Cdk5r1 Overexpression Induces Primary β-Cell Proliferation

Abstract

Decreased β-cell mass is a hallmark of type 1 and type 2 diabetes. Islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. Understanding the pathways controlling islet proliferation may be used to increase functional β-cell mass through transplantation or by enhanced growth of endogenous β-cells. We have shown that the transcription factor Nkx6.1 induces β-cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to define Nkx6.1-independent mechanisms by which Nr4a1 and Nr4a3 induce β-cell proliferation, we demonstrated that cyclin-dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1. Overexpression of Cdk5r1 is sufficient to induce primary rat β-cell proliferation while maintaining glucose stimulated insulin secretion. Overexpression of Cdk5r1 in β-cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. The Cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that Cdk5r1 mediated β-cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the Cdk5 kinase complex is sufficient to induce β-cell proliferation while maintaining glucose stimulated insulin secretion.

Figure 1

Overexpression of Nr4a1 and Nr4a3 results in upregulation of a subset of cell cycle genes unregulated by Nkx6.1. Previous published microarrays of Nr4a1, Nr4a3, or Nkx6.1 overexpressed in primary rat islets were compared for genes induced by Nr4a1 and Nr4a3 but not induced by Nkx6.1. (a) Using Panther pathway analysis (pantherdb.org), 114 genes were found to be upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1, of which 35.2% were found to be involved in the GO category cellular processes. (b) Within the GO category cellular processes, 43.2% of the genes (17 genes) were found to be associated with the GO category cell cycle.

Figure 2

Overexpression of Nr4a1 and Nr4a3 induces Cdk5r1 expression in primary rat islets. Rat islets were treated with AdCMV-GFP, AdCMV-Nkx6.1, AdCMV-Nr4a1, or AdCMV-Nr4a3. (a) Cdk5r1 mRNA levels increase is induced by Nr4a1 and Nr4a3 but not by Nkx6.1, while (b) Cdk5 mRNA levels are unchanged by overexpression of Nkx6.1, Nr4a1, or Nr4a3. Data represent the mean ± SEM of six independent experiments. (c) Representative western blot and quantitation demonstrate that islets transduced with AdCMV-Nr4a1 or AdCMV-Nr4a3 induce upregulation of Cdk5r1 protein levels. Data represent the mean ± SEM of six independent experiments. (d) Representative western blot and quantitation demonstrate that islets transduced with AdCMV-Nr4a1 and AdCMV-Nr4a3 have no additive increase on Cdk5r1 protein levels. Data represent the mean ± SEM of three experiments for western blots. ^∗ p ≤ 0.05; ^∗∗∗ p ≤ 0.001. p value represents the comparison between Nr4a1- or Nr4a3-treated and GFP-treated islets.

Figure 3

Overexpression of Cdk5r1 is sufficient to induce primary rat islet proliferation. (a) Islets were transduced with AdCMV-GFP or AdCMV-Cdk5r1. Protein was harvested 96 hours after viral transduction. A 6-fold increase was observed in Cdk5r1 protein levels in islets transduced with AdCMV-Cdk5r1, as compared to the observed low endogenous level in primary rat islets. Data represent the mean ± SEM of six independent experiments representing the comparison between untreated islets and islets transduced with AdCMV-Cdk5r1. (b) Incorporation of [³H-methyl]-thymidine in rat islets. Rat islets transduced with AdCMV-Cdk5r1 have increased proliferation, while islets treated with AdCMV-GFP or AdCMV-Cdk5 have no induction of proliferation. Data represent the mean ± SEM of four independent experiments representing the comparison between untreated islets and islets transduced with AdCMV-Cdk5r1. (c) Islets were transduced with AdCMV-GFP, Cdk5r1, or Nkx6.1 or were transduced with AdCMV-Nkx6.1 and either GFP or Cdk5r1. Islets were labeled with ³H-thymidine 72 hours after viral transduction, followed by measurements of proliferation at 96 hours after viral transduction. Data represent the mean ± SEM of four independent experiments representing the comparison between AdCMV-Nkx6.1 treated islets and islets cotransduced with AdCMV-Nkx6.1 and AdCMV-Cdk5r1. ^∗ p ≤ 0.05; ^∗∗∗ p ≤ 0.001.

Figure 4

Overexpression of Cdk5r1 is sufficient to induce β-cell proliferation. (a) Percentage of BrdU+Insulin+ islet cells cultured with BrdU for 96 hours. (b) Representative image (40x magnification) of dispersed rat islets transduced with AdCMV-GFP labeled with BrdU. (c) Representative image (40x magnification) of dispersed rat islets transduced with AdCMV-Cdk5r1 labeled with BrdU. Arrows indicate BrdU+Insulin+ nuclei. DAPI is blue, insulin is green, and BrdU is red. Data represent the mean ± SEM of four independent experiments. ^∗∗ p ≤ 0.01. p value represents the comparison between Cdk5r1- and GFP-treated islets.

