Gilles Chaumaz

Down‐Regulation of Osteoclast Differentiation by Daidzein via Caspase 3

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 17β‐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 10−8 M of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], with or without 10−8 M of daidzein, 10−8 M of 17β‐estradiol for 9‐11 days. Multinucleated tartrate‐resistant acid phosphatase‐positive (TRAP+) cells that resorbed bone (osteoclasts) developed in the presence of 1,25(OH)2D3. The number of osteoclasts formed in response to 1,25(OH)2D3 was reduced by 58 ± 8% by daidzein and 52 ± 5% by estrogen (p < 0.01); these effects were reversed by 10−6 M of ICI 182,780. The area resorbed by mature osteoclasts was reduced by 39 ± 5% by daidzein and 42 ± 6% by estradiol (p < 0.01). Both compounds also inhibited the 1,25(OH)2D3‐induced differentiation of osteoclast progenitors (mononucleated TRAP+ cells), 53 ± 8% by daidzein and 50 ± 7% by estradiol (p < 0.05). Moreover, daidzein and estradiol promoted caspase‐8 and caspase‐3 cleavage and DNA fragmentation of monocytic bone marrow cells. Caspase‐3 cleavage was reversed by 10−8 M of ICI 182,780. Both compounds up‐regulated the expression of nuclear estrogen receptors ER‐α and ER‐β. Thus, daidzein, at the same concentration as 17β‐estradiol, inhibits osteoclast differentiation and activity. This may be caused by, at least in part, greater apoptosis of osteoclast progenitors mediated by ERs.

Luminal Cathepsin G and Protease-Activated Receptor 4 A Duet Involved in Alterations of the Colonic Epithelial Barrier in Ulcerative Colitis

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.

Signaling Cross-talk from Gβ4 Subunit to Elk-1 in the Rapid Action of Androgens

Androgens act on transcription via intracellular androgen receptors (ARs), but they also have rapid AR-independent 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 5α-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); Gö6976, Gö6983, 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 Gβ4 subunit coupled to phospholipase C-β2, 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.

Acute stress increases colonic paracellular permeability in mice through a mast cell-independent mechanism: involvement of pancreatic trypsin.

AIMS Increased colonic paracellular permeability (CPP) is a key feature of gastro-intestinal disorders as irritable bowel syndrome and inflammatory bowel diseases. Stress stimulates exocrine pancreatic secretion through cholinergic pathways, and trypsin is known to increase CPP. Consequently we have investigated in this work whether trypsin released into the gut lumen following an acute stress may participate to the short-term increase in CPP. MAIN METHODS Mice were treated with atropine or a non-selective CRF (corticotropin-releasing factor) receptor antagonist (alpha-helical CRF (9-41)), before being submitted to a 2-h stress session. Then, CPP and protease activity in colonic contents (total proteolytic, trypsin activity, and mouse mast cell protease (MMCP)-1 levels) were determined. The effects of colonic contents from sham-stressed or stressed animals on CPP were evaluated in mice colonic tissues mounted in Ussing chambers, in presence or not of soybean trypsin inhibitor (SBTI) or FSLLRY, a protease-activated receptor-2 (PAR2) antagonist. KEY FINDINGS Acute stress significantly increased CPP, proteolytic and trypsin activities, and MMCP-1 levels. Atropine inhibited stress-induced impairment of CPP and strongly diminished total proteolytic and trypsin activities in stressed animals, but not MMCP-1 levels. Colonic contents from stressed animals increased CPP in mice tissues, this effect being inhibited by SBTI and PAR2 antagonist. SIGNIFICANCE Acute stress activates cholinergic pathways, to trigger exocrine pancreatic secretion. Trypsin, released in these conditions, may be responsible for colonic barrier alterations through the activation of PAR2.

Signaling networks from Gβ1 subunit to transcription factors and actin remodeling via a membrane‐located ERβ‐related protein in the rapid action of daidzein in osteoblasts

Although estrogen replacement has been the main therapy to prevent and treat osteoporosis, there are concerns about its safety. Phytoestrogens have attracted attention to their potential impacts in osteoporosis prevention and treatment. Among phytoestrogens, the isoflavone daidzein (Dz) acts on transcription via the intracellular estrogen receptors (ER), mainly ERβ, in osteoblasts, but mimics only part of the estrogen effects. Since estradiol also exerts rapid effects in osteoblasts, we investigated the multistep processes involved in the rapid actions of low (1–100 pM) doses of daidzein. Dz bound to a membrane moiety, related to ERβ since the calcium response to Dz was blocked by an anti‐ERβ antibody directed against the C‐terminus, but not by a double‐stranded siRNA specific for ERβ. This protein was coupled to a pertussis toxin (PTX)‐sensitive Gβ1 subunit whose transducer was PLC‐β2, which triggered a rapid (5 sec) mobilization of calcium from the endoplasmic reticulum. Dz phosphorylated within 15 sec ERK1/2 whose phosphorylation involved two routes: Gβ1/PLC‐β2/PKC/c‐Raf‐1/MEK1/2 and Gβ1/PI3K/cSrc/c‐Raf‐1/MEK1/2 as shown using several inhibitors. Dz induced rapid (1 min) changes in the actin cytoskeleton via the two routes. The rapid (20 sec) phosphorylation of Elk‐1 and CREB by Dz involved Gβ1 and ERK1/2. All the processes were insensitive to the estradiol antagonist ICI 182,780. In conclusion, the rapid effects of Dz seem to be biologically relevant for the function of osteoblast in bone since the isoflavone activates transcription factors linked to early genes controlling cellular proliferation and differentiation, and modulates actin cytoskeleton which controls cell adhesion, division, or secretion. J. Cell. Physiol. 209: 786–801, 2006.

