No abstract

Phytoestrogens are plant-derived compounds with estrogen-like activity. Phytoestrogen-rich diets may prevent postmenopausal osteoporosis and these molecules maintain bone mass in ovariectomized animals. We compared the effects of the isoflavone daidzein, which has no action on tyrosine kinases, and 17␤-estradiol on the development and activity of osteoclasts in vitro. Nonadherent porcine bone marrow cells were cultured on dentine slices or on culture slides in the presence of 10

Flavonols, in contrast to soybean isoflavones, are the most abundant phytoestrogens in western diets, being present in onions, beans, fruits, red wine, and tea. They may protect against atherosclerosis, inhibit certain cancer cell types, and reduce bone resorption. The most widely distributed flavonol is quercetin, which occurs mainly as its glycoside, rutin, but data are very scarce regarding the precise mechanism of action of these compounds on bone-resorbing cells at concentrations similar to those detected in human plasma. We have therefore investigated the effects of nanomolar concentrations of quercetin and rutin on the development and activity of osteoclasts in vitro compared with the effects of 17beta-estradiol. Nonadherent porcine bone marrow cells were cultured on dentine slices in the presence of 10 nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), with or without 10 nM quercetin, 10 nM rutin or 10 nM 17beta-estradiol for 11 days. Multinuclear TRAP+ cells that resorbed dentine (osteoclasts) developed in the presence of 1,25(OH)2D3, but their number was significantly reduced by quercetin, rutin, and 17beta-estradiol (P < 0.05). Like 17beta-estradiol, both flavonols also significantly reduced resorption (P<0.05) as assessed by the size of pits resorbed on dentine slices. Osteoclasts and osteoclast progenitors contained estrogen receptor alpha (ERalpha), ERbeta, and RANK proteins. Both flavonols increased nuclear ERbeta protein and decreased ERalpha protein of osteoclast progenitors. Moreover, rutin reduced RANK protein, whereas 17beta-oestradiol and quercetin promoted apoptosis by cleavage of caspase-8 and caspase-3. All the effects of flavonols were reversed by 1 microM ICI 182,780, an estrogen antagonist. Thus, the anti-resorbing properties of flavonols are mainly mediated by ER proteins through the inhibition of RANK protein or the activation of caspases.

Impairment of the colonic epithelial barrier and neutrophil infiltration are common features of inflammatory bowel disease. Luminal proteases affect colonic permeability through protease-activated receptors (PARs). We evaluated: (i) whether fecal supernatants from patients with ulcerative colitis (UC) trigger alterations of colonic paracellular permeability and inflammation, and (ii) the roles of cathepsin G (Cat-G), a neutrophil serine protease, and its selective receptor, PAR 4 , in these processes. Expression levels of both PAR 4 and Cat-G were determined in colonic biopsies from UC and healthy subjects. The effects of UC fecal supernatants on colonic paracellular permeability were measured in murine colonic strips. Involvement of Cat-G and PAR 4 was evaluated using pepducin P4pal-10 and specific Cat-G inhibitor (SCGI), respectively. In addition, the effect of PAR 4 -activating peptide was assessed. UC fecal supernatants, either untreated or pretreated with SCGI, were infused into mice, and myeloperoxidase activity was determined. PAR 4 was found to be overexpressed in UC colonic biopsies. Increased colonic paracellular permeability that was triggered by UC fecal supernatants was blocked by both SCGI (77%) and P4pal-10 (85%). Intracolonic infusion of UC fecal supernatants into mice increased myeloperoxidase activity. This effect was abolished by SCGI. These observations support that both Cat-G and PAR 4 play key roles in generating and/or amplifying relapses in UC and provide a rationale for the development of new therapeutic agents in the treatment of this disease.

Androgens act on transcription via intracellular androgen receptors (ARs), but they also have rapid ARindependent effects. We have identified the multistep processes involved in the rapid actions of androgens in male osteoblasts, which also possess the classical AR. Incubating cells with 5␣-dihydroxytestosterone (100 pM, DHT) rapidly increased (1 min) the phosphorylation of the transcription factor Elk-1, and this was inhibited by pertussis toxin (PTX). DHT activated ERK1/2, a substrate of Elk-1, within 15 s but had no effect on p38 MAPK or JNK/SAPK. The inhibitors PD98059 (MEK1/2); Gö 6976, Gö 6983, and chelerythrine (protein kinase C); wortmannin and LY294002 (phosphatidylinositol 3-kinase); PP1 (Src); and PTX all blunted the DHT-stimulated phosphorylation of ERK1/2. DHT increased the phosphorylation of c-Raf-1 within 5 s; this was blocked by conventional protein kinase C and phosphatidylinositol 3-kinase inhibitors. The first activated membrane protein was the PTX-sensitive G␤ 4 subunit coupled to phospholipase C-␤2, which triggered a rapid (5 s) increase in intracellular calcium and diacylglycerol formation. The androgen antagonist cyproterone acetate did not modify the responses to DHT. Lastly an anti-AR antibody directed against the ligand binding domain recognized a protein at the plasma membrane. The cascade of rapid effects triggered by androgens may involve the classical AR at the plasma membrane or an uncharacterized form of AR that is insensitive to nuclear antagonists.