HNF1α and CDX2 transcriptional factors bind to cadherin-17 (CDH17) gene promoter and modulate its expression in hepatocellular carcinoma.

Cadherin-17 (CDH17) belongs to the cell adhesion cadherin family with a prominent role in tumorigenesis. It is highly expressed in human hepatocellular carcinoma (HCC) and is proposed to be a biomarker and therapeutic molecule for liver malignancy. The present study aims to identify the transcription factors which interact and regulate CDH17 promoter activity that might contribute to the up-regulation of CDH17 gene in human HCC. A 1-kb upstream sequence of CDH17 gene was cloned and the promoter activity was studied by luciferase reporter assay. By bioinformatics analysis, deletion and mutation assays, and chromatin immunoprecipitation studies, we identified hepatic nuclear factor 1α (HNF1α) and caudal-related homeobox 2 (CDX2) binding sites at the proximal promoter region which modulate the CDH17 promoter activities in two HCC cell lines (Hep3B and MHCC97L). A consistent down-regulation of CDH17 and the two transcriptional activators (HNF1α and CDX2) expression was found in the liver of mouse during development, as well as in human liver cancer cells with less metastatic potential. Suppression of HNF1α and CDX2 expression by small interfering RNA (siRNA) significantly down-regulated expressions of CDH17 and its downstream target cyclin D1 and the viability of HCC cells in vitro. In summary, we identified the minimal promoter region of CDH17 that is regulated by HNF1α and CDX2 transcriptional factors. The present findings enhance our understanding on the regulatory mechanisms of CDH17 oncogene in HCC, and may shed new insights into targeting CDH17 expression as potential therapeutic intervention for cancer treatment.

Preimplantation embryos cooperate with oviductal cells to produce embryotrophic inactivated complement-3b.

Human oviductal epithelial (OE) cells produce complement protein 3 (C3) and its derivatives, C3b and inactivated complement-3b (iC3b). Among them, iC3b is the most potent embryotrophic molecule. We studied the production of iC3b in the oviductal cell/embryo culture system. In the immune system, C3 convertase converts C3 into C3b, and the conversion of C3b to iC3b requires factor I (fI) and its cofactors, such as factor H or membrane cofactor protein. Human oviductal epithelium and OE cells expressed mRNA and protein of the components of C3 convertase, including C2, C4, factor B, and factor D. The OE cell-conditioned medium contained active C3 convertase activity that was suppressed by C3 convertase inhibitor, H17 in a dose and time-dependent manner. Although the oviductal epithelium and OE cells produced fI, the production of its cofactor, factor H required for the conversion of C3b to iC3b, was weak. Thus, OE cell-conditioned medium was inefficient in producing iC3b from exogenous C3b. On the contrary, mouse embryos facilitated such conversion to iC3b, which was taken up by the embryos, resulting in the formation of more blastocysts of larger size. The facilitatory activity was mediated by complement receptor 1-related gene/protein Y (Crry) with known membrane cofactor protein activity on the trophectoderm of the embryos as anti-Crry antibody inhibited the conversion and embryotrophic activity of C3b in the presence of fI. In conclusion, human oviduct possesses C3 convertase activity converting C3 to C3b, and Crry of the preimplantation embryos may be involved in the production of embryotrophic iC3b on the surface of the embryos.

Characterization of an acrosome protein VAD1.2/AEP2 which is differentially expressed in spermatogenesis.

The release of enzymes from the acrosome of the sperm head (acrosome reaction) starts the fertilization process and enables the spermatozoa to penetrate the zona pellucida of the oocytes. Defective acrosome reaction is one of the important causes of infertility in men. To investigate the molecular regulation of spermatogenesis in vivo, we used differential display reverse transcription-polymerase chain reaction to identify stage-specific genes in a retinol-supplemented vitamin-A deficiency (VAD) rat model and identified the VAD1.2 (acrosome-expressed protein 2, AEP2) gene, which was expressed strongly in the rat testis from post-natal day 32 to adult stage. The mouse VAD1.2 mRNA shared 85% and 67% sequence homology, and 74% and 38% amino acid homology, respectively, with the rat and human counterparts. VAD1.2 transcript was abundantly expressed in the rat seminiferous tubules at stage VIII-XII, and the protein was detected in the acrosome region of the round and elongated spermatids of mouse, human, monkey and pig. VAD1.2 co-localized with lectin-PNA to the acrosome region of spermatids. Interestingly, the expression of VAD1.2 protein in human testis diminished in patients with hypospermatogenesis, maturation arrest, undescended testis and Sertoli cell-only syndrome. Co-immunoprecipitation experiments followed by western blotting and mass spectrometry (MS-MS) identified syntaxin 1, beta-actin and myosin heavy chain (MHC) proteins as putative interacting partners. Taken together, the stage-specific expression of VAD1.2 in the acrosome of spermatids and the binding of VAD1.2 protein with vesicle forming (syntaxin 1) and structural (beta-actin and MHC) proteins suggest that VAD1.2 maybe involved in acrosome formation during spermiogenesis.

Junction interaction in the seminiferous epithelium: regulatory roles of connexin-based gap junction.

Anchoring junction, tight junction (TJ), and gap junction (GJ) constitute three major junction types in mammalian testes. Connexin is the well-studied GJ protein. It forms the building block of connexon, which is composed of six connexin units. Connexon forms the functional GJ when pairing with counter-connexon from neighboring cells. In the testis, at least eleven connexins are associated with the Sertoli and germ cells of the seminiferous epithelium and the Leydig cells of the interstitium, modulating spermatogenesis and steroidogenesis, respectively. Significantly, connexins are recently speculated to act as regulators of other junctions in the testes using pan-connexin peptide model. This demonstrates that the loss of connexin function leads to a preferential degradation of occludin-based TJ, but not N-cadherin-based adherens junction (AJ), in the testis, despite the intermingled relationship of these three junctions at the site of blood-testis barrier. In the clinical aspects, connexins are shown to relate to male infertility and testicular dysfunctions. A panel of molecules and proteins and their associated protein kinases are actively participating in the regulation of connexin-mediated GJ and fine-tuning connexin-associated functions in the testis. Herein, we summarize the latest findings of connexins in the testis in the aspects of fertility, and testicular diseases, with emphasis on the unexplored roles of connexins in regulating other junction types. This can shed light on future studies in implicating the putative roles of connexins in the physiological functions of reproduction and the clinical aspects of male infertility. In addition, understanding the roles of connexins can advance the diagnosis and treatment of testicular dysfunction and infertility.