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Regulation of calreticulin gene expression by calcium.

Waser M, Mesaeli N, Spencer C, Michalak M - J. Cell Biol. (1997)

Bottom Line: Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein.Changes in the concentration of extracellular 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 the calreticulin gene in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Group in Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.

ABSTRACT
We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (-115 to -260 and -685 to -1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, 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 treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular 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 the calreticulin gene in vitro and in vivo.

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Activation of the calreticulin promoter by A23187 and  thapsigargin treatment. (A) NIH/3T3 were stably transfected  with either pCC1 or pLC1 and pSVLβ-galactosidase as described  in Materials and Methods. Cells were incubated for 16 h with 7  μM A23187, 100 nM thapsigargin, or an appropriate volume of  DMSO (control cells), as described in Materials and Methods.  (B) Different fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT  expression vector) as described in Materials and Methods. Vectors pCC0, pCC1, pCC2, pCC3, pCC4, and pCC5 contained the  2,142-, 1,763-, 685-, 415-, 260-, and 115-bp DNA fragments of the  calreticulin promoter, respectively. NIH/3T3 were transiently  transfected with different pCC vectors and pSVLβ-galactosidase  as described in Materials and Methods. Cells were incubated for  16 h with 7 μM A23187 (open bars) or 300 nM thapsigargin (filled  bars) and lysed, and reporter enzyme assays were carried out. n/d,  not detectable. Data shown are means ± SD of four separate experiments performed in triplicate.
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Figure 3: Activation of the calreticulin promoter by A23187 and thapsigargin treatment. (A) NIH/3T3 were stably transfected with either pCC1 or pLC1 and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or an appropriate volume of DMSO (control cells), as described in Materials and Methods. (B) Different fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT expression vector) as described in Materials and Methods. Vectors pCC0, pCC1, pCC2, pCC3, pCC4, and pCC5 contained the 2,142-, 1,763-, 685-, 415-, 260-, and 115-bp DNA fragments of the calreticulin promoter, respectively. NIH/3T3 were transiently transfected with different pCC vectors and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187 (open bars) or 300 nM thapsigargin (filled bars) and lysed, and reporter enzyme assays were carried out. n/d, not detectable. Data shown are means ± SD of four separate experiments performed in triplicate.

Mentions: Fig. 3 shows that NIH/3T3 cells stably transfected with pCC1 and pSVβ-galactosidase produced five- to sevenfold more CAT protein after treatment for 16 h with 7 μM A23187 or 100 nM thapsigargin. NIH/3T3 cells were also stably transfected with pLC1 and pSVβ-galactosidase. When these cells were treated with 7 μM A23187 or 100 nM thapsigargin, a threefold increase in luciferase activity was observed (Fig. 3 A). Similar results were obtained with mouse fibroblasts that were transiently transfected with pCC1 or pLC1. The reason for this difference between the CAT and luciferase reporter systems is not clear, but it may be related to an inhibitory effect of Ca2+ on luciferase activity (unpublished observations).


Regulation of calreticulin gene expression by calcium.

Waser M, Mesaeli N, Spencer C, Michalak M - J. Cell Biol. (1997)

Activation of the calreticulin promoter by A23187 and  thapsigargin treatment. (A) NIH/3T3 were stably transfected  with either pCC1 or pLC1 and pSVLβ-galactosidase as described  in Materials and Methods. Cells were incubated for 16 h with 7  μM A23187, 100 nM thapsigargin, or an appropriate volume of  DMSO (control cells), as described in Materials and Methods.  (B) Different fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT  expression vector) as described in Materials and Methods. Vectors pCC0, pCC1, pCC2, pCC3, pCC4, and pCC5 contained the  2,142-, 1,763-, 685-, 415-, 260-, and 115-bp DNA fragments of the  calreticulin promoter, respectively. NIH/3T3 were transiently  transfected with different pCC vectors and pSVLβ-galactosidase  as described in Materials and Methods. Cells were incubated for  16 h with 7 μM A23187 (open bars) or 300 nM thapsigargin (filled  bars) and lysed, and reporter enzyme assays were carried out. n/d,  not detectable. Data shown are means ± SD of four separate experiments performed in triplicate.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141645&req=5

Figure 3: Activation of the calreticulin promoter by A23187 and thapsigargin treatment. (A) NIH/3T3 were stably transfected with either pCC1 or pLC1 and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or an appropriate volume of DMSO (control cells), as described in Materials and Methods. (B) Different fragments of the calreticulin promoter were subcloned into the promoterless reporter plasmids pCATbasic (CAT expression vector) as described in Materials and Methods. Vectors pCC0, pCC1, pCC2, pCC3, pCC4, and pCC5 contained the 2,142-, 1,763-, 685-, 415-, 260-, and 115-bp DNA fragments of the calreticulin promoter, respectively. NIH/3T3 were transiently transfected with different pCC vectors and pSVLβ-galactosidase as described in Materials and Methods. Cells were incubated for 16 h with 7 μM A23187 (open bars) or 300 nM thapsigargin (filled bars) and lysed, and reporter enzyme assays were carried out. n/d, not detectable. Data shown are means ± SD of four separate experiments performed in triplicate.
Mentions: Fig. 3 shows that NIH/3T3 cells stably transfected with pCC1 and pSVβ-galactosidase produced five- to sevenfold more CAT protein after treatment for 16 h with 7 μM A23187 or 100 nM thapsigargin. NIH/3T3 cells were also stably transfected with pLC1 and pSVβ-galactosidase. When these cells were treated with 7 μM A23187 or 100 nM thapsigargin, a threefold increase in luciferase activity was observed (Fig. 3 A). Similar results were obtained with mouse fibroblasts that were transiently transfected with pCC1 or pLC1. The reason for this difference between the CAT and luciferase reporter systems is not clear, but it may be related to an inhibitory effect of Ca2+ on luciferase activity (unpublished observations).

Bottom Line: Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein.Changes in the concentration of extracellular 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 the calreticulin gene in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Group in Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.

ABSTRACT
We have isolated and characterized a 12-kb mouse genomic DNA fragment containing the entire calreticulin gene and 2.14 kb of the promoter region. The mouse calreticulin gene consists of nine exons and eight introns, and it spans 4.2 kb of genomic DNA. A 1.8-kb fragment of the calreticulin promoter was subcloned into a reporter gene plasmid containing chloramphenicol acetyltransferase. This construct was then used in transient and stable transfection of NIH/ 3T3 cells. Treatment of transfected cells either with the Ca2+ ionophore A23187, or with the ER Ca2+-ATPase inhibitor thapsigargin, resulted in a five- to sevenfold increase of the expression of chloramphenicol acetyltransferase protein. Transactivation of the calreticulin promoter was also increased by fourfold in NIH/3T3 cells treated with bradykinin, a hormone that induces Ca2+ release from the intracellular Ca2+ stores. Analysis of the promoter deletion constructs revealed that A23187- and thapsigargin-responsive regions are confined to two regions (-115 to -260 and -685 to -1,763) in the calreticulin promoter that contain the CCAAT nucleotide sequences. Northern blot analysis of cells treated with A23187, or with thapsigargin, 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 treated with A23187 and thapsigargin in vivo. This increase in gene expression required over 4 h of continuous incubation with the drugs and was also sensitive to treatment with cycloheximide, suggesting that it is dependent on protein synthesis. Changes in the concentration of extracellular 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 the calreticulin gene in vitro and in vivo.

Show MeSH
Related in: MedlinePlus