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• W2045470441 abstract "Induction of differentiation of HL-60 human myeloid cells profoundly affected expression of calreticulin, a Ca2+-binding endoplasmic reticulum chaperone. Induction with Me2SO or retinoic acid reduced levels of calreticulin protein by ∼60% within 4 days. Pulse-chase studies indicated that labeled calreticulin decayed at similar rates in differentiated and undifferentiated cells (t12∼4.6 days), but the biosynthetic rate was <10% of control after 4 days. Differentiation also induced a rapid decline in calreticulin mRNA levels (90% reduction after 1 day) without a decrease in transcript stability (t12 ∼5 h). Nuclear run-on analysis demonstrated rapid down-regulation of gene transcription (21% of control at 2 h). Differentiation also greatly reduced the Ca2+ content of the cells (25% of control), although residual Ca2+ pools remained sensitive to thapsigargin, ionomycin, and inositol trisphosphate. Progressive decreases were also observed in levels of calnexin and ERp57, whereas BiP/GRP78 and protein disulfide isomerase were only modestly affected. Ultrastructural studies showed a substantial reduction in endoplasmic reticulum content of the cells. Thus, terminal differentiation of myeloid cells was associated with decreased endoplasmic reticulum content, selective reductions in molecular chaperones, and diminished intracellular Ca2+ stores, perhaps reflecting an endoplasmic reticulum remodeling program as a prominent feature of granulocytic differentiation. Induction of differentiation of HL-60 human myeloid cells profoundly affected expression of calreticulin, a Ca2+-binding endoplasmic reticulum chaperone. Induction with Me2SO or retinoic acid reduced levels of calreticulin protein by ∼60% within 4 days. Pulse-chase studies indicated that labeled calreticulin decayed at similar rates in differentiated and undifferentiated cells (t12∼4.6 days), but the biosynthetic rate was <10% of control after 4 days. Differentiation also induced a rapid decline in calreticulin mRNA levels (90% reduction after 1 day) without a decrease in transcript stability (t12 ∼5 h). Nuclear run-on analysis demonstrated rapid down-regulation of gene transcription (21% of control at 2 h). Differentiation also greatly reduced the Ca2+ content of the cells (25% of control), although residual Ca2+ pools remained sensitive to thapsigargin, ionomycin, and inositol trisphosphate. Progressive decreases were also observed in levels of calnexin and ERp57, whereas BiP/GRP78 and protein disulfide isomerase were only modestly affected. Ultrastructural studies showed a substantial reduction in endoplasmic reticulum content of the cells. Thus, terminal differentiation of myeloid cells was associated with decreased endoplasmic reticulum content, selective reductions in molecular chaperones, and diminished intracellular Ca2+ stores, perhaps reflecting an endoplasmic reticulum remodeling program as a prominent feature of granulocytic differentiation. endoplasmic reticulum inositol 1,4,5- trisphosphate 2,3, types 1, 2, and 3 receptors for IP3 non-fat dry milk phosphate-buffered saline phorbol 12-myristate 13-acetate sarco-endoplasmic reticulum calcium ATPase Calreticulin is a major Ca2+-binding protein present in the lumen of the endoplasmic reticulum (ER)1 of virtually all cell types (1Krause K.-H. Michalak M. Cell. 1997; 88: 439-443Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 2Meldolesi J. Pozzan T. Trends Biochem. Sci. 1998; 23: 10-14Abstract Full Text PDF PubMed Scopus (447) Google Scholar, 3Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (663) Google Scholar). The cDNA sequence predicts an ∼47-kDa acidic protein with a zonal structure featuring a globular N-terminal domain, a proline-rich P domain, and a highly acidic C domain terminating in a KDEL motif, the ER retention signal (3Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (663) Google Scholar, 4Baksh S. Michalak M. Michalak M. Calreticulin. R. G. Landes Co., Austin, TX1996: 11-30Crossref Google Scholar). Biophysical studies show calreticulin to be a highly asymmetric molecule consistent with this predicted