Abstract
Rosiglitazone is a well-known anti-diabetic drug that increases insulin sensitivity via peroxisome proliferator-activated receptor γ (PPARγ) activation, but unfortunately it causes bone loss in animals and humans. A previous study showed that prolyl hydroxylase domain protein (PHD) plays a role in rosiglitazone-induced adipocyte differentiation. Based on the inverse relationship between adipocyte and osteoblast differentiation, we investigated whether PHD is involved in the effects of rosiglitazone on osteoblast differentiation. Rosiglitazone inhibited osteoblast differentiation in a concentration-dependent manner, and in parallel induced three PHD isoforms (PHD1, 2, and 3). PHD inhibitors and knockdown of each isoform prevented the inhibitory effects of rosiglitazone on osteoblast differentiation and increased the expression of Runx2, a transcription factor essential for osteoblastogenesis. MG-132, a proteasomal inhibitor also prevented the rosiglitazone-induced degradation of Runx2. Furthermore, both increased PHD isoform expressions and reduced osteoblast differentiation by rosiglitazone were prevented by PPARγ antagonists, indicating these effects were mediated via PPARγ activation. In vivo oral administration of rosiglitazone to female ICR mice for 8 weeks reduced bone mineral densities and plasma alkaline phosphatase (ALP) activity, and increased PHD expression in femoral primary bone marrow cells and the ubiquitination of Runx2. Together, this suggests that the rosiglitazone-induced suppression of osteoblast differentiation is at least partly induced via PPARγ-mediated PHD induction and subsequent promotion of the ubiquitination and degradation of Runx2.
Highlights
Cellular differentiation is a critical requirement for body homeostasis, and is tightly coordinated by the regulation of several transcription factors and intracellular signals
The transcriptional suppression of Runx2 by peroxisome proliferator-activated receptor γ (PPARγ) activation might contribute to the inhibitory effect of rosiglitazone on osteoblastogenesis, these results suggest that posttranslational modification of Runx2 by PHDs and the subsequent stimulated ubiquitination of Runx2 could play a significant role in the suppression of osteoblast differentiation by rosiglitazone
The present study shows that rosiglitazone suppresses osteoblast differentiation via PPARγmediated PHD upregulation, which in turn leads to increased ubiquitination and degradation of Runx2, an essential transcription factor for osteoblast differentiation
Summary
Cellular differentiation is a critical requirement for body homeostasis, and is tightly coordinated by the regulation of several transcription factors and intracellular signals. Bone homeostasis is maintained by balance between the activities of osteoblasts and osteoclasts, and imbalance between these cells results in metabolic diseases, such as, osteoporosis and PLOS ONE | DOI:10.1371/journal.pone.0139093. Osteoblasts are responsible for bone formation, which leads to mineralization and further differentiation into osteocytes. Over the last two decades, many factors have been found to regulate osteoblast differentiation. Runt-related transcription factor-2 (Runx2), osterix, Msh homeobox-2 (Msx2), bone morphogenetic protein 2 (BMP2), Wnt and Hedgehog have been shown to be required for osteoblastogenesis [4]. Adipocytes originate from mesenchymal progenitor cells, the biological activities of osteoblasts and adipocytes are related. Factors that control osteoblastogenesis have been shown to inhibit adipogenesis, and vice versa [5]
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