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Identification of new adipokines that potentially link obesity to insulin resistance represents a major challenge. We recently showed that NOV/CCN3, a multifunctional matricellular protein, is synthesized and secreted by adipose tissue, with plasma levels highly correlated with BMI. NOV involvement in tissue repair, fibrotic and inflammatory diseases, and cancer has been previously reported. However, its role in energy homeostasis remains unknown. We investigated the metabolic phenotype of NOV(-/-) mice fed a standard or high-fat diet (HFD). Strikingly, the weight of NOV(-/-) mice was markedly lower than that of wild-type mice but only on an HFD. This was related to a significant decrease in fat mass associated with an increased proportion of smaller adipocytes and to a higher expression of genes involved in energy expenditure. NOV(-/-) mice fed an HFD displayed improved glucose tolerance and insulin sensitivity. Interestingly, the absence of NOV was associated with a change in macrophages profile (M1-like to M2-like), in a marked decrease in adipose tissue expression of several proinflammatory cytokines and chemokines, and in enhanced insulin signaling. Conversely, NOV treatment of adipocytes increased chemokine expression. Altogether, these results show that NOV is a new adipocytokine that could be involved in obesity-associated insulin-resistance., (© 2016 by the American Diabetes Association.)

The main hallmark of chronic kidney disease (CKD) is excessive inflammation leading to interstitial tissue fibrosis. It has been recently reported that NOV/CCN3 could be involved in kidney damage but its role in the progression of nephropathies is poorly known. NOV/CCN3 is a secreted multifunctional protein belonging to the CCN family involved in different physiological and pathological processes such as angiogenesis, inflammation and cancers. The purpose of our study was to determine the role of NOV/CCN3 in renal inflammation and fibrosis related to primitive tubulointerstitial injury. After unilateral ureteral obstruction (UUO), renal histology and real-time PCR were performed in NOV/CCN3-/- and wild type mice. NOV/CCN3 mRNA expression was increased in the obstructed kidneys in the early stages of the obstructive nephropathy. Interestingly, plasmatic levels of NOV/CCN3 were strongly induced after 7 days of UUO and the injection of recombinant NOV/CCN3 protein in healthy mice significantly increased CCL2 mRNA levels. Furthermore, after 7 days of UUO NOV/CCN3-/- mice displayed reduced proinflammatory cytokines and adhesion markers expression leading to restricted accumulation of interstitial monocytes, in comparison with their wild type littermates. Consequently, in NOV/CCN3-/- mice interstitial renal fibrosis was blunted after 15 days of UUO. In agreement with our experimental data, NOV/CCN3 expression was highly increased in biopsies of patients with tubulointerstitial nephritis. Thus, the inhibition of NOV/CCN3 may represent a novel target for the progression of renal diseases.

Objective: Evidence points to a founder of the multifunctional CCN family, NOV/CCN3, as a circulating molecule involved in cardiac development, vascular homeostasis and inflammation. No data are available on the relationship between plasma NOV/CCN3 levels and cardiovascular risk factors in humans. This study investigated the possible relationship between plasma NOV levels and cardiovascular risk factors in humans., Methods: NOV levels were measured in the plasma from 594 adults with a hyperlipidemia history and/or with lipid-lowering therapy and/or a body mass index (BMI) >30 kg/m(2). Correlations were measured between NOV plasma levels and various parameters, including BMI, fat mass, and plasma triglycerides, cholesterol, glucose, and C-reactive protein. NOV expression was also evaluated in adipose tissue from obese patients and rodents and in primary cultures of adipocytes and macrophages., Results: After full multivariate adjustment, we detected a strong positive correlation between plasma NOV and BMI (r = 0.36 p<0.0001) and fat mass (r = 0.33 p<0.0005). According to quintiles, this relationship appeared to be linear. NOV levels were also positively correlated with C-reactive protein but not with total cholesterol, LDL-C or blood glucose. In patients with drastic weight loss induced by Roux-en-Y bariatric surgery, circulating NOV levels decreased by 28% (p<0.02) and 48% (p<0.0001) after 3 and 6 months, respectively, following surgery. In adipose tissue from obese patients, and in human primary cultures NOV protein was detected in adipocytes and macrophages. In mice fed a high fat diet NOV plasma levels and its expression in adipose tissue were also significantly increased compared to controls fed a standard diet., Conclusion: Our results strongly suggest that in obese humans and mice plasma NOV levels positively correlated with NOV expression in adipose tissue, and support a possible contribution of NOV to obesity-related inflammation.

