Sam68 is a regulator of Toll-like receptor signaling

Abstract

Recognition of pathogens by Toll-like receptors (TLR) activate multiple signaling cascades and expression of genes tailored to mount a primary immune response, inflammation, cell survival and apoptosis. Although TLR-induced activation of pathways, such as nuclear factor kappaB (NF-κB) and mitogen-activated protein kinases (MAPK), has been well studied, molecular entities controlling quantitative regulation of these pathways during an immune response remain poorly defined. We identified Sam68 as a novel regulator of TLR-induced NF-κB and MAPK activation. We found that TLR2 and TLR3 are totally dependent, whereas TLR4 is only partially dependent on Sam68 to induce the activation of NF-κB c-Rel. Absence of Sam68 greatly decreased TLR2- and TLR3-induced NF-κB p65 activation, whereas TLR4-induced p65 activation in a Sam68-independent manner. In contrast, Sam68 appeared to be a negative regulator of MAPK pathways because absence of Sam68 enhanced TLR2-induced activation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK). Interestingly, TLR2- and TLR3-induced gene expression showed a differential requirement of Sam68. Absence of Sam68 impaired TLR2-induced gene expression, suggesting that Sam68 has a critical role in myeloid differentiation primary response gene 88-dependent TLR2 signaling. TLR3-induced gene expression that utilize Toll/Interleukin-1 receptor-domain-containing adapter-inducing beta interferon pathway, depend only partially on Sam68. Our findings suggest that Sam68 may function as an immune rheostat that balances the activation of NF-κB p65 and c-Rel, as well as MAPK, revealing a potential novel target to manipulate TLR signaling.

Figure 1

Sam68 is critical for TLR-induced signaling in fibroblasts. (a) WT and Sam68 KO MEFs (1 × 10⁵) were stimulated with FSL-1 (300 ng/ml) or Pam3CSK4 (200 ng/ml) for 1 and 3 h. (b) WT and Sam68 KO MEFs (1 × 10⁵) were stimulated with IL-1β (100 ng/ml) for 15–120 min. (c) WT and Sam68 KO MEFs (2 × 10⁵) were stimulated with Pam3CSK4 (200 ng/ml) for the indicated time points. Cells were collected, and cytoplasmic and nuclear fractions were prepared. Western blots were probed to identify nuclear NF-κB translocation of NF-κB p65 and c-Rel. Actin and lamin were used as loading controls for cytoplasm and nucleus, respectively, and PLCγ1 was used as fractionation purity control to show the lack of cytoplasmic contamination in the nucleus. (d, e) WT and Sam68 KO MEFs (2 × 10⁵) were serum starved (0% FBS) overnight and stimulated with Pam3CSK4 or IL-1β for the indicated time points. Cells were collected and lysed in buffer containing phosphatase inhibitors. Protein samples were probed for p-ERK1, p-ERK2, p-JNKp54, p-JNKp46 and total ERK/JNK. b represents one of 2 independent experiments and a, c, d and e are representative of ⩾3 independent experiments. ERK, extracellular signal-regulated kinases; IL, interleukin; JNK, c-Jun N-terminal kinases; KO, knockout; MEF, Mouse embryonic fibroblasts; NF-κB, nuclear factor kappaB; TLR, Toll-like receptor; WT, wild type.

Figure 2

TLR-induced activation of NF-κB in macrophages requires Sam68. (a–b) WT and Sam68 KO RAW264.7 macrophages (1 × 10⁶) were stimulated with (a) Pam3CSK4 (200 ng/ml), (b) poly I:C (10 μg/ml) and LPS (1 μg/ml) for up to 2 h. Cytoplasmic and nuclear fractions were analyzed as indicated to identify nuclear NF-κB translocation. (c) WT, Sam68 KO and Sam68⁺ RAW264.7 macrophages (1 × 10⁶) were stimulated with Pam3CSK4 (200 ng/ml) and poly I:C (10 μg/ml) for 30 min and 1 h. Cytoplasmic and nuclear fractions were probed as indicated. (d) WT and Sam68 KO primary BMDMs (1 × 10⁶) were stimulated with Pam3CSK4 (200 ng/ml) and FSL-1 (300 ng/ml) for the indicated time points. Cytoplasmic and nuclear fractions were probed for NF-κB p65 and c-Rel to determine their nuclear translocation. Actin, lamin and hnRNPA1 were used as loading controls and PLCγ1 was used as nuclear fractionation purity control. BMDMs, bone marrow-derived macrophages; KO, knockout; LPS, lipopolysaccharide; NF-κB, nuclear factor kappaB; WT, wild type.

Figure 3

Loss of Sam68 enhances basal and TLR-induced MAPK activation. WT and Sam68 KO RAWs (2.5 × 10⁵) were plated overnight and switched to serum-free DMEM media for an additional day. Cells were then stimulated with (a) Pam3CSK4 (200 ng/ml), (b) poly (I:C) (10 μg/ml) and (c) LPS (1 μg/ml) for up to 30 min. Cells were collected and lysed in Triton lysis buffer containing protease and phosphatase inhibitors. Total lysates were probed for MAPK activation using phospho-JNK and phospho-ERK antibodies. Samples were probed for loading controls using total ERK and actin. a–c are representative of 3 independent experiments. ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinasesKO, knockout; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; TLR, Toll-like receptor; WT, wild type.

Figure 4

TLR2- but not TLR3-induced gene expression is ablated in Sam68-deficient macrophages. Gene expression of key NF-κB target genes was determined by quantitative real-time PCR. WT and Sam68 KO RAW macrophages (2 × 10⁵) were stimulated with Pam3CSK4 (200 ng/ml) or poly (I:C; 10 μg/ml) for 1 and 3 h. Total cellular RNA was isolated and 1 μg was converted to cDNA. Expression of the genes (a, d) TNF, VCAM1, IL10, (b, e) CXCL10 and (c, f) CCL5 was determined. Values are expressed as fold induction over the mock treatment using the ΔΔCt method. All values are normalized to the housekeeping gene L32. All samples were run in duplicate and ligand stimulation conditions represent n⩾2 independent samples. Statistical analysis was performed in GraphPad (La Jolla, CA, USA) for Student's t-test *P<0.05. cDNA, complementary DNA; KO, knockout; NF-κB, nuclear factor kappaB; TLR, Toll-like receptor; WT, wild type.

Figure 5

Schematic model of the role of Sam68 in TLR2 and TLR3 signaling pathways. In normal cells, Sam68 is required for TLR2 and TLR3-induced NF-κB activation and inflammatory gene expression. In the absence of Sam68, NF-κB nuclear translocation and subsequent inflammatory gene induction are substantially reduced in response to TLR2 activation that signals through MyD88. Although NF-κB nuclear translocation is substantially reduced following TLR3 stimulation, TLR3-induced gene expression is largely unaffected in Sam68-deficient cells. Both TLR2 and TLR3-induced MAPK activation is enhanced in Sam68-deficient cells. NF-κB independent pathways, including MAPK and IRFs, may have a role in gene expression in Sam68-deficient cells in response to TLR3 activation. Black arrow—normal signaling, bold black arrow—enhanced signaling and gray arrow—diminished signaling. IRFs, interferon regulatory factors; MAPK, mitogen-activated protein kinases; MyD88, myeloid differentiation primary response gene 88; NF-κB, nuclear factor kappaB; TLR, Toll-like receptor.