MSM inhibits RANKL-induced signaling in BMMs.

<p>BMMs were incubated with various concentration of MSM for 1 h, together with controls without MSM exposure, were then incubated with or without RANKL (100ng/ml) for 10 min. Cell lysates were immunoblotted for the indicated proteins. (A) MSM inhibits RANKL-induced activation of ERK. (B) MSM suppresses RANKL-induced activation of Gab2, PLCγ2, and Syk. (C) MSM suppresses RANKL-induced IKK phosphorylation, IκB degradation, and NF-κB activation. Tata binding protein (TBP) was used as nuclear protein loading control. (D) NF-κB DNA binding was detected by EMSA. Data shown are representative of three independent experiments.</p

MSM attenuates RANKL-induced osteoclastic marker gene expression by blocking STAT3.

<p>(A) RAW264.7 cells were incubated with or without MSM for 1 h and then either exposed (or not) to RANKL (100ng/ml) for 10 min. Cell lysates were then blotted and immunostained with p-STAT3 and STAT3 antibodies. (B) RAW264.7 cells were transfected with STAT3 shRNA or a non-targeting shRNA for 48 h, then stimulated with RANKL (100 ng/ml) for 10 min. Cell lysates were prepared for western blot with antibodies as indicated. The relative levels of protein were determined using densitometry and normalized to β-actin. (C) RAW264.7 cells were transfected as in (B) and then stimulated with RANKL (100 ng/ml) for 24 h, with total RNA isolated using Qiagen. Expression of osteoclastic marker genes and STAT3 were examined using real-time PCR with GAPDH used as an internal control. Data shown are representative of three independent experiments. Asterisks indicate a significant increase by t-test (**p <0.01, ***p < 0.001).</p

MSM suppresses RANKL-induced osteoclast marker gene and protein expression.

<p>BMMs were cultured in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml) for the indicated number of days, with or without various concentrations of MSM. (A and B) Expression levels of the RANKL-induced osteoclast marker proteins examined by western blot analyses. (C) Expression of mRNAs for RANKL and OPG in bone marrow mesenchymal stem cells (MSCs). (D) Expression of mRNA for RANKL-induced osteoclast marker genes examined by RT-PCR analyses. Beta-actin and GAPDH were used as loading controls. Data shown are representative of three independent experiments.</p

Involvement of STAT5b in MSM-induced osteogenic marker genes in MSCs.

<p>(A) Bone marrow mesenchymal stem cells were cultured in the osteogenic medium at 5 days for ALP, 14 days for osteonectin (ON) and bone sialoprotein (BSP), and 21 days for osteocalcin (OCN) and osterix mRNA expression after the treatment with various concentrations (0, 10 and 20 mM) of MSM. RT-PCR was performed using the cDNA and primers for ALP, ON, BSP, OCN, osterix and 18S. Total RNA was isolated from the MSCs using an RNeasy kit. 18S was used as a control. (B) Bone marrow Mesenchymal stem cells and (C) C3H10T1/2 cells were cultured in osteogenic medium at 5 days for ALP and Runx2, 14 days for OPN and BSP, and 21 days for OCN and osterix mRNA expression after the treatment with 20 mM MSM. After culture, real-time PCR was performed. (D) Osteogenic differentiation marker genes (ALP, BSP, OCN, OPN, Osterix and Runx2) and STAT5b gene expression was analyzed at day 5, 14 and 21 after MSM treatment in C3H10T1/2 cells transfected with STAT5b siRNA or non-target siRNA. The effect of STAT5b knockdown on osteogenic marker genes was analyzed by real-time PCR. GAPDH was used as the internal control. The relative levels of mRNA were determined using densitometric analysis and normalized to the amount of GAPDH. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (*p<0.05, **p<0.01, ***p<0.001).</p

Effects of methylsulfonylmethane (MSM) on the expression of growth hormone (GH) signaling-related proteins in osteoblast-like cells and MSCs.

<p>MG-63 (A) and UMR-106 (B) cells were treated with the indicated MSM concentrations for 24 h. (C) Mesenchymal stem cells were cultured in osteogenic medium with various concentrations of MSM for 21 days. (D) UMR-106 cells were left untreated or pretreated with 50 µM AG490 for 4 h then treated with MSM for 24 h. Protein extracts (20 µg) were separated by 10% SDS-PAGE, and Western blots were performed. β-actin was used as a protein loading control. (E) The relative levels of IGF-1R, GHR and Jak2 protein were determined using densitometric analysis and normalized to the amount of β-actin. This picture is representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p

Effects of methylsulfonylmethane (MSM) on osteoblast differentiation in primary murine bone marrow mesenchymal stem cells (MSCs).

