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 ... -  《-》

Polyphenol extract from Tagetes erecta L. flowers stimulates osteogenesis via β-catenin activation.

Osteoporosis, a prevalent bone disorder, results in reduced bone mineral density and mass. With minimal side effects, medicinal plant-based natural remedies are increasingly explored for osteoporosis. However, the osteogenic potential of Tagetes erecta L. flower, traditionally used for cardiovascular and renal diseases, has not yet been studied. This study investigates the osteogenic effects of the polyphenol-enriched extract from T. erecta L. flowers (TE) and its main components on osteoblast differentiation, with an emphasis on anti-osteoporotic activity. The osteogenic activity of TE was assessed in MC3T3-E1 preosteoblast cells, analyzing osteogenic alkaline phosphatase (ALP) activity via a colorimetric assay and mineralization through Alizarin Red S staining over 14 d. Expression levels of osteogenic markers-transcription factor osterix (SP7), runt-related transcription factor 2 (RUNX2), and ALP-were quantified through quantitative reverse transcription-polymerase chain reaction and western blotting. In vivo effects were evaluated using zebrafish larvae for bone formation and anti-osteoporotic properties. Vertebral development was visualized by staining mineralized structures with calcein or Alizarin Red S. Prednisolone (PDS) was administered to zebrafish larvae to model osteoporosis. Furthermore, molecular docking simulations were conducted to assess the binding affinity of TE components to the ATP-binding pocket of glycogen synthase kinase-3β (GSK-3β), and their inhibitory potential on GSK-3β kinase activity was quantified by in vitro kinase assays. Cellular thermal shift assay (CETSA) was performed to monitor direct bindings of TE and its main components to GSK3-3β. TE promoted vertebral and cranial bone formation in zebrafish larvae, elevating key osteogenic genes, such as sp7, runx2a, runx2b, and alpl. Among TE components, kaempferol and patuletin significantly enhanced vertebral formation, while isorhamnetin showed moderate effects. Patulitrin and quercetagetin did not increased vertebral formation. In MC3T3-E1 cells, TE increased ALP activity, mineralization, and the expression of SP7, RUNX2, and ALP. It also induced GSK-3β phosphorylation at serine 9 and promoted β-catenin nuclear translocation. Inhibition of β-catenin signaling reversed TE-induced osteogenic effects. Molecular docking suggested strong GSK-3β binding by TE components, with patuletin showing notable inhibition GSK-3β activity (half-maximal inhibitory concentration = 379.3 ng/mL) and enhancing vertebral formation. CETSA confirmed that TE and its main components, kaempferol and patuletin, degrades GSK-3β. Additionally, TE alleviated PDS-induced osteoporosis in both cellular and zebrafish models. By targeting GSK-3β and activating β-catenin-mediated pathways, TE shows promise as a novel anti-osteoporotic agent. This study highlights the potential of TE for therapeutic use in bone health, warranting further clinical trials to confirm its applicability.

Sanjaya SS ，Park J ，Choi YH ，Park HS ，Sadanaga T ，Jung MJ ，Kim GY ... -  《-》

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 ... -  《-》

Fermented Oyster Extract Promotes Osteoblast Differentiation by Activating the Wnt/β-Catenin Signaling Pathway, Leading to Bone Formation.

Molagoda IMN ，Karunarathne WAHM ，Choi YH ，Park EK ，Jeon YJ ，Lee BJ ，Kang CH ，Kim GY ... -  《Biomolecules》

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 ... -  《-》