Interleukin-1 signal transduction

Life Sciences, Vol. 59, No. 2, pp. 61-83, 1996 Copyright 0 1996 Elsevier Science Inc. F’rinted in the USA. All rights reserved 0024-3205/96 $15.00 + .OO

PII SOO24-3205(96)00135-X

ELSEVIER

INTERLEUKIN-1

SIGNAL TRANSDUCTION

Jennifer L. Bankers-Fulbright, Kimberly R. Kalli and David J. McKean”

Department

of Immunology,

Rochester, (Received

Mayo Clinic

MN 55905 U.S.A.

in final form May 6, 1996)

Summary Interleukin-1 (IL-l) is primarily an inflammatory cytokine, although it is capable of mediating a wide variety of effects on many different cell types. Nearly every known signal transduction pathway has been reported to be activated in response to IL-I. However, the significance of many of these signaling events is unclear, due to the use of different and sometimes unique cell lines in studying IL-l-initiated signal transduction. Complicating matters further is the lack of association in many studies between identified IL-l-induced signals and subsequent biological responses. In this article, we review what is known about IL-1 receptor signaling and, whenever possible, correlate signaling events to biological responses. Key Wovds: IL-I receptor, signal transduction, gene regulation

The cytokine interleukin-1 (IL-l) plays a central role in the regulation of inflammatory and immune responses, and has been implicated in the pathogenesis of several diseases including diabetes and rheumatoid arthritis (1,2). Although originally described as a co-mitogen required for thymocyte proliferation, IL-I is currently recognized as a cytokine capable of a wide spectrum of effects on numerous cell types. In addition to its well-characterized activities on thymocytes, IL-l also affects other cells of the immune system. IL-l induces K light chain upregulation in pre-B cells, promotes B cell proliferation, and works in combination with other cytokines to induce proliferation of hematopoietic progenitor cells (3,4). However, IL-l is much more than simply an “interleukin” providing communication between cells of the immune system. The effects of IL-l are also observed in the central nervous system, where IL-l can induce slow-wave sleep and fever (5). Additionally, IL- 1 can stimulate bone resorption by osteoclasts, the synthesis of acute phase response proteins by hepatocytes, collagenase production in connective tissue, and has many other activities too numerous to list here. Overall, IL-l is a key player in the body’s response to and recovery from injury. An excellent review of the many biological effects of IL- I has recently been published (6).

Author: D.J. McKean, Ph.D., Guggenheim 3, Mayo Clinic, 200 1st St. SW, Rochester, MN 55905. Phone: (507) 284-8178; FAX: (507) 284-1637; E-mail: [email protected]

‘Corresponding

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IL-1 is actually a family of cytokines that are approximately 18-26% homologous at the amino acid level (7). Two of these members, IL- 1a and IL- 1p, are receptor agonists. IL- 1a and p are synthesized as 3 1 kDa precursors (proIL-1 a and p) that are proteolytically cleaved to generate mature 17 kDa forms of IL- 1. Whereas both proIL- 1a and the mature form of IL- 1a are biologically active, only the mature form of IL-l p is functional. Although the degree of amino acid homology between IL- 1a and p is not impressive, these two proteins have similar three dimensional structures, bind to the same receptors, and have comparable biological activities (8,9,10). The third member of the IL- 1 family is the IL- 1 receptor antagonist (IL-lra). As the name suggests, IL-lra can block the binding and, consequently, the biological activities of IL- 1a and p. Interestingly, a point mutation can convert human IL- 1CLto an IL- lra-like molecule, suggesting that there are critical contact sites in the IL-l receptors (or associated proteins) that are responsible for distinguishing between the IL-l agonists and antagonist (11). Like its ligand, the IL-l receptor (IL-1R) family consists of multiple proteins that have relatively low amino acid homology: the 80 kDa type I IL-1R (IL-lRI), the 68 kDa type II IL-1R (IL-IRII), and a recently described IL-1R accessory protein (IL-1R AcP) (12,13,14). The level of amino acid homology of IL-1RI and IL-l RI1 between species is very high, suggesting strong evolutionary pressure to maintain both receptors. Although it initially appeared that IL- 1RI and IL- lRI1 might have unique tissue distribution, subsequent studies have shown that many cells coordinately express both receptors to varying degrees (13,15). The extracellular domains of the type I and type I1 IL- 1Rs are approximately 28% homologous at the amino acid level and consist of three immunoglobulin-like domains, all of which are required for IL- 1 binding. All three IL- 1 isoforms can bind to both IL- 1Rs, but IL- 1RI1 preferentially binds IL- 1 p. Although there is no evidence that IL- 1RI and IL- 1RI1 form a receptor complex to bind IL-l (16) a recent report suggests that IL-1R AcP may be involved in IL-1 binding (14). The IL- 1R AcP associates with IL- 1RI when complexed with IL- 1a or IL- 1 p, but not when bound to the IL-lra, and increases the affinity of the IL-1RI for IL-l p. The effect of this putative accessory protein on signal transduction by the receptor complex remains to be elucidated. Only the IL- 1RI transduces a signal in response to IL- 1 (15,17). In contrast, the IL- 1RI1 does not transduce a signal upon ligation and has the potential to act as a decoy receptor, preventing IL- 1 from binding to the IL- 1RI (18). This dissimilarity in signaling ability is due to differences in the cytoplasmic domains of the two receptors. The IL-1RI has 215 amino acids in its intracellular domain whereas the IL-lRI1 has only 29. Interestingly, the amino acid sequence of the IL-1RI does not give any indication as to how this receptor signals. It has no endogenous kinase activity and no obvious docking sites for potential transducers. Thus, signal transduction through IL- 1RI has been proposed to involve a conformational change upon ligand binding followed by association and activation of a signal transducing protein(s). The identity of these putative transducers is currently unknown. The incredible sensitivity of this system necessitates extreme efficiency and amplification of signal transduction. Typically, there are less than 200 IL- 1RI on the cell surface and, of these, less than 10% need to be ligated to initiate a biological response (1.5,19). In addition, IL- 1 acts on many different cell types and may not induce the same type of intracellular signals in every situation. Because of these complexities, ‘there is no consensus on the types of second messengers generated by IL-l. However, it is generally agreed that IL- 1 stimulation does not increase intracellular calcium levels nor directly initiate phosphatidylinositol hydrolysis. Otherwise, nearly every other signal transduction system has been reported to be activated in response to IL- I. It is important to keep in mind that these studies frequently utilized unique cell types. Additionally, these studies often examined isolated events in signal transduction cascades and/or have failed to link putative IL-l-induced signals with biologically relevant responses. In this review, we summarize the numerous reports on IL-1 signaling, with special attention given to the most recent literature. In addition, we have correlated IL-1 signaling with its known biological effects whenever possible.

