In differentiated Caco2/AQ GSK1120212 order cells treatment with 1,25D3 caused 2.4-fold induction of CaSR expression after 6 h. The maximal effect of 1,25D3 on CaSR transcriptional

activation in these cells was observed at 24 h (7.6-fold; Fig. 2 and Fig. 3). In the less differentiated cells Coga1A 1,25D3-induced CaSR transcription was 2.9-fold after 12 h and 4.2-fold after 24 h compared with the control group (Fig. 2B). 1,25D3 increased CaSR translation as well. Immunofluorescence staining demonstrated upregulation of the CaSR protein in Caco2/AQ after 24 h and Coga1A after 48 h (Fig. 3C and D). We treated Caco2/AQ and Coga1A cells with TNFα and IL-6 for 6, 12, 24, and 48 h. In Caco2/AQ treatment with the proinflammatory cytokine TNFα caused only modest upregulation of CaSR expression. Treatment with IL-6 was accompanied by a 3.5-fold induction after 6 h compared with control. Combined treatment with TNFα and IL-6 induced CaSR mRNA expression in Caco2/AQ 10.3-fold (p < 0.05) after 24 h and 10.2-fold (p < 0.05) after 48 h. However, the combination of all three compounds either had no effect or reduced CaSR expression ( Fig. 3A). In Coga1A cells, treatment with TNFα induced CaSR robustly, especially at 48 h (134-fold, Buparlisib cell line p < 0.01). Treatment with IL-6 caused only marginal increases in CaSR mRNA expression. Furthermore, we observed upregulation of CaSR expression

in the groups treated with TNFα/IL-6 (68.5-fold) and TNFα/1,25D3 (121.2-fold, p < 0.05) at 48 h. Similar results were observed in the groups that were treated with TNFα/IL-6/1,25D3 at 6 and 48 h (18.8-fold, p < 0.05 and 47.7-fold, p < 0.05; Fig. 3B). To address the question whether alterations on CaSR mRNA expression were translated into protein, we performed immunofluorescence staining. Fig. 3C and D demonstrates the upregulation of the CaSR protein upon treatments with the proinflammatory cytokines using the rabbit polyclonal anti-CaSR antibody. Protein expression

data were confirmed using the mouse monoclonal anti-CaSR antibody (data not shown). Both antibodies gave the same results. Recent studies have demonstrated that murine CaSR activates the NLPR3 inflammasome, which in turn induces maturation and release of the inflammatory cytokine interleukin 1β, amplifying Liothyronine Sodium the inflammatory signal [19] and [20]. Inversely, mice double knockout for CaSR−/−/PTH−/− had increased inflammatory response after administration of dextran sodium sulfate compared with control mice expressing the receptor [21]. This suggests an important role for the CaSR in inflammation. Therefore, it is essential to understand how the expression of the CaSR is modulated in the colon. It has been demonstrated previously that activation of VDREs by 1,25D3 and translocation of NF-κB to the nucleus after the treatment with interleukin 1β led to induction of CaSR expression in rat parathyroid, thyroid, and kidneys [9] and [10].