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

Abstract

Transforming growth factor (TGF)-␤1 stimulates extracellular PP i (ePP i ) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was upregulated by TGF-␤1 activation of ERK1/2 and Ca 2؉ -dependent protein kinase C (PKC␣). 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-␤1 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-␤1 elevated cCa 2؉ and ePP i levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa 2؉ dose-dependent manner. TGF-␤1 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 PKC␣, 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-␤1. TGF-␤1 promotes input of eCa 2؉ through opening of L-and T-VOCs, to potentiate ERK1/2 and PKC␣ signaling cascades, resulting in an enhanced activation of Ank promoter and ePP i production in chondrocyte.The balance between extracellular inorganic pyrophosphate (ePP i ) 3 and extracellular inorganic phosphate (eP i ) is critical for homeostasis of articular cartilage. Indeed, an increase in ePP i supports calcium pyrophosphate dihydrate crystal (CPPD) depositions responsible for articular chondrocalcinosis (ACC) (1). Production of ePP i by articular chondrocytes is dependent on PC-1 (plasma cell membrane glycoprotein-1; also known as ENPP1), an ectoenzyme that produces ePP i from nucleotide triphosphates (2), and on the transporter ANK, which exports PP i across the cell membrane (3). Although ePP i level can be controlled by tissue-nonspecific alkaline phosphatase, which hydrolyzes ePP i into eP i , no expression of this enzyme was detected in healthy articular cartilage compared with osteoarthritic cartilage (4). Therefore, CPPD formation only depends on the expression level of PC-1 and ANK in mature articular cartilage. Transforming growth factor (TGF)-␤1 is a well known inducer of ePP i production by articular chondrocytes (1). We demonstrated previously that ANK was the major contributor of this TGF-␤1-induced ePP i production; meanwhile, PC-1 played a minor role (5). Furthermo...

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