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L., Soreq, H., Nir, U., \\‘allach. D., Perricaudet, hl., Tiollais, P. & Revel, M. (19SO), Two interferon mRN.-\s in humanfibroblasts: In viva translationand Esctlerictliacoli cloningstudies.Proc. narl. .-Icad. Sci. (\Vash.). 77, 7152-7156. \\‘oloski, B.ll.R.N.J., Smith, E.11.. Meyer, \\‘.J., III, Fuller, G.M. 8: Blalock, J.E. (1985), Corticotropinreleasing activity of monokines. Science, 230, 1035-1037. Yasukawa, I;., Hirano, T., \Vatanabe, Y., Muratani, K., blatsuda, T. & Kishimoto, T. (1987), Structure and expression of human B-cell stimulatory factor 2 (BSF-L/IL-6) gene. EMBO J., 6, 2939-2935. Zhang, Y., Lin, J.X. & \‘ilCek, J. (1988), Synthesisof

NF-IL6

Y

interleukin-6 (interferon F?.‘B-cell stimulatory fnstor 2) in humanfibroblastsistriggeredby an incrca\in intracellular cyclic AMP. J. biol. Ckem., 263, 6177-6182. Zhang, Y., Lin, J.X. & VilCek, J. (1990),Interleukin-6 induction by tumor necrosisfactor and interleukin-1 in humanfibroblastsinvolvesactivation of a nuclearfactor binding to a KB-like sequence.Mol. cell. Biol., 10, 3818-3923. Zilberstein, A., Ruggieri, R., Korn, J.H. & Revel, hl. (1956),Structure and expressionof cDNA and genes for human interferon-beta-2, a distinct speciesinducible by growth stimulatory cytokines. EMBO J., 5, 2529-2537.

and gene regulation S. Akira

Institure for Molecular and Cellular Biology, Osaka University, l-3 Yamadaoka, Suita, Osaka 565 (Japan)

Introduction NF-IL6 was initially identified as a nuclear facbinding to a 14-bp palindromic sequence (ACATTGCACAATCT) within an IL 1-responsive element in the human IL6 gene (Isshiki et al., 1990). The gene encoding NF-IL6 was cloned from a Xgtl 1 cDNA expression library of LPS-stimulated human peripheral monocytes by a South-Western method (Akira et al., 1990). Interestingly, the cloned NF-IL6 contained a region highly homologous to the Cterminal portion of C/EBP, the first nuclear factor

tor

proposed to contain a leucine zipper structure (LandSchultz et al., 1988). The highly homologous region includes a basic domain and a leucine zipper structure essential for DNA binding and dimerization, respectively. NF-IL6 recognizes the same nucleotide sequences as C/EBP. Both proteins recognize a variety of the divergent nucleotide sequenceswith differ-

ent

affinity

and

the

consensus

sequence

is

However, expression of these two proteins is quite different. C/EBP is ex-

TNF or IL6, indicating that NF-IL6 may be involved in acute phase, immune and inflammatory responses. Indeed, evidence in support of this has recently been provided. NF-IL6

and acute phase gene regulation

Production of acute phase proteins is regulated by IL6 mainly at a transcriptional level in the liver. Site-directed mutagenesis studies of the promoter regions of hepatic acute phase genes, including haptoglobin, haemopexin, CRP, and aZmacroglobulin have revealed two types of IL6 c&acting response elements ; one is the hexanucleotide CTGGGA and the other is a group of the sequences which seem to be dissimilar to each other but are recognized by the same set of proteins, including the IL6-inducible protein (IL6-dependent-DNA binding protein, IL6DPB). We noticed that the latter sequences include the

T(T/G)NNGNNAA(T/G).

recognition sequence of NF-IL6.

pressed in liver and adipose tissues and is supposed to regulate several hepatocyte- and adipocyte-specific genes. BY contrast, NF-IL6 is expressed at an undetectable or a minor level in all normal tissues, but it is drastically induced by stimulation of LPS, ILl,

binant NF-IL6 binds to these IL6 RE. Recently, Cortese and his colleagues (Poli et al., 1990) have cloned a cDNA coding for IL6DBP and it turned out to be a rat homolog of NF-IL6. When the NF-IL6 gene was introduced into a hepatoma cell line Hep3B, basal production as well as IL6-mediated induction of hap-

