Activation Of TGF-beta Signaling Pathway Research Articles

Identification and characterization of microRNAs, especially gga-miR-181b-5p, in chicken macrophages associated with avian pathogenic E. coli infection

ABSTRACT Avian pathogenic E. coli (APEC) is a common pathogen in the poultry industry, which can cause substantial economic losses. Recently, emerging evidence showed that miRNAs were involved in various viral and bacterial infections. To elucidate the role of miRNAs in chicken macrophages in response to APEC infection, we attempted to investigate the miRNAs expression pattern upon APEC infection via miRNA-seq, and to identify the molecular mechanism of the important miRNAs by using RT-qPCR, western blotting, dual-luciferase reporter assay, and CCK-8. The results showed that a total of 80 differentially expressed (DE) miRNAs were identified in comparison of APEC vs. wild-type group, which corresponded to 724 target genes. Moreover, the target genes of the identified DE miRNAs were mainly significantly enriched in the MAPK signalling pathway, autophagy-bird, mTOR signalling pathway, ErbB signalling pathway, Wnt signalling pathway, and TGF-beta signalling pathway. Remarkably, gga-miR-181b-5p is able to participate in host immune and inflammatory responses against APEC infection via targeting of TGFBR1 to modulate the activation of TGF-beta signalling pathway. Collectively, this study provides a perspective of miRNA expression patterns in chicken macrophages upon APEC infection. These findings provide insight into miRNAs against APEC infection, and gga-miR-181b-5p might be a potential target for treating APEC infection.

S-07-5: ASPROSIN INDUCES VASCULAR ENDOTHELIAL- TO-MESENCHYMAL TRANSITION IN DIABETIC LOWER EXTREMITY PERIPHERAL ARTERY DISEASE

Objective: Altered adipokine secretion in dysfunctional adipose tissue facilitates the development of atherosclerotic diseases including lower extremity peripheral artery disease (PAD). Asprosin is a recently identified adipokine and displays potent regulatory role in metabolism, but the relationship between asprosin and lower extremity PAD remains uninvestigated. In this study, we investigated the effect of asprosin accounting for the lower extremity vascular injury in diabetic patients based on the metabolic profiles of PAD. Design and method: 33 type 2 diabetes mellitus (T2DM) patients (DM), 51 T2DM patients with PAD (DM+PAD) and 30 healthy normal control (NC) volunteers were recruited and the blood samples were collected for detecting the circulatory asprosin level and metabolomic screening. RNA sequencing was performed using the aorta tissues from the type 2 diabetic db/db mice and its control db/m mice. Moreover, human umbilical vein endothelial cells (HUVECs) were treated with asprosin to determine its impact on the endothelial-to-mesenchymal transition (EndMT). Results: The circulating levels of asprosin in DM+PAD group were significantly higher than that of NC group and the DM group. Circulating asprosin level was remarkably negatively correlated with ankle-brachial index (ABI), even after adjusting for age, sex, body mass index (BMI) and other traditional risk factors of PAD (P = 0.049). Logistic regression analysis revealed that asprosin is an independent risk factor for PAD (OR:3.944, 95%CI:1.656 − 9.393, P = 0.002) and receiver-operator characteristic (ROC) curve determined a cut-off value at 188.70 ng/ml with a good sensitivity (74.5%) and specificity (74.6%) of asprosin to distinguish PAD (Area under curve:0.788, 95%CI:0.694 − 0.863, P < 0.001). Data from metabolomics displayed a typical characteristics of de novo amino acid synthesis in collagen protein production by myofibroblasts in which TGF-beta signaling pathway might plays a critical role in patients with PAD. In addition, activation of TGF-beta signaling pathway also appeared in the aortic tissue of db/db mice, suggesting a causal relationship between TGF-beta signaling pathway and artery injury. Asprosin directly induces EndMT in HUVECs in a TGF-beta-dependent manner as TGF-beta signaling pathway inhibitor SB431542 erased the promotional effect of asprosin on EndMT. Conclusions: Elevated circulatory asprosin level is an independent risk factor of lower extremity PAD and might serve as a diagnostic marker. Mechanistically, asprosin directly induces EndMT that participates in vascular injury via activation of TGF-beta signaling pathway.

Abstract 1904: Transforming growth factor-beta mediated epithelial to mesenchymal transition contributes to in vivo resistance to sorafenib in hepatocellular carcinoma

