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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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Effects of BAPTA on A23187- and thapsigargin-dependent activation of calreticulin promoter. NCB1 cells were  loaded with 10 μM BAPTA/AM for 30 min in a media containing  different drugs for 16 h and lysed, and reporter gene analysis was  carried out as described in Materials and Methods. (Open bars)  Cells grown and incubated with 7 μM A23187 or 100 nM thapsigargin in DME supplemented with 100 μM EGTA; (dotted bars)  cells loaded with BAPTA/AM followed by incubation with either  A23187 or thapsigargin (control cells were not treated with  BAPTA/AM); (filled bars) cells loaded with BAPTA/AM and incubated with either A23187 or thapsigargin in Ca2+-depleted  DME. (Cross-hatched bars) Cells loaded with 10 μM EGTA/AM  for 30 min followed by 16-h incubation with either A23187 or  thapsigargin in DME (control cells were not treated with EGTA/ AM); data are shown as a mean ± SD.
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Figure 8: Effects of BAPTA on A23187- and thapsigargin-dependent activation of calreticulin promoter. NCB1 cells were loaded with 10 μM BAPTA/AM for 30 min in a media containing different drugs for 16 h and lysed, and reporter gene analysis was carried out as described in Materials and Methods. (Open bars) Cells grown and incubated with 7 μM A23187 or 100 nM thapsigargin in DME supplemented with 100 μM EGTA; (dotted bars) cells loaded with BAPTA/AM followed by incubation with either A23187 or thapsigargin (control cells were not treated with BAPTA/AM); (filled bars) cells loaded with BAPTA/AM and incubated with either A23187 or thapsigargin in Ca2+-depleted DME. (Cross-hatched bars) Cells loaded with 10 μM EGTA/AM for 30 min followed by 16-h incubation with either A23187 or thapsigargin in DME (control cells were not treated with EGTA/ AM); data are shown as a mean ± SD.

Mentions: To test whether extracellular concentrations of Ca2+ affect the drug-mediated activation of the calreticulin promoter, we incubated NCB1 cells in a Ca2+-depleted medium supplemented with EGTA. We found that A23187- and thapsigargin-dependent activation of the promoter was independent of changes in the extracellular Ca2+ concentration (Fig. 8). These results further indicate that depletion of Ca2+ from ER stores is involved in the activation of the calreticulin promoter.


Regulation of calreticulin gene expression by calcium.

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

Effects of BAPTA on A23187- and thapsigargin-dependent activation of calreticulin promoter. NCB1 cells were  loaded with 10 μM BAPTA/AM for 30 min in a media containing  different drugs for 16 h and lysed, and reporter gene analysis was  carried out as described in Materials and Methods. (Open bars)  Cells grown and incubated with 7 μM A23187 or 100 nM thapsigargin in DME supplemented with 100 μM EGTA; (dotted bars)  cells loaded with BAPTA/AM followed by incubation with either  A23187 or thapsigargin (control cells were not treated with  BAPTA/AM); (filled bars) cells loaded with BAPTA/AM and incubated with either A23187 or thapsigargin in Ca2+-depleted  DME. (Cross-hatched bars) Cells loaded with 10 μM EGTA/AM  for 30 min followed by 16-h incubation with either A23187 or  thapsigargin in DME (control cells were not treated with EGTA/ AM); data are shown as a mean ± SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2141645&req=5

Figure 8: Effects of BAPTA on A23187- and thapsigargin-dependent activation of calreticulin promoter. NCB1 cells were loaded with 10 μM BAPTA/AM for 30 min in a media containing different drugs for 16 h and lysed, and reporter gene analysis was carried out as described in Materials and Methods. (Open bars) Cells grown and incubated with 7 μM A23187 or 100 nM thapsigargin in DME supplemented with 100 μM EGTA; (dotted bars) cells loaded with BAPTA/AM followed by incubation with either A23187 or thapsigargin (control cells were not treated with BAPTA/AM); (filled bars) cells loaded with BAPTA/AM and incubated with either A23187 or thapsigargin in Ca2+-depleted DME. (Cross-hatched bars) Cells loaded with 10 μM EGTA/AM for 30 min followed by 16-h incubation with either A23187 or thapsigargin in DME (control cells were not treated with EGTA/ AM); data are shown as a mean ± SD.
Mentions: To test whether extracellular concentrations of Ca2+ affect the drug-mediated activation of the calreticulin promoter, we incubated NCB1 cells in a Ca2+-depleted medium supplemented with EGTA. We found that A23187- and thapsigargin-dependent activation of the promoter was independent of changes in the extracellular Ca2+ concentration (Fig. 8). These results further indicate that depletion of Ca2+ from ER stores is involved in the activation of the calreticulin promoter.

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