Key points are not available for this paper at this time.
Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5′-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated ∼2–3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis. Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5′-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated ∼2–3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis. The calcium-sensing receptor (CASR) 3The abbreviations used are: CASR, calcium-sensing receptor; PTH, parathyroid hormone; 1,25(OH)2D, 1,25-dihydroxyvitamin D; IL, interleukin; TT cells, human thyroid C-cells; HKC, human proximal tubule cells; IL-6R, interleukin-6 receptor; JAK, Janus kinase; STAT, signal transducers and activators of transcription; Sp1/3, specificity protein 1/3; MAPK, mitogen-activated protein kinase; SRF, serum-response factor; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Earle's medium; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; UTR, untranslated region; oligo, oligonucleotide. is expressed in the parathyroid hormone (PTH) producing chief cells of the parathyroid gland, the calcitonin-producing C-cells of the thyroid, and the cells lining the kidney tubule. The CASR, a plasma membrane G protein-coupled receptor, senses small changes in circulating calcium concentration and modulates intracellular pathways that alter PTH and calcitonin secretion or renal cation handling, thereby playing an essential role in blood mineral ion homeostasis. The relationship between extracellular ionized calcium and PTH concentrations is represented by an inverse sigmoidal curve. The activity and/or expression level of the CASR dictates the extracellular calcium set point (defined as the extracellular calcium concentration at which PTH secretion from the parathyroid gland or calcium reabsorption across the kidney tubule is half-maximal). Increases in extracellular calcium directly stimulate calcitonin secretion. The importance of the CASR in orchestrating the endocrine control of blood calcium concentrations has been underscored by the identification of naturally occurring mutations in the CASR gene that cause human disease. Inactivating mutations result in hypercalcemia, and activating mutations result in hypocalcemia (1Pollak M.R. Brown E.M. Chou Y.-W.H. Hebert S.C. Marx S.J. Steinmann B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1297-1303Abstract Full Text PDF PubMed Scopus (916) Google Scholar, 2Pollak M.R. Brown E.M. Estep H.L. McLaine P.N. Kifor O. Park J. Hebert S.C. Seidman C.E. Seidman J.G. Nat. Genet. 1994; 8: 303-307Crossref PubMed Scopus (539) Google Scholar, 3Hendy G.N. D'Souza-Li L. Yang B. Canaff L. Cole D.E.C. Hum. Mutat. 2000; 16: 281-296Crossref PubMed Scopus (238) Google Scholar). Hypocalcemia is common in critically ill patients, especially those with sepsis and major burn injury (4Zivin J.R. Gooley T. Zager R.A. Ryan M.J. Am. J. Kidney Dis. 2001; 37: 689-698Abstract Full Text PDF PubMed Scopus (192) Google Scholar), and in nonacutely ill patients undergoing surgery (5Lepage R. Legare G. Racicot C. Brossard J.H. Lapointe R. Dagenais M. D'Amour P. J. Clin. Endocrinol. 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Care Med. 2000; 28: PubMed Scopus Google Scholar) that in parathyroid CASR levels were in a of burn injury in which circulating cytokine levels be to be analyses are to more the mechanisms the IL-6 levels to altered calcium the that as increase CASR expression in those for the control of calcium thereby to hypocalcemia and showed in in the that parathyroid, thyroid, and kidney CASR and protein increased intraperitoneal injection of L. G.N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). was with decreased circulating PTH, 1,25-dihydroxyvitamin D and The CASR gene has two promoters (P1 and P2) yielding alternative transcripts containing or 5′-untranslated that to containing the start Brown E.M. Hebert S.C. F. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, N. S. M. T. T. T. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, L. G.N. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). that both the CASR gene promoters have elements that by L. G.N. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In this that by CASR expression in parathyroid, thyroid and kidney reducing PTH secretion and renal calcium and increasing calcitonin contributes to altered calcium homeostasis. have that IL-6 CASR expression in and for the the mechanisms the cytokine CASR gene promoter The resulting in CASR expression could be to the hypocalcemia of critically ill IL-6 was from The human thyroid TT was from the and the human proximal kidney tubule cells were a of Dulbecco's modified Earle's serum and were from and were from and were from and were from and and Sp1 response elements were from constructs were as human Sp1 in human H. S. J. 1999; PubMed Scopus Google Scholar), human in and human Stat3 in Stats and were the constructs as with the The were and and are in and were a containing and vitamin were in and were the Care and were at or with or was by and the serum was and at The were with the were and the parathyroid and thyroid were and were for calcium and PTH PTH and and was from cells or to the of CASR the CASR a of a CASR L. M. M. G.N. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) was and and the glyceraldehyde-3-phosphate that a the was After of the the were in with a and the were used with a was with of by with a L. Canaff L. N. Cole D.E.C. G.N. Hum. Mutat. 2001; 18: PubMed Scopus Google Scholar). were on and to transcription were a G.N. PubMed Scopus (25) Google Scholar). were from to HKC or TT cells with IL-6 or serum in were PBS, at and with After on were at were with of DTT, in and at were at in DTT, PMSF, of or of and for was with to the of containing gene or no were and The were as human CASR a in human CASR in human CASR a in human a from HKC and human GAPDH, a from HKC and in The were with of in and for a of h. In of were used for were to or the signal was with the and of the of transcription was by of the or cells were in PMSF, for at The were at for at and the were at were and were in with in for and with CASR an L. Yang B. Canaff L. M. M. Brown E.M. Cole D.E.C. G.N. J. Clin. Endocrinol. Metab. 2002; PubMed Scopus Google Scholar), a CASR to or the the same a was as with the for with the which was were by the were with a as CASR of the reporter as containing the P1 promoter, and the of to is the A of the of the reporter gene in has been The and were for A in which a Stat element in is mutated was the as with the as S. M. Canaff L. O. G.N. Hum. Genet. PubMed Scopus Google Scholar). The used were as and are in used for CASR gene promoter P1 P2 P1 and P2 in a The of the reporter as containing the P2 promoter, and the of to of the gene in has been The was by for in which Sp1 and two the transcription are mutated was by the 1999; Scopus Google Scholar) as The used were as with a site in with mutated in to and with mutated in and with site in to The were and with and to that was with and the to cells were in with TT cells were in with and and B. mitogen-activated protein cells were in to and with or P1 or P2 promoter constructs and the to cells were for stimulated or not with and of the or were to the to After were for and cells were in in DMEM, cells and The cells were with of with of CASR promoter and of The cells were in and with or cytokine for h. The cells were in and in of on The were for and by activity was in a of and activity was to of HKC were stimulated with IL-6 for of PBS, and at for at were in a in of DTT, PMSF, and were by for at in PMSF, DTT, and After a at were for PMSF, was and were at of were for on with of in were or and were for at of were and for a used for are in were at in which were and used for and/or promoters P1 a was at the a was at the P2 a was at the P2 a was at the element of the human A gene is a was at the element of the human A gene is in a cells were with IL-6 for to and by in DTT, with and was by were with of to the or mutated CASR P1 Stat1/3 or CASR P2 Sp1 elements for for h. of and of protein) were to and with and of with in a The were at for and were and with and were to with Stat1 or Stat3 or Sp1 or are expressed as The from the in IL-6 response studies were to of The of from was the and transfection were by A of was to a PTH, 1,25(OH)2D, and in the of IL-6 on extracellular calcium homeostasis, the cytokine was to and circulating PTH, 1,25(OH)2D, and calcium levels were a After a intraperitoneal injection of IL-6 in serum PTH, 1,25(OH)2D, and calcium levels were decreased at and 24 and Kidney CASR in in circulating PTH, and calcium levels by IL-6 could be to altered CASR expression in for of extracellular calcium homeostasis, CASR levels by the After the injection of IL-6 in parathyroid, thyroid, and kidney CASR levels level to at and the levels were elevated at 24 The to levels were and and of no on CASR levels not and Kidney CASR in the changes in CASR levels were in changes in CASR protein was on of thyroid and the the CASR in both and The is and is D and The mobility are likely to be S. M. Canaff L. O. G.N. Hum. Genet. PubMed Scopus Google Scholar). After injection of IL-6 in thyroid and kidney CASR protein levels and related to levels by 24 and of no on CASR protein levels not CASR protein levels were not of the of Increases CASR the changes in CASR levels