Calcium-sensing Receptor Gene Transcription Is Up-regulated by the Proinflammatory Cytokine, Interleukin-1β

The calcium-sensing receptor (CASR) in parathyroid, thyroid, and kidney is essential for calcium homeostasis. Hypocalcemia is common in critically ill patients having increased circulating proinflammatory cytokines, although the causes are unknown. We hypothesized that the cytokines increase CASR expression and reduce the set point for parathyroid hormone suppression by extracellular calcium, leading to hypocalcemia and hypoparathyroidism. Here, we show in vivo in the rat that parathyroid, thyroid, and kidney CASR mRNA and protein increased after injection of interleukin-1β. This was associated with decreased circulating parathyroid hormone, calcium, and 1,25-dihydroxyvitamin D levels. Interleukin-1β stimulated endogenous CASR gene transcripts and transfected promoter reporter activity in human thyroid C-cells (TT cells) and kidney proximal tubule (HKC) cells. Cotransfection of NF-κB proteins enhanced activity of the reporter constructs, whereas cotransfection with inhibitor-κB or application of an NF-κB nuclear localization sequence peptide abrogated responsiveness to cytokine or NF-κB proteins. Mutagenesis of some, but not all, of the potential κB elements in the 5′ part of the CASR gene led to loss of responsiveness to cytokine. These elements conferred cytokine responsiveness to a heterologous promoter, and in electrophoretic mobility shift assays, NF-κB complexes formed on the same three κB elements. In summary, the CASR gene has several functional κB elements that mediate its upregulation by proinflammatory cytokines and probably contribute to altered extracellular calcium homeostasis in the critically ill.

and the cells lining the kidney tubule (1,2). By virtue of its ability to sense small changes in circulating calcium concentration and couple this information to intracellular pathways that modify PTH and calcitonin secretion or renal cation handling, the CASR plays an essential role in maintaining mineral ion homeostasis. An inverse sigmoidal curve describes the relationship between extracellular ionized calcium and PTH concentration. The activity and/or expression level of the CASR dictates the extracellular calcium set point, this being defined as the extracellular calcium concentration for half-maximal stimulation of PTH secretion from the parathyroid gland or calcium reabsorption across the kidney tubule.
The human CASR gene, located on chromosome 3q13. 3-21 (3), has two promoters (P1 and P2) yielding alternative transcripts containing either exon 1A or exon 1B 5Ј-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site (4 -6). Transcriptional start sites for both promoters have been identified; that for P1 lies downstream of a TATA box, whereas that for P2, which lacks a TATA box, lies in a GC-rich region (6). By both nuclear run-on and promoter/reporter gene transfection studies, P2 appears to be the stronger promoter (6). Vitamin D response elements that confer responsiveness to the hormonally active secosteroid, 1,25-dihydroxyvitamin D (1,25(OH) 2 D) are present in both promoters (6). Other functional cis-acting elements in the CASR gene have yet to be identified.
Hypocalcemia is common in critically ill patients, especially those with sepsis and major burn injury (7)(8)(9)(10) and in nonacutely ill patients undergoing surgery (11,12). Animal studies indicate that administration of endotoxin, bacteria, and proinflammatory cytokines results in a lowering of ionized calcium concentrations (13,14). The mechanisms underlying the hypocalcemia are not known. Several factors may be involved, including decreased secretion of PTH (15), although both increased and decreased concentrations of PTH have been reported (16). There may be resistance to the action of PTH in the kidney and bone, leading to increased urinary calcium excretion and altered bone formation. There is hypomagnesemia and metabolism and function of vitamin D is impaired (15). It was speculated that the hypomagnesemia contributed to the defective PTH secretion and target organ resistance. However, the hypoparathyroidism is persistent following magnesium repletion in burn-injured children, making this explanation unlikely (17). Calcitonin precursors are increased in the circulation of critically ill patients with sepsis and could contribute to the hypocalcemia (16,18,19). In addition, the serum concentra-tions of proinflammatory cytokines such as interleukin-1␤ (IL-1␤) and tumor necrosis factor-␣ (TNF-␣) are markedly increased in patients with burn injury or sepsis and are inversely related to the calcium concentration (16). Serum procalcitonin levels increase after endotoxin injection in normal human subjects (20).
Nielsen et al. (21) showed that the cytokine IL-1␤ increased CASR mRNA levels in cultured bovine parathyroid gland fragments. Murphey et al. (22) used a sheep model of burn injury to demonstrate that the hypocalcemia and hypoparathyroidism that follow severe burn injury are related to up-regulation of parathyroid gland CASR expression. This would be expected to reduce the set point for PTH suppression by extracellular calcium. We hypothesized that the hypocalcemia of critically ill patients is mediated, in part, by proinflammatory cytokines such as IL-1␤ and TNF-␣ acting to up-regulate CASR gene transcription in tissues important for calcium homeostasis.
Cytokines, like IL-1␤ and TNF-␣, work through NF-B, which was discovered as a nuclear factor necessary for immunoglobulin light chain transcription in B cells (23). NF-B exists in the cytoplasm of most cells as an inactive form bound to an inhibitor, IB. Upon receipt of a signal generated by activation of cytokine receptors, IB is phosphorylated, and NF-B is released from IB and translocates to the nucleus to up-regulate specific gene transcription. NF-B is responsible for the inducible expression of many immune and inflammatory response (and other) proteins (24 -26).
