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

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.

Genetic, structural, binding, and functional studies attest to their common origin, with the PL (PL I and II) and PRL genes having evolved by a duplication event from a common ancestral gene in rodents. The major site of synthesis of PRL is the lactotroph cell of the anterior pituitary, although it is also expressed in several extrapituitary tissues. PL is synthesized in the giant trophoblast cell of the placenta during pregnancy. PRL and PL not only have related functions, but also bind and signal through a common receptor, the prolactin receptor (PRL-R), which belongs to the class I cytokine receptor family (1,2). The PRL-R is expressed in numerous tissues, including the pancreatic beta cell, during development and in adult life (3)(4)(5).
Lactogens are important regulators of pancreatic islet development, beta cell proliferation, and beta cell function, in normal physiology and in pregnancy. The phenotype of the PRL-R knock-out mice unequivocally established the importance of lactogen signaling in normal islet development and function (6). These mice display a 25-40% reduction in islet mass, reduced insulin content, a blunted glucose stimulated insulin secretion response and intolerance to a glucose challenge, underscoring the importance of lactogens in normal islet physiology. Conversely, lactogens can also enhance beta cell proliferation and function. This has been demonstrated both in vitro, in primary beta cells from different species and in beta cell lines (7)(8)(9), as well as in vivo, in the setting of pregnancy, continuous hormone infusion in rats (9,10), and in transgenic RIP-mPL1 mice overexpressing PL in their beta cells (11,12). These mice are hypoglycemic, hyperinsulinemic, and display islet hyperplasia as a result of increased beta cell proliferation and hypertrophy.
Lactogens acting through the PRL-R have been repeatedly shown to improve survival in a number of different cell types against diverse cell death-inducing agents (13)(14)(15)(16)(17)(18). However, surprisingly, virtually nothing is known regarding their role in the regulation of beta cell survival. In this context, we have recently demonstrated that PL expression in the beta cell of RIP-mPL1 mice confers resistance in vivo to the diabetogenic and cytotoxic effects of a beta cell toxin, STZ, implying a protective role of mPL1 in beta cells (11,12). However, the signaling pathways and molecular mechanisms by which lactogens improve beta cell survival are not known.
In the current report we demonstrate a direct protective effect of lactogens on rodent pancreatic beta cells, both primary and insulinoma cells, against at least two distinct inducers of cell death, STZ and DEX. STZ in large doses can cause both beta cell necrosis and apoptosis, through DNA damage, activation of poly-ADP-ribose polymerase, and NAD ϩ depletion (19), whereas DEX mainly induces apoptosis through the mitochondrial pathway (20). We show that both STZ and DEX induce activation of the executioner caspase-3, in INS-1 cells, and that lactogens can significantly reduce caspase-3 activation. These studies demonstrate for the first time that the JAK2/ STAT5 pathway is critical for lactogen-mediated protection against DEX-induced cell death in insulinoma cells and in primary beta cells. To elucidate the mechanism by which lactogens protect beta cells, we examined the effect of lactogens on the expression of members of the Bcl family of proteins. A significant and specific increase in the level of the anti-apoptotic member Bcl-X L was observed in both primary beta cells and insulinoma cells treated with lactogens. Importantly, preventing PRL-induced expression of Bcl-X L by Bcl-X L -specific siRNA significantly inhibited the protective effect of lactogens against DEX-induced beta cell death, a first direct demonstration of the importance of Bcl-X L up-regulation in mediating the protective effect of lactogens in any cell type.

EXPERIMENTAL PROCEDURES
Generation of Transgenic Mice-The generation of RIP-mPL1 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). This line was bred onto a CD-1 background for more than eight generations for these studies. Genotyping was performed by tail DNA PCR using primers for human growth hormone, and glyceraldehyde-3-phosphate dehydrogenase, which serves as a control (12). All studies were performed with the approval of, and in accordance with, guidelines established by the University of Pittsburgh Institutional Animal Care and Use Committee.
