Taurine transporter (TauT) deficiency impairs ammonia detoxification in mouse liver.

Hepatic ammonia handling was analyzed in taurine transporter (TauT) KO mice. Surprisingly, hyperammonemia was present at an age of 3 and 12 months despite normal tissue integrity. This was accompanied by cerebral RNA oxidation. As shown in liver perfusion experiments, glutamine production from ammonia was diminished in TauT KO mice, whereas urea production was not affected. In livers from 3-month-old TauT KO mice protein expression and activity of glutamine synthetase (GS) were unaffected, whereas the ammonia-transporting RhBG protein was down-regulated by about 50%. Double reciprocal plot analysis of glutamine synthesis versus perivenous ammonia concentration revealed that TauT KO had no effect on the capacity of glutamine formation in 3-month-old mice, but doubled the ammonia concentration required for half-maximal glutamine synthesis. Since hepatic RhBG expression is restricted to GS-expressing hepatocytes, the findings suggest that an impaired ammonia transport into these cells impairs glutamine synthesis. In livers from 12-, but not 3-month-old TauT KO mice, RhBG expression was not affected, surrogate markers for oxidative stress were strongly up-regulated, and GS activity was decreased by 40% due to an inactivating tyrosine nitration. This was also reflected by kinetic analyses in perfused liver, which showed a decreased glutamine synthesizing capacity by 43% and a largely unaffected ammonia concentration dependence. It is concluded that TauT deficiency triggers hyperammonemia through impaired hepatic glutamine synthesis due to an impaired ammonia transport via RhBG at 3 months and a tyrosine nitration-dependent inactivation of GS in 12-month-old TauT KO mice.

Human and Murine Interleukin 23 Receptors Are Novel Substrates for A Disintegrin and Metalloproteases ADAM10 and ADAM17.

IL-23 (interleukin 23) regulates immune responses against pathogens and plays a major role in the differentiation and maintenance of TH17 cells and the development of autoimmune diseases and cancer. The IL-23 receptor (IL-23R) complex consists of the unique IL-23R and the common IL-12 receptor ß1 (IL-12Rß1). Differential splicing generates antagonistic soluble IL-23R (sIL-23R) variants, which might limit IL-23-mediated immune responses. Here, ectodomain shedding of human and murine IL-23R was identified as an alternative pathway for the generation of sIL-23R. Importantly, proteolytically released sIL-23R has IL-23 binding activity. Shedding of IL-23R was induced by stimulation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), but not by ionomycin. PMA-induced shedding was abrogated by an ADAM (A disintegrin and metalloprotease) 10 and 17 selective inhibitor, but not by an ADAM10 selective inhibitor. ADAM17-deficient but not ADAM10-deficient HEK293 cells failed to shed IL-23R after PMA stimulation, demonstrating that ADAM17 but not ADAM10 cleaves the IL-23R. Constitutive shedding was, however, inhibited by an ADAM10 selective inhibitor. Using deletions and specific amino acid residue exchanges, we identified critical determinants of ectodomain shedding within the stalk region of the IL-23R. Finally, interaction studies identified domains 1 and 3 of the IL-23R as the main ADAM17 binding sites. In summary, we describe human and murine IL-23R as novel targets for protein ectodomain shedding by ADAM10 and ADAM17.

The interleukin-6 receptor Asp358Ala single nucleotide polymorphism rs2228145 confers increased proteolytic conversion rates by ADAM proteases.

The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with a 2-fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level leads to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.

MYC paralog-dependent apoptotic priming orchestrates a spectrum of vulnerabilities in small cell lung cancer.

MYC paralogs are frequently activated in small cell lung cancer (SCLC) but represent poor drug targets. Thus, a detailed mapping of MYC-paralog-specific vulnerabilities may help to develop effective therapies for SCLC patients. Using a unique cellular CRISPR activation model, we uncover that, in contrast to MYCN and MYCL, MYC represses BCL2 transcription via interaction with MIZ1 and DNMT3a. The resulting lack of BCL2 expression promotes sensitivity to cell cycle control inhibition and dependency on MCL1. Furthermore, MYC activation leads to heightened apoptotic priming, intrinsic genotoxic stress and susceptibility to DNA damage checkpoint inhibitors. Finally, combined AURK and CHK1 inhibition substantially prolongs the survival of mice bearing MYC-driven SCLC beyond that of combination chemotherapy. These analyses uncover MYC-paralog-specific regulation of the apoptotic machinery with implications for genotype-based selection of targeted therapeutics in SCLC patients.