NF-B is a dimer of members of the Rel family of proteins, which includes p50, p65, and c-Rel. Each one has a conserved NH 2 -terminal Rel homology domain responsible for DNA binding, dimerization, and interaction with IB family members. Most NF-B proteins are transcriptionally active, but some combinations are inactive or repressive. Thus, p50/p65, p50/c-Rel, p65/p65, and p65/c-Rel are transcriptional activators, whereas the p50/p50 homodimer is a repressor. Each Rel protein contacts one-half of the DNA binding site, which is a variation on the 10-bp consensus sequence 5Ј-GGG(T/A)(A/ C)TTTCC-3Ј. A variable COOH-terminal domain found in activating but not in the repressive Rel proteins is probably responsible for transactivation (24,25).
In the present study, we have shown that IL-1␤ up-regulates parathyroid, thyroid, and kidney CASR mRNA (and corresponding CASR protein in thyroid and kidney) levels in vivo in the rat and that the proinflammatory cytokine stimulates the endogenous CASR gene transcription in human thyroid and kidney cell lines. In addition, we have shown that IL-1␤ and TNF-␣ up-regulate CASR gene transcription via NF-B and identified functional B response elements in both promoters of the human CASR gene.
Animals and Experimental Procedures-Normal male Sprague-Dawley rats (Charles River Laboratories, Inc., St. Constant, Canada) weighing 180 -200 g when received, were fed a standard rodent chow (Ralston Purina Co., LaSalle, Canada) containing 1.01% calcium, 0.74% phosphorus, and 3.3 IU vitamin D 3 /g. All animal experiments were carried out in compliance with, and were approved by, the institutional animal care and use committee. Rats were injected intraperitoneally, at 24, 16, 12, 8, or 4 h before death, with either vehicle (propylene glycol, 0.2 ml/100 g, body weight) or 0.75 g of IL-1␤/100 g, body weight. Blood was obtained by cardiac puncture, and the serum was separated and stored at Ϫ20°C. The rats were anesthetized with pentobarbital, the kidneys were taken, and the parathyroid and thyroid glands were microdissected separately and quick frozen. Sera were analyzed for calcium (Sigma kit) and PTH (rat intact PTH ELISA kit, Immunotopics, San Clemente, CA) and 1,25(OH) 2 D 3 (immunoextraction and radioimmunoassay kit; IDS Ltd., Bolton, UK).
RNA Extraction-Total RNA was prepared from cells or tissues using TRIzol (Invitrogen) according to the manufacturer's instructions.
Ribonuclease Protection Assay of Rat CASR mRNA-For the CASR riboprobe, a 232-bp fragment of a rat CASR cDNA (28) was PCRamplified (forward primer, 5Ј-ACCTTGAGTTTTGTTGCCCA-3Ј (in exon 3); reverse primer, 5Ј-GGAATGGTGCGGAGGAAGGATT-3Ј (in exon 4)) and cloned into the PCR2.1 vector. For the actin riboprobe that protects a 126-nucleotide transcript, the pTRI-␤-actin-125 rat vector (Ambion Inc., Austin, TX) was used. After linearization of the vectors, the antisense probes were in vitro transcribed with T7 polymerase incorporating [␣-32 P]UTP using a MAXI script kit, and the gel-purified riboprobes were used with an RPA III kit (Ambion Inc.). Each probe (2.5 ϫ 10 5 cpm) was hybridized overnight with 2-25 g of total RNA followed by digestion with a ribonuclease A:T1 mix (29). Protected fragments were resolved on 6% acrylamide denaturing gels and exposed to x-ray film.
Western Blot Analysis of the CASR-Tissues were lysed in triple detergent buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% NaN 3 , 0.1% SDS, 1 mM EDTA, 100 g/ml phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 2 g/ml aprotonin, 0.1% Nonidet P-40, 0.5% sodium deoxycholate) for 5 min at 0°C. The lysates were spun at 1200 ϫ g for 2 min at 4°C, and the supernatants were stored at Ϫ80°C. Aliquots were electrophoresed through 4 -12% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were rinsed in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20, blocked with 5% dried milk powder in TBST for 3 h, and incubated with affinity-purified CASR antibody AS2011, raised against a peptide comprising CASR amino acids 215-235 coupled to keyhole limpet hemocyanin. As a control, immunoblotting was carried out as described above with the antibody preadsorbed for 1 h with the peptide (10 g/ml) against which it was raised. Antibody antigen complexes were detected by chemiluminescence using the Lumi-glo kit (Invitrogen). Membranes were reprobed with a tubulin antibody as a loading control.
