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Nucleotides are increasingly recognized as nonredundant extracellular signals for chemotaxis, cell growth, and cytokine release. Effects of extracellular nucleotides are mediated by P2 receptors, among which the P2X(7) subtype is attracting increasing attention for its involvement in apoptosis, cell growth, and cytokine release. Recent studies showed that P2X(7) is overexpressed in chronic lymphocytic leukemia and breast and prostate cancer. The aim of the present study was to better understand the clinical significance of P2X(7) receptor expression in normal and cancer human thyroid tissues. P2X(7) receptor message and protein expression and functional activity were tested in two cell lines (FB1 and FB2) established from either anaplastic or papillary primary thyroid cancer and in several histological samples of human papillary cancer. We show here that human thyroid papillary carcinoma, whether of the classical or follicular variant, expresses the P2X(7) receptor (P2X(7)R) to a much higher level than normal thyroid tissue. The P2X(7)R was similarly up-regulated in FB1 and FB2 cell lines. In contrast to normal thyroid cells, both cell lines responded to extracellular nucleotide stimulation with a large increase in intracellular Ca(2+) and secretion of IL-6. Ca(2+) increase was attenuated and release of IL-6 was fully blocked by P2X(7)R inhibitors. Finally, the thyroid carcinoma cell lines had at least a 3-fold higher intracellular ATP concentration and maintained at least a 3-fold higher extracellular ATP level, compared with control cells. These data suggest that an enhanced P2X(7)R function might be a feature of human thyroid cancer.

We investigated the presence of P2 receptors (P2Rs) in human thyrocytes and their possible involvement in the modulation of cytokine release. P2Rs expression was assessed by RT-PCR and, when possible, by immunoblotting. Human primary thyrocytes express the mRNA for the following P2X and P2Y subtypes: P2X(3), P2X(5), P2X(6), P2X(7), and P2Y(1), P2Y(2), P2Y(4), and P2Y(11). Stimulation with extracellular nucleotides of fura-2-loaded thyrocytes triggered an intracellular Ca(2+) signal, suggesting expression of functional receptors. Thyrocytes spontaneously released the proinflammatory cytokine IL-6. The ATP-hydrolyzing enzyme apyrase reduced basal IL-6 release, whereas extracellular ATP dose-dependently increased IL-6 secretion. Uridine 5'-triphosphate was also an effective stimulus, whereas benzoyl-ATP was ineffective, suggesting a P2Y- rather than P2X-modulated response. Finally, TSH reduced both the intracellular Ca(2+) ([Ca(2+)](i)) rise and IL-6 release triggered by P2Rs stimulation. In conclusion, we provide functional, pharmacological, and biochemical evidence that human primary thyrocytes express P2YR and P2XR subtypes, coupled to increases in ([Ca(2+)](i)) and secretion of IL-6. P2R-dependent modulation of IL-6 release from human thyrocytes suggests a novel mechanism whereby an inflammatory and/or immune-mediated damage can be initiated and amplified in the thyroid.