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. 2018 Oct 25;19(11):3332.
doi: 10.3390/ijms19113332.

The Bromodomain Inhibitor N-Methyl pyrrolidone Prevents Osteoporosis and BMP-Triggered Sclerostin Expression in Osteocytes

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The Bromodomain Inhibitor N-Methyl pyrrolidone Prevents Osteoporosis and BMP-Triggered Sclerostin Expression in Osteocytes

Barbara Siegenthaler et al. Int J Mol Sci. .

Abstract

(1) Background: In an adult skeleton, bone is constantly renewed in a cycle of bone resorption, followed by bone formation. This coupling process, called bone remodeling, adjusts the quality and quantity of bone to the local needs. It is generally accepted that osteoporosis develops when bone resorption surpasses bone formation. Osteoclasts and osteoblasts, bone resorbing and bone forming cells respectively, are the major target in osteoporosis treatment. Inside bone and forming a complex network, the third and most abundant cells, the osteocytes, have long remained a mystery. Osteocytes are responsible for mechano-sensation and -transduction. Increased expression of the osteocyte-derived bone inhibitor sclerostin has been linked to estrogen deficiency-induced osteoporosis and is therefore a promising target for osteoporosis management. (2) Methods: Recently we showed in vitro and in vivo that NMP (N-Methyl-2-pyrrolidone) is a bioactive drug enhancing the BMP-2 (Bone Morphogenetic Protein 2) induced effect on bone formation while blocking bone resorption. Here we tested the effect of NMP on the expression of osteocyte-derived sclerostin. (3) Results: We found that NMP significantly decreased sclerostin mRNA and protein levels. In an animal model of osteoporosis, NMP prevented the estrogen deficiency-induced increased expression of sclerostin. (4) Conclusions: These results support the potential of NMP as a novel therapeutic compound for osteoporosis management, since it preserves bone by a direct interference with osteoblasts and osteoclasts and an indirect one via a decrease in sclerostin expression by osteocytes.

Keywords: N-methyl pyrrolidone; bromodomain; osteocyte; osteoporosis; sclerostin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cell viability dependent on the N-methyl pyrrolidone (NMP) concentration. (A) Differentiating IDG-SW3 cells were treated with NMP in a range from 0.5–5 mM over a time period of 9 days. (B) UMR-106 cells were treated with NMP in a range from 0.5–5 mM over 48 h. WST-1 cell viability assessment at collection time revealed no toxicity at NMP concentrations up to 5 mM.
Figure 2
Figure 2
NMP decreases sclerostin mRNA and protein expression. (A) Immortalized mouse IDG-SW3 cells were stimulated with 2 mM (dark bars) and 5 mM (light bars) NMP during the whole period of differentiation. (B) Rat osteosarcoma UMR-106 cells were incubated with 2 mM (dark bars) and 5 mM (light bars) NMP over a time period of 24 h. (C) UMR-106 cells were treated for 8 h with the indicated NMP concentrations in normal culture medium. Whole cell lysates were subjected to Western Blot analysis indicating an NMP concentration-dependent reduction in sclerostin expression. The star indicates statistical significance at α = 0.05, while “ns” indicates non-significance at the same threshold.
Figure 3
Figure 3
NMP prevents the BMP-2 induced increase in sclerostin expression. Stimulation of UMR-106 cells with 500 ng/mL bone morphogenetic protein 2 (BMP-2) increases sclerostin protein at 8 h (A) whereas a combination of BMP-2 and 2 mM NMP decreases sclerostin protein. Illustration of qRT-PCR of the same treatment is presented in (B).
Figure 4
Figure 4
NMP prevents the osteoporosis induced increase in sclerostin expression. Female rats were subjected to ovariectomy (OVX) or control surgery (Sham) and treated with vehicle (PBS) or NMP and femurs were collected for histology. (A) Location of image acquisition of histological sections is marked by the red square. (B) Sham PBS animals with sclerostin producing osteocytes (brown color). (C) OVX animals injected with the vehicle control (PBS). (D) OVX animals injected with the chemical NMP. Scale bars in B-D represent 50 μm. (E) High and low-magnifications of histological sections stained for sclerostin expression for all three groups as indicated: Sham PBS, OVX PBS and OVX NMP. Scale in the upper panel is 200 µm and in the lower panel 50 µm. The area of the histology of the lower panel is indicated in the respective upper panel by the dashed black rectangle. (F) Percentage of sclerostin positive cells. Stars indicate statistical significance at α = 0.05.
Figure 5
Figure 5
The high affinity bromodomain inhibitor JQ1 decreases sclerostin expression. UMR-106 cells treated for 8 h with a titration of the bromodomain inhibitor JQ1 do not change their metabolic activity (A). JQ1 treatment leads to a concentration dependent decrease in SOST mRNA (B) and Sclerostin protein (C) expression. Error bars indicate standard deviation from three independent experiments. “ns” indicates non-significance at α = 0.05.

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