Unidad de Investigacion, Servicio de Nefrologia, Red in ren, Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Hospital Universitario Reina Sofia, Córdoba, Spain.
Am J Physiol Renal Physiol. 2010 May;298(5):F1197-204. doi: 10.1152/ajprenal.00529.2009. Epub 2010 Feb 24.
We have previously demonstrated that the activation of rat parathyroid calcium-sensing receptor (CaSR) upregulates VDR expression in vivo (Garfia B, Cañadillas S, Luque F, Siendones E, Quesada M, Almadén Y, Aguilera-Tejero E, Rodríguez M. J Am Soc Nephrol 13: 2945-2952, 2002; Rodriguez ME, Almaden Y, Cañadillas S, Canalejo A, Siendones E, Lopez I, Aguilera-Tejero E, Martin D, Rodriguez M. Am J Physiol Renal Physiol 292: F1390-F1395, 2007). The present study was designed to characterize the signaling system that mediates the stimulation of parathyroid VDR gene expression by extracellular calcium. Experiments were performed in vitro by the incubation of rat parathyroid glands and in vivo with normal and uremic (Nx) rats receiving injections of CaCl(2) or EDTA to obtain hypercalcemic or hypocalcemic clamps. A high calcium concentration increased VDR expression. The addition of arachidonic acid (AA) to the low-calcium medium produced an increase in VDR mRNA of the same magnitude as that observed with high calcium. The addition of ionophore to the low-calcium medium also increased VDR mRNA expression. High calcium or the addition of AA to the low-calcium medium induced the activation (phosphorylation) of ERK1/2-MAPK. The specific inhibition of the ERK1/2-MAPK activity prevented the stimulation of VDR expression by high calcium or AA. These results suggest that AA regulates parathyroid VDR gene expression through the activation of the ERK1/2-MAPK. CaSR activation induced the activation of transcription factor Sp1, but not of NF-κB p50 or p65 or activator protein-1. The addition of AA to the low-calcium medium increased specific DNA-binding activity of Sp1 to almost the same level as high calcium, which was prevented by the inhibition of ERK1/2. Furthermore, mithramycin A (a Sp1 inhibitor) prevented the upregulation of VDR mRNA by high calcium. Finally, both sham and Nx hypercalcemic rats showed similar increased levels of VDR mRNA compared with sham and Nx hypocalcemic rats. Our results demonstrate that extracellular calcium stimulates VDR expression in parathyroid glands through the elevation of the cytosolic calcium level and the stimulation of the PLA(2)-AA-dependent ERK1/2-pathway. Furthermore, the transcription factor Sp1 mediates this effect.
我们之前已经证明，大鼠甲状旁腺钙敏感受体 (CaSR) 的激活可在体内上调 VDR 表达（Garfia B、Cañadillas S、Luque F、Siendones E、Quesada M、Almadén Y、Aguilera-Tejero E、Rodríguez M. J Am Soc Nephrol 13: 2945-2952, 2002；Rodriguez ME、Almaden Y、Cañadillas S、Canalejo A、Siendones E、Lopez I、Aguilera-Tejero E、Martin D、Rodríguez M. Am J Physiol Renal Physiol 292: F1390-F1395, 2007）。本研究旨在探讨细胞外钙刺激甲状旁腺 VDR 基因表达的信号转导系统。通过体外孵育大鼠甲状旁腺和体内给予正常和尿毒症 (Nx) 大鼠氯化钙或 EDTA 注射以获得高钙或低钙夹，进行了实验。高钙浓度增加了 VDR 表达。将花生四烯酸 (AA) 添加到低钙培养基中，可使 VDR mRNA 的表达增加到与高钙相同的程度。将离子载体添加到低钙培养基中也可增加 VDR mRNA 的表达。高钙或在低钙培养基中添加 AA 诱导 ERK1/2-MAPK 的激活（磷酸化）。ERK1/2-MAPK 活性的特异性抑制可防止高钙或 AA 刺激 VDR 表达。这些结果表明 AA 通过激活 ERK1/2-MAPK 来调节甲状旁腺 VDR 基因表达。CaSR 激活诱导转录因子 Sp1 的激活，但不诱导 NF-κB p50 或 p65 或激活蛋白-1 的激活。将 AA 添加到低钙培养基中可使 Sp1 的特定 DNA 结合活性增加到与高钙几乎相同的水平，而这可被 ERK1/2 的抑制所阻止。此外，米托蒽醌 A（Sp1 抑制剂）可防止高钙引起的 VDR mRNA 上调。最后，与低钙相比，假手术和 Nx 高钙血症大鼠的 VDR mRNA 水平均明显升高。我们的结果表明，细胞外钙通过升高细胞内钙水平和刺激 PLA2-AA 依赖性 ERK1/2 途径刺激甲状旁腺中的 VDR 表达。此外，转录因子 Sp1 介导了这种作用。
