Uncovering the differentially expressed genes and pathways involved in the progression of stable coronary artery disease to acute myocardial infarction using bioinformatics analysis.

OBJECTIVE: Coronary artery disease (CAD) is the main cause of mortality worldwide. How stable coronary artery disease (SCAD) progresses to acute myocardial infarction (AMI) is not known. This study was aimed to explore the differentially expressed genes (DEGs) and pathways involved in the progression of SCAD to AMI. MATERIALS AND METHODS: Publicly available gene-expression profiles (GSE71226, GSE97320, GSE66360) were downloaded from the Gene Expression Omnibus (GEO) database and integrated to identify DEGs. The GSE59867 dataset was further used to verify the result of screened DEGs. Functional-enrichment analyses, protein-protein interaction network, microRNA-transcription factor (TF)-mRNA regulatory network, and drug-gene network were visualized. RESULTS: Sixty common DEGs (CDEGs) were screened between the SCAD-Control group and AMI-Control group in the integrated dataset. Four upregulated DEGs were selected from GSE59867. Twenty hub genes were discovered, and three significant modules were constructed in the PPI network. The intersection of functional and pathway-enrichment analyses of 60 CDEGs and the module DEGs indicated that they were mainly involved in "inflammatory response", "immune response", and "cytokine-cytokine receptor interaction". A miRNA-TF-mRNA regulatory network comprised 87 miRNAs, 16 upregulated target DEGs and 7 TFs. CONCLUSIONS: We identified several important genes and miRNAs involved in the progression of SCAD to AMI: platelet activating factor receptor (PTAFR), aquaoporin-9 (AQP9), toll-like receptor-4 (TLR4), human constitutive androstane receptor-3 (HCAR3), leucine-rich-α2 glycoprotein-1 (LRG1), mothers Against Decapentaplegic Homolog 4 (SMAD4) and miRNA-149-5p, miRNA-6778-3p, and miRNA-520a-3p. Inflammation and the immune response had important roles in the progression from SCAD to AMI.

Tumor-associated macrophages promote pancreatic ductal adenocarcinoma progression by inducing epithelial-to-mesenchymal transition.

In this study, we investigated the role of tumor-associated macrophages (TAMs) in the progression of pancreatic ductal adenocarcinoma (PDAC). PDAC patients with higher levels of CD68+ TAMs exhibited shorter overall survival. In Transwell assays, PDAC cells incubated with TAMs or conditioned media from TAM cells (TAM-CM) showed higher migration and invasion rates than controls. PET/CT scan analysis of orthotopic PDAC model mice revealed greater primary tumor growth and liver metastasis in the TAM-CM treatment group than the controls. H&E staining of liver tissues showed significantly higher numbers of metastatic nodules in the TAM-CM treatment group. Heat inactivation of TAM-CM significantly reduced Transwell migration by PDAC cells, suggesting the involvement of one or more secreted proteins in PDAC progression. Transcriptome sequencing analysis of PDAC cells treated with TAM-CM revealed significant enrichment of transforming growth factor-ß (TGF-ß) signaling pathway genes. Western blot and qRT-PCR analysis showed that TAM-CM enhanced PDAC migration cells by inducing epithelial-to-mesenchymal transition through the TGF-ß-Smad2/3/4-Snail signaling axis. The pro-tumorigenic effects of TAMs or TAM-CM were abolished by TGF-ß signaling pathway inhibitors and neutralizing TGF-ß antibody. These results demonstrate that TAMs promote PDAC progression through the TGF-ß signaling pathway.

The Interplay between Transcriptional Factors and MicroRNAs as an Important Factor for Th17/Treg Balance in RA Patients.

MicroRNAs regulate gene expression of transcriptional factors, which influence Th17/Treg (regulatory T cells) balance, establishing the molecular mechanism of genetic and epigenetic regulation of Treg and Th17 cells is crucial for understanding rheumatoid arthritis (RA) pathogenesis. The study goal was to understand the potential impact of the selected microRNAs expression profiles on Treg/Th17 cells frequency, RA phenotype, the expression profile of selected microRNAs, and their correlation with the expression profiles of selected transcriptional factors: SOCS1, SMAD3, SMAD4, STAT3, STAT5 in RA; we used osteoarthritis (OA) and healthy controls (HCs) as controls. The study was conducted on 14 RA and 11 OA patients, and 15 HCs. Treg/Th17 frequency was established by flow cytometry. Gene expression analysis was estimated by qPCR. We noticed correlations in RA Th17 cells between miR-26 and SMAD3, STAT3, SOCS1; and miR-155 and STAT3-and in RA Treg cells between miR-26 and SOCS1; miR-31, -155 and SMAD3; and miR-155 and SMAD4. In RA Tregs, we found a negative correlation between miR-26, -126 and STAT5a. The expression level of miR-31 in Th17 cells from RA patients with DAS28 ≤ 5.1 is higher and that for miR-24 is greater in Tregs from patients with DAS28 > 5.1. MiR-146a in Tregs is higher in rheumatoid factor (RF) positive RA patients.

