Calcyclin mediates serum response element (SRE) activation by an osteoblastic extracellular cation-sensing mechanism.
The molecular mechanism of sensing extracellular cations in osteoblasts is controversial. Using an expression-cloning strategy, the calcium-binding protein calcyclin was found to mediate the response of MC3T3-E1 osteoblasts to extracellular cations, but not the calcimimetic NPS-568, indicating the presence of another cation-sensing mechanism. Further understanding of calcyclin function in osteoblasts may identify novel targets for regulating bone formation.


BACKGROUND
Extracellular calcium and other cations seem to regulate the function of osteoblasts through a distinct calcium-sensing mechanism that is coupled to activation of c-fos gene transcription. The identity of this calcium-sensing mechanism is unknown.


METHODS
To identify molecules that participate in this extracellular cation-sensing pathway, we developed an expression cloning strategy in COS-7 cells using cation stimulation of a serum response element (SRE) luciferase reporter derived from the c-fos promoter to screen a mouse MC3T3-E1 osteoblast cDNA library.


CONCLUSIONS
We identified calcyclin (S100A6), a calcium-binding protein of the EF-hand type belonging to the S100 family, as being responsible for transferring a cation-sensing response from osteoblasts to COS-7 cells. Transfection of the calcyclin cDNA into COS-7 and HEK-293 cells confirmed that the overexpression of calcylin caused these cells to gain the ability to sense extracellular cations, including aluminum, gadolinium, calcium, and magnesium. Conversely, we found that an antisense calcyclin construct reduced calcyclin expression and partially inhibited the cation-sensing response in MC3T3-E1 osteoblasts. These results implicate calcyclin in the activation of SRE and establish a role for calcyclin as an accessory protein involved in the cation-sensing pathway in osteoblasts.
Center for Bone and Mineral Disorders, Duke University Medical Center, Durham, North Carolina 27710, USA.
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D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010006:Osteoblasts / Q000378:metabolism*; D010627:Phenethylamines; D011437:Propylamines; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000378:metabolism*; D027602:Serum Response Element; D015398:Signal Transduction; D014018:Tissue Distribution; D014162:Transfection
D000814:Aniline Compounds; D002412:Cations; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C107873:N-(2-chlorophenylpropyl)-1-(3-methoxyphenyl)ethylamine; D016376:Oligonucleotides, Antisense; D010627:Phenethylamines; D011437:Propylamines; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C489134:S100a6 protein, mouse; C055351:S100A6 protein, human; D008156:Luciferases; D002118:Calcium