A mechanism of benefit of soy genistein in asthma: inhibition of eosinophil p38-dependent leukotriene synthesis.

BACKGROUND: Dietary intake of the soy isoflavone genistein is associated with reduced severity of asthma, but the mechanisms responsible for this effect are unknown. OBJECTIVE: To determine whether genistein blocks eosinophil leukotriene C(4) (LTC(4)) synthesis and to evaluate the mechanism of this effect, and to assess the impact of a 4-week period of soy isoflavone dietary supplementation on indices of eosinophilic inflammation in asthma patients. METHODS: Human peripheral blood eosinophils were stimulated in the absence and presence of genistein, and LTC(4) synthesis was measured. 5-lipoxygenase (5-LO) nuclear membrane translocation was assessed by confocal immunofluorescence microscopy. Mitogen-activated protein (MAP) kinase activation was determined by immunoblot. Human subjects with mild-to-moderate persistent asthma and minimal or no soy intake were given a soy isoflavone supplement (100 mg/day) for 4 weeks. The fraction of exhaled nitric oxide (FE(NO)) and ex vivo eosinophil LTC(4) production were assessed before and after the soy isoflavone treatment period. RESULTS: Genistein inhibited eosinophil LTC(4) synthesis (IC(50) 80 nm), blocked phosphorylation of p38 MAP kinase and its downstream target MAPKAP-2, and reduced translocation of 5-LO to the nuclear membrane. In patients with asthma, following 4 weeks of dietary soy isoflavone supplementation, ex vivo eosinophil LTC(4) synthesis decreased by 33% (N=11, P=0.02) and FE(NO) decreased by 18% (N=13, P=0.03). CONCLUSION: At physiologically relevant concentrations, genistein inhibits eosinophil LTC(4) synthesis in vitro, probably by blocking p38- and MAPKAP-2-dependent activation of 5-LO. In asthma patients, dietary soy isoflavone supplementation reduces eosinophil LTC(4) synthesis and eosinophilic airway inflammation. These results support a potential role for soy isoflavones in the treatment of asthma.

Sulfate transport in porcine thyroid cells. Effects of thyrotropin and iodide.

In porcine thyroid cells, thyroglobulin sulfation is controlled by thyrotropin (TSH) and iodide, which contribute to regulating the intracellular sulfate concentration, as we previously established. Here, we studied the transport of sulfate and its regulation by these two effectors. Kinetic studies were performed after [(35)S]sulfate was added to either the basal or apical medium of cell monolayers cultured without any effectors, or with TSH with or without iodide. The basolateral uptake rates were about tenfold higher than the apical uptake rates. TSH increased the basolateral and apical uptake values (by 24 and 9%, respectively, compared with unstimulated cells), and iodide inhibited these effects of TSH. On the basis of results of the pulse-chase experiments, the basolateral and apical effluxes appeared to be well balanced in unstimulated cells and in cells stimulated by both TSH and iodide: approximately 40-50% of the intracellular radioactivity was released into each medium, whereas in the absence of iodide, 70% of the intracellular radioactivity was released on the basolateral side. The rates of transepithelial sulfate transport were increased by TSH compared with unstimulated cells, and these effects decreased in response to iodide. These results suggest that TSH and iodide may each control the sulfate transport process on two sides of the polarized cells, and that the absence of iodide in the TSH-stimulated cells probably results in an unbalanced state of sulfate transport.

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis.

Thyroglobulin (Tg) binds to cell surfaces through various binding sites of high, moderate and low affinity. We have previously shown that binding with low to moderate affinity is pH dependent, selective, but not tissue specific. To identify the regions of Tg involved in this cell surface binding, we studied the binding of (125)I-labeled cyanogen bromide peptides from human Tg to cell surfaces of thyroid cells (inside-out follicles) and of CHO cells. Electrophoretic analysis of cell homogenates after binding of native or of reduced and alkylated (125)I-labeled peptides showed that three peptides, P1, P2 and P3, were always associated with the cells. Sequence analysis allowed the identification of P1 (Ser-2445 to Met-2596 or Met-2610) and P2 (Phe-2156 to Met-2306). P3 proved to be a mixture of several peptides among which two were identified: P3-1 (Cys-1306 to Met-1640) and P3-2 (Cys-2035 to Met-2413) which includes P2. P1, P2 and P3-2 are entirely (P1) or partly (P2 and P3-2) located in the C-terminal domain of Tg homologous with acetylcholinesterase. The smallest peptides, P1 and P2, were purified by preparative electrophoresis. They both displayed strong binding properties towards cell surfaces. Inhibition experiments of (125)I-labeled Tg binding by P1 or P2 indicated that they were involved in Tg binding to cell surfaces. All the other peptides tested for their binding abilities were either not or only poorly involved in Tg binding to cell surfaces, which suggested that P1 and P2 are major Tg sites of binding to cell surfaces. These two peptides are not involved in the binding of Tg to the known Tg 'receptors' described in the literature, to which recycling, transcytosis and regulation functions have been ascribed. Thus they are potential tools to identify cell surface components involved in the process of Tg endocytosis leading to lysosomal degradation.

