Thomas Farruggella

Choice of STATs and other substrates specified by modular tyrosine-based motifs in cytokine receptors.

Many members of the cytokine receptor superfamily initiate intracellular signaling by activating members of the Jak family of tyrosine kinases. Activation of the same Jaks by multiple cytokines raises the question of how these cytokines activate distinct intracellular signaling pathways. Selection of particular substrates--the transcriptional activator Stat3 and protein tyrosine phosphatase PTP1D--that characterize responses to the ciliary neurotrophic factor-interleukin-6 cytokine family depended not on which Jak was activated, but was instead determined by specific tyrosine-based motifs in the receptor components--gp130 and LIFR--shared by these cytokines. Further, these tyrosine-based motifs were modular, because addition of a Stat3-specifying motif to another cytokine receptor, that for erythropoietin, caused it to activate Stat3 in a ligand-dependent fashion.

Detection of receptors for interleukin-6, interleukin-11, leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor in bone marrow stromal/osteoblastic cells.

The functional receptor complexes assembled in response to interleukin-6 and -11 (IL-6 and IL-11), leukemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF), all involve the signal transducer gp130: IL-6 and IL-11 induce homodimerization of gp130, while the rest heterodimerize gp130 with other gp130-related beta subunits. Some of these cytokines (IL-6, IL-11, and CNTF) also require a specificity-determining alpha subunit not directly involved in signaling. We have searched for functional receptor complexes for these cytokines in cells of the bone marrow stromal/osteoblastic lineage, using tyrosine phosphorylation of the beta subunits as a detection assay. Collectively, murine calvaria cells, bone marrow-derived murine cell lines (+/+LDA11 and MBA13.2), as well as murine (MC3T3-E1) and human (MG-63) osteoblast-like cell lines displayed all the previously recognized alpha and beta subunits of this family of receptors. However, individual cell types had different constellations of alpha and beta subunits. In addition and in difference to the other cell types examined, MC3T3-E1 cells expressed a heretofore unrecognized form of gp130; and MG-63 displayed an alternative form (type II) of the OSM receptor. These findings establish that stromal/osteoblastic cells are targets for the actions of all the members of the cytokine subfamily that shares the gp130 signal transducer; and suggest that different receptor repertoires may be expressed at different stages of differentiation of this lineage.

The protein tyrosine phosphatase SHP-2 negatively regulates ciliary neurotrophic factor induction of gene expression

Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.

A sulphite-oxidase-deficient rat model: Metabolic characterization

Rats were made deficient in sulphite oxidase by the administration of a high-tungsten/low-molybdenum regimen as described first by Johnson et al. (J. biol. Chem. 1974, 249, 859). The specific protocol used resulted initially in an exponential decrease in hepatic sulphite-oxidase activity with a half-life of 4 days and an eventual steady-state enzyme level approximately 1% of the normal adult level. A clear inverse relationship was demonstrated between hepatic sulphite-oxidase activity and tissue and/or urine concentrations of sulphite and of two sulphite metabolites, S-sulphonate (RS-SO3−) and and inorganic thiosulphate (S2O3− −). As rat tissues became depleted of sulphite-oxidase activity, sulphite produced endogenously from the catabolism of sulphur-containing amino acids gradually became apparent. The first chemical evidence of an increased systemic concentration of sulphite occurred when the sulphite-oxidase activity had declined to about 7% of the normal adult level; at this point, slight but significant increases in urinary S2O3− − and RS-SO3− concentrations were observed. The additional decline of sulphite oxidase to 1% of the normal level resulted in substantial increases in the excretion of both of these metabolites. In addition, large increases in aortic RS-SO3− concentrations relative to pretreatment levels and smaller increases in plasma and pinna RS-SO3− concentrations were observed as the rats approached a steady-state enzyme level. The ability of these sulphite-oxidase-deficient rats (1% of normal activity) to clear sulphite following intragastric sulphite administration was compared with that of normal rats. The results showed that deficient rats were much less efficient at this process and thus required lower exogenous doses to produce equivalent systemic exposures.

