The potassium channels IK and IK1, responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on IK and IK1 through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10-8 M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. IK and IK1 activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal IK activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of IK1 was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on IK and IK1. We conclude that basal IK and IK1 are positively maintained by steady-state Akt and ERK activities. K+ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K+ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt. .

Insulin-like 6 immunoreactivity (irINSL6) was detected in Leydig cells of the mouse testis. In the brain, labeled somata were detected mainly in the caudal hypothalamus and midbrain. Double labeling the brainstem sections revealed that irINSL6 somata were 5-hydroxytryptamine (5-HT) positive. The presence of irINSL6 in discrete populations of hypothalamic and brainstem neurons and in Leydig cells of the testis suggests a diverse biological function of this novel peptide.

Maintenance of the hematopoietic system is dependent upon the proper regulation and orchestrated functions of the hematopoietic stem cell (HSC) pool. A number of extrinsic signaling pathways and intrinsic regulators have been found to regulate HSC processes. However a full understanding of the ability of HSC to balance the processes of self-renewal, quiescence, and lineage specification is not yet clear. We therefore set out to identify novel HSC regulators by comparative gene expression analysis of whole genome transcriptomes generated for long-term (LT)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34-), short-term (ST)-HSC (Hoechst low/- Lin- Sca1+ cKit+ CD34+), and non-HSC (Hoechst+) of the bone marrow. These studies identified IGF2 as one of the most differentially expressed genes within LT-HSC, suggesting a potential role for IGF2 in the regulation of HSC. Using a combination of lentiviral-mediated overexpression and knockdown experiments, we found IGF2 to confer enhanced self-renewal in vitro and in vivo. Overexpression of IGF2 resulted in an increased percentage of multi-lineage colonies within colony-forming unit (CFU) assays without affecting lineage specification. In vivo, serial bone marrow transplantation revealed that IGF2 within HSC enhances short-term and long-term donor contribution. Analysis of the expression of key cell cycle regulators revealed that IGF2 induced upregulation of p57 expression specifically within HSC. This upregulation could be attributed to differences in the methylation status of the p57 promoter in HSC compared to other progenitor and mature blood cell populations. p57, a member of the Cip/Kip family of cyclin dependent kinase inhibitors, has recently been shown to be required for the regulation of HSC quiescence and long-term self-renewal. Analysis of bone marrow obtained from primary and secondary transplant recipients showed that overexpression of IGF2 resulted in an increased percentage of quiescent HSC. Treatment of HSC overexpressing IGF2 with LY294002, a PI3K-Akt inhibitor, prevented IGF2-mediated upregulation of p57 expression. These findings demonstrate that within HSC, IGF2 induces p57 expression through activation of the PI3K-Akt pathway to regulate HSC quiescence. We have identified a novel role for IGF2 in HSC function, providing new insights into the biology of HSC and opening potential platforms for the development of better therapies involving HSC-mediated hematopoietic reconstitution.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19612号 / 医博第4119号 / 新制||医||1015(附属図書館) / 32648 / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 渡邉 大, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20798号 / 医博第4298号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 辻川 明孝, 教授 清水 章, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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