Deciphering the early-response transcription factor networks in mast cells

IMMUNOLOGY

Institutes of Health, Bethesda, MD 20892-1890, USA; Thomas MacDonald is at the Medical College of St Bartholomew's Hospital, London, UK ECIA 7BE; Herbert Lochs is at the Dept of Internal Medicine IV at the Charitd of the Humboldt University, 10117 Berlin, Germany. References 1 K6hne, G., Schneider, T. and Zeitz, M. (1996) Bailliere's Clin. Gastroenterol.10, 427-442 2 Kerneis, S., Bogdanova, A., Kraehenbuhl, J.R and Pringault, E. (1997) Science277, 949-952

3 Toy,L.S., Yio, X.Y., Lin, A., Honig, S. and Mayer, L. (1997) J. Clin. Invest. 100, 2062-2071 4 Austrup, F., Vestweber, D., Borges, E. et al. (1997) Nature 385, 81-83 5 Weiner, H.L. (1997) Immunol. Today18, 335-343 6 Marth, T., Strober, W. and Kelsall, B.L. (1996) J. lmmunol. 157, 2348-2357 7 Zeitz, M., Quinn, T.C., Graeff, A.S., Schwarting, R. and James, S.P. (1989) Dig. Dis. Sci. 34, 585-595 8 Powrie, R, Carlino, J., Leach, M.W., Mauze, S. and Coffman, R.L. (1996) J. Exp. Med. 183, 2669-2674

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9 Neurath, M.F., Fuss, I., Kelsall, B.L., Sttiber, E. and Strober, W. (1995) ]. Exp. Med. 182, 1281-1290 10 Duchmann, R., Kaiser, I., Hermann, E., Mayet, W., Ewe, K. and Meyer zum B6schenfelde, K-H. (1995) Clin. Exp. lmmunol. 102, 448-455 11 Romagnani, S. (1996) Clin. Immunol. hnmunopathol. 80, 225-235 12 Pender, S.L., Tickle, S.P., Docherty, A.J., Howie, D., Wathen, N.C. and MacDonald, T.T. (1997) J. lmmunol. 158, 1582-1590 13 Neurath, M.E, Pettersson, S., Meyer zum B~schenfelde, K-H. and Strober, W. (1996) Nat. Med. 2, 998-1004

Deciphering the early-response transcription factor networks in mast cells Hovav Nechushtan and Ehud Razin External stimuli mediate signaling

~

cascades in mast cells that lead ast cells are tissue-localized effector cells of the

to the activation of muitiple

in primary cultures of mouse bone-marrowderived mast cells (BMMCs) 3,4. However,

transcription factors. Here,

whereas c-Kit ligand and IL-3 are among the

Hovav Nechushtan and

most potent inducers of mast cell proliferation, stimulation of mast cells via aggregation of their surface FceRI leads to a temporary reduction in their rate of proliferation 3,4. Thus it seems that the induction of

i m m u n e response. Because they are often the first immune cells to interact with airborne and ingested antigens and noxious agents, they are essential for the induction of local inflammatory responses in many tissues. These cells can

Ehud Razin discuss the role played

initiate the inflammatory process when FceRIlx}und IgE receptors on the cell surface are

expression and mast cell functions.

crosslinked with antigen. Activated mast cells immediately release a variety of amines, proteases, proteoglycans and lipid-derived mediatorsL In addition, the relatively late release of cytokines by these activated cells seems to play an essential role in the inflammatory process. A more delayed response also occurs and is characterized by an increase in the transcription of certain genes. However, little is known of the transcription factors that regulate these late responses in mast cells. Multiple transcription factors are induced in the cytoplasm in activated cells and then shuttled to the nucleus where they exert positive or negative control over gene expression (see Fig. 1). One of the most extensively analyzed transcription factors involved in gene Pl: SO 162' 5699ic~)01

