Impairment of ATP-induced Ca²⁺-signalling in human thyroid cancer cells

Abstract

Extracellular nucleotides like ATP that activate the Ca²⁺-phosphatidylinositol (PI) signalling pathway have been suggested to participate in the regulation of normal human thyroid function. We examined, whether P_2y-purinergic receptors are expressed on human thyroid cancer cells and whether post-receptor Ca²⁺ signalling is altered by malignant transformation. Extracellular ATP caused a biphasic increase in cytosolic free Ca²⁺ ([Ca²⁺]_i) in normal human thyrocytes and in human follicular (FTC) and papillary (PTC) thyroid carcinoma cells. In FTC and PTC cell lines the dose-response curves for ATP-induced changes in [Ca²⁺]_i were shifted to the right when compared with normal thyrocytes, whereas in undifferentiated thyroid carcinoma (UTC) cells even high concentrations of ATP (500 μM) failed to stimulate a rise in [Ca²⁺]_i. By contrast, ATP stimulated inositol 1,4,5-trisphosphate (IP₃) formation and capacitative Ca²⁺ entry was operational as judged by thapsigargin in normal thyrocytes and all thyroid cancer cells. Thus, P_2y-purinergic receptors are expressed on thyroid tumor cells independent of degree of differentiation. In UTC cells, however, impairment in the Ca²⁺-phosphatidylinositol (PI) signalling cascade occurs distal to the formation of IP₃ and proximal to the activation of capacitative Ca²⁺ entry. Disturbed ATP-induced Ca²⁺-signalling and alterations in the Ca²⁺-PI signalling cascade may contribute to decreased expression or loss of specific thyroid functions in thyroid cancer cells.

Introduction

In various experimental thyroid systems including human thyrocytes extracellular ATP acts on specific P_2y-purinoceptors at the plasma membrane that stimulate phospholipase C (PLC), which causes rapid breakdown of phosphatidylinositol bisphosphate (PIP₂) into inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (Okajima et al., 1989, Raspe et al., 1991a, Raspe et al., 1991b, D'Arcangelo et al., 1995, Schöfl et al., 1995). DAG activates PKC and IP₃ causes a rise in cytosolic free Ca²⁺ ([Ca²⁺]_i) by mobilizing Ca²⁺ from intracellular stores. A rise in [Ca²⁺]_i is one of the key signals for the regulation of H₂O₂ production, which is a rate limiting step for iodide organification and thyroid hormone synthesis (Raspe et al., 1991b, Nakamura and Ohtaki, 1990, Corvilain et al., 1991, Bjorkman and Ekholm, 1992, Corvilain et al., 1994, Kimura et al., 1995), while PKC-dependent mechanisms promote thyroid cell proliferation (Dumont et al., 1992). Increased production of H₂O₂ in response to extracellular ATP has been reported from human, porcine and dog thyrocytes and FRTL-5 cells (Raspe et al., 1991b, Nakamura and Ohtaki, 1990, Corvilain et al., 1991, Bjorkman and Ekholm, 1992, Corvilain et al., 1994, Kimura et al., 1995) suggesting that extracellular ATP from various sources may be physiologically important in the regulation of normal thyroid function. Alterations in the expression and function of receptors and of intracellular signalling pathways are a hallmark of malignant transformation of many tumors including thyroid carcinomas (Farid et al., 1994). Thyrotropin (TSH)-receptor expression, for example, depends on the stage of differentiation of thyroid neoplasms and alterations in the cAMP-signal transduction pathway have been described which may lead to enhanced growth and abnormal function in thyroid tumors (Farid et al., 1994, Brabant et al., 1991, Michiels et al., 1994, Dremier et al., 1996). However, little information is available about P_2y-purinergic receptors and the Ca²⁺-phosphatidylinositol (PI) signalling pathway in thyroid tumors. Therefore, P_2y-purinergic receptor expression and post-receptor Ca²⁺-PI signalling was investigated in human thyroid cancer cells.