Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells

Abstract

Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca²⁺-sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (−log EC₅₀ = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca²⁺-free medium. Inhibition of phospholipase-C or inositol trisphosphate release reduced intracellular calcium responses to nickel indicating activation of G_q-signaling. CaSR mRNA and protein expression in epithelial cells was demonstrated by RT-PCR, western blot and immunofluorescence. Transfection of specific siRNA inhibited CaSR expression and suppressed nickel-induced intracellular calcium responses in A549 cells thus confirming nickel-CaSR activation. NPS2390, a CaSR antagonist, abolished the calcium response to nickel. Nickel-induced contraction, proliferation, α₁(I)collagen production and inflammatory cytokines mRNA expression by epithelial cells as measured by traction microscopy, BrdU assay and RT-PCR, respectively. These responses were blocked by NPS2390. In conclusion, micromolar nickel concentrations, relevant to nickel found in the lung tissue of humans exposed to high environmental nickel, trigger intracellular Ca²⁺ mobilization in human airway epithelial cells through the activation of CaSR which translates into pathophysiological outputs potentially related to pulmonary disease.

Introduction

Inhalation of metal dusts and fumes can cause a variety of pathophysiological responses relevant to pulmonary disorders and toxicity [1]. Nickel compounds are prevalent in the environment and inhalation is the primary route of their occupational and ambient exposure [2]. Epidemiological studies show that environmental exposure to nickel has been linked to higher incidence of different respiratory diseases [3] and increased mortality in the United States [4], [5]. Nickel is also a toxic constituent of cigarette smoke [6]. Inhaled nickel is concentrated in the lung which has a tendency to retain significant amounts of nickel [7] giving rise to inflammatory damage [8], possibly through the release of reactive oxygen species and inflammatory cytokines [9], [10]. In addition, nickel can activate mitogen-activated protein kinases (MAPK) [11] and nuclear factor-κB [12] in lung epithelial cells.

At millimolar concentrations, nickel is considered a nonspecific Ca²⁺-channel blocker in different cell systems including human lung epithelial cells [13]. However, lower concentrations of nickel, in the range more relevant in terms of human exposure [7], [8], [10], [11], have been reported to trigger transient increases of intracellular calcium concentration ([Ca²⁺]_i) in rodent osteoclasts [14], hepatocytes [15] and Leydig cells [16]. This nickel (micromolar)-induced calcium signal was attributed to the activation of an extracellular calcium-sensing receptor (CaSR) recently characterized as a member of the G-protein-coupled receptor superfamily [17], [18]. Activation of CaSR causes an increase in [Ca²⁺]_i that arises from G_q-mediated activation of phospholipase C (PLC) triggering the formation of inositol-1,4,5-triphosphate (IP₃). CaSR may also signal through G_i proteins [17].

Although CaSR has been identified in rat lung [19], its presence and effects on the human respiratory system have not been thoroughly investigated. Since intracellular calcium is recognized as an important second messenger which may be involved in the toxicity of heavy metals including nickel [20], we examined in the present work the nickel-induced Ca²⁺ signal and derived functional outputs in human airway epithelial cells. We found that stimulation of CaSR by micromolar concentrations of nickel induces a range of potentially important pro-inflammatory effects which may contribute to the pathological and toxicological effects linked to high environmental nickel.