~1

by some of these transcription factors in modulating gene

AP-1 components per se is not sufficient for triggering mast cell proliferation. Interestingly, the m R N A levels of at least one Jun

regulation in response to extracellular signals is AP-1. This transcription factor has been found to mediate gene regulation by various

family member, JunD, in mast cells are practically unaffected by external stimuli 4. In fibroblasts and erythroleukemia cells,

cellular stimuli such as growth factors, cytokines and tumor promoters. AP-1 comprises either homo- or heterodimeric complexes of Jun and Fos proteins, which differ slightly in DNA-binding specificity and trans-activation functions 2.

c-Jun serves as a growth promoter: inhibition of expression of c-Jun by the addition of either a blocking antibody to c-Jun or m R N A antisense reduces proliferation of these cells5& By contrast, application of

A P - I in m a s t cells Activation of mast cells by aggregation of their FceRI or by exposure to c-Kit ligand or interleukin 3 (IL-3) in each case induces the accumulation of both c-Fos and c-Jun m R N A

c-Jun antisense oligodeoxynucleotides to IL-3-treated mast cells significantly enhanced IL-3-induced mast cell proliferation rates 7. Therefore, c-Jun is apparently an endogenous suppressor of mast cell growth. This suggests that, for example, interruption of c-Jun's function could contribute to the autonomous proliferation of mast cells as ~

998 Isevie %tepee L:~

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Fig. I. Early-response transcription factor networks in mast cells. Leucine zipper elements are depicted as 'tails' and the basic helix-loop-helix elements as two 'barrels" connected by a thin connector. As depicted, different combinations of basic helix-loop-helix-leucine zipper proteins can bind to one another as homo- or heterodimers in mast cells (see text for details); c-Fos has been shown to bind USF2, presumably through its teucine zipper; and AP-1 has been found to modulate NF-AT transcriptional activity. Abbreviations: MITF, mi transcription factor; NF-AT, nuclear factor of activated T cells; USF, upstream stimulating factor. seen in mastocytosis. Elucidating the mechanisms whereby c-Jun negatively controls cellular proliferation may provide significant insights into the molecular events associated with mast cell proliferation. In contrast to the effect of IL-3 and Fc~RI aggregation, IL-4, a cytokine that has no direct effect on BMMC proliferation but synergizes with IL-3 in promoting growth of these cells, failed to induce AP-1 DNAbinding activity in mast cells8. Whether this difference in AP-1 DNA-binding activity is one of the reasons for the different proliferative responses of mast cells to these two growth factors is not yet known. In addition to protein tyrosine phosphorylations, agents that activate mast cells9

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induce the regulation of protein kinase C (PKC) activity, resulting in serine or threonine phosphorylation of multiple protein substrates. The family of PKC isozymes regulates a wide variety of physiological processes in diverse cell types. These processes include tumor promotion, membrane receptor function, cell differentiation and proliferation ~°. The effect of activating different PKC isozymes varies according to the stimulus and the ceil type. Since the various PKC isozymes differ in their requirement for Ca 2+, diacytglyceroland lipids, these factors, along with substrate specificity and topological organization, may be crucial in determining which isozyme is activated by a given stimulus 1°. Evidence for the require-

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ment of PKC activity in gene expression has accumulated over the past 20 years. In T cells, for instance, it was dearly demonstrated that cyclic AMP alters PKC-induced transcription regulation of members of the Jun and Fos familyn. Furthermore, PKCs have been implicated in the regulation of mast cell growth and in the expression of AP-1 components in response to Fc~RI aggregation3,12. The AP-1 transcription complex is known to modulate the effect of various transcription factors. For example, altered ratios of c-Fos to c-Jun resulting from glucocorticoid treatment reverse the effect of glucocorticoids upon the activity of the proliferin promoter 13. The nuclear factor of activated T cells (NF-AT) is a protein complex that cooperates with AP-1 under most circumstances ~4. The nomenclature for this transcription factor is rather misleading since, like AP-1, there are several transcription complexes that can bind to the NF-AT recognition motif with or without cooperat,ing with AP-1 (Ref. 15). The role of FceRIIgE-induced NF-AT has been investigated in different systems by different laboratories. In rat basophilic leukemia cells and rat-derived BMMCs, it was shown that aggregation of FceRI induced the binding of a complex to the NF-AT DNA binding site and furthermore that an oligonucleotide of the AP-1 recognition sequence or a combination of anti-Jun and anti-Fos antibodies could prevent this binding 16. This binding could be reproduced by a combination of PKC activation and a Ca 2+ ionophore. These results demonstrate that NF-AT was induced in mast cells by Fc~RI-bound IgE crosslinking and that Fos and Jun were important for the binding of the NF-AT complex 16. Several NF-AT-regulated genes have been identified in mast cells by Baumruker and his colIeagues15,17.They showed that in mast cells this transcription factor has an important role in the induction of tumor necrosis factor c~ (TNF-c0, IL-5 and the chemokine monocyte chemotactic protein 3 (MCP-3; MARC). Interestingly, NF-AT was shown to bind to the MARC promoter without the AP-t cofactor, with binding probably independent of PKC activation but dependent upon p21 ras (Ref. 17). These results imply that the activation of different

