PGRMC2 is an intracellular haem chaperone critical for adipocyte function

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Abstract

Haem is an essential prosthetic group of numerous proteins and a central signalling molecule in many physiologic processes1,2. The chemical reactivity of haem means that a network of intracellular chaperone proteins is required to avert the cytotoxic effects of free haem, but the constituents of such trafficking pathways are unknown3,4. Haem synthesis is completed in mitochondria, with ferrochelatase adding iron to protoporphyrin IX. How this vital but highly reactive metabolite is delivered from mitochondria to haemoproteins throughout the cell remains poorly defined3,4. Here we show that progesterone receptor membrane component 2 (PGRMC2) is required for delivery of labile, or signalling haem, to the nucleus. Deletion of PGMRC2 in brown fat, which has a high demand for haem, reduced labile haem in the nucleus and increased stability of the haem-responsive transcriptional repressors Rev-Erbα and BACH1. Ensuing alterations in gene expression caused severe mitochondrial defects that rendered adipose-specific PGRMC2-null mice unable to activate adaptive thermogenesis and prone to greater metabolic deterioration when fed a high-fat diet. By contrast, obese-diabetic mice treated with a small-molecule PGRMC2 activator showed substantial improvement of diabetic features. These studies uncover a role for PGRMC2 in intracellular haem transport, reveal the influence of adipose tissue haem dynamics on physiology and suggest that modulation of PGRMC2 may revert obesity-linked defects in adipocytes.

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Fig. 1: PGRMC2 controls the intracellular distribution of labile haem.
Fig. 2: PATKO mice are sensitive to cold.
Fig. 3: PGRMC2 regulates haem-sensitive transcription and mitochondrial function in BAT.
Fig. 4: PGRMC2 controls systemic glucose homeostasis.

Data availability

Source data tables are provided for Figs. 14 and Extended Data Figs. 18. Full scans of all western blots are shown in the Supplementary Information. RNA-seq data are available in the Gene Expression Omnibus under accession number GSE124621. All other data supporting the findings in this study are available from the corresponding author upon request.

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Acknowledgements

We thank I. Hamza for labile haem reporter plasmids; A. Kralli, A. Saghatelian, P. Tontonoz, R. L. Wiseman, L. Gerace, J. Z. Long, N. Mitro and J. Hogenesch for critical input; M. R. Wood and T. Fassel for assistance with electron microscopy and N. Hah for help with RNA-seq studies. R.S. thanks the UCLA QCBio Collaboratory community directed by M. Pellegrini. This work was funded by NIH grants DK099810 and DK114785 (E.S. and B.F.C.), DK121196 and S10OD016357 (E.S.), and OD016564 (J.K.P. and J.J.P.). B.P.K. and V.A. were supported by fellowships 15POST25100007 and 17POST33660833 from the American Heart Association.

Author information

A.G. and E.S. conceived the project, designed research and analysed data. A.G. and B.P.K. performed in vivo experiments. A.G., C.G., V.A. and B.P.K. carried out cell-based assays. A.G. and J.Y.L. performed gene-expression and biochemical analyses. A.S.K. prepared PGRMC2 proteins. S.M. prepared apo-Rev-Erbα protein. J.R.M.-B. and W.R.W. carried out mass spectrometry experiments. A.G. and R.S. performed bioinformatic analysis. C.G.P. synthesized CPAG-1. J.J.P. and J. K.P. provided Pgrmc2 and Pgrmc1 floxed mice. R.C.-C. and B.C. contributed to energy balance studies. L.A.S., D.K., C.G.P., G.S. and B.F.C. provided advice and reagents. A.G. and E.S. wrote the manuscript and integrated comments from the other authors.

Correspondence to Enrique Saez.

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Competing interests

The authors declare no competing interests.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Peer review information Nature thanks Urs Albrecht, Edward Chouchani, Iqbal Hamza and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 PGRMC2 binds haem and, with PGRMC1, coordinates its intracellular distribution.

a, Absorbance spectra of mouse PGRMC2 protein shows peaks of haem–protein complexes in the 390–450-nm range. Dotted spectra indicate haem–protein complexes after 10 mM dithionite reduction of the iron moiety. b, LC–MS/MS spectra of haemin standard (left) and PGRMC2 protein (right) with collision energy of 40 V. c, Isotope envelope of haemin calculated on the basis of isotope natural abundance for C34H32ClFeN4O4 (left), PGRMC2 protein (centre) and haemin standard (right). d, Purified mouse PGRMC2(3×M) mutant (Y131F/K187A/Y188F) does not bind haem. e, The Soret peak typical of haemoproteins is absent in PGRMC2(3×M). f, Representative fluorescence imaging of cells expressing targeted HRP or APX labile haem reporters, showing their localization to mitochondria, endoplasmic reticulum, nucleus and cytosol. g, Levels of Pgrmc2 and Pgrmc1 mRNA in siRNA-transfected HEK293T cells (n = 3 biologically independent samples). h, Interaction of PGRMC1 with PGRMC2 is not observed when PGRMC2 is immunoprecipitated using an antibody that recognizes the haem-binding domain at the C terminus of PGRMC2. Representative results from two (ae, h) or three (f, g) independent experiments. Data presented as mean ± s.d., ***P < 0.001 versus scrambled basal; two-way ANOVA with multiple comparisons and a Tukey’s post-test. Source Data

Extended Data Fig. 2 Pgrmc2 is enriched in adipose tissue and regulates BAT function.

a, PGRMC2 protein levels increase during adipocyte differentiation. 3T3-L1 preadipocytes were induced to differentiate and protein extracts prepared at the indicated time points. PPARγ and CEBPδ are markers of mature adipocytes and preadipocytes, respectively. Representative results from three independent experiments. b, Profile of Pgrmc2 mRNA expression across mouse tissues (n = 5 biologically independent samples). c, Whole-body and inguinal subcutaneous fat weight of chow-fed wild-type and PATKO mice housed at 30 °C (WT, n = 8; PATKO, n = 9). d, OCR, core body temperature, CO2 production rate, respiratory exchange ratio (RER), and activity oscillations of PATKO mice housed at 30 °C (WT, n = 5; PATKO, n = 6). e, Levels of plasma noradrenaline, glucose and non-esterified fatty acids (NEFA) in wild-type and PATKO mice on cold challenge (WT, n = 5; PATKO, n = 7). f, Increased oxygen consumption upon acute injection of the β3-agonist CL316,243 (1 mg kg−1) is reduced in PATKO mice housed at 30 °C, despite comparable motor activity (n = 5 biologically independent samples). g, Adipose-specific PGRMC1 and PGRMC2 double-knockout mice (DKO) housed at 30 °C are cold-intolerant (WT, n = 13; DKO, n = 8 biologically independent samples). Survival curves of wild-type and DKO mice exposed to 4 °C (homeothermia is at 31 °C). Mice were exposed to 4 °C at ZT5. Data presented as mean ± s.e.m. *P < 0.05 and ***P < 0.001 versus wild type; two-tailed Student’s t-test (e, f) or two-way ANOVA with multiple comparisons and a Tukey’s post-test (g). Source Data

Extended Data Fig. 3 Effect of Pgrmc2 deletion in BAT.

BAT from chow-fed wild-type and mutant mice housed at 30 °C was analysed. a, Levels of succinyl-CoA, glycine and aminolevulinic acid (ALA) in BAT quantified using targeted metabolomics (n = 5 biologically independent samples per group). b, PATKO mice show reduced expression of Alas1 and Alas2 in BAT (n = 3 biologically independent samples per group). c, Nuclear labile haem is significantly lower in BAT of fat-specific PGRMC1 and PGRMC2 DKO mice housed at 30 °C (n = 4 biologically independent samples per group). Similar to PATKO mice, BAT of DKO mice is discoloured. Representative results from two independent experiments. d, Expression of Rev-Erbα and BACH1 targets (Bmal1 and Fth1, respectively) in BAT of PATKO mice housed at 30 °C (WT, n = 5; PATKO, n = 6). e, Circadian oscillation of clock components is not altered in PATKO BAT (n = 3 biologically independent samples per group per time point). f, Gene ontology (GO) category analysis (biological process) of significantly downregulated genes in RNA-seq analysis of BAT from wild-type and PATKO mice housed at 30 °C (n = 4 biologically independent samples per group). P values determined by standard accumulative hypergeometric statistical test. g, Circos plot of haem-related DEGs showing that the majority (28 out of 45) of them belong to the top-3 downregulated biological processes. Number in parentheses below each biological process represents the total number of DEGs in PATKO BAT in that category. Blue lines refer to downregulated DEGs and red lines to upregulated DEGs. Data presented as mean ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 versus wild type determined by two-tailed Student’s t-test. Source Data

Extended Data Fig. 4 Primary brown adipocytes recapitulate the mitochondrial defects of PATKO BAT.

a, Wild-type and PGRMC2-null primary brown adipocytes differentiated in vitro imaged on day eight. Lipid stained with Nile red (red) and nuclei stained with Hoechst (blue). Scale bar, 100 μm. b, Protein levels of adipocyte markers during the course of differentiation. c, PGRMC2-null brown adipocytes have impaired mitochondrial respiration (n = 3). dh, Lack of PGRMC2 in brown adipocytes results in a defective mitochondrial response to endogenous (d), synthetic pan β-adrenergic agonists (e) and pan β3-adrenergic agonists (f), and to downstream activators of adrenergic signalling (g, h) (n = 5). i, Induction of noradrenaline-responsive genes is similar in wild-type and PGRMC2-null brown adipocytes (n = 3) exposed to 100 nM noradrenaline for 2 h. j, OXPHOS proteins and UCP1 are reduced in primary brown PATKO adipocytes. k, PGRMC1 and PGRMC2 DKO primary brown adipocytes differentiated in vitro show severe mitochondrial dysfunction, an inability to increase oxygen consumption on noradrenaline exposure (n = 3), and reduced UCP1 and OXPHOS proteins. l, m, Overexpression of human wild-type PGRMC2, but not of a haem-binding mutant (3×M (Y137F/K193A/Y194F)), can rescue mitochondrial function and the response to noradrenaline in PATKO adipocytes (l, n = 4; m, WT–mCherry, WT–WT, PATKO–WT, n = 8; WT–3×M, PATKO-3×M, n = 7; PATKO-mCherry, n = 6). n, Ucp1 mRNA expression is restored when human wild-type PGRMC2, but not the haem-binding mutant 3×M, is expressed in PATKO cells (n = 3). o, Levels of mouse and human Pgrmc2 mRNA in primary adipocytes used in ln (n = 3). In ao, n represents biologically independent samples. Representative results from two (jo) or three (ai) independent experiments. Data presented as mean ± s.d. *P < 0.05, **P < 0.01 and ***P < 0.001 versus wild type; ###P < 0.001 versus vehicle; two-way ANOVA with multiple comparisons and a Bonferroni’s post-test. Source Data

Extended Data Fig. 5 PGRMC2-mediated transport of endogenous labile haem regulates mitochondrial function in primary brown adipocytes.

