Background/aim Tumor necrosis factor alpha (TNFα, a.k.a. TNF) is a pleiotropic cytokine that exerts most of its effects through type 1 TNF receptor (TNFR1). Following TNF binding, TNFR1 recruits TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). This interaction triggers formation of signalosome complexes which have been claimed to induce apoptosis (via downstream caspase activations), inflammation (via NF-kappaB) and stress pathways (JNK & p38). However, the mechanism underlying TNF-induced ERK and AKT activation is not completely revealed. TNFR1 is known to constitutively bind c-Src and JAK2, and these enzymes were previously demonstrated to modulate TNF signaling. Therefore, we hypothesized that TNFR1 could be tyrosine phosphorylated by JAK2 and/or c-Src and TNF-induced ERK and Akt activation may be mediated by this phosphorylation. Materials and methods Site-directed mutagenesis (SDM) was performed to substitute the two putative Tyrosine phosphorylation sites on TNFR1 (Y360 and Y401) with alanine (A) or with aspartic acid (D), to inhibit or mimic constitutive phosphorylation, respectively. In 293T cells transfected with mutated or wild type TNFR1, ERK and Akt activations were determined by western blot. TNFR1 interaction with c-Src, JAK2, p85 and Grb2 was examined by co-IP. NF-kB activation was measured by luciferase assay, while proliferation was measured by MTT and apoptosis was evaluated by colorimetric caspase 8/3 assays. For determination of necrosis rates, cellular DNA fragmentation ELISA was performed. Results In this report, we show that TNFR1 is phosphorylated by JAK2 tyrosine kinase at Y401 and by c-Src at Y360 and Y401. Phosphorylation of Y360 and Y401 augments the interaction of Grb2 and PI3Kp85 with TNFR1. We also demonstrate that phosphomimetic mutations of Y360D and Y401D enhance ERK and Akt activation. Conclusion TNFR1 is tyrosine phosphorylated by both c-Src and JAK2, triggering a “noncanonical” pathway, that activates ERK and Akt.

TNFR1 signaling is positively regulated by Jak-2 and c-Src via tyrosine phosphorylation

TNF is a pleiotropic cytokine and shows its biological function by binding to its receptors called TNFR1 and TNFR2. While TNFR1 induces apoptosis by activation of caspase-8 via the "death domain", it also activates IKKα/β, MKK3/6, MKK4/7 by activation of TAK1. Although the TNFR1 signaling pathway is known by in large, it is not known how AKT and MAPKs p38, ERK1/2, and JNK1/2 are activated. The presence of a proline-rich PPAP region, (P448PAP451, a binding site for the SH3 domain-containing proteins) very close to the C-terminus promoted us to determine whether this region has any role in the TNFR1 signal transduction. To test this, the codons of P448 and P451 were changed to that of Alanin, GCG, via site-directed mutagenesis, and this plasmid was named as TNFR1-SH3-P/A. Subsequently, ectopically expressed the wild type TNFR1 and TNFR1-SH3-P/A in 293T cells and determined the levels of TNF-α-mediated phosphorylations of ERK, p38, JNK and AKT, NF-kB, and caspase-8 activation. While ectopic expression of our mutant diminished TNFα-mediated phosphorylations of p38, JNK, ERK and AKT, it increased NF-kB, and caspase-8 activations. In conclusion, TNFα-mediated ERK, AKT, JNK, p38 activations are affected by TNFR1 SH3 domain modifications.

Positive regulation of TNFR1 signaling via SH3 recognition motif.

BACKGROUND Tinnitus is an auditory phantom sensation in the absence of an acoustic stimulus, which affects nearly 15% of the population. Excessive noise exposure is one of the main causes of tinnitus. To now, the knowledge of the genetic determinants of susceptibility to tinnitus remains limited. RESULTS We performed a two-stage genome-wide association study (GWAS) and identified that two single nucleotide polymorphisms (SNPs), rs2846071 located in the intergenic region at 11q13.5 (odds ratio [OR] = 2.14, 95% confidence interval [CI] = 1.96-3.40, combined P = 4.89 × 10- 6) and rs4149577 located in the intron of TNFRSF1A gene at 12p13.31 (OR = 2.05, 95% CI = 1.89-2.51, combined P = 6.88 × 10- 6), are significantly associated with the susceptibility to noise-induced tinnitus. Furthermore, the expression quantitative trait loci (eQTL) analyses revealed that rs2846071 is significantly correlated with the expression of WNT11 gene, and rs4149577 with the expression of TNFRSF1A gene in multiple brain tissues (all P < 0.05). The newly identified candidate gene WNT11 is involved in Wnt pathway, and TNFRSF1A in the tumor necrosis factor pathway, respectively. Pathway enrichment analyses also showed that these two pathways are closely relevant to tinnitus. CONCLUSIONS Our findings highlight two novel loci at 11q13.5 and 12p13.31 conferring susceptibility to noise-induced tinnitus. and suggest that the WNT11 and TNFRSF1A genes might be the candidate causal targets of 11q13.5 and 12p13.31 loci, respectively.

