immediate-early genes induced by antigen receptor stimulation

Immediate-early genes induced by antigen receptor stimulation

Kathleen Kelly and Ulrich Siebenlist

National Institutes of Health, Bethesda, USA

Immediate early genes induced by triggering of the TCR frequently encode proteins that act in signal transduction cascades. Recent advances concerning several immediate-early proteins have been made, including signal-induced regulation of NF-KB by IK-B, the role of Nur77 in T-cell selection and

apoptosis, and the function of PAC-1 in regulating the Ras/ERK pathway.

Current Opinion in Immunology 1995, 7:327-332

Introduction

Following the triggering of TCR, s expressed by T cells, a genetic program is induced that regulates the subsequent growth and differentiation of these cells. This differentiative response is complex, and involves an ordered cascade of gene transcriptional and translational events (Fig. 1). The first genes transcribed following activation have been termed immediate-early (IE) genes as their expression is rapidly activated within minutes to a few hours following receptor ligation without requiring de novo protein synthesis. Not unexpectedly, IE genes encompass several functional categories. In general, many IE genes appear to encode proteins that can act as pleiotropic regulators of cellular activation, such as transcription factors and proteins involved in signal transduction cascades (Table 1). In this review, we will briefly discuss several transcription factors encoded by IE genes. In particular, recent advances in defining the action of the IE transcription factor Nur77 in T-cell apotosis will be presented. Finally, the role of the IE phosphatase PAC-1 (phosphatase of activated cells) in downregulating signal transduction eminating from the triggering of TCRs will be reviewed.

Although many of the IE genes induced in T cells are frequently inducible in other cell types as well (such as fibroblasts, monocytes and regenerating liver) these genes may nevertheless encode functions that are used in a cell-specific fashion. IE genes often mediate signals that can result in multiple possible outcomes, depending on the distinct cellular milieu that receives and interprets these signals. Such an example is Nur77, a ubiquitous IE gene product, which appears to be involved in apoptotic responses in only immature T cells.

Another principle which emerges is that many IE genes encode signal transduction molecules that are already present and functional in resting cells, albeit sustained by low mKNA levels. This is exemplified by NF-rd3

which is a collection of transcription factor complexes that are preformed in resting T cells where they function as critical transducers of signals from the cytoplasm to the nucleus; these complexes are also products of IE genes.

A final feature of the IE genetic response is the built-in negative control of activation, which is subserved by some of the induced proteins (Fig. 2). Thus I~B-0t, the inhibitor of NF-K.B, is immediately induced upon activation. Another example of negative control mediated by a different functional class of IE genes is PAC-1, a mitogen-activated protein (MAP) kinase phosphatase that acts to downregulate extracellular signal-related kinase (ERK)I and EKK2 activation following triggering of TCRs.

Immediate-early transcription factors

Among the products of IE genes in T cells are many transcription factors. These include most, but not all, members of the Jun/Fos family that consists of basic region, leucine-zipper (bZip) transcriptional activators (in particular c-Jun, JunB, c-Fos, Fra-1 and FosB); these proteins generate activating protein-1 (AP-1) activity in cells through homodimerization and heterodimerization [1,2]. The Jun proteins are already present in resting cells and can be activated via phosphorylation by the JNK kinase; activated Jun then participates in stimulating transcription of its own gene [3]. The Jun/Fos proteins have what appear to be T cell specific functions through combinatorial association with the transcription factor nuclear factor of activated T cells (NFAT)p [4]. Together, Jun-Fos dimers and NFATp form a potent transcriptional activation complex which binds to specific DNA regulatory sites of many cytokine genes in T cells in order to transactivate their expression in response to signals. Although NFATp is constitutively expressed in T cells and is activated by a calcium/calcineurin phosphatase-mediated signal,

Abbreviations AP-l--activating protein-1 ; ERK--extracellular signal-regulated kinase; IE--immediate-early; IL--interleukin;

MAP--mitogen-activated protein; NFAT nuclear factor of activated T cells; PAC--phosphatase of activated cells; TCR---T-cell receptor.

© Current Biology Ltd ISSN 0952-7915 327

328 Lymphocyte activation and effector functions

Resting T cell Competent T cell

9 Ag/MHC Mitogen

IE gene induction within -4 hr X

, ~> E Cytokines/ growth factors Receptors N I L-2 R Oncogenes/ ~ iL-2 transcription factors " " Signal transducers

-20 hr

Proliferating T cell Effector functions

~>

© 1995 Current Opinion in Immunology

the necessary levels of Jun/Fos required for complex formation appear to be achieved only after expression of these proteins is induced.

