Calreticulin Inhibits Vitamin D’s Action on the PTH Gene in Vitro and May Prevent Vitamin D’s Effect in Vivo in Hypocalcemic Rats

Alin Sela-Brown, John Russell, Nicholas J. Koszewski, Marek Michalak, Tally Naveh-Many and Justin Silver

Minerva Center for Calcium and Bone Metabolism (A.S-B., T.N-M, J.S.) Nephrology Services Hadassah University Hospital and Hebrew University Medical School Jerusalem, Israel 91120
Department of Medicine (J.R.) Albert Einstein College of Medicine Bronx, New York 10461
Department of Medicine (N.J.K.) University of Kentucky Medical School Lexington, Kentucky 40536
Molecular Biology of Membranes Research Group (M.M.) University of Alberta Edmonton, Alberta T6G 2S2, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
1,25-dihydroxyvitaminD3 [1,25-(OH)2D3] and PTH both act to increase serum calcium. In addition, 1,25-(OH)2D3 decreases PTH gene transcription, which is relevant both to the physiology of calcium homeostasis and to the management of the secondary hyperparathyroidism of patients with chronic renal failure. In chronic hypocalcemia there is secondary hyperparathyroidism with increased levels of PTH mRNA and serum PTH despite markedly increased levels of 1,25-(OH)2D3. We have studied the role of calreticulin in this resistance to 1,25-(OH)2D3. Weanling rats fed a low-calcium diet were hypocalcemic and had increased PTH mRNA levels despite high serum 1,25-(OH)2D3 levels. 1,25-(OH)2D3 given by continuous minipump infusion to normal rats led to the expected decrease in PTH mRNA. The hypocalcemic rats had an increased concentration of calreticulin in the nuclear fraction of their parathyroids, but not in other tissues. Gel shift assays showed that a purified vitamin D receptor and retinoid X receptor-ß bound to the PTH promoter’s chicken and rat vitamin D response element (VDRE), and this binding was inhibited by added pure calreticulin. Transfection studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT) construct showed that 1,25-(OH)2D3 decreased CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin expression vector in the sense orientation prevented the transcriptional effect of 1,25-(OH)2D3, but a calreticulin vector in the antisense orientation had no effect. These results show that calreticulin prevents the binding of vitamin D receptor-retinoid X receptor-ß to the PTH VDRE in gel retardation assays and prevents the transcriptional effect of 1,25-(OH)2D3 on the PTH gene. This is the first report of calreticulin inhibiting a down-regulatory function of a sterol hormone and may help explain the refractoriness of the secondary hyperparathyroidism of many chronic renal failure patients to 1,25-(OH)2D3.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Calreticulin is a calcium-binding protein mainly present in the lumen of the endoplasmic reticulum of the cell that also has functions in other cellular compartments (1). Calreticulin’s cytoplasmic role was shown in fibroblasts from mice with homozygous deletions in the calreticulin gene (2). These mice had defects in integrin-mediated cell adhesion to extracellular matrix as well as the transient elevation in cytoplasmic calcium after integrin engagement (2). This cytoplasmic function is related to calreticulin’s interaction with the conserved amino acid sequence KLGFFKR found in the {alpha}-subunit of integrins (3, 4). Calreticulin may also have a nuclear function in that it modulates transcriptional activity of steroid hormone nuclear receptors (5, 6). It binds the same conserved protein motif KXFF(K/R)R found in the DNA-binding domain of nuclear hormone receptors of sterol hormones. In this way it prevents them from interacting with their DNA-responsive elements. There is recent evidence that the nuclear function may be dependent upon a calreticulin-dependent signaling from the endoplasmic reticulum (7), in addition to the sterol hormone-dependent enhanced nuclear localization of calreticulin (8). Calreticulin inhibited glucocorticoid- and androgen-mediated gene transcription (5, 6). In addition, it prevented the binding and action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on the osteopontin gene in vitro (9).