Mouse Primary Islet Cell and Rat Insulinoma (INS-1) Cell Cultures-Islets were isolated from normal and transgenic mice as described previously (12). Briefly, 3 ml of 1.7 mg/ml Collagenase P (Roche Applied Sciences) in Hanks' buffered saline solution (HBSS) is injected into the pancreatic duct. Subsequently, pancreata are removed and digested at 37°C for 17 min, before sieving through a 500-m wire mesh to separate undigested tissue. The digested pancreas is pelleted and rinsed with HBSS, and islets separated by density gradient in Histopaque (Sigma). After several washes, islets are handpicked under a microscope grid and cultured overnight in RPMI medium containing 5 mM D-glucose supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin (complete medium) (21). For cell culture, islets are rinsed with phosphate-buffered saline (PBS), trypsinized for 8 -10 min at 37°C, and islet cell cultures plated on 12-mm glass coverslips placed in 24-well plates. Cells from 50 -70 islet equivalents (IE) (1 IE ϭ 125 m-diameter islet) were plated on each coverslip in 50 l of complete medium, and incubated at 37°C for 2 h, to allow cells to attach to the glass surface, after which another 1 ml of complete medium was added for an additional 24 h. Thereafter, cells were incubated overnight in fresh RPMI containing 5 mM D-glucose, 1% bovine serum albumin (BSA), without FBS, as the latter contains large amounts of lactogens (22). Islet cell cultures were treated either with 1.5 mM STZ (Sigma) made in 10 mM sodium citrate pH 4, or vehicle as control, for 14 h. For DEX treatment, cells were cultured in medium containing 1% horse serum with addition of 500 nM DEX (Sigma) made in ethanol, or vehicle as control, every 24 h for 2 days. Cells were rinsed with PBS and subsequently fixed in freshly prepared 2% paraformaldehyde for 30 min at room temperature.
Rat insulinoma cells (INS-1) kindly provided by Dr. Doris Stoffers (University of Pennsylvania School of Medicine) were grown in RPMI 1640 medium containing 11 mM D-glucose supplemented with 10% FBS, 100 units/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 M ␤-mercaptoethanol (INS-1 medium). Prior to any treatment, the cells were precultured in serum-free medium (SFM) containing 1% BSA for 16 -18 h, before addition of 200 ng/ml of ovine PRL (Sigma) made in PBS with 0.1% BSA, or PBS with 0.1% BSA alone as control. Eight hours after the addition of PRL, cells were treated as specified in the individual experiments.
INS-1 Cell Viability Measured by the MTT Assay-Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Sigma) assay in INS-1 cells (4 ϫ 10 4 ) split in 96-well plates. After preculture, cells were treated with the individual cell death inducers as follows, 0.5 and 1.0 mM STZ for 14 h, or 10 and 100 nM DEX for 24 h, and subsequently analyzed. For the signaling pathway inhibitor experiments, cells were treated with inhibitors 60 min prior to preculturing with PRL. After 8 h of PRL treatment cell death was induced by 100 nM DEX for 18 h. MTT assay was performed by incubating the cells with 0.5 mg/ml MTT for 2 h at 37°C in 5% CO 2 . Formazan produced in the cells was dissolved with isopropyl alcohol, and absorbance was read at 570 and 690 nm.
siRNA Treatment-INS-1 cells (8 ϫ 10 4 ) grown on glass coverslips in a 24-well plate, were transfected 2 days later with 50 nM of scrambled, JAK2 or Bcl-X L siRNA (Dharmacon, Chicago, IL). The siRNA oligonucleotides were diluted in SFM with HEPES buffer containing Lipofectamine (Invitrogen, Carlsbad, CA), incubated at room temperature for 30 min, then added to the cells and kept for 6 h at 37°C, after which transfection medium was removed and fresh culture medium was added. 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.
