Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes.
ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-beta1 on Ank mRNA level. These data show that TGF-beta1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca2+-dependent PKC pathways in chondrocytes.
UMR 7561 CNRS-Nancy-Université, Laboratoire de Physiopathologie et Pharmacologie Articulaires, France. frederic.cailotto@hotmail.fr
Frederic
F
Cailotto
Arnaud
A
Bianchi
Sylvie
S
Sebillaud
Narayanan
N
Venkatesan
David
D
Moulin
Jean-Yves
JY
Jouzeau
Patrick
P
Netter
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002805:Chondrocalcinosis / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D011756:Diphosphates / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D028061:Phosphate Transport Proteins; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D018631:ras Proteins / Q000378:metabolism
C498769:Ankh protein, mouse; D017931:DNA Primers; D011756:Diphosphates; D008565:Membrane Proteins; D028061:Phosphate Transport Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases; D018631:ras Proteins