Raf kinase inhibitory protein inhibits b-cell proliferation - CiteSeerX

Raf kinase inhibitory protein inhibits b-cell proliferation Lizhi Zhang, MD, Zheng Fu, PhD, Charles Binkley, MD, Thomas Giordano, MD, Charles F. Burant, MD, PhD, Craig D. Logsdon, PhD, and Diane M. Simeone, MD, Ann Arbor, Mich

Background. Raf-1 kinase inhibitory protein (RKIP) was recently identified as a physiologic endogenous inhibitor of the extracellular signal-regulated kinase (ERK) pathway. The expression and role of RKIP within the pancreas are unknown. Methods. RKIP expression in normal pancreas and human insulinomas was examined by using paraffin-embedded sections. Co-localization of RKIP within islet cell subtypes was performed by using double immunofluorescence staining with antibodies directed toward RKIP and endocrine markers. To examine the role of RKIP in b-cell proliferation, stable expression of sense (ss) and antisense (as) RKIP was established in HIT-T15 b cells. The effect of RKIP on the ERK-signaling pathway in b cells was determined by Western blotting with the use of phospho-specific antibodies directed against mitogenactivated protein kinase kinase (MEK) and ERK. The role of RKIP in b-cell proliferation was assessed by using MTS assay and FACS analysis. Results. RKIP was expressed only within pancreatic islet cells. Immunofluorescent double staining revealed that RKIP was expressed in most b cells and a subset of pancreatic polypeptide–expressing cells. Based on the known function of RKIP, we hypothesized that RKIP expression would be downregulated in insulinomas: 8 of 9 human insulinomas demonstrated no RKIP staining, with decreased expression in 1 of 9 insulinomas. Studies using asRKIP and ssRKIP demonstrated that RKIP blocked activation of MEK and ERK by Raf-1 in b cells. We also showed that RKIP inhibited b-cell proliferation by altering cell cycle distribution, rather than by promoting apoptosis. Conclusions. RKIP is important in b-cell proliferation, and its downregulation may play a role in islet neoplasia. (Surgery 2004;136:708-15.) From the Departments of Surgery, Pathology, Internal Medicine, and Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Mich

ENDOCRINE CELLS IN THE PANCREAS are believed to originate from ductal epithelial-like cells.1-4 In mice, endocrine cells initially appear individually in these ductal-like cells during development. The endocrine cells later form clusters in the interstitial tissues and then organize into well-defined islet structures with centrally located b cells surrounded by the other endocrine cell subtypes.5,6 Neogenesis of islets continues through the neonatal period and ceases shortly after weaning.5,7 In the early neonatal period, mitosis of all 4 types of islet endocrine cells is observed.8 In adulthood, b cells of the islets of Langerhans have a limited potential for proliferaAccepted for publication December 27, 2003. Supported by NlH grant DK02137-01 (D.S.) and the American College of Surgeons Faculty Research Fellowship (D.S.). Reprint requests: Diane M. Simeone, MD, Associate Professor of Surgery, TC 2922D, Box 0331, University of Michigan Medical Center, 1500 E Medical Center Dr, Ann Arbor, MI 48109. 0039-6060/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.surg.2003.12.013

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tion.9 Although their proliferative ability is limited, kinetic analyses of pancreatic endocrine cells during postnatal growth suggest a dynamic of b-cell replication and apoptosis.9 There are 2 proposed methods of b-cell proliferation: replication of already differentiated b cells10 or neogenesis from nonendocrine cells or stem cells.11,12 The contribution of these 2 mechanisms to changes in adult b-cell mass remains to be defined. Certainly, a better understanding of the mechanisms involved in b-cell proliferation and differentiation may lead to new strategies for treatment of diabetes. Mitogen-activated protein (MAP) kinases are serine/threonine kinases that are activated by a variety of cell surface receptors. MAP kinases function in signal cascade pathways that control many regulatory processes in eukaryotic cells, including growth, differentiation, and cell survival. The best studied MAP kinases are the extracellular signal-regulated protein kinases (ERKs), which are stimulated by growth factors.13,14 In the ERK-signaling cascade, the small G protein Ras is activated by a variety of growth