Adenovirus (Ad) Transduction-cDNA of a carboxyl-truncated STAT5a variant, Stat5a⌬740, which displays a dominantnegative (DN) effect on both Stat5a and Stat5b-mediated transcription was used to block PRL-induced STAT5 signaling (23). Adenovirus containing the DN-STAT5 or green fluorescent protein (GFP) cDNA were prepared and their concentration determined by optical density at 260 nm and by plaque assay. Primary mouse islet cell cultures were transduced with the adenoviral constructs at a multiplicity of infection (MOI) of 100 pfu/cell as previously described (24).
Analysis of Cell Death by Immunostaining-As STZ induces necrosis and apoptosis, STZ-induced beta cell death was analyzed in primary mouse islet cells by immunofluorescence costaining for insulin, using an anti-insulin antibody (Zymed Lab-oratories Inc., South San Francisco, CA) at 1:50 dilution, and propidium iodide (PI, Sigma) to stain the nuclei (21), and the percentage of condensed, pyknotic insulin-positive nuclei were quantitated. 150 Ϯ 17 insulin-positive cells were counted per coverslip in a total of 10 coverslips each, for normal and transgenic mice. DEX-induced beta cell death was quantitated by co-staining with insulin and terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end-labeling (TUNEL, Roche Applied Sciences), as recommended by the manufacturer, with simultaneous nuclear staining using Hoechst33258. The percentage of TUNEL and insulin-positive cells was quantitated in 8 -14 samples each, counting an average of 600 Ϯ 87 insulin-positive cells per sample for Fig. 2, and in 4 samples each counting an average of 1769 Ϯ 59 insulin-positive cells per sample for Fig. 6. DEX-induced INS-1 cell death was measured by cleaved caspase-3 staining. INS-1 cells (4 ϫ 10 4 ) were split on glass coverslips in a 24-well plate, precultured as described above, and treated with 100 nM DEX for 18 -24 h before fixing with 2% paraformaldehyde for 30 min at room temperature. For the signaling experiments cells were treated with the different inhibitors 60 min prior to PRL treatment. For staining, cells were incubated in rabbit cleaved caspase-3 antibody diluted in PBS containing 1% BSA, 1% normal goat serum, 0.3% Triton X-100, and 0.02% sodium azide overnight at 4°C. After incubation in fluorescein isothiocyanate-conjugated secondary antibody, nuclei were stained with 20 g/ml Hoechst 33258.
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. To examine the expression of Bcl proteins, islets from normal and RIP-mPL1 transgenic mice were either harvested immediately after isolation (Day 0) or cultured in medium containing 10% horse serum (which has lower lactogen levels than FBS) in filter inserts for 1 day after isolation (Day 1). INS-1 cells were treated as indicated in the individual experiments.  OCTOBER 19, 2007 • VOLUME 282 • NUMBER 42

JOURNAL OF BIOLOGICAL CHEMISTRY 30709
Total INS-1 cell or whole islet extracts (20 -30 g) prepared using lysis buffer (21) were blotted on polyvinylidene difluoride membranes, incubated with each primary antibody overnight at 4°C and then with the corresponding peroxidase-conjugated secondary antibodies, after which chemiluminescence was detected using the ECL system (Amersham Biosciences, Piscataway, NJ). The different primary antibodies used were Bax Statistical Analysis-The data are expressed as the mean Ϯ S.E. 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).

