SUMMARY Adipocyte differentiation is characterized by an increase in mitochondrial metabolism. However it is not known whether the increase in mitochondrial metabolism is essential for differentiation or a byproduct of the differentiation process. Here, we report that primary human mesenchymal stem cells undergoing differentiation into adipocytes display an early increase in mitochondrial metabolism, biogenesis, and ROS generation. This early increase in mitochondrial metabolism and ROS generation was dependent on mTORC1 signaling. Mitochondrial targeted antioxidants inhibited adipocyte differentiation which was rescued by the addition of exogenous hydrogen peroxide. Genetic manipulation of mitochondrial complex III revealed ROS generated from this complex is required to initiate adipocyte differentiation. These results indicate that mitochondrial metabolism and ROS generation are not simply a consequence of differentiation, but are a causal factor in promoting adipocyte differentiation.

Mammalian cells increase transcription of genes for adaptation to hypoxia through the stabilization of hypoxia-inducible factor 1α (HIF-1α) protein. How cells transduce hypoxic signals to stabilize the HIF-1α protein remains unresolved. We demonstrate that cells deficient in the complex III subunit cytochrome b, which are respiratory incompetent, increase ROS levels and stabilize the HIF-1α protein during hypoxia. RNA interference of the complex III subunit Rieske iron sulfur protein in the cytochrome b–null cells and treatment of wild-type cells with stigmatellin abolished reactive oxygen species (ROS) generation at the Qo site of complex III. These interventions maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Antioxidants maintained hydroxylation of HIF-1α protein and prevented stabilization of HIF-1α protein during hypoxia. Exogenous hydrogen peroxide under normoxia prevented hydroxylation of HIF-1α protein and stabilized HIF-1α protein. These results provide genetic and pharmacologic evidence that the Qo site of complex III is required for the transduction of hypoxic signal by releasing ROS to stabilize the HIF-1α protein.

Background: Although reactive oxygen species (ROS) are integral for TGF-␤ signaling, the source of ROS is not clear. Results: Inhibition of TGF-␤-induced mitochondrial ROS generation attenuates profibrotic gene expression. Conclusion: ROS generated by complex III of the electron transport chain are required for TGF-␤-mediated transcription in normal human lung fibroblasts. Significance: Mitochondrial ROS might be a novel target to prevent TGF-␤-mediated induced fibrosis.

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von HippelLindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span.Human cells have a finite capacity to replicate, and after a critical number of cell divisions, they reach a state in which further division cannot occur, termed replicative senescence (25). Multiple mechanisms are postulated to explain the process of replicative senescence, including telomere attrition and accumulation of oxidative damage to DNA, lipids, and proteins from free radicals (2, 34). The latter hypothesis is known as the free radical theory. A seminal observation to support the free radical theory was made 30 years ago by Packer and Fuehr, who demonstrated that low oxygen concentration (hypoxia) extends replicative life span of cultured primary human fibroblasts (39). The canonical interpretation of this finding was that hypoxia decreases the generation of reactive oxygen species (ROS) due to limiting oxygen levels. This results in diminished oxidative damage to DNA, lipids, and proteins, thus prolonging replicative senescence and extending the life span of cells. However, we have previously reported that the levels of intracellular ROS paradoxically increase under hypoxia (7). Therefore, the increase in replicative life span observed during hypoxia is not consistent with the free radical theory.The source of the increased ROS generated under hypoxia is the mitochondria (5,8,7,24,31). Hypoxia increases ROS via the transfer of electrons from ubisemiquinone to molecular oxygen at the Q o site of complex III of the mitochondrial electron transport chain (3). It has previously been demonstrated that these ROS are both necessary and sufficient to activate the transcription factor hypoxia-inducible factor (HIF) (7,8). HIFs are transcription factors that regulate physiological responses to hypoxia, including placental development, and pathophysiological processes such as cancer (44). HIFs are basic helix-loop-helix transcription factors comprised of the constitutively stable HIF-␤/arylhydrocarbon receptor nuclear translocator subunit and the oxygen-regulated HIF-␣ subunit. Under normal oxygen conditions, HIF-␣ is hydroxylated at two proline residues within the oxygen-dependent degr...

The molecular mechanisms of pulmonary fibrosis are poorly understood. Previous reports indicate that activation of TGF-␤1 is essential for the development of pulmonary fibrosis. Here, we report that the proapoptotic Bcl-2 family member Bid is required for the development of pulmonary fibrosis after the intratracheal instillation of bleomycin. Mice lacking Bid exhibited significantly less pulmonary fibrosis in response to bleomycin compared with WT mice. The attenuation in pulmonary fibrosis was observed despite similar levels of inflammation, lung injury, and active TGF-␤1 in bronchoalveolar lavage fluid 5 days after the administration of bleomycin in mice lacking Bid and in WT controls. Bleomycin induced similar levels cell death in vitro in alveolar epithelial cells isolated from WT and bid ؊/؊ mice. By contrast, alveolar epithelial cells from bid ؊/؊ mice were resistant to TGF-␤1-induced cell death. These results indicate that Bcl-2 family members are critical regulators for the development of pulmonary fibrosis downstream of TGF-␤1 activation.apoptosis ͉ Bcl-2 ͉ TGF-␤