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Published online before print December 18, 2007

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(Journal of Leukocyte Biology. 2008;83:648-662.)
© 2008 by Society for Leukocyte Biology

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

Florinda Battaglia^*, Silvana Delfino^*, Elisa Merello, Maura Puppo^*, Roberto Piva, Luigi Varesio^* and Maria Carla Bosco^*,1

^* Laboratories of Molecular Biology and
Neurosurgery, G. Gaslini Institute, Genova, Italy; and
Department of Pathology and Center for Experimental Research and Medical Studies (CeRMS), University of Turin, Torino, Italy

¹Correspondence: Laboratorio di Biologia Molecolare, Istituto Giannina Gaslini, Padiglione 2, L.go Gerolamo Gaslini 5, 16147 Genova Quarto, Italy. E-mail: mcbosco1{at}virgilio.it

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-B-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-B motif; 2) three copies of the wild-type NF-B-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-B binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-B family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-B complexes binding the cognate B site on the CCL20 promoter upon hypoxia exposure, and NF-Bp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-Bp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the –92/–82 B site. Moreover, nuclear expression of the other NF-B 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-B pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.

Key Words: monocytes • chemokines • gene regulation • transcription • inflammation

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