/pdf/genome-wide-association-study-identifies-new-loci-associated-1o4fwckn1a.pdf

Genome-wide association study identifies new loci associated with noise-induced tinnitus in Chinese populations.

Additional file 1: Supplementary Figure 1. The principal components analyses (PCA) of the population in the discovery stage in this study and reference populations from the 1,000 Genomes Project. Supplementary Figure 2. The genotypes of rs2846071 are significantly associated with the expression levels of WNT11 in several types of brain tissues from GTEx. Supplementary Figure 3. Colocalization analyses of the association signals from GWAS and brain eQTL data at the 11q13.5 and 12p13.31 loci. Supplementary Figure 4. Chromatin state segmentations for rs1800692 and rs4149570 using the ENCODE data. Supplementary Figure 5. The genotypes of rs4149577 are significantly associated with the expression levels of TNFRSF1A in several types of brain tissue from GTEx. Supplementary Figure 6. Proxy plots for 11q13.5 and 12p13.31 regions in Chinese Han Chinese and European populations. Supplementary Figure 7. Linkage disequilibrium plots for 11q13.5 and 12p13.31 regions in Chinese Han Chinese and European populations. Supplementary Figure 8. Power to detect the genetic effects of rs2846071 and rs4149577. Supplementary Table 1. Summary of the case/control populations used in this study. Supplementary Table 2. Summary of the genotyped and imputed SNPs in the discovery stage. Supplementary Table 3. Summary of the SNPs that have been reported to be associated with tinnitus in previous studies. Supplementary Table 4. Summary of the top 22 SNPs in the discovery stage. Supplementary Table 5. Primers used for SNPs genotyping in the replication stage. Supplementary Table 6. Summary of the association results in the replication stage. Supplementary Table 7. Stratification analyses of rs2846071 and rs4149577 by age. Supplementary Table 8. The predicted functional relevance of rs2846071, rs4149577 and the other SNPs in strong or moderate LD with them. Supplementary Table 9. Pathway analyses based on i-GSEA4GWAS. Supplementary Table 10. The allele and genotype frequencies of rs2846071 and rs4149577 in different populations. 

Additional file 1 of Genome-wide association study identifies new loci associated with noise-induced tinnitus in Chinese populations

Purinergic G-protein-coupled receptors are ancient and the most abundant group of G-protein-coupled receptors (GPCRs). The wide distribution of purinergic receptors in the cardiovascular system, together with the expression of multiple receptor subtypes in endothelial cells (ECs) and other vascular cells demonstrates the physiological importance of the purinergic signaling system in the regulation of the cardiovascular system. This review discusses the contribution of purinergic P2Y receptors to endothelial dysfunction (ED) in numerous cardiovascular diseases (CVDs). Endothelial dysfunction can be defined as a shift from a "calm" or non-activated state, characterized by low permeability, anti-thrombotic, and anti-inflammatory properties, to a "activated" state, characterized by vasoconstriction and increased permeability, pro-thrombotic, and pro-inflammatory properties. This state of ED is observed in many diseases, including atherosclerosis, diabetes, hypertension, metabolic syndrome, sepsis, and pulmonary hypertension. Herein, we review the recent advances in P2Y receptor physiology and emphasize some of their unique signaling features in pulmonary endothelial cells.

https://www.mdpi.com/1422-0067/21/18/6855/pdf

P2Y Purinergic Receptors, Endothelial Dysfunction, and Cardiovascular Diseases.

Tumor necrosis factor alpha (TNF-α) plays a paramount role in homeostasis by inducing tumor cytotoxicity and activating immune system. The signaling complexes formed by TNFR1 to activate JNK, p38, and nuclear factor-kappa B pathways and to subsequently induce apoptosis and necroptosis are well known. However, this "canonical TNF-α signaling" does not explain how ERK, AKT, and STAT3 can be activated by TNF-α. In addition, little to nothing is known about negative regulation of TNFR1 signaling. Because cyclic AMP-activated kinase (PKA) shows anti-TNF and anti-inflammatory activities, we postulated that PKA might affect TNF-α signaling by directly phosphorylating TNFR1. In line with this, we identified 2 putative PKA-phosphorylation motifs RRRT411 and REAT417 within the death domain of TNFR1, and investigated whether "canonical" and "noncanonical" TNFR1 signaling is regulated by modifications of T411 and T417. In this study, we demonstrate for the first time that PKA directly binds to and phosphorylates TNFR1 after TNF-α stimulation. To further support our hypothesis, we generated alanine and phosphomimetic (aspartic acid) mutants of TNFR1 at positions T411 and T417, ectopically expressed these mutants, and determined their influence on TNF-α-induced activations of ERKs, AKT, STAT3, p38α, and JNK1/2. Our results clearly showed that phosphomimetic mutants significantly suppressed and alanine mutants augmented TNF-α-induced phosphorylations of ERKs, AKT, Stat3, p38α, and JNKs. These findings strongly suggest that PKA-mediated phosphorylation of T411 and T417 of TNFR1 interferes with both "canonical" and "noncanonical" TNF-α signaling. [Figure: see text].

Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1

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Negative Regulation of TNFR1 Signaling Via PKA-Mediated Phosphorylation of TNFR1