IMMUNOLOGY

promoters by NF-AT has distinct requirements for AP-1. Weiss's group carried out a comparative study of the binding of proteins to a sequence from the IL-4 promoter that is crucial in T cells for the activationdependent transcription of this gene 18. These studies identified two different kinds of NF-AT in mast cells, which were of distinct molecular mass compared with NF-AT derived from T cells. Furthermore, AP-1 bound to this DNA sequence only in activated T cells and not in activated mast cells. Although different cellular systems and different DNA sequences were used, these data suggest that different genes may use distinct NF-AT-binding sites and selectively recruit different members of the NF-AT family.

USF2 in mast cells c-Jun-containing protein complexes have been immunoprecipitated from mast cells to obtain information on the specific interactions of c-Fos and c-Jun proteins following FceRI aggregation due to IgE-Ag stimulation19. Surprisingly, although total c-Fos protein levels were substantially increased by IgE-Ag induction, an increase in the levels of c-Jun-bound c-Fos was not detected; thus, at least in these systems, most of the IgE-induced c-Fos protein was not bound to c-Jun in the AP-1 complex. Intriguingly, some of the newly synthesized c-Fos was found associated with the upstream stimulating factor 2 (USF2)19. The ability of c-Fos to interact with USF2 may allow receptorspecific regulation of gene expression by increasing the diversity of DNA-binding protein complexes that regulate gene transcription. USF2 is a transcription factor that is characterized by a leucine zipper adjacent to the basic helix-loop-helix domain. It has been proposed to play an important role in the regulation of cellular proliferation and the transcription of many genes. IL-3 induces USF2 protein synthesis in murine mast cells, but it does not significantly affect the level of USF2 mRNA in these cells 2°. Polysomal fractionation and RNA analysis have indicated that this translational regulation is mostly the result of increased USF2 translational efficiency. Moreover, PKC inhibitors prevented both the induction of USF2 protein synthesis and

the increase in USF2 translational efficiency in IL-3-activated mast cells. Considering the proposed role of USF in proliferation, it seems that translational regulation of USF2 might have an important role in cellular growth regulation. Recently an important role for translational regulation in T-cell activation was proposed based on an assay that detected a shift in the translation of around 5% of T-cell mRNA following activation2L While it seems highly likely that a similar translational induction of a variety of genes occurs in activated mast cells, it appears that translational activation of key transcription factors can provide the cell with a mechanism for transcriptional induction of many more genes at a later phase. Another general mechanism that has an important role in the rapid activation of transcription factors is the translocation of transcription factors into the nucleus. Immunohistochemical studies indicate that activation of mast cells through their FceRI receptor or IL-3 receptor induces the nuclear translocation of USF2 and that inhibition of this translocation by cell-permeable peptides leads to a decrease in cellular viability22,

MITF in mast cells Mice harboring a mutation in the mi gene display a variety of abnormalities, including microphthalmia, depletion of skin melanocytes, deafness, a defect in osteoclasts, and a major decrease in mast cell number and function23. Kitamura and coworkers showed that although m i / m i mice are mast-cell deficient, splenic cells from these mice develop into mast cells when cultured with conditioned medium 24. However, these cells are different from mast cells grown from normal mice in many important aspects. For example, when mast cells from m i / m i mice are co-cultured with fibroblasts, they fail to enter the S phase and gradually disappear from the tissue culture plates 25. Furthermore, they express only low levels of c-Kit and nerve growth factor 140 kDa receptors - two growth factor receptors with important roles in mast cell development25,26. Despite the possible crucial role played by the protein encoded by the mi gene - the