Transcytosis of IL-11 and Apical Redirection of gp130 Is Mediated by IL-11α Receptor.

Interleukin (IL)-11 signaling is involved in various processes, including epithelial intestinal cell regeneration and embryo implantation. IL-11 signaling is initiated upon binding of IL-11 to IL-11R1 or IL-11R2, two IL-11α-receptor splice variants, and gp130. Here, we show that IL-11 signaling via IL-11R1/2:gp130 complexes occurs on both the apical and basolateral sides of polarized cells, whereas IL-6 signaling via IL-6R:gp130 complexes is restricted to the basolateral side. We show that basolaterally supplied IL-11 is transported and released to the apical extracellular space via transcytosis in an IL-11R1-dependent manner. By contrast, IL-6R and IL-11R2 do not promote transcytosis. In addition, we show that transcytosis of IL-11 is dependent on the intracellular domain of IL-11R1 and that synthetic transfer of the intracellular domain of IL-11R1 to IL-6R promotes transcytosis of IL-6. Our data define IL-11R as a cytokine receptor with transcytotic activity by which IL-11 and IL-6:soluble IL-6R complexes are transported across cellular barriers.

Proteolytic Cleavage Governs Interleukin-11 Trans-signaling.

Interleukin (IL)-11 has been shown to be a crucial factor for intestinal tumorigenesis, lung carcinomas, and asthma. IL-11 is thought to exclusively mediate its biological functions through cell-type-specific expression of the membrane-bound IL-11 receptor (IL-11R). Here, we show that the metalloprotease ADAM10, but not ADAM17, can release the IL-11R ectodomain. Chimeric proteins of the IL-11R and the IL-6 receptor (IL-6R) revealed that a small juxtamembrane portion is responsible for this substrate specificity of ADAM17. Furthermore, we show that the serine proteases neutrophil elastase and proteinase 3 can also cleave the IL-11R. The resulting soluble IL-11R (sIL-11R) is biologically active and binds IL-11 to activate cells. This IL-11 trans-signaling pathway can be inhibited specifically by the anti-inflammatory therapeutic compound sgp130Fc. In conclusion, proteolysis of the IL-11R represents a molecular switch that controls the IL-11 trans-signaling pathway and widens the number of cells that can be activated by IL-11.

Inhibition of protein kinase II (CK2) prevents induced signal transducer and activator of transcription (STAT) 1/3 and constitutive STAT3 activation.

The Janus kinase / signal transducer and activator of transcription (Jak/STAT) pathway can be activated by many different cytokines, among them all members of the Interleukin (IL-)6 family. Dysregulation of this pathway, resulting in its constitutive activation, is associated with chronic inflammation and cancer development. In the present study, we show that activity of protein kinase II (CK2), a ubiquitously expressed serine/threonine kinase, is needed for induced activation of STAT1 and STAT3 by IL-6 classic and trans-signaling, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). Inhibition of CK2 efficiently prevented STAT phosphorylation and inhibited cytokine-dependent cell proliferation in a Jak1-dependent manner. Conversely, forced activation of CK2 alone was not sufficient to induce activation of the Jak/STAT signaling pathway. Inhibition of CK2 in turn inhibited Jak1-dependent STAT activation by oncogenic gp130 mutations. Furthermore, CK2 inhibition diminished the Jak1- and Src kinase-dependent phosphorylation of a constitutively active STAT3 mutant recently described in human large granular lymphocytic leukemia. In conclusion, we characterize CK2 as an essential component of the Jak/STAT pathway. Pharmacologic inhibition of this kinase is therefore a promising strategy to treat human inflammatory diseases and malignancies associated with constitutive activation of the Jak/STAT pathway.