Thiazide diuretics affect osteocalcin production in human osteoblasts at the transcription level without affecting vitamin D₃ receptors

Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblastlike cell line MG-63. HCTZ dose-dependently (1-100 μM) inhibited 1,25- dihydroxyvitamin D₃ [1,25(OH)₂D₃]-induced OC release by these cells (maximal effect, -40-50% andp < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (-4.7.2 ± 4.0%; p < 0.0001 by paired ANOVA with 100 μM HCTZ). HCTZ (100 μM) also stimulated calcium (Ca²⁺) uptake (8.26 ± 1.78 pmol/mg protein/15 minutes vs. 13.6 ± 0.4.9 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca²⁺ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(β-amino ethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, -22.5 ± 6.9%), suggesting that thiazide-dependent Ca²⁺ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)₂D₃ in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D₃ receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1-100 μM) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca²⁺ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate.