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/β-catenin signaling pathway as indicated by (i) increased stability of β-catenin in the cells, (ii) enhanced β-catenin transcriptional activity, demonstrated by a luciferase-based β-catenin-activated reporter assay, and (iii) augmented Wnt/β-catenin target gene expression. NHEKs depleted for β-catenin had a significantly reduced susceptibility to calcium-induced differentiation. Knockdown of axin 2, an antagonist of β-catenin stability, enhanced the biosynthesis of keratin-1 and loricrin in the cells. Our findings establish a directional crosstalk between CaSR and Wnt/β-catenin signaling in keratinocyte differentiation via Wnt5a that acts as an autocrine stimulus in this process.