Calcium prevents retinoic acid-induced disruption of the spectrin-based cytoskeleton in keratinocytes through the Src/PI3K-p85α/AKT/PKCδ/β-adducin pathways

We have previously reported that spectrin increases dramatically in amount and is assembled into the cytoskeleton in differentiating keratinocytes both in vitro and in vivo (Zhao et al., PLoS ONE 6 (12) (2011) e28267). We demonstrate here that extracellular calcium (Ca2+) enhances differentiation of keratinocytes and that this is associated with increased spectrin expression and formation of a spectrin-based cytoskeleton. While Retinoic acid (RA) also enhanced keratinocyte differentiation, it abrogated the spectrin-based cytoskeleton in keratinocytes. Furthermore, RA substantially inhibited expression of both Src and PI3K-p85α and consequently the amounts of specific phosphorylation of both of these proteins. RA also enhanced AKT expression and dramatically induced phosphorylation of AKT(Thr308), accompanied by phosphorylation of both PKCδ(Thr505) and β-adducin(Ser662) and upregulated cyclin D2 and down-regulated cyclin B1. On the other hand, Ca2+ overcame the inhibitory effects of RA on expression of Src, PI3K-p85α and cyclin B1 by maintaining high levels of phosphorylation of both Src(Tyr527) and PI3K-p85α and preventing phosphorylation of AKT(Thr308), PKCδ(Thr505) and β-adducin(Ser662). These data highlight the importance of Ca2+ in both spectrin expression and the organizational integrity of the spectrin-based cytoskeleton in differentiating keratinocytes and assist in elucidating the signalling pathways involved.