Elsevier

Cellular Signalling

Volume 14, Issue 4, April 2002, Pages 327-340
Cellular Signalling

Cadmium-induced DNA synthesis and cell proliferation in macrophages: The role of intracellular calcium and signal transduction mechanisms

https://doi.org/10.1016/S0898-6568(01)00268-6Get rights and content

Abstract

Cd2+ exposure increases the risk of cancer in humans and animals. In this report, we have studied the effect of Cd2+ on signal transduction and Ca2+ mobilization in murine macrophages. At micromolar concentrations, Cd2+ significantly increased cell division as judged by [3H]thymidine uptake and cell counts. Cd2+-treated cells continued to proliferate even after more than 4 weeks in culture. Cd2+ (1 μM) treatment induced a 1.5- to 2-fold increase in cytosolic free Ca2+, [Ca2+]i, which was transitory and/or oscillatory. The sources of this Ca2+ included both inositol 1,4,5-trisphosphate (IP3)-sensitive and -insensitive stores. Macrophage treatment with 1-(6-((17β-3-methoxyestra-1,2,5(10)-triene-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), decreased Cd2+-induced formation of IP3 in a concentration-dependent manner (Kd∼2 μM). This caused a concomitant, partial decrease in the effect of Cd2+ on [Ca2+]i. Cd2+ itself crosses the macrophage membrane in part via L-type Ca2+ channels, but it also interacts with a cell surface membrane protein(s) coupled to a pertussis toxin-sensitive G protein. Use of selective inhibitors of signal transduction and the quantitation of the levels of phosphorylated MAPK/ERK-activating kinase-1 (MEK1), extracellular signal-regulated kinase-1 (ERK1), and p38 mitogen-activated protein kinase (MAPK) suggests that the effects of Cd2+ are mediated by the p21ras-dependent MAPK, but not the phosphoinositide 3 (PI 3)-kinase signalling pathway. The effect of activating these pathways includes increased availability of the transcription factor NFκB as well as activation of the early genes c-fos and c-myc.

Introduction

Cd2+ is a xenobiotic with no known physiologic function. In addition to industrial exposure, humans may come in contact with Cd2+ from contaminated food and cigarette smoke [1], [2]. Besides its toxicity, Cd2+ exposure is associated with an increased risk of cancers such as lung and prostate [3]. In vitro, low doses of Cd2+ stimulate DNA synthesis, cell multiplication, and malignant transformation [1], [4], [5]. While low doses of Cd2+ are associated with cell transformation, in the millimolar range, Cd2+ is quite toxic and inhibits cell growth [1], [6]. Such exposure is associated with diminished DNA synthesis, DNA strand breaks, and chromosomal aberrations [1], [6]. Cd2+ interacts at the cell membrane and is taken up by mechanisms that include Ca2+ channels and the divalent cation transporter [1], [7], [8], [9], [10]. Cd2+ exposure results in induction of a number of genes necessary for synthesis of metallothionein, glutathione, heat shock proteins, and heme oxygenase-1 [1], [11], [12], [13], [14], [15], [16]. Cd2+ exposure also increases transcription of c-jun, c-myc, c-fos, erg-1, and gadd 153 [1], [11], [13], [17], [18], [19], [20].

In the present study, we examined the effects of Cd2+ exposure on signal transduction and Ca2+ regulation in murine peritoneal macrophages. These cells do not demonstrate a high proliferation rate in vitro and macrophage tumors are very rare. Exposure of these cells to Cd2+ (1 μM) caused a 1.5- to 2-fold increase in [3H]thymidine uptake with a concomitant 1.5- to 2-fold increase in cell protein and cell number. Macrophages exposed to Cd2+ were kept in culture for 4 or more weeks. These cells demonstrated significant morphological abnormalities and continued to proliferate. Cd2+ entry into macrophages is primarily mediated by L-type Ca2+ channels. Cd2+ also binds to a cell surface membrane protein(s) whose interaction with Cd2+ activates a pertussis toxin-sensitive G protein and subsequently phosphatidylinositol-specific phospholipase C (PI-PLC). Cd2+ treatment of macrophages causes a rise in cytosolic free Ca2+, [Ca2+]i, which is mediated by both inositol 1,4,5-trisphosphate (IP3)-sensitive and -insensitive stores. Finally, the effects of Cd2+ on cell proliferation are dependent on the p21ras-dependent mitogen-activated protein kinase (MAPK) pathway, but do not require the phosphoinositide 3 (PI 3)-kinase downstream signalling pathway.

Section snippets

Materials

The sources of thioglycollate, Trypan blue, cell culture materials, [3H]thymidine, BAPTA/AM, Fura-2/AM, thapsigargin, staurosporin, genestein, and fatty acid-free bovine serum albumin (BSA) have previously been described [21], [22]. Xestospongin C was from Calbiochem (Richmond, CA). 1-(6-((17β-3-Methoxyestra-1,2,5(10)-triene-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), wortmannin, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002),

Cd2+ and macrophage division

At lower concentrations of Cd2+ (0.1–1 μM), [3H]thymidine uptake was enhanced to a maximum of 1.5- to 2-fold (Fig. 1A). Above this concentration range, [3H]thymidine uptake decreased. [3H]thymidine uptake may indicate enhanced DNA synthesis, but there are other potential mechanisms of enhanced uptake independent of new synthesis of nucleic acid. We, therefore, also studied the effect of Cd2+ concentration on total cellular protein content and cell number. Over the concentration range of 0.1–1

Discussion

The industrial uses of Cd2+ have increased dramatically in the last several decades and now include nickel/cadmium batteries, electroplating, pigments, and plastics [1], [2], [3], [6]. Cd2+ also enters the environment as a result of mining and smelting of various other metals [1], [2], [3], [6]. Meat, fish, and even fruits contain detectable levels of Cd2+ [1], [2], [3], [6], [54], [55]. Shellfish are particularly prone to accumulate Cd2+ where tissue levels may reach 1000 μg/ml. Cigarette

Acknowledgements

This work was supported by a grant from the National Heart, Lung, and Blood Institute, R37 HL-24066.

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