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The SHB adapter protein is required for normal maturation of mesoderm during in vitro differentiation of embryonic stem cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 45, 34484-34491 p.Article in journal (Refereed) Published
Abstract [en]

Definitive mesoderm arises from a bipotent mesendodermal population, and to study processes controlling its development at this stage, embryonic stem (ES) cells can be employed. SHB ((S) under bar rc (h) under bar omology 2 protein in beta-cells) is an adapter protein previously found to be involved in ES cell differentiation to mesoderm. To further study the role of SHB in this context, we have established ES cell lines deficient for one (SHB+/-) or both SHB alleles (SHB-/-). Differentiating embryoid bodies (EBs) derived from these ES cell lines were used for gene expression analysis. Alternatively, EBs were stained for the blood vessel marker CD31. For hematopoietic differentiation, EBs were differentiated in methylcellulose. SHB-/- EBs exhibited delayed down-regulation of the early mesodermal marker Brachyury. Later mesodermal markers relatively specific for the hematopoietic, vascular, and cardiac lineages were expressed at lower levels on day 6 or 8 of differentiation in EBs lacking SHB. The expression of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 was also reduced in SHB-/- EBs. SHB-/- EBs demonstrated impaired blood vessel formation after vascular endothelial growth factor stimulation. In addition, the SHB-/- ES cells formed fewer blood cell colonies than SHB+/+ ES cells. It is concluded that SHB is required for appropriate hematopoietic and vascular differentiation and that delayed down-regulation of Brachyury expression may play a role in this context.

Place, publisher, year, edition, pages
2006. Vol. 281, no 45, 34484-34491 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-97230DOI: 10.1074/jbc.M604084200ISI: 000241767600069PubMedID: 16971391OAI: oai:DiVA.org:uu-97230DiVA: diva2:172071
Available from: 2008-05-09 Created: 2008-05-09 Last updated: 2012-12-17
In thesis
1. The role of Shb in ES cell differentiation, angiogenesis and tumor growth
Open this publication in new window or tab >>The role of Shb in ES cell differentiation, angiogenesis and tumor growth
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors and intracellular signaling proteins. Previous studies have implied a wide spectrum of Shb-mediated cellular responses, which motivated me to further investigate the role of Shb in differentiation and angiogenesis. Embryonic stem (ES) cells differentiate into endoderm and mesoderm from a bipotent mesendodermal cell population. Interregulatory signals between these germlayers are required for further specification. ES cells overexpressing Shb with an inactive SH2 domain (R522K-Shb) altered the expression of endodermal genes as a consequence of upregulated FGF expression. This response was enhanced by addition of activin A, suggesting a synergistic mechanism operative between FGF and activin A signaling in endoderm specification. To investigate a role for Shb in mesodermal specification, Shb knockout ES cells were established. These cells showed a reduced ability to form blood vessels after VEGF stimulation and delayed downregulation of genes associated with mesendoderm, indicating a reduced capacity for these cells to enter later stages.

To assess a role for Shb in tumor cell apoptosis, Shb expression was silenced in angiosarcoma endothelial cells. FAK-phosphorylation was reduced in Shb knockdown cells and this made them more susceptible to apoptotic stimuli both in vitro and in vivo.

Shb knockout microvasculature in mouse kidney, liver, and heart showed irregular endothelial linings with cytoplasmic projections toward the lumen, a feature that was also related to increased vascular permeability. VEGF treatment failed to stimulate vascular permeability in Shb knockout mice.

In order to elucidate whether these features relate to reduced angiogenesis, tumor growth was examined. Tumors grown in knockout mice showed reduced growth capacity and lower vessel density. In conclusion, Shb is a multifunctional adaptor protein that may be involved in several cellular responses both during embryonic development and adult life.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 49 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 355
Keyword
Cell biology, Shb, tyrosine kinase signaling, ES, endoderm, mesoderm, apoptosis, microvasculature, tumor growth, Cellbiologi
Identifiers
urn:nbn:se:uu:diva-8746 (URN)978-91-554-7205-4 (ISBN)
Public defence
2008-05-31, Auditorium minus, Gustavianum, Akademigatan 3, Uppsala, 13:00
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Available from: 2008-05-09 Created: 2008-05-09Bibliographically approved

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Welsh, Michael

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