RESUMO
Growing evidence shows that the inhibitory effect of inflammatory cytokines on new bone formation by osteogenic precursor cells is a critical cause of net bone-density reduction. Melatonin has been proven to be a potential therapeutic candidate for osteoporosis. However, whether it is capable of antagonizing the suppressing effect of inflammatory cytokines on osteogenic precursor cells is so far elusive. In this study, using the cell culture system of human bone marrow stromal cells and MC3T3-E1 preosteoblasts, we recorded the following vital observations that provided insights of melatonin-induced bone formation: 1) melatonin induced bone formation in both normal and inflammatory conditions; 2) Wnt4 was essential for melatonin-induced bone formation in inflammatory stimulation; 3) melatonin- and Wnt4-induced bone formation occurred via activation of ß-catenin and p38-JNK MAPK pathways by interaction with a distinct frizzled LDL receptor-related protein complex; 4) melatonin suppressed the inhibitory effect of NF-κB on osteogenesis in a Wnt4-dependent manner; and 5) melatonin induced Wnt4 expression through the ERK1/2-Pax2-Egr1 pathway. In summary, we showed a novel mechanism of melatonin-induced bone formation in an inflammatory environment. Melatonin-induced Wnt4 expression is essential for its osteoinductive effect and the inhibitory effect of NF-κB on bone formation. Our novel findings may provide useful information for its potential translational application.-Li, X., Li, Z., Wang, J., Li, Z., Cui, H., Dai, G., Chen, S., Zhang, M., Zheng, Z., Zhan, Z., Liu, H. Wnt4 signaling mediates protective effects of melatonin on new bone formation in an inflammatory environment.
Assuntos
Melatonina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Proteína Wnt4/fisiologia , Animais , Cálcio/metabolismo , Linhagem Celular , Receptores Frizzled/fisiologia , Regulação da Expressão Gênica , Humanos , Inflamação , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , NF-kappa B/fisiologia , Osteoblastos/fisiologia , Osteogênese/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Receptores de LDL/fisiologia , Receptores Wnt/efeitos dos fármacos , Receptores Wnt/fisiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE AND DESIGN: To elucidate the influence of 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP), a canonical Wnt/ß-catenin pathway activator, on the inflammatory response of TLR-engaged innate cells in vitro. MATERIAL OR SUBJECT: Primary human monocytes. TREATMENT: AMPMB (0-10 µM), LPS (0-1.0 µg/ml), Pam3CSK4, FSL-1, or S. typhimurium flagellin (0-0.25 µg/ml). METHODS: TLR-induced cytokine release (TNF, IL-6, IL-12 p40) was monitored by ELISA while Wnt-related signals (GSK3ß, p65, IκB, ß-catenin) were assessed by Western blot, pharmaceutical inhibition and gene silencing. RESULTS: AMBMP induced the rapid phosphorylation of NFκB p65 at Ser(536) and abrogated total IκB, accompanied by a subsequent increase in pro-inflammatory cytokine production (TNF, IL-6, IL-12 p40) in otherwise naive monocytes. However, in TLR2, -4 and -5-engaged monocytes, AMBMP-suppressed cytokine production. In the context of LPS stimulation, this occurred concomitant with the phosphorylative inactivation of GSK3ß at Ser(9), ß-catenin accumulation and abrogation of NFκB p65 phosphorylation. AMBMP-mediated suppression of the TLR4 -induced inflammatory response was reversed by two pharmaceutical Wnt/ß-catenin pathway inhibitors, IWP-2 and PNU-74654 and by Wnt3a silencing. CONCLUSIONS: Herein, we show that AMBMP induces canonical Wnt signaling events and acts as a suppressor of inflammation in surface TLR-engaged primary human monocytes.
