Myoungkun Jeoung

Kalirin is Under-Expressed in Alzheimer's Disease Hippocampus

To identify genes aberrantly expressed in the brain of individuals with Alzheimers Disease (AD), we analyzed RNA extracts from the hippocampus and cerebellum from 19 AD patients and 15 age- and sex-matched control subjects. Our analysis identified a number of genes that were over-expressed or under-expressed specifically in AD hippocampus. Among these genes, kalirin was the most consistently under-expressed in AD hippocampus, which was verified by semi-quantitative RT-PCR and real time PCR. Kalirin is predominantly expressed in the brain, particularly in the hippocampus, and plays crucial roles in neuronal stability and growth. Our observation is the first to relate kalirin to AD and a human disease. In addition to kalirin, the genes for voltage-gated Ca++ channel gamma subunit 3 and visinin-like protein 1 (a Ca++ sensor protein) were under-expressed, whereas inositol 1,4,5-triphosphate 3-kinase B was over-expressed in AD hippocampus. Collectively, these differential expressions could severely impair calcium homeostasis. Remarkably, these aberrant gene expressions in AD hippocampus were not observed in AD cerebellum. Furthermore, housekeeping genes such as ribosomal protein genes are not affected by AD. These results provide new insights into the biochemistry of AD.

Identification of a Novel Role for Endothelins within the Oviduct

Endothelins were first identified as potent vasoactive peptides; however, diversity in the biological function of these hormones is now evident. We have identified a novel role for endothelins: a requirement for these peptides within the oviduct during fertilization and/or early embryo development. In vivo, treatment after ovulation with a dual endothelin receptor antagonist (tezosentan) decreased the number of two-cell embryos that could be collected from within the oviducts. In vitro fertilization experiments showed that gamete viability and their ability to fertilize were not affected by treatment with this antagonist, suggesting that the effect observed in vivo was mediated by the oviduct itself. Expression of mRNA for all three isoforms of the endothelins and both receptor subtypes was detectable within the oviduct. Expression of mRNA for endothelin-3 was regulated by gonadotropins in epithelial cells of the oviduct and increased specifically within the isthmus of this structure. Immunostaining revealed localization of both endothelin receptors A and B to the columnar epithelial cells within the oviduct, suggestive of a local role for endothelins in the regulation of epithelial function and ultimately oviductal secretions. A microarray analysis revealed three likely endothelin-regulated protein networks for future analysis: the TGFbeta, IL-10, and CCAAT/enhancer-binding protein superfamilies. Overall, these results suggest a novel and requisite role for endothelins within the oviduct during fertilization and/or early embryo development.

Production and binding of endothelin-2 (EDN2) in the rat ovary: endothelin receptor subtype A (EDNRA)-mediated contraction.

Endothelin-2 (EDN2)-mediated contraction has been proposed as a final mechanical signal facilitating ovulation. The objectives herein were to determine (1) whether ovarian endothelins were increased before ovulation; (2) whether a specific endothelin-converting enzyme (ECE) was mediating their production; (3) which receptor was facilitating ovarian contraction; and (4) whether receptor-specific antagonism affected ovulation. Follicular development was induced in immature rats with 10 IU pregnant mare serum gonadotrophin (PMSG) and the ovulatory cascade was initiated 48 h later with 10 IU human chorionic gonadotrophin (hCG). In Experiment 1, an immunoassay revealed that the ovarian concentration of endothelin peptide was increased 7-fold 12 h after hCG when compared with 48 h after PMSG (P < 0.05). In Experiment 2, real-time PCR indicated that mRNA for Ece1, but not Ece2, was increased in granulosa cells collected 12 h after hCG when compared with those collected before the ovulatory stimulus (P < 0.05). In Experiment 3, isometric tension analysis revealed that the contractile effect of EDN2 was mediated by endothelin receptor A (EDNRA), not B (EDNRB). In Experiment 4, no effect was observed on the rate of ovulation when rats were treated with an antagonist specific to EDNRA (BQ123) or EDNRB (BQ788), or when mice were treated with BQ123, BQ788 or BQ123 + BQ788. In conclusion, endothelin peptide is produced before ovulation and the contractile action of EDN2 within the ovary is facilitated via EDNRA. In addition, findings of this study indicate synergistic interactions among contractile factors affect ovulatory outcome, while the role of EDNRB alone in the process of ovulation requires further investigation.