NOV/CCN3 is one of the founding members of the CCN (Cyr61 CTGF NOV) family. In the avian retina, CCN3 expression is mostly located within the central region of the inner nuclear layer. As retinal development progresses and this retinal layer differentiates and matures, CCN3 expression forms a dorsal-ventral and a central-peripheral gradient. CCN3 is produced by two glial cell types, peripapillary cells and Müller cells, as well as by horizontal, amacrine, and bipolar interneurons. In retinal neurons and Müller cell cultures, CCN3 expression is induced by activated BMP signaling, whereas Notch signaling decreases CCN3 mRNA and protein levels in Müller cells and has no effect in retinal neurons. In Müller cells, the CCN3 expression detected may thus result from a balance between the Notch and BMP signaling pathways., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Sustained neuroinflammation strongly contributes to the pathogenesis of pain. The clinical challenge of chronic pain relief led to the identification of molecules such as cytokines, chemokines and more recently matrix metalloproteinases (MMPs) as putative therapeutic targets. Evidence points to a founder member of the matricial CCN family, NOV/CCN3, as a modulator of these inflammatory mediators. We thus investigated the possible involvement of NOV in a preclinical model of persistent inflammatory pain., Methods: We used the complete Freund's adjuvant (CFA)-induced model of persistent inflammatory pain and cultured primary sensory neurons for in vitro experiments. The mRNA expression of NOV and pro-inflammatory factors were measured with real-time quantitative PCR, CCL2 protein expression was assessed using ELISA, MMP-2 and -9 activities using zymography. The effect of drugs on tactile allodynia was evaluated by the von Frey test., Results: NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1β- and TNF-α-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-α-induced MMP-9 expression through β1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia., Conclusions: This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.

Increasing evidence suggests that CCN matricellular proteins play important roles in inflammation. One of the major cell types that handle inflammation in the brain is the astrocyte, which, upon activation, dramatically increases its production of cytokines and chemokines. Here, we report that NOV/CCN3, added to primary cultured rat brain astrocytes, markedly increased the expression of CCL2 and CXCL1 chemokines, as indicated by ELISA and RT-qPCR assays. This effect was selective, as the production of thirteen other cytokines and chemokines was not affected by NOV. NOV expression by astrocytes was demonstrated by immunocytochemistry and Western blot analysis, and astrocyte transfection with NOV small interfering RNA (siRNA) markedly decreased CXCL1 and CCL2 production, indicating that endogenous NOV played a major role in the control of astrocytic chemokine synthesis. NOV was shown to mediate several of its actions through integrins. Here, we observed that siRNAs against integrins beta1 and beta5 decreased basal and abrogated NOV-stimulated astrocyte expression of CCL2 and CXCL1, respectively. Using a panel of kinase inhibitors, we demonstrated that NOV action on CCL2 and CXCL1 production involved a Rho/ROCK/JNK/NF-kappaB and a Rho/qROCK/p38/NF-kappaB pathway, respectively. Thus, distinct integrins and signaling mechanisms are involved in NOV-induced production of CCL2 and CXCL1 in astrocytes. Finally, astrocytic expression of NOV was detected in rat brain tissue sections, and NOV intracerebral injection increased CCL2 and CXCL1 brain levels in vivo. Altogether, our data shed light on the signaling pathways operated by NOV and strongly suggest that NOV mediates astrocyte activation and, therefore, might play a role in neuroinflammation., ((c) 2010 Wiley-Liss, Inc.)

