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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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Nuclear run-on analysis of the calreticulin gene in NIH/ 3T3 cells. Nuclei were isolated from cells treated for 4 h with  DMSO (Control), 10 μM A23187 (A23187), or 100 nM thapsigargin (Thapsigargin), and transcription was allowed to proceed in  the presence of [32P]UTP as described in Materials and Methods.  RNA products from equal numbers of nuclei per sample were hybridized to immobilized single-stranded DNA probes that detect  sense (S) or antisense (AS) transcript arising from calreticulin,  actin, G3PDH, H2b, and c-myc genes. CRT-1, calreticulin 5″  probe; CRT-2, calreticulin 3″ probe; actin-1, γ-actin 3″ probe; actin-2, γ-actin 5″ probe.
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Figure 5: Nuclear run-on analysis of the calreticulin gene in NIH/ 3T3 cells. Nuclei were isolated from cells treated for 4 h with DMSO (Control), 10 μM A23187 (A23187), or 100 nM thapsigargin (Thapsigargin), and transcription was allowed to proceed in the presence of [32P]UTP as described in Materials and Methods. RNA products from equal numbers of nuclei per sample were hybridized to immobilized single-stranded DNA probes that detect sense (S) or antisense (AS) transcript arising from calreticulin, actin, G3PDH, H2b, and c-myc genes. CRT-1, calreticulin 5″ probe; CRT-2, calreticulin 3″ probe; actin-1, γ-actin 3″ probe; actin-2, γ-actin 5″ probe.

Mentions: To measure transcription rates of the endogenous calreticulin gene and to determine if the accumulation of calreticulin mRNA was the result of increased transcription due to A23187 and thapsigargin, nuclear run-on transcription assays were carried out. NIH/3T3 cells were treated for 4 h with either A23187 or thapsigargin. Nuclei were prepared from the drug-treated cells and RNA transcripts initiated in vivo were elongated in vitro in the presence of [32P]UTP. The radiolabeled run-on transcripts were hybridized to single-stranded DNAs complementary to either specific calreticulin or control mRNAs (sense probes) or to antisense RNAs from the same regions (antisense probes). The probes used detected two regions of the mouse calreticulin gene (5′ and 3′ regions). Single-stranded probes for the two regions of the γ-actin gene, G3PDH gene, histone H2b gene, and c-myc gene (intron 1 and exon 1 regions) were included as controls for levels of transcription. Fig. 5 shows that both A23187 and thapsigargin induced transcription of calreticulin gene and that the transcription pattern was consistent with A23187- and thapsigargin-dependent accumulation of calreticulin mRNA (Fig. 5). The relative abundance of the calreticulin signal was determined using Phosphorimager analysis of the blots shown in Fig. 5. Comparison of the levels of calreticulin gene transcription between control and drug-treated cells relative to that observed for G3PDH revealed an approximately twofold increase in the calreticulin signal (Fig. 5). When levels of calreticulin gene transcription were compared between control and drug-treated cells relative to γ-actin, H2b, and c-myc gene transcription, an approximately sixfold increase in calreticulin signal was observed. Control genes (G3PDH, H2b, and c-myc) were also induced in the presence of A23187 and thapsigargin but to a lesser extent than that observed for calreticulin gene (Fig. 5). These results indicate that treatment of the cells with the Ca2+ ionophore A23187, or with thapsigargin, induces the expression of calreticulin at the transcriptional level in vivo.


Regulation of calreticulin gene expression by calcium.

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

Nuclear run-on analysis of the calreticulin gene in NIH/ 3T3 cells. Nuclei were isolated from cells treated for 4 h with  DMSO (Control), 10 μM A23187 (A23187), or 100 nM thapsigargin (Thapsigargin), and transcription was allowed to proceed in  the presence of [32P]UTP as described in Materials and Methods.  RNA products from equal numbers of nuclei per sample were hybridized to immobilized single-stranded DNA probes that detect  sense (S) or antisense (AS) transcript arising from calreticulin,  actin, G3PDH, H2b, and c-myc genes. CRT-1, calreticulin 5″  probe; CRT-2, calreticulin 3″ probe; actin-1, γ-actin 3″ probe; actin-2, γ-actin 5″ probe.
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Related In: Results  -  Collection

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Figure 5: Nuclear run-on analysis of the calreticulin gene in NIH/ 3T3 cells. Nuclei were isolated from cells treated for 4 h with DMSO (Control), 10 μM A23187 (A23187), or 100 nM thapsigargin (Thapsigargin), and transcription was allowed to proceed in the presence of [32P]UTP as described in Materials and Methods. RNA products from equal numbers of nuclei per sample were hybridized to immobilized single-stranded DNA probes that detect sense (S) or antisense (AS) transcript arising from calreticulin, actin, G3PDH, H2b, and c-myc genes. CRT-1, calreticulin 5″ probe; CRT-2, calreticulin 3″ probe; actin-1, γ-actin 3″ probe; actin-2, γ-actin 5″ probe.
Mentions: To measure transcription rates of the endogenous calreticulin gene and to determine if the accumulation of calreticulin mRNA was the result of increased transcription due to A23187 and thapsigargin, nuclear run-on transcription assays were carried out. NIH/3T3 cells were treated for 4 h with either A23187 or thapsigargin. Nuclei were prepared from the drug-treated cells and RNA transcripts initiated in vivo were elongated in vitro in the presence of [32P]UTP. The radiolabeled run-on transcripts were hybridized to single-stranded DNAs complementary to either specific calreticulin or control mRNAs (sense probes) or to antisense RNAs from the same regions (antisense probes). The probes used detected two regions of the mouse calreticulin gene (5′ and 3′ regions). Single-stranded probes for the two regions of the γ-actin gene, G3PDH gene, histone H2b gene, and c-myc gene (intron 1 and exon 1 regions) were included as controls for levels of transcription. Fig. 5 shows that both A23187 and thapsigargin induced transcription of calreticulin gene and that the transcription pattern was consistent with A23187- and thapsigargin-dependent accumulation of calreticulin mRNA (Fig. 5). The relative abundance of the calreticulin signal was determined using Phosphorimager analysis of the blots shown in Fig. 5. Comparison of the levels of calreticulin gene transcription between control and drug-treated cells relative to that observed for G3PDH revealed an approximately twofold increase in the calreticulin signal (Fig. 5). When levels of calreticulin gene transcription were compared between control and drug-treated cells relative to γ-actin, H2b, and c-myc gene transcription, an approximately sixfold increase in calreticulin signal was observed. Control genes (G3PDH, H2b, and c-myc) were also induced in the presence of A23187 and thapsigargin but to a lesser extent than that observed for calreticulin gene (Fig. 5). These results indicate that treatment of the cells with the Ca2+ ionophore A23187, or with thapsigargin, induces the expression of calreticulin at the transcriptional level in vivo.

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