Estrogen signaling in healthy and tumor brain.

Sex-specific differences in brain organization and function are widely explored in multidisciplinary studies, ranging from sociology and biology to digital modelling. In addition, there is growing evidence that natural or disturbed hormonal environments play a crucial role in the onset of brain disorders and pathogenesis. For example, steroid hormones, but also enzymes involved in steroidogenesis and receptors triggering hormone signaling are key players of gliomagenesis. In the present review we summarize the current knowledge about steroid hormone, particularly estrogens synthesis and signaling, in normal brain compared to the tumor brain. We will focus on two key molecular players, aromatase and the G Protein-Coupled Estrogen Receptor, GPER.

Molecular Characterization of the Dual Effect of the GPER Agonist G-1 in Glioblastoma.

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite conventional treatment, consisting of a chirurgical resection followed by concomitant radio-chemotherapy, the 5-year survival rate is less than 5%. Few risk factors are clearly identified, but women are 1.4-fold less affected than men, suggesting that hormone and particularly estrogen signaling could have protective properties. Indeed, a high GPER1 (G-protein-coupled estrogen receptor) expression is associated with better survival, especially in women who produce a greater amount of estrogen. Therefore, we addressed the anti-tumor effect of the GPER agonist G-1 in vivo and characterized its molecular mechanism of action in vitro. First, the antiproliferative effect of G-1 was confirmed in a model of xenografted nude mice. A transcriptome analysis of GBM cells exposed to G-1 was performed, followed by functional analysis of the differentially expressed genes. Lipid and steroid synthesis pathways as well as cell division processes were both affected by G-1, depending on the dose and duration of the treatment. ANGPTL4, the first marker of G-1 exposure in GBM, was identified and validated in primary GBM cells and patient samples. These data strongly support the potential of G-1 as a promising chemotherapeutic compound for the treatment of GBM.

Identification of Gender- and Subtype-Specific Gene Expression Associated with Patient Survival in Low-Grade and Anaplastic Glioma in Connection with Steroid Signaling.

Low-grade gliomas are rare primary brain tumors, which fatally evolve to anaplastic gliomas. The current treatment combines surgery, chemotherapy, and radiotherapy. If gender differences in the natural history of the disease were widely described, their underlying mechanisms remain to be determined for the identification of reliable markers of disease progression. We mined the transcriptomic and clinical data from the TCGA-LGG and CGGA databases to identify male-over-female differentially expressed genes and selected those associated with patient survival using univariate analysis, depending on molecular characteristics (IDH wild-type/mutated; 1p/19q codeleted/not) and grade. Then, the link between the expression levels (low or high) of the steroid biosynthesis enzyme or receptors of interest and survival was studied using the log-rank test. Finally, a functional analysis of gender-specific correlated genes was performed. HOX-related genes appeared to be differentially expressed between males and females in both grades, suggesting that a glioma could originate in perturbation of developmental signals. Moreover, aromatase, androgen, and estrogen receptor expressions were associated with patient survival and were mainly related to angiogenesis or immune response. Therefore, consideration of the tight control of steroid hormone production and signaling seems crucial for the understanding of glioma pathogenesis and emergence of future targeted therapies.

GPER Agonist G-1 Disrupts Tubulin Dynamics and Potentiates Temozolomide to Impair Glioblastoma Cell Proliferation.

Glioblastoma (GBM) is the most common brain tumor in adults, which is very aggressive, with a very poor prognosis that affects men twice as much as women, suggesting that female hormones (estrogen) play a protective role. With an in silico approach, we highlighted that the expression of the membrane G-protein-coupled estrogen receptor (GPER) had an impact on GBM female patient survival. In this context, we explored for the first time the role of the GPER agonist G-1 on GBM cell proliferation. Our results suggested that G-1 exposure had a cytostatic effect, leading to reversible G2/M arrest, due to tubulin polymerization blockade during mitosis. However, the observed effect was independent of GPER. Interestingly, G-1 potentiated the efficacy of temozolomide, the current standard chemotherapy treatment, since the combination of both treatments led to prolonged mitotic arrest, even in a temozolomide less-sensitive cell line. In conclusion, our results suggested that G-1, in combination with standard chemotherapy, might be a promising way to limit the progression and aggressiveness of GBM.