Human CASR Gene Promoter Constructs-The construction of the P1-luciferase reporter plasmid (designated here as P1-WT) containing the P1 promoter, exon 1A, and the 5Ј part of exon 2 to nucleotide Ϫ1 (nucleotide ϩ1 is the A of the ATG initiation codon), upstream of the luciferase reporter gene in pGL3 basic, has been described previously (6). The P1-Mut and 1A-Mut constructs were generated by the overlap extension site-specific mutagenesis method (31), using P1-WT as template. For P1-Mut, the primer sets used were as follows: F1 (5Ј-AG-CATTTGCTCAGCTCCTTCTCTA-3Ј with an EspI site in boldface type), RM (5Ј-AGTCAAGGCCCGTTAACTGTATAG-3Ј with mutated residues in boldface type to generate product 1), and FM (5Ј-CTATA-CAGTTAACGGGCCTTGACT-3Ј with mutated residues in boldface type), and R2 (5Ј-CAAACTGTCAACAGTAGGAAATT-3Ј with a HincII site in boldface type to generate product 2). The overlapping fragments were mixed, denatured, and amplified with primers F1 and R2 to generate product 3 that was cleaved with EspI and HincII and cloned into the EspI/HincII-digested P1-WT plasmid to create P1-Mut. For 1A-Mut, the primer sets used were as follows: F1 (5Ј-AGCATTTGCT-CAGCTCCTTCTCTA-3Ј with an EspI site in boldface type, RM (5Ј-CCCTTGCGTCCATGGACAGAACAA-3Ј with mutated residues in boldface type to generate product 1), FM (5Ј-TTGTTCTGTCCATG-GACGCAAGGG-3Ј with mutated residues in boldface type), and R2 (5Ј-CACAGCGCGCTGCTTGGAGGCC-3Ј with a BssHII site in boldface type to generate product 2). The overlapping fragments were mixed, denatured, and amplified with primers F1 and R2 to generate product 3 that was cleaved with EspI and BssHII and cloned into the EspI/BssHII-digested P1-WT plasmid to create 1A Mut. The P1/1A Mut was created by subcloning the HincII-BssHII fragment from 1A Mut into HincII/BssHII-cleaved P1 Mut.
The construction of the P2-luciferase reporter plasmid (designated here as P2-WT) containing the P2 promoter, exon 1B, and the 5Ј part of exon 2 to nucleotide Ϫ1, upstream of the luciferase reporter gene in pGL3 basic, has been described previously (6). To create the P2-Mut plasmid, the overlap extension method was used with primer sets F1 (5Ј-GCGCCCTAAGCTTCTTTCCATCG-3Ј with a HindIII site in boldface type) and RM (5Ј-CCGAGCCATTTGTGAACGGCTCTC-3Ј with mutated residues in boldface type) to generate product 1 and FM (5Ј-GAGAGCCGTTCACAAATGGCTCGG-3Ј with mutated residues in boldface type) and R2 (5Ј-AGAGAGCTTAGCTGACTCTTCAG-3Ј with an EspI site in boldface type) to generate product 2. The overlapping fragments were mixed, denatured, and amplified with primers F1 and R2 to generate product 3 that was cleaved with HindIII and EspI and cloned into the HindIII/EspI-digested P2-WT to create P2-Mut.
To construct the P1-heterologous promoter luciferase reporter plasmids, for SV40 P1 NF-B WT and SV40 P1 NF-B Mut, 110-bp products containing an NF-B element were amplified using P1-WT and P1-Mut as templates with forward primer (5Ј-atgctGCTAGCCCATTGAA-GAAAAGAAATGGAAACT-3Ј) and reverse primer (5Ј-tctgaCTCGAG-GTGATAACATCTACCTCAGGGGGTCCT-3Ј), where the nucleotides in boldface type are an NheI or XhoI site, and the lowercase nucleotides are those added to ensure complete restriction enzyme cleavage. The PCR products were digested with NheI and XhoI and cloned into the pGL3-Promoter vector (Promega) upstream of the SV40 promoter to generate constructs SV40 P1 NF-B WT and SV40 P1 NF-B Mut. To generate constructs SV40 1A NF-B WT and SV40 1A NF-B Mut, 116-bp products containing an NF-B element were amplified using P1-WT and P1-Mut as templates with forward primer (5Ј-atgctGCTAG-CATTTTCTGTAACAAATGATCCTGCTAA-3Ј) and reverse primer (5Ј-tctgaCTCGAGAACACCAAAAATATTATCCCCTTTAA-3Ј), where the nucleotides in boldface type represent an NheI or XhoI site. The PCR products were digested with NheI and XhoI and cloned into the pGL3-Promoter vector upstream of the SV40 promoter to create constructs SV40 1A NF-B WT and SV40 1A NF-B Mut. To construct SV40 P2 NF-B WT and SV40 P2 NF-B Mut, 108-bp products containing an NF-B element were amplified using P2-WT and P2-Mut as templates with forward primer (5Ј-atgctGCTAGCCAAGGAGTAGGGTCAGG-GAAGAG-3Ј) and reverse primer (5Ј-tctgaCTCGAGAGACGGCGAGC-CCCGCGCTTAGGTC-3Ј), where the nucleotides in boldface type represent an NheI or XhoI site. The PCR products were digested with NheI and XhoI and cloned into the pGL3-Promoter vector upstream of the SV40 promoter to create constructs SV40 P2 NF-B WT and SV40 P2 NF-B Mut.
To construct SV40 E2 NF-B WT, a 112-bp product containing a putative NF-B element was amplified using P2-WT as template with forward primer (5Ј-atgctGCTAGCTCTGTAGACATGTGTGTCCCCAC-TGCAG-3Ј) and reverse primer (5Ј-tctgaCTCGAGGGTTCTGCCGTCT-CTCCAGGGCAAGG-3Ј), where the nucleotides in boldface type represent an NheI or XhoI site. The PCR product was digested with NheI and XhoI and cloned into the pGL3-Promoter vector upstream of the SV40 promoter to create construct SV40 E2 NF-B WT.
Cell Culture-HKC cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. TT cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and 5% horse serum. All maintenance medium contained 100 units/ml penicillin, 100 g/ml streptomycin, and 250 ng/ml amphotericin B.