three domain structure. Calreticulin binds Ca2+with both high and low affinities and with high capacity. A single high affinity site (Kd ∼1 μm) is located within the P domain of the molecule and multiple low affinity sites (Kd ∼2 mm, 20–25 mol of Ca2+/mol of protein) are present in the highly acidic C domain (3Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (663) Google Scholar, 4Baksh S. Michalak M. Michalak M. Calreticulin. R. G. Landes Co., Austin, TX1996: 11-30Crossref Google Scholar). The N domain contains at least one site for binding Zn2+, which appears to involve 4 of the histidine residues in this region (5Baksh S. Spamer C. Oikawa K. McCubbin W.D. Heilmann C. Kay C.M. Michalak M. Biochem. Biophys. Res. Commun. 1995; 209: 310-315Crossref PubMed Scopus (17) Google Scholar, 6Baksh S. Burns K. Andrin C. Michalak M. J. Biol. Chem. 1995; 270: 31338-31344Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Binding of these ions affects the conformation and stability of the protein (7Corbett E.F. Michalak K.M. Oikawa K. Johnson S. Campbell I.D. Eggleton P. Kay C. Michalak M. J. Biol. Chem. 2000; 275: 27177-27185Abstract Full Text Full Text PDF PubMed Google Scholar).Numerous and apparently unrelated functions have been ascribed to calreticulin since it was first identified, but its roles in the regulation of Ca2+ homeostasis and as a molecular chaperone of nascent glycoproteins are the two functions that have been most extensively characterized. Calreticulin appears to regulate intracellular Ca2+ homeostasis through more than one mechanism. The high Ca2+-binding capacity of calreticulin suggests that it acts as a Ca2+ store or buffer within the ER, and there is evidence that in at least some cell types it is a main source of inositol 1,4,5-trisphosphate (IP3)-releasable Ca2+ (3Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (663) Google Scholar, 5Baksh S. Spamer C. Oikawa K. McCubbin W.D. Heilmann C. Kay C.M. Michalak M. Biochem. Biophys. Res. Commun. 1995; 209: 310-315Crossref PubMed Scopus (17) Google Scholar, 8Enyedi P. Szabadkai G. Krause K.-H. Lew D.P. Spat A. Cell Calcium. 1993; 14: 485-492Crossref PubMed Scopus (22) Google Scholar, 9Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.-H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). In support of these observations, overexpression of calreticulin in some cells is accompanied by a significant increase in the Ca2+ storage pool, as well as an alteration in IP3-mediated Ca2+ release and influx (9Mery L. Mesaeli N. Michalak M. Opas M. Lew D.P. Krause K.-H. J. Biol. Chem. 1996; 271: 9332-9339Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 10Xu W. Longo F.J. Wintermantel M.R. Jiang X.Y. Clark R.A. DeLisle S. J. Biol. Chem. 2000; 275: 36676-36682Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Moreover, the thapsigargin- and ionomycin-sensitive Ca2+ pools were markedly reduced in calreticulin-null mouse embryonic stem cells, defects that were corrected by ectopic expression of calreticulin (11Nakamura K. Zuppini A. Arnaudeau S. Lynch J. Ahsan I. Krause R. Papp S., De Smedt H. Parys J.B. Müller-Esterl W. Lew D.P. Krause K.H. Demaurex N. Opas M. Michalak M. J. Cell Biol. 2001; 154: 961-972Crossref PubMed Scopus (226) Google Scholar). In contrast, calreticulin-deficient murine embryonic stem cells and fibroblasts exhibited normal levels of IP3-releasable Ca2+ (12Coppolino M.G. Woodside M.J. Demaurex N. Grinstein S., St- Arnaud R. Dedhar S. Nature. 1997; 386: 843-847Crossref PubMed Scopus (348) Google Scholar), suggesting that dependence of Ca2+ release on calreticulin may vary among cell types and signaling pathways. Calreticulin may also regulate Ca2+ levels by direct interaction with the ER uptake mechanism. Luminal Ca2+ released into the cytosol through the IP3 receptor channel is taken back up into the ER by the sarco-endoplasmic reticulum calcium ATPases (SERCA). One isoform of this family of Ca2+ pumps (SERCA2b) has an additional transmembrane segment and a C-terminal domain that extends into the lumen of the ER and contains a putative N-glycosylation site. In elegant studies using the Xenopus oocyte model, Camacho and colleagues (13Camacho P. Lechleiter J.D. Cell. 1995; 82: 765-771Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 14John L.M. Lechleiter J.D. Camacho P. J. Cell Biol. 1998; 142: 963-973Crossref PubMed Scopus (180) Google Scholar) showed that calreticulin inhibited the activity of SERCA2b and altered the temporal and spatial patterns of IP3-mediated Ca2+ release. This activity was dependent on Ca2+ concentration and was mediated by the lectin-binding P domain of calreticulin, rather than its high capacity Ca2+-binding C domain. Thus, calreticulin may modulate agonist-stimulated Ca2+ mobilization through multiple pathways.In addition to its role in Ca2+ signaling, calreticulin has an important function as a lectin-like chaperone for newly synthesized glycoproteins (3Michalak M. Corbett E.F. Mesaeli N. Nakamura K. Opas M. Biochem. J. 1999; 344: 281-292Crossref PubMed Scopus (663) Google Scholar, 15Nauseef W.M. McCormick S.J. Clark R.A. J. Biol. Chem. 1995; 270: 4741-4747Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 16Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 17Ellgaard L. Helenius A. Curr. Opin. Cell Biol. 2001; 13: 431-437Crossref PubMed Scopus (333) Google Scholar). In this respect, it displays many features in common with calnexin, another ER molecular chaperone. Calnexin and calreticulin share regions of structural homology and show lectin-like selectivity for mono-glucosylated N-linked glycoproteins (16Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 18Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 19Spiro R.G. Zhu Q. Bhoyroo V. Söling H.D. J. Biol. Chem. 1996; 271: 11588-11594Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 20Vassilakos A. Michalak M. Lehrman M.A. Williams D.B. Biochemistry. 1998; 37: 3480-3490Crossref PubMed Scopus (227) Google Scholar). The two proteins differ in topography; calreticulin is a soluble luminal ER protein, and calnexin has a transmembrane segment and a cytoplasmic domain. The interactions of calreticulin with unfolded glycoproteins and with other ER chaperones are Ca2+-dependent, suggesting a potential functional link between the chaperone and Ca2+-modulating roles of calreticulin (6Baksh S. Burns K. Andrin C. Michalak M. J. Biol. Chem. 1995; 270: 31338-31344Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 20Vassilakos A. Michalak M. Lehrman M.A. Williams D.B. Biochemistry. 1998; 37: 3480-3490Crossref PubMed Scopus (227) Google Scholar, 21Corbett E.F. Oikawa K. Francois P. Tessier D.C. Kay C. Bergeron J.J. Thomas D.Y. Krause K.H. Michalak M. J. Biol. Chem. 1999; 274: 6203-6211Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Moreover, the 5′-flanking sequence of the calreticulin gene and the genes for the chaperone proteins BiP/GRP78 and GRP94 show regions of sequence homology, suggesting that they are coordinately regulated (22McCauliffe D.P. Yang Y.-S. Wilson J. Sontheimer R.D. Capra J.D. J. Biol. Chem. 1992; 267: 2557-2562Abstract Full Text PDF PubMed Google Scholar).In phagocytic leukocytes, calreticulin serves several important functions. It acts as a molecular chaperone for the enzyme myeloperoxidase (15Nauseef W.M. McCormick S.J. Clark R.A. J. Biol. Chem. 1995; 270: 4741-4747Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), a catalytic component of oxygen-dependent microbicidal systems. In addition, membranous structures containing calreticulin, as well as the Ca2+-ATPase SERCA2b, accumulate in the actin-rich filamentous regions surrounding developing phagocytic vacuoles (23Stendahl O. Krause K.-H. Krischer J. Jerström P. Theler J.-M. Clark R.A. Carpentier J.-L. Lew D.P. Science. 1994; 265: 1439-1441Crossref PubMed Scopus (127) Google Scholar), suggesting involvement of these vesicles in the modulation of Ca2+-dependent phagolysosomal functions. Calreticulin has also been reported to be released from activated neutrophils (24Kishore U. Sontheimer R.D. Sastry K.N. Zaner K.S. Zappi E.G. Hughes G.R.V. Khamashta M.A. Strong P. Reid K.B.M. Eggleton P. Biochem. J. 1997; 322: 543-550Crossref PubMed Scopus (69) Google Scholar) and may bind and alter the activity of the C1q component of complement (25Kovacs H. Campbell I.D. Strong P. Johnson S. Ward F.J. Reid K.B. Eggleton P. Biochemistry. 