The chicken PTH vitamin D3 response element (cPTH VDRE) is located between -74 and -60 of the cPTH gene 5'-flanking region. It is composed of two imperfect direct repeats, GGGTCA and GGGTGT, that are separated by a 3-bp interval (10). Vitamin D receptor (VDR) binds to this responsive element primarily as a heterodimer, in the presence of retinoid X receptor-ß (RXRß) (10). In the rat PTH promoter there are two VDREs, of different affinities (11). The rat high PTH high-affinity VDRE (VDRE1) is located at -793 to -779 and is GGTTCA GTG AGGTAC (11). In transfection experiments the two negative VDREs were required for maximal inhibition of CAT transcription by 1,25-(OH)2D3 (11). In bovine parathyroid cells there is an additive response of retinoic acid with 1,25-(OH)2D3 to decrease PTH mRNA levels (12). In addition, Farrow and co-workers (13, 14) have identified DNA sequences upstream of the bovine PTH gene that bind the 1,25-(OH)2D3 receptor. Demay et al. (15) identified DNA sequences in the human PTH gene that bound the 1,25-(OH)2D3 receptor. When placed upstream to a heterologous viral promoter, the sequences contained in this 25-bp oligonucleotide mediated transcriptional repression in response to 1,25-(OH)2D3 in GH4C1 cells but not in ROS 17/2.8 cells. The down-regulatory element in the PTH promoter differs from up-regulatory elements both in sequence composition and in the requirement for particular cellular factors other than the VDR for repressing PTH transcription (15, 16, 17). The transcription of the PTH gene is markedly decreased by 1,25-(OH)2D3 both in vitro (18, 19) and in vivo (20, 21). This effect of 1,25-(OH)2D3 is used clinically in patients with chronic renal failure to decrease the synthesis and secretion of PTH (22, 23).

Therefore, the regulation of PTH gene transcription is relevant not only in understanding the physiology of the parathyroid gland, but also in the pathophysiology of diseases involving the parathyroid in which there is parathyroid hyperplasia such as chronic renal failure. There are many patients (>40% in some series) with chronic renal failure in whom administration of 1,25-(OH)2D3 does not decrease serum PTH levels (24). This may be related to a number of factors, such as decreased parathyroid levels of the VDR (25) and the calcium-sensing receptor (26, 27, 28), monoclonality of the parathyroid hyperplasia (29), hyperphosphatemia (23), or other factors that have not yet been defined.

Chronic hypocalcemia is an experimental model that simulates the resistance of the parathyroid to 1,25-(OH)2D3 (30). Chronic hypocalcemia leads to increased PTH gene expression despite very high serum 1,25-(OH)2D3 levels, which would be expected to decrease PTH mRNA levels (30). We have studied the role of calreticulin in this model in vitro and in vivo. We show in vivo that hypocalcemic rats had increased amounts of calreticulin protein in the nuclear fraction of their parathyroids and not in other tissues. In vitro, calreticulin blocked DNA binding of VDR-RXRß heterodimers to the cPTH VDRE. Overexpression of calreticulin in transiently transfected opossum kidney (OK) cells inhibited the effect of 1,25-(OH)2D3 on the cotransfected PTH promoter. These results suggest that calreticulin may prevent the transcriptional effect of 1,25-(OH)2D3 on the PTH promoter. This effect of calreticulin may help explain why 1,25-(OH)2D3 is ineffective in controlling the secondary hyperparathyroidism of many patients with chronic renal failure.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hypocalcemia Increases Calreticulin Protein in the Parathyroid
Weanling rats were fed a low-calcium diet for 2 weeks. This led to a marked hypocalcemia (6.5 ± 0.6: 11 ± 1.1 mg/dl (n = 4); P < 0.01) and increased serum 1,25-(OH)2D3 levels (845 ± 56: 145 ± 26 pg/ml; P < 0.01) as compared with controls. There was a marked increase in PTH mRNA levels in these hypocalcemic rats (Fig. 1AGo) despite the increased levels of endogenous 1,25-(OH)2D3 similar to our previous results (31). However, 1,25-(OH)2D3 given to normal rats as a single injection ip (20) or as a continuous dose by osmotic minipumps for 7 days resulted in a decrease in serum PTH and PTH mRNA levels (Fig. 1BGo). It is not known what prevents the parathyroid cell from responding to the high levels of endogenous 1,25-(OH)2D3 in the situation of hypocalcemia (30). Calreticulin has been shown to inhibit the binding of sterol hormone receptors to their DNA responsive elements and prevent their action on their target genes. We therefore studied whether calreticulin was involved in the paradoxical increase in PTH mRNA by a low serum calcium despite the high levels of 1,25-(OH)2D3.