Context: Childhood adrenocortical tumors (ACTs) have a fetal adrenal phenotype and overexpress steroidogenic factor-1 (SF-1). Nephroblastoma overexpressed (NOV)/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3 mRNA is significantly down-regulated in childhood ACTs., Objective: The objective of the study was to measure NOV protein levels in childhood ACTs and characterize NOV expression regulation and biological function in human adrenocortical cells., Design and Setting: Protein extracts from ACT and normal adrenal cortex samples, human adrenocortical carcinoma H295R, primary adrenocortical tumors and fetal adrenal cultures, tissue culture supernatants, and cell lysates from H295R cells overexpressing SF-1 in an inducible fashion were used., Main Outcome Measures: NOV protein levels were measured by enzyme-linked immunoassay and immunoblot. Transient transfection assays were used to study the activity of NOV promoter. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling, caspase assays, and flow cytometry were used to assess the proapoptotic activity of NOV on cells in culture., Results: NOV mRNA and protein expression is lower in childhood ACTs than in normal adrenal cortex. No significant difference was observed between adenomas and carcinomas. SF-1 overexpression down-regulates NOV at the transcriptional level. NOV has a selective proapoptotic activity toward human adrenocortical cells. The C-terminal domain of NOV is responsible for its proapoptotic effect. NOV protein is expressed in DAX-1-positive human fetal adrenal cells., Conclusions: NOV is a selective proapoptotic factor for human adrenocortical cells. Reduced expression of NOV in ACTs may play an important role in the process of childhood ACT tumorigenesis, accounting at least in part for the defect of apoptotic regression of the fetal adrenal that has been proposed to be responsible for tumor formation.

Remodeling of extracellular matrix (ECM) is an important physiological feature of normal growth and development. Recent studies have emphasized the role of matrix metalloproteinases (MMP-2 and MMP-9) in normal mouse nephrogenesis. We have demonstrated previously in the rat that in utero exposure to maternal diabetes impairs renal development leading to a 30% reduction in the nephron number. Transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) are known to mediate high glucose effects on matrix degradation. The aim of the present study was to address the expression of type IV collagenase and TGF-beta1/CTGF systems in rat kidney during normal development and after in utero exposure to maternal diabetes. Both MMP-2 and MMP-9 mRNA metanephric expressions and activities were dramatically downregulated in kidneys issued from diabetic fetuses and in metanephros cultured in the presence of high glucose concentration. TGF-beta1 and CTGF expressions were significantly enhanced in diabetic fetal kidneys and in high glucose cultured metanephroi. Conditioned media obtained from metanephroi grown with high glucose concentration upregulated functional TGF-beta activity in transfected ATDC5 cells. In conclusion, in impaired nephrogenesis resulting from in utero exposure to maternal diabetes, alteration of both type IV collagenase and TGF-beta1/CTGF systems may lead to abnormal remodeling of ECM, which may, in turn, induce defects in ureteral bud branching leading to the observed reduction in the nephron number with consequences later in life: progression of chronic renal disease and hypertension.

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.

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.

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.

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.

NOVH belongs to the CCN (CTGF/CYR61/NOV) family of proteins, some of which have chemotactic, mitogenic, adhesive, and angiogenic properties. Whereas ctgf and cyr61 are growth factor-inducible, immediate-early genes, nov is expressed in growth-arrested or quiescent cells. As nov expression has been shown to be altered in both avian and human nephroblastomas and to be a target of WT1 regulation, NOV may play important roles in normal nephrogenesis and the development of Wilms' tumors. The aim of this study was to determine whether changes in novH expression were associated with tumorigenesis in tissues other than those of the kidney. We showed by Northern blotting and immunohistochemistry that among human adult endocrine tissues, the adrenal gland is a major site of novH expression, and that in adult and fetal adrenal tissue, novH is primarily expressed in the adrenal cortex. Studies with 12 benign and 18 malignant adrenocortical tumors revealed that the levels of novH mRNA and protein decreased significantly (P < 0.004) with progression of adrenocortical tumors from a benign to a malignant state. Although the localization of NOVH did not change, the N-glycosylation profile of benign and malignant tumors differed considerably from that of normal adrenocortical tissue, and these differences may affect the biochemical properties of the molecule. The properties of NOVH here provide the first evidence that this member of the CCN family could be involved in adrenocortical tumor development.