1998; 37: 17865-17874Crossref PubMed Scopus (56) Google Scholar). A recent report (26Ogden C.A. DeCathehneau A. Hoffmann P.R. Bratton D. Ghebrehiwet B. Fadok V.A. Henson P.M. J. Exp. Med. 2001; 194: 781-795Crossref PubMed Scopus (938) Google Scholar) demonstrates that C1q-calreticulin interactions are involved in the uptake of apoptotic cells by phagocytes.Because of its importance in the biology and function of myeloid cells, we have investigated the biosynthesis and regulation of calreticulin in this cell type. Our previous studies (27Denning G.M. Leidal K.G. Holst V.A. Iyer S.S. Pearson D.W. Clark J.R. Nauseef W.M. Clark R.A. Blood. 1997; 90: 372-381Crossref PubMed Google Scholar) of the biosynthesis and post-translational processing of calreticulin in the HL-60 and PLB-985 myeloid cell lines showed that the primary translation product undergoes co-translational signal peptide cleavage and post-translational N-linked glycosylation to form the mature molecule. In the current study, we have investigated the transcription and translation of calreticulin and characterized the mechanisms of the dramatic down-regulation of its expression during in vitroinduction of myeloid cell differentiation.RESULTSInduction of Differentiation Reduces the Level and Biosynthetic Rate of Calreticulin ProteinProtein LevelMe2SO induces granulocytic differentiation of myeloid cell lines (29Collins S.J. Ruscetti F.W. Gallagher R.E. Gallo R.C. J. Exp. Med. 1979; 149: 969-974Crossref PubMed Scopus (500) Google Scholar). To determine the effect of differentiation on calreticulin protein level, HL-60 cells were incubated for 4 days in complete medium supplemented with 1.25% Me2SO; aliquots were removed at 0, 2, and 4 days, and cell lysates were analyzed by immunoblotting with monospecific rabbit anti-human calreticulin (27Denning G.M. Leidal K.G. Holst V.A. Iyer S.S. Pearson D.W. Clark J.R. Nauseef W.M. Clark R.A. Blood. 1997; 90: 372-381Crossref PubMed Google Scholar). HL-60 cell calreticulin protein levels decreased over time (Fig. 1A), with quantitative analysis of 5 independent experiments (Fig.1A, table inset) indicating more than a 50% decline at 4 days. In a separate experiment, five different sets of HL-60 cells were analyzed in parallel at 0 and 4 days after Me2SO addition (Fig. 1B). There was a consistent decrease in calreticulin levels to a mean of 38.8% (±3.7, p < 0.001) of the zero time controls.Protein Catabolic RatePossible mechanisms that could account for the observed decreases in steady-state calreticulin levels accompanying the induction of differentiation were an increase in the catabolic rate or a decrease in the biosynthetic rate. To determine whether differentiation influenced the rate of disappearance of calreticulin, undifferentiated and 4-day Me2SO-induced HL-60 cells were metabolically pulse-labeled with [35S]methionine for 30 min and then chased in fresh complete medium with unlabeled methionine for up to 7 days. For the Me2SO-induced cells, the chase medium included Me2SO, although essentially similar results were obtained if Me2SO was omitted from the chase medium. Fixed volume aliquots of the cultures were removed at intervals for cell centrifugation, immunoprecipitation, SDS-PAGE, and fluorography. Following densitometry and normalization to the corresponding levels obtained immediately after labeling (Fig.2), we determined that labeled calreticulin in the undifferentiated and Me2SO-treated cells decayed at approximately equal rates during the chase period. The calculated calreticulin t12 for untreated cells was 4.5 days and for Me2SO-treated cells was 4.6 days. Thus, an increased rate of loss of calreticulin does not appear to contribute to the reduction of cellular levels of calreticulin observed during HL-60 cell differentiation. An interesting feature of this experiment was the unusually high stability of calreticulin. Our data suggest a fractional catabolic rate in HL-60 myeloid cells of only about 13% per day. In additional experiments, we determined both cell-associated and extracellular 35S-calreticulin. The results indicated that release of calreticulin