Analysis of SOST expression using large minigenes reveals the MEF2C binding site in the evolutionarily conserved region (ECR5) enhancer mediates forskolin, but not 1,25-dihydroxyvitamin D3 or TGFβ1 responsiveness

Abstract

Transcribed from the SOST gene, sclerostin is an osteocyte-derived negative regulator of bone formation that inhibits osteoblastogenesis via antagonism of the Wnt pathway. Sclerostin is a promising therapeutic target for low bone mass diseases and neutralizing antibody therapies that target sclerostin are in development. Diverse stimuli regulate SOST including the vitamin D hormone, forskolin (Fsk), bone morphogenic protein 2 (BMP-2), oncostatin M (OSM), dexamethasone (Dex), and transforming growth factor (TGF)β1. To explore the mechanisms by which these compounds regulate SOST expression, we examined their ability to regulate a SOST reporter minigene containing the entire SOST locus or mutant minigene containing a deletion of the −1 kb to −2 kb promoter proximal region (−1 kb), ECR2, ECR5, or two point mutations in the MEF2 binding site of ECR5 (ECR5/MEF2). Previous reports suggest that both the PTH and TGFβ1 effects on SOST are mediated through ECR5 and that the action of PTH is mediated specifically via the MEF2 binding site at ECR5. Consistent with these reports, the suppressive effects of Fsk were abrogated following both ECR5 deletion and ECR5/MEF2 mutation. In contrast, we found that TGFβ1 negatively regulated SOST and that neither ECR5 nor ECR5/MEF2 was involved. Surprisingly, none of these four deletions/mutations abrogated the suppressive effects of the vitamin D hormone, OSM, Dex, or TGFβ1, or the positive effects of BMP-2. These data suggest that we need to move beyond ECR5 to understand SOST regulation.

Osterix and RUNX2 are Transcriptional Regulators of Sclerostin in Human Bone

“…A number of factors, including OSX and RUNX2, shown in this study, but also vitamin D, TGF and BMPs act through the binding to motifs in the 5' region of the [30,31]. However, elements located distal to the SOST gene, such as the ECR5 sequence included in the so-called Van Buchem´s region, seem to mediate the regulatory effects of factors such as parathryroid hormone and MEF2 on sclerostin production [32] In conclusion, our results confirm that RUNX2 and OSX modulate the transcription of the sclerostin human gene and common RUNX2 polymorphisms may influence bone mass by still unknown mechanisms.…”

Role and mechanism of action of sclerostin in bone

“…Moreover, mechanical loading negatively regulates Sost expression by mechanisms that might involve prostaglandin E2 production, nitric oxide, and periostin expression (11;49–51). In vitro studies using a reporter construct carrying the proximal Sost promoter also identified forskolin and oncostatin M as potential regulators of Sost expression (52;53). In addition to PTH, other systemic hormones have a strong impact on Sost regulation.…”

“…Vitamin D can also regulate Sost expression, although contradictory data has been published. Thus, using Saos-2 cells, Wijenayaka et al showed that 1α,25-dihydroxyvitamin D3 induces the expression of Sost/Sclerostin (57), whereas St John et al found that it decreases the transcriptional activity of the Sost promoter (52). Similarly conflicting clinical data on the effects of vitamin D status on circulating sclerostin levels have been reported (58–62).…”

Regulation of Bone Remodeling by Parathyroid Hormone

“…Having identified that MEF2C is a crucial determinant of osteocytic SOST expression, an obvious question that emerges is whether PTH blocks MEF2C-driven SOST expression. Early studies using heterologous reporter systems suggested that cAMP signaling might regulate MEF2C activity in the setting of the +45 kb SOST enhancer (Leupin et al 2007;St John et al 2015a). In many biologic systems, upstream signals regulate MEF2 transcriptional activity via nucleocytoplasmic shuttling of class IIa HDAC proteins, which serve as potent inhibitors of MEF2-driven gene expression (Haberland et al 2009).…”