Inhibition of sphingosine 1-phosphate protects mice against chondrocyte catabolism and osteoarthritis.

OBJECTIVE: Cartilage loss observed in osteoarthritis (OA) is prevented when osteoclasts in the subchondral bone are inhibited in mice. Here, we investigated the role of the osteoclast secretome and of the lipid mediator sphingosine 1-phosphate (S1P) in chondrocyte metabolism and OA. MATERIALS AND METHODS: We used SphK1LysMCre and wild type mice to assess the effect of murine osteoclast secretome in chondrocyte metabolism. Gene and protein expressions of matrix metalloproteinase (Mmp) were quantified in chondrocytes and explants by RT-qPCR and Western blots. SphK1LysMCre mice or wild type mice treated with S1P2 receptor inhibitor JTE013 or anti-S1P neutralizing antibody sphingomab are analyzed by OA score and immunohistochemistry. RESULTS: The osteoclast secretome increased the expression of Mmp3 and Mmp13 in murine chondrocytes and cartilage explants and activated the JNK signaling pathway, which led to matrix degradation. JTE013 reversed the osteoclast-mediated chondrocyte catabolism and protected mice against OA, suggesting that osteoclastic S1P contributes to cartilage damage in OA via S1P/S1P2 signaling. The activity of sphingosine kinase 1 (SphK1) increased with osteoclast differentiation, and its expression was enhanced in subchondral bone of mice with OA. The expression of Mmp3 and Mmp13 in chondrocytes was low upon stimulation with the secretome of Sphk1-lacking osteoclasts. Cartilage damage was significantly reduced in SphK1LysMCre mice, but not the synovial inflammation. Finally, intra-articular administration of sphingomab inhibited the cartilage damage and synovial inflammation. CONCLUSIONS: Lack of S1P in myeloid cells and local S1P neutralization alleviates from osteoarthritis in mice. These data identify S1P as a therapeutic target in OA.

[Advantages and special features of hospital preparations of parenteral nutrition in neonatalogy]. / Avantages et spécificités des préparations hospitalières de nutrition parentérale en néonatalogie.

INTRODUCTION: The care of premature infants requires specific, suitable parenteral nutrition, in which the dosage must be frequently adjusted. METHOD: A comparative analysis of four industrial standard parenteral nutrition formulations NP 100®, Pediaven AP-HP Nouveau-né 1®, Pediaven AP-HP Nouveau-né 2® and Numetah G13% E® and of two hospital preparations made specifically in hospital pharmacies produced by two separate university hospitals (Nutrine® HCL and Formule standardisée début de nutrition) was conducted. The comparison between the formulations focused on electrolytic compositions and protein/energy ratio. RESULTS: Formule standardisée début de nutrition and Pediaven AP-HP Nouveau-né 1® are free from (i) sodium and potassium, (ii) potassium respectively. Almost equivalent sodium concentration (19-27 mM) and more variable potassium concentration (∼9-26 mM) characterize the other formulations. Protein/energy ratio of Numetah G13% E®, Nutrine® HCL and Formule standardisée début de nutrition is 58% higher than that of NP 100®, Pediaven AP-HP Nouveau-né 1® and Pediaven AP-HP Nouveau-né 2®. DISCUSSION: Formule standardisée début de nutrition and Pediaven AP-HP Nouveau-né 1® are in accordance with the recommendations about hydro-electrolytic supplies during transition phase. Nutrine® HCL complies best to the recommendations about hydro-electrolytic account during stabilization phase. CONCLUSION: Hydro-electrolytic composition and protein/energy ratio of standard hospital parenteral nutrition formulations comply best to nutritional needs of premature infants.

The transcriptional cofactor Lbh regulates angiogenesis and endochondral bone formation during fetal bone development.

