Ymer acts as a multifunctional regulator in nuclear factor-κB and Fas signaling pathways

Abstract

The nuclear factor (NF)-κB family of transcription factors regulates diverse cellular functions, including inflammation, oncogenesis and apoptosis. It was reported that A20 plays a critical role in the termination of NF-κB signaling after activation. Previously, we showed that Ymer interacts and collaborates with A20, followed by degradation of receptor-interacting protein (RIP) and attenuation of NF-κB signaling. Here we show the function of Ymer in regulation of several signaling pathways including NF-κB on the basis of results obtained by using Ymer transgenic (Ymer Tg) mice. Ymer Tg mice exhibited impaired immune responses, including NF-κB and mitogen-activated protein kinase (MAPK) activation, cell proliferation and cytokine production, to tumor necrosis factor (TNF)-α, polyI:C or lipopolysaccharide (LPS) stimulation. Ymer Tg mice were more resistant to LPS-induced septic shock than wild-type mice. Transgene of Ymer inhibited the onset of glomerulonephritis in lpr/lpr mice as an autoimmune disease model. In contrast to the inflammatory immune response to LPS, Fas-mediated cell death was strongly induced in liver cells of Ymer Tg mice in which Ymer is abundantly expressed. These findings suggest that Ymer acts as a regulator downstream of several receptors and that Ymer functions as a positive or negative regulator in a signaling pathway-dependent manner.

Figure 1

Impaired immune response of Ymer Tg mice to TNFα and LPS stimulations. (A) Splenocytes (1 × 10⁶) isolated from WT and Ymer Tg mice were stimulated with LPS (100 ng/mL), and cell proliferation was measured by an MTS assay. (B) Four days after injection with TNFα (50 μg/kg) or LPS (100 μg/kg), spleens were dissected out from WT and Ymer Tg mice. (C) Cell numbers in LPS-stimulated spleen in Ymer Tg mice. Cell numbers of splenocytes in (B) were counted (saline-treated WT, n = 4; saline-treated Tg⁴¹³, n = 4; saline-treated Tg⁵⁴⁵, n = 3; LPS-treated WT, n = 5; LPS-treated Tg⁴¹³, n = 3; LPS-treated Tg⁵⁴⁵, n = 4). (D) Flowcytometric analysis of LPS-treated splenocytes in Ymer Tg mice. Splenocytes in (B) were analyzed by flowcytometry using anti-B220, anti-TCRβ and anti-Mac-1 Abs. Percentages of each population are indicated in the panels.

Figure 2

Resistance to septic shock in Ymer Tg mice. (A) Immunoblot analysis of Ymer Tg mice treated with TNFα or LPS. Three days after intraperitoneal injection with TNFα or LPS, cell lysates from the livers of WT and Ymer Tg mice were used for immunoblot analysis with Abs against PARP, RIP, IKK and P-IKK. (B) Survival study of Ymer Tg mice by LPS. WT and Ymer Tg mice were injected with LPS (50 mg/kg) and then observed for 24 h (WT, n = 4; Tg⁴¹³, n = 4; Tg⁵⁴⁵, n = 5). (C) Production of inflammatory cytokines in Ymer Tg mice. Expression levels of IL-1β, IL-6, TNFα, IFNα and iNOS were measured by qRT-PCR at 4 h after LPS injection. (D) Secretion of IL-6 in sera of Ymer Tg mice. Four hours after LPS injection, IL-6 concentrations of sera from WT and Ymer Tg mice were measured by ELISA (WT, n = 3; Tg⁴¹³, n = 4; Tg⁵⁴⁵, n = 5).