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Figure 1. PTH mRNA Levels Are Increased by Hypocalcemia and Decreased by 1,25-(OH)2D3

A, PTH mRNA levels are increased by dietary hypocalcemia. Rats were maintained on a control diet or a low-calcium diet for 2 weeks after weaning. RNA was run on gels for Northern blots, and each lane represents thyroparathyroid RNA from a single rat. Hybridization was performed for PTH mRNA and 18S RNA as a control gene. B, PTH mRNA levels are decreased by 1,25-(OH)2D3. 1,25-(OH)2D3 was given to normal rats by constant infusion by minipumps (50 pmol/day) for 7 days. PTH mRNA is shown, and the ethidium bromide-stained membrane shows the 28S and 18S rRNA. Each lane represents thyroparathyroid RNA from a single rat.

 
We studied the levels of calreticulin protein in parathyroid protein extracts from rats fed a normal or a low-calcium diet. A Western blot of nuclear and cytoplasmic proteins showed that in the nuclear fraction of the parathyroids of rats fed a low-calcium diet, there was a 2-fold increase in calreticulin level (Fig. 2Go). There was no change in the cytoplasmic calreticulin in these rats. Similar results were obtained in two repeat experiments of different parathyroid protein preparations. Other tissues, such as liver, kidney, and brain showed no differences in calreticulin concentration in their nuclear fractions between control and hypocalcemic rats (not shown). Because of this increase in calreticulin concentration in the nuclear fraction of the parathyroids of hypocalcemic rats, we studied whether calreticulin affected the binding of the VDR to the PTH-VDRE.



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Figure 2. Nuclear Calreticulin Protein Is Increased in Parathyroids of Hypocalcemic Rats

Western blot for calreticulin protein in rat parathyroids fed a normal or a low-calcium diet. Calreticulin protein was increased in the nuclear fraction (N) of hypocalcemic rats compared with control rats, but there was no change in calreticulin protein in the cytoplasmic fraction (C) of these rats.

 
Calreticulin Prevents Binding of the VDR-RXRß Heterodimer to the PTH VDRE Oligonucleotide
The VDR binds as a heterodimer with the RXRß to VDREs (10). We therefore studied the effect of calreticulin on VDR-RXRß heterodimer binding to the VDRE of the cPTH promoter. In gel shift assays VDR and RXRß bound an oligonucleotide corresponding to the chicken VDRE (cVDRE) (Fig. 3Go) or rat VDRE (not shown) forming two complexes (Fig. 3Go). VDR added to the cVDRE displayed binding (not shown) that was enhanced when the VDR was added together with RXRß (Fig. 3Go). Surprisingly, RXRß alone also bound the VDRE. MacDonald et al. (12) showed that when 1,25-(OH)2D3 was added to bovine parathyroid cells in primary culture, there was an additive effect of 9-cis-retinoic acid to decrease PTH mRNA levels, which would be compatible with the enhanced binding of the combined VDR and RXRß to the cVDRE. The binding of the VDR-RXRß heterodimer to the cPTH-VDRE was inhibited by pure calreticulin in a dose-dependent manner (Fig. 3Go). Calreticulin has three distinct structural domains, referred to as the N, P, and C domains (5). Burns et al. (5) showed that calreticulin prevented the glucocorticoid receptor binding to the glucocorticoid response element, and that this was mediated by the N domain and not the P domain of calreticulin. The C domain of calreticulin led to enhanced binding of the glucocorticoid receptor to the glucocorticoid response element (5) and the reason for that finding was not clear. In the present study, the addition of both the glutathione-S-transferase-N (GST-N) or GST-P fusion proteins (Fig. 3Go) or the GST protein (not shown) did not affect the binding of VDR-RXRß to the cVDRE oligonucleotide in mobility shift assays (Fig. 3Go). The reason why both the N and the P domains of the calreticulin protein did not inhibit binding of the VDR-RXRß to the VDRE while the full-length calreticulin did inhibit binding is not clear. It may be because of S-S bridges or folding differences between the full-length calreticulin and its separate domains. Our results, therefore, do not allow us to draw a conclusion as to which domains in calreticulin interact with the VDR-RXRß complex.



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Figure 3. Calreticulin Inhibits Binding of the VDR-RXRß Heterodimer to the cVDRE Oligonucleotide

VDR-RXRß was incubated without or with increasing amounts of intact calreticulin or the calreticulin N and P domains linked to GST after which the cVDRE probe was added. VDR-RXRß formed a complex with the cVDRE resulting in two bands indicated by the arrows. Added calreticulin, in increasing concentrations, inhibited the binding of the heterodimer to the cVDRE. Addition of GST-N domain of calreticulin or GST-P domain of calreticulin did not affect the binding.