Activation of T cells and many other cells leads to the immediate induction of members of the Egr family of transcription factors that contain zinc-fingers (Egr-l-Egr-4, see Table 1 for other names) [5-8]. All members of this family share highly homologous zinc fingers which recognize the same DNA target sequence, although the remaining parts of these proteins are quite divergent. The biological functions of Egr proteins remain somewhat elusive. The Egr factors have been imphcated in positive transcriptional control of several genes, although some data indicates a potential for negative control as well [5,6]. Most hkely, the precise functions and predicted competitive interactions of Egr proteins may depend on the cellular environment and the context in which a bound Egr factor finds itself in a given promoter/enhancer.

IL-2 TGF-[~ IE gene receptors cAMP

BB

Go, ~> S

+

I~:-B

PAC-1 © 1995 Current Opinion in Immunology,

Fig. 2. The role of IE gene products in signal transduction. Transcrip- tion/translation of IE proteins is induced by signaling pathways initiated by triggering of the TCR. One role of IE proteins (e.g. hcB-0t and PAC-1) is to downregulate these primary signaling pathways. IE proteins also act in subsequent signaling pathways, such as the IL-2 pathway, or possibly in signaling pathways regulating progression beyond the restriction point at the GJS phase boundary. TGF, transforming growth factor.

Several of the proteins encoding NF-KB activity are among the IE gene products in T cells [9,10,11°]. These include the p50 and p52 subunits (and their precursors p105 and pl00, respectively) and c-Rel. RelB is not significantly expressed in T cells and expression of p65

Fig. 1. Sequential events in T-cell activation. IE gene transcription occurs mostly within the first four hours after triggering of the TCR with antigen (Ag)/MHC or mitogen. Representative functional categories of IE proteins are listed (including regulators of immune function, proliferation and apoptosis). Subsequent cell cycle progression to S phase driven by IL-2 interaction with its receptor (R) is shown.

is only minimally induced. NF-KB generated from low constitutive m R N A levels in resting cells is necessary to induce transcription of many IE genes, including its own genes [11°]. Induced synthesis of NF-KB is essential to sustain signaling through NF-KB over time to allow full expression of effector functions in T cells.

IKB-ot, the inhibitor of NF-KB, is also immediately induced by activated NF-KB itself [12°]. The induced synthesis of IKB-ct is necessary for the transient nature of NF-KB activation as initial signaling leads to rapid and near-total degradation of the pre-existing IKB-0t [12°,13,14"]. In the absence of further signaling, newly generated IKB-ct quickly represses already activated NF-KB, most likely to limit unintended tissue damage by NF-KB-dependent expression of immune- and stress-responsive proteins. In contrast to IKB-Ct, the recently cloned IKB-~ cannot be induced in its expression by NF-KB [15"]. Therefore, agents like IL-1 and hpopolysaccharide which inactivate IKB-~ (tumor necrosis factor-Or and phorbol 12-myristate 13-acetate [PMA] do not) may allow for a more prolonged activation of NF-KB as this inhibitor is unable to participate in the rapid downmodulation.

The ubiquitous IE protein Nur77 (see Table 1 for other names that have resulted from independent cloning efforts) is necessary for TCR-mediated apoptosis of immature thymocytes and of T-cell hybridomas, suggesting that it is involved in the negative selection of T cells during thymus development [16°',17°°]. Nur77 is a member of the large family of steroid/thyroid hormone receptors. Although many of these zinc-finger containing transcription factors are known to be regulated in their activities by steroid/thyroid ligands, Nur77 is one of several orphan receptors with no known ligand.

Nur77 can be induced by signals generated from a variety of distinct receptors in diverse cell types, suggesting a general role in signal responsiveness. Its distinct function during TCR-mediated apoptosis in immature T cells correlates with more prolonged and quantitatively higher levels of induced mRNA and protein following an apoptotic signal, as opposed to a proliferative signal. The requirement of Nur77 for TCR-mediated apoptosis (but not for dexamethasone-induced apoptosis) was demonstrated by blocking apoptosis with a dominant

Table 1. Proteins with signaling functions* encoded by select immediate-early genes in T ceils. (a) Transcription factors; (b) membrane receptors/intracellular signaling proteins.