Abstract Sorafenib, a multikinase inhibitor targeting Raf kinase and vascular endothelial growth factor receptor, is currently the only approved agent for hepatocellular carcinoma (HCC). However, the majority of HCC patients are inherently resistant to sorafenib treatment. To study molecular mechanisms underlying the resistance to sorafenib in vivo, we studied immunocompromised mice implanted with xenografts of human HCC cells subcutaneously, and identified tumors that showed primary resistance to sorafenib. Primary cultured cells were established from sorafenib-resistant xenografts, and verified for in vitro and in vivo sensitivity to sorafenib. The differentially expressed genes of the resistant cells versus control cells were analyzed by cDNA microarray. The expressions of candidate markers were confirmed by Western blotting, and their significances were verified by specific pathway inhibitors. A subline of Huh7 cells was established by primary culture from a sorafenib-resistant HCC-xenograft (designated as Huh7.1.SR). Compared to Huh7 cells primarily cultured from HCC-xenograft treated with vehicle alone (designated as Huh7.1.v), Huh7.1.SR had a moderate increase of resistance to sorafenib in vitro, with 50%-inhibitory concentration (IC50) increased from 5 to 10 μM. Subcutaneous xenograft studies confirmed that Huh7.1.SR retained the phenotype of in vivo resistance to sorafenib. The IPA (ingenuity pathway analysis) of differentially expressed genes of Huh7.1.SR versus Huh7.1.v cells revealed several pathway networks centering at transforming growth factor beta receptor (TGFBR), hepatocyte nuclear factors, NOTCH, pro-inflammatory cytokines, and nuclear factor kappa B. Huh7.1.SR had an increased expression of TGF-beta and p-Smad2, indicating that the TGF-beta pathway was activated. Huh7.1.SR cells expressed phenotypic features of epithelial to mesenchymal transition (EMT), including higher expression of vimentin, lower expression of E-cadherin, and increased cell migration. The expressions of Slug and Twist, two well-known transcriptional factors of inducing EMT, also increased in Huh7.1.SR cells. Treatment of SD-208, an inhibitor of TGF-beta receptor, partially reversed the phenotypic features of EMT in Huh7.1.SR cells. Moreover, concomitant treatment of SD-208 improved the sensitivity to sorafenib in Huh7.1.SR cells. Our data indicate that activation of TGF-beta signaling pathway is an important mechanism mediating the resistance to sorafenib in HCC in vivo. (The study was supported by the grant NSC 100-2325-B-002-043- from National Science Council of Taiwan) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1904. doi:1538-7445.AM2012-1904

Enhanced Expression of Transcription Factor GATA-4 in Inflammatory Bowel Disease and Its Possible Regulation by TGF-β1

Transforming growth factor beta 1 (TGF-beta1) promotes epithelial healing in inflammatory bowel disease. We hypothesized that GATA-4, a transcription factor cooperating with TGF-beta signaling pathway, is upregulated by TGF-beta1 in the inflamed intestinal epithelium. Normal and inflamed intestinal samples were subjected to immunohistochemistry for GATA-4/6 and the TGF-beta signaling pathway components Smad2/3/4. Proliferation and apoptosis were analyzed using Ki-67 and in situ DNA 3'-end labeling assays and Bax and Bcl-2 immunohistochemistry. Furthermore, GATA-4 was assessed in intestinal Caco-2 cells stimulated with TGF-beta1, or interleukin-6 and tumor necrosis factor alpha. GATA-4 was detected in only 20% of normal intestinal samples, but was upregulated in corresponding inflamed regions. GATA-6 expression remained unchanged during inflammation. TGF-beta1 and Smad3/4, but not Smad2, were expressed concomitantly with GATA-4 in inflamed bowel mucosa. In intestinal Caco-2 cells, TGF-beta1 upregulated GATA-4 and Smad2/3/4, whereas treatment with control cytokines had no effect. Inflammation was associated with increased epithelial cell apoptosis and the enhancement of Bcl-2, but not Bax. We surmise GATA-4 expression is upregulated in inflamed intestine correlating with the activation of TGF-beta signaling pathway. We speculate that TGF-beta1 drives GATA-4 expression during intestinal inflammation, these two components cooperating to promote epithelial healing.

Role of EGF Receptor Activation in Angiotensin II–Induced Renal Epithelial Cell Hypertrophy

For determination of the molecular mechanisms underlying the induction of epithelial cell hypertrophy by angiotensin II (Ang II), a well-characterized porcine renal proximal tubular cell line LLCPKcl4, which does not express endogenous Ang II receptor subtypes, was transfected with cDNA encoding Ang II subtype 1 receptor (AT1R/Cl4). Ang II transactivated the EGF receptor (EGFR) in these AT1R/Cl4 cells, which was blocked by the selective AT1R antagonist losartan but not by the selective AT2R antagonist PD123319. Ang II did not transactivate EGFR in empty vector-transfected LLCPKcl4 cells (Vector/Cl4). Ang II elicited release of soluble heparin-binding EGF-like growth factor (HB-EGF) from AT1R/Cl4 cells, and Ang II-induced EGFR activation was prevented by pretreatment with the specific HB-EGF inhibitor CRM197 or the metalloproteinase inhibitors batimastat or phenanthroline, none of which had any effect on EGFR activation by exogenously administered EGF. Ang II stimulated protein synthesis and cell hypertrophy in AT1R/Cl4 cells without increasing cell number, and signaling studies revealed that Ang II stimulated phosphorylation of the 40S ribosomal protein S6 and the eukaryotic translation initiation factor 4E-binding protein 1, the two downstream target proteins of the mammalian target of rapamycin, which is a central regulator of protein synthesis and cell size. Ang II-induced mammalian target of rapamycin activation, [3H]leucine incorporation, and cellular hypertrophy were inhibited by pretreatment with either batimastat or CRM197 or by pretreatment with rapamycin or the EGFR tyrosine kinase inhibitor AG1478. Ang II also stimulated Smad 2/3 phosphorylation, which was blocked by a selective TGF-beta receptor I kinase inhibitor but not by CRM197. With blockade of TGF-beta receptor, Ang II-mediated hypertrophy was converted into cell proliferation, which was blocked by CRM197. In summary, this is the first demonstration that HB-EGF shedding-dependent EGFR transactivation, along with activation of TGF-beta signaling pathways, mediates Ang II-induced renal tubular epithelial cell hypertrophy.