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. 2021 Aug;297(2):100941.
doi: 10.1016/j.jbc.2021.100941. Epub 2021 Jul 7.

Diet-dependent natriuretic peptide receptor C expression in adipose tissue is mediated by PPARγ via long-range distal enhancers

Fubiao Shi  1 , Zoltan Simandi  2 , Laszlo Nagy  3 , Sheila Collins  4
Affiliations

Diet-dependent natriuretic peptide receptor C expression in adipose tissue is mediated by PPARγ via long-range distal enhancers

Fubiao Shi et al. J Biol Chem. 2021 Aug.

Abstract

The cardiac natriuretic peptides (NPs) are well established as regulators of blood pressure and fluid volume, but they also stimulate adipocyte lipolysis and control the gene program of nonshivering thermogenesis in brown adipose tissue. The NP "clearance" receptor C (NPRC) functions to clear NPs from the circulation via peptide internalization and degradation and thus is an important regulator of NP signaling and adipocyte metabolism. It is well known that the Nprc gene is highly expressed in adipose tissue and dynamically regulated upon nutrition and environmental changes. However, the molecular basis for how Nprc gene expression is regulated is still poorly understood. Here, we identified the nuclear receptor transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) as a transcriptional regulator of Nprc expression in mouse adipocytes. During 3T3-L1 adipocyte differentiation, levels of Nprc expression increase in parallel with PPARγ induction. Rosiglitazone, a classic PPARγ agonist, increases, whereas siRNA knockdown of PPARγ reduces, Nprc expression in 3T3-L1 adipocytes. By using chromosome conformation capture and luciferase reporter assays, we demonstrate that PPARγ controls Nprc gene expression in adipocytes through its long-range distal enhancers. Furthermore, the induction of Nprc expression in adipose tissue during high-fat diet feeding is found to be associated with increased PPARγ enhancer activity. Our findings define PPARγ as a mediator of adipocyte Nprc gene expression and establish a new connection between PPARγ and the control of adipocyte NP signaling in obesity.

