Chromosome 19 open reading frame 80 is upregulated by thyroid hormone and modulates autophagy and lipid metabolism - PubMed (original) (raw)

Chromosome 19 open reading frame 80 is upregulated by thyroid hormone and modulates autophagy and lipid metabolism

Yi-Hsin Tseng et al. Autophagy. 2014 Jan.

Abstract

The thyroid hormone, T 3, regulates cell growth, differentiation and development through binding to the nuclear thyroid hormone receptor (THR), a member of the steroid/TR superfamily of ligand-dependent transcriptional factors. T 3 modulates lipid metabolism in liver, although the detailed molecular mechanisms are unclear at present. Here, by a microarray analysis, we identified a novel chromosome 19 open reading frame 80 (C19orf80) which was activated by T 3. T 3 stimulation led to upregulation of both mRNA and protein levels of C19orf80. Immunofluorescence analysis revealed a vesicle-like pattern of C19orf80 around lipid droplets or within the lysosome-associated compartment in cells. Furthermore, T 3 treatment as well as C19orf80 overexpression specifically activated the autophagic response and lipid metabolism, as observed from lipidated LC3 (LC3-II) and levels of oxygen consumption rate, respectively. Reciprocally, knockdown of C19orf80 obstructed T 3-activated autophagy and lipolysis. Moreover, treatment with autolysosome maturation inhibitors, ammonium chloride and chloroquine, not only suppressed the T 3-activated autophagic process but also lipid metabolism. Our results collectively suggested that T 3 regulates lipid metabolism through a C19orf80-activated autophagic process.

Keywords: autophagy; chromosome 19 open reading frame 80; lipid droplet; lysosome; thyroid hormone.

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Figures

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Figure 1. TR upregulates C19orf80 expression in HCC cell lines. (A) Affymetrix microarray analysis was performed to determine relative fold induction of T3 in HepG2-THRA/THRB cells. Each dot represents a specific gene probe. The C19orf80 tag (220437_at) is indicated. (B) qRT-PCR of C19orf80 mRNA expression levels in HepG2-Neo and HepG2-THRA cells (***P < 0.001, n = 3). Error bars: s.e.m. (C) Immunoblots of HepG2-THRA, HepG2-THRB, HepG2-Neo and Huh7 cell lysates. (D) Schematic representation of the C19orf80 promoter with potential TRE binding sites (+1, translation start site). Serial deletion or mutated fragments of C19orf80 5′-flanking DNA were cloned into pGL3 or pA3tk-luc reporter plasmids, as indicated. T3 induction fold is presented as mean values ± s.e.m. (n = 3). (E) Putative TRE sequences of C19orf80 are indicated. (F) ChIP assay of the C19orf80 5′-flanking region (−3060 to ~−2895). The promoter region of GAPDH was used as the negative control.

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Figure 2. T3 induces the formation and maturation of autolysosomes. (A) Immunofluorescence analysis of C19orf80 with LAMP2 in HepG2-THRA cells (inserts, magnified image of arrowhead indicated). (B) Cells were stained with acidotropic dye, Lyso-ID red, to highlight acidic vesicles. Fluorescence signals were captured and analyzed using ImageJ software. (C) Immunoblots of HepG2-THRA cell lysates. (D) Transmission electron microscopy analysis of HepG2-THRA cells. N, nucleus; M, mitochondria; LD, lipid droplet; AV, autophagic vacuole. (E) HepG2-THRA cells were transfected with mRFP-GFP-LC3 expression plasmids overnight. Culture media were replaced for a further 48 h, and fluorescence images captured. (F) The percentage of acidic vesicular LC3 (RFP+/GFP− signal, arrowhead) was calculated (***P < 0.001, **P < 0.01, n = 3). Error bars: s.e.m. (A, B, and E) Scale bar: 20 μm.

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Figure 3. T3-induced maturation of autolysosomes is blocked by NH4Cl and CQ. (A) HepG2-THRA cells were treated with inhibitors (NH4Cl: 20 mM, CQ: 50 μM), as indicated, and lysates subjected to immunoblot analysis. (B) HepG2-THRA cells were transfected with mRFP-GFP-LC3 overnight. Culture media were treated as for (A), and fluorescence images captured. (C) The percentage of acidic vesicular LC3 (RFP+/GFP− signal) was calculated (***P < 0.001, **P < 0.01, n = 3). Error bars: s.e.m. Scale bar: 20 μm.

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Figure 4. C19orf80 induces an increase in acidic vesicles and promotes autophagy (A) Lyso-ID staining of Huh7-C19orf80 cells. Fluorescence signals were analyzed. (B) Immunoblots of Huh7-C19orf80 cell lysates. Cells were pretreated with inhibitors (NH4Cl: 20 mM, CQ: 50 μM) for 24 h. (C) mRFP-GFP-LC3 expression plasmids were transfected into C19orf80-overexpressing Huh7 and control cells for 72 h. (D) The percentage of acidic vesicular LC3 (RFP+/GFP− signal, arrowhead) was calculated (***P < 0.001, **P < 0.01, n = 3). Error bars: s.e.m. (A and C) Scale bar: 20 μm.

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Figure 5. C19orf80 knockdown compromises T3-induced autophagy. (A) C19orf80 knockdown and cognate control HepG2-THRA cells were stained with Lyso-ID. Fluorescence signals were captured and analyzed. (B) Immunoblots of C19orf80 knockdown and control cells. Cells were pretreated with CQ (50 μM) for 24 h. Relative fold of T3-induced LC3-II lipidation is presented (LC3-II/ACTB, +T3/−T3). (C) mRFP-GFP-LC3 expression plasmids were transfected into C19orf80 knockdown and control cells overnight. Culture media were replaced for a further 48 h, and fluorescence images captured. (D) The percentage of acidic vesicular LC3 (RFP+/GFP− signal) was calculated (***P < 0.001, **P < 0.01, n = 3). Error bars: s.e.m. Scale bar: 20 μm.

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Figure 6. T3-induced C19orf80 is associated with lipid droplets. (A) Immunofluorescence analysis of C19orf80 with PLIN3 in HepG2-THRA cells. BODIPY staining of T3-induced C19orf80 in HepG2-THRA cells (B) or FLAG-tagged C19orf80 in Huh7 cells (C). The insert shows magnified image of region indicated by arrowhead. Scale bar: 20 μm.

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Figure 7. T3-induced C19orf80 is associated with lipid metabolism. (A–C) HepG2-THRA cells were cultured in the absence or presence of 10 nM T3 for 24 h. Acidic inhibitors were added for a further 24 h, as indicated. (D–F) Huh7-C19orf80 and vector control cells were plated for 48 h. (G–I) C19orf80 knockdown and cognate control HepG2-THRA cells were cultured in the absence or presence of 10 nM T3 for 48 h. Lipid droplets were stained with BODIPY (A, D, and G), and triglyceride (TG nmole/μg) (B, E, and H) and oxygen consumption rate (pmole/min/μg protein) (C, F, and I) detected. (***P < 0.001, **P < 0.01, *P < 0.05 n = 3). Error bars: s.e.m. Scale bar: 20 μm.

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