The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH-fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.

We previously established that the expression of the human nov gene (novH) was altered in Wilms' tumors and that levels of novH and WT1 mRNA were inversely correlated in individual Wilms' tumors. Insofar as novH has been shown to be a target for WT1 regulation, novH might play an important role during normal nephrogenesis and in the development of Wilms' tumors. We now show that during normal nephrogenesis novH protein is tightly associated with differentiation of glomerular podocytes. NovH expression is not restricted to renal differentiation but is also detected in endothelium and neural tissue of the kidney. Our results establish that alteration of novH expression in sporadic and heritable Wilms' tumors is associated with dysregulated expression of both novH mRNA and protein. In general, the highest novH expression was noted in the Wilms' tumor, genitourinary anomalies, aniridia, and mental retardation (WAGR)-associated Wilms' tumors. Expression in the Denys-Drash syndrome (DDS)-associated Wilms' tumors fell within the variable spectrum observed in sporadic Wilms' tumor cases. As in developing kidney podocytes, novH protein was also prominent in the abnormal hypoplastic podocytes from DDS cases and in kidney podocytes adjoining Wilms' tumors. In Wilms' tumors exhibiting heterotypic differentiation, novH protein was expressed at high levels in tumor-derived striated muscle and at lower levels in tumor-derived cartilage. These observations taken together indicate that novH may represent both a marker of podocytic differentiation in kidney and a marker of heterotypic mesenchymal differentiation in Wilms' tumors. In addition, absence or very low levels of WT1 are correlated with higher novH expression, and its variable expression in cases with mutant WT1 (sporadic and DDS) suggests that the potential activation and repression transcriptional functions possessed by WT1 are likely dependent on the specific mutation incurred.

The nov gene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mouse nov gene have been isolated and characterized. The mouse nov gene is highly conserved with the human and chick nov genes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. The nov gene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.

The nov gene encodes a putative Insulin-like-Growth Factor-Binding-Protein (IGFBP) of a novel type which is structurally related to a family of growth-factors likely to play a role in the control of cell proliferation. In the kidney, nov is expressed essentially at the embryonic stage and alterations of nov expression, relative to the normal kidney, have been detected in both avian nephroblastomas and human Wilms' tumors. The levels of human nov (novH) and WT1 mRNA in individual Wilms' tumors have been shown to be inversely correlated, suggesting that the expression of novH could be under the negative control of WT1. We have now established the nucleotide sequence of the 5' flanking region and identified two transcription start sites by RNase protection assays and primer extension. We report that in transient cotransfection experiments the transcription activity of novH promoter constructs was repressed by two isoforms of WT1 proteins (WT1 and WT1+KTS). Repression of the novH promoter required both intact zinc finger regions and the NH2 transcription repression domain of WT1. Inasmuch as the minimal region of novH promoter required to mediate WT1 repression in vivo failed to bine recombinant WT1 protein in in vitro footprinting assays this repression may be mediated by either (i) low affinity sites cooperative interactions or (ii) indirectly via protein-protein interactions with another factor(s). Furthermore, constitutive expression of wild type WT1 into 293 cells resulted in a decrease of endogenous NOVH protein levels, suggesting that novH may be a physiological target for WT1. The downregulation of novH expression by WT1 might represent a key element in normal and tumoral nephrogenesis.

We have cloned and sequenced the nov gene (novH) which is the homolog of the chicken nov proto-oncogene overexpressed in avian nephroblastomas. The novH gene is highly conserved and encodes a putative IGF-binding protein similar to that of chicken. We report that relative to autologous normal kidney expression of novH is elevated in Wilms tumors containing predominantly stromal elements and is inversely correlated in these tumors to the expression of WT1. Our results suggest that the regulation of IGFII expression by WT1 and increase of novH in Wilms tumors might be interrelated and represent a key element in tumor development in human.

