GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing
Dong-Min Yu, … , Jeffrey B. Cheng, Richard C. Wang
Dong-Min Yu, … , Jeffrey B. Cheng, Richard C. Wang
Published September 18, 2023
Citation Information: J Clin Invest. 2023;133(21):e170706. https://doi.org/10.1172/JCI170706.
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Research Article Dermatology Immunology

GLUT3 promotes macrophage signaling and function via RAS-mediated endocytosis in atopic dermatitis and wound healing

Abstract

The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow–derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane–localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4Rα subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport–independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Authors

Dong-Min Yu, Jiawei Zhao, Eunice E. Lee, Dohun Kim, Ruchika Mahapatra, Elysha K. Rose, Zhiwei Zhou, Calvin Hosler, Abdullah El Kurdi, Jun-Yong Choe, E. Dale Abel, Gerta Hoxhaj, Kenneth D. Westover, Raymond J. Cho, Jeffrey B. Cheng, Richard C. Wang

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Figure 6

GLUT3 promotes IL-4R subunit endocytosis and M2 polarization through its interaction with RAS.

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GLUT3 promotes IL-4R subunit endocytosis and M2 polarization through its...
(A and B) Western blot (WB) of IL-4Rα and γc chain in the plasma membrane (PM) and endosomal fractions from WT, LysM-Cre Glut3fl/fl (GLUT3 KO) BMDMs (A), and THP-1 cells transduced with control or GLUT3 shRNA (B). Na+/K+-ATPase and EEA1, fractionation controls. Mean of IL-4Rα and γc chain levels relative to EEA1 from quantification of WB (n = 3 biological replicates; Supplemental Figure 9, C and F). (C) HEK293T cells were transfected with the indicated GLUT allele, and GLUT1 (Thermo Fisher Scientific, MA1-37783) or GLUT3 (Abcam, ab15311) was immunoprecipitated. RAS was detected by Western blotting. Normal mouse/rabbit IgG, IP controls. (D) HEK293T cells were transfected with the indicated GLUT allele and immunoprecipitation performed after serum starvation (serum-free, SF) or refeeding as indicated. (E) HEK293T cells were transfected with the indicated GLUT3 allele (Supplemental Figure 9I) and GLUT3 alleles were FLAG immunoprecipitated; RAS was detected by Western blotting. IgG indicates a normal mouse IgG control. (F) The indicated GST fusion protein was bound to glutathione-agarose and incubated with GDP- or GTP-bound (GMP-PNP) KRAS as indicated. Bound proteins were eluted and assessed by Western blotting. The GST blot was stripped and probed for RAS to ensure even loading. (G) Levels of p-STAT6 after expression of indicated shRNA-resistant GLUT3 allele and shRNA of endogenous GLUT3 in THP-1 cells. (H) THP-1 were transduced with the indicated shRNA and shRNA-resistant GLUT3 allele and then the indicated M2 marker (MRC1, TGM2) was assessed by qRT-PCR after IL-4 stimulation (24 hours). P values were calculated by 1-way ANOVA with Dunnett’s test. ***P ≤ 0.001, ****P ≤ 0.0001.