SKI and SMAD4 are essential for IL-21-induced Th17 differentiation.

Th17 cells are essential for the pathogenesis of inflammatory and autoimmune diseases. In the presence of TGF-ß, the differentiation of Th17 cells can be induced by inflammatory cytokines, especially IL-6, which is mainly produced by antigen presenting cells (APCs); or IL-21, which is derived from T cells. IL-21 is required for IL-6-induced Th17 cell differentiation. However, the key regulators and underlying mechanisms for IL-21-induced Th17 differentiation is still elusive. Here we show that SMAD4 is a key regulator in IL-21-induced Th17 differentiation. SMAD4 deficient naïve T cells can differentiate into Th17 cells in the absence of TGF-ß signaling, and these Th17 cells are pathogenic during EAE. SMAD4 represses Rorc mRNA transcription to constrain IL-21-induced Th17 differentiation in the absence of TGF-ß signaling. While in the presence of TGF-ß, SMAD4 losses its suppressive ability due to the degradation of SKI. Mutation of Y429A or A432E on SMAD4 disrupts the interaction of SKI from SMAD4 and eliminates SMAD4 mediated suppression of Th17 differentiation. SMAD4 is indispensable for SKI binding to Rorc promoter region to regulate Th17 differentiation. Moreover, activin can induce Th17 differentiation in combination with IL-21, and the process is also subjected to the control of SKI and SMAD4. This study therefore elucidates critical mechanism for IL-21-induced Th17 differentiation to indicate SKI and SMAD4 as potential therapeutic targets for treating autoimmune diseases.

Loss-of-Function in SMAD4 Might Not Be Critical for Human Natural Killer Cell Responsiveness to TGF-ß.

We characterized the NK cell phenotype and function in three family members with Hereditary Hemorrhagic Telangiectasia (HHT) due to heterozygous SMAD4 mutations. Loss-of-function mutation in this gene did not induce developmental effects to alter CD56bright or CD56dim NK cell subset proportions in peripheral blood; and did not result in major differences in either their IL-15-induced proliferation, or their cytokine secretion response to TGF-ß1. These data suggest that SMAD4 plays a redundant role in downstream TGF-ß signaling in NK cells.

SMAD4 Loss in Colorectal Cancer Patients Correlates with Recurrence, Loss of Immune Infiltrate, and Chemoresistance.

PURPOSE: SMAD4 has shown promise in identifying patients with colorectal cancer at high risk of recurrence or death.Experimental Design: A discovery cohort and independent validation cohort were classified by SMAD4 status. SMAD4 status and immune infiltrate measurements were tested for association with recurrence-free survival (RFS). Patient-derived xenografts from SMAD4-deficient and SMAD4-retained tumors were used to examine chemoresistance. RESULTS: The discovery cohort consisted of 364 patients with stage I-IV colorectal cancer. Median age at diagnosis was 53 years. The cohort consisted of 61% left-sided tumors and 62% stage II/III patients. Median follow-up was 5.4 years (interquartile range, 2.3-8.2). SMAD4 loss, noted in 13% of tumors, was associated with higher tumor and nodal stage, adjuvant therapy use, fewer tumor-infiltrating lymphocytes (TIL), and lower peritumoral lymphocyte aggregate (PLA) scores (all P < 0.04). SMAD4 loss was associated with worse RFS (P = 0.02). When stratified by SMAD4 and immune infiltrate status, patients with SMAD4 loss and low TIL or PLA had worse RFS (P = 0.002 and P = 0.006, respectively). Among patients receiving 5-fluorouracil (5-FU)-based systemic chemotherapy, those with SMAD4 loss had a median RFS of 3.8 years compared with 13 years for patients with SMAD4 retained. In xenografted mice, the SMAD4-lost tumors displayed resistance to 5-FU. An independent cohort replicated our findings, in particular, the association of SMAD4 loss with decreased immune infiltrate, as well as worse disease-specific survival. CONCLUSIONS: Our data show SMAD4 loss correlates with worse clinical outcome, resistance to chemotherapy, and decreased immune infiltrate, supporting its use as a prognostic marker in patients with colorectal cancer.

