成纤维细胞生长因子(FGF)-23和钙敏感受体(CaSR)先前已被假定为肠道钙吸收的负反馈调节的一部分,以防止过量的钙吸收及其毒性。然而,这种反馈调节的潜在机制仍然难以捉摸,特别是它是否需要FGF-23的转录。在这里,我们通过将肠上皮样Caco-2单层暴露于30mMCaCl2和1,25-二羟基维生素D3[1,25(OH)2D3]诱导钙高吸收状态(CHS),然后确定FGF-23mRNA水平和跨上皮钙通量。我们发现CHS上调FGF-23转录,其被CaSR抑制剂(Calhex-231和NPS2143)逆转,但对CaSR转录没有影响。尽管10nM1,25(OH)2D3能够增强跨上皮钙通量,CHS中高于正常水平的钙淹没导致钙通量降低,与FGF-23蛋白表达增加一致。给予钙调神经磷酸酶抑制剂(≤10μMCN585和环孢菌素A),CaSR作用的介质,以控制其靶蛋白的转录和生产,发现部分阻止FGF-23蛋白质的产生和CHS对钙转运的负面影响,而对FGF-23mRNA表达无影响。直接接触FGF-23,但不接触FGF-23+PD173074(FGFR1/3抑制剂),还完全消除了Caco-2单层中1,25(OH)2D3增强的钙转运。然而,CHS和CaSR抑制剂对钙调磷酸酶(PPP3CB)或其靶标的mRNA水平没有影响(即,NFATc1-4)。总之,高根尖钙和1,25(OH)2D3诱导的CHS暴露引发了负反馈机制,以防止进一步的钙吸收。CaSR及其下游介质,钙调磷酸酶,可能有助于监管过程,部分通过增强FGF-23的产生来抑制钙转运。我们的研究,因此,证实了CaSR-自分泌FGF-23轴作为预防过量钙摄取的局部反馈回路的生理意义。
Fibroblast growth factor (FGF)-23 and calcium-sensing receptor (CaSR) have previously been postulated to be parts of a negative feedback regulation of the intestinal calcium absorption to prevent excessive calcium uptake and its toxicity. However, the underlying mechanism of this feedback regulation remained elusive, especially whether it required transcription of FGF-23. Herein, we induced calcium hyperabsorptive state (CHS) by exposing intestinal epithelium-like Caco-2 monolayer to 30 mM CaCl2 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after which FGF-23 mRNA levels and transepithelial calcium flux were determined. We found that CHS upregulated FGF-23 transcription, which was reverted by CaSR inhibitors (Calhex-231 and NPS2143) but without effect on CaSR transcription. Although 10 nM 1,25(OH)2D3 was capable of enhancing transepithelial calcium flux, the higher-than-normal calcium inundation as in CHS led to a decrease in calcium flux, consistent with an increase in FGF-23 protein expression. Administration of inhibitors (≤10 μM CN585 and cyclosporin A) of calcineurin, a mediator of CaSR action to control transcription and production of its target proteins, was found to partially prevent FGF-23 protein production and the negative effect of CHS on calcium transport, while having no effect on FGF-23 mRNA expression. Direct exposure to FGF-23, but not FGF-23 + PD173074 (FGFR1/3 inhibitor), also completely abolished the 1,25(OH)2D3-enhanced calcium transport in Caco-2 monolayer. Nevertheless, CHS and CaSR inhibitors had no effect on the mRNA levels of calcineurin (PPP3CB) or its targets (i.e., NFATc1-4). In conclusion, exposure to CHS induced by high apical calcium and 1,25(OH)2D3 triggered a negative feedback mechanism to prevent further calcium uptake. CaSR and its downstream mediator, calcineurin, possibly contributed to the regulatory process, in part by enhancing FGF-23 production to inhibit calcium transport. Our study, therefore, corroborated the physiological significance of CaSR-autocrine FGF-23 axis as a local feedback loop for prevention of excessive calcium uptake.