Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage.

Multicellular organisms balance oxygen delivery and toxicity by having oxygen pass through several barriers before cellular delivery. In human cell culture, these physiologic barriers are removed, exposing cells to higher oxygen levels. Human cells cultured in ambient air may appear normal, but this is difficult to assess without a comparison at physiologic oxygen. Here, we examined the effects of culturing human cells throughout the spectrum of oxygen availability on oxidative damage to macromolecules, viability, proliferation, the antioxidant and DNA damage responses, metabolism, and mitochondrial fusion and morphology. We surveyed 4 human cell lines cultured for 3 d at 7 oxygen conditions between 1 and 21% O2. We show that oxygen levels and cellular benefit are not inversely proportional, but the benefit peaks within the physioxic range. Normoxic cells are in a perpetual state of responding to damaged macromolecules and mitochondrial networks relative to physioxic cells, which could compromise an investigation. These data contribute to the concept of an optimal oxygen availability for cell culture in the physioxic range where the oxygen is not too high to reduce oxidative damage, and not too low for efficient oxidative metabolism, but just right: the Goldiloxygen zone.-Timpano, S., Guild, B. D., Specker, E. J., Melanson, G., Medeiros, P. J., Sproul, S. L. J., Uniacke, J. Physioxic human cell culture improves viability, metabolism, and mitochondrial morphology while reducing DNA damage.

Signaling adaptor ShcD suppresses extracellular signal-regulated kinase (Erk) phosphorylation distal to the Ret and Trk neurotrophic receptors.

Proteins of the Src homology and collagen (Shc) family are typically involved in signal transduction events involving Ras/MAPK and PI3K/Akt pathways. In the nervous system, they function proximal to the neurotrophic factors that regulate cell survival, differentiation, and neuron-specific characteristics. The least characterized homolog, ShcD, is robustly expressed in the developing and mature nervous system, but its contributions to neural cell circuitry are largely uncharted. We now report that ShcD binds to active Ret, TrkA, and TrkB neurotrophic factor receptors predominantly via its phosphotyrosine-binding (PTB) domain. However, in contrast to the conventional Shc adaptors, ShcD suppresses distal phosphorylation of the Erk MAPK. Accordingly, genetic knock-out of mouse ShcD enhances Erk phosphorylation in the brain. In cultured cells, this capacity is tightly aligned to phosphorylation of ShcD CH1 region tyrosine motifs, which serve as docking platforms for signal transducers, such as Grb2. Erk suppression is relieved through independent mutagenesis of the PTB domain and the CH1 tyrosine residues, and successive substitution of these tyrosines breaks the interaction between ShcD and Grb2, thereby promoting TrkB-Grb2 association. Erk phosphorylation can also be restored in the presence of wild type ShcD through Grb2 overexpression. Conversely, mutation of the ShcD SH2 domain results in enhanced repression of Erk. Although the SH2 domain is a less common binding interface in Shc proteins, we demonstrate that it associates with the Ptpn11 (Shp2) phosphatase, which in turn regulates ShcD tyrosine phosphorylation. We therefore propose a model whereby ShcD competes with neurotrophic receptors for Grb2 binding and opposes activation of the MAPK cascade.

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions.

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.