Orally Bioavailable GSK-3α/β Dual Inhibitor Increases Markers of Cellular Differentiation In Vitro and Bone Mass In Vivo

Abstract

GSK-3, a component of the canonical Wnt signaling pathway, is implicated in regulation of bone mass. The effect of a small molecule GSK-3 inhibitor was evaluated in pre-osteoblasts and in osteopenic rats. GSK-3 inhibitor induced osteoblast differentiation in vitro and increased markers of bone formation in vitro and in vivo with concomitant increased bone mass and strength in rats.Introduction: Inactivation of glycogen synthase kinase -3 (GSK-3) leads to stabilization, accumulation, and translocation of ␤-catenin into the nucleus to activate downstream Wnt target genes. To examine whether GSK-3 directly regulates bone formation and mass we evaluated the effect of 603281-31-8, a small molecule GSK-3 ␣/␤ dual inhibitor in preosteoblastic cells and in osteopenic rats. Materials and Methods: Murine mesenchymal C3H10T1/2 cells were treated with GSK-3 inhibitor (603281-31-8) and assayed for ␤-catenin levels, activity of Wnt-responsive promoter, expression of mRNA for bone formation, and adipogenic markers and alkaline phosphatase activity. In vivo, 6-month-old rats were ovariectomized (OVX), allowed to lose bone for 1 month, and treated with GSK-3 inhibitor at 3 mg/kg/day orally for 60 days. At the end of treatment, BMD was measured by DXA, bone formation rate by histomorphometry, vertebral strength (failure in compression), and the expression levels of osteoblast-related genes by real-time PCR. Results: Treatment of C3H10T1/2 cells with the GSK-3 inhibitor increased the levels of ␤-catenin accompanied by activation of Wnt-responsive TBE 6 -luciferase reporter gene. This was associated with an increased expression of mRNA for bone sialoprotein (1.4-fold), collagen ␣ 1 (I) (∼2-fold), osteocalcin (1.2-fold), collagen ␣ 1 (V) (1.5-fold), alkaline phosphatase (∼160-fold), and runx2 (1.6-fold), markers of the osteoblast phenotype and bone formation activity. Alkaline phosphatase mRNA expression paralleled alkaline phosphatase activity. The mRNA levels of collagens ␣ 1 (I), ␣ 1 (V), biglycan, osteonectin, and runx-2 increased on treatment with the GSK-3 inhibitor in rat femur compared with the OVX control. DXA analyses revealed significant increases in BMC and BMD in cancellous and cortical bone of OVX rats treated with GSK-3 inhibitor. This was associated with increased strength (peak load, energy, and stiffness) assessed by lumbar vertebra load to failure in compression. Histomorphometric analyses showed that 603281-31-8 robustly increased bone formation but did not exclude a small effect on osteoclasts (resorption). Conclusions: An orally active, small molecule GSK-3 inhibitor induced osteoblast differentiation and increased markers of bone formation in vitro, and increased markers of bone formation, bone mass, and strength in vivo, consistent with a role for the canonical Wnt pathway in osteogenesis.

Glycogen synthase kinase-3α/β inhibition promotes in vivo amplification of endogenous mesenchymal progenitors with osteogenic and adipogenic potential and their differentiation to the osteogenic lineage

“…Elucidating how Wnts act as instructive cues for the recruitment, maintenance, and maturation of MSCs and their differentiated progenies is of primary interest given the potential use of these cells in regenerative medicine. Prior studies have implicated canonical Wnt signaling in the regulation of bone metabolism at several levels, in amplifying the undifferentiated MSC pool, (7,8) commitment of undifferentiated MSCs to the osteoblast lineage, (13,14) and stimulation of their differentiation. (14) However, differences in cell preparations, use of models that did not take into account the cellular context, or use of nonphysiologic loss-and gain-of-function approaches are some of the reasons for the emergence of contrasting data.…”

“…Prior studies have implicated canonical Wnt signaling in the regulation of bone metabolism at several levels, in amplifying the undifferentiated MSC pool, (7,8) commitment of undifferentiated MSCs to the osteoblast lineage, (13,14) and stimulation of their differentiation. (14) However, differences in cell preparations, use of models that did not take into account the cellular context, or use of nonphysiologic loss-and gain-of-function approaches are some of the reasons for the emergence of contrasting data. Moreover, the presence of large numbers of Wnts, Wnt receptors, coreceptors, and soluble inhibitors with loosely defined actions has made it difficult to define the function of Wnt signaling in (E) A representative example of a horizontal section of tibia after X-ray mCT scanning of a BALB/c mouse treated for 14 days with vehicle or AR28.…”

“…While there is agreement that inhibition of GSK-3 leads to enhanced bone mass, (14,16) the mechanism by which this occurs is controversial-whether this is due to enhanced drive to differentiation to osteoblasts and/or inhibition of osteoclast differentiation. We have shown that inhibition of GSK-3 blocks preadipocyte differentiation and enhances osteoblast differentiation in vivo despite progenitors with both potentials being amplified.…”

“…(12) Moreover, Wnt/b-catenin signaling was reported to shift MSC cell fate toward osteoblastogenesis at the expense of adipogenesis (13) and increases bone mass in osteopenic rats. (14) There are a number of possible reasons behind the controversial findings. In vitro studies have used MSC-like cell lines (13)(14)(15) or primary human MSCs isolated and expanded in vitro under different culture conditions, (9,10,12) mostly in the absence of environmental signals.…”

“…(14) There are a number of possible reasons behind the controversial findings. In vitro studies have used MSC-like cell lines (13)(14)(15) or primary human MSCs isolated and expanded in vitro under different culture conditions, (9,10,12) mostly in the absence of environmental signals. In vivo studies relied on lossand gain-of-function approaches, (13,16) leading to the presence or absence of factors well beyond physiologic levels, or relied on the use of nonselective GSK-3 inhibitors such as lithium chloride.…”