changes at the transcription were on of human TT cells and HKC cells with and IL-6 for and h. CASR gene and transcripts were stimulated and gene transcription in both not gene transcription was by IL-6. IL-6 CASR gene Activities of CASR P1 and P2 by the of IL-6 on the activity of CASR gene constructs were used in which human CASR P1 or P2 promoters transcription of a reporter gene HKC cells, the of P1 and P2 were and that of the and In the TT thyroid the of P1 and P2 were and that of the The of IL-6 stimulated reporter activity of P1 and P2 constructs in a in HKC and and TT cells not with an increase at the proinflammatory cytokine the activity of both CASR gene IL-6 the Activities of and CASR P1 and P2 regions of the CASR promoters that responsiveness to a series of constructs of the P1 promoter with and or the P2 promoter with and a reporter gene were The or deleted constructs were HKC TT that were stimulated or not with IL-6. the P1 activity increased with constructs to to the the of a between and IL-6 stimulated the activity of constructs the P2 constructs containing of the P2 transcription start IL-6 promoter activity and IL-6 receptor of an IL-6 and a signal which is the for the cytokine of IL-6 to receptor and that and to the and to elements thereby activating gene transcription 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Jr., Nat. 2002; PubMed Scopus Google Scholar). Stat1 and Stat3 IL-6 The can to the the activity of a of transcription factors as the but other that response elements. transcription factors like serum-response and Sp1/3 can to factors by IL-6 and regulate gene expression S. G. F. J. PubMed Scopus Google Scholar). Several to IL-6 in the CASR of the CASR gene with K. K. K. B. M. A. M. M. T. PubMed Scopus Google Scholar) STAT, SRF, and Sp1 elements in P1 and/or P2 promoters and/or the 5′-untranslated regions in CASR promoter P1 is a Stat1 element in and in CASR promoter P2 are no Stat elements of are Sp1 elements that at the transcription start site. The the IL-6 of the CASR P1 and P2 activity was for activity and/or activity of the CASR the P1 and P2 and deleted constructs were HKC cells TT that were stimulated or not with IL-6 in the or of the the CASR P1 constructs in the of the promoter activity was that in the of the for the CASR P2 constructs in the of the promoter activity was that in the of the to IL-6 although the activity level for P1 and was in the than in the of the the was no A and the to activity of the CASR not by IL-6. Stat1 and Stat3 CASR P1 via the Stat in the role of in the IL-6 of the CASR P1 promoter, or mutated at the Stat1 element in were or with a or a HKC cells and the cells were stimulated or not with IL-6. the a increase in activity was stimulated by and this increase was by of or The of the by IL-6 was reduced to that of the and with or the the Stat1 element in is very for the IL-6 of promoter P1, and Stat1 or Stat3 can to the IL-6 Sp1 for and CASR P2 the role of Sp1 in the activity and IL-6 of the CASR P2 promoter, the or constructs the in which the Sp1 clustered at the transcription site were were or with an Sp1 expression or with an expression HKC cells that were stimulated or not with IL-6. of the Sp1 with or to in activity of of with or to in activity of The of and were stimulated by IL-6 or with of The of these promoter constructs by IL-6 was by with The was and of Sp1 or no and the was to IL-6 not Sp1 is essential for activity and is critical for the IL-6 of the CASR P2 Stats for the IL-6 of the CASR P2 P2 promoter has no Stats could be IL-6 by with other transcription factors that to response elements in the the role of Stats in the IL-6 of the CASR P2 promoter, the or constructs were or with or expression HKC cells that were stimulated or not with IL-6. of of the dominant-negative constructs with to a in the with IL-6 The was activity Stat1 and Stat3 are critical for the IL-6 of the CASR P2 on a Stat in of the CASR were with Stat1 elements in CASR P1 and HKC with the CASR gene element and the in which were by Stat1 and/or Stat3 were with and Stat1/3 with The of the reduced the of the of an oligonucleotide was not on the Stat1 element in CASR gene P1 not on Sp1 at the of CASR P2 were with a Sp1 element or a of Sp1 elements a of and downstream of the transcription site of the CASR gene P2 promoter and HKC The that with CASR gene Sp1 elements and Sp1 element electrophoretic and were in a by the of Sp1 or The of the CASR P2 reduced the of the and CASR gene Sp1 in a of an oligonucleotide not or an oligonucleotide a protein no not on a Sp1 element of the transcription site of the CASR gene P2 promoter not of a Stat on the CASR P1 Stat1/3 Stat1 or Stat3 to the P1 Stat1/3 element