Depletion of PAK1 enhances ubiquitin-mediated survivin degradation in pancreatic β-cells

Abstract

Functional β-cell mass deficiency in diabetes results from imbalanced β-cell death and replication, and decreased PAK1 protein levels in human islets from donors with type 2 diabetes implicates a possible role for PAK1 in maintaining β-cell mass. Here, we aim to address the linkage between PAK1 and Survivin, a protein essential for β-cell replication. PAK1 knockout (KO) mouse islets exhibited decreased expression of Survivin protein. MIN6 β-cells with siRNA-mediated suppression of PAK1 also had decreased Survivin protein and exhibited an increased level of ubiquitinated-Survivin. However, no significant changes in Survivin mRNA were found in islets from PAK1 KO mice and PAK1-depleted MIN6 β-cells. The decreased Survivin level in MIN6 cells subjected to hyperglycemic stress was prevented by expression of exogenous PAK1. Moreover, overexpressing Survivin restored proliferation of β-cells that was impaired by the loss of PAK1. These data implicate a role for PAK1 in regulating Survivin protein stability in the β-cell and suggest PAK1 as a potential molecular target for the restoration of β-cell mass.

Keywords: MIN6; PAK1; Survivin; Ubiquitination; mouse islet; pancreatic β-cell; replication.

Figure 1. Decreased [³H]thymidine incorporation in PAK1-depleted MIN6 cells. Thirty-six hours after PAK1 depletion with siRNA, [³H]Methyl thymidine was added as described in methods. The amount of [³H]thymidine incorporated into DNA was measured by liquid scintillation counting and normalized to total cellular protein. Data represent the average ± SE for 3 independent experiments. *p < 0.05, vs. siCon.

Figure 2. Survivin expression is decreased in PAK1^−/− KO islets. Islets were isolated from PAK1^−/− KO and wild type (WT) littermate mice and homogenized. Proteins were resolved by 12% SDS-PAGE for immunoblotting with antibodies as indicated. Data represent the average ± SE for 3 pairs of mice; *p < 0.05, vs. WT.

Figure 3. Depletion of PAK1 decreased Survivin expression in pancreatic MIN6 cells. MIN6 cells were transfected with PAK1 siRNA (siPAK1) or control (siCon) oligonucleotides with pCMV6 vector or pCMV6-myc-PAK1 plasmid. After 48 h incubation, cells were washed twice and incubated with glucose-free MKRBB for 2 h before harvesting. Whole cell detergent lysates were prepared and subjected to 12% SDS-PAGE for immunoblotting with antibodies indicated. Data represent the average ± SE for 3 independent experiments; *p < 0.05, vs. siCon

Figure 4. Survivin mRNA expression in islets from PAK1^−/− KO mice and PAK1-depleted MIN6 cells. (A) Islets were isolated from PAK1^−/− KO and littermate PAK1^+/+ WT mice for use in Q-PCR analysis as described in methods. Data were quantified relative to GAPDH from three batches of islets, *p < 0.05 vs. WT. (B) MIN6 cells were transfected with PAK1 siRNA (siPAK1) or control (siCon) oligonucleotides. After 48 h incubation, cells were harvested for Q-PCR analysis as described in methods. Data were quantified relative to GAPDH from three independent experiments, *p < 0.05 vs. siCon.

Figure 5. Depletion of PAK1 enhanced Survivin-ubiquitination in pancreatic β-cells. (A) MIN6 cells were transiently transfected with siPAK1 or siCon Oligos. Thirty-six hours after transfection, cells were cultured with proteasome inhibitor MG132 (10 µM) for 6 h. Whole cell detergent lysates were prepared and subjected to 12% SDS-PAGE for immunoblotting with antibodies indicated. Data represent the one of the two independent experiments yielding similar results. (B) MIN6 cells were treated as in A. One mg cleared detergent lysates were combined with 1 µg of rabbit anti-Ubiquitin antibody as described in methods. The immunoprecipitates were subjected to immunoblotting with anti-survivin antibody. Data represent the one of the three independent experiments yielding similar results.

Figure 6. Glucotoxic/hyperglycemic stress reduced Survivin protein levels in pancreatic β-cells. MIN6 cells were transfected with pCMV6 vector or pCMV6-myc-PAK1 plasmid. After 48 h incubation, cells were washed twice with and incubated for 2 h in freshly prepared modified Krebs-Ringer bicarbonate buffer. Cells were stimulated with 20 mM D-glucose for 20 min. Cells were then washed twice before harvesting, and whole cell lysates were subjected to immunoblotting with antibodies indicated. Data represent the average ± SE for 3 independent experiments; *p < 0.05 vs. basal.

Figure 7. (A) Decreased [³H]methyl-thymidine incorporation in islets from PAK1^+/− Het mice. [³H]Methyl-thymidine was added to pools of about 200 islets for 18 h. The islets were washed three times with cold medium. The amount of [³H]thymidine incorporated into DNA was measured by liquid scintillation counting and normalized to total cellular protein. Data represent the average ± SE for 3 independent experiments. *p < 0.05, vs. WT. (B) Overexpressing Survivin restored impaired-proliferation of MIN6 cells induced by loss of PAK1 in pancreatic MIN6 β-cells. MIN6 cells were transfected with PAK1 siRNA (siPAK1) or control (siCon) oligonucleotides with MIEG3 vector or Survivin plasmid as described in the Materials and Methods. Thirty-six hours after transfection, [³H]methyl-thymidine was added for 2 h, and the amount of [³H]thymidine incorporated into DNA was measured as described in the Materials and Methods. Data are the average ± SE from 3 independent experiments. *p < 0.05 vs. siCon; # p < 0.05 vs. siPAK1.