Transfection and Reporter Assay-For transient transfection, cells were trypsinized, plated in 6-well dishes in Dulbecco's modified Eagle's medium, 10% fetal bovine serum (1-4 ϫ 10 5 cells/well), and incubated overnight. The next day, cells were transfected with 30 g/well of Superfect reagent with 1 g of CASR promoter construct and 0.5 g of pCH110 per well. The following day, cells were serum-starved in Dulbecco's modified Eagle's medium overnight and cultured with or without cytokines for 10 h. The cells were washed in PBS and lysed in 250 l of lysis buffer (1% Triton X-100, 15 mM MgSO 4 , 4 mM EGTA, 1 mM DTT, 25 mM glycyl-glycine) on ice. Luciferase activity was measured in an EG & G Berthold luminometer using 45 l of cell lysate and Dluciferin. Luciferase activity was normalized to ␤-galactosidase activity. Nuclear Extracts of HKC Cells-Cells were washed, scraped into 1 ml of PBS, and centrifuged at 1500 ϫ g for 10 min at 4°C. Cell pellets were processed in a loose Dounce tissue homogenizer in 2 volumes of buffer (25 mM Tris, pH. 7.9, 0.3 mM DTT, 420 mM NaCl, 10 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and protease inhibitors). Nuclear pellets were obtained by centrifugation (25,000 ϫ g for 20 min at 4°C); resuspended in 20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT; and Dounce homogenized. After a 20-min centrifugation at 25,000 ϫ g, nuclear extracts (supernatants) were dialyzed for 5 h against 20 nM HEPES, pH 7.9, 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM DTT. Protein content was determined, and aliquots were stored at Ϫ80°C.
Electrophoretic Mobility Shift Assay-Two micrograms of nuclear extract were incubated for 20 min on ice in the absence or presence of 10 Ϫ8 M 1,25(OH) 2 D 3 and 1 g of poly(dI⅐dC) in binding buffer (25 mM Tris-HCl, pH 8.0, 50 mM KCl, 15% glycerol, 0.5 mM DTT). Antibodies were then added or not, and samples were incubated for 20 min at room temperature. Five femtomoles of 32 P-end-labeled double-stranded oli-gonucleotides were added and incubated for a further 20 min. Oligonucleotides used for EMSA are detailed in Table I. Samples were electrophoresed at 8 V/cm on 6% nondenaturing polyacrylamide gels equilibrated in 0.25 M Tris, pH 8.3, 1.9 M glycine, 10 mM EDTA; dried; and autoradiographed.
Statistics-Data are expressed as mean Ϯ S.E. The results from the in vivo IL-1␤ response studies were initially subjected to analysis of variance. The significance of differences from base line was then determined using Dunnett's multiple comparison test. For the nuclear run-on assays and luciferase transient transfection assays, comparisons were performed by Student's t test. A p value of Ͻ0.05 was taken to indicate a statistically significant difference.

RESULTS
Interleukin-1␤ Decreases Serum PTH, Calcium, and 1,25(OH) 2 D Levels in Vivo-To examine the effect of IL-1␤ on extracellular calcium homeostasis, the cytokine was administered to rats and circulating PTH, and calcium levels were monitored over a 24-h period. After a single intraperitoneal injection of IL-1␤ in rats, serum PTH and 1,25(OH) 2 D 3 levels were significantly decreased at 12, 15, and 24 h, and serum calcium levels were significantly decreased at 15 and 24 h relative to basal levels (Fig. 1).
Interleukin-1␤ Up-regulates Parathyroid, Thyroid, and Kidney CASR mRNA Levels in Vivo-To assess whether the alter- ations in circulating PTH and calcium levels brought about by IL-1␤ could be due to altered CASR expression in those tissues important for regulation of extracellular calcium homeostasis, we first measured CASR mRNA levels by ribonuclease protection analysis throughout the 24-h period. After the injection of IL-1␤ in rats, parathyroid, thyroid, and kidney CASR mRNA levels rose significantly above basal level to peak at 15 h, and the levels were still elevated at 24 h (Fig. 2, A-C). The peak values relative to basal level varied from one tissue to the other, being 2.2-fold (parathyroid), 2.8-fold (thyroid), and 1.5fold (kidney). Injection of vehicle had no effect on CASR mRNA levels (data not shown).
Interleukin-1␤ Up-regulates Thyroid and Kidney CASR Protein Levels in Vivo-To evaluate whether the changes observed in CASR mRNA levels were reflected in corresponding changes in CASR protein levels, these were measured by immunoblot analysis. The complex pattern observed is due to the CASR existing in both monomeric and dimeric forms. The monomeric core glycosylated (immature) species is 140 kDa, and the mature, fully glycosylated species is 160 kDa (Fig. 2, D and F). The higher molecular mass forms are likely to be dimers (32). After the injection of IL-1␤ in rats, thyroid and kidney CASR protein levels (related to the ␤-tubulin levels) rose ϳ3-fold or ϳ2-fold, respectively, over basal level at 24 h (Fig. 2, E and G). Injection of vehicle had no effect on CASR protein levels (data not shown). Parathyroid CASR protein levels were not assessed because of the small amount of tissue available.