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G Proteins, PK.4 and PKC G proteins, CAMP-dependent kinase (PKA), and protein kinase C (PKC) have all been reported to be directly activated by IL-1 in different cell types. However, much of the evidence for the involvement of these proteins in IL- 1 signal transduction comes from the use of pharmacological inhibitors that have incompletely understood specificity. In other cases, IL- 1-initiated events in unique cell lines have failed to be reproduced in other model systems. However, the potential participation of these transducers in IL-l signaling cannot be casually dismissed. Evidence for and against the controversial involvement of these three signal transducers in IL- 1 receptor signaling is summarized below.

G Proteins G proteins are heterotrimeric signaling molecules that regulate a variety of ion channels and enzymes through the exchange and hydrolysis of guanine nucleotides (reviewed in 20). They are typically activated by cell surface receptors that have seven membrane spanning domains. Each G protein is made up of an a subunit, which binds guanine nucleotides and has GTPase activity, and a second subunit comprised of two proteins, py. In an inactive state, the subunits are associated with each other, and a is in the GDP-bound state. Upon activation, GDP is exchanged for GTP, resulting in a and py dissociation. When dissociated, each of the subunits can regulate the activity of effector proteins. Which effecters are activated depends on which a and py subunits are present in that particular G protein. There are over 20 types of a subunits that can be divided into four major classes: a,, ai, OLD, and a,*. The Q, subunits have been reported to stimulate adenylyl cyclase activity, whereas the ai subunits inhibit this enzyme. The a, and elz subunits have been implicated in phospholipase C (PLC) activation and regulation of sodium and potassium ion exchange, respectively. Additionally, there are 5 known p and 6 known y proteins, allowing for a potential of 30 different py combinations. py subunits have been reported to positively regulate adenylyl cyclase, phosphoinositide 3-kinase (PI3-kinase), PLCp, phospholipase A, (PLA,), and possibly even the mitogen-activated protein kinase (MAPK) pathway (21,22). All of these activities are coordinated by the GTPase activity of the a subunit. When the a subunit hydrolyzes the GTP back to GDP, the subunits reassociate and are once again inactive. IL- 1 signaling was first reported to involve G-proteins in the late 1980s and early 1990s. IL-l stimulation of the murine EL4 cell line was associated with increased membrane binding of GTP-y-S (a non-hydrolyzable GTP analog) and increased GTPase activity in membrane preparations (23). Additionally, a number of groups reported the pertussis toxin sensitivity of IL-1 signal transduction in different cell types (23-26). Pertussis toxin is a holotoxin that consists of a binding and a catalytic subunit. Through the activity of its catalytic subunit, the toxin can irreversibly inactivate the ai class of G proteins. These observations suggested that a G,-like protein was involved in IL-l signal transduction. The evidence for G-protein involvement in IL-l signal transduction seemed strengthened by the ability of pertussis toxin treatment to inhibit IL-l-induced IL-2 gene transcription in EL4 cells (23,26), induction of K light chain synthesis in 7OZ/3 pre-B cells (24), and induction of CAMP in 7OZ/3 cells, YT cells, fibroblasts and human synovial cells (24). Subsequent reports cast doubt on the involvement of G proteins in IL- 1 signal transduction. Ray and colleagues (27) described the absence of IL-l-induced GTP hydrolysis in EL4 and 7OZ/3 cells. Since treatment of these cells with GTP-y-S resulted in higher receptor affiity for IL-l, they suggested that IL- 1 receptor function may indeed be regulated by guanine nucleotides, but not through conventional G proteins. This alternate explanation of the involvement of GTP in IL-1 signal transduction was further supported by O’Neill and colleagues (28). They reported that treatment of EL4 cells and human fibroblasts with only the binding subunit of pertussis toxin (which does not inactivate G pro-

