Characterization of distinct heat shock- and thapsigargin-induced cytoprotective proteins in FRTL-5 thyroid cells.
Heat shock induces the expression of proteins with molecular weights of 70-72 kd and 90 kd, whereas thapsigargin induces the expression of a glucose-regulated protein 78 kd (GRP-78) in certain cells. In this study we examined the induction and cytoprotective effects of heat shock- and thapsigargin-induced proteins in FRTL-5 rat thyroid cells. New protein synthesis was assessed in [35S]methionine-labeled cells and quantitated densitometrically. The expression of specific stress proteins was identified using Western blots, whereas cytoprotection provided by these proteins was evaluated by trypan blue exclusion. Exposure to heat shock (45 degrees C, 15 minutes) induced the expression of proteins with molecular weights at the range of low 70 kD and low 90 kD that peaked between 2-6 hours and returned to baseline within 24 hours. Treatment of cells with thapsigargin (200 nM, 15 minutes) induced the expression of different molecular weight proteins, most likely GRP-78 and -94, that peaked at 4-6 hours and lasted for 24 hours. Neither the removal of growth factors (thyroid-stimulating hormone and insulin) for 5 days nor the elimination of extracellular Ca2+ with EGTA or clamping of the intracellular Ca2+ with BAPTA for 15 minutes affected expression of the heat shock- and the thapsigargin-induced stress proteins. In contrast, protein kinase C inhibitors H7 and GF109203X abolished the expression of all three groups of stress proteins. Both heat shock- and thapsigargin-inuced proteins completely protected cells from subsequent thermal injury (47 degrees C, 35 minutes). The induction of cytoprotective proteins by heat shock and thapsigargin requires the presence of protein kinase C but is Ca(2+)- and growth factor-independent.
Smallridge
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RC
The Endocrinology Division, Mayo Clinic Jacksonville, Florida 32224, USA. smallridge.robert@mayo.edu
Gist
I D
ID
Tsokos
G C
GC
Kiang
J G
JG
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000008:administration & dosage / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis*; D002460:Cell Line; D019610:Cytoprotection; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature; D007328:Insulin / Q000008:administration & dosage; D007700:Kinetics; D018832:Molecular Chaperones / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology*; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000008:administration & dosage; D014343:Trypan Blue
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D007328:Insulin; D018832:Molecular Chaperones; D019284:Thapsigargin; D013972:Thyrotropin; D011493:Protein Kinase C; D014343:Trypan Blue; D002118:Calcium