Hyunggee Kim

Three Different Turkey Luteinizing Hormone Receptor (tLH-R) Isoforms I: Characterization of Alternatively Spliced tLH-R Isoforms and Their Regulated Expression in Diverse Tissues

Abstract Using combinations of reverse transcription-polymerase chain reaction (RT-PCR) and 5′- and 3′-rapid amplification of cDNA ends, three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms were characterized from ovarian mRNA. The first cDNA (tLH-Rintact) showed 98% and 72–75% similarity with chicken and mammalian LH-R sequences, respectively. The second cloned cDNA isoform (tLH-Rinsert) contained an in-frame TGA stop codon within an 86-base pair insertion that was located in the extracellular domain of the seven-transmembrane region. The tLH-Rinsert isoform could encode a truncated soluble protein isoform that lacked the transmembrane region. The third cDNA isoform truncated the transmembrane region (tLH-Rtrunc) and was derived by the deletion of the last exon by incomplete splicing. Generation of multiple transcripts by alternative splicing was elucidated by partial characterization of tLH-R genomic sequences. The differentially regulated expression of the tLH-R mRNA isoforms in nongonadal tissues and ovarian stromal tissues during various reproductive stages was quantified and analyzed by Northern blot and/or RT-PCR. Alternatively spliced tLH-R isoforms were differentially expressed in a tissue-specific manner in most of the tissues examined. The steady-state levels of tLH-R mRNA isoforms were relatively high in the hypothalamus and optic nerve and relatively low in the cortex, pituitary, and cerebellum when compared to levels in ovarian follicles. In nongonadal reproductive tissues, the steady-state levels of tLH-R mRNA isoforms were relatively high in the uterus and infundibulum and relatively low in the isthmus, oviduct, and magnum. In addition, in the nongonadal peripheral tissues, the steady-state levels of tLH-R isoforms were relatively high in the thyroid gland and relatively low in the spleen, adrenal gland, kidney, skin, bursa, and muscle. The present study suggests that the alternative splicing of LH-R transcripts occurs in a tissue-specific manner and has been evolutionarily conserved (similar results were obtained in chicken and swine). These results raise fundamental questions as to the function of LH-R isoforms in nongonadal tissues.

Alterations in p53 and E2F-1 function common to immortalized chicken embryo fibroblasts

A number of non-virally and non-chemically immortalized chicken embryo fibroblast (CEF) cells have been established recently in continuous cell culture. All immortal CEF cells tested showed common genetic alterations in the expression patterns of p53 and E2F-1 mRNA and protein which were down- and up-regulated, respectively. The biological effects of differentially regulated p53 and E2F-1 were determined by reporter gene transcriptional activity assays, DNA binding assays, and Northern blot analysis of the expression patterns of down-stream genes. In addition, expression of most of the cyclin genes was up-regulated in immortal CEF cells, which may be associated with the rapid cell division rates and serum-independent growth patterns seen in immortal CEF cells. The telomeric lengths and chromosome integrity were maintained in all immortal CEF cell lines without detectable telomerase activity. Although the functional inactivations of the p53 and Rb regulatory pathways are known to be common events for cellular immortalization, the genetic changes leading to alteration of p53 and E2F-1 function through transcriptional and post-transcriptional regulation seem to be unique in immortal CEF cells.

Events in the immortalizing process of primary human mammary epithelial cells by the catalytic subunit of human telomerase.

The in vitro immortalization of primary human mammary epithelial (HME) cells solely by the exogenous introduction of the catalytic subunit of human telomerase (hTERT) has been achieved. Early passage hTERT-transfected HME (T-HME) cells continuously decreased the length and density of telomeres even in the presence of telomerase activity, with a significant number of cells staining positive for senescence-associated beta-galactosidase (SA-beta-gal). Subsequently, with the increase in cell passages, the copy number of the exogenously transfected hTERT gene and the percentage of SA-beta-gal positive cells were found to decrease. Eventually, a single copy of the exogenous hTERT gene was observed in the relatively later passage T-HME cells in which telomere length was elongated and stabilized without obvious activation of endogenous hTERT and c-Myc expression. In T-HME cells, the expression of two p53 regulated genes p21(WAF) and HDM2 increased (as in primary senescent HME cells), and was found to be further elevated as the function of p53 was activated by treatment with DNA-damaging agents. p16(INK4a) was shown to be significantly higher in the primary senescent HME and the early passage T-HME cells when compared with the primary presenescent HME cells, with a dramatic repression of p16(INK4a) observed in the later passage T-HME cells. In addition, the expression of E2F1 and its transcription factor activity were found to be significantly higher in the later passage T-HME cells when compared with the earlier passage T-HME cells. Together, our results indicate that in vitro immortalization in HME cells may require the activation of the function of telomerase and other genetic alterations such as the spontaneous loss of p16(INK4a) expression.

