Calcitonin stimulates expression of the rat 25-hydroxyvitamin D3-24-hydroxylase (CYP24) promoter in HEK-293 cells expressing calcitonin receptor: identification of signaling pathways

Abstract

Regulation of the gene for renal 25-hydroxyvitamin D-24-hydroxylase (CYP24) is important for controlling the level of circulating 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ). We report here for the first time that the peptide hormone calcitonin significantly stimulates expression of a rat CYP24 promoter-luciferase construct in both transiently and stably transfected kidney HEK-293 cells. A GC box at −114/−101 and a CCAAT box at −62/−51 have been identified that underlie both basal expression of the CYP24 promoter and the calcitonin inductive response. Data from overexpression studies suggested that Sp1 and NF-Y are the proteins that function through the GC and CCAAT boxes respectively. ERK1/2 signaling pathways were not involved in the calcitonin-mediated response, since stimulation of the promoter was unaffected by the pharmacological ERK1/2 inhibitor PD98059 and by a dominant negative mutant of ERK1/2 (ERK1K71R). In contrast, calcitonin induction but not basal expression was dependent on protein kinase A and protein kinase C (PKC) activities with the inhibitors H89 and calphostin C lowering induction by 50-60%. The atypical PKC, PKC contributes to calcitonin induction, but not to basal expression of the CYP24 promoter, since overexpression of a dominant negative clone PKC K281 M lowered induction by 50%. Cotransfection of a dominant negative form of Ras resulted in calcitonin-mediated induction being reduced also by about 50%. A Ras-PKC signaling pathway for calcitonin action is proposed, which acts through the GC box. The findings have been extrapolated to the in vivo situation where we suggest that induction of renal CYP24 by calcitonin could be important under hypercalcemic conditions thus contributing to the lowering of circulating 1,25(OH) 2 D 3 levels.

CYP24A1 Regulation in Health and Disease

“…Phosphorylation of Sp-1 in the presence of 1,25(OH) 2 D 3 is dependent on PI3-kinase and PKCx activation, suggesting a potential regulatory role of the PKCx isozyme in Sp-1 phosphorylation by 1,25(OH) 2 D 3 , acting via the GC box in HEK293 cells [125]. CALCITONIN A similar pathway is reported to contribute to the substantial induction of CYP24A1 by the peptide hormone calcitonin in HEK293 cells [124]. Calcitonin is primarily secreted by the thyroid parafollicular C cells in response to increasing calcium concentrations and is involved in calcium homeostasis by inhibiting bone resorption and modulating calcium excretion from the kidney [126].…”

“…CYP24A1 REGULATION IN HEALTH AND DISEASE of the CYP24A1 promoter is regulated by an inverted CCAAT box at À62/À51 and a GC box at À114/À101 located downstream of the EBS in vitro [124,125]. The ubiquitous transcription factors Sp-1 and NF-Y acting through the GC and CCAAT box, respectively, contribute to the activation of basal transcriptional activity of the CYP24A1 promoter [124,125]. Molecular components involved in Sp-1/GC and NF-Y/CCAAT regulation of CYP24A1 basal expression are presently not known; however, recent evidence indicates that the PI-3-kinase-PKCx module is not required [125].…”

“…Calcitonin is primarily secreted by the thyroid parafollicular C cells in response to increasing calcium concentrations and is involved in calcium homeostasis by inhibiting bone resorption and modulating calcium excretion from the kidney [126]. Comparable with other regulators of basal CYP24A1 expression, calcitonin-mediated induction of the CYP24A1 promoter is dependent on the transcription factors Sp-1 and NY-F acting through the GC and CCAAT box sites, respectively [124]. PKC and PKA signaling pathways are involved in calcitonin-mediated induction of CYP24A1, possibly acting through Sp-1 and NY-F [124].…”

FGF23/Klotho New Regulators of Vitamin D Metabolism

“…Calcitonin stimulates CYP24A1 transcription through involvement of Ras-PKCzeta isoform pathway [21]. 1,25(OH) 2 D stimulates the transcription of CYP24A1 through its binding to the heterodimer VDR-RXR which recognizes VDRE sites on the CYP24A1 gene promoter [22].…”

Vitamine D : Métabolisme, régulation et maladies associées

“…Les apports en phosphates et la PTH modulent l'activité et l'expression de cette enzyme de manière opposée à leur effet sur CYP27B1 (Figure 2). De plus, une étude récente propose une régulation par la calcitonine de l'expression du gène codant CYP24A1 via la voie de signalisation Ras-PKC zêta (protéine kinase C d'isoforme zêta) [18] ( Figure 2) (Figure 3). Par ailleurs, VDR aurait un rôle direct dans l'inhibition de l'expression de la CYP27B1 dans les reins.…”