Only bioactive forms of PTH (n-oxPTH and Met18(ox)-PTH) inhibit synthesis of sclerostin - evidence from in vitro and human studies

Abstract

Sclerostin (SOST) is produced by osteocytes and is known as a negative regulator of bone homeostasis. Parathyroid hormone (PTH) regulates calcium, phosphate as well as vitamin D metabolism, and is a strong inhibitor of SOST synthesis in vitro and in vivo. PTH has two methionine amino acids (positions 8 and 18) which can be oxidized. PTH oxidized at Met18 (Met18(ox)-PTH) continues to be bioactive, whereas PTH oxidized at Met8 (Met8(ox)-PTH) or PTH oxidized at Met8 and Met18 (Met8, Met18(di-ox)-PTH) has minor bioactivity. How non-oxidized PTH (n-oxPTH) and oxidized forms of PTH act on sclerostin synthesis is unknown. The effects of n-oxPTH and oxidized forms of PTH on SOST gene expression were evaluated in UMR106 osteoblast-like cells. Moreover, we analyzed the relationship of SOST with n-oxPTH and all forms of oxPTH in 516 stable kidney transplant recipients using an assay system that can distinguish in clinical samples between n-oxPTH and the sum of all oxidized PTH forms (Met8(ox)-PTH, Met18(ox)-PTH, and Met8, Met18(di-ox)-PTH). We found that both n-oxPTH and Met18(ox)-PTH at doses of 1, 3, 20, and 30 nmol/L significantly inhibit SOST gene expression in vitro, whereas Met8(ox)-PTH and Met8, Met18(di-ox)-PTH only have a weak inhibitory effect on SOST gene expression. In the clinical cohort, multivariate linear regression showed that only n-oxPTH, but not intact PTH (iPTH) nor oxPTH, is independently associated with circulating SOST after adjusting for known confounding factors. In conclusion, only bioactive PTH forms such as n-oxPTH and Met18(ox)-PTH, inhibit SOST synthesis.

Keywords: KTRs; Non-oxidized; Oxidized; PTH; SOST.

Fig. 1

The effects of n-oxPTH and various forms of oxPTH on SOST gene expression in UMR106 rat osteoblast-like cells (a–d). We cultured the cells for the first 24 h, then, treated them with or without PTH 1–34 derivatives for another 24 h at 1, 3, 20, and 30 nmol/L, respectively. The experiment was repeated six times and the levels of SOST expression were measured by qRT-PCR. a and b Both n-oxPTH and Met18(ox)-PTH peptides, at all dosages caused a significant decrease in SOST gene expression when compared with negative controls (P < 0.001), however n-ox PTH had a maximum inhibitory effect already at 1 nmol/L; an extent of inhibition, that was not reached even with a dose of 30 nmol/L Met18(ox)-PTH peptides. c A similar trend was also observed in the Met18(di-ox)-PTH peptides, despite a relatively slight inhibition in comparison to negative controls (P < 0.05). d However, the Met8(ox)-PTH peptides inhibited the SOST mRNA synthesis even to a lesser extent and only at 20 and 30 nmol/L (P < 0.05), but not at 1 and 3 nmol/L, when compared with negative controls. The x-axis shows the different concentrations. The y-axis represents the relative SOST gene expression. Data was analyzed using the Kruskal–Wallis test followed by Dunn’s post-hoc test. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. control group. ^# P < 0.05, 1 nmol/L vs. 3 nmol/L Met8(ox)-PTH peptides. Abbreviations: iPTH, intact PTH; n-oxPTH, non-oxidized PTH; oxPTH, oxidized PTH; PTH, parathyroid hormone; SOST, sclerostin

Fig. 2

Histogram showing the frequency distribution of plasma iPTH (a), oxPTH (b), n-oxPTH (c), and Sclerostin (d) in 516 stable kidney transplant recipients. The actual concentrations of iPTH, oxPTH, and n-oxPTH in the study population are not normally distributed, except for SOST concentration, which is similar to a normal distribution. Abbreviations: iPTH, intact PTH; n-oxPTH, non-oxidized PTH; oxPTH, oxidized PTH; PTH, parathyroid hormone; SOST, sclerostin

Fig. 3

Scatterplot showing the correlation between SOST and n-oxPTH plasma concentrations in kidney transplant recipients. As actual plasma SOST and n-oxPTH concentrations were moderately skewed in the study, we calculated ln-SOST and ln-n-oxPTH. After data transformation, both ln-SOST and ln-n-oxPTH were close to being normally distributed. We used Pearson correlation analysis to assess the associations between ln-SOST and ln-n-oxPTH. Our findings showed that ln-SOST was significantly negatively associated with ln-n-oxPTH (P < 0.05). Abbreviations: n-oxPTH, non-oxidized PTH; PTH, parathyroid hormone; SOST, sclerostin