Actually,

recom-

INTERLEUKIN toglobin were significantly augmented, suggesting that NF-IL6 works on the endogenous gene for haproglobin as a positive transcription factor (Natsuka et al., 1991). Expression of NF-IL6 and C/EBP is reciprocally regulated by IL6 in the liver. NF-IL6 mRNA is rapidly and drastically induced after IL6 stimulation, preceding the induction of haptoglobin mRNA, whereas constitutively expressed C/EBP mRNA is dramatically decreased with treatment of IL6 (Isshiki et al., 1991). A reciprocal expression of NF-IL6 and C/EBP in the presence of IL6 may explain the induction of the positive acute phase proteins and the decrease of the negative acute phase proteins including albumin seen in inflammation. Involvement of NF-IL6 in macrophage-specific gene expression

NF-IL6 is drastically and abundantly induced in all tissues after stimulation with LPS or inflammatory cytokines such as ILl, TNF, and IL6. Many cytokines, including ILl, TNF, IL6, IL8 and G-CSF, are released from macrophages by LPS, or inflammatory cvtokines. Such evidence implied that NF-IL6 may be Involved in the genes activated in macrophages. In fact, NF-IL6 binding motifs were identified in the functional regulatory regions of the genes for G-CSF, TNF, ILI, IL6, IL8 and lysozyme. To gain further insight into the involvement of NF-IL6 in regulation of the genes activated in macrophages, expression of NF-IL6 was studied in several leukaemic cell lines capable of differentiating to mature macrophages. In all cell lines examined, NF-IL6 mRNA increased markedly along with macrophage differentiation, whereas C/EBPmRNA remained constant (Natsuka et a/., 1992). In this respect, noteworthy is the recent finding that C/EBP-like protein(s) plays a key role in the lineage-specific activation of the gene for chicken myelomonocytic growth factor (cMGF), a distant relative of both the mammalian G-CSF and IL6 (Sterneck et al., 1992). Several lines of recent evidence have demonstrated that C/EBP mRNA increases markedly during differentiation of 3T3-Ll preadipocytes to adipocytes and that C/EBP can transactivate the promoters of the adipocyte-specific genes, 422 (aP2), SCDl and GLUT4 (insulin-responsive glucose transporter) (Christy ef al., 1989). Furthermore, adipocytedifferentiated 3T3-Ll cells exhibited a rapid TNFinduced decrease in. C/EBP protein le\.el and a *.:ciprocal increase in NF-IL6, preceding TNF,nduced phenotlrpic de-differentiation of the cells !?or er ol., 1992). Taken together, these facts indica[e an important role for the acute phase-induced change in the NF-IL6/C/EBP ratio in determining the expression of those genes whose products are posili\,ely or negatively regulated in inflammator) and ;icute phase responses.

6

735

Post-translational modification of NF-IL6

Some evidence indicated that NF-IL6 was regulated at a post-translational level as well. In the hepatoma cell line HepG2, a gel retardation assay revealed a drastic quantitative increase in NF-IL6 binding activities after IL6 stimulation. However, Western blot analysis did not show any change in amounts of the proteins interacting with the NF-IL6 specific antibody before and after IL6 stimulation. This indicates that the increased binding activities may be due to some modification of the pre-existing NF-IL6 protein in HepG2 cells. Our preliminary data indicate that IL6 stimulation causes increased phosphorylation of specific sites of NF-IL6 protein in Hep3B cells. Poli el al. (1990) also demonstrated that post-translational modifications play an important role in the activation of transcription through IL6DBP (a rat homolog of NF-IL6) as well as the increased DNA binding activities after IL6 stimulation. Recently, Metz and Ziff (1991) have presented an interesting finding that increased NF-IL6 binding following forskolin treatment in the rat pheochromocytoma PC12 cell line is due to a translocation of NF-IL6 from the cytoplasm to the nucleus. In contrast to the case of PC 12 cells, NF-IL6 is already present in nuclei of HeLa cells, and hepatoma cells.

Homo- and -hetero-dimer formation C/EBP family members

between the

There are four members of the C/EBP family. These include C/EBP, NF-IL6 (also known as LAP, IL6DBP, AGP/EBP), Ig/EBP-1, and NF-IL69 (CYEBPG) (Descombes er al., 1990; Chang ef al., 1990; Roman er al., 1990; Kinoshita et a/., 1992; Cao et al., 1991). These members of C/EBP family recognize the same nucleotide sequences, but exhibit distinct patterns of espression and may be functionally different. C/EBP is espressed in adipose, liver, and placental tissues \vhich play a vital role in energ) metabolism. The expression of NF-IL6 and NF-IL63 is normally suppressed but drastically and rapidly induced in many tissues by LPS or several inflammatory cytokines including ILl, TNF and IL6. In contrast, Ig/EBP-1 mRNA is ubiquitously expressed and is not influenced by LPS treatment. Combinatorial control of gene espression as a consequence of the formation of homodimeric or heterodimeric transcription factors is now \videly acccpted to be an important mechanism in increase of the repertoire of transcription factors. The formation of heterodimeric cornpleses brt\veen members of the C/EBP family has been also demonstrated. Differential utilization of hnmo- and hererodimers among the C/EBP family members as \vell as modification ((fs. phosphorylation) of each member may modulate rhe

