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

Small molecules are attractive therapeutics to amplify and direct differentiation of stem cells. They also can be used to understand the regulation of their fate by interfering with specific signaling pathways. Mesenchymal stem cells (MSCs) have the potential to proliferate and differentiate into several cell types, including osteoblasts. Activation of canonical Wnt signaling by inhibition of glycogen synthase kinase 3 (GSK-3) has been shown to enhance bone mass, possibly by involving a number of mechanisms ranging from amplification of the mesenchymal stem cell pool to the commitment and differentiation of osteoblasts. Here we have used a highly specific novel inhibitor of GSK-3, AR28, capable of inducing ß-catenin nuclear translocation and enhanced bone mass after 14 days of treatment in BALB/c mice. We have shown a temporally regulated increase in the number of colony-forming units-osteoblast (CFU-O) and -adipocyte (CFU-A) but not colony-forming units-fibroblast (CFU-F) in mice treated for 3 days. However, the number of CFU-O and CFU-A returned to normal levels after 14 days of treatment, and the number of CFU-F was decreased significantly. In contrast, the number of osteoblasts increased significantly only after 14 days of treatment, and this was seen together with a significant decrease in bone marrow adiposity. These data suggest that the increased bone mass is the result of an early temporal wave of amplification of a subpopulation of MSCs with both osteogenic and adipogenic potential, which is driven to osteoblast differentiation at the expense of adipogenesis.

Exposure to mercuric chloride during the induction phase and after the onset of collagen-induced arthritis enhances immune/autoimmune responses and exacerbates the disease in DBA/1 mice.

In susceptible mice, mercuric chloride induces a systemic autoimmune response that is characterized by elevated serum levels of immunoglobulin G1 (IgG1) and immunoglobulin E (IgE), production of anti-nucleolar antibodies (ANolAs) and the formation of renal IgG deposits. We have previously shown that mercury can also enhance immune/autoimmune responses in mouse strains genetically prone to develop spontaneous autoimmune disease. Here, we investigated whether mercury can enhance the severity of murine collagen-induced arthritis (CIA), an inducible (acquired) autoimmune disease that cannot be induced by mercury itself. While mercury administered prior to the induction phase of CIA exerted little, if any, influence, administration of mercury during the induction phase and following onset aggravated the symptoms of this disease and increased the serum levels of IgE and IgG2a antibodies directed against collagen type II (CII). Furthermore, while animals injected with mercury alone exhibited a significant decrease in the ratio of the levels of interferon-gamma (IFN-gamma) to interleukin-4 (IL-4) mRNA in their spleens, this ratio was increased in mice with CIA, with or without administration of mercury. Finally, the production of anti-nuclear antibodies, a hallmark of autoimmunity in response to mercury, was observed in all mice with CIA treated with this heavy metal. Our findings suggest that exposure to mercury during the development of CIA may influence immunological factors in such a way as to synergistically promote disease development.

Expression of the long form of human FLIP by retroviral gene transfer of hemopoietic stem cells exacerbates experimental autoimmune encephalomyelitis.

Subsidence of inflammation and clinical recovery in experimental autoimmune encephalomyelitis (EAE) is postulated to involve apoptosis of inflammatory cells. To test this concept, we examined the effects of overexpressing the long form of human FLICE-inhibitory protein, a potent inhibitor of death receptor-mediated apoptosis, in myelin oligodendrocyte glycoprotein-induced EAE in DBA/1 mice. We found that overexpression of the long form of human FLICE-inhibitory protein by retroviral gene transfer of hemopoietic stem cells led to a clinically more severe EAE in these mice compared with control mice receiving the retroviral vector alone. The exacerbated disease was evident by an enhanced and prolonged inflammatory reaction in the CNS of these animals compared with control mice. The acute phase of EAE was characterized by a massive infiltration of macrophages and granulocytes and a simultaneous increase in TNF-alpha production in the CNS. In the chronic phase of the disease, there was a prolonged inflammatory response in the form of persistent CD4(+) T and B cells in the CNS and a peripheral Th1 cytokine bias caused by elevated levels of IFN-gamma and reduced levels of IL-4 in the spleen. Our findings demonstrate that death receptor-mediated apoptosis can be important in the pathogenesis of EAE and further emphasize the need for effective apoptotic elimination of inflammatory cells to achieve disease remission.