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mi transcription factor (MITF) - in mast cell development and function, its mRNA and protein synthesis in these cells is only beginning to be characterized. By using a specific rabbit polyclonal anti-MITF antibody, IL-3, IL-4 or aggregation of mast cell FceRI were shown to induce the synthesis of MITF in these cells 27. None of these stimuli significantly affected the level of MITF mRNA in the mast cells at any of the time points tested. In addition, using this specific antiMITF antibody, an increase in MITF protein synthesis was demonstrated during differentiation of mast cells from their bone marrow cell precursors. Moreover, a complex containing MITF bound to USF2 was detected only in activated mast cells. Thus, stimulation of mast cells by a variety of stimuli elicits a signaling pathway that regulates MITF expression.

Future directions Elaboration of the details in the response of transcription factors in activated mast cells has only just begun. A complex network of transcription factors seems to be involved. Further study of this network might provide us with a better understanding of mast cell activation and hopefully also with the necessary information for formulating more specific and effective drugs for certain illnesses. Modulation of the transcription factors discussed here, and therefore mast cell protein expression, might have important clinical consequences. Since mast cells have an important role in the skin, conjuctiva and lungs, therapeutic intervention targeted at these tissues might avoid significant systemic side effects and still prove to be of clinical relevance. One approach might be to apply antisense nucleotides, which have been used successfully in mast cells 7,28. Another approach might be to use cellpermeable peptides in order to inhibit the activities of selected transcription factors 29. Chemical inhibitors of transcription factors, which have been employed, for example, to modulate NF-AT activity3°, may also prove valuable. Topical or regional application of these types of modulators might speed up their introduction into the clinic as therapeutic agents.

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Hovav

Nechushtan

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and

Ehud

Razin

([email protected]) are at the Dept of Biochemistry, Hebrew University-Hadassah

Medical

School, P O Box 12272, Jerusalem 91120, Israel.

References 1 Costa, J.J., Weller, P.F. and Galli, S.J. (1997) ]. Am. Med. Assoc. 278, 1815-1822 2 Karin, M., Liu, Z.G. and Zandi, E. (1997) Curr. Opin. Cell. Biol. 9, 240-246 3 Baranes, D. and Razin, E. (1991) Blood 78, 2354-2366 4 Tsai, M., Tam, S.Y. and Galli, S.J. (1993) Eur. J. Immunol. 23, 867-872 5 Kovary, K. and Bravo, R. (1991) Mol. Cell. Biol. 11, 2451-2459 6 Smith, M.J. and Prochownik, E.V. (1992) Blood 79, 19665-19672 7 Chaikin, E., Hakeem, I. and Razin, E. (1994) J. Biol. Chem. 269, 8498-8503 8 Chaikin, E., Hakeem, I. and Razin, E. (1995) Int. Arch. Allergy Immunol. 107, 57-59 9 Nechushtan, H. and Razin, E. (1996) Crit. Rev. Oncol. Hematol. 23, 131-150

10 Newton, C.A. (1997) Curr. Opin. Cell Biol. 7, 161-167 11 Tamir,A. and Isakov, N. (1994) J. Immunol. 152, 3391-3399 12 Razin, E., Szallasi, Z., Kazanietz, M., Blumberg, P.M. and Rivera, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7722-7726 13 Diamond, M.I., Miner, J.N.,Yoshinaga, S.K. and Yamamoto, K.R. (1990) Science 249, 1266-1272 14 lain, J.P., McCaffrey, G., Valge, A.V. and Rao, A. (1992) Nature 356, 801-804 15 Baumruker, T., Pendl, G.G. and Prieschl, E.E. (1997) Int. Arch. Allergy Immunol. 113, 39--41 16 Hutchinson, L.E. and McCbske~; M.A. (1995) J. Biol. Chem. 270, 16333-16338 17 Prieschl, E.E., Gouilleux-Gruart, V., Walker, C., Harrer, N.E. and Baumruker, T. (1995) J. Immunol. 154, 6112-6119 18 Weiss, D.L., Hural, J., Tara, D., Timmerman, L.A., Henkel, G. and Brown, M.A. (1996) Mol. Cell. Biol. 16, 228-235 19 Lewin, I., Nechushtan, H., Ke, Q. and Razin, E. (1993) Blood 82, 3745-3751 20 Zang, Z.C., Nechushtan, H., Jacob-Hirsch, J.,

