Survivin Regulation of Beta-Cell Mass Expansion Figure 7. Beta-cell proliferation is partially inhibited in RIPCre+Survivinfl/fl mice after PDL. (a) Representative Ki67 and insulin immunostaining on consecutive pancreatic sections (upper panel) and quantification (lower panel) reveal that PDL markedly stimulates beta-cell proliferation in the ligated tail of pancreas both in the mutant mice and controls at day 7. However, the increment is significantly attenuated in the mutant mice as compared to that in controls (left lower panel), especially within larger islets (right lower panel), while the increment is relatively more preserved in small islets (middle lower panel) (original magnification, 6400; n = 5 per genotype). (b) Fold change in the expression of the indicated genes related to cell cycle as determined by quatitative PCR in day 7 PDL or sham-operated pancreas in the mutant mice compared with controls (n = 5 per genotype). (c)Western blots and quantification show a decrease in p21, Caspase-3, Aurora B kinase and phospho-AKT expression in islets of day 7 PDL pancreas in mutant mice (gray bars) compared to controls (black bars) (n = 5 per genotype). (d) Representative insulin and TUNEL co- staining in the ligated tail of pancreas (left panel; original magnification, 6400; apoptotic beta cells are indicated by arrows) and quantification (right panel) show low rate of beta cell apoptosis at day 7 following ligation or a sham operation with no significant difference between the mutant mice and controls (n = 5 per genotype). +/+, RIPcre+survivin+/+ mice; 2/2, RIPcre+survivinfl/fl mice. WT, wildtype; KO, knockout. PDL, black bars; sham operation, white bars. The data represent as means 6 SE. *p,0.05, **p,0.01 vs sham operation; #P,0.05, ##p,0.01 vs control. doi:10.1371/journal.pone.0041976.g007 would avoid the confounding effects of preexisting defects in glucose homeostasis. The female mutant mice indeed exhibited glucose intolerance, compromised beta-cell mass expansion, blunted increase in islet and beta-cell number, and impaired beta-cell proliferation following PDL compared to control wild- type littermates after the same procedure. Importantly, PDL-stimulated beta-cell proliferation was blunted, but not completely abolished within the ligated tail of the pancreas in the mutant mice. This may be due to beta-cell neogenesis. PDL has been shown to activate the progenitors in the ductal lining to differentiate in Ngn3 dependent way and give rise to glucose responsive beta cells that subsequently proliferate to increase the functional beta-cell mass [5]. Gene expressions related to islet neogenesis such as Ngn3, NeuroD1, Nkx2.2, Nkx6.1, Pdx1 and MafA are similar between the mutant mice and controls, suggesting that survivin deletion in beta cells does not impair islet neogenesis after PDL. Although the increment of total islet number was markedly attenuated in RIPcre+survivinfl/fl mice compared to wildtype littermates 7 days after PDL, this was largely due to impairment in the rise in the number of larger islets. In contrast, rise in the number of smaller islets were relatively preserved, thereby not significantly contributing to the defect of islet mass expansion observed in mutant mice. These data support our observation that small islets represent newly formed beta cells that likely do not require survivin for its formation or proliferation, whereas expansion of already-formed islets that arise from replication of preexisting beta cells appears to require survivin. The similar phenomenon was found in the FoxM1 knockout mice after 60% PPx [28]. FoxM1 was not required for proliferation of beta cells within small endocrine cell clusters located in the regenerating portion of the pancreas but was specifically required for proliferation of preexisting beta-cells within largerislets. FoxM1 transactivates genes that coordinate the G1/S and G2/M transitions, karyokinesis and cytokinesis [26]. As the known FoxM1 target, survivin fits well within the molecular pathway downstream of Foxm1 and likely functions to assist them in the modulating of beta-cell replication. In our study, FoxM1 gene expression was upregulated by 1.7 fold in survivin mutant mice after PDL compared with wild-type controls. However, defect in beta-cell proliferation indicated that the cell cycle promotion effect of FoxM1 might be mediated through survivin-dependent pathway. References Survivin plays an important role in cell cycle regulation. During G1, survivin transcription increases and reaches a peak in G2/M [34,35]. During mitosis, survivin has been shown to co-localize with caspase-3 and p21 at the centrosome [36]. As a chromosomal passenger protein, survivin is involved in chromosome segregation, central spindle formation, cytokinesis and spindle checkpoint maintenancethrough its interactions with other passenger proteins, inner centromere protein and AuroraB kinase [37]. Thus, disruption of survivin may lead to abnormal centrosome duplica- tion and formation of multipolar spindles, multinucleation and polyploidy. In keeping with these observations, our in vivo data reveal that survivin-deficient beta cells have many enlarged nuclei, together with decreased expression of p21, Caspase-3 and AuroraB kinase. The decreased gene expression of Rb1 and Ccne1 could cause the delay on G1/S transition during cell cycle. In addition, levels of phosphorylated Akt were significantly decreased in the mutant islets, indicating that the phosphatidyli- nositol 3-kinase–AKT pathway may be involved in survivin mediated beta-cell proliferation. Therefore, resurgence of survivin in response to PDL may be critical in recruiting more beta cells to enter cell cycle that is necessary for islet expansion. Taken together, our data show that resurgence of survivin expression in the pancreatic beta cells after PDL is essential for beta-cell mass expansion largely through beta-cell proliferation. The preexisting beta cells seemingly exhibit a stronger require- ment for survivin than new beta cells formed by neogenesis. This study highlights the importance of survivin-dependent replication of preexisting beta cells as a mechanism of beta-cell mass expansion. Given that beta cell expansion appears to occur through proliferation of existing beta cells, survivin may indeed be a viable therapeutic target for beta cell expansion in diabetes. Supporting Information Table S1 (DOC) Primers for qRT-PCR. Author Contributions Conceived and designed the experiments: X. Wu MW. Performed the experiments: X. Wu QZ X. Wang JZ KX. Analyzed the data: X. Wu KX. Contributed reagents/materials/analysis tools: HO RW MW. Wrote the paper: X. Wu RW MW. 1. Finegood DT, Scaglia L, Bonner-Weir S (1995) Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model. Diabetes 44: 249–256. 2. Ackermann AM, Gannon M (2007) Molecular regulation of pancreatic beta-cell mass development, maintenance, and expansion. J Mol Endocrinol 38: 193–206. 3. Dor Y, Brown J, Martinez OI, Melton DA (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429: 41– 46. 4. Teta M, Rankin MM, Long SY, Stein GM, Kushner JA (2007) Growth and regeneration of adult beta cells does not involve specialized progenitors. Dev Cell 12: 817–826. 5. Xu X, D’Hoker J, Stange G, Bonne´ S, De Leu N, et al. (2008) Beta cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell 132: 197–207. 6. Srinivasula SM, Ashwell JD (2008) IAPs: what’s in a name? Mol Cell 30: 123– 135. PLoS ONE | www.plosone.org 10 August 2012 | Volume 7 | Issue 8 | e41976