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teins) inhibited IL-I-induced

IL-2 production in EL4 cells and PGE, synthesis in fibroblasts as effectively as the intact toxin. In addition, treatment of EL4 cells with either the intact toxin or the binding subunit affected neither IL- 1 receptor affinity nor IL- 1-induced activation of the transcription factor NFKB. The authors concluded that inhibition of IL-l signal transduction by pertussis toxin was mediated by the binding subunit rather than the enzymatic subunit of the toxin and, therefore, did not indicate the involvement of a G protein. A recent report by Zumbihl and colleagues (233) has resolved some of these issues. Commercially available pertussis toxin binding subunit preparations have small amounts of holotoxin contamination. By using lower concentrations of the preparations, this group showed that the residual intact toxin was responsible for the apparent effect of the pertussis toxin binding subunit on IL-l signal transduction. Additionally, when EL4 cells were stimulated with intact pertussis toxin or a non-functional toxin, only the holotoxin inhibited IL-l-induced IL-2 production. However, this group also noted that pertussis toxin treatment of EL4 cells did not affect the number of IL- 1 binding sites, IL- l/IL- 1R internalization, NFKB activation, or IL-2Ra expression. Thus the authors conclude that IL-1 signaling can be differentiated into at least two pathways characterized by their sensitivity to pertussis toxin.

cAMPlPK4 The CAMP-dependent kinase (PKA) is a serinelthreonine kinase that can be activated by many different stimuli in mammalian cells (reviewed in 29). The cAMP/PKA signal transduction pathway is typically initiated by the activation of heterotrimeric G proteins that regulate adenylyl cyclase, which generates CAMP. CAMP activates one or more of the 12 possible PKA isoforms, which subsequently phosphorylate their target proteins. There are many potential cellular substrates for PKA, including the transcription factor NFKB (30). Since 1988, many groups have reported that IL-l stimulation leads to increased intracellular CAMP levels. IL-l-induced CAMP production has been reported in human and mouse fibroblasts (3 l-34), K562 cells (35), 70213 cells, YT cells, murine thymocytes (3 l), and murine TH2 cells (36). In these cell types, inducers of CAMP (i.e. forskolin) or CAMP analogs mimicked IL-l-stimulated activities (35,37,38). Caution must be used when interpreting these results, however, since pharmacologic inducers of CAMP cause long-lasting, high intracellular levels of CAMP if they are left in the cell culture. This treatment may not accurately mimic cytokine-induced CAMP upregulation, which is typically modest and transient. Additionally, increases in intracellular CAMP are not always observed following IL-1 stimulation (27,3845), and have even been reported to be detrimental to IL-lmediated induction of IL-2 in EL4 cells (26). Although it is possible that the levels of CAMP generated by IL- 1 may be too small or too compartmentalized to measure in the above systems, studies in BALB/c 3T3 fibroblasts (34), osteoblastic cells (46) and rat mesangial cells (47) indicate that CAMP can augment IL-l signaling, suggesting that IL-l receptors may utilize a separate pathway. Since the major consequence of increased CAMP levels is the activation of PKA, it is not surprising that IL-l has also been reported to activate PKA in some model systems that show IL-l-induced increases in CAMP (32,42,48,49). Interestingly, where IL-1 does not detectably upregulate CAMP levels, IL-l dependent PKA activation is still observed. In EL4 cells, which do not respond to IL-1 with elevated CAMP, inhibition of PKA activity was reported to completely block IL-l induction of the transcription factor AP-1 (50,5 1). Additionally, two recent reports indicate that IL-l-induced adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells is independent of CAMP or adenylate cyclase, yet dependent on PKA activity (52). These reports conclude that a novel mechanism of PKA activation may be used by the IL-l signaling pathway. Thus, examination of PKA activity itself following IL- 1 stimulation may be more relevant than the measurement of IL-l -induced adenylate

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cyclase activation. However, some studies using PKA inhibitors report no effect on IL-l signal transduction. These results have been observed in IL-l-stimulated IL-6 production by astrocytes (53), c-myc expression in DlOA cells (49), and human chorionic gonadotropin (hcg) release by choriocarcinema cell lines (54). Thus, it is unlikely that PKA activation is a universal component of the IL- 1 signaling pathway.

