Presence of type IIB procollagen in mouse articular cartilage and growth plate is revealed by immuno-histochemical analysis with a novel specific antibody.

Type II collagen is the major fibrillar collagen in cartilage. It is synthesized in the form of precursors (procollagens) containing N- and C-terminal propeptides. The two main isoforms of type II procollagen protein are type IIA and type IIB procollagens, generated in a developmentally regulated manner by differential splicing of the primary gene transcript. Isoform IIA contains exon 2 and is produced mainly by chondroprogenitor cells while isoform IIB lacks exon 2 and is produced by differentiated chondrocytes. Thus, expression of IIA and IIB isoforms are reliable markers for identifying the differentiation status of chondrocytes but their biological function in the context of skeletal development is still not yet fully understood. Specific antibodies against IIA and IIB procollagen isoforms are already available. In this study, a synthetic peptide spanning the junction between exon 1 and exon 3 of the murine sequence was used as an immunogen to generate a novel rabbit polyclonal antibody directed against procollagen IIB. Characterization of this antibody by Western-blotting analysis of murine cartilage extracts and ELISA tests demonstrated its specificity to the type IIB isoform. Furthermore, by immunohistochemical studies, this antibody allowed the detection of procollagen IIB in embryonic cartilage as well as in articular cartilage and growth plate of young adult mice. Interestingly, this is the first antibody that has allowed the detection of procollagen IIB at both the intra- and extracellular level. This antibody therefore represents an interesting new tool for monitoring the spatial and temporal distribution of IIB isoforms in skeletal tissues of mouse models and for tracking the trafficking and processing of type IIB procollagen.

The epigenetic players and the chromatin marks involved in the articular cartilage during osteoarthritis.

Epigenetics defines the modifications of the genome that do not involve a change in the nucleotide sequence of DNA. These modifications constitute a mechanism of gene regulation poorly explored in the context of cartilage physiology. They are now intensively studied by the scientific community working on articular cartilage and its related pathology such as osteoarthritis. Indeed, epigenetic regulations can control the expression of crucial gene in the chondrocytes, the only resident cells of cartilage. Some epigenetic changes are considered as a possible cause of the abnormal gene expression and the subsequent alteration of the chondrocyte phenotype (hypertrophy, proliferation, senescence…) as observed in osteoarthritic cartilage. Osteoarthritis is a joint pathology, which results in impaired extracellular matrix homeostasis and leads ultimately to the progressive destruction of cartilage. To date, there is no pharmacological treatment and the exact causes have yet to be defined. Given that the epigenetic modifying enzymes can be controlled by pharmacological inhibitors, it is thus crucial to describe the epigenetic marks that enable the normal expression of extracellular matrix encoding genes, and those associated with the abnormal gene expression such as degradative enzyme or inflammatory cytokines encoding genes. In this review, only the DNA methylation and histone modifications will be detailed with regard to normal and osteoarthritic cartilage. Although frequently referred as epigenetic mechanisms, the regulatory mechanisms involving microRNAs will not be discussed. Altogether, this review will show how this nascent field influences our understanding of the pathogenesis of OA in terms of diagnosis and how controlling the epigenetic marks can help defining epigenetic therapies.

Tumor Suppressive Role of miR-342-5p in Human Chondrosarcoma Cells and 3D Organoids.

