Wnt5a/β-Catenin Signaling Drives Calcium-Induced Differentiation of Human Primary Keratinocytes

It is well established that a gradient of extracellular calcium within the epidermis regulates the differentiation of keratinocytes. However, the molecular mechanisms implicated in this process are not fully understood. RNA interference of the calcium-sensing receptor (CaSR) showed that CaSR is essential in calcium-induced differentiation of normal human epidermal keratinocytes (NHEKs) by increasing the levels of free intracellular calcium, which upregulates the expression of Wnt5a but not Wnt3a, Wnt4, and Dkk-1 in the cells. Subsequently, autocrine Wnt5a promotes the differentiation of NHEKs, determined by increased biosynthesis of keratin-1 and loricrin, whereas proliferation is suppressed. Addition of both Wnt5a and calcium to NHEKs activated the Wnt/beta-catenin signaling pathway as indicated by (i) increased stability of beta-catenin in the cells, (ii) enhanced beta-catenin transcriptional activity, demonstrated by a luciferase-based beta-catenin-activated reporter assay, and (iii) augmented Wnt/beta-catenin target gene expression. NHEKs depleted for beta-catenin had a significantly reduced susceptibility to calcium-induced differentiation. Knockdown of axin 2, an antagonist of beta-catenin stability, enhanced the biosynthesis of keratin-1 and loricrin in the cells. Our findings establish a directional crosstalk between CaSR and Wnt/beta-catenin signaling in keratinocyte differentiation via Wnt5a that acts as an autocrine stimulus in this process.