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d6rl1 FOR I/A! Ih’ I.\lklUh:OLOG

expression of the different genes which are involved in the process of signal transduction and cell differentiation. References Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kiinoshita, S., Nishio, Y., Nakajima. T., Hirano. T. & Kishimoto, T. (1990), A nuclear factor for IL-6 expression (NF-IL6) is a member of C/EBP family. EMBO J., 9, 1897-1906. Cao, Z., Umek, R.M. B: hIcKnight, S.L. (1991), Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-Ll cells. Genes Develop., 5, 1538-1552. Chang, C.J., Chen, T.T., Lei, H.Y., Chen, D.S. & Lee, S.C. (1990). Molecular cloning of a transcription factor, AGP/EBP, that belongs to members of the C/EBP family. Mol. cell. Biol., 10, 6642-6653. Christy, R.J.. Yang, V.W., Ntambi, J.M., Geiman, D.E., Landschulz, W.H., Friedman, A.D., Nakabeppu, Y., Kelly, T.J. & Lane, M.D. (1989), Differentiationinduced gene expression in 3T3-Ll preadipocytes : CCAAT/enhancer binding protein interacts with and activates the promoters of two adipocyte-specific genes. Genes Develop., 3, 1323-1335. Descombes, P., Chojkier, M., Lichtsteiner, S., Falvey, E. & Schibler, U. (1990), LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Devefop., 4, 1541-1551. Isshiki, H., Akira, S., Tanabe, O., Nakajima, T., Shimamoto, T., Hirano, T. & Kishimoto, T. (1990), Constitutive and IL-l inducible factors interact with the IL-1 responsive element in the IL-6 gene. Mol. cell. Biol., 10, 2757-2764. Isshiki, H., Akira, S., Sugita, T., Nishio, Y., Hashimoto, S., Pawlowski, T., Suematsu, S. & Kishimoto, T. (1991), Reciprocal expression of NF-IL6 and C/EBP

J’

in hepatocyte: possible inwlvement of SF-IL6 in acute phase protein _ecneespression. ,\.t’w Biol., 3. 63-70. Kinoshita. S.. Akira, S. 6: Kishimoto, T. (1992). A member of the C/EBP family, NF-IL6$, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc. nat. Acad. SC;. (Wash.), 87, 1473-l-176. Landschulz, W.H.. Johnson, P.F., Adashi, E.Y., Graves, B.J. & McKnight, S.L. (198S), Isolation of a recombinant copy of the gene encoding C/EBP. Gerres Develop., 2, 786-800. Metz, R. 6r Ziff, E. (1991). CAMP stimulates the C/EBPrelated transcription factor rNFIL-6 to rrans-locate to the nucleus and induce c-fos transcription. Genes Develop., 5, 1754-1766. Natsuka, S., Isshiki, H., Akira, S. & Kishimoto, T. (1991), Augmentation of the haptoglobin production in Hep3B cell line by a nuclear factor NF-IL6. FEBS Letters, 291, 58-62. Natuska, S., Akira, S., Nishio, Y., Hashimoto, S., Sugita, T., Isshiki, H. & Kishimoto, T. (1992), Macrophage differentiation specific expression of NF-IL6, a transcription factor for IL-6. Blood, 79, 460-466. Poli, V., Mancini, F.P. & Cortese, R. (1990), IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell, 63, 643-653. Roman, C., Ptatero, J.S., Shuman, J. & Calame, K. (1990). Ig/EBP-1: a ubiquitously espressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Develop., 4, 1404-1415. Ron, D., Brasier, A.R., McGehee, R.E. & Habener, J.F. (1992), Tumor necrosis factor-induced reversal of adipocytic phenotype of 3T3-Ll cells is preceded by a loss of nuclear CCAAT/enhancer binding protein (C/EBP). J. clin. Invest., 89, 223-233. Sterneck, E., Muller, C., Katz, S. & Leutz, A. (1992), Autocrine growth induced by kinase type oncogenes in myeloid cells requires AP-I and NF-M, a myeloid specific, C/EBP-like factor. EMBO J., 11, 115-126.

NF-IL6 and gene regulation.

46111FORUM 734 IA’ I&~IV~UI\‘OLOG L., Soreq, H., Nir, U., \\‘allach. D., Perricaudet, hl., Tiollais, P. & Revel, M. (19SO), Two interferon mRN.-\s...
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