Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to ␣-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates ␣-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long‐term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2‐associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300‐fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen‐induced stress response was dependent upon p53 since siRNA‐mediated knockdown of p53 in wild‐type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long‐term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen‐induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long‐term expansion in vitro. STEM CELLS 2012;30:975–987

Background:Little is known about the role of JNK mitochondrial signaling in cardiomyocyte cell death. Results: Global and mitochondrial inhibition of JNK protects against I/R injury thus reducing infarct volume. Conclusion: Blocking JNK mitochondrial translocation or JNK inhibition may be an effective treatment for I/R-induced cardiomyocyte death. Significance: These findings suggest a new molecular target for JNK inhibition.