IGFR pathway regulates axial patterning during early neural development in mammal

Posters / Growth Hormone & IGF Research 22S1 (2014) S25–S52

and mIGFBP-6 decreased migration to 62 ± 5% and 66 ± 3% respectively (p<0.001). In these cells, coincubation with IGF-II significantly increased migration in the presence of wt but not mIGFBP-6. We have previously shown that MAP kinase pathways are involved in IGFBP-6-induced rhabdomyosarcoma cell migration, so we compared activation of these pathways in HEY and SKOV3 cells. Wt and mIGFBP-6 increased Erk phosphorylation by 62-100% in both cell lines (p<0.05). WtIGFBP-6 also increased Jnk phosphorylation by 138-153% in both cell lines (p<0.05), but the effect of mIGFBP-6 was less clear. The effects of specific Erk and Jnk pathways inhibitors on IGFBP-6-induced migration are underway. Conclusions: IGFBP-6 has opposing effects on migration of HEY and SKOV3 ovarian cancer cells, but activates MAP kinase pathways in both. Delineating the pathways underlying the differential effects on migration will increase our understanding of ovarian cancer metastasis and shed new light on the IGFindependent effects of IGFBP-6, which may in turn lead to the development of an optimized IGFBP-6-based therapeutic that is antitumorigenic and not promigratory.

GP2-7 The IGF/IGFR pathway regulates axial patterning during early neural development in mammal N. Takata1, E. Sakakura2, T. Kasukawa3, M. Eiraku2, Y. Sasai1, on behalf of the Organogenesis and Neurogenesis in CDB. 1 Organogenesis and Neurogenesis Group, RIKEN, Center for Developmental Biology, Kobe, Japan, 24-Dimensional Tissue Analysis Unit, RIKEN, Center for Developmental Biology, Kobe, Japan, 3 Large Scale Data Managing Unit, RIKEN Center for Life Science Technologies, Kobe, Japan Introduction: The creation of a three germ layer containing embryo results from inductive signaling interactions between progenitor tissues, thereby causing tissue specification and regionalization. These signals must be precisely controlled in order to properly drive developmental processes in a time- and site-specific manner. In contrast, embryonic stem cells (ESCs) can generate all three germ layers in vitro. When cultured as floating aggregates under serum-free conditions, ESCs differentiate into neuroectodermal progenitors preferentially without the exogenous morphogen gradients and instructive cues found in vivo. It is unknown, therefor, how in vitro derived neural tissues is able to recapitulate the complex processes of in vivo neural development. Methods: in vitro differentiation ESC culture, microarray. Results: In order to explain in vitro neuroectodermal-cell specification, we have taken advantage of the mouse ESC culture systems and performed a microarray analysis, using ES cell lines with fluorescent reporter knocked in at neuroectodermal gene. We found that IGF related genes significantly changed among huge data sets and also confirmed that neural genes expressed in the neuroectodermal cell. Next we explored IGF related gene function during neural development by addition of agonists or antagonists of IGF signaling. We found that activation or inhibition of IGF signaling changed neural tissue fate in our in vitro system. Conclusion: Together, these results demonstrate a means to analyze neuronal regional specification in vitro. Future studies into neural development will strive to utilize this system in order to elucidate the mechanisms that control the specification and morphogenesis of neural tissues as well as strive to compare in vitro tissue organization mechanisms versus their in vivo counterparts.

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GP4-1 Loss of PI 3-kinase-binding protein PI3KAP/XB130 suppresses thyroid function and induces enlargement of thyroid glands in mice D. Yamanaka1,2, F. Hakuno2, S. Minami1, K. Hashimoto3, K. Suzuki4, K. Chida2, S.-I. Takahashi2. 1Department of Bioregulation, Nippon Medical School, Kawasaki, Japan, 2Departments of Animal Sciences and Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan, 3 Department of Preemptive Medicine and Metabolism, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan, 4Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan Bioactivities of IGFs are often potentiated in the presence of other factors. We have previously reported that PI 3-kinase-binding protein PI3KAP/XB130 is necessary for cell proliferation induced synergistically by thyroid-stimulating hormone (TSH) and IGF-I in a thyroid cell line FRTL-5. However, roles of PI3KAP/XB130 in vivo remained unclear. Here, we generated PI3KAP/XB130 knockout (KO) mice and analyzed its roles in the regulation of thyroid function. Firstly, we found that KO mice unexpectedly showed increased size of thyroid glands compared to wild type (WT) mice. Histological analysis of KO thyroid glands demonstrated increases in follicular lumen area, number of cells per follicle and thyroid cell size. These results suggested that PI3KAP/XB130 deficiency led to some compensatory changes in thyroid glands, unlike cultured thyroid cells in which PI3KAP/XB130 knockdown inhibited cell proliferation. To further examine potential mechanisms for these phenotypes, we analyzed serum indices related to thyroid function. Serum TSH levels were elevated and serum T4 levels were reduced in KO mice, whereas serum T3 levels were normal. We further investigated gene expression involved in thyroid hormone synthesis. Thyroidal mRNA levels of thyroglobulin were unchanged, and mRNA levels of Na+/I- symporter and thyroid peroxidase were increased in KO mice. The changes in expression of these genes could not explain reduction of serum T4 in KO mice. Analysis of expression of thyroid hormone-responsive genes in liver showed that mRNA levels of Spot14, type 1 deiodinase and malic enzyme in KO mice were comparable to those in WT mice, implying that thyroid dysfunction in KO mice had little influence on peripheral tissues. Taking these results together, the present study demonstrated that deletion of PI3KAP/XB130 gene downregulated thyroid function and caused compensatory increases in TSH levels, leading to enlargement of thyroid glands. GP4-7 ProIGF-II not matureIGF-II promotes estrogen independent breast cancer growth and metastasis in a SCID and Nude mouse model: differential signallng mechanisms thru IGF and Insulin receptors and phosphorylation/activation of estrogen and progesterone receptors T. Jian1, S. Kalla-Singh2, V.K. Radhakrishnan2, M.A. De León3, D. DeLeon2. 1Radiation Biology, LLUSM, Loma Linda, United States, 2 CHDMM, LLUSM, Loma Linda, United States, 3CHDMM, LLUSM, Loma Linda, United States Minor Outlying Islands Hormone independent breast cancer tumor growth is associated with a more aggressive and chemoresistant phenotype. Our research group have shown that there is a significant difference in signaling between mature and proIGF-II in breast cancer cells. The purpose of this study is to determine the ability of matureIGFII and proIGF-II to establish and promote breast cancer development in an animal model. We transfected MCF-7 cell line with a vector containing proIGF-II, matureIGF-II or vector only. We then