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Neddylation and Its Target Cullin 3 Are Essential for Adipocyte Differentiation - PubMed

  • ️Mon Jan 01 2024

Neddylation and Its Target Cullin 3 Are Essential for Adipocyte Differentiation

Hongyi Zhou et al. Cells. 2024.

Abstract

The ongoing obesity epidemic has raised awareness of the complex physiology of adipose tissue. Abnormal adipocyte differentiation results in the development of systemic metabolic disorders such as insulin resistance and diabetes. The conjugation of NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to target protein, termed neddylation, has been shown to mediate adipogenesis. However, much remains unknown about its role in adipogenesis. Here, we demonstrated that neddylation and its targets, the cullin (CUL) family members, are differentially regulated during mouse and human adipogenesis. Inhibition of neddylation by MLN4924 significantly reduced adipogenesis of 3T3-L1 and human stromal vascular cells. Deletion of NAE1, a subunit of the only NEDD8 E1 enzyme, suppressed neddylation and impaired adipogenesis. Neddylation deficiency did not affect mitotic cell expansion. Instead, it disrupted CREB/CEBPβ/PPARγ signaling, essential for adipogenesis. Interestingly, among the neddylation-targeted CUL family members, deletion of CUL3, but not CUL1, CUL2, or CUL4A, largely replicated the adipogenic defects observed with neddylation deficiency. A PPARγ agonist minimally rescued the adipogenic defects caused by the deletion of NAE1 and CUL3. In conclusion, our study demonstrates that neddylation and its targeted CUL3 are crucial for adipogenesis. These findings provide potential targets for therapeutic intervention in obesity and metabolic disorders.

Keywords: adipogenesis; metabolism; neddylation; obesity; post-translational modification.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1

Neddylation and its Cullin family targets are differentially regulated during adipogenesis. (A,B) Protein expression in day (D) 0 preadipocytes, Day 2 differentiating, and Day 8 mature adipocytes during DMI-induced 3T3-L1 adipocyte differentiation. Three independent experiments. (C) Protein expression during different days of DMI+pioglitazone-induced MEF adipocyte differentiation. Two independent experiments. (D) Protein expression in day 0 human SVCs isolated from mediastinal adipose biopsy and 14 days (D14) after adipocyte differentiation. Representative data from four independent human donors were shown. Arrowheads indicated neddylated CULs detected by the NEDD8 antibody. Due to the complexity of the data, quantification of Western blots was not performed.

Figure 2
Figure 2

Inhibition of neddylation by MLN4924 blocks 3T3-L1 adipogenesis. (A) 3T3-L1 preadipocytes were treated with vehicle or indicated doses of MLN4924 (MLN) from Day 0-2, together with standard DMI hormone cocktails. On Day 8 (D8) of adipocyte differentiation, cells were harvested for oil-red O (ORO) staining, Western blot (WB), and RT-PCR. (B) Western blot and (C) RT-PCR of adipocyte marker proteins (n = 3). *, p < 0.05; **, p < 0.005 vs vehicle-treated MLN 0 group. Two-way ANOVA with Tukey’s multiple comparisons test. (D) Day 8 3T3-L1 adipocytes were stained with ORO (scale bar: 200 µm), and (E) cellular TG contents were quantified by enzymatic assays (n = 3). Data were normalized to cellular proteins. **, p < 0.005 vs. vehicle-treated MLN 0 group. One-way ANOVA with Dunnett’s multiple comparison test. Three independent experiments.

Figure 3
Figure 3

Inhibition of neddylation by MLN4924 blocks adipocyte differentiation of human stromal vascular cells. (A) hSVCs were treated with vehicle (0 µM) or indicated doses (0.25, 0.5, and 1.0 µM) of MLN4924 (MLN) from Day 0-6, together with human adipocyte differentiation media. Cells were then changed to human adipocyte maintenance media until Day 14 for oil red O (ORO) staining, Western blot (WB), and RT-PCR. (B) ORO staining and (C) cellular TG contents after normalization to cellular proteins (n = 4). **, p < 0.005 vs. vehicle-treated MLN 0 group. One-way ANOVA with Dunnett’s multiple comparison test. (D) RT-PCR analysis of adipocyte differentiation markers (Pparγ2, Cebpα, and Plin1) (n = 4). *, p < 0.05; **, p < 0.005 vs. vehicle- treated MLN 0 group. Two-way ANOVA with Tukey’s multiple comparisons test. (E) Western blot of adipocyte markers (PPARγ and PLIN1) at D14 mature adipocytes. Three independent experiments.

Figure 4
Figure 4

CRISPR/Cas9-mediated NAE1 deletion in 3T3-L1 cells blocks adipogenesis. (A) Targeting strategy of specific gRNA1 and gRNA2 against mouse Nae1 exons. (B) Western blot in Day 0 (D0) 3T3-L1 cells stably infected with lentiviruses generated from pLentiCRISPR v2 vector-targeted control cells (V) and pLentiCRISPR v2 expressing gRNA1 (NAE1KO1, KO1) and gRNA2 (NAE1KO2, KO2) against Nae1. (C) Representative pictures of ORO staining (scale bar = 200 µm), (D) cellular TG content (normalized to protein level) (n = 3). **, p < 0.005 vs. vector-targeted cells. One-way ANOVA with Dunnett’s multiple comparison test. (E) RT-PCR (n = 3) and (F) Western blot analyses of adipocyte marker proteins. *, p < 0.05 vs. vector-targeted cells. Two-way ANOVA with Tukey’s multiple comparisons test. (C–F) Performed in Day 8 (D8) V and NAE1KO1 and NAE1KO2 3T3-L1 cells. Three independent experiments.