Astrocytoma: A Hormone-Sensitive Tumor?

Astrocytomas and, in particular, their most severe form, glioblastoma, are the most aggressive primary brain tumors and those with the poorest vital prognosis. Standard treatment only slightly improves patient survival. Therefore, new therapies are needed. Very few risk factors have been clearly identified but many epidemiological studies have reported a higher incidence in men than women with a sex ratio of 1:4. Based on these observations, it has been proposed that the neurosteroids and especially the estrogens found in higher concentrations in women's brains could, in part, explain this difference. Estrogens can bind to nuclear or membrane receptors and potentially stimulate many different interconnected signaling pathways. The study of these receptors is even more complex since many isoforms are produced from each estrogen receptor encoding gene through alternative promoter usage or splicing, with each of them potentially having a specific role in the cell. The purpose of this review is to discuss recent data supporting the involvement of steroids during gliomagenesis and to focus on the potential neuroprotective role as well as the mechanisms of action of estrogens in gliomas.

The Role of ERα36 in Development and Tumor Malignancy.

Estrogen nuclear receptors, represented by the canonical forms ERα66 and ERß1, are the main mediators of the estrogen-dependent pathophysiology in mammals. However, numerous isoforms have been identified, stimulating unconventional estrogen response pathways leading to complex cellular and tissue responses. The estrogen receptor variant, ERα36, was cloned in 2005 and is mainly described in the literature to be involved in the progression of mammary tumors and in the acquired resistance to anti-estrogen drugs, such as tamoxifen. In this review, we will first specify the place that ERα36 currently occupies within the diversity of nuclear and membrane estrogen receptors. We will then report recent data on the impact of ERα36 expression and/or activity in normal breast and testicular cells, but also in different types of tumors including mammary tumors, highlighting why ERα36 can now be considered as a marker of malignancy. Finally, we will explain how studying the regulation of ERα36 expression could provide new clues to counteract resistance to cancer treatments in hormone-sensitive tumors.

Dual Epigenetic Regulation of ERα36 Expression in Breast Cancer Cells.

Breast cancer remains the major cause of cancer-induced morbidity and mortality in women. Among the different molecular subtypes, luminal tumors yet considered of good prognosis often develop acquired resistance to endocrine therapy. Recently, misregulation of ERα36 was reported to play a crucial role in this process. High expression of this ERα isoform was associated to preneoplastic phenotype in mammary epithelial cells, disease progression, and enhanced resistance to therapeutic agents in breast tumors. In this study, we identified two mechanisms that could together contribute to ERα36 expression regulation. We first focused on hsa-miR-136-5p, an ERα36 3'UTR-targeting microRNA, the expression of which inversely correlated to the ERα36 one in breast cancer cells. Transfection of hsa-miR136-5p mimic in MCF-7 cells resulted in downregulation of ERα36. Moreover, the demethylating agent decitabine was able to stimulate hsa-miR-136-5p endogenous expression, thus indirectly decreasing ERα36 expression and counteracting tamoxifen-dependent stimulation. The methylation status of ERα36 promoter also directly modulated its expression level, as demonstrated after decitabine treatment of breast cancer cell and confirmed in a set of tumor samples. Taken together, these results open the way to a direct and an indirect ERα36 epigenetic modulation by decitabine as a promising clinical strategy to counteract acquired resistance to treatment and prevent relapse.

Low-Dose Alkylphenol Exposure Promotes Mammary Epithelium Alterations and Transgenerational Developmental Defects, But Does Not Enhance Tumorigenic Behavior of Breast Cancer Cells.

