Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.
Dufd1 (DUF729 domain containing 1) is related to Mtfr1 (mitochondrial fission regulator 1), a gene involved in the regulation of antioxidant activity in the mouse testis. The present study was undertaken to better understand their role in regulating mitochondrial architecture and function in the mouse. We show that Dufd1 is expressed as a 2 kb mRNA and has a more specific tissue pattern compared to Mtfr1, with highest level of expression in testes, lower level in spleen, and negligible levels in other organs and/or tissues. In the male gonad, Dufd1 mRNA expression increases during postnatal development, similarly to Mtfr1. In situ hybridization and real-time PCR analyses show that Dufd1 is expressed in the seminiferous tubules by middle-late pachytene spermatocytes and spermatids. In transfected cells, the Dufd1-tagged protein is located in mitochondria, associated with the tips of mitochondrial tubules and to tubules constrictions, and induces mitochondrial fission although with a lesser efficiency than Mtfr1. We also found that both endogenous Dufd1 and Mtfr1 proteins are associated with membrane-enriched subcellular fractions, including mitochondria. Inhibition of Mtfr1 and/or Dufd1 expression, in a testicular germ cells line, severely impairs O(2) consumption and indicates that both genes are required for mitochondrial respiration. Accordingly, analysis of testes mitochondria from Mtfr1-deficient mice reveals severely reduced O(2) consumption and ATP synthesis compared to wt animals. These data show that, in murine testis, Dufd1 and Mtfr1 have redundant functions related to mitochondrial physiology and represent genes with a potential role in testicular function.
Stalled cell division in precursor bone cells and reduced osteoblast function are considered responsible for the microgravity-induced bone loss observed during spaceflight. However, underlying molecular mechanisms remain unraveled. Having overcome technological difficulties associated with flying cells in a space mission, we present the first report on the behavior of the potentially osteogenic murine bone marrow stromal cells (BMSC) in a 3D culture system, flown inside the KUBIK aboard space mission ISS 12S (Soyuz TMA-8 + Increment 13) from March 30 to April 8, 2006 (experiment "Stroma-2"). Flight 1g control cultures were performed in a centrifuge located within the payload. Ground controls were maintained on Earth in another KUBIK payload and in Petri dishes. Half of the cultures were stimulated with osteo-inductive medium. Differences in total RNA extracted suggested that cell proliferation was inhibited in flight samples. Affymetrix technology revealed that 1,599 genes changed expression after spaceflight exposure. A decreased expression of cell-cycle genes confirmed the inhibition of cell proliferation in space. Unexpectedly, most of the modulated expression was found in genes related to various processes of neural development, neuron morphogenesis, transmission of nerve impulse and synapse, raising the question on the lineage restriction in BMSC.
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