11-Deoxy,16,16-Dimethyl Prostaglandin E₂ Induces Specific Proteins in Association with Its Ability to Protect Against Oxidative Stress

Abstract

Prostaglandins (PGs) act locally to maintain cellular homeostasis and stimulate stress response signaling pathways. These cellular effects are diverse and are tissue-dependent. PGE(2), and the synthetic analogue, 11-deoxy,16,16-dimethyl PGE(2) (DDM-PGE(2)), protect renal proximal tubular epithelial (LLC-PK1) cells against cellular injury induced by the potent nephrotoxic and nephrocarcinogenic metabolite of hydroquinone, 2,3,5-tris-(glutathion-S-yl)hydroquinone. Although this cytoprotective response (in LLC-PK1 cells) is mediated through a thromboxane or thromboxane-like receptor coupled to AP-1 signaling pathways, the mechanism of cytoprotection is unknown. In this study, we utilized HPLC-electrospray ionization tandem mass spectrometric (ESI MS/MS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometric (MALDI TOF) analysis of proteins isolated from DDM-PGE(2)-stimulated LLC-PK1 cells to identify candidate cytoprotective proteins. DDM-PGE(2) selectively stimulated the synthesis of several proteins in LLC-PK1 cells. Peptide sequencing by ESI-MS/MS of in-gel tryptic protein digests revealed the identity of eight proteins: endothelial actin binding protein, myosin, elongation factor 2 (EF-2), elongation factor 1alpha-1 (EF-1alpha), heat shock protein 90beta (HSP90beta), glucose-regulated protein 78 (GRP 78), membrane-organizing extension spike protein, and actin. Both ESI-MS/MS and MALDI-MS analysis resulted in the same protein identification. Western analysis confirmed the temporal induction of the majority of these proteins, including EF-2, EF-1alpha, HSP90beta, GRP78, and actin. The collective expression of these proteins suggests that DDM-PGE(2)-mediated cytoprotection may involve alterations in cytoskeletal organization and/or stimulation of an endoplasmic reticulum (ER) stress response. The present studies provide insights into potential downstream targets of PG signaling.

N-glycosylation of ovomucin from hen egg white

“…Loading volume was 20 μl and a molecular weight standard (Bio-Rad) was loaded to a separate well. SDS-PAGE was performed [27] In-gel trypsin digestion Excised gel pieces containing the protein of interest with and without PNGase F treatments (destaining was carried out as for the PNGase F treated sample), were digested following a modified protocol [28] based on the method of Shevchenko et al [29]. Briefly, the proteins in the gel pieces were reduced with 10 mM DTT, alkylated with 50 mM iodoacetamide, washed with 100 mM ammonium bicarbonate and acetonitrile, and dried in a SpeedVac.…”

Grp78 is essential for 11-deoxy-16,16-dimethyl PGE2-mediated cytoprotection in renal epithelial cells

“…These proteins included elongation factor 2, heat shock protein 90␤, elongation factor 1␣, glucose-regulated protein 78 (Grp78), and actin. The known functions of these proteins suggest that cytoprotection may involve changes in the cytoskeleton, maintenance of cell polarity, activation of the unfolded protein response, or possibly increases in proximal tubule cell regenerative capacity (41,50).…”

“…Among the DDM-PGE 2 proteins identified, Grp78 was of particular interest since induction of Grp78 and other endoplasmic reticulum (ER) molecular chaperones confers protection to epithelial cells against various insults (25,50). In addition, overexpression of Grp78 prevents ER stress-mediated transcriptional activation of the ER stress response and protects cells against death caused by calcium depletion from the ER (8).…”

Proteomics in renal research

“…1D SDS-PAGE followed by ESI/ MS/MS or matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS was utilized to identify downstream effectors of prostaglandin signaling in cultured porcine renal proximal tubule cells (LLC-PK 1 ) labeled with [ and treated with DDM-PGE 2 (88,117). Peptide sequencing of upregulated proteins led to the identification of glucose-regulated protein/BiP (GRP78), an endoplasmic reticular protein related to the heat shock protein 70 class of proteins.…”