 at the membrane. The addition of lithium to cells at the log phase of hMSC growth reduced the rate of proliferation in a dose-dependent manner and resulted in an overall increase in the level of cytosolic β-catenin.
The osteogenic potential of MSCs in the presence or absence of Dkk-1 was investigated. MSCs were differentiated to an osteogenic phenotype using two standard protocols; one mediated by dexamethasone and one mediated by bone morphogenic protein 2 (BMP2). In the presence of Dkk-1 (100 ng mL −1 ), MSCs underwent extensive apoptosis in both of the osteo-inductive conditions tested (FIG. 41). As a result of the apoptosis, the overall net activity of the osteogenic marker, alkaline phosphatase (ALP) per culture was significantly reduced (FIG. 42). The level of ALP production per surviving cell remained constant in the case of Dex-induced mineralization (FIG. 43), but varied with donor in the case of BMP2 induction. For the two donors tested, Dkk-1 reduced the level of ALP production per surviving cell and in one donor, the surviving cells appeared to compensate for the loss of a major fraction of the monolayer by up-regulating ALP production (FIG. 44). In any event, the overall effect of Dkk-1 treatment was to reduce the net production of alkaline phosphatase.
The effect of LiCl on osteogenesis in an osteogenic differentiation assay was assessed. Lithium has been known to inhibit GSK3β, a critical enzyme in the canonical Wnt pathway. Not wishing to be bound to any particular theory, inhibition of GSK3β has the opposite effect of Dkk-1 in present system. To investigate the effect of lithium directly on osteogenesis, the hMSCs were grown to confluency and treated for up to 30 days with an osteogenic medium containing a 100 fold lower than standard concentration of dexamethasone and 10 mM LiCl or 10 mM KCl. Lower levels of dexamethasone were used to improve the detection of differences in osteogenesis induced by LiCl. Under these conditions, the hMSC monolayer detached from the plastic and spontaneously curled into a roughly spherical cellular aggregate. In the presence of LiCl, the aggregate was formed after about 7 days of treatment whereas in the presence of KCl, the effect was seen after about 12 days. The aggregates were fixed, paraffin embedded, sectioned and stained for calcified deposits (Alizarin Red S). On sectioning and staining of the sections with Alizarin Red S, it was apparent that mineralization was much more evident in the LiCl treated aggregates than in the control with dense patches of mineral detected throughout the sections of the LiCl-treated cells (FIG. 45). Quantification of mineral by colorimetric measurement of Alizarin Red S staining demonstrated that LiCl treated cultures produced mineralized matrix more rapidly than the controls (FIG. 46).
Analysis of gene expression by the differentiating hMSCs revealed that transcripts commonly associated with osteogenesis increased over time in osteogenic medium both in the presence and absence of LiCl but there was a striking up-regulation of alkaline phosphatase transcription. Alkaline phosphatase transcription was maximal after 10 days in lithium treated cultured compared with 30 days in the controls (FIG. 47).
GSK3β Inhibitors for the Treatment of Osteolytic Lesions in Multiple Myeloma.
Multiple myeloma (MM) is a uniformly fatal malignancy of antibody-secreting plasma cells (PCs). In about 80% of patients, painful osteolytic lesions accompany the malignancy. Activation of the canonical Wnt pathway leads to the inhibition of glycogen synthetase kinase-3 (GSK3β), resulting in an increase in cytosolic β-catenin that regulates gene expression and drives hMSCs to an osteogenic phenotype. It has been demonstrated that the canonical Wnt signaling antagonists, Dkk-1 and Frizzled related protein B (FrzB) contribute to the inhibition of Wnt-mediated hMSC differentiation. MM cells express high levels of these inhibitors in patients and also upregulate the formation of osteoclasts. Up-regulation of osteoclast activity coupled with the inhibition of osteogenesis is therefore a cause of osteolytic lesion formation.
