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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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A23187- and thapsigargin-dependent increase in calreticulin mRNA levels (A) and protein levels (B). (A) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187,  100 nM thapsigargin, or DMSO. Total RNA was then isolated  and electrophoretically separated on a formaldehyde-agarose gel,  blotted onto Hybond N nylon membrane, and hybridized with  cDNA probes encoding mouse calreticulin and G3PDH, as described in Materials and Methods. (Top) Autoradiogram of a  Northern blot probed with calreticulin and G3PDH cDNA.  (Lane 1) Control cells; (lane 2) A23187-treated cells; (lane 3)  thapsigargin-treated cells. (Bottom) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of Northern blots. (B)  NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin  and cellular extracts were prepared as described by Mery et al.  (1996). The proteins were separated by SDS-PAGE, transferred  electrophoretically to nitrocellulose membranes, and incubated  with goat anti-calreticulin antibody as described by Milner et al.  (1991). For each condition, three different amounts of cells were  analyzed (lanes 1 and 4, 30,000 cells per well; lanes 2 and 5,  100,000 cells per well; lanes 3 and 6, 150,000 cells per well), and  the immunoblots were scanned by densitometry. (Top, lanes 1, 2,  and 3) Control, untreated cells; (lanes 4, 5, and 6) thapsigargin-treated cells. (Bottom) The slope of the cell number as a function  of OD was obtained by a linear fit to give a relative cellular content of calreticulin (Mery et al., 1996). TG, thapsigargin.
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Figure 4: A23187- and thapsigargin-dependent increase in calreticulin mRNA levels (A) and protein levels (B). (A) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or DMSO. Total RNA was then isolated and electrophoretically separated on a formaldehyde-agarose gel, blotted onto Hybond N nylon membrane, and hybridized with cDNA probes encoding mouse calreticulin and G3PDH, as described in Materials and Methods. (Top) Autoradiogram of a Northern blot probed with calreticulin and G3PDH cDNA. (Lane 1) Control cells; (lane 2) A23187-treated cells; (lane 3) thapsigargin-treated cells. (Bottom) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of Northern blots. (B) NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin and cellular extracts were prepared as described by Mery et al. (1996). The proteins were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose membranes, and incubated with goat anti-calreticulin antibody as described by Milner et al. (1991). For each condition, three different amounts of cells were analyzed (lanes 1 and 4, 30,000 cells per well; lanes 2 and 5, 100,000 cells per well; lanes 3 and 6, 150,000 cells per well), and the immunoblots were scanned by densitometry. (Top, lanes 1, 2, and 3) Control, untreated cells; (lanes 4, 5, and 6) thapsigargin-treated cells. (Bottom) The slope of the cell number as a function of OD was obtained by a linear fit to give a relative cellular content of calreticulin (Mery et al., 1996). TG, thapsigargin.

Mentions: Treatment of nontransfected cells with A23187 and thapsigargin also led to altered expression of the endogenous calreticulin gene. Specifically, we used Northern blot analysis to measure the relative mRNA levels in NIH/3T3 cells treated with these drugs. Fig. 4 A shows that an approximately four- to fivefold increase in the abundance of calreticulin mRNA was observed in cells incubated with these drugs. There was an approximately fourfold increase in calreticulin protein in NIH/3T3 cells incubated with thapsigargin (Fig. 4 B). This suggests that changes in the level of calreticulin mRNA resulted in changes in calreticulin expression.


Regulation of calreticulin gene expression by calcium.

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

A23187- and thapsigargin-dependent increase in calreticulin mRNA levels (A) and protein levels (B). (A) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187,  100 nM thapsigargin, or DMSO. Total RNA was then isolated  and electrophoretically separated on a formaldehyde-agarose gel,  blotted onto Hybond N nylon membrane, and hybridized with  cDNA probes encoding mouse calreticulin and G3PDH, as described in Materials and Methods. (Top) Autoradiogram of a  Northern blot probed with calreticulin and G3PDH cDNA.  (Lane 1) Control cells; (lane 2) A23187-treated cells; (lane 3)  thapsigargin-treated cells. (Bottom) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of Northern blots. (B)  NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin  and cellular extracts were prepared as described by Mery et al.  (1996). The proteins were separated by SDS-PAGE, transferred  electrophoretically to nitrocellulose membranes, and incubated  with goat anti-calreticulin antibody as described by Milner et al.  (1991). For each condition, three different amounts of cells were  analyzed (lanes 1 and 4, 30,000 cells per well; lanes 2 and 5,  100,000 cells per well; lanes 3 and 6, 150,000 cells per well), and  the immunoblots were scanned by densitometry. (Top, lanes 1, 2,  and 3) Control, untreated cells; (lanes 4, 5, and 6) thapsigargin-treated cells. (Bottom) The slope of the cell number as a function  of OD was obtained by a linear fit to give a relative cellular content of calreticulin (Mery et al., 1996). TG, thapsigargin.
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Figure 4: A23187- and thapsigargin-dependent increase in calreticulin mRNA levels (A) and protein levels (B). (A) Nontransfected NIH/3T3 cells were incubated for 16 h with 7 μM A23187, 100 nM thapsigargin, or DMSO. Total RNA was then isolated and electrophoretically separated on a formaldehyde-agarose gel, blotted onto Hybond N nylon membrane, and hybridized with cDNA probes encoding mouse calreticulin and G3PDH, as described in Materials and Methods. (Top) Autoradiogram of a Northern blot probed with calreticulin and G3PDH cDNA. (Lane 1) Control cells; (lane 2) A23187-treated cells; (lane 3) thapsigargin-treated cells. (Bottom) The abundance of calreticulin mRNA (calreticulin mRNA/G3PDH mRNA ratio) was determined using Phosphorimager analysis of Northern blots. (B) NIH/3T3 cells were incubated for 16 h with 100 nM thapsigargin and cellular extracts were prepared as described by Mery et al. (1996). The proteins were separated by SDS-PAGE, transferred electrophoretically to nitrocellulose membranes, and incubated with goat anti-calreticulin antibody as described by Milner et al. (1991). For each condition, three different amounts of cells were analyzed (lanes 1 and 4, 30,000 cells per well; lanes 2 and 5, 100,000 cells per well; lanes 3 and 6, 150,000 cells per well), and the immunoblots were scanned by densitometry. (Top, lanes 1, 2, and 3) Control, untreated cells; (lanes 4, 5, and 6) thapsigargin-treated cells. (Bottom) The slope of the cell number as a function of OD was obtained by a linear fit to give a relative cellular content of calreticulin (Mery et al., 1996). TG, thapsigargin.
Mentions: Treatment of nontransfected cells with A23187 and thapsigargin also led to altered expression of the endogenous calreticulin gene. Specifically, we used Northern blot analysis to measure the relative mRNA levels in NIH/3T3 cells treated with these drugs. Fig. 4 A shows that an approximately four- to fivefold increase in the abundance of calreticulin mRNA was observed in cells incubated with these drugs. There was an approximately fourfold increase in calreticulin protein in NIH/3T3 cells incubated with thapsigargin (Fig. 4 B). This suggests that changes in the level of calreticulin mRNA resulted in changes in calreticulin expression.

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