title: Calcium input potentiates the transforming growth factor (TGF)-beta1-dependent signaling to promote the export of inorganic pyrophosphate by articular chondrocyte.
abstract: Transforming growth factor (TGF)-β1 stimulates extracellular PP(i) (ePP(i)) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-β1 activation of ERK1/2 and Ca(2+)-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePP(i) metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa(2+)) or cytosolic Ca(2+) (cCa(2+)) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePP(i) levels (radiometric assay), and cCa(2+) input (fluorescent probe). Voltage-operated Ca(2+)-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa(2+) and ePP(i) levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa(2+) dose-dependent manner. TGF-β1 effects were suppressed by cCa(2+) chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca(2+). SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-β1. TGF-β1 promotes input of eCa(2+) through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePP(i) production in chondrocyte.
authors.affiliation: Laboratoire de Physiopathologie, Pharmacologie et Ingénierie Articulaires, Faculté de Médecine, UMR 7561 CNRS-Nancy-Université, Vandœuvre-Lès-Nancy, France. frederic.cailotto@hotmail.fr
authors.forename: Frederic
authors.initials: F
authors.lastname: Cailotto
authors.forename: Pascal
authors.initials: P
authors.lastname: Reboul
authors.forename: Sylvie
authors.initials: S
authors.lastname: Sebillaud
authors.forename: Patrick
authors.initials: P
authors.lastname: Netter
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Jouzeau
authors.forename: Arnaud
authors.initials: A
authors.lastname: Bianchi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism; D002358:Cartilage, Articular / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D002805:Chondrocalcinosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology; D011756:Diphosphates / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism* / Q000494:pharmacology; D050807:ets-Domain Protein Elk-1 / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C498769:Ankh protein, mouse; D015220:Calcium Channels; D011756:Diphosphates; C494114:Elk1 protein, rat; D007476:Ionophores; D028061:Phosphate Transport Proteins; D016329:Sp1 Transcription Factor; D053773:Transforming Growth Factor beta1; D050807:ets-Domain Protein Elk-1; D015759:Ionomycin; D051571:Protein Kinase C-alpha; D048052:Mitogen-Activated Protein Kinase 3; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; D018631:ras Proteins; D002118:Calcium