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Overexpression of the Shb SH2 domain-protein in insulin-producing cells leads to altered signaling through the IRS-1 and IRS-2 proteins
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.
2002 (English)In: Molecular Medicine, Vol. 8, no 11, 695-704 p.Article in journal (Refereed) Published
Abstract [en]

Background

Overexpression of the Src homology 2 domain protein Shb in _-cells of transgenic mice has been shown to promote an increased _-cell mass. To investigate the mechanisms by which Shb controls the _-cell mass, we have presently studied the effects of Shb overexpression on the IRS-1–induced signaling pathway in mouse islet _-cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns.

Materials and Methods

Shb overexpression was achieved in RINm5F cells by selection of stable clones or by FACS purification of transiently transfected cells. For Shb overexpression in primary mouse islet cells, a Shb-transgene mouse was used. Cell proliferation and death rates were determined using flow cytometry. Serum-, insulin-, and IGF-1-stimulated signaling events were studied by immunoblot, immunoprecipitation, and in vitro kinase procedures.

Results

Transient Shb overexpression in RINm5F cells resulted in increased proliferation. Both Shb-overexpressing RINm5F cells and islet cells from transgenic mice (islet Shb) exhibited increased basal tyrosine phosphorylation of IRS-1. Shb overexpression resulted also in the assembly and activation of a multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK, and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpressing cells. Finally, Shb overexpression did not affect insulin-induced phosphorylation of the PI3K-antagonist PTEN.

Conclusion

It is concluded that the Shb-induced alterations in the IRS-1/PI3K/Akt pathway may be relevant to the understanding of growth and death patterns of insulin-producing cells.

Place, publisher, year, edition, pages
2002. Vol. 8, no 11, 695-704 p.
National Category
Basic Medicine
Research subject
Cell Research
Identifiers
URN: urn:nbn:se:uu:diva-93972OAI: oai:DiVA.org:uu-93972DiVA: diva2:167637
Available from: 2006-01-27 Created: 2006-01-27 Last updated: 2013-02-06Bibliographically approved
In thesis
1. Role of MAP Kinases in the Life and Death of Beta-cells
Open this publication in new window or tab >>Role of MAP Kinases in the Life and Death of Beta-cells
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The development of diabetes mellitus depends on the balance between beta-cell proliferation and death. As mitogen-activated protein kinases (MAPK) may control this balance, the aim of this study was to investigate the events leading to MAPK activation in beta-cells and the consequences of these events. Overexpression of the SH2-domain containing adaptor protein Shb resulted in the assembly and activation of multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpression cells. This was paralleled by an attenuated activation of the MAP kinases ERK1/2. Thus, Shb-induced alterations in the IRS-1/PI3K/Akt/ERK pathway might explain the increased proliferation and apoptosis of beta-cells overexpressing Shb.

The importance of the MAP kinase p38 in nitric oxide- and cytokine-induced beta-cell death was also investigated. Knock-down of p38 expression resulted in a lowered cell death rate in response to a nitric oxide donor. In transient transfections MKK3 over-expression resulted in increased p38 phosphorylation in RIN-5AH cells. In addition, a short-term MKK3 expression resulted in increased cytokine-induced cell death. A nitric oxide synthase inhibitor abolished the MKK3-potentiating effect on cytokine-induced cell death and inhibitors of phosphatases enhanced MKK3-stimulated p38 phosphorylation. Finally, as the dominant negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide.

In further support for an MKK3/6-indepedent mechanism, the adaptor protein TAB1 significantly increased the cytokine- and nitric oxide-stimulated phosphorylation of p38. The TAB1-mediated activation of p38 was paralleled by a compensatory inhibition of ERK and JNK. In summary, p38 MAPK, activated mainly by TAB1, promotes, at least in part, beta-cell death in response to cytokines or nitric oxide.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 102
Keyword
Cell biology, diabetes, beta-cell, p38 MAPK, nitric oxide, apoptosis, Cellbiologi
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6317 (URN)91-554-6450-5 (ISBN)
Public defence
2006-02-21, Room B21, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2006-01-27 Created: 2006-01-27Bibliographically approved

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Welsh, NilsWelsh, Michael

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