(a) c-Jun, JunB, c-Fos, FosB, Fra-t

p50/p105 (NF-KB1, AT243t), p52/p100 (NF-KB2, AT189), c-Rel I

Nut77 (NGFI-B, Tisl, N10, AT416, TR3, Nak-1), NOT (Rnr-1, Nurrt, HZF-3), Minor/AT229 (Nut88)

Egr-1 (Krox-24, NGFI-A, AT225, Zif/268, TisS), Egr-2 (Krox-20, AT591), Egr-3 (Pilot), Egr-4 (AT133, NGFI-C)

HLXi (HB24), HoxB7 (Hox-2.3)

c-Myc

IRF-t

HLH462

(b) Gem (AT270)

Pac-t (AT120)

Traf-1 (AT563)

CD69 (AT237)

IL-2 receptor-or

Jak-3

*Examples of T-cell activation molecules that play a role in intracellular signal transduction. Several other important functional categories of IE genes exist, such as chemokines/cytokines, that are beyond the scope of this review (for examples, see [20]). ~ 'AT' clones were originally described in [45].

negative-acting clone of Nur77 [16°°], or by blocking it with an antisense construct of Nur77 [17°°].

It is likely that the biological function of this protein is regulated not only by the level and persistence of its expression, but also by other factors. It is known that Nur77 DNA binding activity can be modulated via phosphorylation, although it is not known if such regulation occurs during apoptosis of T cells or mitogenesis. Furthermore, an inhibitor of Cdk kinases (which regulate the cell-cycle) can physically interact with Nur77 and may thus modulate it in a cell-cycle dependent manner (A Winoto, personal communication). Finally, the immunosuppressant cyclosporin A can block the DNA-binding activity of Nur77, apparently mediating its effect through a domain of Nut77 distinct from the DNA-binding region [18°]. It is thus possible that cyclosporin A blocks the activity of another factor

Immediate-early genes Kelly and Siebenlist 329

that is required to interact with Nut77 for DNA-binding in vivo.

Transgenic mice expressing the dominant negative form of Nur77 appear to block negative selection during T-cell development (A Winoto, personal communication). Furthermore, overexpression of Nur77 in T cells of transgenic animals severely reduces the generation of double- and single-positive T cells, presumably by promoting apoptosis of T cells in the thymus subsequent to the double-negative stage of development (K Bravo, personal communication).

Nur77 is a member of a subfamily of orphan steroid receptors which also includes NOT [19] and Minor (previously isolated as AT229; S Irving, personal communication; see Table 1 for other names). All three members are encoded by IE genes in T cells. The DNA-binding regions of these proteins are highly conserved and they bind to the same DNA recognition element, suggesting potential competitive interactions between these proteins. It is thus hkely that the circuitry that regulates the precise relative level and activity of each individual member may be critical to the overall biological outcome of a signal. The Nur77 family is reminiscent of the Egr family.

The Ras-ERK pathway and downregulation by PAC-1

In T cells, triggering of the T C R induces several signaling cascades which ultimately lead to the early events in T-cell activation including the induction of IE genes [20]. The most recent evidence shows that two major pathways synergize to mediate signal transduction eminating from the TCR; one initiated by an increase in intracellular Ca 2+, and the other involving the activation of the guanine nucleotide binding protein, p21 ras [21°]. An important pathway that lies downstream of Ikas activation sequentially involves activation of Raf, MAP kinase kinase (MEK), and the 'classical' MAP kinases, ERK1 and ERK2 [22 °°] (Fig. 3). ERK activation is mediated by dual phosphorylation within the motif Thr-Glu-Tyr by MEK [23]. A large fraction of the activated ERK translocates into the nucleus [24 °] and phosphorylates transcription factors such as TCF/Elk 1. This pathway is evolutionarily conserved, and its delineation has come from several organisms and differentiated cell types [22°°,25°].

What is the role of the ERK pathway in T-cell activation? The use of constitutively active, and dominant negative, forms of Raf-1 have shown it to be an upstream activator of ERK in T cells [26] and to be required for IL-2 gene expression [27°]. The fact that both Raf-1 and p21 ras function are essential for Jurkat activation is consistent with the model that p21ras couples the TCK to Raf-1 and hence to the MAP kinase cascade, following the paradigm that has been observed in many other systems. There is a suggestion, however, that Raf-1 may have at least one other effector than MEK

330 Lymphocyte activation and effector functions

I=p44=ERK 1 MAPK ~ I1=p41 =ERK 2

(Ser/Thr kinase)

iiiiiiiiiiiiiii i ̧

© 1995 Current Opinion in Immunology I ~ ' 1

Fig. 3. Diagrammatic representation of the major signal transduction pathway generated by triggering of TCR and resulting in ERK] and ERK2 activation. TEY, Thr-Glu-Tyr.

[28]. Using an in vivo model system, E1KK activation clearly appears to be required for the positive selection of thymocytes as shown in transgenic animals expressing a dominant negative form of MEK at high levels in the thymus [29°'].

