Hypoxia transcriptionally induces macrophage-inflammatory protein-3alpha/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-kappaB.

Hypoxia, a condition of low oxygen tension, occurring in many pathological processes, modifies the mononuclear phagocyte transcriptional profile. Here, we demonstrate hypoxic up-regulation of the CCL20 chemokine in primary human monocytes (Mn) and macrophages. mRNA induction was paralleled by protein secretion and dependent on gene transcription activation. Functional studies of the CCL20 promoter using a series of 5'-deleted and mutated reporter constructs demonstrated the requirement for the NF-kappaB-binding site located at position -92/-82 for gene transactivation by hypoxia, as 1) transcription was abrogated by a 3-bp mutation of the NF-kappaB motif; 2) three copies of the wild-type NF-kappaB-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-kappaB binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-kappaB family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-kappaB complexes binding the cognate kappaB site on the CCL20 promoter upon hypoxia exposure, and NF-kappaBp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-kappaBp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the -92/-82 kappaB site. Moreover, nuclear expression of the other NF-kappaB family members was inhibited in hypoxic Mn. In conclusion, this study characterizes a previously unrecognized role for hypoxia as a transcriptional inducer of CCL20 in human mononuclear phagocytes and highlights the importance of the NF-kappaB pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.

Topotecan inhibits vascular endothelial growth factor production and angiogenic activity induced by hypoxia in human neuroblastoma by targeting hypoxia-inducible factor-1alpha and -2alpha.

Neuroblastoma produce angiogenic peptides, and the extent of angiogenesis correlates with tumor progression and poor clinical outcome. Hence, angiogenic factor inhibition represents an important therapeutic option. One of the major drives to tumor angiogenesis is hypoxia, a decrease in oxygen tension that characterizes the tumor microenvironment. We investigated the effects of the topoisomerase I inhibitor, topotecan, on vascular endothelial growth factor (VEGF) induction by hypoxia in advanced-stage human neuroblastoma cells. Topotecan counteracted hypoxic induction of VEGF and decreased angiogenic activity of conditioned medium from hypoxic cultures in vivo in the chick chorioallantoic membrane. Promoter-driven reporter studies showed the role of both hypoxia-inducible factor (HIF)-1alpha and -2alpha in VEGF transcription activation by hypoxia, because (a) overexpression of either protein by cotransfection with expression vectors resulted in VEGF promoter transactivation, which was abrogated by mutation in the HIF-binding site, and (b) targeted knockdown of HIF-1alpha/2alpha by RNA interference inhibited hypoxia-stimulated VEGF transcriptional activity and protein secretion. Topotecan-inhibitory effects on VEGF induction by hypoxia were mediated through suppression of both HIF-1alpha and HIF-2alpha protein accumulation and transactivation properties, which was specific and required ongoing RNA transcription. A similar pattern of results was obtained in cells treated with the hypoxia-mimetic agent, desferrioxamine. These data provide the first evidence that topotecan is a potent inhibitor of HIF-1alpha and HIF-2alpha subunits in hypoxic neuroblastoma cells, leading to decreased VEGF expression and angiogenic activity. An important clinical implication of these findings is that therapies targeted to the HIF pathway have the potential to inhibit neuroblastoma angiogenesis and growth.

The hypoxic synovial environment regulates expression of vascular endothelial growth factor and osteopontin in juvenile idiopathic arthritis.

OBJECTIVE: Synovial angiogenesis, a critical determinant of juvenile idiopathic arthritis (JIA) pathogenesis, is sustained by various mediators, including vascular endothelial growth factor (VEGF) and osteopontin (OPN). We characterized the contribution of the local hypoxic environment to VEGF and OPN production by monocytic cells recruited to the synovium in JIA. METHODS: Paired synovial fluid (SF) and peripheral blood (PB) samples were collected from 20 patients with JIA. Mononuclear cells (MC) were isolated, and monocytic cells were purified by adherence, maintained in a hypoxic environment, or subjected to reoxygenation. VEGF and OPN protein concentrations were tested in SF, plasma, and culture supernatants by ELISA, and mRNA expression was assessed in freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction. Synovial tissue was obtained at synovectomy from 4 patients with JIA, and analyzed by immunohistochemistry for VEGF, OPN, CD68, and hypoxia-inducible factor-1alpha (HIF-1alpha). RESULTS: VEGF mRNA expression was increased in SFMC and SF monocytic cells compared to matched PBMC and PB monocytic cells or SF lymphocytes, correlating with significantly higher protein levels in SF relative to plasma samples. Accordingly, OPN mRNA expression in SF monocytic cells was associated with significant increase of SF protein. Immunohistochemistry revealed the presence of both factors in synovial tissues at the level of the lining and sublining layers, which colocalized with intense CD68 and HIF-1alpha staining, suggesting production by hypoxic synovial monocytic cells. VEGF and OPN expression was abrogated upon SF monocytic cell reoxygenation and maintained by exposure to prolonged hypoxia. CONCLUSION: Hypoxic synovial monocytic cells are a likely source of VEGF and OPN in JIA. These data point to a role for hypoxia in the perpetuation of synovitis in JIA.

Hypoxic synovial environment and expression of macrophage inflammatory protein 3gamma/CCL20 in juvenile idiopathic arthritis.

OBJECTIVE: Synovial inflammation is a major determinant of juvenile idiopathic arthritis (JIA) pathogenesis and is mediated by local chemokine secretion. Monocytic cells are an important source of chemokines. The purpose of this study was to investigate expression of CCL20, a macrophage inflammatory protein, in synovial fluid (SF) and SF-derived monocytic cells from JIA patients and its regulation by hypoxia, a common feature of the inflamed synovial environment. METHODS: Mononuclear cells and monocytic cells were isolated from paired SF and peripheral blood (PB) samples from JIA patients and were maintained in a hypoxic environment or subjected to reoxygenation. CCL20 concentrations in SF, PB, and culture supernatants were measured by enzyme-linked immunosorbent assay. CCL20 expression was assessed in both freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha were detected in the synovial tissue and cells of JIA patients by immunohistochemistry and Western blotting. RESULTS: CCL20 concentrations were significantly higher in SF compared with PB samples (P < 0.0001). SF mononuclear cells, but not matched PB mononuclear cells, constitutively expressed CCL20 messenger RNA. The SF monocytic cell fraction produced higher amounts of CCL20 than did SF lymphocytes, and CCL20 expression was associated with HIF positivity. Reoxygenation abrogated HIF and CCL20 expression, which was maintained in SF monocytic cells exposed to prolonged hypoxia. CONCLUSION: CCL20 is released into the SF of JIA patients, and SF monocytic cells are a major source of this chemokine. The hypoxic synovial microenvironment may directly contribute to the persistent inflammation associated with JIA by increasing CCL20 production by SF monocytic cells, thus representing a potential therapeutic target.