Interleukin-20 inhibits the osteogenic differentiation of MC3T3-E1 cells via the GSK3β/β-catenin signalling pathway.

To investigate the effects of interleukin-20 (IL-20) on the osteogenic differentiation of MC3T3-E1 cells. The pre-osteoblast line MC3T3-E1 was treated with different concentrations of IL-20 (0, 2, 20 and 100 ng/mL), and the cell viability was detected by the CCK8 assay. To assess the influence of IL-20 on osteogenic differentiation, alkaline phosphatase (ALP) activity and Alizarin red staining were performed at predetermined times. The expression levels of Runt-related transcription factor 2 (RUNX2), Osterix (Osx), glycogen synthase kinase-3β (GSK-3β) and β-catenin were detected by qRT-PCR and Western blotting analyses. 5 nmol/L lithium chloride (LiCl) was used as GSK-3β inhibitor. IL-20 promoted cell proliferation but decreased ALP activity and mineralization. Moreover, IL-20 downregulated the expression of RUNX2, Osx and β-catenin but upregulated the level of GSK-3β. The results suggest that IL-20 could inhibit the osteogenic differentiation of MC3T3-E1 cells via the GSK3β/β-catenin signalling pathway.

Chen X ，Liu Y ，Meng B ，Wu D ，Wu Y ，Cao Y ... -  《-》

Anthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3β and subsequently activating β-catenin.

The bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. This study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. Cell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. AHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3β (GSK-3β); particularly, isovitexin-4'-O-glucoside most strongly binds to the peptide backbone of GSK-3β at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3β at SER9 (an inactive form) and released β-catenin into the nucleus. Pretreatment with FH535, a Wnt/β-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. AHs stimulate osteogenic activities through the inhibition of GSK-3β and subsequent activation of β-catenin, leading to anti-osteoporosis effects.

Karunarathne WAHM ，Molagoda IMN ，Lee KT ，Choi YH ，Jin CY ，Kim GY ... -  《-》

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways.

Bone marrow stromal cells (BMSCs) are multipotent precursors that give rise to osteoblasts, and contribute directly to bone formation. Connexin 43 (Cx43) is the most ubiquitous gap junction protein expressed in bone cell types, and plays crucial roles in regulating intercellular signal transmission for bone development, differentiation and pathology. However, the precise role and mechanism of Cx43 in BMSCs are less known. Here, we investigate the function of Cx43 in osteogenic differentiation of BMSCs in vitro. BMSCs were isolated by whole bone marrow adherent culture. Knock down of Cx43 was performed by using lentiviral transduction of Cx43 shRNA. BMSCs were induced to differentiate by culturing in a-MEM, 10% FBS, 50 µM ascorbic acid, 10 mM beta-glycerophosphate, and 100 nM dexamethasone. Alkaline phosphatase (ALP) activity and alizarin red S staining were used to evaluate osteogenic differentiation in calcium nodules. Target mRNAs and proteins were analyzed by using real-time quantitative PCR (qPCR) and western blotting. Cx43 expression markedly increased during osteogenic differentiation. Osteogenic differentiation was suppressed following lentiviral-mediated knockdown of Cx43 expression, as judged by decreased levels of Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP), osteocalcin (Bglap), Osterix (Osx), alkaline phosphatase (ALP) activity and the number of calcium nodules in response to osteogenic differentiation stimuli. Knock down of Cx43 reduced the level of phosphorylation of GSK-3beta at Ser9 (p-GSK-3beta), resulting in decreased beta-catenin expression and activation. Furthermore, treatment of Cx43-knockdown cells with lithium chloride (LiCl), a GSK-3beta inhibitor, reduced osteogenic differentiation and decreased GSK-3beta levels, as well as partially rescued levels of both total and activated beta-catenin. These findings indicate that Cx43 positively modulates osteogenic differentiation of BMSCs by up-regulating GSK-3beta/beta-catenin signaling pathways, suggesting a potential role for Cx43 in determining bone mass and bone mineral density by modulating osteogenesis.

Lin FX ，Zheng GZ ，Chang B ，Chen RC ，Zhang QH ，Xie P ，Xie D ，Yu GY ，Hu QX ，Liu DZ ，Du SX ，Li XD ... -  《-》

Fisetin promotes osteoblast differentiation and osteogenesis through GSK-3β phosphorylation at Ser9 and consequent β-catenin activation, inhibiting osteoporosis.

Fisetin is a bioactive flavonol that inhibits osteoclastogenesis and promotes osteoblastogenesis. However, the osteogenic activity of fisetin needs to be comprehensively elucidated. In the present study, we observed that fisetin significantly increased alkaline phosphatase (ALP) activity and bone mineralization in MC3T3-E1 preosteoblasts, accompanied by a significant increase in runt-related transcription factor 2 (RUNX2), ALP, collagen type Ⅰ alpha 1 (Col1α1), osterix (OSX), osteocalcin (OCN), and bone morphogenetic protein 4 (BMP4) expression. Furthermore, fisetin promoted vertebral formation in zebrafish larvae, with the highest fisetin concentration comparable with that observed in β-glycerophosphate treatment. Fisetin also inhibited prednisolone (PDS)-induced anti-osteoblastic genes, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase-6 (ACP6), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK). Fisetin potently mitigated the PDS-induced inhibition of ALP activity and bone mineralization, as well as vertebral resorption in zebrafish larvae. Moreover, we confirmed that fisetin-induced osteogenic effect was activated through phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, consequently releasing β-catenin from the destructive complex to promote its nuclear translocation. β-Catenin inhibition by FH535 and the stabilization of GSK-3β by DOI hydrochloride remarkably inhibited fisetin-induced osteogenic activities, indicating that the GSK-3β/β-catenin signaling pathway plays a vital role in fisetin-induced osteogenesis. Collectively, our findings suggest that fisetin stimulates osteogenic activity and could be used as an effective strategy to prevent bone resorption.

Molagoda IMN ，Kang CH ，Lee MH ，Choi YH ，Lee CM ，Lee S ，Kim GY ... -  《-》

Microtubule actin crosslinking factor 1 promotes osteoblast differentiation by promoting β-catenin/TCF1/Runx2 signaling axis.

Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered.

Hu L ，Su P ，Yin C ，Zhang Y ，Li R ，Yan K ，Chen Z ，Li D ，Zhang G ，Wang L ，Miao Z ，Qian A ，Xian CJ ... -  《-》