Calcium Input Potentiates the Transforming Growth Factor (TGF)-β1-dependent Signaling to Promote the Export of Inorganic Pyrophosphate by Articular Chondrocyte

摘要

Transforming growth factor (TGF)-β1 stimulates extracellular PPi (ePPi) 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 Ca²⁺-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePPi metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-β1 under extracellular (eCa²⁺) or cytosolic Ca²⁺ (cCa²⁺) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePPi levels (radiometric assay), and cCa²⁺ input (fluorescent probe). Voltage-operated Ca²⁺-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-β1 elevated cCa²⁺ and ePPi levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa²⁺ dose-dependent manner. TGF-β1 effects were suppressed by cCa²⁺ 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 Ca²⁺. 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 eCa²⁺ through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePPi production in chondrocyte.