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Homeostatic regulation of growth-associated increase in the labile intracellular pool of zinc in 3T3 cells Simpson, Madeline

Abstract

The size of the labile intracellular pool of zinc (LIPZ) is increased during growth factorstimulated proliferation in 3T3 cells, but the mechanisms involved are unknown. We hypothesized that this increase in the size of the LIPZ is the result of altered cellular zinc homeostasis, ensuring an adequate supply of zinc to sustain cell proliferation. The objectives of this study were to study the role of metallothionein (MT), zinc importers (DCT1, ZIP1 and ZIRTL), zinc exporters (ZnT-1, -2, and ZnT-4), and zinc net retention in the proliferationassociated increase in the size of the LIPZ. 3T3 fibroblasts were cultured in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS) for 72 h (1 x 10⁴/T75; n = 6), followed by induction of quiescence with 1% FBS for 48 h. The cells were then cultured in DMEM supplemented with 10% FBS for 48 h, with or without growth factors [platelet-derived growth factor (100 ng/mL), epidermal growth factor (50 ng/mL), and insulin-like growth factor-1 (20 ng/mL)]. The size of the LIPZ was assessed by relative brightness of Zinquin-dependent fluorescence using a UV fluorescent microscope. Levels of MT were assessed using the ¹⁰⁹Cd-hemoglobin affinity assay. Abundance of transporter mRNA was determined by reverse transcription-polymerase chain reaction. Zinc net retention was determined by incubating cells with ⁶⁵zinc beginning at the time of growth factor treatment. Growth factor treatment increased cell number (48%) and relative brightness of Zinquin-dependent fluorescence, as well as redistributing the LIPZ to the area surrounding the nucleus, but had no effect on total cellular zinc concentration and MT protein levels. Growth factor treatment increased the abundance of DCT1 (27%), ZIRTL (33%), ZnTl (24%) and ZnT4 (67%) mRNA, but did not affect the abundance of ZIP1 and ZnT2 mRNA. Zinc net retention were increased by growth factor treatment at 45 min, 6 and 48 h, but were not affected at 0, 5, 15, 30, and 60 min, and 12 h. These data suggest that the increase in the size of the LIPZ in response to growth stimulation is via increased zinc net retention through selective upregulation of zinc transporter expression, and is likely required for DNA synthesis.

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