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factors and, when activated, binds to Raf-1 kinase. This induces activation of Raf-1, which then phosphorylates and activates ERKs. Activated ERKs can then translocate to the nucleus and regulate gene expression by the phosphorylation of transcription factors. Several reports have suggested that ERKs play an important role in signaling proliferative responses of b cells.15,16 Recently, Raf-1 kinase inhibitory protein (RKIP), isolated in a yeast 2-hybrid screen as a Raf-1– associated protein,17 has been shown to exhibit an inhibitory function within the ERK-signaling pathway.17,18 RKIP binds to Raf-1 and MEK in vitro and in vivo, interfering with the activation of the Raf/ MEK/ERK–signaling pathway. Stimulation by growth factors induces the release of RKIP from Raf-1, allowing activation of MEK and ERK. The aim of this study was to evaluate the expression of RKIP in the pancreas and to specifically determine the role of RKIP in the proliferation of b cells. We found that RKIP was expressed only within the islets of the pancreas, and co-localized with insulin- and pancreatic polypeptide (PP)–producing cells. RKIP was downregulated in human insulinomas and inhibited proliferation in a b-cell line. These data suggest that RKIP has a unique role in regulation of proliferation within the pancreatic islet. MATERIAL AND METHODS Pancreatic tissues and cultured cells. Paraffinembedded normal human pancreatic tissue and human insulinomas came from the University of Michigan Medical Center and conformed to the policies and practices of the University of Michigan Institutional Review Board. The HIT-T15 SV40transformed hamster b-cell line was obtained from American Type Culture Collection (ATCC, Manassas, Va) and grown in F12K Kaighn’s modification medium supplemented with 10% horse serum, 2.5% fetal bovine serum, 2 mmol/L Lglutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin. The cells were maintained in a humidified incubator with 5% CO2 at 378C. Subcloning of RKIP. The complementary DNA (cDNA) of human RKIP was obtained from ATCC and subcloned into pcDNA3.1(+) and pcDNA3.1(-) expression vectors (Invitrogen, San Diego, Calif) by using BamHI and EcoRI sites, which express sense (ss) and antisense (as) RKIP that are driven by a cytomegalovirus (CMV) promoter. To distinguish between pcDNA3.1(+)ssRKIP and pcDNA3.1(-) asRKIP plasmids, we analyzed the orientation of RKIP cDNA in plasmid clones by sequencing and

