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.
Battaglia
Florinda
F
Laboratory of Molecular Biology, Istituto Giannina Gaslini, Padiglione 2, Largo Gerolamo Gaslini 5, Genoa Quarto, Italy.
Delfino
Silvana
S
Merello
Elisa
E
Puppo
Maura
M
Piva
Roberto
R
Varesio
Luigi
L
Bosco
Maria Carla
MC
D002467:Cell Nucleus / Q000502:physiology; D054418:Chemokine CCL20 / Q000235:genetics*; D017931:DNA Primers; D005786:Gene Expression Regulation; D006801:Humans; D000860:Hypoxia; D007963:Leukocytes, Mononuclear / Q000502:physiology; D019402:Macrophage Inflammatory Proteins / Q000235:genetics*; D016328:NF-kappa B / Q000502:physiology*; D010586:Phagocytes / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014158:Transcription, Genetic
C108355:CCL20 protein, human; D054418:Chemokine CCL20; D017931:DNA Primers; D019402:Macrophage Inflammatory Proteins; D016328:NF-kappa B