Dietary Garcinol Arrests Pancreatic Cancer in p53 and K-ras Conditional Mutant Mouse Model.

Pancreatic cancer (PC) patients have poor prognosis and survival rate. Gemcitabine, the drug of choice has a dismal 15% response rate. Earlier, we reported that Garcinol alone and in combination with gemcitabine showed a dose-dependent favorable response on PC cell lines. This study probes the in vivo effects of dietary Garcinol on PC progression in transgenic PC mice (KPC; K-ras and p53 conditional mutant). KPC male mice were divided into: KC- Control diet; KGr-0.05% Garcinol diet; KGm-Gemcitabine injected; KGG - Garcinol diet + Gemcitabine injected groups. Changes in tumor progression, toxicity, or cell morphology were monitored by magnetic resonance imaging, Fore-stomach, and blood smear, respectively. Pancreatic Intraepithelial Neoplasia (mPanIN) grading with hematoxylin and eosin (H&E) staining was conducted on pancreas and validated by immunohistochemistry. The KGr group showed improved survival, no observable toxicity with marked reduction in papilloma formation in the fore-stomach, and a higher ratio of NK and NKT cells compared to Non-NK lymphocytes. Additionally, the KGr, KGm, and KGG groups showed reduction in tumor volumes and reduced number of advanced mouse PanIN3. Dietary Garcinol alone and in combination with gemcitabine retarded the progression of PC in transgenic PC mice, arresting the cancer in the earlier stages, improving prognosis and survival.

SMAD4 promotes TGF-ß-independent NK cell homeostasis and maturation and antitumor immunity.

SMAD4 is the only common SMAD in TGF-ß signaling that usually impedes immune cell activation in the tumor microenvironment. However, we demonstrated here that selective deletion of Smad4 in NK cells actually led to dramatically reduced tumor cell rejection and augmented tumor cell metastases, reduced murine CMV clearance, as well as impeded NK cell homeostasis and maturation. This was associated with a downregulation of granzyme B (Gzmb), Kit, and Prdm1 in Smad4-deficient NK cells. We further unveiled the mechanism by which SMAD4 promotes Gzmb expression. Gzmb was identified as a direct target of a transcriptional complex formed by SMAD4 and JUNB. A JUNB binding site distinct from that for SMAD4 in the proximal Gzmb promoter was required for transcriptional activation by the SMAD4-JUNB complex. In a Tgfbr2 and Smad4 NK cell-specific double-conditional KO model, SMAD4-mediated events were found to be independent of canonical TGF-ß signaling. Our study identifies and mechanistically characterizes unusual functions and pathways for SMAD4 in governing innate immune responses to cancer and viral infection, as well as NK cell development.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

Background & Aims: Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor ß (TGFß) family signaling through SMAD4 in colonic epithelial cells. Methods: The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFß1 and bone morphogenetic protein 2. Results: Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFß signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions: TGFß suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

TGF-ß/SMAD4 mediated UCP2 downregulation contributes to Aspergillus protease-induced inflammation in primary bronchial epithelial cells.

Elevated levels of mitochondrial reactive oxygen species (ROS) can lead to the development of airway inflammation. In this study, we investigated the role of Aspergillus proteases-which contribute to the pathogenesis of Aspergillus-induced diseases such as allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, and atopic asthma-and their mechanisms of action in airway inflammation using primary human bronchial epithelial cells, and evaluated the inflammatory responses mediated by mitochondrial ROS. We found that Aspergillus proteases regulated the expression of multifunctional inflammatory cytokines such as interleukin (IL)- 1ß, - 6, and - 8, and transforming growth factor (TGF)-ß, which stimulated cytokine production and chemokines involved in leukocyte migration and activated an inflammatory cascade. Expression of these factors and activator protein (AP)- 1 were decreased by treatment with the mitochondrial ROS scavenger Mito-TEMPO, suggesting that mitochondria are important sources of ROS in the context of inflammatory response by Aspergillus protease. The regulation of mitochondrial ROS influenced the production of proinflammatory mediators by preventing mitochondrial ROS-induced AP-1 activation in airway epithelial cells. In addition, Aspergillus protease-mediated mitochondrial ROS production was associated with downregulation of uncoupling protein (UCP)- 2 expression by TGF-ß-SMAD4 signaling, which may play a regulatory role in mitochondrial ROS formation during fungal protease-mediated epithelial inflammation. This improved understanding of the allergenic fungal protease-induced inflammatory mechanism in the bronchial epithelium will help in developing intervention strategies for the regulation of inflammatory response in allergic airway diseases.