The protein kinase C (PKC) family is made up of at least 10 related isoforms that phosphorylate target proteins on serine and threonine residues (reviewed in 55, 56). These proteins can be classified into three subgroups based on structural differences: classical (cPKC), novel (nPKC), and atypical (aPKC) isoforms. Although the activation of PKC mimics the effects of IL-l signaling in many cell types, it remains controversial which, if any, of the PKC isoforms are involved in IL-1 signal transduction. Upon activation, PKC undergoes a cellular redistribution and binds to phospholipids in the inner plasma membrane. Although activation of all known PKC family members is dependent upon phosphatidylserine (PS), the PKC subgroups differ in their additional activation requirements. cPKC isoforms require diacylglycerol (DAG) and calcium in addition to PS, and can bind phorbol esters. Alternatively, members of the nPKC and aPKC subgroups do not require calcium for activation, and at least one of the two members of the aPKC group, PKCC, does not bind phorbol esters. Downstream consequences of PKC activation include activation of the transcription factors NFKB and NF-IL6 (30,57), both of which also can be induced by IL-l. Two PKC isoforms, PKCC and PKCa, have been implicated in the activation of the mitogen activated protein kinase (MAPK) pathway. PKC< has been reported to activate MAPK kinase (MAPKK or MEK) in vitro, and PKCa can directly phosphorylate and activate Raf-1, which in turn can activate MEK (58,59). Most of the evidence that IL- 1 activates PKC comes from reports stating that cells stimulated with phorbol ester, which activates the classic and novel isoforms of PKC, exhibit some of the phosphorylation events seen after IL-l stimulation (60). Additionally, IL-I has been reported to generate diacylglycerol (DAG) in some cell types and to induce the translocation of PKC to the plasma membrane in a T cell line (36). Inhibitors of PKC activity (H7 and staurosporine) have been reported to inhibit IL-l-induced GM-CSF and cycolooxygenase upregulation in fibroblasts (61,62). However, the PKC inhibitors H7 and staurosporine are not specific for PKC, so interpretations based only on this treatment should be viewed skeptically. Although the above reports suggest that IL- 1 signaling may involve PKC activation at some step, other well-controlled analyses have failed to detect IL- 1-induced PKC activity. For instance, IL-l can augment IL-2 production in LBRM cells chronically treated with phorbol ester to deplete PKC, suggesting that classical and novel PKC isoforms are not required for IL-l signaling in these cells (39,63). Additionally, IL- 1a and TPA costimulation of Jurkat T cells expressing IL-1Rl results in the synergistic activation of NFKB, indicating that distinct signaling pathways are activated by each (64). Since the atypical PKC isoforms do not require DAG for activation and are not affected by phorbol ester treatment, it is possible that IL- 1 activates one of these isoforms in the cell types in which IL-l signaling is not mimicked by phorbol esters. Interestingly, the atypical PKC isoform C has recently been implicated in NFKB activation (58).

LiDid Metabolism IL-l has been reported to use lipids as second messengers in a variety of cell types including macrophages, T cells and mesangial cells. In the past year, studies of IL-l-induced lipid metabolism have focused primarily on the sphingomyelin signaling pathway. However, other lipid mediators, including DAG, also have been reported to be rapidly generated in a number of cell types following stimulation

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with IL-1 (65,66). These lipids have many activities including the activation of kinases and phosphatases, and the generation of other potential second messengers (67).

Sphingomvelin

Pathwav

In the last few years, the sphingomyelin pathway has been identified as a signal transduction pathway utilized by IL-1 (reviewed in 65,68). Sphingomyelin is present primarily in the outer leaflet of the plasma membrane and can be hydrolyzed by sphingomyelinase to yield ceramide, a second messenger that diffuses readily through the lipid bilayer. Ceramide has been reported to activate both a kinase and a phosphatase, suggesting a critical role in regulation of the phosphorylation state of a cell following IL-l stimulation (reviewed in 68). The ceramide-activated protein kinase (CAPK) is a 97-kD member of the rapidly growing family of proline-directed serinejthreonine kinases. Interestingly, this kinase, like IL- 1, has been reported to induce phosphorylation of the epidermal growth factor receptor (EGF-R) on threonine (69). The ceramide-activated protein phosphatase (CAPP) is exclusively cytosolic (70). Other downstream events associated with ceramide release include increased transcription of the cyclooxygenase gene (71) and activation of the transcription factor NF-rB (72). To utilize ceramide as a second messenger, IL- 1 must activate a sphingomyelinase. Currently, two types of this enzyme have been identified. The first, an acidic sphingomyelinase (a-SMase), is localized to the lysosomal compartment and requires DAG for activation (73). The second, found in the plasma membrane, is a neutral sphingomyelinase (n-SMase) which is DAG independent and requires magnesium. Although both enzymes generate ceramide upon activation, the co-localization of both sphingomyelin and the n-SMase in the plasma membrane make this enzyme more likely to be part of a receptor-initiated signal transduction cascade. However, cytokines have been reported to activate both neutral (74) and acidic (73,75) SMase. Studies examining IL-1 signaling in Niemann-Pick Disease Type A fibroblasts, which lack a-SMase but not n-SMase, show that sphingomyelin turnover and thymidine incorporation (76) as well as NF-KB activation (77) occur normally, indicating that a-SMase is not essential for IL-l signal transduction in these cells. Additionally, only n-SMase is reported to be activated by IL-1 in EL4 cells (78). However, since membrane-permeable ceramide analogs can mimic some aspects of IL-1 signaling in some cells but not in others (78,79), the overall importance of the sphingomyelinase pathway in IL- 1 signal transduction remains controversial.