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restriction enzyme mapping. Sequence analysis showed 100% homology to the published sequence for RKIP cDNA. Transfection and selection. HIT-T15 cells were transfected with 1 lg of pcDNA3.1(-)asRKIP or pcDNA3.1(+)ssRKIP, respectively, by using Lipofectamine Plus Reagent (Gibco BRL, Galthersburg, Md) according to the manufacturer’s instructions. Selection was initiated 48 hours after transfection by adding 500 lg G418/mL (Gibco BRL, Galthersburg, Md) to the supplemented culture medium. Selection medium was changed every 2 days for 4 weeks until all nontransfected cells died. Resistant cell clones were isolated and expanded for further characterization. The empty vectors, pcDNA3.1(-) and pcDNA3.1(+), were also transfected into HIT-T15 cells and served as negative controls. Immunoblot analysis. Whole cell lysates were prepared by incubating the cells in ice-cold lysis buffer (50 mmol/L Tris-HCl [pH 7.8], 2 mmol/L EDTA, 50 mmol/L NaF, 1% Triton X-100, 5 lg/mL leupeptin, 5 lg/mL pepstatin, and 0.5 mmol/L PMSF). The cells were sonicated for 5 seconds, and the lysates centrifuged at 14,000 3 g for 15 minutes at 48C. The supernatant was removed and assayed for protein concentration with the use of Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, Calif). Equal amounts of protein (20-50 lg) were resolved by 10% or 15% SDS-PAGE and transferred to nitrocellulose membranes. Immunoblot analysis was performed with the use of rabbit polyclonal anti-RKIP (Santa Cruz Biotechnology, Santa Cruz, Calif), mouse anti-actin (Sigma, St. Louis, Mo) at titers of 1:3000, and rabbit polyclonal anti– phospho-ERK, anti–phospho-MEK, anti-ERK and anti-MEK antibodies (Santa Cruz Biotechnology). Images were visualized with the use of ECL Detection System (Amersham, Arlington Heights, Ill). Film images were scanned with an Agfa Arcus II (Bayer Corp, Ridgefield Park, NJ) to create a digital image. Immunohistochemistry. Human pancreatic tissue and insulinoma tissue were fixed in 10% buffered formalin and embedded in paraffin by using standard techniques. For staining of insulin and RKIP, standard immunohistochemical staining procedures were performed. Briefly, after blocking with 5% horse serum at room temperature for 20 minutes, sections were incubated with guinea pig anti-insulin (1:1000) (Linco Research Inc, St. Charles, Mo) or rabbit anti-RKIP (1:500) (Santa Cruz Biotechnology) overnight at 48C. After rinsing, biotinylated secondary antibodies and horseradish peroxidase–conjugated antibodies were applied

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Fig 1. RKIP expression in the human pancreas. Paraffinembedded sections of normal human pancreas were incubated with anti-RKIP antibody and developed with DAB. Note the absence of RKIP staining outside of the islet. A, original magnification 320; B, original magnification 340.

according to the manufacturer’s instructions. Peroxidase activity was visualized by applying diaminobenzidine solution, containing 0.05% H2O2, for 2 to 10 minutes at room temperature. Sections were then counterstained with hematoxylin, dehydrated, cleared, and mounted. For the double immunofluorescence staining, the tissue sections were incubated overnight at 48C in a mixture of rabbit anti-RKIP or goat anti-RKIP antibodies (Santa Cruz Biotechnology) with antibodies against insulin, glucagon (both at 1:1000; Linco Research Inc, St. Charles, Mo), somatostatin (1:500; Dako, Carpinteria, Calif), and pancreatic polypeptide (1:500; Linco Research), respectively. After being washed with phosphate-buffered solution (PBS), the sections were incubated for 2 hours at room temperature with a mixture of FITClabeled antirabbit IgG and Texas red-labeled antiguinea pig IgG or Texas red-labeled antigoat IgG (Vector Labs, Burlingame, Calif) for double staining. Slides were rinsed with PBS and mounted

