ANK is a multipass transmembrane protein transporter, thought to play a role in the export of intracellular inorganic pyrophosphate (iPPi), and so to contribute to the pathophysiology of chondrocalcinosis. As TGF-beta1 was shown to favour 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 PCR and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective role of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinases inhibitors and dominant negative/overexpression plasmids 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 level, as well as ePPi production. Using siRNA technology, we showed that Ank contributed for approximately 60 % and PC-1 for nearly 20 % to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of extracellular signal-regulated kinase (ERK) pathway, but neither of p38 mitogen-activated protein kinase (p38-MAPK) nor of protein kinase A (PKA). In line with the general protein kinase C (PKC) inhibitor calphostin C, Go6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKC delta inhibitor). These data suggest a regulatory role for calcium in TGF-beta 1 induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta 1 on Ank expression was inhibited by the suppression of 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 Ank gene, which requires activation of Ras, Raf-1, ERK and Ca2+-dependent PKC pathways in chondrocyte.