Lbh is thought to act as a transcriptional cofactor and is highly conserved among species. Here we show that Lbh is expressed in chondrocytes, cells of the perichondrium, and the primary spongiosa in fetal growth plates of mice and chickens. Lbh overexpression in chick wings, using the RCAS-retroviral vector strategy, results in shortened skeletal elements and delayed hypertrophic chondrocyte maturation and bone formation. Additionally, osteoclast and endothelial cell invasion are delayed in the Lbh-overexpressing bones. Finally, we find a dramatic suppression of Runx2 and VEGF mRNAs in chondrocytes and osteoblasts that overexpress Lbh. Strikingly, this abnormal bone development in infected limbs can be rescued by concurrent overexpression of Runx2. These results suggest that during endochondral bone formation, Lbh may negatively regulate vascular invasion and formation of the early ossification center at least in part by interfering with Runx2 and/or VEGF expression.

The implementation of a quality assurance procedure for the Veterinary Services of France.

Due to the increasing complexity of food production systems and the concerns that these systems raise, there has been increasing demand from the general public for more State control of these processes. In France, it is the official Veterinary Services who are responsible for food safety and who must respond to these demands. The Veterinary Service is formulating a quality assurance procedure in accordance with standard EN 45004-ISO 17020, which determines the requirements that inspection bodies must follow to be recognised, at national, European and international level, as competent and reliable. As part of this procedure, the Veterinary Service will review requirements in terms of organisation, functions, qualifications and resources. The progress of inspection service orders, from their conception by the Central Administration, to their implementation by decentralised services, must be carefully managed. It is essential that service orders be implemented effectively and systematically by using recognised methods and issuing adequate inspection reports. The training and qualifications of inspectors are very important: their skills must remain up-to-date so that there is always a network of qualified staff, that is, staff who have an understanding of production processes and who have recognised competences in terms of initial training, continuous professional development and adequate experience. The quality systems implemented will only meet expectations if they are continuously monitored by means of regular evaluations. For this reason, both internal and external audits are performed. These new practices contribute to establishing a basis for the improvement of internal evaluation. In order to facilitate the implementation of a quality assurance procedure for inspection services, several tools, that are linked with the information system of the government department responsible for food, are, or will be, at the disposal of the decentralised Veterinary Services, i.e. a national database, mail and service order processing software, and inspection procedures.

Phosphorylation of MafA is essential for its transcriptional and biological properties.

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation.

Characterization of a novel quiescence responsive element downregulated by v-Src in the promoter of the neuroretina specific QR1 gene.

The neuroretina is a functional unit of the central nervous system which arises through successive steps of division, growth arrest and differentiation of neuroectodermal precursors. Postmitotic quail neuroretina (QNR) cells are conditionally induced to divide upon infection with temperature sensitive mutants of Rous sarcoma virus (RSV), since QNR cell division can be arrested by either inactivating p60v-Src at the nonpermissive temperature (41 degrees C) or by serum deprivation at 37 degrees C. We are studying the transcriptional control of QR1, a neuroretina specific gene, whose expression is down-regulated in proliferating cells at 37 degrees C and is fully restored when these cells are made quiescent. We previously showed that this quiescence specific upregulation implicates a promoter region named A box, which binds Maf transcription factors. We report the identification of the C box, a second promoter sequence that activates QR1 transcription in non dividing cells. This sequence is able to form two DNA-protein complexes, one of which (C4) is predominantly detected in growth arrested NR cells. We identified the DNA binding site for C4 and described mutations that abolish both C4 binding and promoter activity in quiescent cells. Moreover, we show that a multimerized C box is able to stimulate a heterologous promoter in non dividing cells and constitutes, therefore, a novel quiescence responsive enhancer. Finally, we report that QR1 transcriptional response to cell quiescence requires cooperation between the C box and A box.

Induction of postmitotic neuroretina cell proliferation by distinct Ras downstream signaling pathways.