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in fluorescence mounting medium (Vector Labs). Images were obtained with the use of a Carl Zeiss fluorescence microscope (Carl Zeiss, New York, NY). Proliferation assay. Cell proliferation was measured with the use of CellTiter 96 AQ nonradioactive cell proliferation assay (MTS; Promega, Madison, Wis). Briefly, cells were plated in 96-well plates at a density of 2000 cells/well in 100 lL medium. Cells were allowed to grow for 2, 4, 6, or 8 days, then 20 lL/well of combined MTS solution was added. After an incubation of 2 hours at 378C in a humidified 5% CO2 atmosphere, the absorbance was measured at 490 nm with the use of an ELISA plate reader. Experiments were performed in triplicate. Flow cytometric assay. Parental and transfected HIT-T15 cell lines were grown to 60% to 80% confluence. The cells were trypsinized and washed once with PBS, then counted and resuspended in 0.5 mL PBS at a concentration of 13106 cells/mL. propidium iodide (PI) DNA labeling was performed after ethanol fixation for analysis of cell cycle distribution. Briefly, 0.5 mL cold 100% ethanol was added, and the cells were incubated for 20 minutes. The cells were collected after a brief centrifuge, the ethanol was removed, and 0.5 mL PI-RNAse solution (50 lg/mL PI + 100 lg/mL RNAse Type I in PBS) was added. The cells were then incubated for a minimum of 20 minutes in the dark and were analyzed by flow cytometry. For detection of apoptotic cells, an ApoAlert Annexin V-FITC Apoptosis kit was used (BD Biosciences, Palo Alto, Calif). Cells were induced to undergo apoptosis with serum starvation or treatment with TNFa (400 U/mL) for 48 hours. Cells were collected and stained with annexinV-FITC according to the manufacturer’s instructions. Statistical analysis. Statistical analysis was performed by using analysis of variance and a post hoc comparison among groups with Fisher’s protected least squares difference test. Statistical significance was accepted as P < .05. RESULTS RKIP expression in pancreas. While RKIP has been reported to have a wide tissue distribution, with the protein enriched in the CNS and muscle,19,20 the expression of RKIP in the pancreas has not been previously examined. To determine the expression of RKIP in the pancreas, immunohistochemical staining was performed by using multiple sections of paraffin-embedded sections from 6 normal human pancreata. RKIP immuno-


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Fig 2. RKIP expression within endocrine cell subtypes of the islets of Langerhans. A-D, Immunofluorescent localization of islet hormones was performed in human pancreatic sections. A, Insulin; B, glucagon; C, somatostatin; and D, PP. E-H, RKIP staining of islet. I-L, Merged images of the double labeling. A doublelabeled PP cell is indicated with arrow (panel L). Original magnification 340. The experiment was performed in duplicate.

reactivity was detected only in pancreatic islets, with no staining evident in acinar or ductal cells (Fig 1). The endocrine cell subtypes expressing RKIP were identified by immunofluorescent double staining by using an anti-RKIP antibody and antibodies directed against insulin, glucagon, somatostatin, and PP (Fig 2). RKIP was expressed in most b cells and a subset of PP-expressing cells, (Fig 2, L, arrow), but not in cells expressing glucagon or somatostatin. Similar results were seen in mouse pancreatic sections (data not shown). RKIP expression in human insulinomas. Insulinomas are tumors of b-cell origin. Although they are rare, they are the most common type of pancreatic endocrine neoplasm. Based on the known function of RKIP and its expression in b cells, we hypothesized that RKIP expression would be downregulated in insulinomas. To examine the expression of RKIP, we stained 9 human insulinomas with an anti-RKIP antibody. Staining

with an anti-insulin antibody confirmed that the tumors expressed insulin (Fig 3, A, black arrowhead). Eight of the 9 tumors demonstrated no RKIP staining (Fig 3, B, black arrow), whereas RKIP staining could be seen in normal islets adjacent to the tumor (Fig 3, B, black arrowhead). In 1 of the 9 insulinomas, RKIP expression was present in approximately 5% to 10% of the insulinoma cell population (Fig 3, C). The effect of RKIP on the MAP kinase– signaling pathway. To study the functional role of RKIP in b cells, the HIT-T15 SV-40 transformed hamster b-cell line was used. HIT-T15 cells were stably transfected with constructs expressing antisense (asRKIP) and sense (ssRKIP) RKIP. Two stable cell lines were used in each group. Results were similar in each pair of stable cell lines; therefore, in some experiments, results were pooled. As negative controls, HIT-T15 cells were transfected with empty vector alone. To determine