Interleukin-1␤ Increases CASR Gene Transcription-To assess whether the changes in CASR mRNA levels were occurring at the level of gene transcription, nuclear run-on assays were performed on extracts of human TT cells and HKC cells cultured with and without IL-1␤ for 8 and 12 h. CASR gene exon 1A, exon 1B, and exon 2 transcripts were all stimulated Ͼ2-fold, as was COX-2 gene transcription in both cell types ( Fig. 3; data not shown). Glyceraldehyde-3-phosphate dehydrogenase gene transcription was unaffected by IL-1␤. Thus, IL-1␤ specifically stimulates endogenous CASR gene transcription.

FIG. 3. Induction of CASR gene transcription by IL-1␤.
Nuclear run-on assays were performed as described under "Experimental Procedures" on nuclei of human TT cells (A) and HKC cells (B). Autoradiographs of representative experiments repeated three times are shown. Densitometry was performed, and relative transcription rates were calculated, taking that for CASR exon 1A as 100%.

FIG. 4. Activity of the human CASR P1 and P2 promoters is up-regulated by cytokines.
A, CASR promoter P1, exon 1A, promoter P2, exon 1B, and exon 2 showing the alternative splicing of exons 1A and 1B to exon 2. Portions of CASR promoters P1 or P2 with appropriate 5Ј-untranslated region sequences were cloned upstream of the luciferase reporter gene in PGL3 basic as described. HKC or TT cells were transfected with the luciferase reporter constructs, either promoterless (PGL3) or P1 or P2 as described under "Experimental Procedures." B, HKC cells were stimulated or not with IL-1␤. C, HKC cells were stimulated or not with TNF-␣. D, TT cells were stimulated or not with IL-1␤. B-D, luciferase activity was measured. *, p Ͻ 0.05 relative to without added cytokine.

Transcriptional Activities of Human CASR P1 and P2 Are
Up-regulated by IL-1␤ and TNF-␣-To analyze their transcriptional activities, constructs were used in which human CASR P1 and P2 promoters drive transcription of a luciferase reporter gene (Fig. 4A). When transfected into HKC cells, the basal activities of P1 and P2 were 9-and 32-fold that of the promoterless control, respectively (Fig. 4, B and C), consistent with our previous studies (6). In the TT thyroid C-cells, the basal activities of P1 and P2 were 10-and 33-fold that of the control, respectively (Fig. 4D). The addition of IL-1␤ or TNF-␣ (5 ng/ml) during the transient transfection experiments stimulated reporter activity of P1 2-fold and of P2 2.5-fold in both HKC and TT cells (Fig. 4, B-D). Thus, proinflammatory cytokines stimulate the activity of both CASR gene promoters.

IL-1␤ Acts on CASR Gene Transcription via an NF-B-dependent
Pathway-Cytokines like IL-1␤ and TNF-␣ work through DNA binding homo-or heterodimers of members of the NF-B/Rel multigene family, which include p50, p65 (RelA), and c-Rel. NF-B dimers are present in the cytoplasm of most cells in an inactive form bound to the inhibitory protein, IB. Upon receipt of a signal generated by activation of cytokine receptors, IB is phosphorylated, ubiquitinated, and degraded. The freed NF-B complex then translocates into the nucleus to regulate transcription of specific genes by interacting with cis-acting B response elements with most combinations of NF-B proteins activating transcription.
To test whether the NF-B pathway is involved in the upregulation of CASR gene transcription by IL-1␤, HKC cells were transfected with the promoterless PGL3 (negative control), P1 WT, P2 WT, or (NF-B) 6 (positive control) luciferase reporter constructs. The transfected cells were pretreated with a synthetic peptide containing a membrane-permeable region fused to the NF-B NLS, which specifically prevents the nuclear translocation of NF-B subunits, or with an inactive analogue as control and then stimulated with IL-1␤. The transcriptional activities of P1, P2, and (NF-B) 6 were stimulated by IL-1␤ in cells pretreated with the inactive form of the peptide but not in those cells pretreated with the NF-B NLS peptide (Fig. 5A). Neither the NF-B inhibitory peptide nor the inactive control peptide altered the unstimulated expression (i.e. basal expression in the absence of IL-1␤) of the P1 and P2 constructs or the pGL3 promoter vector (representing a basic TATA-luciferase construct) (data not shown). These results indicate that IL-1␤ acts on CASR gene transcription via an NF-B-dependent pathway.