Ras-induced cell transformation is mediated through distinct downstream signaling pathways, including Raf, Ral-GEFs-, and phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. In some cell types, strong activation of the Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) cascade leads to cell cycle arrest rather than cell division. We previously reported that constitutive activation of this pathway induces sustained proliferation of primary cultures of postmitotic chicken neuroretina (NR) cells. We used this model system to investigate the respective contributions of Ras downstream signaling pathways in Ras-induced cell proliferation. Three RasV12 mutants (S35, G37, and C40) which differ by their ability to bind to Ras effectors (Raf, Ral-GEFs, and the p110 subunit of PI 3-kinase, respectively) were able to induce sustained NR cell proliferation, although none of these mutants was reported to transform NIH 3T3 cells. Furthermore, they all repressed the promoter of QR1, a neuroretina growth arrest-specific gene. Overexpression of B-Raf or activated versions of Ras effectors Rlf-CAAX and p110-CAAX also induced NR cell division. The mitogenic effect of the RasC40-PI 3-kinase pathway appears to involve Rac and RhoA GTPases but not the antiapoptotic Akt (protein kinase B) signaling. Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. Activation of either Ras downstream pathway resulted in ERK activation, and coexpression of a dominant negative MEK mutant or mKsr-1 kinase domain strongly inhibited proliferation induced by the three Ras mutants or by their effectors. Similar effects were observed with dominant negative mutants of Rac and Rho. Thus, both the Raf-MEK-ERK and Rac-Rho pathways are absolutely required for Ras-induced NR cell division. Activation of these two pathways by the three distinct Ras downstream effectors possibly relies on an autocrine or paracrine loop, implicating endogenous Ras, since the mitogenic effect of each Ras effector mutant was inhibited by RasN17.

mafA, a novel member of the maf proto-oncogene family, displays developmental regulation and mitogenic capacity in avian neuroretina cells.

Transcription factors of the Maf proto-oncogene family have been shown to participate in the regulation of several differentiation specific genes. We previously reported that a member(s) of this family is involved in the regulation of the neuroretina specific gene, QR1, through a promoter region, designated the A box, that is closely related to the Maf recognition element (MARE). We undertook an identification of Maf family genes expressed in the quail neuroretina (QNR) and we report the isolation of mafA, a gene encoding a novel member of the large Maf proteins subgroup. Expression of this gene is developmentally regulated in the neuroretina. MafA is able to bind to MARE sequence and to heterodimerize with v-Maf, MafB, Jun and Fos, but not with the small MafF and MafK proteins. Accordingly, it is able to transactivate the QR1 promoter A box. We also show that increased expression of mafA induces sustained proliferation of postmitotic QNR cells.

Characterization of a leucine zipper-containing protein identified by retroviral insertion in avian neuroretina cells.

We reported previously that post-mitotic chicken embryonic neuroretina (NR) cells are induced to proliferate following in vitro infection with RAV-1, a retrovirus that does not carry an oncogene. NR cell multiplication results from the frequent activation and subsequent retroviral transduction of two related serine/threonine protein kinases, the c-mil/c-raf or c-Rmil/B-raf genes. We also showed that a very early event in the activation of these proto-oncogenes is the synthesis of chimeric mRNAs containing viral and cellular sequences joined by a splicing mechanism. In the current study, we have examined the ability of RAV-1 to induce proliferation of quail NR cells. By using the reverse transcription-polymerase chain reaction technique, we identified, in several proliferating quail NR cultures infected with RAV-1, a chimeric mRNA containing cellular sequences joined to the RAV-1 splice donor site. These cellular sequences are derived from a gene designated R10, which is expressed through a 1.9-kilobase (kb) mRNA detected in several embryonic tissues. A second transcript of 2.3 kb is specifically expressed in the NR, where both transcripts are developmentally regulated. The R10 cDNA encodes a 251-amino acid polypeptide that contains a leucine zipper motif. It exhibits significant similarity with the putative D52/N8L protein, encoded by an mRNA reported previously to be overexpressed in human breast and lung carcinomas. By using polyclonal antibodies specific for its amino-terminal and leucine zipper-containing regions, we identified the R10 gene product as a cytoplasmic protein of 23 kDa in cultured avian fibroblasts. A second protein of 30 kDa is detected in post-mitotic NR cells that express the 2.3-kb transcript. We also show, by in vitro transcription/translation and immunoprecipitation, that the R10 protein can readily form homodimers, presumably through its leucine zipper motif.