Up-regulation of the endoplasmic reticulum molecular chaperone GRP78 during hibernation in thirteen-lined ground squirrels

Abstract

Hibernating mammals endure conditions of low body temperature and oxidative stress that would be highly injurious to humans and most other mammals. Stress conditions frequently trigger the production of molecular chaperones; in the endoplasmic reticulum the glucose-regulated protein-78 (GRP78) helps to minimize protein misfolding under stress. The present study evaluated the GRP78 response in seven organs of hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Transcript levels of grp78, assessed by RT-PCR, were significantly higher (3.5- to 4.1-fold) in brown adipose tissue and brain of hibernating squirrels compared with euthermic control animals but remained low or stable in all other tissues. GRP78 protein content, assessed by Western blotting, was also elevated in brown adipose and brain during hibernation by 1.4-1.6 fold. A 2490 bp cDNA sequence was retrieved that contained the full length open reading frame of ground squirrel grp78 and the translated protein sequence of 654 amino acids shared 98-99% identity with GRP78 from other mammalian sources. Selected specific amino acid substitutions were found in the ground squirrel sequence that may aid GRP78 function under the near 0 degrees C body temperatures of the hibernating state. Electrophoretic mobility shift and supershift assays showed that the activating transcription factor, ATF4, binds to the promoter region of the grp78 gene in ground squirrel brain and may be responsible for grp78 up-regulation during hibernation. Changes in grp78 gene and protein expression appear to aid stress tolerance in two highly oxygen-dependent organs that are critical to whole animal survival during hibernation.