Keon-Bong Oh

ER stress-inducible ATF3 suppresses BMP2-induced ALP expression and activation in MC3T3-E1 cells

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts.

SMILE inhibits BMP-2-induced expression of osteocalcin by suppressing the activity of the RUNX2 transcription factor in MC3T3E1 cells

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. Several recent studies have suggested that SMILE is a novel co-repressor that is involved in nuclear receptor signaling; however, the role of SMILE in osteoblast differentiation has not yet been elucidated. This study demonstrates that SMILE inhibits osteoblast differentiation by regulating the activity of Runt-related transcription factor-2 (RUNX2). Tunicamycin, an inducer of endoplasmic reticulum stress, stimulated SMILE expression. Bone morphogenetic protein-2-induced expression of alkaline phosphatase and osteocalcin, both of which are osteogenic genes, was suppressed by SMILE. The molecular mechanism by which SMILE affects osteocalcin expression was also determined. An immunoprecipitation assay revealed a physical interaction between SMILE and RUNX2 that significantly impaired the RUNX2-dependent activation of the osteocalcin gene. A ChIP assay revealed that SMILE repressed the ability of RUNX2 to bind to the osteocalcin gene promoter. Taken together, these findings demonstrate that SMILE negatively regulates osteocalcin via a direct interaction with RUNX2.

No expression of porcine endogenous retrovirus after pig to monkey xenotransplantation

This study was performed to investigate the expression of two porcine endogenous retrovirus (PERV) elements, PERV gag and full-length conserved PERV, in blood cells collected periodically from organ-recipient monkeys that underwent pig to non-human primate xenotransplantation. The heart and kidney-respectively acquired from α-1,3-galactosyltransferase knockout (GT-KO) pigs that survived for24 and 25 days-were xenografted into cynomolgus monkeys. The two PERV elements expressed in the xenografted GT-KO pig organs were not present in the blood cells of the recipient monkeys. In the present study, we deduced that PERVs are not transmitted during GT-KO pig to monkey xenotransplantation.

Identification and promoter analysis of PERV LTR subtypes in NIH-Miniature pig

Porcine endogenous retroviruses (PERVs) are integrated into the genomes of all pigs. Since some PERVs can also infect human cells, they represent a potential risk for xenotransplantation involving pig cells or organs. The long terminal repeat (LTR) elements of PERVs show promoter activity that can affect human functional genes; therefore, we examined these elements in this study. We detected several expressed LTRs in the NIH-miniature pig liver, among which we identified 9 different subtypes. When these LTRs were compared, distinct structures that contained several insertion and deletion (INDEL) events and tandem repeats were identified in the U3 region. The transcriptional activity of the 9 LTR subtypes was analyzed in the PK15 porcine cell line and in the HepG2 and Hep3B human liver cell lines, and transcriptional regulation was found to be different in the 3 cell lines. The D LTR subtype was found to have stronger promoter activity than all other types in 4 different human cell lines (HepG2, Hep3B, U251, and 293). Using computational approaches, the D type was shown to contain 4 transcription factor-binding sites distinct from those in the U3 regions of the other subtypes. Therefore, deletion mutants were constructed and examined by a transient transfection luciferase assay. The results of this analysis indicated that the binding site for the Hand1:E47 transcription factor might play a positive role in the transcriptional regulation of PERV LTR subtype D in human liver cell lines.

Small heterodimer partner-interacting leucine zipper protein inhibits adipogenesis by regulating peroxisome proliferator-activated receptor γ activity.

AIMS Adipocytes play a critical role in energy balance. Growth of fat tissue is achieved via an increase in adipocyte mass and the formation of newly differentiated adipocytes from precursor cells. Understanding the cellular and molecular mechanisms of adipocyte differentiation is crucial for the study of obesity- and fat-related diseases. The present study was designed to study whether small heterodimer partner-interacting leucine zipper protein (SMILE), a novel co-repressor, could regulate differentiation of adipocyte in 3T3-L1 cells. MATERIALS AND METHODS Treatment of endoplasmic stress inducers, thapsigargin and tunicamycin, inhibited adipocyte differentiation, stimulated Smile mRNA expression, and repressed the expression of adiponectin (Adipoq) in 3T3-L1 pre-adipocyte. Overexpression of SMILE in 3T3-L1 cells decreased the expression of the mRNA encoding Adipoq, a major marker of adipocytes, significantly. Furthermore, knockdown of SMILE recovered the thapsigargin-mediated repression of Adipoq transcription. Co-immunoprecipitation experiments revealed that SMILE interacted physically with PPARγ in 3T3-L1 cells. In addition, chromatin immunoprecipitation experiments revealed that SMILE suppressed the binding affinity of PPARγ for the Adipoq promoter. KEY FINDINGS We demonstrate that SMILE controls adipocyte differentiation by regulating the transactivity of peroxisome proliferator-activated receptor γ (PPARγ). SIGNIFICANCE These findings demonstrate that SMILE represses adipocyte differentiation by regulating PPARγ transactivity; hence, SMILE is a potential regulator of PPARγ-related diseases.

Methylation and expression changes in imprinted genes H19 and Igf2 during serial somatic cell nuclear transfer using piglet fibroblasts

Cloned pigs produced by somatic cell nuclear transfer (SCNT) are important as a potential alternative source of organs. Although SCNT has created new possibilities for targeted gene modification, the successful cloning of pigs is rare. Here, we successfully conducted serial SCNT for three generations. We determined that the piglet genome was inherited from donor cell nuclei using microsatellite analysis of each generation. The methylation of differentially methylated regions (DMRs) in H19 was gradually reduced over the three generations of serial SCNT. By contrast, methylation of the insulin-like growth factor 2 (Igf2) DMR increased in the F1 generation, compared to the F0, and remained at the higher level in the F2 and F3 generations. The methylation patterns of housekeeping genes such as GAPDH and β-actin were unchanged in the serially cloned pigs. In addition, expression levels of H19 and Igf2 were variable for each generation of serial SCNT piglets, but there was no clear relationship between the methylation and gene expression patterns. Our study conclusively demonstrates that the methylation patterns of DMRs in H19 and Igf2 were altered, compare to the F0 donor, during serial SCNT, but housekeeping genes were unaffected.

