PKCdelta in Insulin Secreting Cells Figure 5. Effects of rottlerin and phorbol ester on cellular distribution of p21Cip1/WAF1. Representative pictures of immunocytochemical staining for p21Cip1/WAF1 in INS-1E cells after 16 h serum starvation (control), after 90 min treatment with the PKCd inhibitor rottlerin (10 mM) and after stimulation with PMA (1 mM for 2 min) in the presence or absence of the PKCd inhibitor rottlerin (10 mM) for 90 min. doi:10.1371/journal.pone.0028828.g005 and 0.6% in KO) [22]. These observations suggest that p21Cip1/WAF1 does not induce proliferation. Indeed, cell cycle inhibitors rather influence the velocity of proliferation while induction of b-cells proliferation occurs only under special conditions such as in new born and pregnant and lactating animals or after 90% pancreatectomy. PKCd has been found to be involved in a variety of cellular events. Although several reports indicate a pro-apoptotic role, PKCd was also shown to exert anti-apoptotic and proliferative effects in various cell types. Such opposing effects of PKCd may be cell type- or stimulus-specific or mediated by spatio-temporal differences of PKCd activation. There is evidence that, similar to p21Cip1/WAF1, different functional effects of PKCd are connected with the diverse compartmentalization of the enzyme. Upon stimulation with phorbol ester or fatty acids, PKCd redistributes between a cytosolic, a membrane-bound and a cytoskeleton- associated compartment in b-cells [36]. The pro-apoptotic effect of PKCd is linked to its nuclear accumulation [37,38]. Furthermore, cleavage of PKCd by caspase-3 releases a constitutively active fragment that promotes apoptosis [39]. Our data suggest that a substantial amount of PKCdWT in transgenic cells is phosphor- ylated at Ser643, an autophosphorylation site and, consequently, is stimulated under control culture conditions. This increased activity does not induce apoptotic cell death suggesting that additional factors generated under stress conditions are needed to turn PKCd into a pro-apoptotic kinase (Fig. 7). Reduced PKCd activity significantly augmented apoptosis consistently in PKCdKN INS-1E cells (up to 2-fold), in PKCdKN transgenic mouse b-cells (by 60%), as well as after down regulation of PKCd by RNA interference in control INS-1E cells (2-fold). Although not discussed in detail, in a study by Cantley et al. using PKCdKO mice, the rate of apoptosis was 80% higher in knockout cells than in control cells [11]. The physiological impact of this finding remains unclear, especially as islet size and insulin content were not reduced in PKCdKO mice when compared to wild type mice [40]. In agreement, mice that express PKCdKN exclusively in b- cells show no reduction in islet size and insulin content [14,41]. In addition to cell cycle regulation, emerging evidence suggests that p21Cip1/WAF1 exerts other functions in diverse cellular processes, including cell differentiation and survival. The impaired replication and increased apoptosis of b-cells of p21Cip1/WAF1 transgenic mice mirror our observations obtained in insulin secreting cells with reduced PKCd activity and may thus result from prolonged nuclear accumulation of p21Cip1/WAF1 [29]. Interestingly, similar to PKCdKO mice, these mice show improved recovery from streptozotocin-induced hyperglycemia, which has been attributed to an increased regeneration of insulin- producing cells [11,29]. In line, our previous study disclosed protection of mice with b-cell specific expression of PKCdKN against HFD-induced hyperglycemia. Apparent contradictory to this assumption are two studies which link stress-induced expression of p21Cip1/WAF1 to reduced insulin mRNA and b-cell failure. In one study oxidative stress-induced expression or PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28828