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 RIP-mPL1 transgenic mice protects beta cells from the diabetogenic and cytotoxic effects of STZ in vivo (11,12). To directly demonstrate the pro-survival effect of lactogens and to identify the mechanisms involved in lactogen-mediated beta cell survival, we established an in vitro cell system. For this purpose, primary cell cultures prepared from islets isolated from normal and transgenic mice were subjected to STZ treatment and beta cell death was quantitated by co-staining with insulin and PI (Fig. 1A). The numbers of condensed pyknotic beta cell nuclei, an indicator of cell death, were significantly (2-fold) lower in transgenic versus normal islet cells (Fig. 1, A and B) indicating a protective effect of lactogens in vitro. We further examined the effect of lactogens on STZinduced cell death in a rat insulinoma cell line, INS-1, acutely treated with or without PRL. There was a significant ϳ35% decrease in INS-1 cell death in PRL-treated cells compared with controls as measured by the MTT assay (Fig. 1C). STZ also induced activation of cleaved caspase-3, a marker for cell death, in INS-1 cells. Again, PRL-treatment reduced levels of active caspase-3, and the reduction was significant at 0.5 mM STZ (Fig.  1, D and E). Thus, these results indicate that lactogens can indeed protect beta cells from STZ-mediated cell death in vitro, and that INS-1 cells may serve as a useful model system for studying intracellular signaling pathways and molecular mechanisms mediating the pro-survival effect of lactogens in beta cells.

Lactogens Protect Mouse Primary Beta Cells and INS-1 Cells against DEX-induced Cell Death in Vitro-
To determine whether lactogens can protect beta cells against another cell death inducer which is pathophysiologically more relevant, the glucocorticoid, DEX, which acts through the glucocorticoid receptor, was examined. Glucocorticoid levels become elevated under various physiological and pathophysiological conditions, such as stress (25), late gestation (26), and during its use as a therapeutic agent (27,28). In addition to causing insulin resistance and beta cell dysfunction (28,29), there is mounting evidence that glucocorticoids also induce beta cell death mainly through the mitochondrial apoptotic pathway (30). Therefore, we examined the ability of lactogens to protect beta cells against DEX-induced cell death. Normal islet cells precultured with/ without exogenous PRL, or mPL1 transgenic islet cells, were treated with DEX, (500 nM for 48 h) and beta cell death was quantitated by TUNEL and insulin co-staining ( Fig. 2A). Basal cell death in the absence of DEX (Fig. 2B, gray bars) was minimal and not significantly different between normal, transgenic or PRL-treated normal islet cells. DEX caused a 2.5-fold increase in beta cell death in normal islet cell cultures (Fig. 2B). DEX-induced beta cell death was significantly and completely blocked in both RIP-mPL1 transgenic and in PRL-treated normal islet cells (Fig. 2B) assay, which was strikingly inhibited by PRL at both DEX concentrations (Fig. 2C). The mode of cell death induced by DEX is mainly through apoptosis (20,30), as demonstrated by the 5-7fold increase in the percentage of active caspase-3-positive INS-1 cells treated with 10 and 100 nM DEX (Fig. 2, D and E). PRL significantly inhibited (2-4-fold) the percentage of active caspase-3-positive cells in DEX-induced INS-1 cells (Fig. 2, D  and E). Taken together, the results thus far demonstrate that lactogens protect beta cells against at least two different inducers of cell death, STZ and DEX, and that this protective effect is mediated at least in part through inhibition of caspase-3 activation.
