Induction of ReIA(p65) and IicBo Subunit Expression during Differentiation of Human Peripheral Blood Monocytes to Macrophages1

We evaluated the expression and DNA binding activity of nuclear factor (NF)-scB subunits in human peripheral blood monocytes and in monocyte-derived macrophages (MDMs). Constitutive DNA binding activity consisting of p50 homodimers was detected in nuclear extracts from both cell types. An additional complex composed of p50/ReIA(p65) heterodimers appeared only in nuclear extracts from 7-day MDMs. lmmunoblot analysis showed that the p50 subunit was constitutively expressed in monocytes and MDMs. In contrast, the ReIA(p65) subunit was barely detectable in monocytes, but ‘its level increased markedly in MDMs. Analysis of ReIA(p65) mRNA revealed that the stability of ReIA(p65) mRNA was significantly higher in MDMs, compared with monocytes. In MDMs, an upregulation of l.cBa synthesis as well as the appearance of a novel M 40,000 form of licBa were also observed. These results suggest that macrophage differentiation results in the expression of active p50/ReIA(p65) heterodimers with the capacity to activate target gene expression. The parallel induction of IacBa synthesis may allow for the continuous presence of a cytoplasmic reservoir of p50/ReIA(p65) complexes that are readily available for inducer-mediated stimulation. Introduction The NF3-icB/Rel family of transcription factors is involved in the activation of a wide variety of genes important in the Received 5/30/96; revised 6/30/96; accepted 11/12/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 This work was supported by Grant 940C, XI Progetto di Aicerca sull’AIDS, from theltalian Ministry of Health and by grantsfrom the Cancer Research Society, Inc. and the Medical Research Council of Canada (to J. H. and M. A. W.). A. A. was supported by a studentship from FCAA. M. P. was supported by a fellowship from the Italian Ministry of Health. 2 To whom requests for reprints should be addressed. Phone: (396) 49903238; Fax: (396) 4453369-49902082. 3 The abbreviations used are: NF-KB, nuclear factor-KB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MDM, monocyte-denved macrophage; AT-PCA, reverse transcription-PCA; TIR, transferrin receptor; WCE, whole-cell extract; LPS, lipopolysaccharide; PMA, phorbol 12-mynstate 1 3-acetate; TNF, tumor necrosis factor IL, interleukin. regulation of immune functions including cytokines, cell surface immunoreceptors, adhesion molecules, and acute phase proteins (1 , 2). Several viral enhancers also contain NF-KB recognition sites in their promoter elements and use this host-regulatory pathway to selectively activate viral gene expression (1 , 2). The NF-KB/ReI family includes several members: p50 (NF-KB1) and its precursor p1 05, p52 (NFKB2) and its precursor p1 00 (also called lyt-1 0), ReIA(p65), c-ReI (p85), and ReIB (3). Recently, an alternatively spliced form of ReIA, termed i ReIA (p65i ) was also identified (3). All DNA-binding subunits [p50, p52, ReIA(p65), c-Rel, and ReIB] exhibit extensive homology in the NH2-terminal 300 amino acids, a region that contains DNA binding, dimerization, and nuclear localization domains of the protein (4). In addition, ReIA(p65) and c-Rel also contain a strong transcriptional activation region in the COOH-terminal domain of the protein (5). The activity of NF-KB/Rel proteins is finely controlled at multiple regulatory levels including intracellular localization, posttranslational processing, splicing, and transcription (6). NF-icB heterodimers are generally retained in an inactive form in the cytoplasm through their interaction with a group of inhibitory molecules, the IKBS (7). Several forms of 1KB, including IKBa/MAD-3 (and its avian homologue, pp4O), lKB , bcl-3, and IKBy, as well as the p105 precursor, have now been described (7). Among these 1KB family members, IKBa is best characterized (8, 9) and has been shown to interact with high affinity with ReIA(p65) and c-Rel and to inhibit p50/ReIA(p65) and p52/ReIA(p65) heterodimers, ReIA(p65) homodimers, and NF-KB dimers that contain c-Rel (7, 1 0). In many cell types, specific signals such as cytokines, phorbol esters, virus infection, and mitogens induce the release of IKBa from NF-KB, therefore permitting transport of DNA-binding NF-KB dimers to the nucleus (7, 1 1 , 12). Phosphorylation and degradation of IKBa are the necessary steps required for dissociation of the heterotrimeric form of NF-KB (1 1 , 13, 1 4). Furthermore, the activation of NF-KB DNA binding activity stimulates the de novo synthesis of lKBa because the promoter of IKBa contains NF-scB sites and is highly responsive to NF-KB induction (1 5-1 8). Therefore, an autoregulatory loop is established that ultimately permits the restoration of latent NF-KB/IKBa complexes in the cytoplasm (15-1 8). Notably, disruption of the NF-KB/IKBa autoregulatory loop by overexpression of lKBa antisense RNA results in malignant transformation, suggesting that IKBa may represent a potential growth-suppressor activity (19). NF-KB binding activity is regulated during B-cell differentiation (20). In pre-B cells, NF-KB complexes are maintained in an inactive form in the cytosol, whereas NF-KB activity is constitutively expressed at high levels in the nucleus of mature B cells. During the pre-B cell to B-cell transition, mod-