NF-B Subunit p65 Stimulates the CASR Promoters-To test the ability of individual NF-B subunits to up-regulate CASR promoter activity, we examined the effect of overexpression of NF-B subunits p50 and p65 both singly and in combination on the transcriptional activity of the P1 and P2 promoters in HKC cells in the absence and the presence of IL-1␤. In this experiment, the effects observed are due to the combination of endogenous and exogenous NF-B proteins. For the P1 promoter, in the absence of IL-1␤, co-transfection of p65 either alone or with p50 produced a ϳ60% increase in luciferase activity over that obtained with the mock-transfected control (Fig. 5B). In the presence of IL-1␤, the addition of exogenous p65 either alone or in combination with p50 produced a 2-2.3-fold increase in luciferase activity of P1 over that observed with the mocktransfected control (Fig. 5B). For the P2 promoter in the absence of IL-1␤, co-transfection of p65 either alone or with p50 produced a 68% increase in transcriptional activity over control FIG. 5. IL-1␤ stimulates CASR P1 and P2 promoters via an NF-B-dependent pathway. A, HKC cells were transfected with luciferase reporter constructs either promoterless (pGL3) or containing multiple NF-B response elements ((NF-B) 6 -luciferase) or containing the P1 or P2 CASR promoters as described under "Experimental Procedures." Cells were stimulated or not with IL-1␤ in the absence or presence of either the active NLS peptide or an inactive peptide. The NF-B p65 subunit induces and IB inhibits CASR gene transcription. HKC cells were transfected with either the CASR P1 promoter (B) or the CASR P2 promoter (C) luciferase constructs as described under "Experimental Procedures" in the absence (Ϫ) or presence (ϩ) of NK-B subunits p50, p65, or p50/p65 combined or IB as indicated. Cells were stimulated (ϩ) or not (Ϫ) with IL-1␤ as indicated. (Fig. 5C). In the presence of IL-1␤, the addition of exogenous p65 either alone or with the other NF-B proteins led to a 2.3-2.6-fold increase in P2 transcriptional activity (Fig. 5C). For both P1 and P2 promoters, in the presence of IL-1␤, the p65/p50 combination produced a slightly greater -fold increase than p65 alone (Fig. 5, B and C). An additional set of experi-ments in which c-Rel was overexpressed either alone or in combination with p50 or p65 suggested that c-Rel contributed little to the IL-1␤ stimulation of both P1 and P2 promoters (data not shown). Overall, the results indicate that the CASR P1 and P2 promoters are activated by selected combinations of the NF-B proteins.

Overexpression of IB Abolishes IL-1␤ and NF-B Subunit
Stimulation of CASR P1 and P2 Promoters-To further confirm that the NF-B pathway is necessary and sufficient for the IL-1␤ induction of CASR gene transcription, we used an expression vector driving IB production to block signaling of the NF-B pathway. HKC cells were cotransfected with either the CASR P1 or P2 constructs and expression vectors for p65 and/or p50 in the absence or presence of IL-1␤ as described above. In addition, the expression vector for IB was cotransfected. For both the P1 promoter (Fig. 5B) and the P2 promoter (Fig. 5C), the increase in luciferase activity observed in cells stimulated with IL-1␤ only or transfected with NF-B constructs and stimulated or not with IL-1␤ in the absence of exogenous IB (Fig. 5, B and C) was completely abolished by overexpression of IB. Exogenous expression of IB had no effect on the basal activity of CASR P1 and P2 promoters. The results show that the NF-B pathway is necessary and sufficient for the IL-1␤ stimulation of CASR gene transcription.
Several Potential NF-B Response Elements Are Present in the CASR Gene Regulatory Regions-Scanning of the CASR gene with Mat Inspector version 2.2 (Genomatix Software) (33) revealed potential NF-B response elements in both P1 and P2 promoters and the corresponding 5Ј-untranslated regions (Fig. 6).
A Subset of the NF-B Response Elements Confer Cytokine Responsiveness to the CASR Gene-CASR P1 and P2 promoterluciferase reporter constructs were prepared in which the NF-B elements were mutated either individually or in combination (see Fig. 7). To dissect out the contributions of the individual putative B elements, the wild-type or mutated constructs were transfected into HKC cells that were then stimulated or not with IL-1␤. For the CASR P1 construct, mutation of the P1 element alone led to a 50% loss of activity. Mutation of the 1A element alone or in combination with the P1 element led to complete loss of the IL-1␤-stimulated component of transcription (Fig. 8A). Deletion of exon 2 that contains a putative NF-B element was without effect on IL-1␤-stimulated transcription (Fig. 8A). For the CASR P2 construct, mutation of the P2 element led to complete loss of the IL-1␤-stimulated component of transcription (Fig. 8B), whereas deletion of exon 2 was without effect on IL-1␤-stimulated transcription (Fig. 8B). None of the mutations (with the exception of the deletion of exon 2) had any effect on basal transcriptional activity. Both P1 and P2 exon 2-deleted constructs had slightly increased basal and IL-1␤-stimulated transcriptional activity, demonstrating the presence of a putative repressor element in exon 2 (Fig. 8,  A and B).
A Subset of PI and P2 NF-B Elements Confers Cytokine Responsiveness to a Heterologous Promoter-Portions of the P1 and P2 promoters containing their respective NF-B elements (either wild-type or mutated) were cloned upstream of the SV40 promoter driving the luciferase reporter gene in pGL3-Promoter vector (see Fig. 9). Wild-type P1 and P2 sequences containing NF-B elements conferred cytokine responsiveness on the heterologous promoter when transiently transfected into HKC cells (Fig. 10). The -fold stimulation with the cytokines corresponded exactly to that obtained with the P1 and P2 NF-B elements in the context of their natural promoters (compare Figs. 8 and 10). Constructs with mutated NF-B elements had lost the ability to confer cytokine responsiveness to the heterologous promoter (Fig. 10).
Protein-DNA Complexes Form on a Subset of the CASR Gene NF-B Elements-EMSAs were conducted with oligonucleo-tides representing either a consensus NF-B element or the CASR gene NF-B elements (Fig. 11) and HKC nuclear extract. The protein-DNA complexes formed with the CASR gene elements and consensus NF-B element had similar electrophoretic mobilities, and they were shifted in a similar fashion by the addition of antibodies against either p50 or p65 (Fig. 11). This identified the complexes as predominantly p65/p50 heterodimers and p50/p50 homodimers. The complexes formed on three of the four putative NF-B elements in the following order of intensity, exon 1A ϭ P2 Ͼ P1. Complexes did not form on the putative NF-B element in exon 2 (data not shown). The addition of unlabeled oligonucleotides reduced the intensity of the labeled consensus and CASR gene NF-B element-protein complexes in a similar manner (Fig. 11 and data not shown). When the EMSAs were conducted with labeled mutated consensus and CASR gene NF-B elements, the DNA-protein complexes failed to form ( Fig. 11; data not shown).