Figure 5
Figure 5

Neddylation deficiency impairs CREB/CEBPβ/PPARγ signaling during early adipogenesis. (A) Cell cycle protein expression at indicated hours (h) after DMI induction in vector-targeted control (V) and NAE1 gRNA2-knockout (NAE1KO2, KO2) cells. (B) FACS analysis of cell population at different cell cycle stages after DMI treatment for indicated times (0, 16, and 20 h) in V, NAE1 gRNA1-knockout (NAE1KO1, KO1), NAE1KO2 3T3-L1 cells. Two independent experiments. (C) RT-PCR (n = 3) and (D) Western blot in NAE1KO2 3T3-L1 cells at 0, 12, and 24 h after DMI induction. *, p < 0.05. Multiple unpaired t-tests with the Holm–Sidak method. (E) Western blot to analyze signaling pathways in response to DMI induction at 0, 5, and 15 min (mins) in V, KO1, and KO2 3T3-L1 preadipocytes. Data are representative of three independent experiments.

Figure 6
Figure 6

Knockout of CUL3, but not CUL1, 2, and 4A, in 3T3-L1 cells blocks adipogenesis. Vector-targeted control (V), CUL1 gRNA1–knockout (CUL1KO1), and gRNA2-knockout (CUL1KO2) 3T3-L1 preadipocytes were subjected to DMI-induced adipocyte differentiation. (A) Western blot, (B) representative pictures of oil red O (ORO) staining (scale bar = 200 µm), and (C) cellular TG contents quantified by enzymatic assays and normalized to protein levels (n = 5) at day 8 (D8) of adipocyte differentiation. *, p < 0.05; **, p < 0.005. One-way ANOVA with Dunnett’s multiple comparison test. (D–F) Control (V), CUL2 gRNA1–knockout (CUL2KO), CUL3 gRNA1-knockout (CUL3KO), and CUL4A gRNA1-knockout (CUL4AKO) 3T3-L1 preadipocytes were subjected to DMI-induced adipocyte differentiation. (D) Western blot analyses, (E) representative pictures of ORO staining (scale bar = 5 mm), and (F) cellular TG contents quantified by enzymatic assays and normalized to protein levels (n = 3–5) at Day 8. **, p < 0.005. One-way ANOVA with Dunnett’s multiple comparison test. Three independent experiments.

Figure 7
Figure 7

CUL3 deficiency also impairs CREB/CEBPβ/PPARγ signaling during adipocyte differentiation. (A) Western blot to analyze signaling pathways in response to DMI induction for the indicated 0, 5, and 15 min (mins) in vector-targeted control (V) and CUL3KO 3T3-L1 preadipocytes. (B) RT-PCR (n = 3) and (C) Western blot in V and CUL3KO 3T3-L1 cells at 0, 12, 24, and 48 h after DMI induction. *, p < 0.05; **, p < 0.005. Multiple unpaired t-tests with the Holm–Sidak method. Data are representative of three independent experiments.

Figure 8
Figure 8

ROCK inhibitor fails, and pioglitazone minimally rescues the blunted adipogenesis caused by CUL3 and neddylation deficiency. (A) Western blot and quantification to analyze RHOA protein expression in Day 0 vector-targeted control (V) and CUL3KO 3T3-L1 preadipocytes (n = 3). *, p < 0.05. Unpaired t-test. (B,C) V and CUL3KO 3T3-L1 preadipocytes were subject to DMI-induced adipocyte differentiation in the presence of vehicle (Veh) or 10 µM Y27632 until day 8 for (B) Western blot analyses and (C) cellular TG contents (normalized to cellular proteins) (n = 3). (D,E) V and CUL3KO 3T3-L1 cells and (F,G) V, NAE1KO1 (KO1), NAE1KO2 (KO2) 3T3-L1 cells were subject to DMI-induced adipocyte differentiation in the presence of vehicle (Veh) or 1 µM pioglitazone until Day 8 for Western blot analyses and cellular TG contents (normalized to cellular proteins) (n = 3). (C,E,G): **, p < 0.005 vs. V with the same treatment. #: P < 0.05 vs. the same cells treated with the vehicle. Two-way ANOVA with Tukey’s multiple comparisons test. Three independent experiments.

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References

    1. Wang Q.A., Tao C., Gupta R.K., Scherer P.E. Tracking adipogenesis during white adipose tissue development, expansion and regeneration. Nat. Med. 2013;19:1338–1344. doi: 10.1038/nm.3324. - DOI - PMC - PubMed
    1. Rosen E.D., MacDougald O.A. Adipocyte differentiation from the inside out. Nat. Rev. Mol. Cell Biol. 2006;7:885–896. doi: 10.1038/nrm2066. - DOI - PubMed
    1. Tanaka T., Yoshida N., Kishimoto T., Akira S. Defective adipocyte differentiation in mice lacking the C/EBPbeta and/or C/EBPdelta gene. EMBO J. 1997;16:7432–7443. doi: 10.1093/emboj/16.24.7432. - DOI - PMC - PubMed
    1. Tang Q.Q., Otto T.C., Lane M.D. CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis. Proc. Natl. Acad. Sci. USA. 2003;100:850–855. doi: 10.1073/pnas.0337434100. - DOI - PMC - PubMed
    1. Farmer S.R. Transcriptional control of adipocyte formation. Cell Metab. 2006;4:263–273. doi: 10.1016/j.cmet.2006.07.001. - DOI - PMC - PubMed

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