Msx2 mediates the inhibitory action of TNF-alpha on osteoblast differentiation - PubMed (original) (raw)

Msx2 mediates the inhibitory action of TNF-alpha on osteoblast differentiation

Hye-Lim Lee et al. Exp Mol Med. 2010.

Abstract

TNF-alpha, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-alpha signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-alpha-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-alpha down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2(-/-)calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-alpha induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-alpha. TNF-alpha activated the NF-kappaB and the JNK pathways. Inhibition of NF-kappaB or JNK activation reduced the inhibitory effect of TNF-alpha on ALP expression, whereas TNF-alpha-induced Msx2 expression was only suppressed by the inhibition of the NF-kappaB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-alpha on BMP2-regulated osteoblast differentiation and that the TNF-alpha-activated NF-kappaB pathway is responsible for Msx2 induction.

PubMed Disclaimer

Figures

Figure 1

TNF-α suppresses BMP2-induced ALP expression in Runx2-/- cells. (A, B) C2C12 cells were incubated in DMEM supplemented with 5% FBS for 24 h in the presence of the indicated reagents. Runx2+/+ and Runx2-/- cells were incubated in α-MEM supplemented with 10% FBS, 10 mM β-glycerophosphate and 50 µg/ml ascorbic acid for 48 h in the presence of the indicated reagents. Then, ALP staining (A) or RT-PCR (B) was performed. (C) To block new protein synthesis, C2C12 cells were treated with BMP2 and/or TNF-α in the presence of cycloheximide (CHX, 10 µg/ml) for 24 h. The concentrations of TNF-α and BMP2 were 10 ng/ml and 100 ng/ml, respectively, or as otherwise indicated. *, In Runx2-/- cells, the RT-PCR products of the ALP gene were obtained by carrying out the amplification step for five more cycles than in C2C12 and Runx2+/+ cells.

Figure 2

Expression of Msx2 is induced by TNF-α. (A) Over-expressed Msx2 inhibited BMP2-induced ALP expression. C2C12 cells were transiently transfected with pcDNA or the Msx2 expression vector and incubated in the presence of BMP2 for 24 h. RT-PCR was then performed. (B) C2C12 cells were incubated in the presence of the indicated reagents for 24 or 48 h. RT-PCR (left panel) or western blot analysis (right panel, nuclear extracts of 24 h samples) was performed to detect Msx2 induction. Histone H3 was used as a loading control. (C) Runx2+/+ and Runx2-/- cells were incubated in the presence of the indicated reagents for 24 h, and RT-PCR was performed. (D) Runx2 did not exert any effect on TNF-α-induced Msx2 expression. Runx2-/- cells were transiently transfected with pcDNA or the Runx2 expression vector and incubated in the presence of TNF-α for 24 h. RT-PCR was then performed.

Figure 3

TNF-α-inhibited ALP expression is rescued by the knockdown of Msx2. To induce the silencing of Msx2, C2C12 cells were transfected with _si_RNAs for Msx2 (si Msx2) or control _si_RNAs (si control) and further incubated in the presence of the indicated reagents for 24 h. Next, RT-PCR, immunoblot analysis and ALP staining were performed. Lamin B was used as a loading control.

Figure 4

TNF-α induces Msx2 expression via NF-κB activation. (A) Inhibition of NF-κB activation by BAY-11-7082 (BAY, 10 µM) blocked TNF-α-induced Msx2 expression. C2C12 cells were treated with the indicated reagents for 10 min (upper panel) or for 24 h (middle and lower panels), and then immunoblotting, RT-PCR and ALP staining were performed. The levels of phospho-IκBα and total IκBα were examined using whole cell lysates whereas the Msx2 protein level was examined using nuclear extracts. (B) BAY-11-7082 did not exert any cytotoxic effects at a concentration of 10 µM. The cell cytotoxicity/proliferation assay was performed using the CCK-8 reagent as described in Methods. Data represent the mean + SD of quadruplicates. *, P < 0.01 (Student's _t_-test) (C) Over-expression of _dn_IκBα suppressed TNF-α-induced Msx2 expression. C2C12 cells were transiently transfected with pcDNA or the _dnI_κBα expression vector and incubated in the presence of TNF-α for 24 h. Next, RT-PCR and immunoblot analyses were performed.

Figure 5

JNK activation is not involved in TNF-α-induced Msx2 expression. (A, C) C2C12 cells were treated with the indicated reagents for 5 min (A) or for 24 h (C) and then immunoblot analysis and RT-PCR were performed. JNK activation was confirmed using whole cell lysates whereas the Msx2 protein level was examined using nuclear extracts. (B) SP600125 did not exert any cytotoxic effects at a concentration of 10 µM. Data represent the mean + SD of quadruplicates. *, P < 0.01 (Student's _t_-test)

References

1. Alappat S, Zhang ZY, Chen YP. Msx homeobox gene family and craniofacial development. Cell Res. 2003;13:429–442. -PubMed
1. Boyce BF, Li P, Yao Z, Zhang Q, Badell IR, Schwarz EM, O'Keefe RJ, Xing L. TNF-alpha and pathologic bone resorption. Keio J Med. 2005;54:127–131. -PubMed
1. Brockman JA, Scherer DC, McKinsey TA, Hall SM, Qi X, Lee WY, Ballard DW. Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. Mol Cell Biol. 1995;15:2809–2818. -PMC -PubMed
1. Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science. 2002;296:1634–1635. -PubMed
1. Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003;278:45969–45977. -PubMed

Msx2 mediates the inhibitory action of TNF-alpha on osteoblast differentiation - PubMed (original) (raw)