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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 nucleotide sequence of the promoter region of the  mouse calreticulin gene. The nucleotides are numbered with the  putative transcriptional initiation site of the mouse calreticulin  gene at +1 (Smith and Koch, 1989). (Horizontal lines) Putative  binding sites for the DNA binding proteins: AP-2, Sp1, AP-1 and  H4TF-1, and SIF. CCAAT sites (boxed). TATA box (underlined). ATG initiation codon (bold). These sequence data are  available from GenBank/EMBL/DDBJ under accession number  U38249.
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Figure 1: The nucleotide sequence of the promoter region of the mouse calreticulin gene. The nucleotides are numbered with the putative transcriptional initiation site of the mouse calreticulin gene at +1 (Smith and Koch, 1989). (Horizontal lines) Putative binding sites for the DNA binding proteins: AP-2, Sp1, AP-1 and H4TF-1, and SIF. CCAAT sites (boxed). TATA box (underlined). ATG initiation codon (bold). These sequence data are available from GenBank/EMBL/DDBJ under accession number U38249.

Mentions: Plasmid pCM7 was constructed by subcloning a 7-kb HindIII restriction fragment from p1.3 into the HindIII restriction site of pBluescript. This fragment contained 1.8 kb of the 5′-flanking region and the entire coding region of the calreticulin gene. The 1.8-kb promoter fragment was further subcloned into the promoterless chloramphenicol acetyltransferase (CAT)1 reporter expression vector pCATbasic and pXP-1 (luciferase expression vector [De Wet et al., 1987] producing plasmids pCC1 and pLC1, respectively). To generate pCC1, a HindIII/StuI fragment of pCM7 (nucleotides −1,723 to +40 of the calreticulin gene; Fig. 1) was cloned into the blunt-ended XbaI/HindIII sites of pCATbasic. To generate pLC1, an SmaI/StuI fragment was subcloned into the SmaI restriction site of pXP-1.


Regulation of calreticulin gene expression by calcium.

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

The nucleotide sequence of the promoter region of the  mouse calreticulin gene. The nucleotides are numbered with the  putative transcriptional initiation site of the mouse calreticulin  gene at +1 (Smith and Koch, 1989). (Horizontal lines) Putative  binding sites for the DNA binding proteins: AP-2, Sp1, AP-1 and  H4TF-1, and SIF. CCAAT sites (boxed). TATA box (underlined). ATG initiation codon (bold). These sequence data are  available from GenBank/EMBL/DDBJ under accession number  U38249.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: The nucleotide sequence of the promoter region of the mouse calreticulin gene. The nucleotides are numbered with the putative transcriptional initiation site of the mouse calreticulin gene at +1 (Smith and Koch, 1989). (Horizontal lines) Putative binding sites for the DNA binding proteins: AP-2, Sp1, AP-1 and H4TF-1, and SIF. CCAAT sites (boxed). TATA box (underlined). ATG initiation codon (bold). These sequence data are available from GenBank/EMBL/DDBJ under accession number U38249.
Mentions: Plasmid pCM7 was constructed by subcloning a 7-kb HindIII restriction fragment from p1.3 into the HindIII restriction site of pBluescript. This fragment contained 1.8 kb of the 5′-flanking region and the entire coding region of the calreticulin gene. The 1.8-kb promoter fragment was further subcloned into the promoterless chloramphenicol acetyltransferase (CAT)1 reporter expression vector pCATbasic and pXP-1 (luciferase expression vector [De Wet et al., 1987] producing plasmids pCC1 and pLC1, respectively). To generate pCC1, a HindIII/StuI fragment of pCM7 (nucleotides −1,723 to +40 of the calreticulin gene; Fig. 1) was cloned into the blunt-ended XbaI/HindIII sites of pCATbasic. To generate pLC1, an SmaI/StuI fragment was subcloned into the SmaI restriction site of pXP-1.

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