Avni, D., Meyuhas, O. and Razin, E. (1998) Oncogene 16, 763-769 21 Garcia-Sanz, J.A., Mikulits, W., Livingstone, A., Lefkovits, I. and Mullner, E.W. (1998) FASEB J. 12, 299-306 22 Frenkel, S., Kay, G., Nechustan, H. and Razin, E. ]. Immunol. (in press) 23 Moore, K. (1995) Trends Genet. 11,442-448 24 Jippo-Kanemoto, T., Adachi, S., Ebi, Y. et al. (1992) Blood 80, 1933-1939 25 Ikeda, H., Adachi, S., Kasugai, T. et al. (1992) Blood 80, 1454-1462 26 Jippo, T., Ushio, H., Hirota, S. et al. (1994) Blood 84, 2977-2983 27 Nechushtan, H., Zhang, Z., Razin, E. et aL (1997) Blood 89, 2999-3008 28 Lewin, I., Jacob-Hirsch, J., Zang, Z.C. et al. (1996) J. Biol. Chem. 271, 1514-1519 29 Lin, Y.Z., Yao, S.Y., Veach, R.A., Torgerson, T.R. and Hawiger, J. (1995) J. Biol. Chem. 270, 14255 30 Kuromitsu, S., Fukunaga, M., Lennard, A.C., Masuho, Y. and Nakada, S. (1997) Biochem. Phaemacol. 54, 999-1005

Transplantation treatment for diabetes Richard M. Smith and Tom E. Mandel Islet transplantation in the treatment of insulin-dependent he

treatment

of

insulin-

diabetes mellitus is making rapid

d e p e n d e n t (type I) diabetes mellitus (IDDM) is currently

progress. Vascularized pancreas

the focus of m u c h attention,

allotransplantation is now an

with the possibility of prevention or cure at the forefront of research. In patients with established disease the replacement of the

accepted treatment but has major limitations. A recent meeting*

destroyed islets of Langerhans by transplantation is an attractive option but

discussed alternative approaches, particularly islet

still faces m a n y problems, not least being the shortage of h u m a n organ donors.

Pancreas versus islet allotransplantation Pancreas allotransplantation is usually performed in patients with diabetes who also need a renal allograft. It provides excellent glycaemic control but does little to ameliorate their other, usually severe, diabetic

xenotransplantation.

complications (G. Weir, Boston, MA). H o w ever, in IDDM only the islets of Langerhans need replacement. Islet autotransplantation after pancreas resection protects about 70% of patients from developing diabetes. Similarly, in patients with abdominal malignancy rendered diabetic by extensive upper ab-

planted with a live r succesfully prevent diabetes. Thus, islet transplantation is technically possible if the problems of rejection and graft viability can be overcome. Although slowly improving, the pooled Islet Transplant Registry results of islet allografts in IDDM show that only 10% of patients ever become independent of a need for exogenous insulin and even fewer become independent for an extended period. Recent experience from the University of Giessen is more encouraging: of 12 patients transplanted, nine had some islet function at one year and four were independent of exogenous insulin (for further information on clinical islet transplantation visit the International Islet Transplant Registry website h t t p : / / w w w , med.uni-giessen.de/itr/). Weir Pll: 50167 5699(98)01323 r

© 1998 Elsevier Soence Ltd

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dominal resection (including removal of liver and pancreas), islet allografts trans-

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*The meeting 'Transplantation Treatment for Diabetes: Where Next?' was held at Bristol, UK, on 5 June 1998.