Human stem cell osteoblastogenesis mediated by novel glycogen synthase kinase 3 inhibitors induces bone formation and a unique bone turnover biomarker profile in rats

Abstract

Wnt activation by inhibiting glycogen synthase kinase 3 (GSK-3) causes bone anabolism in rodents making GSK-3 a potential therapeutic target for osteoporotic and osteolytic metastatic bone disease. To understand the wnt pathway related to human disease translation, the ability of 3 potent inhibitors of GSK-3 (AZD2858, AR79, AZ13282107) to 1) drive osteoblast differentiation and mineralisation using human adipose-derived stem cells (hADSC) in vitro; and 2) stimulate rat bone formation in vivo was investigated. Bone anabolism/resorption was determined using clinically relevant serum biomarkers as indicators of bone turnover and bone formation assessed in femurs by histopathology and pQCT/μCT imaging. GSK-3 inhibitors caused β-catenin stabilisation in human and rat mesenchymal stem cells, stimulated hADSC commitment towards osteoblasts and osteogenic mineralisation in vitro. AZD2858 produced time-dependent changes in serum bone turnover biomarkers and increased bone mass over 28 days exposure in rats. After 7 days, AZD2858, AR79 or AZ13282107 exposure increased the bone formation biomarker P1NP, and reduced the resorption biomarker TRAcP-5b, indicating increased bone anabolism and reduced resorption in rats. This biomarker profile was differentiated from anabolic agent PTH1-34 or the anti-resorptive Alendronate-induced changes. Increased bone formation in cortical and cancellous bone as assessed by femur histopathology supported biomarker changes. 14 day AR79 treatment increased bone mineral density and trabecular thickness, and decreased trabecular number and connectivity assessed by pQCT/μCT. GSK-3 inhibition caused hADSC osteoblastogenesis and mineralisation in vitro. Increased femur bone mass associated with changes in bone turnover biomarkers confirmed in vivo bone formation and indicated uncoupling of bone formation and resorption.

Activation of the Wnt Pathway through AR79, a GSK3β Inhibitor, Promotes Prostate Cancer Growth in Soft Tissue and Bone

“…In addition to impacting Wnt signaling, in vitro chemical pharmacology screening studies with AR79 have shown that this compound also inhibits homeodomain-interacting protein kinase 2 (HIPK2) with equipotency compared with GSK3β (11). To determine if AR79 impacted HIPK2 in PCa, we exposed multiple PCa cell lines to AR79 and measured changes in phospho-HIPK2 expression.…”

“…In agreement with the role of Wnts on bone production and inhibition of GSK3β on Wnt signaling, an orally available GSK3β inhibitor was shown to increase bone mass in rats (32). AR79 was developed in order to block GSK3β activity and hence induce bone anabolic activity as demonstrated by the stimulation of osteoblastogenesis with AR79 in vitro and a bone anabolic phenotype in rodents in vivo (11). However, prior to clinical evaluation of its bone anabolic potential, it was deemed important to determine its impact on PCa, which is known to be influenced by Wnt signaling (33, 34).…”

“…Since in vitro pharmacology studies with AR79 indicated that this compound also inhibits homeodomain-interacting protein kinase 2 (HIPK2) with equipotency compared with GSK3β (11) we wanted to determine if off target effects of AR79 on HIPK2 could account for the impact on growth. HIPK2 is known to act as a tumor suppressor through activation of p53 which both (1) mediates induction of apoptosis (41) and (2) inhibits the wnt/β-catenin pathway through miRNA-34 (42).…”

“…AR79 (AstraZeneca, UK) was used at 15 mg/kg and 50 mg/kg orally once daily based on in vivo studies in mice that demonstrated it inhibited GSK-3 activity and increased bone mass in femurs (11). AR 79 was formulated fresh daily in 0.5% hydroxypropylmethyl cellulose (HPMC)(Sigma-H7509) with 0.1% Tween 80 (Sigma-P8074) in water.…”

“…AR79 upregulates β-catenin and bone mineralization in cell assays and produces an increase in bone mass in vivo (11). As AR79 modulates the Wnt pathway, we sought to determine if it could impact the progression of PCa in soft tissue and bone.…”