Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

Abstract

The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 mOsm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53 regulating proteins p38 MAP kinase and the ubiquitin ligase MDM2 were studied as a function of increasing osmolarity. MDM2 protein expression was unchanged at all osmolarities, whereas MDM2 phosphorylation (Ser166) increased at osmolarities up to 537 mOsm and remained constant at higher osmolarities. Phosphorylation of p38 increased at osmolarities up to 687 mOsm which correlated with an increased phosphorylation of p53 (Ser15) and the decreased p53 degradation. Caspase-3 activity increased gradually with hypertonicity and at 737 mOsm both Caspase-3 activity and annexin V binding are high even though p53 expression and activity are low, indicating that initiation of apoptosis under severe hypertonic conditions is not strictly controlled by p53.

Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation

“…It has previously been shown that acute hyperosmotic stress leads to a biphasic stabilization and activation of the transcription factor p53 in mouse NIH3T3 fibroblasts, supposedly through Ser-15 phosphorylation of p53 by the serine/threonine kinase ATM and/or the p38 MAP kinase ( 11 , 27 ). As exposure to Cisplatin likewise elicits cell shrinkage under isotonic conditions, we tested the effect of Cisplatin on p53-mediated signaling.…”

“…Studies by Planells-Cases and coworkers ( 36 ) indicate that Cisplatin induced taurine release and the cytotoxic effects of the chemotherapeutic drugs Staurosporine, Cisplatin and Carboplatin are abolished in LRRC8A and LRRC8D knockout HEK cells ( 36 ). In this context it is noted that osmotic cell shrinkage previously has been shown to induce Caspase-3 activation and apoptosis in EATC via activation of a volume sensory cell death pathway, where p53 is phosphorylated by the protein kinase p38 and hence protected against degradation ( 11 , 27 ). Consequently, it has been suggested that Cisplatin-induced cell death in A2780WT cells via the intrinsic cell death pathway involves activation of the volume sensory cell death pathway due to activation of transporters for ions/organic osmolytes/AVD ( 37 ) and that the reduced Cisplatin sensitivity in A2780CisR partly reflects reduced LRRC8A expression/AVD ( 44 ).…”

Hypertonic Saline Primes Activation of the p53–p21 Signaling Axis in Human Small Airway Epithelial Cells That Prevents Inflammation Induced by Pro-inflammatory Cytokines

“…For the first time, unsupervised analysis of RNaseq data suggested a role for HTS treatment in the modulation of the p53-p21 signaling axis, as we further confirmed through RT-PCR, WB, and fluorescence microscopy. Of note, hyperosmolarity has been very recently associated with increased p53 protein expression in mouse fibroblasts, 28 where p53 protein was stabilized through a post-translational mechanism (phosphorylation at serine 15), decreasing its degradation via MDM2-mediated ubiquitinylation. Here, similar results were observed because RNaseq and proteomics data showed increased transcription and protein expression of genes downstream to p53, above all p21, while only protein levels of p53 (but not its direct mRNA transcription) were significantly up-regulated by HTS alone.…”

Taurine Reduced Epidural Fibrosis in Rat Models after Laminectomy via Downregulating EGR1

“…Given that only if osmolarity change due to taurine administration was more than 737 mOsm, cell viability altered significantly, we did not carried on experiments in equimolar sucrose or similar osmolyte as control [30,31]. According to the results from a CCK-8 assay, fibroblasts treated with up to 100 mM taurine for 48 h exhibited no significant reduction of cell viability (Fig.…”