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, 291 (4), 1591-603

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

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Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Amy H Zhao et al. J Biol Chem.

Abstract

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Keywords: PBR; bone marrow; cancer; mitochondria; photodynamic therapy; protoporphyrin IX.

Figures

FIGURE 1.
FIGURE 1.
Bone marrow histology and evaluation of erythroid cells. A, sections of Tspofl/fl and Tspo−/− mice femur bone showing the marrow. Histological evaluation of the marrow showed no difference in overall hematopoietic cellularity. Scale bar, 200 μm. Inset, bone marrow cytology; there was no difference in relative density of erythroid precursor cells. Scale bar, 15 μm. B, enumeration of erythroid cells showed no differences in the percentage of erythroid precursors between the two groups (n = 3/group).
FIGURE 2.
FIGURE 2.
Expression of genes involved in PPIX synthesis in Tspo−/− bone marrow. A, schematic showing the different enzymatic steps involved in the conversion of ALA to PPIX and heme. The enzymes were ALA synthase (Alas), ALA dehydratase (Alad), hydroxymethyl bilane synthase (Hmbs), uroporphyrinogen III synthase (Uros), uroporphyrinogen decarboxylase (Urod), coproporphyrinogen oxidase (Cpox), protoporphyrinogen oxidase (Ppox), and ferrochelatase (Fech). The intermediates were porphobilinogen (PBG), hydroxymethyl bilane (HMB), uroporphyrinogen III (UROG III), coproporphyrinogen III (CPG III), and protoporphyrinogen IX (PPGIX). B, expression levels of transcripts coding enzymes involved in the conversion of ALA to PPIX in Tspofl/fl and Tspo−/− bone marrow at both baseline and after treatment with ALA. a, b, and c indicate p < 0.05 (n = 6/group).
FIGURE 3.
FIGURE 3.
Heme levels in tissues from Tspo−/− mice. A, PPIX standard and a representative analytical spleen sample showing the emission spectrum of PPIX with excitation at 400 nm; two emission maximums at 605 and 660 nm specific for PPIX are observed in both standard and prepared sample. B–D, normalized baseline heme levels in tissues (liver, spleen, and bone marrow) from Tspofl/fl and Tspo−/− mice. *, p < 0.05 (n = 6/group).
FIGURE 4.
FIGURE 4.
PPIX levels in plasma and tissues from Tspo−/− mice. Panels show normalized PPIX fluorescence in blood plasma (A), liver (B), spleen (C), and bone marrow (D), at baseline and at 1, 4, and 8 h after administration of ALA (n = 6–8/group).
FIGURE 5.
FIGURE 5.
PPIX uptake and localization in Tspofl/fl and Tspo−/− fibroblasts. A, PPIX fluorescence and MitoTracker® Green (Mito) colocalization in Tspofl/fl and Tspo−/− fibroblasts. Scale bar, 50 μm. B, Mander's overlap coefficient for PPIX and MitoTracker® Green localization was not different between Tspofl/fl and Tspo−/− fibroblasts. C, Pearson's coefficient for PPIX and MitoTracker® Green localization was not different between Tspofl/fl and Tspo−/− fibroblasts. D, PPIX uptake by fibroblasts after treatment with increasing concentrations of PPIX (0, 0.5, 1.0, or 1.5 μm) for 4 h was not different between Tspofl/fl and Tspo−/− cells. Mean fluorescence intensity for PPIX was measured by flow cytometry (n = 6 independent primary cultures/group).
FIGURE 6.
FIGURE 6.
Porphyrin-mediated phototoxicity in Tspofl/fl and Tspo−/− fibroblasts. A, spectrum of band pass filter used for photoexcitation allowed transmission of light 450 ± 60 nm. B, photoexcitation-induced cell death was not different between Tspofl/fl and Tspo−/− after treatment with increasing concentrations of PPIX (0, 0.5, 1.0, or 1.5 μm) and subsequent exposure to increasing energies of light (160, 240, or 320 mJ; n = 6 independent primary cultures/group).
FIGURE 7.
FIGURE 7.
PPIX synthesis from ALA in Tspofl/fl and Tspo−/− fibroblasts. A, PPIX concentration in Tspofl/fl and Tspo−/− fibroblast cell lysates before and after treatment with ALA. a and b indicate p < 0.05 (n = 3/group). B, expression levels of transcripts coding enzymes involved in the conversion of ALA to PPIX in Tspofl/fl and Tspo−/− fibroblasts at both baseline and after treatment with ALA. a, b, and c indicate p < 0.05 (n = 6 independent primary cultures/group).
FIGURE 8.
FIGURE 8.