Leishmania donovani mediated higher expression of CCL4 induces differential accumulation of CD4+CD56+NKT and CD8+CD56+NKT cells at infection site.

Sterile cure from visceralized Leishmania donovani (L. donovani) needs Th1 cell support along with the assistance from innate immune cells, NK cells and NKT cells. NKT cells play as a connecting link between innate and adaptive immune cell and support T helper cell function. Earlier, a categorical function of CD56 positive CD4+ or CD8+ NKT cells was reported in visceral leishmaniasis (VL). It was observed in in vitro that CD4+CD56+NKT cells, but not CD8+CD56+NKT cells, were accumulated at the L. donovani infection site. Therefore, in vitro experiments have been carried out to decipher the mechanism behind preferential accumulation of CD4+CD56+NKT cells at infection site. In this study, 1.89 fold higher expression of CCL4/MIP-1ß was noticed in infected macrophages. The higher expression of CCL4 was correlated with preferential accumulation of CCR5+CD4+CD56+NKT cells and apoptosis of CD8+CD56+NKT cells at in vitro infection site. The CD4+CD56+NKT cells were also observed expressing TGF-ß dominantly. Interaction of CCL4 chemotaxis was interrupted by blocking, which led to drift back the TGF-ß producing CD4+CD56+NKT cells and promoted CD8+CD56+NKT cells recruitment in in vitro infection site. CCR5 blockade also reduced CD25 and FoxP3 positive CD4+CD56+NKT cells in in vitro infection site. Therefore, it was concluded that Leishmania promotes strategic expression of CCL4, which alternately attracts CCR5+ cells, mostly expressing regulatory cytokines, at infection site. This reduces the CD8+CD56+NKT cells at infection site through Smad4 mediated TGF-ß expression and activation of caspases. Data indicates that L. donovani induces higher expression of CCL4 in host cell to attract CCR5+ cells under its strategic plan to downregulate host immune response.

SMAD4 impedes the conversion of NK cells into ILC1-like cells by curtailing non-canonical TGF-ß signaling.

Among the features that distinguish type 1 innate lymphoid cells (ILC1s) from natural killer (NK) cells is a gene signature indicative of 'imprinting' by cytokines of the TGF-ß family. We studied mice in which ILC1s and NK cells lacked SMAD4, a signal transducer that facilitates the canonical signaling pathway common to all cytokines of the TGF-ß family. While SMAD4 deficiency did not affect ILC1 differentiation, NK cells unexpectedly acquired an ILC1-like gene signature and were unable to control tumor metastasis or viral infection. Mechanistically, SMAD4 restrained non-canonical TGF-ß signaling mediated by the cytokine receptor TGFßR1 in NK cells. NK cells from a SMAD4-deficient person affected by polyposis were also hyper-responsive to TGF-ß. These results identify SMAD4 as a previously unknown regulator that restricts non-canonical TGF-ß signaling in NK cells.

T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes.

OBJECTIVE: Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies. DESIGN: We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. RESULTS: Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+ and CD4+ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+ T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo. CONCLUSIONS: These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

Smad4 represses the generation of memory-precursor effector T cells but is required for the differentiation of central memory T cells.

The transcriptional regulation underlying the differentiation of CD8(+) effector and memory T cells remains elusive. Here, we show that 18-month-old mice lacking the transcription factor Smad4 (homolog 4 of mothers against decapentaplegic, Drosophila), a key intracellular signaling effector for the TGF-ß superfamily, in T cells exhibited lower percentages of CD44(hi)CD8(+) T cells. To explore the role of Smad4 in the activation/memory of CD8(+) T cells, 6- to 8-week-old mice with or without Smad4 in T cells were challenged with Listeria monocytogenes. Smad4 deficiency did not affect antigen-specific CD8(+) T-cell expansion but led to partially impaired cytotoxic function. Less short-lived effector T cells but more memory-precursor effector T cells were generated in the absence of Smad4. Despite that, Smad4 deficiency led to reduced memory CD8(+) T-cell responses. Further exploration revealed that the generation of central memory T cells was impaired in the absence of Smad4 and the cells showed survival issue. In mechanism, Smad4 deficiency led to aberrant transcriptional programs in antigen-specific CD8(+) T cells. These findings demonstrated an essential role of Smad4 in the control of effector and memory CD8(+) T-cell responses to infection.