Chondrosarcomas are malignant bone tumors. Their abundant cartilage-like extracellular matrix and their hypoxic microenvironment contribute to their resistance to chemotherapy and radiotherapy, and no effective therapy is currently available. MicroRNAs (miRNAs) may be an interesting alternative in the development of therapeutic options. Here, for the first time in chondrosarcoma cells, we carried out high-throughput functional screening using impedancemetry, and identified five miRNAs with potential antiproliferative or chemosensitive effects on SW1353 chondrosarcoma cells. The cytotoxic effects of miR-342-5p and miR-491-5p were confirmed on three chondrosarcoma cell lines, using functional validation under normoxia and hypoxia. Both miRNAs induced apoptosis and miR-342-5p also induced autophagy. Western blots and luciferase reporter assays identified for the first time Bcl-2 as a direct target of miR-342-5p, and also Bcl-xL as a direct target of both miR-342-5p and miR-491-5p in chondrosarcoma cells. MiR-491-5p also inhibited EGFR expression. Finally, only miR-342-5p induced cell death on a relevant 3D chondrosarcoma organoid model under hypoxia that mimics the in vivo microenvironment. Altogether, our results revealed the tumor suppressive activity of miR-342-5p, and to a lesser extent of miR-491-5p, on chondrosarcoma lines. Through this study, we also confirmed the potential of Bcl-2 family members as therapeutic targets in chondrosarcomas.

Marine Collagen Hydrolysates Promote Collagen Synthesis, Viability and Proliferation While Downregulating the Synthesis of Pro-Catabolic Markers in Human Articular Chondrocytes.

Cartilage is a non-innervated and non-vascularized tissue. It is composed of one main cell type, the chondrocyte, which governs homeostasis within the cartilage tissue, but has low metabolic activity. Articular cartilage undergoes substantial stresses that lead to chondral defects, and inevitably osteoarthritis (OA) due to the low intrinsic repair capacity of cartilage. OA remains an incurable degenerative disease. In this context, several dietary supplements have shown promising results, notably in the relief of OA symptoms. In this study, we investigated the effects of collagen hydrolysates derived from fish skin (Promerim®30 and Promerim®60) and fish cartilage (Promerim®40) on the phenotype and metabolism of human articular chondrocytes (HACs). First, we demonstrated the safety of Promerim® hydrolysates on HACs cultured in monolayers. Then we showed that, Promerim® hydrolysates can increase the HAC viability and proliferation, while decreasing HAC SA-ß-galactosidase activity. To evaluate the effect of Promerim® on a more relevant model of culture, HAC were cultured as organoids in the presence of Promerim® hydrolysates with or without IL-1ß to mimic an OA environment. In such conditions, Promerim® hydrolysates led to a decrease in the transcript levels of some proteases that play a major role in the development of OA, such as Htra1 and metalloproteinase-1. Promerim® hydrolysates downregulated HtrA1 protein expression. In contrast, the treatment of cartilage organoids with Promerim® hydrolysates increased the neosynthesis of type I collagen (Promerim®30, 40 and 60) and type II collagen isoforms (Promerim®30 and 40), the latter being the major characteristic component of the cartilage extracellular matrix. Altogether, our results demonstrate that the use of Promerim® hydrolysates hold promise as complementary dietary supplements in combination with the current classical treatments or as a preventive therapy to delay the occurrence of OA in humans.

Repair of full-thickness articular cartilage defects using IEIK13 self-assembling peptide hydrogel in a non-human primate model.

Articular cartilage is built by chondrocytes which become less active with age. This declining function of the chondrocytes, together with the avascular nature of the cartilage, impedes the spontaneous healing of chondral injuries. These lesions can progress to more serious degenerative articular conditions as in the case of osteoarthritis. As no efficient cure for cartilage lesions exist yet, cartilage tissue engineering has emerged as a promising method aiming at repairing joint defects and restoring articular function. In the present work, we investigated if a new self-assembling peptide (referred as IEIK13), combined with articular chondrocytes treated with a chondrogenic cocktail (BMP-2, insulin and T3, designated BIT) could be efficient to restore full-thickness cartilage defects induced in the femoral condyles of a non-human primate model, the cynomolgus monkey. First, in vitro molecular studies indicated that IEIK13 was efficient to support production of cartilage by monkey articular chondrocytes treated with BIT. In vivo, cartilage implant integration was monitored non-invasively by contrast-enhanced micro-computed tomography, and then by post-mortem histological analysis and immunohistochemical staining of the condyles collected 3 months post-implantation. Our results revealed that the full-thickness cartilage injuries treated with either IEIK13 implants loaded with or devoid of chondrocytes showed similar cartilage-characteristic regeneration. This pilot study demonstrates that IEIK13 can be used as a valuable scaffold to support the in vitro activity of articular chondrocytes and the repair of articular cartilage defects, when implanted alone or with chondrocytes.