PPIX synthesis from ALA in MA-10 cells and MA-10:TspoΔ/Δ cells. A, PPIX concentration in MA-10 cells and MA-10:TspoΔ/Δ cell lysates before and after treatment with ALA. a, b, and c indicate p < 0.05 (n = 3/group). B, expression levels of transcripts coding enzymes involved in the conversion of ALA to PPIX in MA-10 cells and MA-10:TspoΔ/Δ cells at both baseline and after treatment with ALA. a, b, and c indicate p < 0.05 (n = 3/group).
FIGURE 9.
FIGURE 9.
TSPO expression levels and PPIX synthesis from ALA in colon cancer cells. A, expression levels of TSPO and mitochondrial protein IDH2 in relation to ACTB in primary Tspofl/fl fibroblasts, MA-10 Leydig cells, and four different colon cancer cell lines (HCT116, HT29, LOVO, and DLD1 cells). Equal total protein (50 μg) was loaded to generate this representative Western blot. B, PPIX concentration in cell lysates before and after treatment with ALA in HCT116, HT29, LOVO, and DLD1 colon cancer cell lines. a, b, and c indicate p < 0.05 (n = 3/group).
FIGURE 10.
FIGURE 10.
Effect of TSPO2 expression on PPIX synthesis from ALA. A, Tspo2 gene expression normalized to endogenous expression in the bone marrow. Expression of Tspo2 was not observed (ø) in MA-10 cells and fibroblasts (blank). Specific ∼8-fold expression of Tspo2 was induced by adenoviral expression in Tspofl/fl and Tspo−/− fibroblasts. Adenovirus expressing tdTomato (tdTom) was examined as a negative control. B, PPIX concentration in Tspofl/fl and Tspo−/− fibroblast lysates after induced expression of tdTom or Tspo2, before and after treatment with ALA. a, b, and c indicate p < 0.05 (n = 3/group).
FIGURE 11.
FIGURE 11.
Analysis of mitochondrial bioenergetics in Tspofl/fl and Tspo−/− fibroblasts. A, baseline OCR and maximal OCR after addition of the protonophore FCCP was significantly lower in Tspo−/− compared with Tspofl/fl fibroblasts. OCR declined upon blocking ATP synthase activity using oligomycin or inhibiting electron transport chain using antimycin A and rotenone, but they were not significantly different between the two genotypes. B, mitochondrial coupling efficiency was not different between Tspofl/fl and Tspo−/− fibroblasts. C, mitochondrial ATP production was significantly lower in Tspo−/− compared with Tspofl/fl fibroblasts. D, proton leak was significantly lower in Tspo−/− compared with Tspofl/fl fibroblasts. E, spare respiratory capacity was significantly lower in Tspo−/− compared with Tspofl/fl fibroblasts. *, p < 0.05 (n = 5–7 independent primary cultures).
FIGURE 12.
FIGURE 12.
Analysis of mitochondrial membrane potential (Δψm) in Tspofl/fl and Tspo−/− fibroblasts. A, representative images of Tspofl/fl and Tspo−/− fibroblasts stained with TMRM and MitoTracker® Green from experiments measuring Δψm, showing baseline fluorescence, after treatment with oligomycin, and FCCP. B, normalized TMRM/MitoTracker® Green fluorescence intensity comparison between Tspofl/fl and Tspo−/− fibroblasts showed significantly decreased Δψm at baseline and after oligomycin treatment. *, p < 0.05 (n = 150 cells/group). C, expression of mitochondrial proteins VDAC1 and IDH2 were not different between Tspofl/fl and Tspo−/− fibroblasts. D, representative z-stacks of MitoTracker® Green stained Tspofl/fl and Tspo−/− fibroblasts used for calculating mitochondrial volume. Mitochondrial volume was not different between Tspofl/fl and Tspo−/− fibroblasts. Mitochondrial mass as measured by MitoTracker® Green labeling intensity was also not different between Tspofl/fl and Tspo−/− fibroblasts (n = 150 cells/group).
FIGURE 13.
FIGURE 13.
Effect of PK11195 treatment on ALA to PPIX bioconversion and Δψm. A, PPIX concentration in Tspofl/fl and Tspo−/− fibroblast cell lysates after treatment with ALA in the presence of 0 (control) or 1 μm PK11195. Treatment with PK11195 did not have an effect PPIX production. B, normalized TMRM/MitoTracker® Green fluorescence intensity comparison between Tspofl/fl and Tspo−/− fibroblasts after treatment with 0 (control) and 100 nm and 1 μm PK11195. Baseline Δψm was significantly lower in Tspo−/− compared with Tspofl/fl fibroblasts. Treatment with PK11195 significantly decreased Δψm at 1 μm concentration in both Tspofl/fl and Tspo−/− fibroblasts. a and b indicate p < 0.05 (n = 4 independent primary cultures/group).

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