REGULATION OF CALRETICULIN GENE-EXPRESSION BY CALCIUM

We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter re gion, The mouse calreticulin gene consists of nine exons and eight int rons, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the cal reticulin promoter was subcloned into a reporter gene plasmid containi ng chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/3T3 cells, Treatment of trans fected cells either with the Ca2+ ionophore A23187, or with the ER Ca2 +-ATPase inhibitor thapsigargin, resulted in a five-to sevenfold incre ase of the expression of chloramphenicol acetyltransferase protein, Tr ansactivation of the calreticulin promoter was also increased by fourf old in NIH/3T3 cells treated with bradykinin, a hormone that induces C a2+ release from the intracellular Ca2+ stores. Analysis of the promot er deletion constructs revealed that A23187- and thapsigargin-responsi ve regions are confined to two regions (-115 to -260 and -685 to -1,76 3) in the calreticulin promoter that contain the CCAAT nucleotide sequ ences, Northern blot analysis of cells treated with A23187, or with th apsigargin, revealed a fivefold increase in calreticulin mRNA levels. Thapsigargin also induced a fourfold increase in calreticulun protein levels. Importantly, we show by nuclear run-on transcription analysis that calreticulin gene transcription is increased in NIH/3T3 cells tre ated with A23187 and thapsigargin in vivo. This increase in gene expre ssion required over 4 h of continuous incubation with the drugs and wa s also sensitive to treatment with cycloheximide, suggesting that it i s dependent on protein synthesis, Changes in the concentration of extr acellular and cytoplasmic Ca2+ did not affect the increased expression of the calreticulin gene. These studies suggest that stress response to the depletion of intracellular Ca2+ stores induces expression of th e calreticulin gene in vitro and in vivo.