Keywords: Nprc; PPARγ; adipocyte; enhancer; gene expression; natriuretic peptide; obesity.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Nprc mRNA expression during 3T3-L1 adipocyte differentiation.A–C, mRNA levels of Nprc (A), Fabp4 (B), and Pparγ1/2 (C) during 3T3-L1 adipocyte differentiation. Data were normalized with 36B4. D, protein levels of NPRC, FABP4, PPARγ1/2, and ADIPOQ during 3T3-L1 adipocyte differentiation (Day 0, 2, 4, and 6). FABP4, fatty acid–binding protein 4; Nprc, NP receptor C; PPARγ1/2, peroxisome proliferator–activated receptor gamma1/2.
Figure 2
Figure 2
Modulation of Nprc expression by rosiglitazone and PPARγ.A, mRNA levels of Nprc and Fabp4 in 3T3-L1 adipocytes after treatment with 1 μM rosiglitazone (Rosi) or vehicle (Veh) for 6 h. B, mRNA levels of Nprc, Fabp4, and Pparγ1/2 mRNA in 3T3-L1 adipocytes after siRNA knockdown of Pparγ (siPparγ). C, protein levels of PPARγ1/2, NPRC, FABP4, and ADIPOQ in 3T3-L1 adipocytes after siPparγ. D, mRNA levels of Nprc and Fabp4 in NIH-3T3 cells (NIH) and NIH-3T3 cells stably expressing PPARγ (NIH-PPARγ) after treatment with 1 μM rosiglitazone (Rosi) or vehicle (Veh) for 6 h. Quantitative PCR data were normalized with 36B4. Student's t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Fabp4, fatty acid–binding protein 4; NIH, National Institutes of Health; Nprc, NP receptor C; ns, not significant; PPARγ, peroxisome proliferator–activated receptor gamma.
Figure 3
Figure 3
ChIP-Seq identified PPARγ enhancers in the upstream distal region of Nprc.A, PPARγ-binding sites in the upstream distal region of the Nprc promoter were identified by ChIP-Seq in 3T3-L1 adipocytes and inguinal white adipose tissue (iWAT) from mice fed with low-fat diet (LFD), high-fat diet (HFD), HFD plus vehicle (Ctrl), and HFD plus rosiglitazone (Rosi) (Soccio et al., 2017 (25)). B, HindIII-digested restriction fragments containing Nprc promoter (Pro) and the PPARγ-binding sites (−9 kb, −44/49 kb, −51 kb, −54 kb, −58 kb, 62 kb, and −71 kb) are illustrated for the chromosomal conformation capture (3C) analysis as described for Figure 4. The arrowheads indicate the primers used for 3C PCR in each fragment. ChIP-Seq, chromatin immunoprecipitation sequencing; Nprc, NP receptor C; PPARγ, peroxisome proliferator–activated receptor gamma.
Figure 4
Figure 4
Interaction of PPARγ-binding fragments with Nprc promoter.A, procedure of chromosomal conformation capture (3C) analysis (modified from Cope and Fraser, 2009 (48)). B, 3C analysis with C3H10T1/2 adipocytes to determine the interaction between Nprc promoter and PPARγ-binding sites within each HindIII-restriction fragment (−9 kb, −44/49 kb, −54 kb, −58 kb, −62 kb, and −71 kb; see also Fig. 3B for details). Ercc3 was positive control for constitutive enhancer interaction. β-actin was DNA input control. Ligase+, HindIII-digested chromatin with intramolecular religation. Ligase−, HindIII-digested chromatin without intramolecular religation. BAC, bacterial artificial chromosome DNA covering Nprc (RP23-305L10) and Ercc3 (RP23-148C24) genes was digested with HindIII and randomly religated as positive control. Arrowhead indicates nonspecific amplification products. Nprc, NP receptor C; PPARγ, peroxisome proliferator–activated receptor gamma; PPRE, PPARγ response element.
Figure 5
Figure 5
Rosiglitazone increases Nprc distal enhancer activity but not proximal promoter activity alone.A, Nprc promoter (Pro), distal PPARγ enhancers (−9 kb, −44 kb, −49 kb, −51 kb, −54 kb, −58 kb, and 62 kb), and the three PPARγ response elements (−49 kb—P1, P2, and P3) are shown in gray and were cloned for luciferase reporter analysis. The sequences of the consensus PPRE motif and the Nprc PPREs are listed in inset. B, luciferase activity of Nprc promoter (−2233 bp to +1 bp, mNprc-2.2k). TK promoter alone (TK) and TK promoter with a PPARγ enhancer from the Angptl4 gene (Angptl4+2.3k) were used as the negative and positive controls, respectively. C, luciferase activity of the Nprc distal enhancer fragments (−9 kb, −44 kb, −49 kb, −51 kb, −54 kb, −58 kb, and −62 kb). D, enhancer activity of the Nprc distal PPREs in the −49 kb fragment (P1–P3). For reporter assays, NIH-3T3 and NIH-3T3 stably expressing PPARγ (NIH-3T3-PPARγ) cells were transfected with reporter plasmids and treated with vehicle (Veh) or 1 μM rosiglitazone (Rosi) for 48 h. Luciferase activity was normalized to protein concentrations. E, dual-luciferase assay of the −49kb-3xP2. 293FT cells were transfected with indicated reporter plasmids in combination with GFP or PPARγ and treated with vehicle (Veh) or 1 μM rosiglitazone (Rosi) for 24 h. Data were representative of at least three independent experiments. Student's t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. NIH, National Institutes of Health; Nprc, NP receptor C; PPARγ, peroxisome proliferator–activated receptor gamma; PPRE, PPARγ response element.
Figure 6
Figure 6
HFD induces Nprc expression and PPARγ enhancer activity in adipose tissue.A, levels of Nprc mRNA and the −49 kb PPARγ enhancer RNAs (eNprc-49k) in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) of wildtype mice fed with control diet (CD) or HFD. B, levels of Fabp4 and the −5.4 kb PPARγ enhancer RNA (eFabp4-5.4kb) in BAT and iWAT of mice fed with CD and HFD. C, levels of Npra mRNA in the BAT and iWAT of mice fed with CD and HFD. Student's t test, ∗p < 0.05 and ∗∗∗∗p < 0.0001. Fabp4, fatty acid–binding protein 4; HFD, high-fat diet; Npra, NP receptor A; Nprc, NP receptor C; ns, not significant; PPARγ, peroxisome proliferator–activated receptor gamma.
Figure 7
Figure 7
Obesity induces NPRC expression in adipocytes. Obesity induces NPRC expression in adipocytes potentially though a PPARγ-dependent mechanism. The increase in NPRC lowers the NPRA/NPRC ratio and reduces adipocyte natriuretic peptide signaling and results in decreased energy expenditure and impaired insulin sensitivity. NPRA, NP receptor A; NPRC, NP receptor C; PPARγ, peroxisome proliferator–activated receptor gamma.
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