 
We have shown that in vitro calreticulin interferes with the binding of the VDR-RXRß heterodimer to the VDRE oligonucleotide. A similar effect had been shown for the osteopontin gene shown by Wheeler et al. (9) who demonstrated that calreticulin prevented binding of the VDR-RXR heterodimer to the mouse osteopontin VDRE. Together with the present results, this shows that calreticulin inhibits DNA binding by the VDR in vitro. This effect may be due to binding of the calreticulin protein to the KXFF(K/R)R-like motif present in the DNA-binding domain of the receptor. This is the first demonstration that calreticulin can interfere with a down-regulatory effect of a sterol hormone on gene transcription. To determine a functional role for the effect of calreticulin on the response of the PTH promoter to 1,25-(OH)2D3, we performed transfection experiments.

Calreticulin Inhibits the Transcriptional Effect of 1,25-(OH)2D3 on the PTH VDRE
Opossum kidney (OK) cells were transiently transfected with the cVDRE-CAT construct. In the presence of 1,25-(OH)2D3 (1 x 10-8 M), CAT expression, as assessed by ribonuclease (RNase) protection assay, was reduced by 75% compared with cells that were not treated with 1,25-(OH)2D3 (Fig. 4Go, A and C) and (10). Overexpression of calreticulin, by using a sense orientation expression vector cotransfected with the cPTH gene promoter-CAT construct, completely abolished the effect of 1,25-(OH)2D3 on the PTH promoter (Fig. 4Go, B and C). Cotransfection of the VDRE-CAT plasmid with a plasmid transcribing antisense calreticulin had no effect on the down-regulation of CAT transcription by 1,25-(OH)2D3 (Fig. 4Go, B and C). Similar results were obtained with the rat PTH VDRE (not shown). In all experiments, expression of a cotransfected cytomegalovirus (CMV)-ß-galactosidase plasmid was used as a control, and the results were normalized for ß-galactosidase expression (Fig. 4Go). To verify that the calreticulin cDNA was being expressed, a portion of the RNA from the transfected cells was analyzed by Northern blots. A calreticulin transcript of the correct size was easily detectable in cells with the overexpressed calreticulin plasmids, whereas it was undetectable in nontransfected cells (not shown). These results show that overexpression of calreticulin prevents the effect of 1,25-(OH)2D3 to down-regulate CAT expression from a PTH-VDRE promoter. This is the first demonstration that calreticulin can prevent a down-regulatory effect of 1,25-(OH)2D3. It has been shown that calreticulin prevents the up-regulation by 1,25-(OH)2D3 of the osteopontin gene transcription (9). Calreticulin’s effect on the VDRE has been shown to be specific to the VDRE as it had no effect on the transcription of other promoter elements, such as the cAMP response element (9). There are other examples in which calreticulin overexpression has been shown to inhibit a biological effect, such as the decreased expression of dexamethazone-sensitive cytochrome P450 protein and mRNA (5). In addition, the overexpression of calreticulin and androgen receptor in Vero fibroblasts transfected with the androgen-responsive element-CAT construct, inhibited CAT activity induced by androgens (6). The present results, therefore, support the notion that calreticulin can modify the action of sterol hormones on their target genes.



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Figure 4. Calreticulin Inhibits the Transcriptional Effect of 1,25-(OH)2D3 on the PTH Promoter

A, Ribonuclease protection analysis of gene transcripts for CAT and ß-galactosidase of total RNA from OK cells that were transiently transfected with cPTH VDRE-CAT plasmid and the ß-galactosidase plasmid and then treated without (-D3) or with (+D3) 1,25-(OH)2D3 (1 x 10-8 M). B, The same as in panel A but in addition the cells were cotransfected with a plasmid that expressed the antisense transcript for calreticulin (lanes 1 and 2) or cotransfected with a plasmid that expressed the sense orientation of the calreticulin cDNA (lanes 3 and 4). The labels -D and +D indicate the absence or presence of 10-8 M 1,25-(OH)2D3 in the cell incubation medium, respectively. C, Quantification of the effect of calreticulin antisense and sense expression on the regulation of cPTH VDRE CAT gene transcription by 1,25-(OH)2D3. The results are expressed as CAT expression normalized for ß-galactosidase expression measured by RNase protection assays. The mean ± SEM are shown for four separate experiments in which each experimental condition was run in triplicate. Significant differences (P < 0.05) between experimental and control levels are denoted by an asterisk.