The rapid destabilization of p53 mRNA in immortal chicken embryo fibroblast cells

The steady-state levels of p53 mRNA were dramatically lower in immortal chicken embryo fibroblast (CEF) cell lines compared to primary CEF cells. In the presence of cycloheximide (CHX), the steady-state levels of p53 mRNA markedly increased in immortal CEF cell lines, similar to levels found in primary cells. The de novo synthetic rates of p53 mRNA were relatively similar in primary and immortal cells grown in the presence or absence of CHX. Destabilization of p53 mRNA was observed in the nuclei of immortal, but not primary, CEF cells. The half-life of p53 mRNA in primary cells was found to be a relatively long 23 h compared to only 3 h in immortal cells. The expression of transfected p53 cDNA was inhibited in immortal cells, but restored upon CHX treatment. The 5′-region of the p53 mRNA was shown to be involved in the rapid p53 mRNA destabilization in immortal cells by expression analysis of 5′- and 3′-deleted p53 cDNAs as well as fusion mRNA constructs of N-terminal p53 and N-terminal deleted LacZ genes. Together, it is suggestive that the downregulation of p53 mRNA in immortal CEF cells occurs through a post-transcriptional destabilizing mechanism.

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.

Effects of Active Immunization with Inhibin α Subunit on Reproductive Characteristics of Turkey Hens

Abstract The hypothesis for the present study is that the active immunization of female turkeys with inhibin (INH) would neutralize endogenous INH, and increase levels of circulating follicle stimulating hormone (FSH) and the number of preovulatory follicles, and subsequently enhance egg production. Two experiments were conducted with female turkeys in their first (30 wk of age) and second (62 wk of age) laying cycles. Treatment groups included control turkeys immunized with keyhole limpet hemocyanine (KLH) and experimental turkeys immunized with recombinant turkey inhibinα conjugated to KLH (rtINH), vasoactive intestinal peptide (VIP) conjugated to KLH or rtINH+VIP. Egg production increased (P < 0.05) in VIP and rtINH+VIP immunized birds, but not in rtINH immunized hens in comparison with a control group. A similar number of ovarian follicles, arranged in the follicular hierarchy of laying hens, was observed in all experimental groups. However, there was a larger number of nongraded yellow follicles in rtINH-immunized (62.5%) and rtINH+VIP-immunized (73.5%) groups compared with that of controls, suggesting overstimulation by FSH. Anterior pituitary FSHβ subunit, LHβ subunit, and prolactin (PRL) mRNA contents were determined by Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) in laying hens at the end of the experimental period. Hens immunized with rtINH showed increased FSHβ subunit mRNA content, but no change in the content of LHβ subunit or PRL mRNA. Hens immunized with VIP or rtINH+VIP had significant increases in both pituitary LHβ subunit and FSHβ subunit mRNA contents, accompanied by a decline in PRL mRNA abundance. The magnitude of the increase in FSHβ subunit to INH immunoneutralization was greater in first-cycle hens than in second-cycle hens. These data suggest that active immunization of female turkeys with INH neutralizes endogenous INH and increases both circulating FSH and the number of preovulatory follicles. However, no significant increase in egg production was observed in INH-immunized hens. The data confirm previous reports that VIP immunoneutralization increases egg production in turkey hens and shows for the first time that it also increases FSHβ subunit and LHβ subunit gene expression.