Nerve growth factor exposure promotes tubular epithelial-mesenchymal transition via TGF-ß1 signaling activation.

Clinical studies showed that renal expression and serum levels of nerve growth factor (NGF) are increased in renal diseases characterized by progressive fibrosis, a pathologic process in which TGF-ß1 mediates most of the key events leading to tubular epithelial-mesenchymal transition (EMT). However, the pathogenic role of high NGF levels has not yet been elucidated. In this study, we found that in tubular renal cells, HK-2, NGF transcriptionally up-regulated TGF-ß1 expression and secretion and enhanced cell motility by activating EMT markers via its receptors, TrkA and p75(NTR). Interestingly, we observed that TGF-ß1-SMAD pathway activation and the up-regulation of EMT markers NGF-induced were both prevented when knockdown of TGF-ß1 gene occurred and that the pretreatment with an antibody anti-NGF reversed the nuclear translocation of pSMAD3/SMAD4 complex. Collectively, our results demonstrated that NGF promotes renal fibrosis via TGF-ß1-signaling activation, suggesting that in kidney diseases high NGF serum levels could contribute to worsen renal fibrosis.

Smad4 promotes differentiation of effector and circulating memory CD8 T cells but is dispensable for tissue-resident memory CD8 T cells.

Tissue-resident memory CD8 T cells are a unique subset of virus-specific CTLs that bolster local immune responses after becoming lodged in previously infected tissues. These cells provide enhanced protection by intercepting returning pathogens before a new infection gets established. In contrast, central memory CD8 T cells circulate in the bloodstream and proliferate in secondary lymphoid organs before replenishing effector and memory CD8 T cell populations in remote parts of the body. Both populations of virus-specific memory CD8 T cells participate in immunity to influenza virus infection; however, the signaling pathways that instruct developing memory CD8 T cells to distribute to specific tissues are poorly defined. We show that TGF-ß promotes the development of pulmonary tissue-resident memory T cells via a signaling pathway that does not require the downstream signaling intermediate Sma- and Mad-related protein (Smad)4. In contrast, circulating memory CD8 T cells have no requirement for TGF-ß but show signs of arrested development in the absence of Smad4, including aberrant CD103 expression. These signaling pathways alter the distribution of virus-specific CTLs in the lungs but do not prevent robust cytokine responses. Our data show that Smad4 is required for normal differentiation of multiple subsets of virus-specific CD8 T cells. In normal circumstances, Smad4 may be activated via a pathway that bypasses the TGF-ß receptor. Improved understanding of these signaling pathways could be used to augment vaccine-induced immunity.

Exchange protein directly activated by cAMP modulates regulatory T-cell-mediated immunosuppression.

The cAMP signalling pathway plays an essential role in immune functions. In the present study we examined the role of the cAMP/EPAC1 (exchange protein directly activated by cAMP) axis in regulatory T-cell (Treg)-mediated immunosuppression using genetic and pharmacological approaches. Genetic deletion of EPAC1 in Tregs and effector T-cells (Teffs) synergistically attenuated Treg-mediated suppression of Teffs. Mechanistically, EPAC1 inhibition enhanced activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) and up-regulated SMAD7 expression while down-regulating expression of SMAD4. Consequently, CD4+ T-cells were desensitized to transforming growth factor (TGF) ß1, a cytokine employed by Tregs to exert a broad inhibitory function within the immune system. Furthermore, deletion of EPAC1 led to production of significant levels of ovalbumin IgG antibodies in a low-dose, oral-tolerance mouse model. These in vivo observations are consistent with the finding that EPAC1 plays an important role in Treg-mediated suppression. More importantly, pharmacological inhibition of EPAC1 using an EPAC-specific inhibitor recapitulates the EPAC1 deletion phenotype both in vivo and in vitro. The results of the present study show that EPAC1 boosts Treg-mediated suppression, and identifies EPAC1 as a target with broad therapeutic potential because Tregs are involved in numerous pathologies, including autoimmunity, infections and a wide range of cancers.