Fetal and neonatal exposure to long-chain alkylphenols has been suspected to promote breast developmental disorders and consequently to increase breast cancer risk. However, disease predisposition from developmental exposures remains unclear. In this work, human MCF-10A mammary epithelial cells were exposed in vitro to a low dose of a realistic (4-nonylphenol + 4-tert-octylphenol) mixture. Transcriptome and cell-phenotype analyses combined to functional and signaling network modeling indicated that long-chain alkylphenols triggered enhanced proliferation, migration ability, and apoptosis resistance and shed light on the underlying molecular mechanisms which involved the human estrogen receptor alpha 36 (ERα36) variant. A male mouse-inherited transgenerational model of exposure to three environmentally relevant doses of the alkylphenol mix was set up in order to determine whether and how it would impact on mammary gland architecture. Mammary glands from F3 progeny obtained after intrabuccal chronic exposure of C57BL/6J P0 pregnant mice followed by F1-F3 male inheritance displayed an altered histology which correlated with the phenotypes observed in vitro in human mammary epithelial cells. Since cellular phenotypes are similar in vivo and in vitro and involve the unique ERα36 human variant, such consequences of alkylphenol exposure could be extrapolated from mouse model to human. However, transient alkylphenol treatments combined to ERα36 overexpression in mammary epithelial cells were not sufficient to trigger tumorigenesis in xenografted Nude mice. Therefore, it remains to be determined if low-dose alkylphenol transgenerational exposure and subsequent abnormal mammary gland development could account for an increased breast cancer susceptibility.

Mammary epithelial cell phenotype disruption in vitro and in vivo through ERalpha36 overexpression.

Estrogen receptor alpha 36 (ERα36) is a variant of the canonical estrogen receptor alpha (ERα66), widely expressed in hormone sensitive cancer cells and whose high expression level correlates with a poor survival prognosis for breast cancer patients. While ERα36 activity have been related to breast cancer progression or acquired resistance to treatment, expression level and location of ERα36 are poorly documented in the normal mammary gland. Therefore, we explored the consequences of a ERα36 overexpression in vitro in MCF-10A normal mammary epithelial cells and in vivo in a unique model of MMTV-ERα36 transgenic mouse strain wherein ERα36 mRNA was specifically expressed in the mammary gland. By a combination of bioinformatics and computational analyses of microarray data, we identified hierarchical gene networks, downstream of ERα36 and modulated by the JAK2/STAT3 signaling pathway. Concomitantly, ERα36 overexpression lowered proliferation rate but enhanced migration potential and resistance to staurosporin-induced apoptosis of the MCF-10A cell line. In vivo, ERα36 expression led to duct epithelium thinning and disruption in adult but not in prepubescent mouse mammary gland. These phenotypes correlated with a loss of E-cadherin expression. Here, we show that an enhanced expression of ERα36 is sufficient, by itself, to disrupt normal breast epithelial phenotype in vivo and in vitro through a dominant-positive effect on nongenomic estrogen signaling pathways. These results also suggest that, in the presence of adult endogenous steroid levels, ERα36 overexpression in vivo contributes to alter mammary gland architecture which may support pre-neoplastic lesion and augment breast cancer risk.

From ERα66 to ERα36: a generic method for validating a prognosis marker of breast tumor progression.

BACKGROUND: Estrogen receptor alpha36 (ERalpha36), a variant of estrogen receptor alpha (ER) is expressed in about half of breast tumors, independently of the [ER+]/[ER-] status. In vitro, ERalpha36 triggers mitogenic non-genomic signaling and migration ability in response to 17beta-estradiol and tamoxifen. In vivo, highly ERalpha36 expressing tumors are of poor outcome especially as [ER+] tumors are submitted to tamoxifen treatment which, in turn, enhances ERalpha36 expression. RESULTS: Our study aimed to validate ERalpha36 expression as a reliable prognostic factor for cancer progression from an estrogen dependent proliferative tumor toward an estrogen dispensable metastatic disease. In a retrospective study, we tried to decipher underlying mechanisms of cancer progression by using an original modeling of the relationships between ERalpha36, other estrogen and growth factor receptors and metastatic marker expression. Nonlinear correlation analyses and mutual information computations led to characterize a complex network connecting ERalpha36 to either non-genomic estrogen signaling or to metastatic process. CONCLUSIONS: This study identifies ERalpha36 expression level as a relevant classifier which should be taken into account for breast tumors clinical characterization and [ER+] tumor treatment orientation, using a generic approach for the rapid, cheap and relevant evaluation of any candidate gene expression as a predictor of a complex biological process.