DISCUSSION
The extracellular CASR acts as a "calciostat," and changes in the amount of the calciostat have important effects on extracellular calcium homeostasis. This is well known from studies of hypercalcemic and hypocalcemic humans harboring mutations (either inactivating or activating mutations, respectively) in the CASR gene (32,34,35) and mice either heterozygous or homozygous for deletion of the casr gene (36). Hence, the demonstration of changes in CASR expression provides the link to alterations in extracellular calcium levels.
In the present study, we have focused on the mechanism underlying the IL-1␤ stimulation of CASR expression. This is important in beginning to define the mechanisms operative in the dysregulated mineral ion homeostasis of the critically ill. Initially, we demonstrated that IL-1␤ administration to rats led to a fall in serum PTH levels and concomitantly serum calcium levels over a 24-h period. We next showed that IL-1␤ up-regulates parathyroid, thyroid, and kidney CASR mRNA levels in vivo. In addition, for the thyroid and kidney, the CASR protein levels were documented to be elevated after a single injection of IL-1␤ in vivo. We then demonstrated that human thyroid C-cell and kidney proximal tubule cell CASR gene transcription is increased by IL-1␤. Transcripts derived from both P1 and P2 promoters of the CASR gene were up-regulated.
In HKC and TT cells, IL-1␤ (and TNF-␣) stimulated transcriptional activity of P1 and P2 reporter gene constructs ϳ2fold and ϳ2.5-fold, respectively. The involvement of the NF-Bsignaling pathway was then demonstrated in two ways. First, co-transfection of exogenous NF-B proteins into HKC cells led to increased activity of the P1 and P2 reporter constructs. Second, co-transfection with inhibitor-B or treatment with a peptide representing the NF-B nuclear localization sequence abrogated the responsiveness of the CASR gene promoters to IL-1␤ or exogenous NF-B proteins.
The 5Ј region of the CASR gene has several consensus B response elements, and we have shown that some are functional and others are not. Mutagenesis of some, but not all, of the putative elements led to loss of responsiveness to IL-1␤, and these elements conferred IL-1␤ responsiveness to a heterologous promoter. In EMSAs with HKC nuclear extract, complexes comprising homo-or heterodimers of p65 and p50 formed on the same three B elements shown to be active in transcriptional assays.
To assess the clinical relevance of the present study, we will review the new data in conjunction with previous animal and human studies as they pertain to three main tissues or organs involved with calcium homeostasis, namely the parathyroid, thyroid, and kidney.
In cultured parathyroid tissue, either bovine gland slices (21) or equine cells (37), IL-1␤ inhibited PTH secretion and increased CASR mRNA levels. The effects of IL-1␤ were counteracted by the IL-1 receptor antagonist, indicating that the cytokine was acting via a specific IL-1 receptor on parathyroid cells. Circulating levels of IL-1␤ are elevated after severe burn injury, which is associated with severe abnormalities of bone and mineral metabolism (38). The use of a sheep model demonstrated that the hypocalcemia that follows severe burn injury is related to up-regulation of parathyroid gland CASR expression (22). In the present studies, administration of IL-1␤ to rats brought about increases in parathyroid gland CASR mRNA levels with decreases in circulating PTH and 1,25(OH) 2 D 3 levels followed closely by serum calcium levels over a 24-h period. Taken together with the results of previous in vitro and in vivo studies, the data of the present study consolidate the notion that IL-1␤ acts to increase parathyroid CASR expression, thereby reducing the set point for PTH suppression by extracellular calcium. The consequences of this are initial hypoparathyroidism and hypocalcemia.
Thyroid calcitonin-producing C-cells express the CASR (39,40), which mediates the extracellular calcium-stimulated secretion of calcitonin (40 -42). Circulating levels of calcitonin, mainly high molecular weight procalcitonin forms, are elevated in critically ill patients (19). The levels are higher in septic (especially septic shock) than in nonseptic patients and are related to the severity of illness. Circulating calcitonin levels correlate with length of stay in intensive care and with nonsurvival versus survival (8). In the present studies, a single injection of IL-1␤ to rats brought about a marked (ϳ3-fold) increase above the basal level of thyroid CASR mRNA levels. This response was more marked than the ϳ2-fold above basal level increase noted in the parathyroid. The greater quantitative response in the thyroid could be due to the combined effect of IL-1␤ and IL-6. The latter cytokine is known to be constitu- tively produced by thyrocytes and up-regulated by IL-1␤ (43), and we have found 2 that IL-6 up-regulates CASR gene expression in the TT thyroid cell line. In addition, all molecular species of the CASR (high molecular weight aggregates, most likely dimers, the monomeric 160-kDa mature form, and the monomeric 140-kDa immature form) were up-regulated in the thyroid, achieving an ϳ3-fold increase over basal level 24 h after stimulation with IL-1␤.
