Actin and myosin contribute to mammalian mitochondrial DNA maintenance.

Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.

Proliferative response of different human osteoblast-like cell models to proinflammatory cytokines.

Children with inflammatory bowel disease are known to be at risk of osteopenia. The cause of this osteopenia is likely to be multifactorial, but the inflammatory process with its characteristic overproduction of cytokines has been implicated. To investigate this possible contribution of the disease activity to the development of osteopenia, we performed in vitro assays of the proliferation of osteoblast-like cells of differing origins in response to the inflammatory cytokines tumor necrosis factor-alpha and IL-1/beta. Osteoblast-like cells derived from pediatric bone explants, adherent stromal cells derived from bone marrow (osteoprogenitors), MG-63 osteosarcoma cells, and SV-40 virally transformed osteoprogenitor cells (HCC1) were studied. Tumor necrosis factor-alpha stimulated the proliferation of cells in primary cultures (i.e. from explants and marrow samples) in a linear, dose-dependent manner. In contrast, inhibition of proliferation was observed with the established cell lines (MG-63 and HCC1). IL-1beta stimulated proliferation of all cells apart from the immortalized human bone marrow cell line, HCC1, in which case potent inhibition was observed. We conclude that proinflammatory cytokines are potent regulators of osteoblast-like cell proliferation, and that the responses are specific to cell type. The opposite results obtained with established cell lines compared with the primary cultures suggest that careful consideration should be given to choosing the most suitable cell line for in vitro studies relating to in vivo mechanisms predisposing to osteopenia.