Elsevier

Bone

Volume 37, Issue 2, August 2005, Pages 148-158
Bone

SOST is a target gene for PTH in bone

https://doi.org/10.1016/j.bone.2005.03.018Get rights and content

Abstract

Intermittent parathyroid hormone (PTH) application is an established pharmacological principle to stimulate bone formation. Yet, the molecular mechanisms underlying this bone anabolic action are not fully understood. Recently, SOST (sclerostin) was identified as a potent osteocyte expressed negative regulator of bone formation in vitro, in murine models and in patients with the bone overgrowth disorders Sclerosteosis and Van Buchem disease. Therefore, we have studied the impact of PTH on SOST regulation. First, we analyzed SOST expression during PTH-induced bone formation in a classical model of local bone formation. 8-month-old mice received intermittently 100 nM hPTH(1–34) or vehicle onto the calvaria for 5 days. PTH stimulated bone formation as assessed by fluorochrome-marker-based histomorphometry. SOST expression was reduced in PTH-treated calvariae 4 h after the last administration as evaluated by real-time quantitative PCR. Next, we observed a decrease of SOST expression in femoral cortical bone of 6-month-old rats following single subcutaneous systemic administration of 80 μg/kg PTH(1–34). Finally, we studied SOST mRNA expression in bone of 11-month-old osteopenic estrogen-deprived (OVX) rats following 8-week systemic intermittent administration of 5 μg/kg PTH(1–34). PTH-treated animals displayed increases in bone mineral density as detected by pQCT, while SOST mRNA levels were decreased compared to vehicle-treated OVX and SHAM controls. PTH decreased SOST expression also in vitro. 100 nM PTH(1–34) inhibited expression in mouse calvaria organ cultures and in osteoblastic UMR-106 cells within 6 h by 95%. An IC50 of 1 nM was determined for PTH(1–34) in UMR-106 cells, whereas the PTH antagonist (d-Trp12,Tyr34)-bPTH(7–34) did not efficiently reduce SOST expression. Furthermore, SOST inhibition by PTH was not blocked by the protein synthesis inhibitor cycloheximide, indicating direct regulation, and PTH did not influence SOST mRNA degradation, implying transcriptional regulation. Finally, we observed full suppression of SOST by the cAMP inducer forskolin, partial inhibition by ionomycin, and no effect with PMA, indicating that PTH action is mainly mediated via the cAMP/PKA pathway. In summary, we have shown that PTH directly inhibits SOST transcription in vivo and in vitro, suggesting that SOST regulation may play a role in mediating PTH action in bone.

Introduction

It is well known that parathyroid hormone (PTH) produces both anabolic and catabolic effects in bone depending on the mode of administration. Continuous treatment stimulates high bone turnover, resulting in net bone loss, whereas intermittent application increases bone mass by enhancing bone formation [1]. Sustained elevation of PTH levels increases the number and activity of bone resorbing osteoclasts. This effect is mediated indirectly through osteoblasts, which abundantly express PTH receptor (PTH1R). Continuous PTH treatment results in regulation of several genes that are involved in bone resorption [2]. For example, PTH regulates expression of the membrane-bound tumor necrosis factor (TNF) ligand family member, RANK ligand (RANKL), and its secreted decoy receptor osteoprotegerin (OPG), which are key factors for the cell–cell coupling of activated osteoblasts to osteoclast differentiation and bone resorption [3], [4], [5]. PTH increases RANKL expression and represses OPG expression, favoring hence interaction of RANKL with its signaling receptor RANK expressed in the osteoclast lineage leading to enhanced bone resorption [4], [6], [7].