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levels in asRKIP-transfected HIT-T15 cells were significantly reduced compared with control tranfectants (Fig 4, A). In contrast, RKIP protein levels in ssRKIP-transfected HIT-T15 cells were significantly increased. To examine the effect of RKIP on the activation steps of the Raf/MEK/ERK cascade, we used asRKIP- and ssRKIP-expressing cells and compared their effects with control HIT-T15 cells. ssRKIP decreased basal phosphorylation of both MEK and ERK by Raf-l, whereas asRKIP increased phosphorylation of MEK and ERK compared with controls (Fig 4, B, C). These data indicate that RKIP is able to block basal levels of MEK and ERK activation by Raf-l in b cells. The role of RKIP in b-cell proliferation. The effect of RKIP on the proliferation of b cells was examined with the use of MTS assay. Control cells and asRKIP- and ssRKIP-expressing HIT-Tl5 cells were grown in culture for 8 days. As shown in Fig 5, A, the proliferation rate of asRKIP-expressing cells was increased compared with control cells, whereas ssRKIP-expressing cells exhibited decreased growth rates. The effect of RKIP on b-cell proliferation was confirmed with FACS analysis of synchronized HITTl5 cells, which revealed that asRKIP-expressing cells had a significant increase and that ssRKIPexpressing cells had a significant decrease in the percentage of cells in the G2 + S phase (Fig 5, B). Apoptosis induced by serum-starvation or TNFa (measured by annexin V staining and caspase-3 levels) was not altered in asRKIP- and ssRKIPexpressing cells lines compared with control cells (data not shown), suggesting that RKIP does not regulate b-cell proliferation via an apoptotic pathway.

Fig 3. RKIP expression in human insulinomas. A, B, Representative pancreatic section of 8 of the 9 insulinomas examined. A, Section of a human insulinoma labeled with an anti-insulin antibody. The arrow indicates the tumor and the arrowhead indicates a normal nearby islet. B, There is loss of expression of RKIP in the insulinoma labeled with an anti-RKIP antibody (arrow), but preserved RKIP labeling in a normal nearby islet (arrowhead). C, In 1 of the 9 insulinomas, RKIP expression was present in approximately 5% to 10% of the insulinoma cell population. Original magnification 320.

the effect of antisense and sense RKIP cDNA transfection on endogenous RKIP protein levels in HIT-T15 cells, we analyzed cell lysates by Western blotting, using an anti-RKIP antibody. RKIP protein

DISCUSSION The Raf-l/MEK/ERK module is a ubiquitously expressed signaling cascade that controls proliferation and differentiation of many cell types. Recently, RKIP has been reported to be an inhibitory protein for the Raf-1/MEK/ERK module17 and to possibly function as a rheostat that sets the sensitivity threshold for the activation of the Raf/1/ MEK/ERK pathway.18 RKIP is expressed in many tissues, including the central nervous system, spleen, testis, ovary, muscle, and stomach;19,20 however, levels of expression vary widely. The presence of RKIP in the pancreas has not been examined previously. Interestingly, we found that RKIP was expressed only in the islet cells in the pancreas by immunohistochemistry. Costaining with antibodies directed toward insulin, glucagon,