Follicle-stimulating hormone interacts with exoloop 3 of the receptor.

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of ∼330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of580KVPLITVSKAK590 was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH α but not FSH β. This is in contrast to photoaffinity labeling of FSH β by the peptide mimic of the N-terminal region of the receptor. Leu583 and Ile584 are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu583 and Ile584 side chains appear to project in opposite directions. Ile584 appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu583 is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu583 improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile584 and Lys590abolished the cAMP and IP induction. Our results open a logical question whether Leu583, Ile584, and Lys590 interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.

Trans-activation, cis-activation and signal selection of gonadotropin receptors

It has been thought that when a hormone binds to a receptor, the liganded receptor activates itself and generates hormone signals, such as the cAMP signal and the inositol phosphate signal (cis-activation). We describe that a liganded LH receptor or FSH receptor molecule is capable of intermolecularly activating nonliganded receptors (trans-activation). Particularly, intriguing is the possibility that a pair of compound heterozygous mutants, one defective in binding and the other defective in signaling, may cooperate and rescue signaling. Furthermore, trans-activation of the binding deficient receptors examined in our studies generates either the cAMP signal or the IP signal, but not both. Trans-activation and selective signal generation have broad implications on signal generation mechanisms, and suggest new therapeutic approaches.

Methodology matters: IVF versus ICSI and embryonic gene expression

The use of assisted reproduction treatment, especially intracytoplasmic sperm injection (ICSI), is now linked to a range of adverse consequences, the aetiology of which remains largely undefined. Our objective of this study was to determine differences in gene expression of blastocysts generated by ICSI as well as ICSI with artificial oocyte activation (ICSI-A) versus the less manipulative IVF, providing fundamental genetic information that can be used to aid in the diagnosis or treatment of those adversely affected by assisted reproduction treatment, as well as stimulate research to further refine these techniques. Murine blastocysts were generated by ICSI, ICSI-A and IVF, and processed for a microarray-based analysis of gene expression. Ten blastocysts were pooled for each procedure and three independent replicates generated. The data were then processed to determine differential gene expression and to identify biological pathways affected by the procedures. In blastocysts derived by ICSI versus IVF, the expression of 197 genes differed (P < 0.01). In blastocysts derived by ICSI-A versus IVF and ICSI-A versus ICSI, the expression of 132 and 65 genes differed respectively (P < 0.01). Procedural-induced changes in genes regulating specific biological pathways revealed some consistency to known adverse consequences. Detailed investigation of procedure-specific dysfunction is therefore warranted.

Orientation of Follicle-stimulating Hormone (FSH) Subunits Complexed with the FSH Receptor β SUBUNIT TOWARD THE N TERMINUS OF EXODOMAIN AND α SUBUNIT TO EXOLOOP 3

Follicle-stimulating hormone (FSH) comprises an α subunit and a β subunit, whereas the FSH receptor consists of two halves with distinct functions: the N-terminal extracellular exodomain and C-terminal membrane-associated endodomain. FSH initially binds to exodomain, and the resulting FSH/exodomain complex modulates the endodomain and generates signal. However, it has been difficult to determine which subunit of FSH contacts the exodomain or endodomain and in what orientation FSH interacts with them. To address these crucial issues, the receptor was Ala-scanned and the hormone subunits were probed with photoaffinity labeling with receptor peptides corresponding to the N-terminal region of the exodomain and exoloop 3 of the endodomain. Our results show that both regions of the receptors are important for hormone binding and signal generation. In addition, the FSH β subunit is specifically labeled with the N-terminal peptide, whereas the α subunit is labeled with the exoloop 3 peptide. These contrasting results show that the FSH β subunit is close to the N-terminal region and that the α subunit is projected toward exoloop 3 in the endodomain. The results raise the fundamental question whether the α subunit, common among the glycoprotein hormones, plays a major role in generating the hormone signal common to all glycoprotein hormones.