<p>(A) Comparison of alkaline phosphatase (ALP) activity of various MSM treatments with varying culture durations. Cells were treated with various concentrations (0, 5, 10 and 20 mM) of MSM for 3, 5, and 7 days. ALP activity was measured by the release of p-nitrophenol from p-nitrophenyl phosphate and was normalized to the protein level. Osteoblastic mineralization was determined by Alizarin Red S staining (B) and von Kossa staining (C) at day 21. Cells were cultured in non osteogenic medium (NO) or osteogenic medium (OM) with various concentrations (0 and 20 mM) of MSM for 21 days (scale bar: 50 µm). Mineral nodules (more than 1 mm in diameter) were counted and expressed in histogram. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p

Methylsulfonylmethane (MSM)-induced binding activity of STAT5b to the insulin-like growth factor-1 receptor (IGF-1R) GAS site.

<p>(A) UMR-106 cells were cultured in serum-free MEM for 24 h and incubated with MSM (20 mM) for 12 or 24 h. Nuclear extracts (NE) were separated and blotted onto a nitrocellulose membrane, which showed an increase in the level of IGF-1R and STAT5b. Tata binding protein (TBP) was used as a nuclear protein loading control. (B) STAT5b DNA binding was detected by electrophoretic mobility shift assay. Nuclear extracts were prepared and incubated with STAT5 probe (from a part of Panomics EMSA kits). The resulting complexes were electrophoresed on a 6% non-denaturing gel. (C) The IGF-1R (−2350/+640) LUC reporter plasmid or (D) the 700 bp IGF-1-pGL2 were cotransfected into UMR 106 cells with the STAT5b expression vector, the β-galactosidase expression plasmid (pCMV β-Gal), and either empty pGL2 vector, and then incubated with 50 µM AG490 for 4 h, then treated with 20 mM MSM for 24 h. Promoter activities were expressed as luciferase normalized to β-galactosidase values. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p

Methylsulfonylmethane (MSM)-enhanced GH signaling requires STAT5b activation in C3H10T1/2 cells.

<p>(A) C3H10T1/2 cells were grown to 50% confluence and transfected with ON-TARGETplus SMARTpool siRNA targeting STAT5b or ON-TARGETplus Non-targeting siRNA using FuGene 6, according to the manufacturer’s instructions. 48 hours after transfection, cells were cultured with serum free osteogenic medium for 24 h and then cultured in osteogenic medium with 20 mM MSM for 24 h after the initiate osteoblast differentiation. Protein extracts (20 µg) were separated by 10% SDS-PAGE, and Western blots were performed. β-actin was used as a protein loading control. (B) The relative levels of protein were determined using densitometric analysis and normalized to the amount of β-actin. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (**p<0.01, ***p<0.001).</p

Effects of methylsulfonylmethane (MSM) on viability in osteoblast-like cells and MSCs.

<p>MG-63 and UMR-106 cells exposed to control condition without MSM or growth facilitated condition with increasing concentration of MSM for 24 h. C3H10T1/2 cells and mesenchymal stem cells grown in the osteogenic media (10 mM sodium β-glycerophosphate and 50 µg/ml ascorbic acid) and exposed to control condition or growth facilitated condition for 21 days. After culture, cell viability was evaluated using MTT assay. Data shown are representative of three independent experiments.</p

Methylsulfonylmethane (MSM) enhances growth hormone (GH) signaling via Jak2/STAT5b activation in osteoblast-like cells.

<p>(A) Jak2 was immunoprecipitated with anti-Jak2 antibody from whole cell extracts of UMR 106 cells left untreated or pretreated with 30 nM GH for 2 h followed by MSM for 24 h. Jak2 precipitation and the phosphorylation status of Jak2 were analyzed by Western blotting with anti-Jak2 and anti-phosphotyrosine (4G10) antibodies. (B) The relative levels of Jak2 phosphorylation were determined using densitometric analysis and normalized to the amount of Jak2. (C) UMR-106 cells were incubated with 50 µM AG490 for 4 h, then left untreated or pretreated with 30 nM GH for 2 h followed by MSM for 24 h. STAT5b precipitation and the phosphorylation status of the precipitated STAT5b were analyzed by Western blot with anti-STAT5b and 4G10 antibodies. This picture is representative of three independent experiments. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (***p<0.001).</p