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. 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. Although bone fracture is among the most common tissue injuries requiring emergent care, human bone can recover integrity through scarless regeneration. Unfortunately, osteoporosis and impaired fracture healing are part of a panoply of aging-related maladies that impede the seamless regeneration of new bone with structural and biomechanical integrity. These in turn are prominent comorbidities associated with chronic disorders such as Alzheimer's disease (AD) (1Roos P.M. Osteoporosis in neurodegeneration.J. Trace Elem. Med. Biol. 2014; 28: 418-421Crossref PubMed Scopus (22) Google Scholar, 2Cornelius C. Koverech G. Crupi R. Di Paola R. Koverech A. Lodato F. Scuto M. Salinaro A.T. Cuzzocrea S. Calabrese E.J. Calabrese V. Osteoporosis and alzheimer pathology: Role of cellular stress response and hormetic redox signaling in aging and bone remodeling.Front. Pharmacol. 2014; 5: 120Crossref PubMed Scopus (38) Google Scholar, 3Pan J.X. Tang F. Xiong F. Xiong L. Zeng P. Wang B. Zhao K. Guo H. Shun C. Xia W.F. Mei L. Xiong W.C. 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Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. Discovery of a small molecule promoting mouse and human osteoblast differentiation via activation of p38 MAPK-beta.Cell Chem Biol. 2019; 26: 926-935.e926Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). DIPQUO additionally promotes matrix mineralization of differentiating human mesenchymal cells, ossification of larval zebrafish vertebrae, and osteoblastogenic regenerative processes in recovering adult zebrafish blastema. These properties together mark DIPQUO as a strong lead candidate bone anabolic compound. Our previous study identified activation of p38 mitogen-activated protein kinase (MAPK)-β as a prime mechanistic driver of DIPQUO-mediated biological activity (5Cook B. Rafiq R. Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. 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Biol. 2018; 6: 170Crossref PubMed Scopus Google Scholar). In previous the of p38 MAPK activation between 2 and DIPQUO treatment of cells, to with in cell and activation of osteogenic effector by DIPQUO In addition, studies that osteogenic differentiation and that of structural attenuated activity, an to Therefore, we that DIPQUO impact a effector pathway of p38 with therapeutic in the development of DIPQUO as a lead candidate bone and as a to the study of bone disorders and aging-related In the we to identify by DIPQUO in a to therapeutic to in lead kinase profiling, and functional we identified GSK3-β signaling as an effector of DIPQUO identify or of DIPQUO, we a of in kinase assays, in which a of on of activity in and adipogenesis and for activation or inhibition by These in and GSK3-β inhibition in both that are in with a inhibition which for with inhibitors. In to the to be by protein kinase and and kinase protein and and and GSK3-β as a effector of First, the cellular thermal shift D. R. M. T. C. L. Y. P. in and tissues the cellular thermal shift PubMed Scopus Google Scholar, D. P. cellular thermal shift A novel for in and mechanistic Pharmacol. 2016; PubMed Scopus Google used to the to GSK3-β by DIPQUO with an chemical (5Cook B. Rafiq R. Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. 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Li P. of glycogen synthase inhibition by promotes of embryonic from mouse One. 2012; PubMed Scopus Google Scholar), and this activity for in C2C12 myoblasts In a to C2C12 with the R. G. Y. S. inhibition by an small molecule bone in 2012; PubMed Scopus Google and to stimulate expression of Although the in for and are and to the which be for and for the of to be to and activity for with DIPQUO and GSK3-β inhibitors activity that both and promoted activity to with stimulation the identified in the screen as GSK3-β via functional expression as a In assays, expression of is by treatment of C2C12 with inhibitors to other candidate to of to in activity via of as in and the of of Therefore, in for which in to of activity of of other kinase with protein kinase and and and or kinase to the resulting in the cell In treatment in In addition, with to between of to expression can with other GSK3-β inhibitors to osteogenic differentiation. C2C12 for with DIPQUO, or a or DIPQUO, or a and by for expression and by for expression and activation promoted by the of DIPQUO attenuated by C2C12 with in in activity are as the in and in and in and and in C. differentiating human skeletal muscle for with DIPQUO and and by for expression of associated with osteoblast with treatment expression with and to are as the and in and for and for and for and and for activation of p38 MAPK by treatment with of and DIPQUO with the MAPK are as the and C2C12 either or for with GSK3-β a with DIPQUO or and by for for activity for are to the protein and as the and in and In are from biological alkaline Wnt signaling glycogen synthase kinase DIPQUO and other GSK3-β inhibitors for to in and to osteogenic of the GSK3-β inhibitors or used to C2C12 for cell and activity to or activity the A and These for the of this study and are well for both that stimulation of by either molecule is to treatment of C2C12 with DIPQUO to or