In vivo intra-luteal implants of prostaglandin (PG) E1 or E2 (PGE1, PGE2) prevent luteolysis in cows. II: mRNA for PGF2α, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4 prostanoid receptors in luteal tissue.

Previously, it was reported that chronic intra-uterine infusion of PGE(1) or PGE(2) every 4h inhibited luteolysis in ewes by altering luteal mRNA for luteinizing hormone (LH) receptors and unoccupied and occupied luteal LH receptors. However, estradiol-17β or PGE(2) given intra-uterine every 8h did not inhibit luteolysis in cows, but infusion of estradiol+PGE(2) inhibited luteolysis. In contrast, intra-luteal implants containing PGE(1) or PGE(2) in Angus or Brahman cows also inhibited the decline in circulating progesterone, mRNA for LH receptors, and loss of unoccupied and occupied receptors for LH to prevent luteolysis. The objective of this experiment was to determine how intra-luteal implants of PGE(1) or PGE(2) alter mRNA for prostanoid receptors and how this could influence luteolysis in Brahman or Angus cows. On day-13 Angus cows received no intra-luteal implant and corpora lutea were retrieved or Angus and Brahman cows received intra-luteal silastic implants containing Vehicle, PGE(1), or PGE(2) and corpora lutea were retrieved on day-19. Corpora lutea slices were analyzed for mRNA for prostanoid receptors (FP, EP1, EP2, EP3 (A-D), EP3A, EP3B, EP3C, EP3D, and EP4) by RT-PCR. Day-13 Angus cow luteal tissue served as pre-luteolytic controls. mRNA for FP receptors decreased in day-19 Vehicle controls compared to day-13 Vehicle controls regardless of breed. PGE(1) and PGE(2) up-regulated FP gene expression on day-19 compared to day-19 Vehicle controls regardless of breed. EP1 mRNA was not altered by any treatment. PGE(1) and PGE(2) down-regulated EP2 and EP4 mRNA compared to day-19 Vehicle controls regardless of breed. PGE(1) or PGE(2) up-regulated mRNA EP3B receptor subtype compared to day-19 Vehicle control cows regardless of breed. The similarities in relative gene expression profiles induced by PGE(1) and PGE(2) support their agonistic effects. We conclude that both PGE(1) and PGE(2) may prevent luteolysis by altering expression of mRNA for prostanoid receptors, which is correlated with changes in luteal mRNA for LH receptors reported previously in these same cows to prevent luteolysis.

Hematopoetic Prostaglandin D Synthase: An ESR1-Dependent Oviductal Epithelial Cell Synthase

Oviductal disease is a primary cause of infertility, a problem that largely stems from excessive inflammation of this key reproductive organ. Our poor understanding of the mechanisms regulating oviductal inflammation restricts our ability to diagnose, treat, and/or prevent oviductal disease. Using mice, our objective was to determine the spatial localization, regulatory mechanism, and functional attributes of a hypothesized regulator of oviductal inflammation, the hematopoietic form of prostaglandin D synthase (HPGDS). Immunohistochemistry revealed specific localization of HPGDS to the oviducts epithelium. In the isthmus, expression of HPGDS was consistent. In the ampulla, expression of HPGDS appeared dependent upon stage of the estrous cycle. HPGDS was expressed in the epithelium of immature and cycling mice but not in the oviducts of estrogen receptor α knockouts. Two receptor subtypes bind PGD₂: PGD₂ receptor and G protein-coupled receptor 44. Expression of mRNA for Ptgdr was higher in the epithelial cells (EPI) than in the stroma (P < 0.05), whereas mRNA for Gpr44 was higher in the stroma than epithelium (P < 0.05). Treatment of human oviductal EPI with HQL-79, an inhibitor of HPGDS, decreased cell viability (P < 0.05). Treatment of mice with HQL-79 increased mRNA for chemokine (C-C motif) ligands 3, 4, and 19; chemokine (C-X-C motif) ligands 11 and 12; IL-13 and IL-17B; and TNF receptor superfamily, member 1b (P < 0.02 for each mRNA). Overall, these results suggest that HPGDS may play a role in the regulation of inflammation and EPI health within the oviduct.

Functional Integration of the Conserved Domains of Shoc2 Scaffold

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.