anaemia are the commonest clinical manifestations of HHT [1]. Hepatic involvement, characterized by different arteriovenous malformations within the liver parenchyma, occurs in up to 74% of patients with HHT [2]. As far as we know, no information has been published on therapeutic alternatives for haemodialysis patients suffering from severe complications of HHT. We report on a 59-year-old woman with chronic renal failure secondary to bilateral renal hypoplasia and undergoing haemodialysis, who received the diagnosis of HHT. She had presented relapsing epistaxis during the last year, with severe anaemia and hypotension. Due to the need for weekly transfusions and the poor tolerance to haemodialysis sessions, we consider treatment with raloxifene (60 mg/day), a selective estradiol receptor modulator. Raloxifene increases the expression of endoglin and ALK1, whose synthesis is defective in HHT patients, at the surface of endothelial cells [3]. A remarkable improvement of HHT symptoms along with a significant decrease in the number and severity of epistaxis was observed after raloxifene treatment. However, 6 months after the onset of raloxifene treatment, the patient presented with signs and symptoms of high-output cardiac failure, with severe ascites and a left pleural effusion. Increase of haemodialysis ultrafiltration along with large-volume paracentesis were started, but the patient showed a poor tolerance to these measures, owing to the presence of anaemia, hypotension and chronic liver disease. Serum biochemistry demonstrated a progressive cholestasis. A computed tomography (CT) scan showed liver cirrhosis with splenomegaly and massive arteriovenous malformations (Figure 1). Prompted by the progressive clinical deterioration of the patient, we decided to start treatment with bevacizumab, a humanized recombinant monoclonal antibody against the vascular endothelial growth factor (VEFG). The VEGF is a key regulator of angiogenesis and is upregulated in a variety of diseases. On the other hand, endoglin and ALK1 are involved in the transforming growth factor-β signalling pathway, which is a potent stimulator of VEFG production. Bevacizumab has been shown to be effective in diseases of abnormal angiogenesis, improving survival and symptomatology [4, 5]. Bevacizumab has been also used for the treatment of liver complications of HHT, inducing a clear improvement [6]. After obtaining the patient's informed consent, six courses of bevacizumab (5 mg/kg) were administered during a 3-month period and raloxifene was maintained without changes. A further reduction in the frequency of epistaxis was observed besides a remarkable improvement of ascites and liver enzymes. As shown in Figure 1, serial CT scans demonstrated a clear diminution of hepatic vascularity and a smooth contour of the liver. Fig. 1. CT scan of the liver with intravenous contrast before and 6 months after bevacizumab treatment, showing a dramatic reduction of vascularity and improvement of liver surface. Many different therapies have been proposed for epistaxis and hepatic involvement in HHT, but none of them have shown conclusive results. Hormonal therapy with estradiol for epistaxis and gastrointestinal management of HHT has shown a variable degree of efficacy depending on the patient. According to our experience, both raloxifene and bevacizumab are very effective alternatives for the treatment of severe HHT in haemodialysis patients.

Imatinib mesylate in scleroderma-associated diffuse skin fibrosis: a phase II multicentre randomized double-blinded controlled trial

Imatinib mesylate is a potent inhibitor of platelet-derived growth factor and transforming growth factor-β signalling pathways which may play a role in systemic sclerosis (SSc)-associated skin changes. We aimed primarily at assessing the efficacy of imatinib mesylate in scleroderma skin fibrosis. We performed a phase II double-blinded trial on patients with scleroderma with either morphoea involving > 20% of body surface area or SSc with extensive skin involvement: modified Rodnan Skin Score (mRSS) ≥ 20/51. Each patient was randomized to receive either imatinib mesylate 400 mg or placebo daily for a total of 6 months, and then was followed up 6 months after therapy discontinuation. Skin fibrosis was assessed by mRSS and measurement of the dermal thickness using skin biopsies performed at inclusion and at 6 months of treatment. In addition, quality of life (Dermatology Life Quality Index and modified Health Assessment Questionnaire for Scleroderma) was recorded at each visit, and pulmonary function before and after intervention. Twenty-eight patients were included in the study with a mean age of 48·9 years (range 30-71): 25 had a diagnosis of a SSc and three of diffuse cutaneous scleroderma. Demographic data, frequency of organ involvement of SSc and mRSS were comparable between groups. At 6 months, the proportion of variation of mRSS from inclusion was not statistically significantly different between the two groups (median +0·10 in imatinib group vs. -0·16 in placebo group, P = 0·098). Similarly, changes in dermal thickness, quality of life and diffusion capacity for carbon monoxide were not significantly different between groups. This study failed to demonstrate the efficacy of imatinib 400 mg daily to improve skin fibrosis of diffuse scleroderma after 6 months of treatment based on validated outcome measurements.

