Amino acid deprivation regulates the stress-inducible gene p8 via the GCN2/ATF4 pathway.

In mammals, the GCN2/ATF4 pathway has been described as the main pathway involved in the regulation of gene expression upon amino acid limitation. This regulation is notably conferred by the presence of a cis-element called Amino Acid Response Element (AARE) in the promoter of specific genes. In vivo, the notion of amino acid limitation is not limited to nutritional context, indeed several pathological situations are associated with alteration of endogenous amino acid availability. This is notably true in the context of tumour in which the alteration of the microenvironment can lead to a perturbation in nutrient availability. P8 is a small weakly folded multifunctional protein that is overexpressed in several kinds of cancers and whose expression is induced by different stresses. In this study we have demonstrated that amino acid starvation was also able to induce p8 expression. Moreover, we brought the evidence, in vitro and in vivo, that the GCN2/ATF4 pathway is involved in this regulation through the presence of an AARE in p8 promoter.

Expression of PPARgamma and beta/delta in human primary osteoblastic cells: influence of polyunsaturated fatty acids.

As previously reported, the age-related association between bone loss and increased marrow adipose volume may involve inhibitory effects of polyunsaturated fatty acids (PUFAs) potentially released by medullary adipocytes on osteoblastic proliferation and cell cycle progression. Because PUFAs have been reported to activate peroxisome proliferator-activated receptors (PPARs), we investigated the expression of these nuclear receptors in human primary osteoblastic (hOB) cells and examined the effects of natural PPAR ligands on hOB cell proliferation. We demonstrated basic expressions of PPARgamma and PPARbeta/delta in hOB cells at the protein level. As already shown for PUFAs, a short-term treatment with 15deoxy-Delta(12,14) -prostaglandin J2 (15dPGJ2) or prostacyclin (PGI2), which are specific ligands for PPARgamma and PPARbeta/delta, respectively, also significantly inhibited hOB cell proliferation. Given that the cell cycle withdrawal resulting from PPARgamma activation was often associated with the induction of cell differentiation, long-term effects of PUFAs and 15dPGJ2 were also assessed on the expression levels of transcription factors. PUFAs and 15dPGJ2 enhanced the expression of PPARgamma in hOB cells. It is of interest to note that PPARgamma protein level was dose-dependently increased, whereas that of Cbfal was decreased by a fatty acid-rich serum. In conclusion, this study shows that PPARgamma and beta/delta are expressed by hOB cells. The results further suggest that the short-term antiproliferative effect of PUFAs may involve PPAR activation in hOB cells, resulting in a cell cycle withdrawal favorable for the long-term differentiating effects of fatty acids. Further studies are now required to confirm the functional role of PPARs in the antiproliferative effects of PUFAs in hOB cells.

Role of polyunsaturated fatty acids in the inhibitory effect of human adipocytes on osteoblastic proliferation.

As previously reported, the association of bone loss with an increase in bone marrow adipose volume may be related to the inhibition of human osteoblastic cell proliferation in the presence of human adipocytes. In the osteoblastic supernatant, fatty acid composition varied after coculture with mature adipocytes, with a marked increase in the proportion of docosahexaenoic acid (22:6 n-3; DHA) (+90 +/- 8%). This suggests that polyunsaturated fatty acids (PUFA) may contribute to the inhibitory effect of adipocytes on osteoblastic cell proliferation. The purpose of the present study was to evaluate the effects of two PUFA, DHA and arachidonic acid (20:4 n-6; AA), on the proliferation of primary human osteoblastic (hOB) cells and human osteosarcoma cell line, MG-63. The effects of cholesterol and oleic acid, a monounsaturated FA (18:1 n-9; OA), both being present in adipocyte lipidic vacuoles, were also investigated. At between 10 and 50 micromol/L, DHA and AA induced a significant dose-dependent decrease in hOB cell proliferation (p < 0.0001 and p < 0.006 for DHA and AA, respectively) when compared with control hOB cells exposed to the vehicle (bovine serum albumin). This inhibition reached -50% with 50 micromol/L of DHA or 20 micromol/L of AA. This effect was not related to cell apoptosis, as shown by terminal deoxynucleotidyltransferase-mediated dUTP-fluorescein nick end labeling (TUNEL) and Hoechst dye staining. In contrast, OA and cholesterol had no effect on hOB cell proliferation, even at a high concentration (200 micromol/L). Similar results were observed with regard to MG-63 cell proliferation. In addition, flow cytometric analysis showed that the number of hOB cells in the S phase of the cycle was twofold lower when treated with 50 micromol/L of DHA or AA. In vitro results indicate that mature adipocytes may contribute to age-related bone loss through the release of polyunsaturated fatty acids, which impair osteoblastic proliferation.