a, b, Inhibition for 48 h of endogenous haem synthesis with 0.5 mM succinylacetone (FBS + SA), but not exogenous haem depletion (haem-depleted FBS), in wild-type primary brown adipocytes phenocopies the mitochondrial defects of PATKO cells (a, n = 8; b, n = 4). c, d, Treatment with succinylacetone (0.5 mM) markedly reduces Ucp1 mRNA and protein levels (n = 3). e, Exogenous haemin (20 μM) does not correct mitochondrial dysfunction in PATKO cells (n = 3). f, PATKO brown adipocytes show higher levels of Rev-Erbα and BACH1 protein. g, Dual knockdown of Rev-Erbα and BACH1 in mature PATKO adipocytes restores mitochondrial respiration (n = 5). h, Pgrmc2, Rev-Erbα (also known as Nr1d1) and Bach1 mRNA in control and knockdown cells. In ah, n represents biologically independent samples. Representative results from two independent experiments. Data presented as mean ± s.d. **P < 0.01 and ***P < 0.001 versus wild type; ###P < 0.001 versus scrambled; two-way ANOVA with multiple comparisons and a Bonferroni’s post-test. Source Data

Extended Data Fig. 6 Body composition of PATKO mice fed a HFD.

Wild-type and PATKO mice were fed HFD for 20 weeks. a, Body weight progression (WT, n = 7; PATKO, n = 9). b, BAT of PATKO mice fed HFD is smaller compared to BAT of HFD-fed wild-type mice. No difference was seen in inguinal WAT (iWAT), epididymal WAT (eWAT) or liver weight (WT, n = 7; PATKO, n = 9). c, PATKO mice fed HFD had higher levels of plasma triglycerides and NEFA (WT, n = 7; PATKO, n = 8). d, H&E staining of liver shows increased steatosis in PATKO mice. Scale bar, 100 μm. Representative images of seven biologically independent samples. e, PATKO mice fed HFD had more lipid accumulation in liver (n = 8). In ae, n represents biologically independent samples. Data presented as mean ± s.e.m.; *P < 0.05 versus wild type; two-tailed Student’s t-test. Source Data

Extended Data Fig. 7 Analysis of adipose depots of PATKO mice fed a HFD.

Wild-type and PATKO mice were fed a HFD for 20 weeks. a, H&E stain images of BAT from wild-type and PATKO mice on a HFD show similar morphology. Insets are magnified on the right. Scale bar, 100 μm. Representative images of seven biologically independent samples. b, Gene expression analysis in BAT shows reduced levels of Fth1 and Bmal1, targets of BACH1 and Rev-Erbα respectively, in PATKO BAT (WT, n = 7; PATKO, n = 8). c, H&E staining of iWAT and eWAT from wild-type and PATKO mice fed HFD do not show clear differences. Scale bar, 100 μm. Representative images of seven biologically independent samples. d, Size analysis of iWAT and eWAT adipocytes from HFD-fed wild-type and PATKO mice. The x axis indicates area in μm2 (n = 5 images of biologically independent samples). e, Gene expression analysis in iWAT reveals a modest increase in expression of genes involved in lipid handling. Similar to BAT, Bmal1 expression is significantly reduced in iWAT of PATKO mice (WT, n = 7; PATKO, n = 9). In ae, n represents biologically independent samples. Data presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 and ***P < 0.001 versus wild type; two-way ANOVA with multiple comparisons and a Bonferroni’s post-test. Source Data

Extended Data Fig. 8 Effect of pharmacological activation of PGRMC2 in DIO mice.

DIO mice were treated with CPAG-1 for 30 days. a, Body weight (left) and food intake (right) progression (n = 8). b, Expression of Pgc-1α and Bmal1 is increased in BAT of treated DIO mice (n = 8). c, H&E staining of iWAT shows no difference between vehicle- and CPAG-1-treated DIO mice. Scale bar, 100 μm. d, Gene expression analysis reveals increased expression of Pgc-1α and Ucp1 in iWAT of CPAG-1-treated DIO mice (n = 8). e, H&E staining shows reduced fibrosis and immune cell infiltration in eWAT of DIO mice treated with CPAG-1. Scale bar, 100 μm. f, Gene expression analysis shows decreased expression of markers of inflammation in eWAT of treated mice (n = 8). g, H&E staining of liver shows that CPAG-1 treatment modestly reduces lipid deposition. Scale bar, 100 μm. h, Hepatic gene expression analysis shows decreased levels of gluconeogenic genes and inflammation markers in liver of treated mice (n = 8). i, Treatment with CPAG-1 for four days significantly increases nuclear labile haem levels in the liver of DIO mice (n = 4). In ai, n represents biologically independent samples. Representative images of eight biologically independent samples per group (d, e, g). Data presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 and ***P < 0.001 versus vehicle; two-way ANOVA with multiple comparisons and a Bonferroni’s post-test. Source Data

