IL-17 inhibits adipogenesis in part via C/EBPα, PPARγ and Krüppel-like factors

Abstract

IL-17 is an inflammatory cytokine associated with anti-microbial host defense and pathogenesis of autoimmune diseases. Obesity is considered to be an inflammatory condition, but how cytokines and fat metabolism are interconnected remains poorly understood. Mesenchymal stem cells can differentiate into adipocytes, which serve as depots for stored fat. Despite the pro-inflammatory properties of IL-17, both IL-17- and IL-17RA-deficient mice are overweight. Consistently, IL-17 suppresses maturation of cells with adipogenic potential. However, the mechanism underlying IL-17-mediated inhibition is not defined. In this study, we addressed this question by evaluating the impact of IL-17 on a variety of transcription factors (TFs) that control adipogenesis, using 3T3-L1 cells to model adipocyte differentiation. Surprisingly, IL-17 does not suppress adipogenesis via C/EBPβ and C/EBPδ, TFs often considered to be central regulators of adipogenesis. Rather, IL-17 suppresses expression of several pro-adipogenic TFs, including PPARγ and C/EBPα. Moreover, we found that IL-17 regulates expression of several members of the Krüppel-like family (KLF). Specifically, IL-17 suppresses KLF15, a pro-adipogenic TF, and enhances expression of KLF2 and KLF3, which are anti-adipogenic. Thus, IL-17 suppresses adipogenesis at least in part through the combined effects of TFs that regulate adipocyte differentiation.

Figure 1. IL-17 suppresses adipogenesis

A. 3T3-L1 cells were cultured alone (“control”) or in adipogenic culture conditions (“Diff.”) in the presence of IL-17 (200 ng/ml) and/or a suboptimal dose of TNFα (2 ng/ml) for 10 days. Cells were stained with Oil Red O to visualize fat formation. B. Lysates from the cells described in panel A were solubilized and evaluated by light spectroscopy at OD₅₁₀. C. Cells were differentiated in duplicate for 10 days with the indicated cytokines and triglyceride content (TG) was assessed.

Figure 2. IL-17 suppression of adipogenesis correlates with C/EBPα and PPARγ but not C/EBPβ and C/EBPδ expression

A. Diagram of TFs that regulate adipocyte differentiation. Pro-adipogenic TFs are indicated in blue, anti-adipogenic TFs are in orange. B–C. Effect of IL-17 on C/EBP and PPARγ expression during adipogenic differentiation. 3T3-L1 cells were cultured alone (“control”) or in adipogenic culture conditions (“Differentiated”) in the presence of IL-17 (200 ng/ml) and/or a suboptimal dose of TNFα (2 ng/ml) for 2 days. Total RNA was extracted and subjected to qRT-PCR in triplicate with primers specific for the indicated genes.

Figure 2. IL-17 suppression of adipogenesis correlates with C/EBPα and PPARγ but not C/EBPβ and C/EBPδ expression

A. Diagram of TFs that regulate adipocyte differentiation. Pro-adipogenic TFs are indicated in blue, anti-adipogenic TFs are in orange. B–C. Effect of IL-17 on C/EBP and PPARγ expression during adipogenic differentiation. 3T3-L1 cells were cultured alone (“control”) or in adipogenic culture conditions (“Differentiated”) in the presence of IL-17 (200 ng/ml) and/or a suboptimal dose of TNFα (2 ng/ml) for 2 days. Total RNA was extracted and subjected to qRT-PCR in triplicate with primers specific for the indicated genes.

Figure 3. A–E. Effect of IL-17 on adipogenic TF expression during adipogenic differentiation

3T3-L1 cells were cultured alone (“control”) or in adipogenic culture conditions (“Differentiated”) in the presence of IL-17 (200 ng/ml) and/or a suboptimal dose of TNFα (2 ng/ml) for 2 days. Total RNA was extracted and subjected to qRT-PCR in triplicate and subjected to qRT-PCR in triplicate with primers specific for the indicated genes. F. KLF15 expression kinetics in differentiation. Expression of KLF15 was assessed by qRT-PCR as bove from 1–5 days of differentiation with or without IL-17. Arrow indicates time of withdrawal of IL-17, as described in the text.

Figure 4. A-F. IL-17 regulation of anti-adipogenic TFs

3T3-L1 cells were cultured alone (“control”) or in adipogenic culture conditions (“Differentiated”) in the presence of IL-17 (200 ng/ml) and/or a suboptimal dose of TNFα (2 ng/ml) for 2 or 8 days. Total RNA was extracted and subjected to qRT-PCR in triplicate with primers specific for the indicated genes.

Figure 5. IL-17 regulation of KLF15 and GR. A. Regulation of GR by IL-17 during adipogenic differentiation

3T3-L1 cells were cultured alone (“control”) or in adipogenic culture conditions (“Differentiated”) in the presence of IL-17 (200 ng/ml) and/or TNFα (2 ng/ml) for the indicated times. Total RNA was extracted and subjected to qRT-PCR. B. IL-17 does not impact the KLF15 intronic enhancer. 3T3-L1 cells were transiently transfected with the reporter construct pGL3-intron-KLF15 enhancer ± IL-17. After 48 h, cell lysates were analyzed for luciferase. C. IL-17 does not regulate the KLF15 proximal promoter. 3T3-L1 cells were transiently transfected with the promoter construct pGL3-promoter-KLF15 with or without IL-17. After 48 h, cell lysates were analyzed for luciferase.