Other LiDid Mediators

m,

Activation

IL-1 has been reported to induce prostaglandin E, (PGE,) production in a number of different cell types (46,80,81). The rate-limiting step of PGE, biosynthesis is the release of arachidonic acid from membrane phospholipids, which is mediated by phospholipase A, (PLA,). Two types of mammalian PLA, exist: a 97-kDa cytosolic form (cPLA,) and a 14-kDa secreted form (sPLA,) (reviewed in 82). sPLA, has been found in inflammatory exudates and non-selectively cleaves fatty acids from the sn-2 position of phospholipids. cPLA, translocates to the plasma membrane upon activation and has been shown to preferentially hydrolyze arachidonyl-phospholipids. This information suggested that cPLA, could be a critical regulation point for IL- 1-induced PGE, production, and several recent reports support this conclusion. Increased mRNA and protein levels of cPLA,, but not sPLA,, have been temporally associated with IL- l-induced PGE, production in normal human synovial cells (8 l), rheumatoid synovial fibroblasts (80,83), and a rat glioma cell line (84). Upregulation of cPLA, gene transcription by IL-l is most likely due to the activation of the transcription factors AP- 1, NFKB and NF-IL6, all of which have consensus response elements in the 5’ enhancer region of the cPLA, gene (85,86).

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Increased synthesis of cPLA, is not the only way that IL-I can affect the activity of this enzyme. Studies in rat mesangial cells showed that IL- 1 regulates the activity of cPLA, in a biphasic manner (85). Stimulation of mesangial cells with IL-1 and calcium ionophore results in the activation of pre-existing cPLA, by phosphorylation. This ability to activate cPLA, without protein synthesis explains the finding that arachidonate is released from these cells as early as 5 minutes after IL- 1 and calcium ionophore stimulation. The authors propose that the regulation of cPLA, activity by IL- 1 has two-steps: activation of pre-existing cPLA,, followed by upregulation of cPLA, by de novo protein synthesis. The kinase that activates cPLA, in rat mesangial cells in vivo in response to IL-l has not been identified, but in vitro studies have shown that both the extracellular signal-regulated kinase 2 (ERK2) and PKC can phosphorylate and increase enzymatic activity of cPLA, (87). DAG Generation DAG can play a critical role in the activation of the c-PKC isoforms and other kinases, as well as in the activation of the a-SMase (73). Increased levels of DAG have been observed following IL-l stimulation in mesangial cells (88), EL-4 cells (25) human T cells (41) and human monocytes (89). In these systems, DAG generation occurs rapidly following IL-I stimulation, usually within seconds, and is transient. However, unlike most DAG-producing signaling cascades, IL-l stimulation does not typically generate DAG from phosphatidylinositol (PI) (65). Phosphatidylcholine (PC) is the main source of DAG in most of the cell types listed above, except for rat mesangial cells which apparently use phosphatidylethanolamine (PE) (88). In contrast to the well-studied phospholipases C that act specifically on PI, no such PC- or PE-specific PLC has been cloned to date. Since PKC isoforms have been reported to be activated by IL- 1 stimulation, it is possible that IL- 1-induced DAG generation is involved in this pathway.

Kinases IL-I has been reported to increase phosphorylation of cell proteins in many different model systems. Increases in serine and threonine phosphorylation are most commonly observed, although there also are several reports suggesting that IL- 1 may activate tyrosine kinases (90,9 1). Increases in phosphorylation are generally seen within 15 minutes of IL-I stimulation and are transient, decreasing to background levels within 30 to 60 minutes. In spite of the temporal association of protein phosphorylation and IL- 1RI ligation, it remains unclear whether the observed events result from primary (receptor transducer) or secondary (kinase cascade) signaling events. Although the identities of most of the cellular proteins reported to be phosphorylated by IL-l remain unknown, several have been identified. These include cPLA, (85), I-plastin (44), the small heat shock protein hsp27 (92) and threonine-669 of the epidermal growth factor receptor (EGF-R) (93). Identification of IL- l-responsive kinase(s) has been difftcult. It is important to remember that increases in phosphorylation can result either from the activation of a kinase or the inactivation of a phosphatase, and there is evidence that both can occur following IL- 1 stimulation of tibroblasts (60,94,95). In the past two years, hsp27 and the EGF-R T669 peptide have been utilized in attempts to identify the kinase(s) involved in IL-l signal transduction. The mitogen-activated protein (MAP) kinase pathway has been implicated in the IL- 1-induced phosphorylation of both hsp27 and EGF-R. MAP kinases are a family of proline-directed serine/threonine kinases that are activated by phosphorylation on threonine and tyrosine residues. There are multiple types of MAP kinase in mammals, including the extracellular signal regulated kinases (ERKs or MAPK), the Jun N-terminal kinases (JNK or SAPKJp54 MAP kinases), and p38 MAP kinase (reviewed in 96, 97). These MAP kinase cascades function in parallel and exhibit limited crosstall< (Figure 1). The ERKs can be stimulated in response to growth factor receptor activation of a tyrosine kinase or G protein, and utilize Ras- dependent pathways. This leads to the sequential activation of Raf and MAP kinase kinase (MAPKK or MEK).