Figure 3

Impaired activation after treatment with LPS or TNFα in Ymer Tg mice. (A) Thioglycollate-induced peritoneal macrophages isolated from WT and Ymer Tg mice were stimulated with LPS (1 μg/mL) for the indicated times, and the cell lysates were analyzed by immunoblotting to detect the activation of IKK and MAPK. The percentages of isolated macrophages were >85%. (B) Cytokine production in thioglycollate-induced peritoneal macrophages from Ymer Tg mice after treatment with LPS. mRNA expression levels of cytokines (IL-6, TNFα and IL-1β) were analyzed by reverse transcription–polymerase chain reactions (RT-PCRs). (C) IL-6 production by thioglycollate-induced peritoneal macrophages from Ymer Tg mice after treatment with LPS. ELISA was performed to measure IL-6 production in the culture supernatant at the indicated times. (D) MEFs isolated from WT and Ymer Tg mice were stimulated with TNFα (10 ng/mL) for the indicated times, and the cell lysates were analyzed by immunoblotting to detect the activation of IKK and JNK. (E) NF-κB luciferase reporter assay using MEFs from Ymer Tg mice. Eight hours after TNFα (10 ng/mL) stimulation, NF-κB-dependent transcriptional activities were measured.

Figure 4

Ymer inhibits glomerulonephritis in lpr/lpr mice. (A) The levels of urinary protein in B6-lpr/+;Ymer ⁵⁴⁵, B6-lpr/lpr and B6-lpr/lpr;Ymer ⁵⁴⁵ mice. Urinary proteins were measured during 4–18 wks of age (B6-lpr/+;Ymer ⁵⁴⁵, n = 9; B6-lpr/lpr, n = 10; B6-lpr/lpr;Ymer ⁵⁴⁵, n = 7). (B) Histochemical analysis of glomerulonephritis. Renal tissues were dissected out at 7–8 months of age and used for histochemical analysis with PAS staining. Arrows indicate the proliferation of mesangium cells in the glomerulus. Scale bars indicate 50 μm. (C) Quantification of glomerulonephritis in Ymer Tg mice crossed with lpr mice. The frequency of inflammatory glomerulus in which cells were proliferated in the mesangial region was quantified using B6-lpr;Ymer hybrid mice (B6-lpr/+;Ymer ⁵⁴⁵, n = 7; B6-lpr/lpr, n = 8; B6-lpr/lpr;Ymer ⁵⁴⁵, n = 5). Each dot indicates the score of glomerulonephritis obtained from individual mice. The Welch t test was used to measure differences among samples.

Figure 5

Acceleration of Fas-induced apoptosis in livers of Ymer Tg mice. (A) Survival study for Fas-induced cell death was performed with anti-Fas Ab (10 μg/mouse) intraperitoneal injection into WT and Ymer Tg mice (WT, n = 5; Tg⁴¹³, n = 6; Tg⁵⁴⁵, n = 6). Survival of the mice was observed for 24 h after injection. Statistical analysis was performed by using the Kaplan-Meier test. (B) Livers treated with anti-Fas Ab were dissected out at 6 h after injection. (C) Livers treated with anti-Fas Ab were dissected out at 4 h after injection and used for histological analysis with HE staining. Scale bars indicate 100 μm in low magnification and 20 μm in high magnification (boxed). (D) Immunoblot analysis of apoptosis-related proteins in anti-Fas Ab-treated livers from Ymer Tg mice. Two hours after injection with anti-Fas Ab, cell lysates from livers were probed with the indicated Abs.

Figure 6

Ymer accelerates the caspase-dependent signal and maintains long-term activation of NF-κB signaling in Fas-stimulated thymocytes. (A) Thymocytes isolated from WT and Ymer ⁵⁴⁵ Tg mice were stimulated with anti-Fas Ab (10 μg/mL) and then probed with the indicated Abs to detect the activation of downstream signaling after stimulation. (B) Time course of cleaved form of PARP in thymocytes from WT and Ymer ⁵⁴⁵ Tg mice after anti-Fas Ab treatment. A relative amount of the cleaved form of PARP at each time in (A) is shown. The value of the chase period at 0 h was defined as 1. (C) Time course of phosphorylated IKKα/β in thymocytes from WT and Ymer ⁵⁴⁵ Tg mice after anti-Fas Ab treatment. Relative amount of phosphorylated IKKα/β at each time in (A) is shown. The value of the chase period at 0 h was defined as 1. (D) Model for biological roles of Ymer. Ymer may have multi-functions for several signaling pathways including TLR4, TNFR and Fas. Ymer attenuates NF-κB signaling downstream of TLR4 and TNFR, whereas it accelerates LPS-induced IFN production and Fas-induced NF-κB and JNK activation for apoptotic cell death.