The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes.

Osteoarthritis (OA) is a degenerative disease of the joints which is associated with an impaired production of the cartilage matrix by the chondrocytes. Here, we investigated the role of Lysine-Specific Demethylase-1 (LSD1), a chromatin remodeling enzyme whose role in articular chondrocytes was previously associated with a catabolic activity and which is potentially involved during OA. Following a loss of function strategy and RNA sequencing analysis, we detail the genes which are targeted by LSD1 in human articular chondrocytes and identify COL9A1, a gene encoding the α1 chain of the cartilage-specific type IX collagen, as negatively regulated by LSD1. We show that LSD1 interacts with the transcription factor SOX9 and is recruited to the promoter of COL9A1. Interestingly, we observe that OA cartilage displays stronger LSD1 immunostaining compared with normal, and we demonstrate that the depletion of LSD1 in OA chondrocytes prevents the decrease in COL9A1 following Il-1ß treatment. These results suggest LSD1 is a new regulator of the anabolic activity of articular chondrocytes potentially destabilizing the cartilage matrix, since it negatively regulates COL9A1, a gene encoding a crucial anchoring collagen molecule. This newly identified role played by LSD1 may thus participate in the alteration of the cartilage matrix during OA.

Hypoxia induces copper stable isotope fractionation in hepatocellular carcinoma, in a HIF-independent manner.

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer, with increasing incidence worldwide. The unrestrained proliferation of tumour cells leads to tumour hypoxia which in turn promotes cancer aggressiveness. While changes in the concentration of copper (Cu) have long been observed upon cancerization, we have recently reported that the isotopic composition of copper is also altered in several types of cancer. In particular, we showed that in hepatocellular carcinoma, tumour tissue contains heavier copper compared to the surrounding parenchyma. However, the reasons behind such isotopic signature remained elusive. Here we show that hypoxia causes heavy copper enrichment in several human cell lines. We also demonstrate that this effect of hypoxia is pH, HIF-1 and -2 independent. Our data identify a previously unrecognized cellular process associated with hypoxia, and suggests that in vivo tumour hypoxia determines copper isotope fractionation in HCC and other solid cancers.

Cartilage tissue engineering: molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction.

BACKGROUND: Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes. SCOPE OF REVIEW: This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions. MAJOR CONCLUSIONS: Current research involves the use of chondrocytes or progenitor stem cells, associated with "smart" biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process. GENERAL SIGNIFICANCE: This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Hypoxia promotes the production and inhibits the destruction of human articular cartilage.

OBJECTIVE: To determine the effects of hypoxia on both anabolic and catabolic pathways of metabolism in human articular cartilage and to elucidate the roles played by hypoxia-inducible factors (HIFs) in these responses. METHODS: Normal human articular cartilage from a range of donors was obtained at the time of above-the-knee amputations due to sarcomas not involving the joint space. Fresh cartilage tissue explants and isolated cells were subjected to hypoxia and treatment with interleukin-1α. Cell transfections were performed on isolated human chondrocytes. RESULTS: Using chromatin immunoprecipitation, we found that hypoxia induced cartilage production in human tissue explants through direct binding of HIF-2α to a specific site in the master-regulator gene SOX9. Importantly, hypoxia also suppressed spontaneous and induced destruction of human cartilage in explant culture. We found that anticatabolic responses were predominantly mediated by HIF-1α. Manipulation of the hypoxia-sensing pathway through depletion of HIF-targeting prolyl hydroxylase-containing protein 2 (PHD-2) further enhanced cartilage responses as compared to hypoxia alone. Hypoxic regulation of tissue-specific metabolism similar to that in human cartilage was observed in pig, but not mouse, cartilage. CONCLUSION: We found that resident chondrocytes in human cartilage are exquisitely adapted to hypoxia and use it to regulate tissue-specific metabolism. Our data revealed that while fundamental regulators, such as SOX9, are key molecules both in mice and humans, the way in which they are controlled can differ. This is all the more important since it is upstream regulators such as this that need to be directly targeted for therapeutic benefit. HIF-specific hydroxylase PHD-2 may represent a relevant target for cartilage repair.