Regulation of thyroid cell volumes and fluid transport: opposite effects of TSH and iodide on cultured cells.

Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 +/- 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.

Sulfated tyrosines of thyroglobulin are involved in thyroid hormone synthesis.

Thyroid hormone synthesis is under the control of thyrotropin (TSH), which also regulates the sulfation of tyrosines in thyroglobulin (Tg). We hypothesized that sulfated tyrosine (Tyr[S]) might be involved in the hormonogenic process, since the consensus sequence required for tyrosine sulfation to occur was observed at the hormonogenic sites. Porcine thyrocytes, cultured with TSH but without iodide in the presence of [(35)S]sulfate, secreted Tg which was subjected to in vitro hormonosynthesis with increasing concentrations of iodide. A 63% consumption of Tyr[S] (1 residue) was observed at 40 atoms of iodine incorporated into Tg, corresponding to a 40% hormonosynthesis efficiency. In addition, hyposulfated Tg secreted by cells incubated with sodium chlorate was subjected to in vitro hormonosynthesis. With 0.5 Tyr[S] residue (31% of the initial content), the efficiency of the hormonosynthesis was 29%. In comparison, when hormonosynthesis was performed by cells, with only 0.25 Tyr[S] residue (16% of the initial content), the hormonosynthesis efficiency fell to 18%. These results show that there exists a close correlation between the sulfated tyrosine content of Tg and the production of thyroid hormones.

Thyrotropin regulates tyrosine sulfation of thyroglobulin.

OBJECTIVE: To study the regulation of thyroglobulin sulfation by thyrotropin (TSH) and iodide. Sulfation, a widespread post-translational modification of proteins, is involved in various biological activities. Thyroglobulin has been reported to be sulfated but, to date, the role of sulfate residues in the metabolism and function of thyroglobulin is not known; moreover, the regulation of thyroglobulin sulfation has not been yet investigated. METHODS: The effect of TSH on thyroglobulin sulfation was studied in porcine thyroid cells cultured on porous collagen-coated filters. Cells cultured with or without TSH and with or without iodide (KI) were incubated for 4 days with radioactive sulfate. The specific radioactivity of thyroglobulin subunit (330kDa) was determined from apical media analyzed by electrophoresis. Enzymatic hydrolysates of the purified thyroglobulin were separated by oligosaccharide affinity chromatography and thin-layer chromatography; alkaline hydrolysates were analyzed only by thin-layer chromatography. RESULTS: Thyroglobulin secreted by TSH-stimulated cells incorporated about twofold less radioactive sulfate. Iodide slightly modified this incorporation. Enzymatic hydrolysates of purified thyroglobulin showed sulfate residues bound essentially to complex oligosaccharide units. Alkaline hydrolysis was necessary to release all sulfated amino acids (tyrosine and serine). In the absence of TSH the proportion of tyrosine sulfate was dramatically increased: 24% compared with 7% (+KI) or 5% (-KI). The ratio of specific radioactivity of thyroglobulin to the specific radioactivity of intracellular inorganic sulfate (determined in each culture condition) gave the number of sulfated residues incorporated: 46 (-TSH) and 31 (+TSH) per mol thyroglobulin. From this distribution, we deduced the number of residues bound to complex oligosaccharide units and to tyrosine. Thus TSH decreased the number of sulfate residues on tyrosine from 11 to 2 per mol thyroglobulin. CONCLUSIONS: TSH regulates the binding of sulfate groups to tyrosine residues. Iodide exerts a slight control over this process.