Müllerian inhibiting substance in the caudate amphibian Pleurodeles waltl.

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-ß family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.

An alkylphenol mix promotes seminoma derived cell proliferation through an ERalpha36-mediated mechanism.

Long chain alkylphenols are man-made compounds still present in industrial and agricultural processes. Their main use is domestic and they are widespread in household products, cleansers and cosmetics, leading to a global environmental and human contamination. These molecules are known to exert estrogen-like activities through binding to classical estrogen receptors. In vitro, they can also interact with the G-protein coupled estrogen receptor. Testicular germ cell tumor etiology and progression are proposed to be stimulated by lifelong estrogeno-mimetic exposure. We studied the transduction signaling pathways through which an alkyphenol mixture triggers testicular cancer cell proliferation in vitro and in vivo. Proliferation assays were monitored after exposure to a realistic mixture of 4-tert-octylphenol and 4-nonylphenol of either TCam-2 seminoma derived cells, NT2/D1 embryonal carcinoma cells or testis tumor in xenografted nude mice. Specific pharmacological inhibitors and gene-silencing strategies were used in TCam-2 cells in order to demonstrate that the alkylphenol mix triggers CREB-phosphorylation through a rapid, ERα36-PI3kinase non genomic pathway. Microarray analysis of the mixture target genes revealed that this pathway can modulate the expression of the DNA-methyltransferase-3 (Dnmt3) gene family which is involved in DNA methylation control. Our results highlight a key role for ERα36 in alkylphenol non genomic signaling in testicular germ cell tumors. Hence, ERα36-dependent control of the epigenetic status opens the way for the understanding of the link between endocrine disruptor exposure and the burden of hormone sensitive cancers.

Temporal and spatial SOX9 expression patterns in the course of gonad development of the caudate amphibian Pleurodeles waltl.

The SOX family of transcription factors is thought to regulate gene expression in a wide variety of developmental processes. Namely, SOX9 expression is conserved in vertebrate sex determination or differentiation. Nevertheless, information about caudate amphibians is lacking. In this study, we provide data on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. Phylogenetic analysis of SOX9 P. waltl ortholog reveals that the deduced protein segregates from the group of anuran and could be more closely related to amniote than to anamniote. However, SOX9 lacks the PQA-rich domain present in amniotes. In larvae, SOX9 is expressed in both sexes in gonad-mesonephros complexes as soon as stage 42, before gonad differentiation. At stage 54(60d) at which testis differentiation is already in progress, analyses of isolated gonads reveal a male-enriched expression of SOX9, which was quantified by real-time PCR. At the end of metamorphosis (stage 56), SOX9 shows a nuclear localization only in the testis. In adults, SOX9 is still expressed in testes and ovaries. In the ovary, SOX9 is found in oocytes from stage I to stage VI but it is never detected in the nucleus. Our results suggest that in P. waltl, like in non mammalian vertebrates, SOX9 could play a role during the late phase of gonad differentiation rather than in sex determination. Its role in germ cells of the adult ovary has still to be elucidated.

Evidence for a conserved role of retinoic acid in urodele amphibian meiosis onset.