The induction of MAPK activity following growth factor stimulation of cells appears to be required for the cell's progression to S phase, at least in fibroblasts [30",31]. EIKK activation has been shown to be critical for the induction of AP-1 activity in fibroblasts [32], presumably as a resuk of ERK-mediated transcriptional induction of c-fos [33]. Assuming a similar scenario in T cells, the regulation of IL-2 expression by the MAPK pathway is not unexpected because the AP-1 transcription factor family interacts with several elements within the IL-2 promoter [34].

Experiments with PC12 pheochromocytoma cells sug- gest that the duration of E1KK activation is critical for cell signaling [22"°]. Clearly, it is essential that MAP kinase activation is a reversible process in order for signal transduction to be properly regulated. Two MAP kinase phosphatases have been described, both of which are IE proteins. PAC-1 [35"] was cloned from phytohemagglutinin (PHA) plus PMA activated T cells, and MKP-1 [36] (originally called 3CH134 or CL100) was cloned from fibroblasts stimulated with serum or oxidative stress. PAC-1 and MKP-1 are similar in their catalytic domains and have been shown to possess phosphatase activity against phosphothreonine and phosphotyrosine on ERK1 and EKK2 [37"',38"']. PAC-1 and MKP-1 are also found predominantly in the nucleus where they are positioned to inactive translocated ERKs [35"].

PAC-1 expression is induced in human peripheral blood T cells by agents, such as PMA, anti-CD2, or anti-CD3 that stimulate the activation of E1KKs [39]. PAC-1 protein is detectable within the first half hour, reaches a plateau by one hour, and is maintained for several hours following triggering of the TC1K. The dephosphorylation and consequent inactivation of ERK2 is proportional to the accumulation of PAC-1 [37"']. The kinetics of PAC-1 expression may explain the observation that IL-2 binding to its receptor activates p21ras function [40] but not E1KK2 [41]. As PAC-1 is present during the interval oflL-2 receptor triggering, it may constitutively dephosphorylate and inactivate EIKK effectively downregulating the E1KK pathway during G 1 progression.

Although PAC-1 and MKP-1 are highly similar in their carboxy-terminal halves that contain the catalytic domain, they share only about 30% amino acid identity in their amino-terminal halves. This suggests the possibihty that the two MAPK phosphatases may have distinct regulatory domains. PAC-1 is expressed predominantly in hematopoietic tissues [35"], whereas MKP-1 is expressed in several tissues [36,42]. Therefore, the presumptive regulatory domains of PAC-1 and MKP-1 may have been selected for unique functions (such as substrate recognition or protein stability) in different cell types. Moreover, it is becoming clear that additional genes exist that encode the enzymes highly related to PAC-1 and MKP-1 [43]. Likewise, there are MAPK-related enzymes that are regulated by dual-specific phosphorylation (the JNK family and HOG) and that are effectors of distinct signal transduction cascades [25",44"]. Following the PAC-1/MKP-1 paradigm, it seems highly hkely that there are IE genes encoding dual specific phosphatases for JNK and HOG that are induced by the same stimuh (e.g. tumor necrosis factor or ultraviolet light) responsible for JNK and HOG activation.

Conclusions

In this brief review we have discussed the function and regulation of several IE genes products, in particular, the transcription factors NF-KB and Nur77 and the tyrosine phosphatase PAC-1, a regulator of the MAP kinase pathway. Many IE gene products are modulated in their activities by interacting factors that can be cell-type specific or that are themselves regulated by additional signals. Some IE gene products serve to negatively control initial signal responses. Future efforts will need to focus on the biological targets of the IE, gene products. This includes the elucidation of targets (and functions) specific to individual members of a family of similar proteins, including those of Nur77, Egr and PAC-1. In addition, the complex interactions between IE gene products and the pre-existing cellular environment on one hand, and the modulating influences of additional signals on the other hand, need to be better understood. Ultimately, it is the sum of these complex interactions

Immediate-ear ly genes Kel ly and Siebenl ist ~331

which result in such fundamental cellular decisions o f whether to proliferate or die. The activities of IE gene products serve as integrators o f many cellular signals and are thus central to this critical decisison-making process.

Acknowledgements

We thank all current and past members of our laboratories for their valuable contributions. We apologize for being unable to cite many important references in this brief and necessarily incomplete review.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • " of outstanding interest

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K Kelly, Laboratory of Pathology, Building 10, Room 2A-33, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: [email protected] Ulrich Siebenlist, Laboratory of lmmunoregulation, Building 10, Room 11B-16, National Institutes of Health, Bethesda, MD 20892, USA. E-mail: [email protected]

immediate-early genes induced by antigen receptor stimulation

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