| Summary: | The genomic activity of 1,25(OH)<sub>2</sub>D<sub>3</sub> is mediated by vitamin D receptor (VDR), whilst non-genomic is associated with protein disulfide isomerase family A member 3 (PDIA3). Interestingly, our recent studies documented that PDIA3 is also involved, directly or indirectly, in the modulation of genomic response to 1,25(OH)<sub>2</sub>D<sub>3</sub>. Moreover, PDIA3 was also shown to regulate cellular bioenergetics, possibly through the modulation of STAT signaling. Here, the role of VDR and PDIA3 proteins in membrane response to 1,25(OH)<sub>2</sub>D<sub>3</sub> and calcium signaling was investigated in squamous cell carcinoma A431 cell line with or without the deletion of <i>VDR</i> and <i>PDIA3</i> genes. Calcium influx was assayed by Fura-2AM or Fluo-4AM, while calcium-regulated element (NFAT) activation was measured using a dual luciferase assay. Further, the levels of proteins involved in membrane response to 1,25(OH)<sub>2</sub>D<sub>3</sub> in A431 cell lines were analyzed via Western blot analysis. The deletion of either <i>PDIA3</i> or <i>VDR</i> resulted in the decreased baseline levels of Ca<sup>2+</sup> and its responsiveness to 1,25(OH)<sub>2</sub>D<sub>3</sub>; however, the effect was more pronounced in A431∆<i>PDIA3</i>. Furthermore, the knockout of either of these genes disrupted 1,25(OH)<sub>2</sub>D<sub>3</sub>-elicited membrane signaling. The data presented here indicated that the VDR is essential for the activation of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), while PDIA3 is required for 1,25(OH)<sub>2</sub>D<sub>3</sub>-induced calcium mobilization in A431 cells. Taken together, those results suggest that both VDR and PDIA3 are essential for non-genomic response to this powerful secosteroid.
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