Cartilage-characteristic matrix reconstruction by sequential addition of soluble factors during expansion of human articular chondrocytes and their cultivation in collagen sponges.

OBJECTIVE: Articular cartilage has a poor capacity for spontaneous repair. Tissue engineering approaches using biomaterials and chondrocytes offer hope for treatments. Our goal was to test whether collagen sponges could be used as scaffolds for reconstruction of cartilage with human articular chondrocytes. We investigated the effects on the nature and abundance of cartilage matrix produced of sequential addition of chosen soluble factors during cell amplification on plastic and cultivation in collagen scaffolds. DESIGN: Isolated human articular chondrocytes were amplified for two passages with or without a cocktail of fibroblast growth factor (FGF)-2 and insulin (FI). The cells were then cultured in collagen sponges with or without a cocktail of bone morphogenetic protein (BMP)-2, insulin, and triiodothyronine (BIT). The constructs were cultivated for 36 days in vitro or for another 6-week period in a nude mouse-based contained-defect organ culture model. Gene expression was analyzed using polymerase chain reaction, and protein production was analyzed using Western-blotting and immunohistochemistry. RESULTS: Dedifferentiation of chondrocytes occurred during cell expansion on plastic, and FI stimulated this dedifferentiation. We found that addition of BIT could trigger chondrocyte redifferentiation and cartilage-characteristic matrix production in the collagen sponges. The presence of FI during cell expansion increased the chondrocyte responsiveness to BIT.

Type II collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes.

miRNAs have been shown to be essential for normal cartilage development in the mouse. However, the role of specific miRNAs in cartilage function is unknown. Using rarely available healthy human chondrocytes (obtained from 8 to 50 year old patients), we detected a most highly abundant primary miRNA H19, whose expression was heavily dependent on cartilage master regulator SOX9. Across a range of murine tissues, expression of both H19- and H19-derived miR-675 mirrored that of cartilage-specific SOX9. miR-675 was shown to up-regulate the essential cartilage matrix component COL2A1, and overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted cells. We thus provide evidence that SOX9 positively regulates COL2A1 in human articular chondrocytes via a previously unreported miR-675-dependent mechanism. This represents a novel pathway regulating cartilage matrix production and identifies miR-675 as a promising new target for cartilage repair.

Lack of oxygen in articular cartilage: consequences for chondrocyte biology.

Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia-mediated chondrocyte function in the native articular cartilage.

Hypoxia. HIF-mediated articular chondrocyte function: prospects for cartilage repair.

In a chronically hypoxic tissue such as cartilage, adaptations to hypoxia do not merely include cell survival responses, but also promotion of its specific function. This review will focus on describing such hypoxia-mediated chondrocyte function, in particular in the permanent articular cartilage. The molecular details of how chondrocytes sense and respond to hypoxia and how this promotes matrix synthesis have recently been examined, and specific manipulation of hypoxia-induced pathways is now considered to have potential therapeutic application to maintenance and repair of articular cartilage.

Hypoxia promotes the differentiated human articular chondrocyte phenotype through SOX9-dependent and -independent pathways.

