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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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The kinetics of activation of the calreticulin promoter  by A23187 and thapsigargin treatment. NCB1 cells (stably expressing CAT under control of the calreticulin promoter and  β-galactosidase) were incubated with 7 μM A23187, 100 nM  thapsigargin, or DMSO (control cells) for the times indicated. At  different time points, cells were either harvested (open bars) or  washed with PBS and then incubated in drug-free media to a total of 16 h of incubation (cross-hatched bars). CAT protein levels  and β-galactosidase activity were measured as described in Materials and Methods. Data are reported as a mean ± SD of four  separate experiments performed in triplicate.
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Figure 6: The kinetics of activation of the calreticulin promoter by A23187 and thapsigargin treatment. NCB1 cells (stably expressing CAT under control of the calreticulin promoter and β-galactosidase) were incubated with 7 μM A23187, 100 nM thapsigargin, or DMSO (control cells) for the times indicated. At different time points, cells were either harvested (open bars) or washed with PBS and then incubated in drug-free media to a total of 16 h of incubation (cross-hatched bars). CAT protein levels and β-galactosidase activity were measured as described in Materials and Methods. Data are reported as a mean ± SD of four separate experiments performed in triplicate.

Mentions: A23187 and thapsigargin modulate intracellular Ca2+ concentration within seconds. To determine the kinetics of the Ca2+-dependent activation of calreticulin promoter, time-dependent expression of CAT was measured in NCB1 cells (NIH/3T3 cells stably transfected with pCC1 and pSVβ-galactosidase). Two experimental protocols were used for this analysis. First, the cells were incubated for 2, 4, 8, 12, and 16 h with A23187 or thapsigargin followed by measurement of CAT expression (Fig. 6, open bars). In the second protocol, the cells were incubated for 2, 4, 8, and 12 h with the drugs followed by incubation in a drug-free medium to a total of 16 h of incubation for each data point (Fig. 6, hatched bars). Fig. 6 shows that for both protocols the overall kinetics and magnitude of induction of CAT expression by A23187 and thapsigargin were similar. Maximal induction of CAT expression in the NCB1 cells required 16 h of continuous incubation with both drugs (Fig. 6), or exposure to the drugs for 4–8 h followed by incubation in a drug-free medium to a total of 16 h (Fig. 6). These results indicate that the Ca2+ store depletion–dependent activation of the calreticulin promoter is very slow and that it may require de novo protein synthesis.


Regulation of calreticulin gene expression by calcium.

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

The kinetics of activation of the calreticulin promoter  by A23187 and thapsigargin treatment. NCB1 cells (stably expressing CAT under control of the calreticulin promoter and  β-galactosidase) were incubated with 7 μM A23187, 100 nM  thapsigargin, or DMSO (control cells) for the times indicated. At  different time points, cells were either harvested (open bars) or  washed with PBS and then incubated in drug-free media to a total of 16 h of incubation (cross-hatched bars). CAT protein levels  and β-galactosidase activity were measured as described in Materials and Methods. Data are reported as a mean ± SD of four  separate experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: The kinetics of activation of the calreticulin promoter by A23187 and thapsigargin treatment. NCB1 cells (stably expressing CAT under control of the calreticulin promoter and β-galactosidase) were incubated with 7 μM A23187, 100 nM thapsigargin, or DMSO (control cells) for the times indicated. At different time points, cells were either harvested (open bars) or washed with PBS and then incubated in drug-free media to a total of 16 h of incubation (cross-hatched bars). CAT protein levels and β-galactosidase activity were measured as described in Materials and Methods. Data are reported as a mean ± SD of four separate experiments performed in triplicate.
Mentions: A23187 and thapsigargin modulate intracellular Ca2+ concentration within seconds. To determine the kinetics of the Ca2+-dependent activation of calreticulin promoter, time-dependent expression of CAT was measured in NCB1 cells (NIH/3T3 cells stably transfected with pCC1 and pSVβ-galactosidase). Two experimental protocols were used for this analysis. First, the cells were incubated for 2, 4, 8, 12, and 16 h with A23187 or thapsigargin followed by measurement of CAT expression (Fig. 6, open bars). In the second protocol, the cells were incubated for 2, 4, 8, and 12 h with the drugs followed by incubation in a drug-free medium to a total of 16 h of incubation for each data point (Fig. 6, hatched bars). Fig. 6 shows that for both protocols the overall kinetics and magnitude of induction of CAT expression by A23187 and thapsigargin were similar. Maximal induction of CAT expression in the NCB1 cells required 16 h of continuous incubation with both drugs (Fig. 6), or exposure to the drugs for 4–8 h followed by incubation in a drug-free medium to a total of 16 h (Fig. 6). These results indicate that the Ca2+ store depletion–dependent activation of the calreticulin promoter is very slow and that it may require de novo protein synthesis.

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