Abstract
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 approximately 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.
Highlights
The calcium-sensing receptor (CASR)3 is expressed in the parathyroid hormone (PTH) producing chief cells of the para
Interleukin-6 Decreases Serum PTH, 1,25(OH)2D, and Calcium Levels in Vivo—To examine the effect of IL-6 on extracellular calcium homeostasis, the cytokine was administered to rats, and circulating PTH, 1,25(OH)2D, and calcium levels were monitored over a 24-h period
Interleukin-6 Up-regulates Parathyroid, Thyroid, and Kidney CASR mRNA Levels in Vivo—To assess whether alterations in circulating PTH, 1,25(OH)2D and calcium levels brought about by IL-6 could be due to altered CASR expression in tissues important for regulation of extracellular calcium homeostasis, we measured CASR mRNA levels by ribonuclease protection assay throughout the 24-h period
Summary
Oligonucleotides used for CASR gene promoter deletion constructs Each forward (F) primer is named according to the first CASR gene nucleotide relative to the transcription start site (ϩ1) of either promoter P1 or P2. Restriction enzyme recognition sites (ACGCGT, MluI; AAGCTT, HindIII) are in boldface type. Nucleotides in lowercase type are those added to promote efficient restriction enzyme digestion. Ribonuclease Protection Assay of Rat CASR mRNA—For the CASR riboprobe, a 232-bp fragment of a rat CASR cDNA [24] was PCR-amplified (forward primer, 5Ј-ACCTTGAGTTTTGTTGCCCA-3Ј (in exon 3), and reverse primer, 5Ј-GGAATGGTGCGGAGGAAGGATT-3Ј (in exon 4)) and cloned into PCR2.1 vector. For the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) riboprobe that protects a 316-nucleotide transcript, the pTRI-GAPDH Rat vector (AM7432: Ambion Inc., Austin, TX) was used.
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