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Protein Kinases

Growth Factors

Cytokines;

U.V.

Kinase Cascade:

Raf

Substrates Response:

MEKK

MEK

JNKK

I

(SEK, MEKK4)

ERK (MAPK)

JNK (SAPK)

TCF: ELK-l;

SAP-l

Activate MAPKAPKZ

c-Jun; ATF-2; ELK-l Augment c-Jun transactivation

Mitogenesis

Stress Response

Fig. I. Intracellular mitogen-activated kinase cascade comprising the ERK and JNK signal transduction pathways. Although this outline identifies the primary interactions within the MAP kinase signaling cascade, it does not identify potential crosstalk interactions between these two pathways. The potential participation of the Rho family GTPases, Rat-l and Cdc42, in signaling events upstream of MEKK is incompletely characterized.

MEK directiy phosphorylates and activates the ERKs, which then phosphorylate substrates that include transcription factor components in the ternary complex factor (TCF) family. ERK also has been reported to upregulate the activities of other kinases, such as MAPK-activated protein kinase 2 (MAPKAPK2) and cPLA,. JNKs can be activated by a variety of stimuli including cytokines and UV irradiation. Members of the Rho family of small GTPases (Rat- 1, Cdc42) have been implicated as early components in the JNK kinase cascade (234-236). Additional components of the JNK pathway include the Map kinase kinase kinase, MEKK, which phosphorylates JNK kinase-1 (JNKK or SEKI). JNKK phosphorylates and activates JNK in viva and in vitro (98), and can also phosphorylate ~38 MAPK in vitro (99). JNK substrates include the transcription factor components cJun, ATF2 and ELK- 1 (100,lO 1,237). The substrate specificity of p38 MAPK has not been defined.

Hs~27 Kinase Hsp27 is a cytosolic protein expressed in a wide variety of tissues. Although its exact function is unknown, hsp27 has been reported to act as a molecular chaperone, aid in cell recovery horn stressful conditions, and play a role in drug resistance (reviewed in 102). Hsp27 is phosphorylated in response

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to a variety of stimuli in a number of different cell types. In fibroblasts (103) endothelial (104,105) and monocytes (106), serine phosphorylation of hsp27 is induced by IL- 1.

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Two groups have partially purified an IL- 1-activated hsp27 kinase from the KB cell line (104,105). Both groups identified a protein of approximately 50 kDa that could be inactivated by protein phosphatase 2A and subsequently reactivated by activated, recombinant ERIC2 (~42 MAPK). These data are consistent with the 50 kDa hsp27 kinase being human MAPKAPK2. Stokoe and colleagues (107) have reported that MAPKAPK2 is the major enzyme responsible for phosphorylation of the small heat shock proteins in mammalian cells. In support of this, Ahlers and colleagues (106) reported that IL- 1 stimulation of monocytes resulted in the activation of MAPKAPK2 and subsequent phosphorylation of hsp27. This phosphorylation was inhibited by tyrosine kinase inhibitors and could be reconstituted by the addition of activated, purified MAPKAPK2. MAPKAPK2 is typically activated in vivo by the ERK subfamily of MAPKs, implying that IL- 1 may utilize the MAP kinase cascade to phosphorylate hsp27. This hypothesis is supported by the ability of activated ERK2 to activate the 50 kDa hsp27 kinase in vitro (104,l OS), and a report that IL- 1 can activate and induce transcription of ERK2 in rat mesangial cells (108). However, Freshney and colleagues (105) indicated that IL-l stimulation of KB cells did not activate ERK2, so a different ERK-like kinase appeared to be responsible for activating hsp27 kinase. Indeed, this group postulated a novel protein kinase cascade involving 35 kDa and 40 kDa proteins purified from IL- 1 stimulated KB cells. This cascade was similar to the MAP kinase cascade in that the 40 kDa protein (like ERK2) phosphorylated the ~50 hsp27 kinase on threonine residues and required phosphorylation of both tyrosine and threonine residues to be active itself. Limited amino acid sequencing of p40 suggests that it is the human homolog of murine p38 MAPK. The 35 kDa protein, which activated the 40 kDa protein, could not phosphorylate recombinant ERK and therefore was not a typical MEK. Together these results suggest the phosphorylation of hsp27 by IL- 1 in KB cells appears to be the result of a MAPK-like cascade. Interestingly, Rouse and colleagues (109) have recently described a novel kinase cascade triggered by stress and heat shock which results in the phosphorylation of hsp27. This cascade involves at least two new proteins called RK (MAPKAPK2 reactivating kinase, 42 kDa) and RK kinase (RKK) which appear to be analogous to the 40 kDa and 35 kDa proteins respectively.

