Epithelial Wnt/β-catenin signaling regulates palatal shelf fusion through regulation of Tgfβ3 expression

The canonical Wnt/β-catenin signaling plays essential role in development and diseases. Previous studies have implicated the canonical Wnt/β-catenin signaling in the regulation of normal palate development, but functional Wnt/β-catenin signaling and its tissue-specific activities remain to be accurately elucidated. In this study, we show that functional Wnt/β-catenin signaling operates primarily in the palate epithelium, particularly in the medial edge epithelium (MEE) of the developing mouse palatal shelves, consistent with the expression patterns of β-catenin and several Wnt ligands and receptors. Epithelial specific inactivation of β-catenin by the K14-Cre transgenic allele abolishes the canonical Wnt signaling activity in the palatal epithelium and leads to an abnormal persistence of the medial edge seam (MES), ultimately causing a cleft palate formation, a phenotype resembling that in Tgfβ3 mutant mice. Consistent with this phenotype is the down-regulation of Tgfβ3 and suppression of apoptosis in the MEE of the β-catenin mutant palatal shelves. Application of exogenous Tgfβ3 to the mutant palatal shelves in organ culture rescues the midline seam phenotype. On the other hand, expression of stabilized β-catenin in the palatal epithelium also disrupts normal palatogenesis by activating ectopic Tgfβ3 expression in the palatal epithelium and causing an aberrant fusion between the palate shelf and mandible in addition to severely deformed palatal shelves. Collectively, our results demonstrate an essential role for Wnt/β-catenin signaling in the epithelial component at the step of palate fusion during palate development by controlling the expression of Tgfβ3 in the MEE.     

Elevated COX2 expression and PGE2 production by downregulation of RXRα in senescent macrophages

Increased systemic level of inflammatory cytokines leads to numerous age-related diseases. In senescent macrophages, elevated prostaglandin E2 (PGE2) production contributes to the suppression of T cell function with aging, which increases the susceptibility to infections. However, the regulation of these inflammatory cytokines and PGE2 with aging still remains unclear. We have verified that cyclooxygenase (COX)-2 expression and PGE2 production are higher in LPS-stimulated macrophages from old mice than that from young mice. Downregulation of RXRα, a nuclear receptor that can suppress NF-κB activity, mediates the elevation of COX2 expression and PGE2 production in senescent macrophages. We also have found less induction of ABCA1 and ABCG1 by RXRα agonist in senescent macrophages, which partially accounts for high risk of atherosclerosis in aged population. Systemic treatment with RXRα antagonist HX531 in young mice increases COX2, TNF-α, and IL-6 expression in splenocytes. Our study not only has outlined a mechanism of elevated NF-κB activity and PGE2 production in senescent macrophages, but also provides RXRα as a potential therapeutic target for treating the age-related diseases.     

Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation

The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3–SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3–SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3–SH2 connector, and SH2 domain of SLAP2 (SLAP2–32). While both domains adopt typical folds, the short SH3–SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding.     

β-Elemene piperazine derivatives induce apoptosis in human leukemia cells through downregulation of c-FLIP and generation of ROS

β-Elemene is an active component of the herb medicine Curcuma Wenyujin with reported antitumor activity. To improve its antitumor ability, five novel piperazine derivatives of β-elemene, 13-(3-methyl-1-piperazinyl)-β-elemene (DX1), 13-(cis-3,5-dimethyl-1-piperazinyl)-β-elemene (DX2), 13-(4-ethyl-1-piperazinyl)-β-elemene (DX3), 13-(4-isopropyl-1-piperazinyl)-β-elemene (DX4) and 13-piperazinyl-β-elemene (DX5), were synthesized. The antiproliferative and apoptotic effects of these derivatives were determined in human leukemia HL-60, NB4, K562 and HP100-1 cells. DX1, DX2 and DX5, which contain a secondary amino moiety, were more active in inhibiting cell growth and in inducing apoptosis than DX3 and DX4. The apoptosis induction ability of DX1 was associated with the generation of hydrogen peroxide (H(2)O(2)), a decrease of mitochondrial membrane potential (MMP), and the activation of caspase-8. Pretreatment with the antioxidants N-acetylcysteine and catalase completely blocked DX1-induced H(2)O(2) production, but only partially its activation of caspase-8 and induction of apoptosis. HL-60 cells were more sensitive than its H(2)O(2)-resistant subclone HP100-1 cells to DX1-induced apoptosis. The activation of caspase-8 by these compounds was correlated with the decrease in the levels of cellular FLICE-inhibitory protein (c-FLIP). The proteasome inhibitor MG-132 augmented the decrease in c-FLIP levels and apoptosis induced by these derivatives. FADD- and caspase-8-deficient Jurkat subclones have a decreased response to DX1-induced apoptosis. Our data indicate that these novel β-elemene piperazine derivatives induce apoptosis through the decrease in c-FLIP levels and the production of H(2)O(2) which leads to activation of both death receptor- and mitochondrial-mediated apoptotic pathways.     