Pleurodeles waltl is a urodele amphibian displaying a ZZ/ZW genetic mode of sex determination. Gonad differentiation can later be modulated by hormone treatment. To investigate germ cell differentiation, we analyzed the expression of the meiosis marker PwDmc1 and show that germ cells enter meiosis in late larval life in females, and 2 months after metamorphosis in males. Organotypic cultures of gonad-mesonephros complexes demonstrated that retinoic acid triggers meiosis entry in P. waltl. In vivo analyses of both PwRaldh2 and PwCyp26b1 expressions, the enzymes required for RA synthesis and degradation respectively, indicate that meiosis onset depends on PwCyp26b1 repression in the gonad during normal or steroid-induced sex-reversed development. Taken together, our results show that RA-dependent meiosis entry could be a conserved mechanism of germ cell differentiation in vertebrates and provide evidence for crosstalk between steroid and RA signaling in the course of sex differentiation. Developmental Dynamics 238:1389-1398, 2009. (c) 2009 Wiley-Liss, Inc.

Lifelong testicular differentiation in Pleurodeles waltl (Amphibia, Caudata).

BACKGROUND: In numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented. METHODS: Testes were obtained from Pleurodeles waltl of different ages reared in our laboratory. Testis evolution was studied by several approaches: morphology, histology, immunohistochemistry and RT-PCR. Surgery was also employed to study testis regeneration. RESULTS: In this species, the testis is linked to the lung. This association consists of connective tissue derived from the mesorchium and the coelomic epithelium surrounding the lung and takes place at the end of larval life. This tissue contains lobules including primordial germ cells with a typical large and polylobular nucleus. The anterior part of the testis remains thin and undifferentiated while the posterior part differentiates in a large first testis lobe where spermatogenesis occurs during the first year of life. The undifferentiated status of the anterior part is attested by the lack of expression of the testis marker Dmrt1 and the meiosis entry marker Dmc1. Three-year-old Pleurodeles waltl possess multiple testes made up of two lobes. The second lobe appears at the caudal extremity of the first one from residual primordial germ cells located near or even inside efferent ducts in the glandular tissue that usually appears following spermatozoa extrusion. Surprisingly, in the case of surgical elimination of the anterior part of the testis, de novo spermatogenesis is stopped in the first lobe which becomes restricted to the glandular tissue. Following first testis lobe removal, the anterior part of the testis regenerates a new testis lobe, a process stimulated in the presence of DHT. CONCLUSION: Pleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood.

Freemartin in the amphibian Pleurodeles waltl: parabiosis between individuals from opposite sex triggers both germ and somatic cells alterations during female gonad development.

Wild type embryos of the newt Pleurodeles waltl were used to realize parabiosis, a useful model to study the effect of endogenous circulating hormones on gonad development. The genotypic sex of each parabiont (ZZ male or ZW female) was determined early from the analysis of the sex chromosome borne marker peptidase-1. In ZZ/ZZ and ZW/ZW associations, gonads develop according to genetic sex. In ZZ/ZW associations, the ZZ gonads differentiate as normal testes while ZW gonads development shows numerous alterations. At the beginning of sex differentiation, these ZW gonads possess a reduced number of germ cells and a reduced expression of steroidogenic factor 1 and P450-aromatase mRNAs when compared to gonads from ZW/ZW associations. During gonad differentiation, conversely to the control situation, these germ cells do not enter meiosis as corroborated by chromatin status and absence of the meiosis entry marker DMC1; the activity of the estradiol-producing enzyme P450-aromatase is as low as in ZZ gonads. At adulthood, no germ cells are observed on histological sections, consistently with the absence of VASA expression. At this stage, the testis-specific marker DMRT1 is expressed only in ZZ gonads, suggesting that the somatic compartment of the ZW gonad is not masculinized. So, when exposed to ZZ hormones, ZW gonads reach the undifferentiated status but the ovary differentiation does not occur. This gonad is inhibited by a process affecting both somatic and germ cells. Additionally, the ZW gonad inhibition does not occur in the case of an exogenous estradiol treatment of larvae.

Evidence for a key role of leptin in osteoarthritis.

