SOX2 plays a crucial role in cell proliferation and lineage segregation during porcine pre‐implantation embryo development

ObjectivesGene regulation in early embryos has been widely studied for a long time because lineage segregation gives rise to the formation of a pluripotent cell population, known as the inner cell mass (ICM), during pre‐implantation embryo development. The extraordinarily longer pre‐implantation embryo development in pigs leads to the distinct features of the pluripotency network compared with mice and humans. For these reasons, a comparative study using pre‐implantation pig embryos would provide new insights into the mammalian pluripotency network and help to understand differences in the roles and networks of genes in pre‐implantation embryos between species.Materials and methodsTo analyse the functions of SOX2 in lineage segregation and cell proliferation, loss‐ and gain‐of‐function studies were conducted in pig embryos using an overexpression vector and the CRISPR/Cas9 system. Then, we analysed the morphological features and examined the effect on the expression of downstream genes through immunocytochemistry and quantitative real‐time PCR.ResultsOur results showed that among the core pluripotent factors, only SOX2 was specifically expressed in the ICM. In SOX2‐disrupted blastocysts, the expression of the ICM‐related genes, but not OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real‐time PCR analysis, pluripotency‐related genes, excluding OCT4, and proliferation‐related genes were decreased in SOX2‐targeted blastocysts. In SOX2‐overexpressing embryos, the total blastocyst cell number was greatly increased but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated that SOX2 is essential for ICM formation and cell proliferation in porcine early‐stage embryogenesis.     

Regulation of ROMK by extracellular cations

The effect of external potassium (K) and cesium (Cs) on the inwardly rectifying K channel ROMK2 (K(ir)1.1b) was studied in Xenopus oocytes. Elevating external K from 1 to 10 mM increased whole-cell outward conductance by a factor of 3.4 +/- 0.4 in 15 min and by a factor of 5.7 +/- 0.9 in 30 min (n = 22). Replacing external Na by Cs blocked inward conductance but increased whole-cell conductance by a factor of 4.5 +/- 0.5 over a period of 40 min (n = 15). In addition to this slow increase in conductance, there was also a small, rapid increase in conductance that occurred as soon as ROMK was exposed to external cesium or 10 mM K. This rapid increase could be explained by the observed increase in ROMK single-channel conductance from 6.4 +/- 0.8 pS to 11.1 +/- 0.8 pS (10 mM K, n = 8) or 11.7 +/- 1.2 pS (Cs, n = 8). There was no effect of either 10 mM K or cesium on the high open probability (P(o) = 0.97 +/- 0.01; n = 12) of ROMK outward currents. In patch-clamp recordings, the number of active channels increased when the K concentration at the outside surface was raised from 1 to 50 mM K. In cell-attached patches, exposure to 50 mM external K produced one or more additional channels in 9/16 patches. No change in channel number was observed in patches continuously exposed to 50 mM external K. Hence, the slow increase in whole-cell conductance is interpreted as activation of pre-existing ROMK channels that had been inactivated by low external K. This type of time-dependent channel activation was not seen with IRK1 (K(ir)2.1) or in ROMK2 mutants in which any one of 6 residues, F129, Q133, E132, V121, L117, or K61, were replaced by their respective IRK1 homologs. These results are consistent with a model in which ROMK can exist in either an activated mode or an inactivated mode. Within the activated mode, individual channels undergo rapid transitions between open and closed states. High (10 mM) external K or Cs stabilizes the activated mode, and low external K stabilizes the inactivated mode. Mutation of a pH-sensing site (ROMK2-K61) prevents transitions from activated to inactivated modes. This is consistent with a direct effect of external K or Cs on the gating of ROMK by internal pH.     

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M^R) and the human hepatic cancer cell line SNU387 (SNU387^R) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387^R cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M^R cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.     

Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.     

First isolation of Streptococcus downei from human dental plaques

In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.     

Characterization of a thermoacidophilic L-arabinose isomerase from Alicyclobacillus acidocaldarius: role of Lys-269 in pH optimum

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.     

Designer TGFβ superfamily ligands with diversified functionality

Transforming Growth Factor--beta (TGFβ) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGFβ superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-βA and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGFβ superfamily ligands through development of novel chimeric TGFβ ligands with diverse biological and clinical values.     

BMPs and their clinical potentials

Bone morphogenetic protein (BMP) signaling in diseases is the subject of an overwhelming array of studies. BMPs are excellent targets for treatment of various clinical disorders. Several BMPs have already been shown to be clinically beneficial in the treatment of a variety of conditions, including BMP-2 and BMP-7 that have been approved for clinical application in nonunion bone fractures and spinal fusions. With the use of BMPs increasingly accepted in spinal fusion surgeries, other therapeutic approaches targeting BMP signaling are emerging beyond applications to skeletal disorders. These approaches can further utilize next-generation therapeutic tools such as engineered BMPs and ex vivo- conditioned cell therapies. In this review, we focused to provide insights into such clinical potentials of BMPs in metabolic and vascular diseases, and in cancer. [BMB reports 2011; 44(10): 619-634].