from the cells contributed only modestly to cellular calreticulin homeostasis (<20%) under our experimental conditions (data not shown).FIG. 2Analysis of the catabolic rate of calreticulin by pulse-chase labeling and immunoprecipitation.HL-60 cells were cultured for 4 days in the presence or absence of Me2SO as in Fig. 1. They were pulse-labeled with [35S]methionine for 30 min and then chased in fresh complete medium with unlabeled methionine for 7 days, and aliquots were removed at intervals, as indicated, for immunoprecipitation, SDS-PAGE, and fluorography. Each lane contained the immunoprecipitate from cells that were pelleted from a fixed volume aliquot of the culture, thereby avoiding the potentially confounding effect of35S-calreticulin dilution during cell division. The content of calreticulin at each point is indicated as a percent of the zero time value, based on densitometric quantitation. The half-life of calreticulin by this method was 4.5 or 4.6 days for untreated or Me2SO-treated cells, respectively. The study shown is representative of three independent experiments. CRT,calreticulin.View Large Image Figure ViewerDownload (PPT)Protein Biosynthetic RateIn view of the lack of any change in the catabolism of calreticulin to account for its decreased levels in differentiated cells, we next investigated whether the biosynthetic rate was changed in response to Me2SO-induced differentiation. Parallel experiments were also carried out with other differentiating agents. Thus, HL-60 cells were cultured in Me2SO or retinoic acid to induce granulocytic differentiation or in PMA or 1,25-dihydroxyvitamin D3 to induce monocytic differentiation. At intervals, aliquots of cells were taken for biosynthetic pulse labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE analysis (Fig. 3). Induction of differentiation by each agent decreased the amount of nascent calreticulin labeled with [35S]methionine, indicating reductions in the biosynthetic rate. Similar results were observed in studies on PLB-985 cells (data not shown). The effect was progressive over time and showed differences in rate and extent with the various differentiating agents. Initially, the effect was most rapid in the Me2SO-treated cells, which exhibited a reduction of calreticulin synthesis to 36% of zero time levels after only 1 day of treatment. In 4 replicate experiments, the mean normalized values for calreticulin biosynthesis rates (relative to zero time controls) after 1, 2, 4, and 6 days of culture in Me2SO were 44.2 (p < 0.05), 33.8 (p < 0.01), 9.5 (p < 0.001), and 2.0% (p < 0.001), respectively. These experiments clearly indicate that the reduction in calreticulin protein levels observed during differentiation of myeloid cells resulted largely from a major decrease in the rate of calreticulin biosynthesis.FIG. 3Analysis of biosynthetic rate of calreticulin by pulse labeling and immunoprecipitation. HL-60 cells were induced to differentiate by growth in 1.25% Me2SO, 1 μm retinoic acid (RA), 80 nm PMA, or 10 nm 1,25-dihydroxyvitamin D3((OH)2D3). Aliquots were taken initially and after 1, 2, 4, and 6 days for pulse labeling ([35S]methionine for 30 min), immunoprecipitation, SDS-PAGE, and fluorography. Each lane contained the immunoprecipitate from 4 × 106 cells. The relative biosynthetic rate of calreticulin at each point is indicated as a percent of the 0 time value, based on densitometric quantitation. The study shown for Me2SO is representative of four independent experiments, whereas the other differentiating agents were studied in one experiment each. CRT, calreticulin.View Large Image Figure ViewerDownload (PPT)Induction of Differentiation Reduces Calreticulin mRNA Level and Transcriptional RateTranscript LevelThe studies described above showed that the induction of differentiation of HL-60 cells by a variety of agents resulted in a marked reduction in the biosynthesis of calreticulin. To determine the effect of myeloid cell differentiation on calreticulin mRNA levels, Northern analysis was carried out on the poly(A)+ RNA from cells induced to differentiate (Fig.4). Calreticulin mRNA was highly expressed in untreated cells but was markedly