Martine Duterque-Coquillaud

Dynamic Compression of Chondrocyte-Agarose Constructs Reveals New Candidate Mechanosensitive Genes

Articular cartilage is physiologically exposed to repeated loads. The mechanical properties of cartilage are due to its extracellular matrix, and homeostasis is maintained by the sole cell type found in cartilage, the chondrocyte. Although mechanical forces clearly control the functions of articular chondrocytes, the biochemical pathways that mediate cellular responses to mechanical stress have not been fully characterised. The aim of our study was to examine early molecular events triggered by dynamic compression in chondrocytes. We used an experimental system consisting of primary mouse chondrocytes embedded within an agarose hydrogel; embedded cells were pre-cultured for one week and subjected to short-term compression experiments. Using Western blots, we demonstrated that chondrocytes maintain a differentiated phenotype in this model system and reproduce typical chondrocyte-cartilage matrix interactions. We investigated the impact of dynamic compression on the phosphorylation state of signalling molecules and genome-wide gene expression. After 15 min of dynamic compression, we observed transient activation of ERK1/2 and p38 (members of the mitogen-activated protein kinase (MAPK) pathways) and Smad2/3 (members of the canonical transforming growth factor (TGF)-β pathways). A microarray analysis performed on chondrocytes compressed for 30 min revealed that only 20 transcripts were modulated more than 2-fold. A less conservative list of 325 modulated genes included genes related to the MAPK and TGF-β pathways and/or known to be mechanosensitive in other biological contexts. Of these candidate mechanosensitive genes, 85% were down-regulated. Down-regulation may therefore represent a general control mechanism for a rapid response to dynamic compression. Furthermore, modulation of transcripts corresponding to different aspects of cellular physiology was observed, such as non-coding RNAs or primary cilium. This study provides new insight into how chondrocytes respond to mechanical forces.

Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures.

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self‐repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)‐2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long‐term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real‐time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP‐2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP‐2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP‐2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT. J. Cell. Biochem. 111: 1642–1651, 2010.

Targeting the DNA-binding activity of the human ERG transcription factor using new heterocyclic dithiophene diamidines

Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.

Identification of four alternatively spliced transcripts of the Ucma/GRP gene, encoding a new Gla-containing protein.

The Ucma protein (Upper zone of growth plate and cartilage matrix associated protein) has recently been described as a novel secretory protein mainly expressed in cartilage and also as a novel vitamin-K-dependent protein named GRP (Gla-rich protein). This protein has the highest Gla content of any protein known to date. In this article, we identify four alternatively spliced variants of Ucma/GRP gene transcripts in mouse chondrocytes. We show that the expression of all four isoforms is associated with the early stages of chondrogenesis. The Ucma/GRP gene encodes four proteins named Ucma/GRP-F1, -F2, -F3, and -F4, which differ by exon 2, exon 4, or both. Among them, only Ucma/GRP-F1 and -F3 were secreted into the culture medium of transfected chondrocytes, while Ucma/GRP-F2 and -F4 accumulated in the cells. Using HeLa cells or freshly isolated embryonic mouse chondrocytes transfected with enhanced green fluorescent protein fusions, microscopy analysis revealed that Ucma/GRP-F1 and -F3 were localized in the Golgi complex, whereas Ucma/GRP-F2 and -F4 formed aggregates. This aggregation was microtubule-dependent since disruption of microtubules with nocodazole reduced Ucma/GRP-F2 and -F4 aggregation in a reversible manner. Using biochemical fractionation and Western blot analysis, Ucma/GRP-F1 and -F3 isoforms were detected in the soluble fraction while Ucma/GRP-F2 and -F4 were found in an insoluble-enriched fraction. We conclude that the co-expression of soluble and insoluble isoforms also Gla-rich and Gla-deleted isoforms may be finely tuned. Imbalance in isoform expression may therefore be involved in skeletal pathology.

Réponse des chondrocytes humains à la bone morphogenetic protein-2 après leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage

AIM OF THE STUDYnCartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic.nnnMATERIALS AND METHODSnChondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored.nnnRESULTSnOur results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages.nnnCONCLUSIONnThe efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.

N-substituted Piperazinopyridylsteroid Derivatives as Abiraterone Analogues Inhibit Growth and Induce Pro-apoptosis in Human Hormone-independent Prostate Cancer Cell Lines

Nine new 17‐(piperazin‐1‐yl)pyridin‐5‐yl)steroids as abiraterone analogues were synthesized. Compounds 5d and 5g showed selective activities against 17α‐hydroxylase/C17,20‐lyase (CYP17A1) and aromatase (CYP19), respectively. IC50 values of 5d were 5.09 and >50uf020 μm, whereas these values for 5g were >50 uf020μm and 7.40 μm, respectively, for CYP17A1 and CYP19. Molecular modelling highlighted that the inhibitor designed to bind cytochrome P450 haem iron is a necessary condition but not the only rationale to explain inhibitory activity. These abiraterone analogues were then evaluated on hormone‐independent prostate cancer cell lines DU‐145 and PC‐3 and on hormone‐dependent breast and prostate cancer cell lines MCF‐7 and LNCaP, respectively. Compounds 5e, 5g and 5i have showed potent activities only on hormone‐independent prostate cancer cell lines DU‐145 and PC‐3 with 60–85% inhibition of both cell viability and growth at 10 nm with pro‐apoptotic mechanism as illustrated in PC‐3 cells by DNA ladder assay and Western blotting of Bax, Casp‐3 and its substrate, the poly (ADP–ribose) polymerase. We conclude that hybrid heterocycle steroids could be good lead compounds in the drug design especially against hormone‐independent prostate cancer.

Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg) during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.