A sulphite-oxidase-deficient rat model: subchronic toxicology.

Abstract Toxicity resulting from exposure to sulphite originating both endogenously and exogenously was investigated in normal rats and in rats made sulphite-oxidase-deficient by molybdenum deficiency abetted by administration of tungstate. The sulphite-oxidase-deficient rats were outwardly as healthy as controls and exhibited normal weight gain and maintenance over the 9-wk test period. The systemic sulphite exposures of normal and deficient rats resulting from various sulphite treatments could be compared by determining the concentrations of tissue S-sulphonate (RS-SO3−) metabolites formed. In general, relatively low intakes of exogenous sulphite (0–3·5 mmol/kg/day) by sulphite-oxidase-deficient rats produced systemic sulphite exposures equivalent to those produced by the ingestion by normal rats of highly sulphited diets (intakes of 13–25 mmol/kg/day). The advantages of the sulphite-oxidase-deficient rat compared to the normal rat as a model for human exposure are discussed. Using these two animal models, it was demonstrated that anaemia and thiamine deficiency, which have been produced previously in sulphite-feeding studies, result solely from the action of high concentrations of sulphite in the diet and/or gut and are not attributable to systemic sulphite exposure. Likewise, prothrombin time and erythrocyte concentrations of glutathione were not affected by high systemic sulphite concentrations in these experiments. A 4 149 incidence of mammary adenocarcinoma was observed in sulphite-oxidase-deficient rats, all in rats aged less than 5 months, compared to 0 143 observed in age-matched rats with normal sulphite oxidase. Although this result was not statistically significant, the rarity of spontaneous tumours of this type among rats of this age suggests that these carcinomas may, in fact, have been treatment related. If indicated, further investigation will be undertaken to determine the role of sulphite-oxidase-deficiency, sulphite and/or tungstate, as well as other elements of the model, in the aetiology of these tumours.

Comparative Effects of Aminoglycosides on Renal Cortical and Urinary Phospholipids in the Rat

Abstract We examined the relationship between the nephrotoxicity potential of four aminoglycosides and the capacity of the drugs to induce a renal cortical phospholipidosis. Sprague-Dawley rats were injected subcutaneously with neomycin, gentamicin, tobramycin, or netilmicin, 100 mg/kg per day, for 1 to 4 days, and phospholipid accumulation in the renal cortex and phospholipid excretion in the urine were measured. The rank order of the drug-induced renal cortical phospholipidosis was netilmicin > tobramycin > gentamicin > neomycin. This order is the reverse of the previously established nephrotoxicity potentials of these drugs. Conversely, the rank order according to peak urinary excretion of phospholipids was gentamicin > neomycin > tobramycin > netilmicin. The rank order of the total urinary phospholipid excretion during the 4 days of the study was neomycin ≥ gentamicin > tobramycin ≥ netilmicin. Urinary phospholipid excretion may prove to be a sensitive indicator of aminoglycoside nephrotoxicity.

Preferential S-sulfonate formation in lung and aorta.

S-sulfonate (S-SO-3) compounds have previously been identified as metabolites of sulfite in the plasma of several species of mammals [6--8]. We now report the formation of non-diffusible and relatively stable S-sulfonates in the aorta and lung lobes of rabbits exposed intravenously to constant arterial sulfite concentrations of approx. 550 microM. Under these conditions the kinetics of S-SO-3 formation were first order with coefficients in the range of 0.3--0.4 h-1 and asymptotic concentrations of approx. 900 and 9000 nmol S-SO-3/g dry wt. of lung and aorta respectively. The kinetics of this reaction in aorta tissue were closely approximated in vitro. Clearance of S-SO-3 from both lungs and aorta appeared to be first order with a half-life of 2--3 days.