reduced in both HL-60 and PLB-985 cells within 1 day of Me2SO treatment, and further reduction was seen after 2 days (Fig. 4A). Following normalization to the levels of β-actin mRNA, it was estimated that Me2SO treatment induced an approximate 80% reduction in the level of calreticulin transcripts within 1 day. In 6 replicate experiments, the mean normalized values for calreticulin message levels (relative to zero time controls) after 1, 2, and 4 days of culture in Me2SO were 19.6, 16.2, and 9.1%, respectively (p < 0.001 for each).FIG. 4Northern hybridization analysis of calreticulin transcript levels during myeloid cell differentiation.A, HL-60 or PLB-985 cells were cultured in the presence of 1.25% Me2SO for 0, 1, or 2 days, as indicated. Poly(A)+ RNA was isolated, separated (3 μg/lane) on 1.2% agarose-formaldehyde gels, transferred, and probed for calreticulin mRNA, followed by stripping and probing for β-actin, which was used as a loading control. After densitometric quantitation, the content of calreticulin transcripts was corrected for actin and expressed as a percent of the zero time values. The studies shown are representative of five independent experiments for HL-60 cells and two experiments for PLB-985 cells. B, HL-60 cells were cultured and analyzed as in A, except that the blots were also probed for p47phox and p67phox. The data for calreticulin mRNA levels are expressed in the same fashion as above. The study shown is representative of two independent experiments. C, HL-60 cells were cultured and analyzed as inA, except that differentiation was induced with either Me2SO (Me) or retinoic acid (R) for 1 or 3 days, as indicated, and the blots were also probed for type 1 IP3 receptor (IP3R). The data for calreticulin and IP3R mRNA levels are expressed in the same fashion as above. The study shown is representative of two independent experiments. CRT, calreticulin.View Large Image Figure ViewerDownload (PPT)Differentiation of HL-60 and PLB-985 cells is accompanied by the induction of a number of myeloid-specific genes, the products of which carry out essential functions in the terminally differentiated granulocytic cell. Two such genes are p47phox and p67phox, cytosolic components of the phagocyte NADPH oxidase (35Gupta J.W. Kubin M. Hartman L. Cassatella M. Trinchieri G. Cancer Res. 1992; 52: 2530-2537PubMed Google Scholar). To place the differentiation-induced changes in calreticulin mRNA in context, they were compared with those for p47phoxand p67phox in HL-60 cells treated with Me2SO (Fig.4B). The cellular level of calreticulin mRNA was again rapidly reduced, whereas the p47phox and p67phoxtranscripts increased from undetectable levels in untreated cells to maximal expression after 2–4 days of Me2SO treatment. Parallel studies were done comparing calreticulin with the type 1 IP3 receptor, a protein that, like calreticulin, is localized to the endoplasmic reticulum and is involved in calcium release (Fig. 4C). We observed that both Me2SO and retinoic acid induced a 3–4-fold increase in IP3R mRNA within 1 day of treatment, whereas calreticulin mRNA was again significantly reduced.The detailed kinetics of the effects of various inducing agents on calreticulin mRNA expression in HL-60 cells were examined in a slot-blot hybridization assay. RNA was isolated from the cells at 0, 3, 6, 9, 12, 24, and 48 h after initiation of treatment and probed for calreticulin and β-actin. After scanning densitometry, calreticulin mRNA was normalized to β-actin levels, and results were expressed as a percentage of the initial value (Fig.5). Me2SO treatment produced the most rapid reduction (50% in ∼5 h) of calreticulin mRNA (as was observed for protein synthetic rates, see Fig. 3). Retinoic acid and PMA were also very active, reducing the mRNA levels by 50% in ∼10 h, whereas 1,25-dihydroxyvitamin D3 was less active in down-regulation of calreticulin transcripts (50% reduction in ∼25–30 h).FIG. 5Time course of calreticulin transcript levels during HL-60 cell differentiation. HL-60 cells were cultured in the various differentiating agents as indicated, and aliquots were removed at the times shown. Poly(A)+ RNA was isolated