Sos1 disruption impairs cellular proliferation and viability through an increase in mitochondrial oxidative stress in primary MEFs

Abstract

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs. Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.

Differential Role of the RasGEFs Sos1 and Sos2 in Mouse Skin Homeostasis and Carcinogenesis

“…1a, left panel). Western blot (WB) analysis confirmed the disappearance of the Sos1 and Sos2 proteins in skin of mice treated under these experimental conditions and confirmed our previous observations in MEFs (18), indicating that there are no compensatory changes of either Sos1 or Sos2 protein expression when the other form is absent (Fig. 1a, right panel).…”

“…Consistently, our analyses under physiological conditions first documented the functional relevance of Sos1 (predominant) and Sos2 for normal skin cell proliferation, and our skin wound repair studies further demonstrated a reduction of cell proliferation and migration of fibroblasts to the wounded area in Sos1/2-DKO mice. Other reports have also demonstrated a significant blockade of migration in both Ras-and Sos-depleted MEFs after wounding (18,26).…”

“…In this regard, upon Ras elimination, keratinocytes cease proliferation and enter into senescence without any signs of apoptosis induction (19). The key role of Sos1 in control of cell proliferation has also been widely documented in other cell lineages, tissues, and organs (5,17,18,29,30).…”

“…Interestingly, Sos1/2 double-knockout (Sos1/2-DKO) animals died precociously, whereas single Sos1-or Sos2-deficient mice were viable, suggesting functional redundancy between Sos proteins in organism homeostasis and survival (17). We also demonstrated that Sos1-null mouse embryonic fibroblasts (MEFs), but not Sos2-null MEFs, showed decreased cell proliferation and migration (18). However, the specific role of Sos1/2 proteins in other different cell lineages and tissues, under physiological and/or pathological conditions, has not been yet examined.…”

Frontline Science: TNF-α and GM-CSF1 priming augments the role of SOS1/2 in driving activation of Ras, PI3K-γ, and neutrophil proinflammatory responses

“…The low recovery neutrophils number has severely restricted the experiments we could conduct; thus, direct assays of Ras activation were impractical. It has been shown previously, however, that in fibroblasts isolated from the same mouse strains, loss of SOS1/2 resulted in an almost complete blockade of EGF or PDGF‐stimulated Ras activation . In unprimed cells, fMLP‐stimulated ERK activation (Fig.…”