In the kidney, the CASR is present along essentially the entire nephron. In the proximal tubule, it is located predominantly at the base of the brush border of the apical membrane of the tubular epithelial cells, although there is some basolateral expression as well. Likewise, the CASR is principally apical in the inner medullary collecting duct. In contrast, high levels of the CASR are found on the basolateral surface of the cortical thick ascending limb epithelial cells. Similarly, the lower levels of the CASR are basolateral in the medullary thick ascending limb and the distal convoluted tubule.
With respect to renal actions that are believed to be mediated by the CASR, in the proximal tubule, the sodium phosphate cotransporter, NPT2, that reabsorbs the bulk of the filtered phosphate, is located in the apical brush-border membrane as well as in subapical vesicles. PTH promotes phosphaturia by stimulating the endocytosis of NPT2-containing vesicles from the proximal tubular apical membrane, followed by lysosomal degradation of the cotransporter. It has been suggested that proximal tubular CASR activation might induce removal of NPT2. Consistent with this idea, protein kinase C activation (as would occur with CASR activation) does promote NPT2 retrieval from proximal tubule apical membranes, resulting in hypophosphatemia (2). Increased proximal tubular CASR expression stimulated by IL-1␤ would result in increased CASR activation for any given serum calcium concentration. Therefore, this mechanism could contribute to the hypophosphatemia that is common in critically ill patients (8,44).
Changes in serum calcium regulate production of 1,25(OH) 2 D by affecting the activity of the proximal tubule mitochondrial cytochrome P 450 25-hydroxyvitamin D-1␣hydroxylase. In thyroparathyroidectomized rats in which PTH and phosphate were maintained at constant levels, an inverse correlation was seen between serum calcium and 1,25(OH) 2 D levels, suggesting that calcium regulates 1,25(OH) 2 D independently of PTH (45)(46)(47). Extracellular calcium directly regulates 1,25(OH) 2 D production in the HKC cell line (48). As indicated in the present study, the proinflammatory cytokine IL-1␤ causes increased CASR expression in parathyroid and kidney, and this, in turn, probably contributes to reduced 25hydroxyvitamin D-1␣-hydroxylase activity, responsible for generating the hormonally active metabolite, 1,25(OH) 2 D, in the kidney proximal tubule. On the one hand, because of the increase in parathyroid CASR levels, there is a reduction in the potent 25-hydroxyvitamin D-1␣-hydroxylase stimulator, PTH. On the other hand, increases in proximal tubular CASR would blunt the effectiveness of hypocalcemia in stimulating the renal 25-hydroxyvitamin D-1␣-hydroxylase, contributing to the hypovitaminosis D often seen in the critically ill (8).
In the present study, careful examination of the effect of IL-1␤ up to 24 h after administration in the rat, documented a doubling in parathyroid CASR mRNA levels at 15 h, although the corresponding response in the kidney was less marked. This is reminiscent of the finding of up-regulation of parathyroid, but not kidney, CASR expression some 48 h post-burn injury in the sheep (22). Thus, it might well be that not all sites of CASR expression in the nephron are equally affected by IL-1␤ (or other cytokines). We have demonstrated a clear cut effect of IL-1␤ on CASR expression in the proximal tubular cells. Assessment of changes in CASR in other portions of the nephron in response to cytokines will require further analysis. However, whereas the CASR in the distal nephron, the cortical thick ascending limb, and distal convoluted tubule plays a key role in regulating calcium and magnesium reabsorption, the indirect evidence from assessment of critically ill patients does not support a marked shift in mineral ion excretion contributing to the hypocalcemia and hypomagnesemia. Urinary excre-2 L. Canaff, and G. N. Hendy, unpublished data.
FIG. 11. Comparison of protein-DNA complexes formed in gel retardation assays with oligonucleotides representing a consensus NF-B element or the putative CASR gene NF-B elements and HKC nuclear extract. Electrophoretic mobility shift assays with double-stranded oligonucleotides representing potential NF-B elements in P1, exon 1A, P2, and exon 2 and a consensus NF-B element (see Table  I) were conducted as described under "Experimental Procedures," and antibodies against p50 or p65 were added as indicated. The p65/p65-, p50/p65-, and p50/p50-containing complexes formed on the consensus NF-B or CASR gene exon 1A, P2, or P1 oligonucleotides are indicated by arrows (3). tion of calcium is often low in critical illness and related to serum-ionized calcium levels (8). In addition, analysis of fractional excretion rates of calcium and magnesium in the sheep model of burn injury experiments (22) and in horses with enterocolitis (49) provided no evidence for altered renal reabsorption of these cations.
In summary, the present studies have provided key insights into how alterations in expression of the CASR may impact on calcium homeostatic mechanisms in the early stages of critical illness. These include 1) an increased sensitivity of the parathyroid gland to circulating calcium concentrations, leading to reduced secretion of PTH; 2) the enhanced secretion of procalcitonin from thyroid C-cells; and 3) the decreased ability of the renal proximal tubule to generate the hormonally active vitamin D metabolite, 1,25(OH) 2 D, that is essential for proper calcium (and phosphate) absorption across the gut. Inflammation promotes increased local blood coagulation that is beneficial but carries the risk of increased systemic coagulation. The mechanism revealed in the present studies may be part of an important counterregulatory system aimed at minimizing the deleterious effects of calcium and cytokines in promoting scattered intravascular coagulation and atherosclerosis that would be anticipated to occur during the inflammatory response.