and analyzed by slot-blot hybridization with a calreticulin probe, following which the blots were stripped and probed for β-actin, which was used a loading control. After densitometric quantitation, the content of calreticulin transcripts was corrected for actin and expressed as a percent of the zero time values. The results shown are from a single study, which is representative of two independent experiments. CRT, calreticulin.View Large Image Figure ViewerDownload (PPT)Transcript StabilityPossible mechanisms that could account for the observed decreases in steady-state levels of calreticulin transcripts accompanying the induction of differentiation were a decrease in transcript stability (i.e. an increase in the catabolic rate) or a decrease in the transcriptional rate. To determine whether calreticulin mRNA stability was affected by Me2SO treatment, studies were carried out using actinomycin D as an inhibitor of transcription. HL-60 cells were cultured in Me2SO for 0, 1, or 2 days and then incubated in fresh medium; actinomycin D was added at various times up to 6 h (see “Experimental Procedures”). At the end of these incubations, poly(A)+ RNA was extracted, and slot-blot hybridization analyses were carried out. Levels of calreticulin and β-actin mRNA were expressed as a percentage of the levels at 0 time, and t12 was calculated from these values. Because thet12 averaged between 3 and 5 h, analysis focused especially on the 4-h time point. Treatment of the cells with Me2SO resulted in significant and similar increases in the normalized 4-h transcript levels for both calreticulin and actin (Table I). In keeping with this, the calculatedt12 values for both calreticulin and actin transcripts were increased by ∼50% (Table I), indicating stabilization of these mRNAs in Me2SO-induced HL-60 cells. Thus, the down-regulation of calreticulin mRNA in differentiating HL-60 cells could not be explained by increased transcript catabolism. Indeed, enhanced mRNA stability provided some counter-balance to decreased transcription (see below).Table IStability of calreticulin mRNA in undifferentiated and Me2SO-induced HL-60 cellsTime in Me2SOTranscript level (4 h after actinomycin D)t1/2CalreticulinActinCalreticulinActindays%h027.3 ± 3.91-aPercent of control; mean ± S.E. for a total of 4–6 independent experiments.35.4 ± 9.83.1 ± 0.21-bMean ± S.E. for a total of 4–6 independent experiments.3.3 ± 0.4160.5 ± 5.038.8 ± 10.34.7 ± 0.53.5 ± 0.2262.9 ± 6.657.4 ± 21.55.0 ± 0.64.8 ± 0.71-a Percent of control; mean ± S.E. for a total of 4–6 independent experiments.1-b Mean ± S.E. for a total of 4–6 independent experiments. Open table in a new tab Transcriptional RateTo determine whether reduction in the rate of gene transcription was responsible for the rapid down-regulation of calreticulin mRNA in differentiating HL-60 cells, nuclear run-on analyses were carried out on undifferentiated and Me2SO-induced cells. As illustrated in the left-hand panel of Fig. 6, transcription of the calreticulin gene was essentially eliminated following 96 h of Me2SO treatment, as was transcription of myeloperoxidase, another gene that is down-regulated in differentiation-induced HL-60 cells. In the same cells, the transcription of the internal control β-actin remained largely unchanged, whereas transcription of p47phox was undetectable initially but was strongly induced at 96 h. To determine more precisely the period during which calreticulin transcription was affected, nuclear run-on analysis was carried out at shorter intervals after initiation of Me2SO treatment. These studies (Fig. 6, right-hand panel) showed that calreticulin gene transcription was down-regulated very rapidly after Me2SO exposure (79% decrease in 2 h). Myeloperoxidase transcription appeared to be reduced even more" @default.
• W2045470441 created "2016-06-24" @default.
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• W2045470441 date "2002-08-01" @default.
• W2045470441 modified "2023-10-11" @default.
• W2045470441 title "Regulation of Calreticulin Expression during Induction of Differentiation in Human Myeloid Cells" @default.
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