OBJECTIVE: To evaluate the contribution of leptin (an adipose tissue-derived hormone) to the pathophysiology of osteoarthritis (OA), by determining the level of leptin in both synovial fluid (SF) and cartilage specimens obtained from human joints. We also investigated the effect of leptin on cartilage, using intraarticular injections of leptin in rats. METHODS: Leptin levels in SF samples obtained from OA patients undergoing either knee replacement surgery or knee arthroscopy were measured by enzyme-linked immunosorbent assay. In addition, histologic sections of articular cartilage and osteophytes obtained during surgery for total knee replacement were graded using the Mankin score, and were immunostained using antibodies to leptin, transforming growth factor beta (TGFbeta), and insulin-like growth factor 1 (IGF-1). For experimental studies, various doses of leptin (10, 30, 100, and 300 microg) were injected into the knee joints of rats. Tibial plateaus were collected and processed for proteoglycan synthesis by radiolabeled sulfate incorporation, and for expression of leptin, its receptor (Ob-Rb), and growth factors by reverse transcriptase-polymerase chain reaction and immunohistochemical analysis. RESULTS: Leptin was observed in SF obtained from human OA-affected joints, and leptin concentrations correlated with the body mass index. Marked expression of the protein was observed in OA cartilage and in osteophytes, while in normal cartilage, few chondrocytes produced leptin. Furthermore, the pattern and level of leptin expression were related to the grade of cartilage destruction and paralleled those of growth factors (IGF-1 and TGFbeta1). Animal studies showed that leptin strongly stimulated anabolic functions of chondrocytes and induced the synthesis of IGF-1 and TGFbeta1 in cartilage at both the messenger RNA and the protein levels. CONCLUSION: These findings suggest a new peripheral function of leptin as a key regulator of chondrocyte metabolism, and indicate that leptin may play an important role in the pathophysiology of OA.

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression.

A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3, is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

Interleukin 17, a nitric oxide-producing cytokine with a peroxynitrite-independent inhibitory effect on proteoglycan synthesis.

OBJECTIVE: To compare the potency of 2 cytokines, interleukin 17 (IL-17) and IL-1beta, on rat cartilage proteoglycan synthesis with special attention to nitric oxide (NO) and peroxynitrite formation. METHODS: Chondrocytes in alginate beads were stimulated with human recombinant (rh) IL-17 (0.03 to 300.0 ng/ml) and/or rhIL-1beta (0.25 to 25.0 ng/ml) in the presence or not of L-NMMA or CuDips. Alternatively, rats were injected with either IL-17 (10.0 micro g) or IL-1beta (1.0 micro g) into each knee joint. NO concentrations were determined by a spectrofluorimetric assay, proteoglycan synthesis by 35SO4-2 incorporation, peroxynitrite generation by immunostaining for 3-nitrotyrosine, and IL-1beta mRNA expression by reverse transcription-polymerase chain reaction. RESULTS: IL-17 inhibited proteoglycan synthesis and increased NO production, both in vitro and in vivo, without inducing expression of IL-1beta mRNA in cartilage. Additive effects were observed when IL-17 was combined with low concentrations of IL-1. Surprisingly, a similar NO synthesis between IL-1 and IL-17 led to a less suppressive effect of IL-17 on cartilage anabolism than with IL-1. Both in vitro and in vivo, peroxynitrite formation was extensive with IL-1beta, but negligible or nonexistent with IL-17. L-NMMA and CuDips completely corrected the suppressive effect of IL-1beta on proteoglycan synthesis, unlike with IL-17. CONCLUSION: These data showed that NO is weakly involved in the IL-17 mediated inhibition of proteoglycan synthesis in rat. NO overload may not be predictive of any inhibitory effect on cartilage anabolism, but instead superoxide is a key regulator of NO contribution to chondrocyte dysfunction. Since IL-17 is a NO-producing cytokine with additive effects when combined with IL-1, it may play a pivotal role in cartilage destruction during rheumatoid arthritis, for which infiltrating cells produce high levels of superoxide and proinflammatory cytokines.