Figure 5

Cdk5r1 overexpression protects INS-1 β-cells from apoptosis. (a) 832/13 INS-1 cells were transduced with AdCMV-GFP, AdCMV-Cdk5, or AdCMV-Cdk5r1. Twenty-four hours after adenoviral transduction, cells were cultured for 18 hours in the presence of camptothecin, thapsigargin, etoposide, or the vehicle control. Cell counts were completed 18 hours after drug treatment. Cell viability was determined by comparing the untreated population to the treated population. Cells overexpressing Cdk5r1 presented greater viability when treated with thapsigargin or etoposide than control cells. Data represent the mean ± SEM of nine independent experiments. p value represents the comparison between Cdk5r1- and GFP-treated 832/13 cells. Cells were transduced with AdCMV-GFP or AdCMV-Cdk5r1 and subsequently treated with camptothecin, thapsigargin, or etoposide. Western blotting for total caspase-3 or cleaved caspase-3 was queried to determine activation of apoptosis pathway. Representative western blot (b) and quantitation (c). Data represent the mean ± SEM of four independent experiments. ^∗ p ≤ 0.01; ^∗∗ p ≤ 0.01.

Figure 6

Overexpression of Cdk5r1 maintains glucose stimulated insulin secretion. (a) Total insulin content was measured in primary rat islets at 96 hours after transduction with AdCMV-GFP, AdCMV-Cdk5, or AdCMV-Cdk5r1. No significant difference was observed in total insulin content. (b) Glucose stimulated insulin secretion was measured 96 hours after transduction with AdCMV-GFP, AdCMV-Cdk5, or AdCMV-Cdk5r1 in media containing 2.5 mM glucose and 16.7 mM glucose for 1 hour. No decrease in glucose stimulated insulin rate was observed. Data represent the mean ± SEM of four independent experiments.

Figure 7

Cdk5r1 mediated β-cell proliferation is dependent on the Cdk5 kinase activity. (a) Islets transduced with AdCMV-GFP, AdCMV-Cdk5, or AdCMV-Cdk5r1 were cultured in the presence of ³H-thymidine for 48 hours. Islets were treated with vehicle or 10 μM roscovitine for the final 24 hours of the labeling period. Data represent the mean ± SEM of four independent experiments. (b) 832/13 cells were transfected with siCDK5 or siCTRL. Twenty-four hours later cells were transduced with AdCMV-GFP or AdCMV-Cdk5r1. Cdk5 mRNA was measured by RT-PCR, demonstrating a significant decrease in Cdk5 levels with siCDK5 transfection. (c) Proliferation was measured by cell counts, demonstrating that knockdown of Cdk5 causes a significant impairment of Cdk5r1 mediated proliferation. Data represent the mean ± SEM of three independent experiments. (c) ^∗ p ≤ 0.05. p value represents the comparison between Cdk5r1- and GFP-treated islets.

Figure 8

Overexpression of Cdk5r1 results in increased pRb phosphorylation. (a) Untransduced islets were compared to islets transduced with AdCMV-GFP, AdCMV-Cdk5, or AdCMV-Cdk5r1, and levels of phosphorylated pRb were measured. Representative western blot from four independent experiments. (b) Untreated islets and islets transduced with AdCMV-GFP, AdCMV-Cdk5, or AdCMV-Cdk5r1 were treated with 10 μM roscovitine and levels of phosphorylated pRb were measured. Representative western blots and quantitation from three independent experiments. ^∗ p ≤ 0.05; ^∗∗ p ≤ 0.01; ^∗∗∗ p ≤ 0.001.

Figure 9

Model of how Cdk5r1 induces β-cell proliferation. Overexpression of Nr4a1 or Nr4a3 induces expression of Cdk5r1 in primary rat β-cells. Cdk5r1 forms a complex with Cdk5, thus activating the Cdk5 kinase. The Cdk5r1/Cdk5 kinase complex phosphorylates pRb. In the unphosphorylated state pRb binds and sequesters E2F family members, thus inhibiting cell cycle progression. Upon phosphorylation of pRB, E2F family members are released and permitted to induce expression of cell cycle genes that are sufficient to induce proliferation. The Cdk5 kinase inhibitor roscovitine blocks phosphorylation of pRb and inhibits Cdk5r1 induced β-cell proliferation.