Tollip, an Intracellular Trafficking Protein, Is a Novel Modulator of the Transforming Growth Factor-β Signaling Pathway

Upon activation, TGF-β type I receptor (TβRI) undergoes active ubiquitination via recruitment of E3 ligases to the receptor complex by Smad7. However, how ubiquitination of TβRI is coupled to intracellular trafficking, and protein degradation remains unclear. We report here that Tollip, an adaptor protein that contains both ubiquitin-associated domains and endosome-targeting domain, plays an important role in modulating trafficking and degradation of TβRI. Tollip was previously demonstrated to possess a functional role in modulating the signaling of interleukin-1 and Toll-like receptors. We identify here that Tollip interacts with Smad7, a major modulatory protein involved in the negative regulation of TGF-β signaling. Overexpression of Tollip antagonizes TGF-β-stimulated transcriptional response, Smad2 phosphorylation, and epithelial-mesenchymal transition. Tollip also interacts with ubiquitinated TβRI, and such interaction requires ubiquitin-associated domains of Tollip. The interaction and intracellular colocalization of Tollip with TβRI is enhanced by Smad7. Overexpression of Tollip accelerates protein degradation of activated TβRI. In addition, Tollip alters subcellular compartmentalization and endosomal trafficking of activated TβRI. Collectively, our studies reveal that Tollip cooperates with Smad7 to modulate intracellular trafficking and degradation of ubiquitinated TβRI, whereby negatively regulates TGF-β signaling pathway.

Characteristics and osteogenic differentiation of stem/progenitor cells in the human dental follicle analyzed by gene expression profiling

The dental follicle is an ectomesenchymal tissue that surrounds developing tooth germ and that contains osteoblastic-lineage-committed stem/progenitor cells. We examined the osteogenic potential of human dental follicle cells (hDFC) by microarray analysis. We first compared the characteristics of hDFC with those of human bone marrow mesenchymal stem cells (hMSC). Like hMSC, hDFC expressed stem cell markers such as STRO-1 and Notch-1 and differentiated not only into the osteoblastic lineage, but also into the adipogenic lineage. We analyzed the gene expression profiles of hDFC and hMSC that were not differentiated toward the osteogenic lineage. The expression of cell markers and growth factor receptors by hDFC and hMSC was similar, whereas the expression pattern of homeobox genes differed between hDFC and hMSC. Next, we investigated gene expression in hDFC during osteogenic differentiation. Gene expression profiles were analyzed in hDFC cultured in osteogenic induction medium (OIM) or in growth medium (GM) for 3 and 10 days. Many genes whose expression was regulated under these conditions were functionally categorized as "transcription" genes. Osteogenic markers were up-regulated in hDFC during osteogenic differentiation, whereas neurogenic markers were down-regulated. The genes whose expression was regulated in hDFC during osteogenic differentiation were further analyzed by ingenuity pathway analysis and real-time polymerase chain reaction. Bone morphogenetic protein and transforming growth factor-β signaling pathways were activated in hDFC cultured in OIM for 3 days. This study indicates that the dental follicle contains stem cells and/or osteoblastic progenitor cells and is a potential cellular resource for bone regeneration therapy.