Aurora Kinase A is critical for the Nkx6.1 mediated β-cell proliferation pathway

Abstract

Type 1 and type 2 diabetes are ultimately characterized by depleted b-cell mass. Characterization of the molecular pathways that control b-cell proliferation could be harnessed to restore these cells. The homeobox b-cell transcription factor Nkx6.1 induces b-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated b-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in b-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary b-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat b-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the b-cell proliferation pathway.

Treatment with Mammalian Ste-20-like Kinase 1/2 (MST1/2) Inhibitor XMU-MP-1 Improves Glucose Tolerance in Streptozotocin-Induced Diabetes Mice

“…These were aurora kinase A (AURKA) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma (PIK3CG). AURKA is involved in regulating β cell proliferation [ 22 ], whereas PIK3CG was shown to positively modulate insulin secretion [ 23 ]. Based on these data, the expected phenotypes of AURKA and PIK3CG inhibition would be a reduction in pancreatic β cell function, which is contrary to our findings.…”

β-Hydroxybutyrate improves β-cell mitochondrial function and survival

“…25 Cells were plated at a concentration of 2 × 10 5 cells/mL in 24-well plates (at 1 ml/well) or in 96-well plates (at 100 μL/well), then cultured for 48 h total with vehicle (water; CON) or 10 mM β-Hydroxybutyrate (βHB). Cellular proliferation was determined by cell counts, 26 MTT assays (Sigma-Aldrich) 24 and Alamar Blue assays (SigmaAldrich).…”

Cdk5r1 Overexpression Induces Primaryβ-Cell Proliferation

“…For experiments using the Cdk5 inhibitor roscovitine, islets were cultured for 48 hours with 10 μ mol/L roscovitine (Santa Cruz) or DMSO (vehicle, Sigma) [ 27 ]. For all islet experiments using adenoviral transduction, viral treatments occurred within four hours of completing the islet isolation procedure as previously published [ 10 , 11 , 26 , 28 – 32 ]. Islets were transduced with adenovirus for 18 hours, after which media and adenoviral vectors were removed.…”

“…All recombinant viruses were shown to be replication incompetent by being E1a deficient, using an RT-PCR screen, as previously described [ 40 ]. For studies involving adenovirus mediated gene manipulation, pools of 200 islets were transduced with ~2 × 10 7 IFU/mL adenovirus (moi~100–200) for 18 hours, as previously described [ 10 , 11 , 29 , 32 , 41 , 42 ]. Media were changed after 18 hours and islets were cultured for up to 96 hours.…”

“…832/13 cells were transfected using Lipofectamine RNAiMAX (Life Technologies) with siRNA duplexes against rat Cdk5, 5′-GUUCAGCCCUCCGGGAGAUTT-3′ [ 44 ], or a previously described scrambled control, 5′-GAGACCCTATCCGTGATTA-3′ [ 32 , 34 ]. Following an overnight incubation, cells were transduced with AdCMV-GFP or AdCMV-Cdk5r1 or left untreated.…”