somatstatin, and PP demonstrated that RKIP is expressed specifically in b- and PP- expressing cells. Based on these co-localization studies and the known role of RKIP in inhibiting ERK signaling, we hypothesized that RKIP expression might be downregulated in insulinomas, in which normal growth regulatory mechanisms are lost. Indeed, we found that RKIP expression was not detectable in 8 of 9 insulinomas and had only a low level of expression in 1 of 9 tumors. The etiology of loss of RKIP expression in the 8 insulinomas is unknown; further studies will need to be performed to determine if RKIP loss is due to deletion/mutation of the RKIP gene or due to epigenetic mechanisms that regulate RNA or proteins levels of RKIP. The role of RKIP in neoplastic development and/or progression in other types of neoplasms is relatively unknown. A single report by Fu and colleagues21 found that RKIP did not influence the tumorigenic properties of human prostate cancer cells, but rather appeared to suppress metastasis by decreasing vascular invasion. We showed that, in ssRKIP-expressing HIT-T15 cells, phosphorylation of MEK and ERK was decreased compared with control cells. This finding is in accordance with previous studies in other cell types in which overexpression of RKIP was found to interfere with the activation of MEK and ERK and the induction of AP-1.17 In contrast, lowering RKIP protein levels by expression of antisense RKIP RNA in b cells induced activation of MEK and ERK. This has been observed in other cell types, in which blocking RKIP function with antisense or a neutralizing antibody induced activation of MEK, ERK, and AP-1 transcription.20 RKIP levels regulated b-cell growth, as evidenced by both MTS assays and FACS analysis, with increased growth evident in asRKIP-expressing cells and decreased growth in ssRKIP-expressing Fig 4. The effect of RKIP levels on MAP kinase activity in b cells. A, HIT-T15 cells were stably transfected with empty vector (asRKIP or ssRKIP), and RKIP protein levels were assessed by Western blotting with an anti-RKIP antibody. b actin served as a loading control. Expression levels of RKIP were quantitated with the use of densitometry and are presented graphically as percentage of control. B, C, MAP kinase activity in HIT-T15 cells stably transfected with empty vector (asRKIP or ssRKIP) was determined by Western blotting with anti–phosphoMEK and anti–phospho-ERK antibodies. Anti-total MEK and ERK antibodies were used as loading controls. Levels of phosphorylation of MEK and ERK were quantitated with the use of densitometry and are presented graphically as percentage of control. Experiments were performed in triplicate (*P < .05 and **P < .01 vs control).

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cells compared with control cell lines. The effect of RKIP on b-cell proliferation had not been examined previously. Two previous reports evaluated the role of MAP kinases in b-cell proliferation. With fetal bovine serum used as a mitogenic stimulus, the MEK inhibitor PD98059 blocked BrdU incorporation in the MIN6 pancreatic b-cell line.15 This finding is in agreement with an earlier report in which the ERK inhibitor PD98059 blocked fetal bovine serum-induced [3H]thymidine incorporation in rat insulinoma cell DNA.22 While it is highly likely that RKIP is regulating bcell growth due to effects on the ERK-signaling pathway, it is possible that RKIP may have additional interactions with other signaling pathways. RKIP was originally identified as a member of a family of phosphatidylethanolamine-binding proteins; however, its ability to bind phospholipids seems to have no bearing on its inhibitory function within the ERK-signaling pathway.17 RKIP has also recently been shown to antagonize the signal transduction pathways that mediate activation of the transcription factor nuclear factor kappa B (NFjB) in response to stimulation with TNFa or interleukin 1.23 Modulation of RKIP expression levels affected NFjB signaling independently of the MAP kinase pathway. Since MAP kinases and NFjB have physiologically distinct roles, RKIP may function, at least in part, to coordinate the regulation of these 2 pathways.23 The role of NFjB in RKIP-mediated growth regulation in the b cell remains to be elucidated. However, we observed no effect of RKIP on b-cell apoptosis, a mechanism strongly influenced by NFjB.

Fig 5. The role of RKIP in b-cell proliferation. A, Downregulation of RKIP promotes b-cell proliferation. HIT-T15 cell lines expressing empty vector (control), ssRKIP, or asRKIP were grown in culture over an 8-day period; then MTS assays were performed. Results were from 3 separate experiments. B, Cell cycle distribution by flow cytometric analysis with propidium iodide staining. Results were expressed as the percentage of cells in G2 + S phase and are from 3 separate experiments (*P < .05 vs control).

CONCLUSION We have demonstrated that RKIP is specifically expressed in b cells and in a subset of PP cells in the human islet. RKIP expression is downregulated in human insulinomas and inhibits b-cell proliferation in the HIT-TI5 b-cell line. These data collectively support a model in which limited b-cell replication in adult islets is due to high levels of expression of RKIP, which inhibits the replicative ability of b cells. Downregulation or loss of RKIP expression results in b-cell proliferation and may contribute to the development of islet neoplasia. REFERENCES 1. Pictet R. Rutter RJ. Development of the embryonic endocrine pancreas. In: Steinerand D, ed. Handbook of Physiology. Vol l. Baltimore: Williams and Wilkins; 1972:25-66. 2. Wessells NK, Cohen JH. Early pancreatic organogenesis: morphogenesis, tissue interaction, and mass effects. Cell Tissue Res 1967;15:237-70.