EGF-R T669 Kinase IL-1 stimulation of fibroblasts results in the phosphorylation of the EGF-R on threonine-669 (T669), which is associated with decreased affinity of the EGF-R for its ligand (93,110). The amino acid sequence surrounding T669 Iits the MAPK substrate consensus motif suggesting that this family of kinases may be activated by IL-l and be responsible for EGF-R phosphorylation. Using a peptide based on the sequence surrounding T669, Kracht and colleagues (Ill) reported in KB cells an IL-I activated, 45 kDa T669 kinase that did not react with antiserum to ERK1/2 (p44/p42 MAP kinases). This unique T669 kinase required both tyrosine and threonine phosphorylation to be active, suggesting that it might be regulated in a manner similar to MAPK. However, MEK could not activate T669 kinase. These characteristics led the group to postulate that their T669 kinase might be a form of the INK subfamily of MAPK, which are also not activated by MEK (reviewed in 96). Subsequently, two papers were published supporting the identification of T669 kinase as a INK (74,112). Kracht and colleagues reported the activation of two T669 kinases in rabbit liver following systemic injection of rabbits with IL-l. Peptide sequences and biochemical properties of the two kinases indicated that both were isoforms of INK. In addition, studies in fibroblasts and HepG2 cells showed that IL- 1 activated a kinase(s) that phosphorylated T669 (but not MBP) and did not phosphorylate ERKl or ERK2 (74). This kinase activity could be immunoprecipitated with an anti-INK antiserum and co-chromatographed with INK on Mono Q and phenyl-Superose. The activation of T669 kinase in

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this system was also Ras-, Raf-, and MEK-independent. Thus at least one member of the INK family appears to play a role in the IL- 1-induced phosphorylation of the EGF-R in several cell types. Based on the recent reports identifying Racl and Cdc42 as upstream activators of INK following EGF stimulation, it will be interesting to see whether IL-1 also utilitzes these small G proteins. It has also been postulated that IL- 1 induces the phosphotylation of the EGF-R by activating CAPK through the sphingomyelin pathway. CAPK appears to be exclusively membrane-bound and membranes containing activated CAPK will specifically phosphorylate the T669 peptide (113). However, the only IL-l regulated T669 kinase in KB cells was found in the cytosol, not in membrane fractions (111). The T669 kinase activity in fibroblasts and HepG2 cells also was freely soluble (79), suggesting that the kinase responsible for EGF-R phosphorylation is not CAPK. Interestingly, Kyriakis and colleagues (114) showed that stimulation of HepG2 cells with bacterial sphingomyelinase could induce INK activity indicating that CAPK may be an upstream activator of IL-l-induced T669 kinase activity. However, this result was not reproducible by Bird and colleagues (79) using C,-ceramide or sphingosine to stimulate HepG2 cells and fibroblasts, and therefore, the involvement of CAPK in EGF-R T669 phosphorylation by IL-l remains controversial.

Other Kinases &Casein

Kinase

A novel, multimeric kinase specific for p-casein has been reported to be activated by IL-1 in MRC-5 fibroblasts, bovine articular chondrocytes and human endothelial cells (103,115). This serine/threonine p-casein kinase is distinct from the hsp27, EGF-R T669, and MAP kinases in substrate specificity and response kinetics. The physiological substrates and activation requirements are unknown. The activity of p-casein kinase peaks at 15 minutes following IL-l stimulation, and remains detectable for several hours post-stimulation. Additionally, p-casein kinase is not inactivated by phosphatases in vitro indicating that the activity of this kinase may not be dependent on phosphorylation (115). Therefore, it has been postulated that p-casein kinase may lie upstream of or parallel to the MAP kinase and hsp27 kinase cascades activated by IL-l. However, the authors concluded that, since this kinase was located mainly in the cytosol, it is probably not coupled directly to IL-1RI (115). IL-lRI-Associated

Kinases

Discovering a directly coupled kinase has long been a goal in IL-1 signal transduction research. Using two murine T helper cell lines, DION and EL4, Martin and colleagues (116) were able to co-precipitate a serine/threonine kinase activity with the type I IL-1 receptor in the presence of IL-1 LXor p. The kinase activity was IL- 1 concentration-dependent and not detectable when IL- 1RI were immunoprecipitated with an antibody that blocked the ligand binding site. In addition, kinase activity was 90% inhibited by staurosporine (an inhibitor of numerous serine/threonine kinases), but not by protein tyrosine kinase or PKC inhibitors. In vitro kinase assays using MBP, Hl histone, p-casein and hsp27 as substrates showed.that only MBP, Hl histone and an endogenous, unidentified 60 kDa protein were phosphorylated after IL-1 stimulation. Although it is possible that this kinase activity could be an upstream activator of the novel hsp27 or T669 kinase cascades described previously, neither of these IL-l inducible cascades have been described previously in T helper cells. Croston and colleagues (144) have also reported an IL-IRI-associated kinase activity (IRAK). IRAK is activated very rapidly by IL- 1 in IL- 1RI-transfected human embryonic kidney 293 cells, reaching maximal levels two minutes after IL-l stimulation. Interestingly, this group has postulated that IRAK is involved in NFKB activation. Using a series of IL-1RI mutants, they showed that the same muta-