Combination of valproic acid and ATRA restores RARβ2 expression and induces differentiation in cervical cancer through the PI3K/Akt pathway

Epigenetic silencing of the tumor suppressor gene, RARβ2, through histone deacetylation has been established as an important process of cervical carcinogenesis. This pivotal role has led to the suggestion that a combination of retinoids selective for RARβ2 with histone deacetylase (HDAC) inhibitors may have therapeutic potential. Valproic acid (VPA), a HDAC inhibitor, has a critical role in the regulation of gene expression through histone acetylation and causes transformed cells to undergo growth arrest, differentiation, and apoptosis. Therefore, we hypothesized that the combination of VPA and ATRA could restore RARβ2 expression, thus resulting in enhanced anti-neoplastic activity in cervical cancer. Here, we show that VPA combined with ATRA led to hyperacetylation of histone H3 and a significant alteration of gene expression in cervical cancer cells, including RARβ2 gene expression, which was upregulated 50- to 90-fold. The combination therapy effectively inhibited the growth of cervical cancer cells more than the single agent treatment both in vitro and in vivo. The additive effects were associated with a significant upregulation of p21(CIP1) and p53 as well as a pronounced decrease in p-Stat3. Furthermore, the combined treatment led to cell cycle arrest predominantly at the G1 phase, and it preferentially induced cell differentiation rather than apoptosis in cervical cancer cells. The differentiation program was determined by the presence of E-cadherinmediated adhesion and activation of the PI3K/Akt pathway. Taken together, these results provide new insight into the mechanisms of enhanced antitumor activity of the HDAC inhibitor and ATRA regimen, thus offering a new therapeutic strategy for cervical cancer patients.     

Genome change in wheat observed through the structure and expression of α/β-gliadin genes

To better understand genome structure and the expression of α/β-gliadin multigenes in hexaploid wheat, bacterial artificial chromosome (BAC) clones containing α/β-gliadin genes from the three loci, Gli-A2, Gli-B2, and Gli-D2, were screened. Based on their restriction fragment patterns, we selected five BAC clones, namely, two clones for Gli-A2, two clones for Gli-B2, and one clone for Gli-D2, to fully sequence. Approximately 200 kb was sequenced for each locus. In total, twelve α/β-gliadin intact genes and four pseudogenes were found, and retrotransposons or other transposons existed in each BAC clone. Dot-plot analysis revealed the pattern of genome segmental duplication within each BAC. We calculated time since duplication of each set of α/β-gliadin genes and insertion of retrotransposons. Duplication of all adjacent genes within the same BAC clone took place before or after allotetrapolyploidization, but duplication of certain genes occurred before diploid differentiation of wheat species. Retrotransposons were also inserted before and after the segmental duplication events. Furthermore, translocation of α/β-gliadin genes from chromosomes 1 to 6 apparently occurred before the diversification of various wheat genomes. Duplication of genome segments containing α/β-gliadin genes and retrotransposons were brought about through unequal crossing-over or saltatory replication and α/β-gliadin genes per se were duplicated without any recombination events. Out of twelve intact α/β-gliadin genes detected from their sequences, nine were expressed, although their patterns of expression were distinct. Since they have similar cis-elements and promoter structures, the mechanisms underlying their distinct gene expression and possible applications are discussed.     

TGF-β1 suppresses IL-6-induced STAT3 activation through regulation of Jak2 expression in prostate epithelial cells

Chronic inflammation plays an important role in the initiation and progression of various human diseases including benign prostatic hyperplasia or prostate cancer. Here we show that the proinflammatory cytokine interleukin-6 (IL-6) has prosurvival effects and chronically activates the Jak2/STAT3 signalling pathway in a model of benign prostatic hyperplasia (BPH-1). We demonstrate that the antiinflammatory cytokine transforming growth factor-β1 (TGF-β1), which also permanently activates its canonical signalling pathway through SMAD proteins in BPH-1 cells, modifies the effects of IL-6 on cell proliferation. Importantly, TGF-β1 inhibits IL-6 signal transduction by decreasing the phosphorylation levels of STAT3. This effect is associated with decreased expression of Jak2 at both mRNA and protein levels. Moreover, we showed that TGF-β1 inhibits IL-6-induced expression of the cancer-associated gene MUC1. These observations demonstrated a novel interaction between TGF-β1 and IL-6 signalling and suggested another mechanism of how defects in TGF-β signalling, frequently associated with prostate pathologies, can contribute to the disruption of tissue homeostasis.     

MicroRNA-197 induces epithelial–mesenchymal transition and invasion through the downregulation of HIPK2 in lung adenocarcinoma

The major cause of cancer-related deaths in patients with lung adenocarcinoma (LAD) is due to distant metastasis. Many reports have indicated that miRNA plays a key role in tumour metastasis. The expression of miR-197 is correlated with LAD progression, however, the mechanism of miR-197 is still unknown in the processing of LAD. A Boyden chamber migration/invasion assay was used for the metastatic function study in vitro. Real-time PCR and Western blot assays were employed to analyse the EMT hallmark changes in both the mRNA and protein levels. \(3^{\prime }\)-UTR reporter luciferase assay was used to show that HIPK2 is a direct target of miR-197. miR-197 enhances LAD cell migration and invasion miR-197. The downregulation of miR-197 suppresses the EMT and migration ability. HIPK2 is a direct functional target of miR-197 in LAD metastasis. In summary, miR-197 controls EMT and metastasis by directly silencing HIPK2. The findings suggest that interfering with the miR-197-dependent regulation of HIPK2 could be a useful approach for the treatment of patients with late stage metastatic LAD.     

Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-β

Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A₂, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A₂ synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-κβ signaling pathway.