The human locus (novH) corresponding to the nov protooncogene overexpressed in avian nephroblastoma has been identified and mapped on chromosome 8q24.1. Another locus sharing homology with novH and corresponding to the connective tissue growth factor (CTGF) gene has also been mapped on chromosome 6q23.1. The chromosomal assignment of nov and CTGF proximal to c-myc and c-myb respectively is of interest because chromosomal abnormalities involving these regions have been associated with different human tumors including Wilms'.

We have previously reported that truncated forms of the v-myb oncogene of avian myeloblastosis virus (AMV) are expressed in transformed chicken embryo fibroblasts (CEF). In this paper, we show that deletion mutants encoding v-myb products altered in either the DNA-binding or the negative regulatory domains are able to induce CEF transformation. In addition, we report that recombinant plasmids expressing gag-myb fusion proteins are maintained as extrachromosomal forms in transfected cells. This observation provides an important clue for a possible role of myb in the DNA replication processes.

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.

We have shown in a previous study that the expression of an as yet unidentified, embryonic gene (nov) encoding a potential IGF binding protein was upregulated in all of eight virally-induced avian nephroblastomas tested. We now report that homologous sequences are conserved in human DNA and are expressed in normal human bone marrow, thymic cells and in one nephroblastoma.

A novel human oncogene, designated VAV, has been recently characterized. This oncogene was generated by a rearrangement within the 5' coding sequences of a normal cellular gene, the VAV proto-oncogene. The normal VAV gene is specifically expressed in hematopoietic cells regardless of their differentiation lineage. We now report that the VAV locus has been localized in the human genome at chromosome 19p12----19p13.2 by analysis of its segregation pattern in rodent-human somatic cell hybrids and by chromosomal in situ hybridization. The VAV locus might be closely linked to the insulin receptor (INSR) locus, as suggested by comigration of INSR and VAV high-molecular-weight DNA fragments after pulsed-field gel electrophoresis. The VAV chromosomal assignment is of interest because chromosome region 19p13 is involved in different karyotypic abnormalities in a variety of malignancies including melanomas and leukemias. The identification of a novel proto-oncogene that maps to that region will enable us to define whether VAV is involved in any of the translocations observed.

Chicken embryo fibroblasts sensitized by ts RSV respond to TGFs present in the media of non-transformed FR3T3 and NRK-4 rat cells and of the same cells transformed by KiMSV or RSV. They also respond to TGFs present in the media of BHK hamster cells transformed by MoMSV, PyV or RSV. Two other indicator rat cell lines, untransformed NRK-4 and FR3T3, sensitized by ts KiMSV, respond to the same TGF-containing media, and this response is increased by exogenous EGF. Normal FR3T3 cells failed to respond to any of the media. The most sensitive target cells were the ts KiMSV-FR3T3 cells at the restrictive temperature (39.5 degrees C). All the media tested on NRK-49F target cells required EGF for their TGF activity which was essentially dependent on prior activation by acidification. These data show that the above media from non-transformed or transformed cells contain beta-TGFs, with no detectable accompanying alpha-TGF activity. The release of and the response to these TGFs are not interdependent. A function of ts mutant src and k-ras viral oncogenes, still expressed at the restrictive temperature, can sensitize non-responsive cells, without there being any specificity towards the TGF producer cells.

Normal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony-forming activity on two widely used target cells, rat NRK-49F and mouse AKR-2B, is essentially revealed only after prior acidification of cell-conditioned media. These TGFs are EGF-dependent when assayed on NRK-49F cells and EGF-independent on AKR-2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.

Transformed cells have been isolated after transfection of chicken embryo fibroblasts (CEF) with the DNA of a recombinant clone (KXA 3457) in which the v-myb sequences are flanked by the two AMV-LTRs. Abnormal myb-specific RNA species and myb-related polypeptides were found to be expressed in these cells, suggesting that transformation of CEF by v-myb might require alterations of the oncogene product.