Negative feedback maintenance of heme homeostasis by its receptor, Rev-erbalpha

Abstract

Intracellular heme levels must be tightly regulated to maintain proper mitochondrial respiration while minimizing toxicity, but the homeostatic mechanisms are not well understood. Here we report a novel negative feedback mechanism whereby the nuclear heme receptor Rev-erbalpha tightly controls the level of its own ligand. Heme binding to Rev-erbalpha recruits the NCoR/histone deacetylase 3 (HDAC3) corepressor complex to repress the transcription of the coactivator PGC-1alpha, a potent inducer of heme synthesis. Depletion of Rev-erbalpha derepresses PGC-1alpha, resulting in increased heme levels. Conversely, increased Rev-erbalpha reduces intracellular heme, and impairs mitochondrial respiration in a heme-dependent manner. Consistent with this bioenergetic impairment, overexpression of Rev-erbalpha dramatically inhibits cell growth due to a cell cycle arrest. Thus, Rev-erbalpha modulates the synthesis of its own ligand in a negative feedback pathway that maintains heme levels and regulates cellular energy metabolism.

Figure 1.

Rev-erbα regulates intracellular heme levels. (A) Overexpression of Rev-erbα. Expression level of total Rev-erbα protein in NIH3T3 cells stably transfected control vector (Control-3T3) or vector expressing Rev-erbα SD55/59 (REV-3T3). (B) Rev-erbα reduces heme levels. Intracellular heme levels were measured in NIH3T3 cells stably expressing either control (Control-3T3) or Rev-erbα SD55/59, a stable form of Rev-erbα (REV-3T3). Mean ± SEM (n = 3). (*) P < 0.05 versus control cells expressing empty vector. (C) Depletion of Rev-erbα increases heme levels. Intracellular heme levels were measured in either control or Rev-erbα-deficient HepG2 cells. Mean ± SEM (n = 3). (*) P < 0.05 versus control shRNA β-galactosidase. (D) Depletion of Rev-erbα. shRNA knockdown of either β-galactosidase (control) or human Rev-erbα in HepG2 liver cells. Rev-erbα mRNA was quantitated by QPCR and normalized to those of GADPH level. Mean ± SD (n = 3). (*) P < 0.05 compared with control by Student's t-test.

Figure 2.

Rev-erbα represses PGC-1α gene expression. (A) PGC-1α is required for Rev-erbα regulation of heme levels. HepG2 cells were infected with adenovirus expressing Rev-erbα shRNA and/or PGC-1α shRNA as indicated. Mean ± SEM is shown. (*) P < 0.05 versus control adenovirus. (B) Rev-erbα depletion induces PGC-1α. PGC-1α gene expression was measured after siRNA knockdown of either scrambled sequence (control) or human Rev-erbα in HepG2 liver cells. (C) Overexpression of Rev-erbα. Rev-erbα was determined by QPCR in mouse livers with tail-injected AAV-GFP (control) and AAV-Rev-erbα 55/59SD. (*) P < 0.05 versus control. (D) Rev-erbα represses PGC-1α expression. PGC-1α and Alas1 were measured in livers from mice transduced by tail injection of AAV-GFP (control) and AAV-Rev-erbα 55/59SD.

Figure 3.

Rev-erbα recruits the HDAC3/NCoR corepressor complex to repress the PGC-1α gene through an intronic Rev-erb regulatory element. (A) Schematic presentation of the PGC-1α Intron1 sequence in which two conserved Rev-erbα-binding monomeric sites are closely located. (ROREd) distal RORE; (ROREp) proximal RORE. (B) Rev-erbα regulation of PGC-1α intron luciferase reporter transfected in HEK 293T cells. The control is pGL-3 promoter vector. PGC-1α luciferase reporter plasmid (0.1 μg) was used in transfection mixture along with 2 μg of pCDNA-Flag-Rev-erbα expression vector. The luciferase activities of all experiments are expressed as the mean ± SD (n = 3). (C) ChIP assay for recruitment of Rev-erbα and HDAC3 in 293T cells. (D,E) HDAC3 knockdown (D) or NCoR knockdown (E) induces endogenous PGC-1α gene expression. After shRNA or siRNA transfection, total RNA was prepared and PGC-1α gene expression was analyzed relative to GAPDH control by quantitative real-time PCR. The fold change was calculated as the relative abundance of PGC-1α mRNA in the cells receiving HDAC3 shRNA or NCoR siRNA divided by the relative abundance of PGC-1α mRNA in the cells receiving control shRNA or siRNA, which were set to 1. Results are expressed as mean ± SD. (*) P < 0.05 by paired Student's t-test.

Figure 4.

Intracellular heme concentration modulates PGC-1α gene expression. (A) Succinylacetone induces the expression of PGC-1α and ALAS1. HepG2 cells were treated with succinylacetone (5 mM) for 16 h. mRNA were quantitated by RT–PCR and normalized to GADPH. (B) Heme represses Alas1 and PGC-1α expression in HepG2 cells. Heme treatment was 10 μM for 16 h. (C) Heme represses Alas1 and PGC-1α in primary mouse hepatocytes. Heme treatment was 6 μM for 16 h. (D) Effect of heme on the expression of PGC-1α gene in cells depleted of Rev-erbα in HepG2 cells. Mean ± SD (n = 3); (*) P < 0.05 compared with DMSO-treated cells transfected with control shRNA. (E) Effect of heme on the occupancy of Rev-erbα, HDAC3, and NCoR at the PGC-1α intronic sequence in HepG2 cells. Results of heme treatment are normalized to DMSO results.

Figure 5.

Rev-erbα inhibits respiration-driven oxygen consumption rate and mitochondrial gene expression. (A) Ectopic expression of Rev-erbα inhibits respiration-driven oxygen consumption rate. Oxygen consumption rates and calculations were performed as in the Materials and Methods. Mean ± SEM (n = 15); (*) P < 0.05. (B) Addition of heme (2.5 μM for 6 h) partially rescues the inhibition of oxygen consumption by Rev-erbα. Mean ± SEM (n = 3); (*) P < 0.05. (C) Mitochondrial gene expression in REV-3T3 cells.

Figure 6.

Ectopic Rev-erbα causes cell cycle arrest and blocks cell growth. (A) Ectopic Rev-erbα blocks cell growth. Cell number experiments are expressed as mean ± SD (n = 3). (*) P < 0.05. (B) The depletion of Rev-erbα promotes cell growth. Cell numbers are expressed as mean ± SD (n = 3). (*) P < 0.05. (C) Ectopic Rev-erbα arrests cells in G2/M phase. The DNA content of NIH3T3 cells was determined by FACS. The percentage of cells in each cell cycle phase was calculated by ModFit Software and is presented in the figure. (*) P < 0.05.

Figure 7.

The Rev-erbα/PGC-1α pathway regulating heme homeostasis. Heme promotes Rev-erbα repression of PGC-1α, thereby reducing ALAS1 gene expression and heme biosynthesis. Conversely, low heme levels reduce Rev-erbα repression, enhancing PGC-1α stimulation of heme synthesis via transcriptional activation of the rate-limiting enzyme ALAS1.