11-Deoxy,16,16-dimethyl prostaglandin E2 induces specific proteins in association with its ability to protect against oxidative stress.
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.
Towndrow
Kelly M
KM
Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, USA.
Jia
Zhe
Z
Lo
Herng-Hsiang
HH
Person
Maria D
MD
Monks
Terrence J
TJ
Lau
Serrine S
SS
D015064:16,16-Dimethylprostaglandin E2 / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D030562:Databases, Protein; D000091342:Endoplasmic Reticulum Chaperone BiP; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018374:LLC-PK1 Cells; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D020648:Peptide Elongation Factor 1 / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D020648:Peptide Elongation Factor 1; D010446:Peptide Fragments; D011506:Proteins; D014357:Trypsin; D015064:16,16-Dimethylprostaglandin E2