Production and maintenance of the pancreatic β-cell mass is a highly regulated process driven by four major mechanisms that include- β-cell replication, β-cell neogenesis, β-cell hypertrophy and β-cell apoptosis [1, 2]. In the rodent, an exponential expansion of the pancreatic β-cell mass begins during the final phase of gestation and lasts through the third week after birth. Correspondingly, in humans, β-cell expansion occurs during the last trimester of pregnancy and continues through the first few months of life [1, 2]. An increase in β-cell mass is required for insulin secretion in the maintenance of metabolic homeostasis , both in the initial transition to a carbohydrate-based diet following weaning and throughout life thereafter . The molecular mechanisms regulating β-cell growth are mostly unknown but are dependent on a variety of growth factors, including glucose, insulin, insulin-like growth factor (IGF-I), and epidermal growth factor (EGF) [5, 6], that provide mitogenic signals to the β-cell in vivo.
Epidermal growth factor receptor (EGFR) is a member of the ErbB receptor family, consisting of 4 transmembrane tyrosine kinase receptors: EGFR (ErbB1, HER1), ErB2 (neu/HER2), ErbB3 (HER3) and ErbB4 (HER4) [7, 8]. All such proteins contain an extracellular domain responsible for ligand binding, a single membrane-spanning domain, and a cytoplasmic tyrosine kinase domain with multiple auto-phosphorylation sites. Binding of a ligand to EGFR leads to the formation of homo- or heterodimers, followed by phosphorylation of tyrosine residues and second messenger recruitment [7, 8]. EGF is a potent growth factor and one of the 11 ligands of this receptor that signals via multiple downstream pathways including: PI3K/AKT, ERK1/2, JNK, JAK/STAT3, and others, dependent on which of the 5 tyrosine residues is phosphorylated .
EGFR signaling is critical for pancreatic development and for β-cell proliferation, as shown by EGFR knock-out and transgenic mouse models. Genetic disruption of EGFR is lethal in the embryonic and peri-embryonic periods and the pancreatic phenotype reveals a reduced pancreas size due to impaired ductal branching, abnormal islet cell localization, and defective differentiation [9–12]. Embryonic cell cultures established from these mice show a 50% reduction of β-cell mass, without impairment of other islet cell types . After birth, tissue-specific attenuation of EGFR signaling in the β-cell using a dominant negative EGFR (EGFR-DN) that lacks 40% of tyrosine kinase activity leads to a failure of postnatal β-cell proliferation and islet mass expansion, resulting in insulin-deficient diabetes by two weeks of life . This suggests that EGFR signaling after birth is critical for β-cell proliferation.
Survivin is the smallest member of a well-conserved protein family known as inhibitor of apoptosis proteins (IAPs) . In cancer cells, survivin has at least two established functions; one as an inhibitor of programmed cell death  and the other as a regulator of cell division . To perform its diverse functions, the survivin protein must shuttle between multiple subcellular compartments, including the cytoplasm, mitochondria, and nucleus . Evidence suggests that survivin can inhibit both the extrinsic and intrinsic (mitochondrial) pathways of programmed cell death by blocking the activity of several caspase proteins [18, 19]. Survivin also forms a complex with a group of chromosomal passenger proteins including Aurora B kinase, INCENP, and Borealin [20, 21] to regulate cell division. Phosphorylation of survivin at threonine 117 by Aurora-B regulates survivin-targeting to the centromere and thus the entire chromosomal passenger complex [22, 23]. Phosphorylation at an additional site, threonine 34, is critical for the anti-apoptotic function of survivin; whereby mutation at this site results in caspase 3 activation and mitochondrial apoptosis .
During embryogenesis (E15.5) in the mouse, survivin is expressed throughout the pancreatic epithelium . Around the end of gestation, it becomes gradually restricted to endocrine cells. Postnatally, its expression becomes further restricted, where eventually (P21) it is expressed exclusively within the pancreatic β-cells . In previous work, we engineered mice harboring a conditional deletion of survivin in pancreatic endocrine cells by mating survivin floxed mice with mice expressing a Cre-recombinase protein under the control of a Pax-6 promoter . These mice developed insulin-deficient diabetes after birth due to a failure of β-cell mass expansion . On a cellular level, we observed a slowing of cell cycle progression through G1/S and G2/M in the survivin null β-cells, which correlated with an increase in expression of the cell cycle inhibitor, p21
. Similar findings were also observed in a Pdx-1Cre;survivin
mouse model . In other related work, transplantation of pancreatic β-cells engineered to ectopically express survivin from a rat-insulin promoter into streptozotocin-treated mice resulted in long-term correction of hyperglycemia and rescue of streptozotocin-induced β-cell death . Together, these data suggest that survivin is important in both the normal expansion of the β-cell mass after birth and in the survival of β-cells following stress-induced apoptosis.
As both EGF and survivin are essential for β-cell proliferation, and as survivin expression is regulated by EGF in cancer cells, we hypothesized that EGF also regulates survivin expression in β-cells and thereby is one of the mechanisms involved in promoting β-cell growth. We chose the well-established insulin-producing β-cell lines, MIN6 and INS-1, as an experimental model system to test this hypothesis. Here, we show that survivin is regulated by several pancreatic β-cell growth factors, including glucose, insulin, and EGF. Induction of survivin by EGF occurs extremely quickly, within 15 minutes of treatment. The mechanism of EGF-induced survivin occurs primarily through activation of the ERK pathway and prolongation of survivin half-life by inhibiting ubiquitin conjugation on the survivin protein. Thus, we have identified a novel mechanism for survivin regulation in pancreatic β-cells that implicates ERK as a critical molecule for its post-translational modification and signaling for protein degradation.