In vitro CpG methylation and garcinol reduce PERV LTR promoter activity

Porcine endogenous retroviruses (PERVs) in the pig genome represent a potential risk of infection in pig-to-human transplantation. Long terminal repeats (LTRs) are known to be strong promoter elements that could regulate the transcription activity of PERV elements. It is possible that DNA methylation controls promoter activity of PERV family. Here, we analyzed CpG dinucleotides and CpG islands of six transcribed PERV LTRs. Promoter activity of the LTRs from the six clones methylated by CpG methyltransferase (M. SssI), and luciferase assay after garcinol treatment (histone acetyltransferase inhibitor) were examined, indicating that promoter activity of the PERV LTRs was significantly decreased.

Identification and characterization of transposable element-mediated chimeric transcripts from porcine Refseq and EST databases

Transposable elements are mobile genomic sequences that comprise a large portion of mammalian genomes. The transposable element fusion phenomenon within porcine genes has not yet been reported; therefore, we investigated transposable element fusion genes in the Sus scrofa genome. Porcine transposable element-mediated chimeric transcripts were identified and characterized. Most transposable elements preferentially inserted themselves into an antisense orientation and into the 3’ end of porcine genes. The transposable element fusion gene between porcine mRNA and ERV class I, one of the LTR retrotransposons, was not detected. This data will be of great use to further studies focused on a better understanding of the biological function of porcine genes in relation to transposable elements.

102 PROTEOMIC ANALYSIS OF CONDITIONED MEDIUM SUPPLEMENTED WITH PORCINE FOLLICULAR FLUID

Follicular fluid (FF) contains growth factors, electrolytes, hormones, amino acids, and unknown factors. Supplementation of porcine FF (pFF) to in vitro maturation (IVM) medium was reported to improve the oocyte maturation, monospermic fertilization and embryonic development. This study aimed at investigating whether pFF supplementation affects the characteristics of donor cells for somatic cell nuclear transfer and the proteomic composition of the culture medium. Ear fibroblast cells from an NIH major histocompatibility complex (MHC) inbred miniature pig were cultured with different culture methods: 1) DMEM + 10% FBS (FBS); 2) DMEM + 10% FBS + 10% pFF (pFF). The conditioned medium was collected at 72 h. After isoelectric focusing (IEF), the equilibrated strips were submitted to SDS-PAGE. Normalized protein spots were considered significantly different between the two groups if expression levels varied by two standard deviations. To identify the protein spots, an Ettan MALDI-TOF method was used. Upon submission of the amino acid sequences, proteins were identified by a homology search using ProteinInfo or BLAST search using the ExPASy Molecular Biology Server. The proportion of G0/G1 stage cells in the pFF group was significantly higher than the proportions in the other groups (P < 0.05). Among 42 differentially expressed spots, 36 proteins were identified in the pFF group. Some molecular functions of the spots were: catalytic or methytransferase activity, eukaryotic cell surface binding, or ferric iron binding. It can be concluded that pFF supplementation of culture medium positively affects cell-cycle synchronization and cell metabolism. Further studies are needed to analyse the function of these important cellular proteins. This work received grant support from the Agenda Program (No. PJ006688) and (No. PJ007189), Rural Development Administration, Republic of Korea.

58 DEVELOPMENT OF α1,3-GALACTOSYLTRANSFERASE KNOCK-OUT CLONED PIG EMBRYOS

This study was performed to increase the developmental rate of cloned embryos with the 1,3-Galactosyltransferase (GalT) gene knocked out (KO). Ovaries were collected from local slaughterhouse and immature oocytes were cultured in TCM-199 + 0.1% PVA + FSH + LH (0.5 μg mL-1) + EGF (10 ng mL-1) + 10% porcine follicular fluid (pFF) at 38.5°C in 5% CO2 humidified chamber for 40 h (1-step) or 20 h (with hormone) +20 h (without hormone; 2-step). After IVM, the oocytes with 1st polar body were enucleated and transferred the GalT KO donor cell originated from miniature pig. The embryos transferred with normal mini-pig ear fibroblast cell were used as control. The reconstructed embryos were fused with 2 electric pulses (DC) of 1.2 kV cm-1 for 30 μs. For the development of cloned embryos, the embryos were cultured in PZM-3 under 5% CO2 in air at 38.5°C for 6 days. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle, and pregnancy diagnosis was determined at 28 days after embryo transfer using ultrasonography. Differences among treatment means were determined by a chi-square test. A probability of P < 0.05 was considered statistically significant. The maturation rate was significantly higher in the 2-step method (89.8 ± 2.75) compared with single maturation method (79.6 ± 8.95; P < 0.05). The blastocyst development of cloned embryos reconstructed with GalT KO donor cell (28.4 ± 2.14) was not different from cloned embryos by normal donor cell (27.4 ± 0.01). The cell number of GalT blastocyst (36.1 ± 11.1) was not different statistically from control (26.9 ± 9.3). The apoptosis rate was also not different in both groups (2.9 to 4.9%). Five surrogates were pregnant and the GalT KO fetuses were still ongoing pregnancy at 45 days after embryo transfer. This work received grant support from the Agenda Program (No. 200901FHT010305146 and No. 200901FHT010305535), Rural Development Administration, Republic of Korea.