High interaction alginate-hyaluronate associations by hyaluronate deacetylation for the preparation of efficient biomaterials.

The paper presents fundamental investigations of alginate-hyaluronate association with significant polymer interactions for preparation of efficient biomaterials. For this purpose, acetamide functions of hyaluronate were partly cleaved by hydrazine at high temperature, yielding amino groups accessible to carboxylic functions of the alginate chain. Alginate-hyaluronate association was studied both in dissolved state by rheological measurements and CD, and in the form of gel slabs prepared after calcium diffusion. Appreciable interaction between carboxylic groups of alginate and the released amino groups of hyaluronate was put into evidence by enhanced values of the viscosity of mixed solutions, and by assessment of the properties of the gel formed: moderate deacetylation allowed gels of improved hardness and viscosity. Nevertheless, high deacetylation was observed to hinder the gel formation by Ca(2+) complexation of alginate, by the significant competition of COOH-NH(2) association. Interaction between alginate and modified hyaluronate results in regular gel structure, with small cavities.

Influence of mature adipocytes on osteoblast proliferation in human primary cocultures.

It has been shown that the bone loss occurring with aging in spongy bone is associated with a reduced osteoblastic bone formation and an increased volume of marrow adipose tissue. This observation suggests a relationship between cells from the osteoblastic and adipogenic lineages. The purpose of the present study was to evaluate the influence of mature adipocytes on osteoblastic proliferation and activity in a model of coculture. Human primary osteoblastic (hBOB) cells were derived from femoral bone explants collected in patients undergoing orthopedic surgery. Human stromal osteoblastic (hMSOB) cells were obtained from bone marrow samples collected by aspiration during orthopedic surgery. Extramedullary and medullary mature adipocytes (hAd) showing similar functions, except for their response to insulin, hAd were isolated from mammary adipose tissue collected in women undergoing tumorectomy. Cells were cocultured, with hAd being separated from osteoblastic cells (hBOB or hMSOB) by a porous membrane (0.4 microm). When hBOB cells were seeded on the upper side of the insert and hAd were floating on the lower side, cell contacts between the two cell types were possible through the pores of the membrane. At the end of the experiment, proliferation of the osteoblastic cells was evaluated by [(3)H]-thymidine incorporation and alkaline phosphatase (AP) activity was measured. After 20 h of coculture, proliferation of the hBOB cells was significantly decreased when compared with control hBOB (-40 +/- 6%, p < 0.05). To establish whether or not the influence of hAd on hBOB proliferation required intercellular communications, hAd and hBOB cells were cocultured far from the porous membrane. Six other independent experiments confirmed an inhibition of hBOB proliferation under both experimental conditions (p < 0.05): -35 +/- 7% with possible intercellular contacts, and -30 +/- 7% without any contact. In contrast, the proliferation of hMSOB cells was not significantly modified after coculture with hAd. In addition, the presence of hAd did not significantly modify the AP activity of hBOB (0.163 +/- 0.143 and 0.181 +/- 0.114 nmol/min per microgram of protein in controls and after coculture, respectively). No reproducible effect of hAd-conditioned medium was noted on hBOB- and hMSOB-cell proliferation or hBOB-cell activity. In conclusion, mature adipocytes induced an inhibition of hBOB-cells proliferation, probably mediated by a factor secreted by hAd. This effect may contribute to the age-related reduction of bone formation and bone loss.