PKCdelta in Insulin Secreting Cells Figure 7. Dual effect of PKCd in insulin-secreting cells. PKCd supports proliferation by phosphorylation of p21Cip/WAF1 which results in nuclear extrusion of the cell cycle inhibitor (white symbols). In the presence of metabolic stress, e.g. prolonged exposure to high concentrations of free fatty acids (FFA) PKCd favors apoptotic cell death through nuclear accumulation and stimulation of FOXO1 (gray symbols). PKCdKN (white and gray stripes) inhibits both the anti-apoptotic and pro-apoptotic effect of PKCd. doi:10.1371/journal.pone.0028828.g007 is the persistent expression of p21Cip1/WAF1, while in our cell systems endogenous p21Cip1/WAF1 expression is transient. Phosphorylation events presumably not only regulate the compart- mentalization but also the protein stability of p21Cip1/WAF, although controversial data have been reported on this issue. It was previously shown that PKB-dependent phosphorylation of p21Cip1/WAF1 at both Thr145 and Ser146 increases the stability of the CDK inhibitor and enhances its anti-apoptotic activity [31,44,45]. However, in our experiments, inhibition of PKB by Akti-1/2 affected neither phosphorylation nor nuclear accumulation of p21Cip1/WAF1, suggest- ing a minor role of PKB in our cell system. Likewise, inhibition of ERK1/2 with PD98059 had no impact on the subcellular distribution and phosphorylation of p21Cip1/WAF1. Although we did not study the effect of PKCd on p21Cip1/WAF1 stability in detail, PKCdWT cells revealed a significantly lower amount of p21Cip1/WAF1 than control or PKCdKN cells, suggesting that Ser146 phosphorylation affects p21Cip1/WAF1 stability. These data are consistent with reports demonstrating that phosphorylation of p21Cip1/WAF1 at Ser146 by PKCd leads to destabilization of the CDK inhibitor [46]. Thus, our data indicate that p21Cip1/WAF1 exerts a dual effect depending on its subcellular distribution (Fig. 7). When trapped in the cytosol due to phosphorylation, p21Cip1/WAF1 might favor proliferation, a notion supported by the increased Ki67 staining in PKCdWT cells. This cytosolic localization of p21Cip1/WAF1 is known to exert anti-apoptotic effects by CDK-dependent or independent mechanisms [47,48]. One mechanism of anti- apoptotic action of p21Cip1/WAF1 involves its direct binding to and inhibition of the pro-apoptotic kinases ASK1 or JNK [31,49,50]. In contrast, nuclear p21Cip1/WAF1 inhibits cell cycle progression and might eventually lead to apoptosis. In addition to the binding to cyclin-CDK complexes, p21Cip1/WAF1 interacts directly with the proliferating cell nuclear antigen (PCNA), and thereby inhibits PCNA-dependent DNA replication [51]. Improper and prolonged nuclear accumulation of p21Cip1/WAF1 may explain the observa- tions that mice overexpressing p21Cip1/WAF1 specifically in b-cells develop age-related hyperglycemia under normal feeding [29]. Whether PKCd-dependent regulation of p21Cip1/WAF1 affects b-cell function in humans needs further experimental evidence. The expression of p21Cip1/WAF1 in human islets and the fact that PKCd reduces nuclear accumulation of p21Cip1/WAF1 in primary mouse islet cells supports the view that the cell cycle inhibitor could play a regulatory role also in adult human b-cells under special proliferative conditions [23]. In conclusion, our study demonstrates that PKCd induces posttranslational modifications of p21Cip1/WAF1 which, in turn, determine its subcellular distribution and function in INS-1E cells. This study reveals that PKCd is not per se a pro-apoptotic kinase and underlines the importance of understanding molecular mechanisms for the evaluation of therapeutic targets in the treatment of diabetes mellitus. Materials and Methods Ethics Statement The use for scientific purposes of isolated human islets was approved by the local ethics committee (University of Tuebingen, Medical Faculty No. 533/2010BO2). All animal experiments were done in accordance with the accepted standard of human care of animals and approved by the local Animal Care and Use Committee (Notification from 12.01.10). Cell preparations, culture and transfection INS-1E cells, kindly provided by C. B. Wollheim (University of Geneva, Switzerland), were cultured in RPMI 1640 (GIBCO) containing 11 mM glucose, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 0.05 mM b-mercaptoethanol and 10% FCS as described [52]. INS-1E cells were stably transduced with retroviruses encoding wild type PKCd (PKCdWT) or the K376R- mutation in the ATP-binding domain (PKCdKN) driven by the rat insulin 1 gene promoter. Cell clones expressing the transgene were selected by geneticin (G418) and subcloned as single cell clones. INS-1E cells were transfected with siRNA against PKCd (20 nM/56105 cells; On-Target plus siRNA, # J-080142-05, Dharmacon, Chicago) using a siRNA transfection reagent (DharmaFECT 3, Dharmacon). As control, siRNA against luciferase (20 nM/56105 cells) was transfected in parallel. Cells were used 2 d after transfection. p21Cip1/WAF1-proficient and deficient mouse embryonic fibroblasts (MEFs) were cultured in DMEM supplemented with 10% FCS and antibiotics. PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28828