Lactogens Protect INS-1 Cells against DEX-induced Cell Death through the JAK2/STAT5 Signaling Pathway-Lactogens activate a number of signaling pathways including JAK2/ STAT5, phosphatidylinositol 3-kinase (PI3K/Akt), and extracellular signal-regulated kinase-1/2 (ERK1/2) (31-33), each potentially important for cell survival. Analysis of these three pathways by Western blot (Fig. 3A) indicates that PRL rapidly activates these signaling pathways in INS-1 cells (Fig. 3B). To determine whether any of these signaling pathways could mediate the protective effect of PRL, specific pharmacological inhibitors for each of these pathways were used at different concen-trations, and their effect on PRL-stimulated INS-1 cell survival against DEX-induced beta cell death was examined using the MTT assay (Fig. 4, A-C). Neither the PI3K inhibitor, wortmannin (Fig. 4A), nor the ERK 1/2 inhibitor, PD98059 (Fig. 4B), at two different concentrations, significantly affected the protective effect of PRL against DEX-induced cell death. In contrast, the JAK2 inhibitor, AG490, reduced the protective effect of PRL in a dose-dependent manner, with complete loss of protection by PRL at the higher AG490 dose of 100 M (Fig. 4C). To further confirm this, we examined whether wortmannin or AG490 could inhibit the protective effect of PRL on DEX-induced INS-1 cell death using a different cell death assay, active caspase-3 staining (Fig. 4, D and E). Again, wortmannin at two different concentrations did not significantly affect PRL-mediated survival (Fig. 4D), whereas, AG490, in a dose-dependent manner inhibited the protective effect of PRL measured by cleaved caspase-3 staining, reaching significance at the higher 50 M dose (Fig. 4E), confirming the findings from the MTT assay (Fig. 4, A and C). Given the inherent caveats of pharmacological inhibitors, we used a different approach, JAK2-specific siRNAs, to down-regulate the JAK2 signaling pathway in INS-1 cells (Fig. 5). There was an ϳ80% reduction in the level of JAK2 protein within 48 h of JAK2 siRNA transfection com-  pared with control untransfected and scrambled siRNA-transfected INS-1 cells (Fig. 5A). Furthermore, PRL was unable to activate the downstream STAT5 signaling pathway as evidenced by the lack of ph-STAT5 in JAK2 siRNA-transfected cells compared with PRL-treated control and scrambled siRNA-transfected cells (Fig. 5A). Although prolonged PRL treatment reduces the level of JAK2 protein (34), as observed in PRL-treated control and scrambled siRNA-transfected cells, PRL is able to activate ph-STAT5 in these cells (Fig. 5A). We next examined the effect of inhibiting the JAK2/STAT5 pathway on PRL-mediated protection against DEX-induced INS-1 cell death by the MTT assay (Fig. 5B). PRL significantly protected INS-1 cells against DEX-induced cell death in scrambled siRNA-transfected cells. However, the protective effect of PRL was completely lost in the JAK2 siRNA-transfected cells (Fig.  5B). Taken together, these results establish the importance of the JAK2 signaling pathway in mediating the protective effect of lactogens in INS-1 cells, and further suggest that the PI3K and ERK1/2 pathways are not involved in PRL-mediated protection against DEX-induced beta cell death.

Lactogens Protect Mouse Primary Beta Cells against DEXinduced Cell Death through the JAK2/STAT5
Signaling Pathway-We initially examined the activation of the JAK2/ STAT5 signaling pathway by PRL in mouse islets. There is a large increase in ph-STAT5 within 10 min of PRL treatment, which is sustained up to 30 min (Fig. 6, A and B). To determine whether this pathway mediates the protective effect of lactogens in primary beta cells, we used an adenoviral construct containing the cDNA of a carboxyl-truncated variant of STAT5a, Stat5a⌬740. This mutant has a DN effect, through sustained DNA binding activity, thus inhibiting transcription mediated by both STAT5a and STAT5b (23). Primary mouse beta cells from normal and RIP-mPL1 transgenic mice were transduced with Ad-DN-STAT5a or Ad-GFP as control. Subsequently normal islet cell cultures were treated either with (PRL) or without (NL) PRL. Cell death was induced in both normal and transgenic (TG) islet cells using DEX for 48 h, with vehicle treatment used as control. Beta cell death, measured by insulin-TUNEL co-staining, showed no significant differences between the four vehicle-treated groups (NL-GFP, NL-DN, TG/PRL-GFP, TG/PRL-DN, data not shown). DEX treatment resulted in 14.2 Ϯ 2.1% beta cell death in the NL-GFP group. As expected, there was a significant reduction, 6.0 Ϯ 2.0%, in DEXinduced beta cell death in the TG/PRL-GFP group (Fig. 6C). However, in the presence of the DN-STAT5a mutant the protective effect of lactogens against DEX-induced beta cell death was completely lost (16.1 Ϯ 2.2% versus 19.9 Ϯ 3.2% beta cell death in NL-DN versus TG/PRL-DN groups respectively, Fig.  6C). These results demonstrate that lactogens mediate their pro-survival effects on primary beta cells through the JAK2/ STAT5 signaling pathway.
