Lactogens Promote Beta Cell Survival through JAK2/STAT5 Activation and Bcl-XL Upregulation

Yuichi Fujinaka,Karen Takane,Hiroko Yamashita,Rupangi C Vasavada

doi:10.1074/jbc.m702607200

Yuichi Fujinaka, Karen Takane + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m702607200

Copy DOI

Abstract

One of the goals in the treatment for diabetes is to enhance pancreatic beta cell function, proliferation, and survival. This study explores the role of lactogenic hormones, prolactin (PRL) and placental lactogen (PL), in beta cell survival. We have previously shown that transgenic mice expressing mouse placental lactogen-1 (mPL1) in beta cells under the rat insulin II promoter (RIP) are resistant to the diabetogenic and cytotoxic effects of streptozotocin (STZ) in vivo. The current study demonstrates that lactogens protect rat insulinoma (INS-1) cells and primary mouse beta cells against two distinct beta cell death inducers, STZ and dexamethasone (DEX), in vitro. Further, we identify the mechanism through which lactogens protect beta cells against DEX-induced death. The signaling pathway mediating this protective effect is the janus-activated-kinase-2/signal transducer and activator of transcription-5 (JAK2/STAT5) pathway. This is demonstrated in INS-1 cells and primary mouse beta cells using three separate approaches, pharmacological inhibitors, JAK2-specific siRNAs and a dominant-negative STAT5 mutant. Furthermore, lactogens specifically and significantly increase the anti-apoptotic protein Bcl-XL in insulinoma cells and mouse islets. Bcl-XL-specific siRNA significantly inhibits lactogen-mediated protection against DEX-induced beta cell death. We believe this is the first direct demonstration of lactogens mediating their protective effect through the JAK2/STAT5 pathway in the beta cell and through Bcl-XL in any cell type.

Highlights

Genetic, structural, binding, and functional studies attest to their common origin, with the placental lactogen (PL) (PL I and II) and PRL genes having evolved by a duplication event from a common ancestral gene in rodents
Lactogens Protect Mouse Primary Beta Cells and INS-1 Cells against STZ-induced Cell Death in Vitro—We have previously shown that overexpression of PL in rat insulin II promoter (RIP)-mouse placental lactogen-1 (mPL1) transgenic mice
Beta cell death is a major factor in the loss of functional beta cell mass that occurs in both type I and type II diabetes, as well as in islet transplantation [35,36,37]

Summary

EXPERIMENTAL PROCEDURES

Generation of Transgenic Mice—The generation of RIPmPL1 transgenic mice has been previously described in detail [11]. 3– 6-month-old male and female mice were used from the transgenic line with the highest transgene expression, L60 [11]. Twenty-four hours after transfection the cells were precultured in SFM containing 1% BSA with/without 200 ng/ml of PRL for 8 h, subsequently treated with 100 nM DEX for 18 h, and either fixed with 2% paraformaldehyde for 30 min at room temperature for cleaved caspase-3 staining or analyzed for cell viability by the MTT assay or for proteins by Western blot analysis. Western Blot Analysis—To analyze the signaling pathways activated by lactogens in islets, CD-1 mouse islets (200 IE/well) were cultured 24 h in filter inserts in complete medium after isolation They were subsequently incubated in SFM overnight, treated with 200 ng/ml of PRL for varying times, washed with cold PBS containing 100 ␮M sodium orthovanadate and frozen. Results were analyzed by using Student’s t test or oneway repeated measures analysis of variance (n.s., no significance; *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.005)

RESULTS

C B cl-XL

DISCUSSION