3. Wessels NK, Evans J. Ultrastructural studies of early morphogenesis and cytodifferentiation in the embryonic mammalian pancreas. Dev Biol 1968;17:413-46. 4. Gu G, Brown JR, Melton DA. Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mech Dev 2003;120:35-43. 5. Deltour L, Leduque P, Paldi A, et al. Polyclonal origin of pancreatic islets in aggregation mouse chimaeras. Development 1991;112:1115-21. 6. Dubois PM. Ontogeny of the endocrine pancreas. Hormone Res 1989;32:53-60. 7. Crisera CA, Maldonado TS, Kadison AS, et al. Transforming growth factor- b in the developing mouse pancreas: a potential regulator of exocrine differentiation. Differentiation 2000;65:255-9. 8. Magami Y, Azuma T, Inokuchi H, et al. Heterogeneous cell renewal of pancreas in mice: [3H]-thyrnidine autoradiographic investigation. Pancreas 2002;24:153-60. 9. Bonner-Weir S. Islet growth and development in the adult. J Mol Endocrinol 2000;24:297-302. 10. Wilkin TI. Neogenesis of islet cells. Diabetes Metab Rev 1998;14:329-35. 11. Bouwens L. 1998. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 1998;43:332-6. 12. Bouwens L, Kloppel G. Islet cell neogenesis in the pancreas. Virchows Arch 1996;427:553-60. 13. Brunet A, Roux D, Lenormand P, et al. Nuclear translocation and p42/p44 mitogen-activated protein kinase is required for growth factor-induced gene expression and cell cycle entry. EMBO J 1999;18:664-74. 14. Cobb MH, Goldsmith El. How MAP kinases are regulated. J Biol Chem 1995;270:14843-6.

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15. Burns CI, Squires PE, Persuad SI. Signaling through the p38 and p42/44 mitogen-activated families of protein kinases in pancreatic b cell proliferation. Biochem Biophys Res Comm 2000;268:541-6. 16. Tanabe K, Okuya S, Tanizawa Y, et al. Leptin induces proliferation of pancreatic cell line MIN6 through activation of mitogen-activated protein kinase. Biochem Biophys Res Comm 1997;241:765-8. 17. Yeung K, Seitz T, Li S, et al. Suppression of Raf-l kinase activity and MAP kinase signaling by RKIP. Nature 1999;401:173-7. 18. Yeung K, Ianosch B, McFerran D, et al. Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the Raf kinase inhibitor protein. Mol Cell Biol 2000;20:3079-85. 19. Bollengier F, and Mahler A. Localization of the novel neuropolypeptide hJ in subsets of tissues from different species. J Neurochem 1988;50:1210-4. 20. Frayne I, Ingram C, Love S, et al. Localization of phosphatidylethanolamine-binding protein in the brain and other tissues of the rat. Cell Tissue Res 1999;298:415-23. 21. Fu Z, Smith PC, Zhang L, Rubin MA, Dunn RL, Yao Z, Keller ET. Effects of raf kinase inhibitor protein expression on suppression of prostate cancer metastasis. J Nat Cancer Inst 2003;95:878-9. 22. Schuppin GT, Pons S, Hugl S, et al. A specific increased expression of insulin receptor substrate 2 in pancreatic beta-cell lines is involved in mediating serum-stimulated beta-cell growth. Diabetes 1998;47:1074-85. 23. Yeung K, Rose DW, Dhillon AS, et al. Raf kinase inhibitor protein interacts with NFjB inducing kinase and TAK-1 and inhibits NFjB activation. Mol Cell Biol 2001;21: 7207-17.