The bone anabolic effect of intermittent application of PTH in animals and humans is known for many years. It has led to the only osteoporosis therapy that is currently clinically accepted to increase bone mass through daily injections of recombinant peptide comprising the first 34 amino acids of human PTH [8]. Despite this well-established pharmacological principle, the molecular mechanisms whereby PTH stimulates bone formation when intermittently applied are at best partially understood. PTH appears to increase bone mass by stimulating bone-forming osteoblasts at all levels of their life cycle including recruitment [9], proliferation [10], [11], differentiation [12], [13], [14], [15], and apoptosis [16]. However, molecular analysis of these cellular effects have only slowly progressed so far and are characterized by controversial in vitro results mainly due to the lack of in vitro systems that truly model the bone anabolic effect of intermittent PTH application. For instance, conflicting results have been obtained regarding the regulation of bone formation marker genes [17], [18] and apoptosis by PTH in cultured osteoblastic cells under different conditions showing both positive and negative regulation [19]. Microarray gene expression analysis using osteoblastic UMR-106 cells has revealed a high complexity of gene regulation by PTH [20]. To date, the proto-oncogene c-fos [21], the transcription factor Cbfa1 [22], and insulin-like growth factor (IGF-I) [23] have been identified as essential genes for the bone anabolic action of PTH mainly by mouse genetic studies. Moreover, rodent studies have shown several genes to be up-regulated in osteoblasts, osteocytes, and megakaryocytes following bone anabolic intermittent PTH treatment [24], [25], [26]. Finally, some evidence suggests that the bone formation response might partially also be linked to bone resorption, although PTH acts directly on bone-forming osteoblasts via PTH1R. For example, IGF-I- and c-fos-deficient mice lacking bone formation responses following PTH treatment have decreased or absent bone resorption, respectively [21], [23]. In summary, PTH exerts complex actions in bone and net positive or negative outcome on bone mass depends critically on the kinetics of PTH exposure. Further identification of novel involved genes and studies of their molecular mechanisms of action are required to elucidate PTH actions in bone.

Recently, the SOST gene and its protein product sclerostin were identified as a novel potent negative regulator of bone formation [27], [28]. SOST is strongly expressed in osteocytes within bone and is structurally most closely related to the DAN/cerberus family of BMP antagonists [29]. As predicted from the structural relationship, SOST was shown to bind to BMPs and to inhibit proliferation and differentiation of osteoblasts. However, it may not act as a classical BMP antagonist predominantly blocking Smad phosphorylation, but use alternative signaling pathways or interfere with unknown BMP-induced factors, co-factors, or other pathways [28]. Consistent with negative regulation of bone formation, overexpression of SOST in transgenic mice produced osteopenia due to reduced bone formation [27]. Most importantly, SOST loss-of-function mutations in humans are the cause of the autosomal recessive bone dysplasias Sclerosteosis and Van Buchem disease, which are characterized by massive bone overgrowth throughout life and increased bone strength and serum markers of bone formation [21], [23], [30], [31].

Therefore, we investigated in the present study whether SOST is regulated during PTH-induced bone formation. We found that PTH quickly and robustly suppresses SOST expression in vivo and in vitro, and that it is a direct target gene of PTH regulated at the level of transcription.

Section snippets

Animals

Animal experimentation was carried out according to regulations effective in the Kanton of Basel-Stadt, Switzerland. 8-month-old female OF/IC mice (Iffa Credo, France) and 6-month-old Wistar rats (BRL, Fuellinsdorf, Switzerland) were housed in groups of seven (mice) and three to five (rats) animals at 25°C with a 12:12-h light–dark cycle. They were fed a standard laboratory diet containing 0.8% phosphorus and 1.1% calcium (NAFAG 890, Basel, Switzerland). Food and water were provided ad libitum.

Calvaria model of local bone formation

Results

Intermittent local application of hPTH(1–34) onto the calvaria (Fig. 1A) of aged mice increased bone formation as expected. Increased uptake of fluorochrome markers into mineralizing matrix, as an indirect marker of bone formation, could already be detected after the last PTH injection at day 5 (Fig. 1B). A small twofold increase (P < 0.1) in bone formation rate—reflecting the amount of mineralized matrix deposited between day two and four—had occurred in the calvaria of animals that had been

Discussion

Intermittent PTH(1–34) administration is currently the only FDA-approved osteoporosis therapy for restoring bone mass in the osteoporotic skeleton. Although the anabolic action of PTH has been studied for many years, the molecular mechanisms are still incompletely elucidated. This study investigated whether the recently identified potent bone formation inhibitor SOST is regulated by PTH.

Indeed, we observed SOST mRNA down-regulation by PTH in in vivo models of PTH-induced bone formation: first,

Acknowledgments

Myma Baptist, Margot Brüderlin, Marcel Merdes, and Anne Studer are gratefully acknowledged for their essential contributions to the in vivo aspects of this study. We thank Heidi Jeker and Johann Wirsching for their excellent technical assistance with in vitro studies and qPCR. We are grateful to Lynda Bonewald for the gift of MLO-Y4 cell RNA. Finally, we thank Klaus Seuwen for critically reading the manuscript.

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