Lactogens Increase Expression of the Anti-apoptotic Member Bcl-X L in Mouse Islets and in INS-1 Cells-Although lactogens enhance expression of different anti-apoptotic members of the Bcl family of proteins in other cell types (15,16), there is nothing known regarding their effects in beta cells. Therefore, extracts prepared from islets isolated from normal and transgenic mice were examined for expression of three different members of the Bcl family of proteins by Western blot analysis (Fig. 5A). Extracts were prepared from islets either immediately after isolation (day 0) or after 1 day in culture (day 1) to allow recovery from the isolation process. Of the three members of the Bcl family examined, only expression of the anti-apoptotic member Bcl-X L was increased in RIP-mPL1 transgenic islets compared with normal islets on both day 0 and day 1 (Fig. 7, A  and B). There was no difference in expression of the other antiapoptotic member Bcl-2 (Fig. 7, A and C), or the pro-apoptotic member Bax (Fig. 7A, quantitation not shown) between normal and transgenic islets. Quantitation of the individual Bcl/Bax ratios shows a significant increase in the Bcl-X L /Bax ratio in transgenic islets at day 0 (Fig. 7B).
To address whether acute lactogen treatment could enhance Bcl-X L expression in beta cells and to examine the time course of induction, INS-1 cells were stimulated with PRL for different periods of time (2-24 h) and expression of Bcl family of proteins was examined by Western analysis. There was a gradual distinct increase in Bcl-X L , a slight increase in Bcl-2 and no change in Bax expression (Fig. 7D). Quantitation of the ratio of Bcl/Bax demonstrates a significant increase in the Bcl-X L /Bax ratio by 6 h with a peak at 18 h (Fig. 7E). However, the Bcl-2/Bax ratio although slightly increased by 2-4 h was never significantly increased compared with untreated INS-1 cells (Fig. 7F). Bax/ tubulin ratio remained unchanged through the time course (data not shown). These results demonstrate a specific increase in Bcl-X L expression by lactogens in beta cells.

Increase in Bcl-X L Expression by Lactogens Is Necessary to Mediate Its Protective Effect against DEX-induced INS-1 Cell Death-
The role of lactogen-mediated up-regulation of Bcl proteins in cell survival has not directly been examined in any cell type. Therefore, to determine whether up-regulation of the antiapoptotic Bcl-X L protein by lactogens is required to mediate the antiapoptotic effect of lactogens in beta cells, Bcl-X L -specific siRNA was used to inhibit its expression. As shown in Fig. 8A, PRL-induced Bcl-X L expression was not affected by 50 nM of control, scrambled siRNA. However, 50 nM Bcl-X L -specific siRNA specifically inhibited PRL-induced expression of Bcl-X L , without affecting expression of other members of the Bcl family. Quantitation of the ratios of the Bcl family of proteins/tubulin from  three separate experiments indicate that Bcl-X L siRNA specifically reduces Bcl-X L expression in PRL-treated cells to levels equal to control untreated cells or scrambled siRNAtransfected cells, without affecting expression of Bcl-2 or Bax (Fig. 8, B-D).
To determine whether PRL-stimulated Bcl-X L expression is required to mediate its protective effect, scrambled or Bcl-X L siRNA transfected INS-1 cells were pre-treated with or without PRL before induction of cell death with DEX. Cell death was quantitated by active caspase-3 staining (Fig. 9A). DEX induces 23.4 Ϯ 2% cell death in scrambled siRNA-transfected PBS-treated INS-1 cells, and, as expected, PRL protects these cells (3.7 Ϯ 0.5%) from DEX-induced cell death (Fig. 9B). The incremental cell death induced by DEX in Bcl-X L siRNA transfected PBS-treated cells was similar (28.3 Ϯ 2.8%) to the cell death induced by DEX in scrambled siRNA-transfected PBS-treated cells. However, most importantly, normalizing Bcl-X L levels using siRNA significantly diminished the protective effect of PRL against DEX-induced INS-1 cell death (Fig. 9B). This is demonstrated by a significant increase in INS-1 cell death in PRL-treated Bcl-X L siRNA-transfected cells (18.3 Ϯ 0.7%) versus PRL-treated scrambled siRNA-transfected cells (3.7 Ϯ 0.5%). Thus, reducing the level of Bcl-X L in PRLtreated cells to control levels through Bcl-X L siRNA (Fig. 8B) significantly reduced, although did not completely abolish, the protective effect of PRL on DEX-induced cell death (Fig. 9B). These results demonstrate that the increase in Bcl-X L expression by lactogens is required to mediate part of the protective effect of lactogens on DEX-induced apoptosis in INS-1 cells.

