Differential regulation of CDX1 and CDX2 gene expression by deficiency in methyl group donors

The CDX2 and CDX1 homeobox genes have respectively a tumour suppressor and proliferative role in the intestinal epithelium. We analyzed DNA methylation and histones modifications associated with CDX2 and CDX1 promoters in two human colon cancer cell lines expressing differentially these genes, Caco2/TC7 [CDX2 positive-CDX1 negative] and HT29 [CDX2 negative-CDX1 negative] cells. Chromatin immunoprecipitation experiments indicated that CDX2 and CDX1 gene expression correlated with a histone modifications pattern characterizing active chromatin (H3K4 trimethylated and H3 acetylated). Bisulfite DNA sequencing and methylation-specific PCR showed that CDX2 and CDX1 promoters display no methylation in HT29 cells even though both genes are not expressed. In contrast, the CDX1 promoter is methylated in Caco2/TC7. DNA demethylation by 5aza-dC or the combination of 5aza-dC plus SAHA, an inhibitor of histone deacetylases, restored CDX1 expression in Caco2/TC7 cells but these treatments were inefficient on both CDX2 and CDX1 in HT29 cells. Thus, in colon cancer cells the changes in chromatin conformation are heterogeneous and repression of CDX2 and CDX1 in HT29 cells is not due to epigenetic mechanisms. In vivo, dietary deprivation of methyl groups in rats upregulated CDX1 mRNA and downregulated to a lesser extent CDX2 mRNA expression. Moreover, methyl group deprivation downregulated CDX2 protein by changing its phosphorylation pattern. The changes in CDX2 and CDX1 expression determined by methyl group deprivation may constitute one of the mechanisms sustaining the protective role attributed to folate in colon cancer.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Suppression of migratory and metastatic pathways via blocking VEGFR1 and VEGFR2

Abstract

Background: Vascular endothelial growth factor (VEGF) A and B are endothelial cell mitogens whose ligation to VEGFR1/VEGFR2 drives tumor angiogenesis and metastasis, and epithelial-mesenchymal transition (EMT). Blockade of these signaling axes could be obtained by disturbing the interactions between VEGFA and/or VEGFB with VEGFR1 and/or VEGFR2.

Methods: A 14-mer peptide (VGB) that recognizes both VEGFR1 and VEGFR2 were investigated for its inhibitory effects on the VEGF‐induced proliferation and migration using MTT and scratch assay, respectively. Downstream signaling pathways were also assessed by quantitative estimation of gene and protein expression using real-time PCR and immunohistochemistry (IHC).

Results: We investigated the inhibitory effects of VGB on downstream mediators of metastasis, including epithelial-cadherin (E-cadherin), matrix metalloprotease-9 (MMP-9), cancer myelocytomatosis (c-Myc), and nuclear factor-κβ (NF-κβ), and migration, comprising focal adhesion kinase (FAK) and its substrate Paxilin. VGB inhibited the VEGF‐induced proliferation of human umbilical vein endothelial cells (HUVECs), 4T1 and U87 cells in a time- and dose-dependent manner and migration of HUVECs. Based on IHC analyses, treatment of 4T1 mammary carcinoma tumor with VGB led to the suppression of p-AKT, p-ERK_1/2, MMP-9, NF-κβ, and activation of E-cadherin compared with PBS-treated controls. Moreover, quantitative real-time PCR analyses of VGB-treated tumors revealed the reduced expression level of FAK, Paxilin, NF-κβ, MMP-9, c-Myc, and increased expression level of E-cadherin compared to PBS-treated controls.

Conclusions: Our results demonstrated that simultaneous blockade of VEGFR1/VEGFR2 is an effective strategy to fight solid tumors by targeting a wider range of mediators involved in tumor angiogenesis, growth, and metastasis.     

Response surface optimization of prodigiosin production by phthalate degrading Achromobacter denitrificans SP1 and exploring its antibacterial activity

Abstract

The role of various parameters like temperature, pH, blood bag concentration, agitation and incubation that influence the production of prodigiosin by Achromobacter denitrificans SP1 was determined. The Plackett–Burman and Box–Behnken experimental designs were employed to statistically optimize and find out the best combinational effect of parameters for the better yield of prodigiosin using blood bag as sole carbon and energy source for the growth of A. denitrificans SP1. The maximum (1.314?mg/ml) prodigiosin production was attained at a temperature of 24?°C, pH (8.8), and blood bag (1?g) as optimum; while the predicted value was 1.319?mg/ml with a correlation coefficient of 0.987; which signifies the fitness of the model. Antimicrobial activity of the prodigiosin was also evaluated and found to be an effective agent against bacterial pathogens including Staphylococcus aureus and Proteus mirabilis. Utilization of the plasticizer di (2-ethylhexyl)phthalate (DEHP) in blood bag and the production of antibacterial prodigiosin makes A. denitrificans SP1, an effective competitor toward the pathogenic bacterial disinfection and wastewater treatment processes.     

Upregulation of fibronectin expression by COX-2 is mediated by interaction with ELMO1

Engulfment and cell motility 1 (ELMO1), a bipartite guanine nucleotide exchange factor (GEF) for the small GTPase Rac 1, was identified as a susceptibility gene for glomerular disease. Here, we reported that ELMO1 interacted with COX-2 in human mesangial cells. Furthermore, we identified ELMO1 as a posttranslational regulator of COX-2 activity. We demonstrated that COX-2 cyclooxygenase activity increased fibronectin promoter activity. The protein-protein interaction between ELMO1 and COX-2 increased the cyclooxygenase activity of COX-2 and, correspondingly, fibronectin expression. We also found that ET625, the dominant negative form of ELMO1 lacking Rac1 activity, interacted with COX-2, increased cyclooxygenase activity of COX-2 and enhanced COX-2-mediated fibronectin upregulation. To further rule out Rac1 as an ELMO1-mediated regulator of COX-2 activity, we employed the constitutive active Rac1, Rac1^Q63E, and demonstrated that Rac1 signaling has no effect on COX-2-mediated fibronectin promoter activity. These results suggest that ELMO1 contributes to the development of glomerular injury through serving as a regulator of COX-2 activity. The interaction of ELMO1 with COX-2 could play an important role in the development and progression of renal glomerular injury.