Proteomic Analysis of Zn Depletion/Repletion in the Hormone-Secreting Thyroid Follicular Cell Line FRTL-5

Abstract

Zinc deficiency predisposes to a wide spectrum of chronic diseases. The human Zn proteome was predicted to represent about 10% of the total human proteome, reflecting the broad array of metabolic functions in which this micronutrient is known to participate. In the thyroid, Zn was reported to regulate cellular homeostasis, with a yet elusive mechanism. The Fischer Rat Thyroid Cell Line FRTL-5 cell model, derived from a Fischer rat thyroid and displaying a follicular cell phenotype, was used to investigate a possible causal relationship between intracellular Zn levels and thyroid function. A proteomic approach was applied to compare proteins expressed in Zn deficiency, obtained by treating cells with the Zn-specific chelator N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene-diamine (TPEN), with Zn repleted cells. Quantitative proteomic analysis of whole cell protein extracts was performed using stable isotope dimethyl labelling coupled to nano-ultra performance liquid chromatography-mass spectrometry (UPLC-MS). TPEN treatment led to almost undetectable intracellular Zn, while decreasing thyroglobulin secretion. Subsequent addition of ZnSO₄ fully reversed these phenotypes. Comparative proteomic analysis of Zn depleted/repleted cells identified 108 proteins modulated by either treatment. Biological process enrichment analysis identified functions involved in calcium release and the regulation of translation as the most strongly regulated processes in Zn depleted cells.

Keywords: calcium channels; endocrine tissues; metal ion; ribosomes; zinc transport.

Figure 1

Secretion of thyroglobulin and expression of ZnT transporters in FRTL-5 cells. (A) Confluent cells were grown for 24 h in the presence (+) or absence (-) of serum (FBS) and/or a hormone cocktail. Supernatants were collected after 2, 4, 8, and 24 h and analyzed by Western blotting with anti-thyroglobulin (Tg) antibodies. (B) Western blot analysis of whole cell lysates, collected at 2, 3, and 4 days after seeding, with anti-ZnT2, ZnT4, and ZnT8 antibodies. Cells were grown in the presence of FBS and hormones.

Figure 2

Distinct localization of the zinc transporter ZnT8 and vesicle-like structures containing labile zinc in FRTL-5 cells. Cells grown in standard culture conditions were co-stained with: (A) anti ZnT8 polyclonal antibody and (B) FluoZin-3-AM. (C) Merged image of panels (A) and (B) with DAPI staining of nuclei. All images represent optical sections obtained by confocal acquisition. Size bar corresponds to 10 µM.

Figure 3

Intracellular localization of the zinc transporter ZnT8 and markers of intracellular vesicular compartments. Each row of panels contains the individual and merged images of double fluorescent staining with anti-ZnT8 (green fluorescence) and a distinct antibody against specific markers of intracellular compartment indicated on the left (red fluorescence). Markers: β1β2, AP2 (plasma membrane—clathrin vesicle trafficking); TGN 46 (Trans-Golgi Network); gm130 (Golgi compartment); β-COP (Golgi compartment—Endoplasmic Reticulum); TF rec (Transferrin receptor—recycling endosomes). Cell nuclei were labeled with DAPI (blue fluorescence). Images represent optical sections obtained by confocal acquisition. Size bar corresponds to 10 µM.

Figure 3

Intracellular localization of the zinc transporter ZnT8 and markers of intracellular vesicular compartments. Each row of panels contains the individual and merged images of double fluorescent staining with anti-ZnT8 (green fluorescence) and a distinct antibody against specific markers of intracellular compartment indicated on the left (red fluorescence). Markers: β1β2, AP2 (plasma membrane—clathrin vesicle trafficking); TGN 46 (Trans-Golgi Network); gm130 (Golgi compartment); β-COP (Golgi compartment—Endoplasmic Reticulum); TF rec (Transferrin receptor—recycling endosomes). Cell nuclei were labeled with DAPI (blue fluorescence). Images represent optical sections obtained by confocal acquisition. Size bar corresponds to 10 µM.

Figure 4

Intracellular Zn depletion by treatment of FRTL-5 cells with increasing concentrations of the Zn-specific chelator TPEN. Fluorimetric analysis of intracellular free Zn levels in FRTL-5 cells under standard culture conditions. Each column represents the average ± SD of three independent experiments. The arbitrary unit fluorescence reflects Zn binding by the Zn-specific fluorescent probe Fluozin-3 (p < 0.01) for TPEN and Zinc treatments—Paired Student’s t-Test. Asterisks “*” represent significant differences among treatments (p < 0.01).

Figure 5

Effect of Zn depletion/repletion on XIAP expression in Zn depleted/repleted FRTL-5 cells. (A) Top panel: immunoblotting of whole cell lysates with anti-XIAP antibodies. Bottom panel: quantitative analysis of two independent experiments performed in triplicate. Each column represents the average of XIAP band intensities normalized to the corresponding values for α-tubulin. Data are expressed as mean ± SD. Statistical significance was evaluated by Welch one-way ANOVA followed by a Tamhane test; different letters indicate significant differences (p < 0.01). (B) Intracellular Zn staining with Fluozin-3 in FRTL-5 cells treated as described in panel C (CTR = control). (C) Study design: Confluent FRTL-5 cells were treated for 2 h with 25 µM TPEN to induce marginal Zn deficiency and then incubated with 25 µM ZnSO₄ for 24 h to replete intracellular Zn stores. After treatments, total proteins were extracted, digested, and analyzed by mass spectrometry.

Figure 6

(A) Venn diagram comparing the distribution of modulated proteins in the three different conditions. (B) log2 fold change for each modulated protein visualized as a heatmap. Green, light red, and light blue boxes on the left side refer to the relevant clusters discussed in the text. (C) Venn diagram showing the interaction among three domains of zinc-related proteins: ZNBP, ZiNc Binding Proteins listed in the Zn proteome interaction network [4]; ZNThy, proteins modulated in thyroid by Zn depletion/repletion in this work; INT: Zinc binding protein INTeractors listed in the Zn proteome interaction network [4]. (D) Representative doubly charged ions of a peptide belonging to an identified protein labeled as light, intermediate, and heavy.

Figure 7

Gene ontology biological processes (A,C) and molecular functions (B,D) significantly enriched by proteins modulated in Zn depletion (TPEN (Zn deprived cells) vs. CTRL (Control cells), panels A, B), and in Zn repletion (REC (Zn repleted cells) vs. TPEN, panels C, D).