DISCUSSION
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). The inducers and pathways mediating beta cell death vary in these different settings. Therefore, expanding our understanding of the growth factors, signaling pathways and molecular mechanisms that regulate beta cell survival under these conditions would greatly enhance our future potential to treat diabetes. In this context, the current studies are the first to demonstrate a direct protective effect of the lactogenic hormones, PRL and PL, on beta cell survival and identify JAK2/STAT5 as the signaling pathway mediating this action. Furthermore, these studies define the increase in Bcl-X L expression induced by PRL as being a critical molecular mechanism through which PRL protects beta cells.
While PRL and PL are known to enhance beta cell function and proliferation in vitro and in vivo (7)(8)(9)(10)(11)(12), there has been no direct study examining their effect on beta cell survival. On the other hand, several in vitro studies have shown that PRL protects many cell types such as mammary and prostate epithelial cells, granulosa cells, decidual cells and the lymphoma cell line Nb2, against varied cell-death inducers including ceramide, nitric oxide, glucocorticoids and growth factor withdrawal (13)(14)(15)(16)(17)(18). In this study we directly demonstrate a protective effect of lactogens on beta cells against STZ-induced cell death in vitro, corroborating our previous studies demonstrating anti-diabetogenic and anti-cytotoxic effects of lactogens in RIP-mPL1 mice against STZ in vivo (11,12). We next determined if lactogens can protect beta cells against the glucocorticoid, DEX, a more pathophysiologically relevant cell death inducer. The rationale for using DEX was 3-fold; first, it induces apoptosis through the mitochondrial pathway in many cell types including the beta cell (20,30); second, lactogens are known to protect other cell types against DEX-induced cell death (14,16); and third, elevated glucocorticoid levels, found during stress, in late gestation, and with wide use therapeutically for anti-inflammatory and immunomodulatory purposes, frequently precipitate diabetes in patients (25)(26)(27)(28)(29). Here, we demonstrate that lactogens protect beta cells against DEX-induced apoptosis. Thus, chronic endogenous expression or acute exogenous treatment of rat insulinoma cells and primary mouse islet cells with lactogens causes a significant decrease in beta cell death, measured by numerous assays, against at least two distinct beta cell death inducers, STZ and DEX, suggesting a generalized pro-survival role of lactogens in beta cells.
PRL and PL acting through a common PRL-R activate several downstream signaling pathways including JAK2/STAT5, PI3K/ Akt, ERK1/2, adenylyl cyclase/cAMP, and protein kinase/intracellular calcium (31)(32)(33). However, which of these pathways mediates the protective effect of PRL on beta cells is not known. Different signaling pathways have been implicated in the pro- survival effect of PRL in other cell types (17,18). For example, in androgen-deprived prostate epithelial cells, PRL improves survival through activation of STAT5a and STAT5b but not ERK1/2 or Akt (17), whereas, in rat decidual cells PRL-mediated survival is through the PI3K/Akt pathway and not the JAK2 pathway (18). To examine which of these pathways mediates the protective effect of lactogens in the beta cell, we used three separate approaches, including pharmacological inhibitors, JAK2-specific siRNAs and a DN-STAT5 mutant. Of the three different inhibitors, only the JAK2 inhibitor AG490, reduced lactogen-mediated protection of INS-1 cells. In general, AG490 is known to be a specific inhibitor of the JAK2 tyrosine kinase in most cell types (38). However, there is evidence suggesting that AG490 can also inhibit the highly related JAK3 tyrosine kinase and its downstream signaling pathways in lymphocytes (39). We therefore examined the importance of the JAK2/STAT5 pathway using different approaches, siRNA in INS-1 cells and a DN mutant in primary beta cells. Together, all three methods demonstrate that lactogens mediate their pro-survival effect through the JAK2/STAT5 pathway in the rat insulinoma cell line as well as in primary beta cells.