oligonucleotide precipitation were of HKC cells with IL-6 for increasing of a complex the CASR P1 Stat1/3 element and Stat1 or Stat3 of the was and at with no increase at not Sp1 was in the complex no on mutated CASR gene of an on the CASR P2 Sp1 Stat1 or Stat3 Sp1 or is to the P2 Sp1/3 element oligonucleotide precipitation were of HKC cells with IL-6 for increasing of a complex the CASR P1 Sp1 elements and Sp1 and and Stat1 and Stat3 of the was and at with no increase at not no on mutated CASR gene that the with P1 and P2 and mutated were the same HKC for of by the In this have the mechanisms underlying the of CASR expression by the proinflammatory IL-6. In of IL-6 to to in serum PTH, 1,25(OH)2D, and calcium that were a that IL-6 parathyroid, thyroid, and kidney CASR levels and for the thyroid and kidney which of could be that the CASR protein levels were thyroid and kidney proximal tubule CASR gene transcription was increased by IL-6 in of both CASR gene P1 and P2 transcripts were In renal proximal tubule HKC and thyroid TT cells, IL-6 stimulated activity of P1 and P2 reporter gene constructs and IL-6 by the IL-6 receptor that of an and a signal which is the for the cytokine of IL-6 to receptor and that and to the and to elements thereby activating gene transcription 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Jr., Nat. 2002; PubMed Scopus Google Scholar). There are of the with Stat1 and Stat3 the that IL-6 The can to the the activity of a of transcription factors as the but other that response elements S. G. F. J. PubMed Scopus Google Scholar). transcription factors like SRF, and Sp1/3 G. 1999; PubMed Scopus Google Scholar) to factors by IL-6 and regulate gene the is for the of IL-6 by activating Stats and/or other the CASR gene promoters with that response element to and elements in to Stat elements. The could the of the that to these elements. in this of the that although of the to activity of both P1 and P2 was not in a major in the IL-6 of of the of the Stat1 and Stat3 are the that IL-6 The studies with and mutated constructs of both CASR P1 and P2 promoters on a Stat1/3 element in and Sp1/3 elements at the transcription start site of promoter P2 as critical for IL-6 of the CASR The of IL-6 with of Stat1 and/or Stat3 dominant-negative constructs and the P1 promoter the of these Stats for the The of IL-6 with of an Sp1 dominant-negative and the P2 promoter constructs the of Sp1 for the downstream even Stat elements are not in the P2 promoter, of Stat1 or Stat3 dominant-negative constructs the IL-6 of the P2 In with HKC and Stat1/3 on the Stat1 element in on Stat1 element within promoter The that Stats on this element the from the promoter-reporter that the IL-6 of the P1 promoter the Stat Stats were as and this cytokine is a Stat1 and of is on Stat elements in to as in the S. C. Cell. 17: PubMed Scopus Google Scholar). of the by IL-6 of Stat1 and Stat3 with the CASR P1 promoter Stat element was by an oligonucleotide precipitation In with HKC Sp1/3 and on the of Sp1 elements the transcription start site of promoter P2. In with the promoter-reporter in which of an Sp1 dominant-negative the IL-6 of promoter this the importance of the Sp1 in the by the In the Sp1 elements are for the activity of the that both Stat and Sp1 elements in that the of the is to between the Stat and Sp1 at response elements. this with Stat1 and Sp1 for of the gene M.R. M.J. J. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and with Stat3 and Sp1 for the protein gene Cell. 18: PubMed Google Scholar). this is not the with the CASR P2 promoter as has no Stat elements. the of the IL-6 of P2 promoter reporter constructs by Stat1 or Stat3 dominant-negative constructs a role for the Stats in the of this promoter by IL-6. of the was in the of not Sp1 and but Stat1 and Stat3 in a complex at the Sp1 element in the CASR P2 A mechanism has been for cytokine of other gene promoters that Stat elements but have Sp1 elements. IL-6 of the gene via between Stat3 and Sp1 S. B. J. J. J. PubMed Scopus Google Scholar), and Stat3 with Sp1 the by of the of gene S. Endocrinol. PubMed Scopus Google Scholar). In studies the altered CASR expression that the endocrine control of blood calcium may be in occurring in critically ill patients in other circulating proinflammatory cytokine levels are In these blood that although the of increased The mechanisms that are may a critical that the of calcium and in and the that the in serum calcium may be as in critically ill patients a of hypocalcemia is with a The the to the of small that and the CASR, be in critically P. J. and for critical of the and for for the cells, and and of for the
Canaff et al. (Wed,) studied this question.