Inkoo Khang

Suppression of Nek2A in mouse early embryos confirms its requirement for chromosome segregation

Nek2, a mammalian structural homologue of Aspergillus protein kinase NIMA, is predominantly known as a centrosomal kinase that controls centriole-centriole linkage during the cell cycle. However, its dynamic subcellular localization during mitosis suggested that Nek2 might be involved in diverse cell cycle events in addition to the centrosomal cycle. In order to determine the importance of Nek2 during mammalian development, we investigated the expression and function of Nek2 in mouse early embryos. Our results show that both Nek2A and Nek2B were expressed throughout early embryogenesis. Unlike cultured human cells, however, embryonic Nek2A appeared not to be destroyed upon entry into mitosis, suggesting that the Nek2A protein level is controlled in a unique manner during mouse early embryogenesis. Suppression of Nek2 expression by RNAi resulted in developmental defects at the second mitosis. Many of the blastomeres in Nek2-suppressed embryos showed abnormality in nuclear morphology, including dumbbell-like nuclei, nuclear bridges and micronuclei. These results indicate the importance of Nek2 for proper chromosome segregation in embryonic mitoses.

Involvement of CLOCK:BMALI heterodimer in serum-responsive mPerI induction

A rapid induction of mouse period1 (mPer1) gene expression is supposed to be critical in the clock gene regulation, especially in the phase resetting of the clock, but its molecular mechanism is poorly understood. Based on the previous finding that the process does not involve de novo synthesis of proteins, we postulated the involvement of CLOCK:BMAL1 heterodimer, a positive regulator of circadian oscillator, in the rapid induction of mPer1 transcription. To test this hypothesis, we utilized CLOCK&Dgr;19, a dominant-negative mutant, to suppress the function of CLOCK:BMAL1 in vitro. Serum-evoked rapid increases of mPer1 mRNA expression and promoter activity were significantly blunted when CLOCK:BMAL1 function was interfered with. Furthermore, DNA binding activity of CLOCK:BMAL1 heterodimer to five E-boxes of mPer1 promoter markedly increased shortly after serum shock. Taken together, these results suggest that CLOCK:BMAL1 heterodimer is not only a core component of negative feedback loop driving circadian oscillator, but also involved in the rapid induction of mPer1 during phase resetting of the clock.

Selective Roles of Protein Kinase C Isoforms on Cell Motility of GT1 Immortalized Hypothalamic Neurones

Recently, we demonstrated that activation of the protein kinase C (PKC) signalling pathway promoted morphological differentiation of GT1 hypothalamic neurones via an increase in β‐catenin, a cell‐cell adhesion molecule, indicating a possible involvement of PKC in cellular motility. In this study, we explored the differential roles of PKC isoforms in GT1 cell migration. First, we transiently transfected GT1 cells with enhanced green fluorescence protein (EGFP)‐tagged actin to monitor the dynamic rearrangement of filamentous‐actin (F‐actin) in living cells. Treatment with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a PKC activator, markedly promoted lamellipodia formation, while safingol (a PKCα‐selective inhibitor) blocked the TPA‐induced lamellipodial actin structure. Both wound‐healing and Boyden migration assays showed that TPA treatment promoted neuronal migration of GT1 cells; however, cotreatment of TPA with safingol or rottlerin (a PKCδ‐selective inhibitor) clearly blocked this TPA effect, indicating that both PKCα and PKCδ may be positive regulators of neuronal migration. By contrast, PKCγ‐EGFP‐expressing GT1 cells exhibited decreased cellular motility and weak staining for actin stress fibres, suggesting that PKCγ may act as a negative mediator of cell migration in these neurones. Among the PKC downstream signal molecules, p130Cas, a mediator of cell migration, and its kinase, focal adhesion kinase (FAK), increased following TPA treatment; phosphorylation of p130Cas was induced in a PKCα‐dependent manner. Together, these results demonstrate that PKCα promotes GT1 neuronal migration by activating focal adhesion complex proteins such as p130Cas and FAK.

Expression and regulation of gonadotropin-releasing hormone (GnRH) and its receptor mRNA transcripts during the mouse ovarian development

The present study examines the expression and regulation of gonadotropin‐releasing hormone (GnRH) and its receptor (GnRH‐R) mRNA levels during mouse ovarian development. A fully processed, mature GnRH mRNA together with intron‐containing primary transcripts was expressed in the immature mouse ovary as determined by Northern blot analysis and reverse transcription‐polymerase chain reaction (RT‐PCR). The size of ovarian GnRH mRNA was similar to that of hypothalamus, but its amount was much lower than that in the hypothalamus. Quantitative RT‐PCR procedure also revealed the expression of GnRH‐R mRNA in the ovary, but the estimated amount was a thous and‐fold lower than that in the pituitary gland. We also examined the regulation of ovarian GnRH and GnRH‐R mRNA levels during the follicular development induced by pregnant mares serum gonadotropin (PMSG) and/or human chorionic gonadotropin (hCG). Ovarian luteinizing hormone receptor (LH‐R) mRNA was abruptly increased at 48 h after the PMSG administration and rapidly decreased to the basal level thereafter. Ovarian GnRH mRNA level was slightly decreased at 48 h after the PMSG administration, and then returned to the basal value. GnRH‐R mRNA level began to increase at 24 h after the PMSG treatment, decreased below the uninduced basal level at 48 h, and gradually increased thereafter. HCG administration did not alter ovarian GnRH mRNA level, while it blocked the PMSG‐induced increase in GnRH mRNA level. Taken together, the present study demonstrates that the expression of GnRH and GnRH‐R mRNA are regulated by gonadotropin during follicular development, suggesting possible intragonadal paracrine roles of GnRH and GnRH‐R in the mouse ovarian development.