In mouse mammary epithelial cells (15) and the Nb2 lymphoma cell line (16), pretreatment with lactogens increases the expression of the anti-apoptotic molecules Bcl2 and Bcl-X L , with a concomitant decrease in the pro-apoptotic molecule Bax, thus increasing the ratio of Bcl/Bax which normally favors cell survival. To better understand the molecular mechanisms through which lactogens mediate their pro-survival effect on beta cells we demonstrate a specific increase in the expression of the anti-apoptotic protein, Bcl-X L , in lactogen-treated insulinoma cells and mouse islets, thus causing an increase in the Bcl-X L /Bax ratio in beta cells. While there is an association between the induction of anti-apoptotic members of the Bcl family of proteins by lactogens and their pro-survival effect in different cell types, including thymocytes and mammary epithelial cells (15,16), there is no direct evidence linking the two phenomena in any cell type. In the present study we demonstrate using Bcl-X L -specific siRNA, that lactogen-induced upregulation of Bcl-X L is necessary to mediate its protective effect on beta cells against DEX-induced cell death. We believe this is the first direct demonstration of the importance of Bcl-X L in mediating the pro-survival effect of lactogens in any cell type thus far.
Based on our results from Fig. 9B, PRL does not completely lose its protective effect upon reduction of Bcl-X L , suggesting that there could be additional molecular pathways through which PRL mediates its pro-survival effect in beta cells. The pro-survival effect of PRL in non-beta cells correlates with down-regulation of the insulin-like growth factor binding protein 5 (40), increased expression of other regulators of apoptosis, such as X-linked inhibitor of apoptosis protein (41), c-Myc, and pim1 (42). In this context, a study examining the transcriptional modulation of genes by PRL in rat islets by DNA microarray analysis identified pro-apoptotic genes such as clusterin, NFKBIA, and TNFRSF1A as being down-regulated by PRL (43). Whether any of these molecules are important in mediating the pro-survival effect of lactogens in beta cells remains to be determined. Thus, the current study demonstrates that the increase in Bcl-X L is required to achieve the full protective effect of lactogens in beta cells, and also supports the idea that other, as yet unidentified, molecular pathways may also be involved.
These studies raise important questions: Do lactogens protect beta cells against other cell death inducers relevant to type I and type II diabetes? Do lactogens mediate their protective effect against different cell death inducers through common signaling pathways and molecular mechanisms? In this context human growth hormone (hGH) was recently shown to protect INS-1 cells against cytokine-induced cell death through the B. activation of STAT5 with a concomitant increase in the Bcl-X L / Bax ratio (44). However, unlike PRL and PL which signal through the PRL-R, hGH can interact with either the GH receptor or the PRL-R in rodent cells, and activation of these two receptors initiates different signaling profiles and quite distinct functional outcomes in beta cells (7,9,45). Therefore, it is unclear which of the two receptors is mediating the pro-survival effects of hGH on the beta cell.
Lactogens have been repeatedly shown to enhance beta cell proliferation and function, and the current studies demonstrate that lactogens can also improve survival of beta cells against different cell death inducers. Therefore, lactogens can now be added to the short list of growth factors with the ability to improve all three critical beta cell parameters: function, proliferation and survival, ideal characteristics of potential candidates for future therapeutic strategies to treat diabetes.