 
The effect of hypocalcemia to increase PTH mRNA levels in vivo is mainly posttranscriptional (31), while the effect of 1,25-(OH)2D3 is transcriptional (20). Therefore, there must be a mechanism whereby the effect of hypocalcemia dominates over that of 1,25-(OH)2D3. Hypocalcemic rats have an increase in their PTH mRNA levels despite increased levels of 1,25-(OH)2D3 that would have been expected to decrease PTH gene transcription. This paradox implies that there is a factor that prevents the effect of 1,25-(OH)2D3 on the PTH gene. It is possible that calreticulin might be that factor. In the present study rats with hypocalcemia had an increased calreticulin concentration in the nuclear fraction of their parathyroids. This result is given a functional relevance by the findings in vitro in the present studies in which calreticulin prevented binding of the VDR-RXRß to the PTH-VDRE and where cotransfected sense calreticulin prevented the effect of 1,25-(OH)2D3 to decrease VDRE-CAT transcription.

Many patients with chronic renal failure treated by chronic dialysis do not respond as expected to administration of 1,25-(OH)2D3 (24). In some patients this may be related to monoclonal replication of the parathyroids (29) or down-regulation of the VDR (25), but this certainly does not apply to all such resistant patients. It is possible that in this subset of patients there is an increase in the calreticulin protein in their parathyroids. The model used in the present study was secondary hyperparathyroidism due to hypocalcemia. This model simulates many of the features of secondary hyperparathyroidism of renal failure such as increased levels of PTH mRNA and serum PTH (30) as well as increased parathyroid cell proliferation (32). In addition, the high levels of serum 1,25-(OH)2D3 provide an opportunity to understand the resistance of the parathyroid to 1,25-(OH)2D3 in many patients with secondary hyperparathyroidism. We have shown that in vivo these hypocalcemic rats have an increase in calreticulin protein in their nuclear fraction. In addition, calreticulin inhibited VDR-RXRß binding to the PTH VDRE. Overexpression of calreticulin inhibited the effect of 1,25-(OH)2D3 on VDRE-CAT expression in transfected cells. These results suggest a functional role for calreticulin in the down-regulation of the PTH gene transcription by 1,25-(OH)2D3.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals
Weanling male Hebrew University strain rats were maintained on either a normal calcium (0.6%) diet or a low-calcium (0.02%) (-Ca) diet (Teklad, Madison, WI) for 2 weeks. For the experiment with 1,25-(OH) 2D3, rats of 150 g body weight were implanted with osmotic minipumps sc and received 1,25-(OH)2D3 (50 pmol/day) or propylene glycol vehicle for 7 days. The thyroparathyroid tissue was removed under pentobarbital anesthesia and blood samples were taken for serum calcium and 1,25-(OH)2D3. All animal studies were conducted in accord with the principles and procedures outlined in the "Guidelines for Care and Use of Experimental Animals." For RNA extractions, thyroparathyroid tissue from individual rats was frozen in liquid nitrogen and stored at -70 C until analysis. RNA was extracted from the thyroparathyroids of each rat and analyzed by Northern blots for PTH mRNA and 18S ribosomal RNA as previously described (33). For protein extraction, pooled microdissected parathyroids from 10 rats in each group, or other tissues from individual rats, were removed, and proteins from the nuclear and cytoplasmic fractions were extracted immediately by the method of Dignam et al. (34).

Western Blot Analysis
Forty micrograms of the nuclear and cytosolic extracts were run on 10% SDS-polyacrylamide gel. The gels were transferred to nitrocellulose membrane and incubated with polyclonal goat anticalreticulin as first antibody (dilution of 1:300 in 1% BSA-1x PBS) and then with horseradish peroxidase-rec Protein G (Zymed Laboratories, South San Francisco, CA) as second antibody. The peroxidase reaction was developed using an ECL detection system (New England Biolabs, Beverly, MA).