Proinflammatory role of vasopressin through V1b receptors in hapten-induced experimental colitis in rodents: implication in IBD

Vasopressin and its receptors modulate several gut functions, but their role in intestinal inflammation is unknown. Our aims were to determine 1) the localization of V1b receptors in human and rodent colon, 2) the role of vasopressin and V1b receptors in experimental colitis using two approaches: V1b⁻(/)⁻ mice and a selective V1b receptor antagonist, SSR149415, and 3) the mechanisms involved. V1b receptors were localized in normal and inflamed colon from humans and rats. Experimental colitis was induced in rats and mice and some groups were treated before or after colitis induction with oral SSR149415 (3-30 mg/kg). Other groups of mice were submitted to dehydration to increase vasopressin plasma levels, prior to colitis induction. Body weight, damage scores, MPO, and TNF-α tissue levels were determined. Finally, colonic segments of wild-type (WT) and V1b⁻(/)⁻ mice were mounted in Ussing chambers and paracellular permeability in response to vasopressin was studied. V1b receptors were expressed in enterocytes and ganglia cells of the enteric nervous system of human and rat intestine. Expression levels were independent from inflammatory status. Colitis was less severe in rodents treated by either preventive or curative SSR149415 and in V1b⁻(/)⁻ mice. 2,4,6-Trinitrobenzene sulfonic acid induced a strong mortality in dehydrated animals that was reversed by preventive SSR149415 or mast cell stabilizer. Vasopressin significantly increased paracellular permeability in WT, but not in V1b⁻(/)⁻ mice. Preincubation of colon tissues with SSR149415 abolished the vasopressin effect. Similarly, vasopressin had no effect in colonic preparations from WT mice pretreated with mast cell stabilizers. Vasopressin, through V1b receptor interaction, has proinflammatory properties linked to mast cell activation and downstream alterations of the colonic epithelial barrier. These findings underline the potential interest of V1b receptor blockers in gut inflammatory diseases.

Extracellular matrix regulates alpha s1-casein gene expression in rabbit primary mammary cells and CCAAT enhancer binding protein (C/EBP) binding activity

Previous studies have shown that both the signal transducer and activator of transcription 5 (STAT5) and the CCAAT enhancer binding proteins (C/EBPs) are involved in the regulation of casein gene expression by mammary epithelial cells. Prolactin (Prl) activation of STAT5 is necessary for casein gene expression. The extracellular matrix (ECM) regulates also casein gene expression. Here, we have investigated whether ECM regulates C/EBPs activity in primary rabbit mammary epithelial cells. Isolated primary mammary cells were cultured on plastic or on floating collagen I gel. Prolactin induced αs 1‐casein gene expression when cells were cultured on collagen but not on plastic. It is noteworthy that activated STAT5 was detected in both culture conditions. Several STAT5 isoforms (STAT5a, STAT5b, and other STAT5 related isoforms, some with lower molecular weight than the full‐length STAT5a and STAT5b) were detected under the different culture conditions. However, their presence was not related to the expression of αs 1‐casein gene. The binding of nuclear factors to a C/EBP specific binding site and the protein level of C/EBPβ differed in cells cultured on plastic or on collagen but these parameters were not modified by Prl. This suggests that C/EBP binding activity was regulated by ECM and not by Prl. Interestingly, these modifications were correlated to the expression of the αs 1‐casein gene. Hence, the activation of the αs 1‐casein gene expression depends on two independent signals, one delivered by Prl via the activation of STAT5, the other delivered by ECM via C/EBP. J. Cell. Biochem. 82:371–386, 2001.

Dietary isoflavones act on bone marrow osteoprogenitor cells and stimulate ovary development before influencing bone mass in pre-pubertal piglets†

Food containing soybeans provide isoflavone phytoestrogens that can preserve bone mass in postmenopausal women, and prevent bone loss in ovariectomized rats. But their effects on bone remain unclear, particularly on bone formation during growth. Two groups of eight pre‐pubertal piglets were fed a basal or an isoflavone‐enriched (S800) diet for 6 weeks. The S800 diet contained 800 mg SoyLife™/kg, providing 2.8 mg isoflavones/kg body weight/day. Several bones were collected and tested for bone strength and density. Bone marrow was collected from humeri together with blood samples and genital tracts. The plasma concentrations of isoflavones were increased in the pigs fed S800, but growth rate, body weight, plasma bone markers, bone mineral density, and strength were all unaffected. In contrast, cultured stromal cells from S800 pigs had more alkaline phosphatase‐rich cells and mineralized nodules, secreted more osteocalcin, osteoprotegerin and RANK‐L, synthesized more osteoprotegerin, and RANK‐L. Cultured mononucleated nonadherent bone marrow cells from S800 pigs developed fewer tartrate‐resistant acid phosphatase mononucleated cells (osteoclast progenitors) when cultured with 1,25(OH)2D3, and resorbed a smaller area of dentine slices. Freshly isolated bone marrow osteoclast progenitors from S800 pigs had more caspase‐3 cleavage activity, and synthesized less RANK. Both osteoclast and osteoblast progenitors had ERα and ERβ, whose syntheses were stimulated by the S800 diet. The S800 piglets had heavier ovaries with more follicles, but their uterus weight was unaffected. We conclude that dietary isoflavones have no detectable effect on the bone mass of growing female piglets, but act on bone marrow osteoprogenitors via ERs—mainly ERβ, and stimulate ovary development. J. Cell. Physiol. 212: 51–59, 2007.