These results demonstrate that SRC3 interacts with RORt in Th17 cells but not in thymocytes

These results demonstrate that SRC3 interacts with RORt in Th17 cells but not in thymocytes. Open in a separate window Figure 1. SRC3 interacts with RORt in Th17 cells but in not thymocytes. not with SRC1, Yoda 1 impairs Th17 differentiation but not thymocyte development. These data suggest that SRC3 works with SRC1 to regulate RORt-dependent Th17 differentiation but is not essential for RORt-dependent thymocyte development. Introduction The transcription factor RORt directs the differentiation of Th17 cells, which secrete IL-17 (1). Th17 cells participate in protective immunity but also mediate pathological immune responses involved in autoimmune conditions, such as multiple sclerosis, colitis, and autism. Thus, inhibiting Th17 cell formation and function may prevent the development and progression of these conditions (2C7). Because RORt is required for the generation of pathogenic Th17 cells, it is an attractive drug target for controlling Th17-mediated immunological disorders (8, 9). However, mice deficient in RORt have been found to exhibit severe defects in thymocyte development, including thymocyte apoptosis, abnormal Yoda 1 cell cycle progression, and accumulation of immature CD8+ cells (10, 11). Thus, broadly targeting RORt could lead to severe unintended side effects. To develop more targeted approaches to inhibit Th17 differentiation, it is important to understand the mechanisms regulating RORt activity. Transcription factors like RORt, which belongs to the steroid nuclear receptor family (11), cannot regulate cellular function unless in the presence of co-factors. Co-factors do not usually have DNA-binding activity and thus depend on transcription factors to carry them to the chromatin to regulate gene expression. The highly conserved steroid receptor co-activator (SRC) family consists of three members, SRC1, SRC2, and SRC3, which are important co-factors for steroid nuclear receptor-mediated transactivation. The SRCs recruit acetyltransferases and methyltransferases that epigenetically modify histones to activate gene expression (12). Our previous study showed that RORt recruits SRC1 to stimulate Th17 differentiation (13). However, mice deficient in SRC1 only show partially impaired Th17 differentiation (13). Furthermore, it was reported recently that SRC3 also regulates Th17 differentiation (14). The highly conserved nature of the SRC family led us to question the relationship between SRC1 and SRC3 in the function of Th17 cells. In this study, we demonstrate that SRC3 is a co-factor for RORt that is necessary for Th17 differentiation but not for thymic T cell development. We detected SRC3-RORt complexes in Th17 cells but not in thymocytes. In addition, CD4+ T cells from mice exhibited defective Th17 differentiation and induction of passive experimental autoimmune encephalomyelitis (EAE) after adoptive transfer. In contrast, mice did not exhibit the defects in thymocyte development observed in RORt-deficient mice. Furthermore, we identified a lysine to arginine mutation in RORt (RORt-K313R) that specifically disrupts the interaction between RORt and SRC3 but not SRC1. Cells expressing RORt-K313R exhibited impaired Th17 differentiation but normal thymocyte development. Therefore, whereas RORt must interact with SRC3 to regulate Th17 differentiation, the SRC3-RORt interaction is not essential for RORt-regulated thymocyte development. Materials & Methods Mice The (mouse strains, described previously (10, 15), were bred and Rabbit Polyclonal to TGF beta Receptor II housed under specific pathogen-free conditions in the Animal Resource Center at the Beckman Research Institute of City of Hope under protocols approved by the Institutional Animal Care and Use Committee. Mice were 10C12 weeks of age for EAE and 6C8 weeks for all other experiments, with littermates age-matched across experimental groups. Antibodies, cytokines and plasmids Antibodies against RORt (Q31C378, BD Bioscience), SRC1 (128E7, Cell Signaling), SRC3 (ab2831, Abcam), and FLAG (M2, Sigma-Aldrich) were used for immunoblot analysis. PE-indotricarbocyanine (Cy7)-conjugated anti-CD8 (53C6.7), PE-conjugated anti-RORt (B2D), allophycocyanin Yoda 1 (APC)-conjugated anti-IL-17A (eBio17B7), PE-conjugated anti-Thy1.2 (53C2.1), PE-conjugated anti-CD24 (M1/69), PE-conjugated anti-TCR (H57C597), PE-Cy5-conjugated anti-CD19 (eBio1D3), PE-conjugated anti-CD11b (M1/70), FITC-conjugated anti-CD4 (GK1.5), APC-conjugated anti-IL-4 (11B11), and APC-conjugated anti-Foxp3 (FJK-16s) were from eBioscience. Monoclonal antibodies against mouse CD3 (145C2C11), CD28 (37.51), IL-4 (11B11), IFN (XMG1.2), and the p40 subunit of IL-12 and IL23 (C17.8), as well as PE-Cy7-conjugated anti-Ly6G (1A8), FITC-conjugated anti-IFN (XMG1.2), PE-conjugated anti-GM-CSF (MP1C22E9), FITC-Cy7-conjugated anti-CD45 (104), and PE-conjugated anti-CD25 (PC61.5) were purchased from Biolegend. Goat anti-hamster antibody was from MP Biomedicals. APC-conjugated anti-CD3 (145C2C11) and FITC-conjugated anti-CD44 (IM7) were from BD Pharmingen. Recombinant mouse IL-12, IL-4, IL-6, IL-23, and TGF were from Miltenyi Biotech. An empty retroviral expression plasmid murine stem cell virus (MSCV)-IRES-GFP and those encodes RORt and SRC1 have been described previously (13). Mouse SRC3 was amplified and inserted into pMSCV-IRES-GFP. Point mutations of RORt were generated using a site-directed mutagenesis kit from Agilent Technologies. Retrovirus Transduction Platinum-E retroviral packaging cells (Cell Biolabs) were plated in a 10-cm dish in 10 ml RPMI-1640 medium plus 10% FBS. 24 h later, cells were transfected with an empty pMSCV vector or the appropriate retroviral expression plasmids with BioT transfection reagent (Bioland). After overnight incubation, the medium was replaced and cultures were maintained for another 24 h. Viral supernatants were collected 48 h and 72 h later, passed through 0.4-m filters (Millipore), and supplemented with 8 g/ml of polybrene (Sigma-Aldrich) and 100 U/ml of recombinant IL-2 (for transducing.