DNA Mobility Shift Assay
Recombinant human (h) VDR and hRXRß were diluted 1:10 in ice-cold KTEDG buffer (400 mM KCl, 20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM EDTA, 10% glycerol, 2 mM dithiothreitol), 1.6 µl of the diluted proteins were used in 1x binding buffer (100 mM KCl, 20 mM Tris, pH 7.5, 1.5 mM EDTA, 5% glycerol, 0.1% NP 40, 2 mM dithiothreitol), at a final volume of 20 µl, which included 1 µg deoxyinosinic-deoxycytidylic acid and the end-labeled VDRE DNA fragment probe (30,000 cpm). The reaction was incubated on ice for 1 h, loaded on a cooled prerun 5% nondenaturating polyacrylamide gel and run for 3 h in 0.5x Tris-borate-EDTA buffer at 4 C. The gels were dried and exposed to x-ray film. The chicken cPTH VDRE (bold letters) consisted of 5'-gatcGGAAGCTTACATGAGGGTCAGGAGGGTGTGCCT-GCAGG-3' 3'-CCTTCGAATGTACTCCCAGTCCTCCCACACGG-ACGTCCctag-5'

and was end-labeled with Klenow large-fragment enzyme using [{alpha}-32P[{rho}{varsigma}{theta}ß]dGTP.

When the binding of the VDR-RXRß heterodimer to the VDRE was performed with increasing concentrations of calreticulin and its N and P domains or the GST protein, they were incubated at 4 C for 15 min before addition of the VDRE probe.

Plasmid Constructions
The cPTH promoter sequence, -55 to +20 was synthesized by PCR and ligated into a commercially available p-CAT expression vector (Promega, Madison WI) using the SalI and XbaI restriction sites present in the multiple cloning site. An oligonucleotide containing the cPTH VDRE, GGGTCA GGA GGGTGT, was placed upstream from the cPTH promoter using the HindIII and SalI sites that were available. The calreticulin expression plasmid in the sense orientation was prepared by subcloning the XhoI-SmaI fragment of the pSVL-CRT plasmid (5) into pMCC-ZP plasmid containing a CMV promoter. The calreticulin expression plasmid in the antisense orientation was prepared as described (5). Plasmids that expressed either the sense or antisense transcripts for the calreticulin gene were synthesized as described previously (5).

Transfection Studies
Opossum kidney (OK) cells were cotransfected by the method of lipofection (35) with the cPTH VDRE-CAT plasmid (2.5 µg), CMV-ß-galactosidase (1 µg) plasmid, and either the sense or antisense plasmid for calreticulin (1 µg). Analysis of CAT and ß-galactosidase gene transcripts after treatment with hormone (1 x 10-8 M) for 24 h were quantified by ribonuclease protection assay using a commercially available kit (Ambion, Austin TX). Total RNA from OK cells transfected with the cPTH-CAT and CMV-ß-galactosidase plasmid constructs was prepared by extraction with phenol-guanidinium thiocyanate. The RNA extracts were incubated with RNase-free DNase I (GIBCO-BRL, Bethesda MD) for 5 min at room temperature and ethanol precipitated. The RNA pellets were incubated with 32P-labeled RNA probes (158 bases for CAT and 370 bases for ß-galactosidase) in 20 µl of buffer containing 80% formamide, 100 mM sodium citrate, pH 6.4, 300 mM sodium acetate, pH 6.4, and 1 mM EDTA, overnight at 43 C. The hybridization mixtures were digested with a combination of RNase A and RNase T1 at 37 C for 30 min. The protected RNA hybrids were precipitated with propanol-guanidinium thiocyanate and separated on 6% nondenaturing polyacrylamide gels. The gels were dried and exposed to x-ray film for 6 h at -80 C. The resulting autoradiograms were quantified by densitometric scanning, and values for the CAT gene transcripts were normalized with respect to the values for ß-galactosidase gene transcripts. To verify that the calreticulin genes were being expressed in the experiments where calreticulin expression vectors were used, a portion of the RNA from the transfected cells was analyzed by Northern blots. In all cases a calreticulin transcript of the correct size was observed.


    ACKNOWLEDGMENTS
 
We thank Ms. Miriam Offner for excellent technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Justin Silver, M.D., Ph.D., Nephrology Services, P.O. Box 12000, Jerusalem, Israel 91120.

Research support was provided for these studies by the Israel Academy of Sciences (to T.N-M), NIH Grant RO1-DK38422 (to J.R.), University of Kentucky Medical Center Research Fund (to N.J.K.), and the Medical Research Council of Canada (M.M.).

Received for publication September 3, 1997. Revision received March 9, 1998. Revision received April 22, 1998. Accepted for publication April 23, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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