The chondrocyte is solely responsible for synthesis and maintenance of the resilient articular cartilage matrix that gives this load-bearing tissue its mechanical integrity. When the differentiated cell phenotype is lost, the matrix becomes compromised and cartilage function begins to fail. We have recently shown that hypoxia promotes the differentiated phenotype through hypoxia-inducible factor 2alpha (HIF-2alpha)-mediated SOX9 induction of the main matrix genes. However, to date, only a few genes have been shown to be SOX9 targets, while little is known about SOX9-independent regulators. We therefore performed a detailed microarray study to address these issues. Analysis involved 35 arrays on chondrocytes obtained from seven healthy, non-elderly human cartilage samples. Genes were selected that were down-regulated with serial passage in culture (as this causes loss of the differentiated phenotype) and subsequently up-regulated in hypoxia. The importance of key findings was further probed using the technique of RNA interference on these human articular chondrocytes. Our results show that hypoxia has a broader beneficial effect on the chondrocyte phenotype than has been previously described. Of special note, we report new hypoxia-inducible and SOX9-regulated genes, Gdf10 and Chm-I. In addition, Mig6 and InhbA were induced by hypoxia, predominantly via HIF-2alpha, but were not regulated by SOX9. Therefore, hypoxia, and more specifically HIF-2alpha, promotes both SOX9-dependent and -independent factors important for cartilage homeostasis. HIF-2alpha may therefore represent a new and promising therapeutic target for cartilage repair.

Hypoxia-inducible factor 2alpha is essential for hypoxic induction of the human articular chondrocyte phenotype.

OBJECTIVE: To uncover the mechanism by which hypoxia enhances cartilage matrix synthesis by human articular chondrocytes. METHODS: The hypoxic response was investigated by exposing normal (nonarthritic) human articular chondrocyte cultures to 20% oxygen and 1% oxygen. Induction of the differentiated phenotype was confirmed at the gene and protein levels. In its first reported application in human articular chondrocytes, the RNA interference method was used to directly investigate the role of specific transcription factors in this process. Small interfering RNA directed against hypoxia-inducible factor 1alpha (HIF-1alpha), HIF-2alpha, and SOX9 were delivered by lipid-based transfection of primary and passaged human articular chondrocytes. The effect of each knockdown on hypoxic induction of the chondrocyte phenotype was assessed. RESULTS: Hypoxia enhanced matrix synthesis and SOX9 expression of human articular chondrocytes at both the gene and protein levels. Although HIF-1alpha knockdown had no effect, depletion of HIF-2alpha abolished this hypoxic induction. Thus, we provide the first evidence that HIF-2alpha, but not HIF-1alpha, is essential for hypoxic induction of the human articular chondrocyte phenotype. In addition, depletion of SOX9 prevented hypoxic induction of matrix genes, indicating that the latter are not direct HIF targets but are up-regulated by hypoxia via SOX9. CONCLUSION: Based on our data, we propose a novel mechanism whereby hypoxia promotes cartilage matrix synthesis specifically through HIF-2alpha-mediated SOX9 induction of key cartilage genes. These findings have potential application for the development of cartilage repair therapies.

NOV/CCN3 impairs muscle cell commitment and differentiation.

NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

NOV/CCN3 induces adhesion of muscle skeletal cells and cooperates with FGF2 and IGF-1 to promote proliferation and survival.

During mammalian development, expression of the Nephroblastoma overexpressed gene (NOV/CCN3) is tightly regulated in skeletal muscles. Ex vivo, ectopic expression of NOV blocks myogenic differentiation. NOV also supports endothelial cell adhesion and angiogenesis through interactions with integrins. Integrins play fundamental roles during myogenesis. In this study, we show that NOV mediates adhesion and spreading of myoblasts. Myoblasts adhesion to NOV does not require proteoglycans and is dependent on integrin beta1, whereas spreading involves another RGD-sensitive integrin. The C-Terminal part of NOV as well as full-length is able to support adhesion of myoblasts; in addition, both increase focal-adhesion kinase (FAK) phosphorylation. Furthermore, NOV is an adhesive substrate that, combined with FGF2 or IGF-1, promotes cell specific proliferation and survival, respectively, in a better way than fibronectin. Taken together, these results identify NOV as an adhesion substrate for myoblasts which, in concert with growth factors, could play a role in the physiology of muscle cells.