Identification and characterization of functional Smad8 and Smad4 homologues from Echinococcus granulosus.

Smad family proteins are essential cellular mediators of the transforming growth factor-ß superfamily. In the present study, we identified two members of the Smad proteins, Smad8 and Smad4 homologues (termed as EgSmadE and EgSmadD, respectively), from Echinococcus granulosus, the causative agent of cystic echinococcosis (CE). Phylogenetic analysis placed EgSmadE in the Smad1, 5, and 8 subgroup of the R-Smad sub-family and EgSmadD in the Co-Smad family. Furthermore, EgSmadE and EgSmadD attained a high homology to EmSmadE and EmSmadD of E. multilocularis, respectively. Both EgSmadE and EgSmadD were co-expressed in the larval stages and exhibited the highest transcript levels in activated protoscoleces, and their encoded proteins were co-localized in the sub-tegumental and tegumental layer of the parasite. As shown by yeast two-hybrid and pull-down analysis, EgSmadE displayed a positive binding interaction with EgSmadD. In addition, EgSmadE localized in the nuclei of Mv1Lu cells (mink lung epithelial cells) upon treatment with human TGF-ß1 or human BMP2, indicating that EgSmadE is capable of being translocated into nucleus, in vitro. Our study suggests that EgSmadE and EgSmadD may take part in critical biological processes, including echinococcal growth, development, and parasite-host interaction.

Immunolocalization of Smad-4 in developing molar roots of alendronate-treated rats.

INTRODUCTION: During root formation, Smad-4 plays a key role during the epithelial-mesenchymal interactions and the Hertwig's epithelial root sheath (HERS) apical proliferation. The root formation and eruption of rat molars is impeded by alendronate treatment due to the inhibition of bone resortion by this drug. The present study aimed to examine the structures affected in the developing root and immunodetect the presence of Smad-4 in rats treated with alendronate. METHODS: Newborn Wistar rats were daily injected 2.5 mg/kg alendronate (ALN) during 9, 12 and 30 days. The controls (CON) were injected with saline. The maxillae were fixed and embedded in paraffin or Spurr resin. Paraffin sections were incubated in Smad-4 antibody that was labelled with DAB. The ultrathin sections were examined in a transmission electron microscope. RESULTS: In ALN, a short portion of root dentine was formed; the epithelial diaphragm (ED) and the dental follicle (DF) were disorganized by the contact of bone trabeculae. The (CON) molar roots developed normally. Smad-4 labelling was detected in the cytoplasm of fibroblasts and cementoblasts adjacent to the cementum in CON; in ALN group, few ED cells presented weak immunolabelling. Ultrastructurally, the ED and DF appeared disrupted due to the presence of thin bone trabeculae between its cells. It resulted in the lack of apical proliferation of HERS and, consequently, arrest of root formation. CONCLUSION: The immunodetection of Smad-4 in the DF cells of ALN specimens indicates that the signalling for the differentiation of these cells into cementum-forming fibroblasts and cementoblasts occurs, despite the impairment of root elongation.

MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4.

Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-ß1 (TGF-ß1) is considered to be the main stimuli factor responsible for the activation of HSC. MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation, differentiation, and apoptosis. The involvement of miRNAs and their roles in TGF-ß1-induced HSC activation remains largely unknown. Our study found that the expression of miR-146a was downregulated in HSC in response to TGF-ß1 stimulation in dose-dependent manner by one-step real-time quantitative PCR. Moreover, we sought to examine whether miR-146a became dysregulated in CCl(4)-induced hepatic fibrosis in rats. Our study revealed that miR-146a was downregulated in liver fibrotic tissues. In addition, The HSC transfected with miR-146a mimics exhibited attendated TGF-ß1-induced α-smooth muscle actin (α-SMA) expression compared with the control. Furthermore, overexpression of miR-146a suppressed TGF-ß-induced HSC proliferation, and increased HSC apoptosis. Bioinformatics analyses predict that SMAD4 is the potential target of miR-146a. MiR-146a overexpression in TGF-ß1-treated HSC did not decrease target mRNA levels, but significantly reduced target protein expression. These results suggested that miR-146a may function as a novel regulator to modulate HSC activation during TGF-ß1 induction by targeting SMAD4.