It has been demonstrated that when MM cells are co-cultured with bone marrow hMSCs, MM cells up-regulate their expression of Dkk-1, while initial studies indicate a decrease in FrzB. Furthermore, IL-6 is produced at high levels by hMSCs, and induces rapid MM proliferation, as well as being a potent inducer of osteoclast activity. These observations demonstrate a cycle of osteogenic inhibition by Dkk-1/FrzB and perpetuation of the plasma cell malignancy through IL-6 induced proliferation in the cell lines tested.
Therapy directed at the inhibition of GSK3β may provide an agent to promote osteoblast differentiation. Inhibitors of GSK3β that mimic positive Wnt signaling was tested to assess the effects of GSK3β inhibitors to promote osteoblast differentiation. With the differentiation of into osteoblast using a GSK3β inhibitor, GSK3β inhibitors can be used to prevent osteolytic lesions that are the major cause of morbidity in MM.
Multiple myeloma patients have high levels of circulating Dkk-1 due to the high levels of expression by the malignant plasma cells. The plasma cells and mesenchymal cells also express interleukin 6 that exacerbates the malignancy and activated osteoclasts which break down bone. Dkk-1 prevents repair of the broken down bone by killing MSCs (osteoblast precursor cells) and also by reducing their ability to differentiate. Osteoblast differentiation is driven by a signal transduction pathway mediated by the Wnt class of secreted ligands; a major step in this signal transduction pathway is the inhibition of glycogen synthetase kinase 3β (GSK3β). Dkk-1 inhibits this pathway at the membrane and prevents inhibition of GSK3β and thus Dkk-1 inhibits osteogeneic differentiation. Two classes of molecules can prevent the effect of Dkk-1: (1) molecules that compete for Dkk-1 binding at the level of the membrane (peptide analogs of Dkk-1) or (2) molecules that inhibit GSK3β directly (e.g. lithium carbonate, lithium chloride, indirubin oxime classes of molecules).
It has been demonstrated that Dkk-1 inhibits osteogenesis of MSCs at two levels; apoptosis and direct inhibition of differentiation. It has also been demonstrated that the GSK3β inhibitor, lithium chloride, and a putative antagonist of Dkk-1 (peptide A) enhances osteogenic differentiation. Therefore either class of molecule can be used in treating osteolytic lesion formation in multiple myeloma.
Human mesenchymal stem cells (hMSCs) from bone marrow stromal differentiate into mesenchymal tissue lineages and are good candidates for cellular therapies. Given that loss-of-function mutations in the Wnt receptor LRP5 result in a low bone mass phenotype (Gong et al., 2001 Cell 107:513-523), and a gain-of-function mutation in the same receptor results in high bone mass (Boyden et al., 2002 N. Engl. J. Med. 346:1513-1521), it has been demonstrated that Wnt signaling is required for osteoblastic differentiation of hMSCs. Furthermore, the Wnt antagonist Dkk-1 inhibits this differentiation and predisposes the cells towards cell cycle entry.
A functional Wnt signaling pathway exists in hMSCs, which express Lrp6, Wnt5a, and β-catenin. While not wishing to be bound to any particular theroy, the data disclosed elsewhere herein depicts an ex vivo model of osteogenesis, in which BMP2 induces expression of Wnt ligands that signals in an autocrine fashion to promote osteogenesis. Accordingly, alkaline phosphatase levels are highly upregulated in cells treated with BMP2, compared to controls, whereas no increase is seen when the treatment includes Dkk-1.
Dkk-1 has been implicated in forming osteolytic bone lesions in multiple myeloma (MM), thus secreted Wnt inhibitors and pharmacological Wnt activators can be used to examine the role of Wnt signaling in the pathology of MM. Treatment with 6-bromoindirubin oxime, PS-341, or lithium chloride maintains alkaline phosphatase expression in the presence of Dkk-1. Therefore, this model of osteogenesis provides an experimentally accessible means to screen for novel drugs which activate Wnt signaling in hMSCs and therefore represents important treatments for bone disease and.<?php
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