New target genes for NOV/CCN3 in chondrocytes: TGF-beta2 and type X collagen.

UNLABELLED: We studied the involvement of NOV/CCN3, whose function is poorly understood, in chondrocyte differentiation. NOV was found to upregulate TGF-beta2 and type X collagen and to act as a downstream effector of TGF-beta1 in ATDC5 and primary chondrocytes. Thus, NOV is a positive modulator of chondrogenesis. INTRODUCTION: NOV/CCN3 is a matricellular protein that belongs to the CCN family. A growing body of evidence indicates that NOV could play a role in cell differentiation, particularly in chondrogenesis. During chick embryo development, NOV expression is tightly regulated in cartilage, and a high expression of NOV has been associated with cartilage differentiation in Wilms' tumors. However, a precise role for NOV and potential target genes of NOV in chondrogenesis are unknown. MATERIALS AND METHODS: ATDC5 cells and primary chondrocytes were either treated with NOV recombinant protein or transfected with a NOV-specific siRNA to determine, using quantitative RT-PCR, the effect of NOV on the expression of several molecules involved in chondrocyte differentiation. Stable ATDC5 clones expressing NOV were also established to show that NOV was a downstream effector of TGF-beta1. RESULTS: We established that NOV/CCN3 expression increases in ATDC5 cells at early stages of chondrogenic differentiation and precedes the appearance of TGF-beta2 and of several chondrocytic markers such as SOX9 or type X collagen. When exogenously administered, NOV recombinant protein up-regulates TGF-beta2 and type X collagen mRNA levels both in ATDC5 cells and in primary mouse chondrocytes but does not influence SOX9 expression. This regulation also occurs at the endogenous level because downregulation of NOV expression is correlated with an inhibition of TGF-beta2 and type X collagen in primary chondrocytes. Furthermore, we found that NOV expression is downregulated when chondrocytes are exposed to TGF-beta1-dedifferentiating treatment in chondrocytes, further providing evidence that NOV may counteract TGF-beta1 effects on chondrocytes. CONCLUSIONS: This study provides the first characterization of two new targets of NOV involved in chondrocyte differentiation, shows that NOV acts with TGF-beta1 in a cascade of gene regulation, and indicates that NOV is a positive modulator of chondrogenesis.

The expression of novH in adrenocortical cells is down-regulated by TGFbeta 1 through c-Jun in a Smad-independent manner.

The human NOV secreted glycoprotein (NOVH) is abundant in the fetal and adult adrenal cortex. The amount of NOVH increases in benign adrenocortical tumors and decreases in malignant adrenocortical tumors, suggesting that NOVH plays a role in tumorigenesis in the adrenal cortex. Transforming growth factor beta1 (TGFbeta1), fibroblast growth factor 2 (FGF2), and insulin growth factors (IGFs) play crucial roles in the physiology of the adrenal cortex. We investigated the effects of these factors on the expression of novH in the NCI H295R adrenocortical cell line. The amounts of NOVH protein and novH transcripts were down-regulated by TGFbeta1 and up-regulated by FGF2, whereas IGFs had no effect. Furthermore, the TGFbeta1-dependent inhibition of novH promoter activity was completely abrogated following site-directed mutation of two activating protein (AP-1) sequences (positions -473 and -447), whereas the stimulatory effect of FGF2 was not affected. Co-transfection with dominant negative forms of c-Jun and MEKK1 also abrogated novH-targeted regulation by TGFbeta1, whereas the overproduction of Smad proteins or dominant negative forms of Smad had no effect. Taken together, these results suggest that c-Jun and MEKK1 signaling but not Smad signaling are involved in the TGFbeta1-dependent decrease in NOVH in NCI H295R cells. In conclusion, our data provide evidence that novH is a new target of TGFbeta1; unlike other members of the CCN (cyr61, ctgf, nov) family, however, its expression is repressed rather than induced.