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The small molecule DIPQUO promotes osteogenic differentiation via inhibition of glycogen synthase kinase 3-beta signaling.

ABSTRACT: Bone fractures are common impact injuries typically resolved through natural processes of osteogenic regeneration and bone remodeling, restoring the biological and mechanical function. However, dysfunctionality in bone healing and repair often arises in the context of aging-related chronic disorders, such as Alzheimer's disease (AD). There is unmet need for effective pharmacological modulators of osteogenic differentiation and an opportunity to probe the complex links between bone biology and cognitive disorders. We previously discovered the small molecule DIPQUO, which promotes osteoblast differentiation and bone mineralization in mouse and human cell culture models, and in zebrafish developmental and regenerative models. Here, we examined the detailed function of this molecule. First, we used kinase profiling, cellular thermal shift assays, and functional studies to identify glycogen synthase kinase 3-beta (GSK3-β) inhibition as a mechanism of DIPQUO action. Treatment of mouse C2C12 myoblasts with DIPQUO promoted alkaline phosphatase expression and activity, which could be enhanced synergistically by treatment with other GSK3-β inhibitors. Suppression of the expression or function of GSK3-β attenuated DIPQUO-dependent osteogenic differentiation. In addition, DIPQUO synergized with GSK3-β inhibitors to stimulate expression of osteoblast genes in human multipotent progenitors. Accordingly, DIPQUO promoted accumulation and activation of β-catenin. Moreover, DIPQUO suppressed activation of tau microtubule-associated protein, an AD-related effector of GSK3-β signaling. Therefore, DIPQUO has potential as both a lead candidate for bone therapeutic development and a pharmacological modulator of GSK3-β signaling in cell culture and animal models of disorders including AD.

SUBMITTER: Cook B

PROVIDER: S-EPMC8138761 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundChronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β₁-induced myofibroblast differentiation is currently largely unknown.PurposeTo determine the contribution of GSK-3 signalling in TGF-β₁-induced myofibroblast differentiation.Experimental approachWe used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively.ResultsStimulation of MRC5 and primary human lung fibroblasts with TGF-β₁ resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β₁-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β₁-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling.Conclusion and implicationWe demonstrate that GSK-3 signalling regulates TGF-β₁-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases.

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The small molecule DIPQUO promotes osteogenic differentiation via inhibition of glycogen synthase kinase 3-beta signaling.

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