HOXC10 Suppresses Browning to Maintain White Adipocyte Identity

Abstract

Promoting beige adipocyte development within white adipose tissue (WAT) is a potential therapeutic approach to staunch the current obesity epidemic. Previously, we identified homeobox-containing transcription factor HOXC10 as a suppressor of browning in subcutaneous WAT. Here, we provide evidence for the physiological role of HOXC10 in regulating WAT thermogenesis. Analysis of an adipose-specific HOXC10 knockout mouse line with no detectable HOXC10 in mature adipocytes revealed spontaneous subcutaneous WAT browning, increased expression of genes involved in browning, increased basal rectal temperature, enhanced cold tolerance, and improved glucose homeostasis. These phenotypes were further exacerbated by exposure to cold or a β-adrenergic stimulant. Mechanistically, cold and β-adrenergic exposure led to reduced HOXC10 protein level without affecting its mRNA level. Cold exposure induced cAMP-dependent protein kinase-dependent proteasome-mediated degradation of HOXC10 in cultured adipocytes, and shotgun proteomics approach identified KCTD2, 5, and 17 as potential E3 ligases regulating HOXC10 proteasomal degradation. Collectively, these data demonstrate that HOXC10 is a gatekeeper of WAT identity, and targeting HOXC10 could be a plausible therapeutic strategy to unlock WAT thermogenic potentials.

Figure 1

Deletion of AHKO results in widespread accumulation of beigelike adipocytes in subcutaneous WAT depots of AHKO mice. A: Targeting strategy for HOXC10 gene knockout. The targeting vector contained homology arms of 2.9 kb (5' arm) and 4.6 kb (3' arm). The flox sites were located in exon 2. B: Relative mRNA levels of Cre and HOXC10 in inguinal WAT (iWAT) of control and AHKO mice (n = 10). C: Relative protein levels of HOXC10 in SVF and fractionated adipocytes isolated from iWAT of five pooled control and AHKO mice (n = 2). HSP90 served as a loading control. D: Representative photograph of iWAT and corresponding circulating leptin levels at 20 weeks of age fed on chow diet. E: H-E staining of iWAT section. Scale bars = 20 μm. Insets illustrate a magnified view of the figures (scale bars = 100 μm). n = 4. F: Relative mRNA levels of common adipocyte genes and brown/beige-selective thermogenic markers; n = 10. G: Body temperature was measured following 7-day cold challenge at 10 weeks of age; n = 7. Glucose (H) and insulin tolerance test (I) of control and AHKO mice between 15 and 16 weeks of age. Data are presented as mean ± SEM. Student t test: *P < 0.05, **P < 0.01, ***P < 0.001 (n = 10). AAC, area above the curve; AUC, area under the curve; Frt, loxP-flippase recognition target.

Figure 2

Mice lacking AHKO exhibit increased energy expenditure. Mean average heat production (A), VO₂ consumption rates (B), RER (C), and activity level (D) determined at 14 weeks of age by indirect calorimetry in metabolic chambers during the 3-day measurement (after an initial 48 h of acclimation period). Dark- and light-phase cumulative means within dark or light phase and each time point were compared by Student t test between AHKO mice and their littermate male control mice. Data are presented as mean ± SEM. *P < 0.05. n = 14. BW, body weight; hr, hour.

Figure 3

β-AR activation leads to enhanced thermogenic function in subcutaneous WAT of AHKO mice. A: Representative H-E staining of inguinal WAT from control and AHKO mice before (top panels) or after (bottom panels) injection with CL to induce beiging. Scale bars = 20 μm. Insets illustrate a magnified view of the figures (scale bars = 100 μm). n = 4. B: Relative mRNA levels of brown-fat selective markers, mitochondrial genes, and Ucp-1–independent thermogenic markers in inguinal WAT of control and AHKO mice treated with CL. Data are presented as mean ± SEM. Student t test: *P < 0.05, **P < 0.01, ***P < 0.001. n = 4.

Figure 4

HOXC10 protein is translationally regulated in subcutaneous WAT by cold exposure and β₃-adrenergic activation. Immunoblot and densitometry analysis of endogenous HOXC10 protein in inguinal WAT isolated from WT C57BL6/J mice held at room temperature (RT) or exposed to 4°C (A) or treated daily with CL (1 mg/kg) or vehicle (saline) for 7 days (B) (n = 4–5 mice/group). HSP90 served as a loading control. Data are presented as mean ± SEM. Student t test: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Physiological regulation of white adipocyte HOXC10 through cAMP-dependent PKA activation. A: Proteasome activity per inguinal WAT from WT mice after adaptation to room temperature (RT) or cold (4°C) for 7 days (n = 6 mice/group). B: In vivo ubiquitination assay on HeLa cells transfected with hemagglutinin (HA)–tagged ubiquitin and Flag-tagged HOXC10 expression vectors for 24 h, treated with forskolin (10 µm), followed by immunoprecipitations (IP) using FLAG antibody (M2) beads. Immunoblots (IB) were done on IP-lysate samples using anti-ubiquitin (to monitor ubiquitin conjugates on HOX10) and anti–Flag-HRP (to monitor successful transfection) antibodies. Flag-tagged p53 (Flag-p53) cotransfected with HA-ubiquitin was a positive (+ve) loading control for polyubiquitination. n = 4 independent in vivo ubiquitination experiments. C: HEK293T cells transfected with Flag-tagged HOXC10, V5-tagged KCTD2, Myc-tagged KCTD5, and HA-tagged KCTD17 expression vectors for 48 h, treated with forskolin (10 µm), and followed by IP using FLAG antibody (M2) beads. Immunoblots were performed on input and IP samples using anti-Flag, anti-V5, anti-Myc, anti-HA, and anti-KCTD5 antibodies. Anti-KCTD5 antibody preferentially detected KCTD5 (26 kDa, black arrowheads), but was also able to detect KCTD2 and KCTD17 (29 kDa and 37 kDa, black and blue arrowheads, respectively). HSP90 was used as a loading control; n = 3 independent replicates. D: Repeat of B above, using HeLa cells transfected with HA-tagged ubiquitin (Ubq), Flag-tagged HOXC10, and KCTD2, 5, and 17 expression vectors for 24 h, followed by IP using FLAG antibody (M2) beads. Immunoblots were done on IP-lysate samples using anti-ubiquitin (to monitor ubiquitin conjugates on HOXC10), anti-Flag, anti-KCTD (to monitor successful transfection), and anti-HSP90 (loading control) antibodies; black arrowheads show the preferentially detected KCTD5 protein band. n = 3 independent in vivo ubiquitination experiments. E, left: Differentiated 3T3-L1-FlagHOXC10 cells were treated with cycloheximide (CHX; 100 µg/ml), forskolin (10 µm), or H89 (50 µmol/L) for the indicated hours (hr). The remaining HOXC10 at different time points was quantified as the percentage of initial HOXC10 level (0 h of CHX treatment). *Statistically different between control and forskolin-treated; #statistically different between forskolin and H89-treated. HSP90 served as a loading control. E, right: Corresponding Ucp-1 mRNA expression following treatment with CHX and forskolin. n = 4 independent CHX chase assays. Data are presented as mean ± SEM. Student t test: *P < 0.05, **P < 0.01, ***P < 0.001. AU, arbitrary units.