activity A and However, of of DIPQUO with or DIPQUO with in and the of activity of either this is that on the Accordingly, of Wnt signaling with the which to GSK3-β activity, the of expression and activity with the effective of DIPQUO to C2C12 cells, human skeletal muscle are mesenchymal progenitors that potential to toward or osteogenic in a on culture M.R. M. S. S. N. of from multipotent muscle PubMed Google Scholar, T. A. A. N. A. H. K. differentiation of human skeletal One. PubMed Scopus Google Scholar). Therefore, as a to and the of DIPQUO to with GSK3-β inhibition in human and cells, promoting osteogenic differentiation. In for with of DIPQUO and in expression of osteoblast marker including and of DIPQUO with GSK3-β inhibitors on a pathway of osteogenic C2C12 with of DIPQUO and and for activation of p38 which we previously is for and is a pathway in skeletal differentiation and patterning (5Cook B. Rafiq R. Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. Discovery of a small molecule promoting mouse and human osteoblast differentiation via activation of p38 MAPK-beta.Cell Chem Biol. 2019; 26: 926-935.e926Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, M.B. Shim J.H. Zou W. Sitara D. Schweitzer M. Hu D. Lotinun S. Sano Y. Baron R. Park J.M. Arthur S. Xie M. Schneider M.D. Zhai B. Gygi S. et al.The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice.J. Clin. Invest. 2010; 120: 2457-2473Crossref PubMed Scopus (278) Google Scholar). that of p38 MAPK is in response to the of DIPQUO and Therefore, biological expression and activity of the osteogenic in activation of osteoblast in human and activation of MAPK signaling through p38 that GSK3-β inhibition DIPQUO and other inhibitors osteogenic in both mouse and human cell culture models. Accordingly, osteogenic are on biological function of GSK3-β signaling. C2C12 with a GSK3-β a to with DIPQUO of activation suppressed through the canonical Wnt pathway to and of GSK3-β from including and R. H. and therapeutic Full Text Full Text PDF PubMed Scopus Google Scholar). a is and in the activation of genes that can cell and differentiation in developmental including P. role of the signaling pathway in formation and maintenance of bone and J. Cell Biol. 2016; PubMed Scopus Google Scholar). DIPQUO as an of GSK3-β activity, we expression and activation of In of C2C12 cells, by of treatment with of additionally with a the used this study as a for promoting osteogenic differentiation Although a accumulation the in a as the of activity, a a factor response used to for with DIPQUO or to controls for and to DIPQUO treatment additionally an with with activity controls identified GSK3-β as a of tau microtubule-associated protein which is in associated with S. D. J.M. D. S. B. S. Alzheimer's of the microtubule-associated protein tau by glycogen synthase in Biol. 4: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.M. of tau by tau to and Cell Sci. PubMed Google Scholar, C. R. S. of Alzheimer's PubMed Scopus Google Scholar). has as a candidate therapeutic including to and effective that tau and impact M. of as a therapeutic on tau 2011; 4: PubMed Scopus Google Scholar). Therefore, we that DIPQUO treatment in cell culture impact tau in as a GSK3-β function of DIPQUO through GSK3-β in this we of In human tau treatment with DIPQUO reduced of a of tau associated with and of in K. and by human tau protein kinase to Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Moreover, the that DIPQUO of tau with the with an for activation (5Cook B. Rafiq R. Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. Discovery of a small molecule promoting mouse and human osteoblast differentiation via activation of p38 MAPK-beta.Cell Chem Biol. 2019; 26: 926-935.e926Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). is on by and on as as other by kinase signaling including T. K. S. and of tau by 2014; PubMed Scopus Google and the MAPK T. J. E. J. B. kinase tau and 2006; PubMed Scopus Google Scholar, Park J.S. tau and oxidative in to of and signaling Chem. PubMed Scopus Google Scholar), p38 A. T. N. T. A of of tau One. 2018; Google Scholar), and kinase E. Y. T. Liu L. Wang L. M. J.A. B. A. in tau through 2007; PubMed Scopus Google Scholar, J. Deng J. W. Z. Alzheimer's in 2012; PubMed Scopus Google Scholar), of which in and other In kinase assays, MAPK identified as or by DIPQUO Although in kinase assays, inhibitors and in with of in expression Accordingly, of suppressed by DIPQUO is by that for and MAPK T. G. K. of of tau and to 2018; PubMed Scopus (38) Google Scholar). Therefore, inhibition of GSK3-β be to a strong However, inhibition of signaling with in with DIPQUO impact tau on either or the that DIPQUO GSK3-β activity of GSK3-β expression with of tau with an of in GSK3-β with controls kinase and functional assays, we identified GSK3-β as a effector of the candidate bone anabolic molecule Although that by DIPQUO, GSK3-β via of expression and activity by treatment with inhibitors and by the of protein Moreover, DIPQUO to with other GSK3-β inhibitors to activity and downstream activation of p38 MAPK signaling in C2C12 myoblasts and stimulate expression of osteoblast genes in is in to previous study (5Cook B. Rafiq R. Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. Discovery of a small molecule promoting mouse and human osteoblast differentiation via activation of p38 MAPK-beta.Cell Chem Biol. 2019; 26: 926-935.e926Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), which used protein as a osteogenic factor and as a and In that we DIPQUO promoted expression of associated with osteoblast and that in parallel to the signaling pathway both activation and Our DIPQUO in with GSK3-β signaling. DIPQUO additionally promoted accumulation of and activity in a inhibition of GSK3-β by DIPQUO suppressed of tau microtubule-associated protein, a of GSK3-β to the of a associated with bone healing and DIPQUO therefore has the potential to as an between and bone disorders to in and in and to as a strong lead candidate molecule to osteogenic by aging-related Although DIPQUO as a GSK3-β is GSK3-β is a inhibition of GSK3-β signaling be a of osteogenic differentiation. There is a of a role for GSK3-β signaling in and cognitive disorders, AD. in mice of GSK3-β in differentiating osteogenic in developmental in skeletogenesis and ossification H. M. B. G. S. G. D.A. F. function in bone skeletal development, and life PubMed Scopus Google Scholar). and inhibitors of GSK3-β in and cognitive disorders additionally on osteogenic differentiation. has to stimulate osteoblast differentiation and bone and A. M.M. on bone PubMed Scopus Google Scholar, L. Y. Wang R. Tang J. Wang T. Liu W. H. Li B. of bone regeneration by pathway via from in Rep. PubMed Scopus Google Scholar), and bone and fracture healing in a of bone R. G. Y. S. inhibition by an small molecule bone in 2012; PubMed Scopus Google Scholar, G. R. A. S. with a fractures bone PubMed Scopus Google Scholar). Therefore, modulation of GSK3-β in a bone biology and cognitive function DIPQUO is of and is from the of inhibitors identified by in of inhibition and with and S. L. of the of a new Dis. 2018; PubMed Scopus Google Scholar). Therefore, is that DIPQUO is a and GSK3-β inhibitors to which in the of the has of mechanism and to in a of disorders for which is effective L. J.M. of by from to signaling 2012; 5: PubMed Scopus Google Scholar). other which through for and which through from development in to and other and chronic in for S. L. of the of a new Dis. 2018; PubMed Scopus Google Scholar). of of GSK3-β as a therapeutic has therefore an on and pharmacological or A of this is in of the wide array of for regulation of GSK3-β GSK3-β is among in and which can be suppressed by including both and protein in and and cell of from E. synthase and 2015; PubMed Scopus Google Scholar). study that in C2C12 of GSK3-β the osteogenic activity of DIPQUO However, we that of GSK3-β is by which DIPQUO activity and that this is an on and modulation of an In we identify affected other GSK3-β and identify osteogenic in the context of inhibition of other kinase Although we modulation of GSK3-β is an important of DIPQUO we do this is or the In DIPQUO potential for therapeutic as a bone anabolic molecule and as a of GSK3-β signaling to of chronic disease associated with bone repair A of identified for potential roles in and adipogenesis with kinase by the on of kinase in and that and with assays, a to on a and a between and is the of kinase DIPQUO in this in a inhibition with a with a inhibition of kinase to a and an kinase inhibitors in kinase for and the development for on a assays, of an to a kinase is by the of a is as the and by an is as of DIPQUO in a and a with the of the used by for on a and the of to assays, a kinase is by an and a with to the kinase of the by from the kinase the and in the of an formation is the DIPQUO in a with of to the the kinase to the a of and a inhibition kinase to the kinase inhibitors in a kinase for by of of and the of to C2C12 mouse mouse fibroblast and human embryonic cell from the and in from Cell and in osteoblast the culture to and fibroblast growth with DIPQUO, or (5Cook B. Rafiq R. Lee H. Banks K.M. El-Debs M. Chiaravalli J. Glickman J.F. Das B.C. Chen S. Evans T. Discovery of a small molecule promoting mouse and human osteoblast differentiation via activation of p38 MAPK-beta.Cell Chem Biol. 2019; 26: 926-935.e926Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), C2C12 with DIPQUO or other inhibitors for activity, or from the DIPQUO and expression human tau and DIPQUO or expression by A GSK3-β an of to and in C2C12 cells, with of activity activity assays, with a with of with culture and with DIPQUO or for Cell and and a to 2 of mouse GSK3-β C2C12 for for with or expression of GSK3-β by with with a from C2C12 or in the and phosphatase from resolved by on or to and to in in and with used as MAPK mouse from Cell and mouse and or and for with a to by C2C12 for with DIPQUO or and cell and such that from in in and in in a a for with in in and on and protein by a and to in the for in a C2C12 for expression treatment with DIPQUO or or as in in a of and the an activity the to to in to a an a and protein by in to and the activity as a of in of protein in from the and from as previously a and the with with controls and to as for be are the for osteogenic of DIPQUO has to as of that of with the of this for of B. C. the and the N. W. Q. Z. S. C. the study and T. E. the study and the and the by the for and by of is the of the and the of the of

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