Extended Data Fig. 9 Evaluation of interaction of CPAG-1 with PGRMC1 and PGRMC2 in live cells.

a, HEK293T cells transfected with expression vectors for either PGRMC1 or PGRMC2 were treated with 10 μM probe 25 (the photoreactive form of CPAG-1) and DMSO, 100 μM haemin or 100 μM CPAG-1 for 30 min followed by UV photocross-linking, lysis and conjugation of labelled proteomes to a tetramethylrhodamine (TAMRA)-azide tag. Labelled proteomes were separated by SDS–PAGE and visualized by in-gel fluorescence scanning. The intensity of the signals indicates the affinity of probe 25 for the overexpressed proteins. The black asterisk marks PGRMC1 protein and the red asterisk marks PGRMC2 protein. Although detectable, PGRMC1 shows very poor labelling with probe 25 relative to PGRMC2. Both interactions can be competed by haemin or CPAG-1. Western blot analysis confirms expression of PGRMC1 and PGRMC2 in transfected cells. Representative results from two independent experiments.

Extended Data Fig. 10 PGRMC2 is an intracellular haem chaperone critical for adipocyte function.

Model of the proposed role for PGRMC2 in haem dynamics in brown adipocytes. PGRMC2 acquires haem from PGRMC1, which forms a complex with FECH, the last enzyme in haem synthesis. PGRMC2, located in the endoplasmic reticulum and the nuclear envelope, facilitates delivery of labile haem to the nucleus. Nuclear labile haem alters expression of genes regulated by haem-responsive transcriptional repressors such as Rev-Erbα and BACH1, which influence mitochondrial bioenergetics. FVLCR1b, a mitochondrial haem exporter identified in erythrocytes, and HRG-1, a plasma membrane haem importer characterized in macrophages, are also shown. FVLCR1b and HRG-1 are both expressed in brown adipocytes, but their role in haem dynamics in this cell type remains to be defined.

Supplementary information

Supplementary Information

Full scans of all western blots presented in this manuscript and sequences, or catalog numbers, of primers and probes used for gene expression analysis.

Reporting Summary

Supplementary Table 1 | RNAseq analysis of BAT from WT and PATKO mice

a, FPKM and p values of BAT transcriptomes (n = 4 biologically independent samples). b, Differentially expressed genes between WT and PATKO BAT. c-f, FPKM (c), biological process analysis (d), annotation (e), and enrichment (f) of genes significantly upregulated between in PATKO BAT. g-j, FPKM (g), biological process analysis (h), annotation (i), and enrichment (j) of genes significantly downregulated in PATKO BAT. k, Heme and iron related genes expressed in BAT. l-m, heme and iron related genes differentially expressed between WT and PATKO BAT and their annotation (m). n, Comparison between heme and iron related DEGs and the top three upregulated and downregulated biological pathways enriched in PATKO BAT. Enrichment analysis performed with standard accumulative hypergeometric statistical test using Metascape.

41586_2019_1774_MOESM4_ESM.xlsx

Supplementary Table 2 | The analysis of transcription factor binding motifsa-b, De novo (a) and known (b) transcription factor binding motifs enriched in enhancers of genes significantly downregulated in PATKO BAT. Analysis was performed with Homer (homer.ucsd.edu).

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Galmozzi, A., Kok, B.P., Kim, A.S. et al. PGRMC2 is an intracellular haem chaperone critical for adipocyte function. Nature 576, 138–142 (2019) doi:10.1038/s41586-019-1774-2

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