Post-transcriptional inactivation of p53 in immortalized murine embryo fibroblast cells

The steady-state levels of p53 mRNA and protein were barely detectable by Northern and Western blot analysis in spontaneously immortalized (10)3 and (10)7 murine embryo fibroblast (MEF) cells. But when cells were treated with cycloheximide (CHX) or emetine, expression levels were restored to those observed in primary and immortal (10)10 MEF cells. However, levels of p53 mRNA were not changed in primary or (10)10 MEF cells by CHX treatment. De novo p53 mRNA synthetic rates were similar in primary, (10)10, (10)3, and (10)7 MEF cells treated with or without CHX. Treatment with actinomycin D (ActD) showed that p53 mRNA in primary and (10)10 MEF cells had a relatively long half-life of 22 h, compared to less than 2 h for (10)3 and (10)7 MEF cells. Pulse-chase analysis of p53 mRNA turnover using CHX and ActD showed that the rapid destabilization of p53 mRNA in (10)3 and (10)7 MEF cells could be regulated at the transcriptional and translational levels. In addition, the destabilization of p53 mRNA appeared to occur in the nucleus for (10)3 and (10)7 cells, but not for primary and (10)10 MEF cells. Taken together, the present study demonstrates that inactivation of the p53 gene occurs at the post-transcriptional level by rapid destabilization of its mRNA in the nucleus of spontaneously immortalized (10)3 and (10)7 MEF cells.

Three different Turkey luteinizing hormone receptor (tLH-R) isoforms II: Characterization of differentially regulated tLH-R messenger ribonucleic acid isoforms in the ovary

Abstract We have recently characterized three different, alternatively spliced, partial turkey LH receptor (tLH-R) cDNA isoforms by the combination of reverse transcription-polymerase chain reaction (RT-PCR) and 5′- and 3′-rapid amplification of cDNA ends. The first cDNA (intact form: tLH-Rintact) showed 98% and 72–75% similarity with chicken and mammalian LH receptor sequences, respectively. The other two cloned cDNA isoforms (insertion and truncated forms: tLH-Rinsert and tLH-Rtrunc) could encode truncated soluble protein isoforms that lack the transmembrane region. Northern blot analysis detected two transcripts of 3.0 kilobases (kb) (tLH-Rintact) and 1.5 kb (tLH-Rtrunc) in the turkey ovary but could not discriminate a third alternatively spliced transcript (tLH-Rinsert) due to the small 86-base pair difference in the size range of approximately 3.0-kb mRNAs. But with the combination of RNase protection assay, RT-PCR, and Northern blot analysis, three different alternatively spliced tLH-R mRNA isoforms were quantified. Differential expression of the tLH-R mRNA isoforms was demonstrated in ovarian stromal tissue during various reproductive stages and in the theca and granulosa layer through follicular development. To gain a better understanding of the physiological significance of the three different tLH-R isoforms, total RNA from the theca layer through follicular development after prolactin (PRL) treatment was analyzed by RT-PCR. PRL treatment for 8–14 days significantly increased the steady-state levels of total tLH-R mRNAs, including tLH-Rinsert and tLH-Rtrunc mRNAs, compared to those in nontreated controls. In contrast, the steady-state levels of tLH-Rintact mRNA during the same period was not significantly changed when compared to that in nontreated controls. The present study shows that tLH-R transcripts are alternatively spliced in a tissue-specific manner in the turkey and that the mechanism may, in part, be controlled hormonally.

Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha.

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHalpha. The steady-state mRNA levels of cDcoHalpha were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHalpha showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHalpha increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHalpha mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHalpha mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHalpha probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHalpha showed similar functional activity as measured by increased expression of a reporter gene, as well as alpha-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHalpha might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states.

Gonad-specific expression of two novel chicken complementary DNA isoforms.

Abstract Differential display reverse transcription polymerase chain reaction was used to isolate a novel cDNA clone (C47) that was initially shown to be downregulated in senescent chicken embryo fibroblast cells. In a tissue environment, C47 transcripts were only detected in gonadal tissue. The expression of the larger isoform (C47L) was essentially restricted to the ovary, and the smaller isoform (C47S) was predominately expressed in the testis. Although levels of the C47L mRNA were relatively high in both the small white and the developing larger follicles, there was very low expression in regressed and postovulated follicles. Nucleotide sequence analysis indicated that two different transcripts of the single-copy C47 gene were generated by differential polyadenylation in the 3′ untranslated region. As a result of a single nucleotide deletion, the C47L mRNA produced a smaller 48-kDa protein, and the C47S mRNA generated a larger 57-kDa protein when both were translated in vitro. Both protein isoforms were shown to contain conserved C2H2 Zn finger motifs and nuclear localization signals suggestive of being putative transcription factors. These results suggest that the C47L and C47S isoforms might play an important role in the regulation and maintenance of ovarian and testicular functions, respectively, in the chicken.