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tions that result in the inability of IL- 1 to induce NFKB also prevent the co-immunoprecipitation of active IRAK. It will be interesting to determine if these kinase activities can be co-precipitated with the IL- 1RI in other cell types and whether other downstream events are adversely affected by the IL1RI mutants that do not associate with IRAK.

TranscriDtion Factors and Regulation of TranscriDtion IL- 1 causes the rapid induction of a wide variety of genes that encode proinflammatory proteins as well as lymphokines that initiate or augment lymphocyte activation. The induction of these genes is regulated by IL- 1-inducible transcription factors that are members of the immediate-early response gene family, including AP- 1, NFKB, and NF-IM. These transcription factors can be activated within minutes of IL-l receptor ligation independent of de n~vo protein synthesis by stimulus-induced post-translational modifications. IL-l also can induce the synthesis of components of these transcription factors later in the activation program. The mechanisms responsible for mediating synergistic transactivating functions by interaction of different IL-l-inducible transcription factors or other transcription factors is incompletely understood.

AP-1 is a dimeric complex consisting of Jun polypeptides (c-Jun, JunB, JunD) and Fos polypeptides (c-Fos, FosB, Fra-1, Fra-2). The complex may consist of Jun homodimers, Jun heterodimers or Jun-Fos heterodimers. Post-translational modifications of the component polypeptides play an important role in regulation of AP- 1 activity. For example, phosphorylation of two or three C-terminal residues in the c-Jun DNA binding domain negatively regulates DNA binding (117), whereas phosphorylation of two N-terminal serine residues stimulates cJun transactivating potential (118). The ability to express specific combinations of dimers possessing different DNA binding affinities or transactivating abilities suggests a potential regulatory role for the stimulus-induced expression and/or activation of different AP- 1 component polypeptides. In addition, the effects of IL- 1 on AP- 1 induction may go far beyond activation of genes with AP-1 responsive elements. AP- 1 can also physically associate with other transcription factors (i.e. NF-AT and Ott) and alter their transactivating activities. IL-l has been reported to regulate AP-1 components post-translationally and by inducing Jun-family gene expression (119,120). In addition, the Fos family members c-Fos, FosB and Fra-1 have been reported to be transcriptionally upregulated in T cells in response to stimulation with IL-l plus TPA ( 12 1). Increased levels of c-Jun (119,122,123), JunB (119,120) and c-Fos (123) mRNA have been observed soon after IL-l stimulation. Recent studies also have demonstrated that IL-l treatment results in the activation of JNK (79,237). As mentioned in the previous section, JNK is responsible for phosphorylating cJun on two N-terminal serine residues, thereby potentiating cJun transactivation function. IL-l also has been reported to stimulate ERK- and JNK-mediated phosphorylation of the TCF family transcription factors Elk- 1 and SAP- 1, which regulate c-Fos expression (237). Although the factors that upregulate transcription of the c-jun and C--OSgenes themselves are incompletely characterized, the AP-1 transcription factor has been reported to play a role in the regulation of these genes (124,125). Alternatively, Muegge and colleagues (123) have reported that IL-l stimulation of a human hepatoma cell line (HepG2) results in the activation of two unique transcription factors that bind to distinct sites, designated junl and jun2, in the c-jun enhancer. The junl and jun2 sites show some sequence similarity to consensus AP- 1 sites, but the IL- 1-induced proteins that bind to these sequences do not react with antibodies to the known Jun and Fos family members. Additionally, the c-jun promoter is activated within 15 minutes in the absence of de nova protein

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synthesis indicating that the junl- and jun2-binding proteins must be post-translationally activated, most likely by phosphorylation. JNK activity induced by IL-l could be responsible for the activation of pre-existing AP-l-like factors that regulate Jun family transcription.

NFKB NFxB was originally identified as a transcriptional regulator of immunoglobulin kappa chain genes. More recently, it has been shown to be critical in the regulation of a large number of genes including IL-2, IL-8 and the HIV LTR. Activation of NFKB can occur within minutes of stimulation because it is constitutively present in the cytosol of resting cells. Cytosolic NFKB is associated with a family of inhibitor proteins called IKB, which block the nuclear localization sequences of NFKB family members. Upon appropriate stimulation of the cell, Idor is phosphorylated and degraded, freeing NFKB to translocate to the nucleus and activate transcription of genes containing NFKB enhancer elements. The transcription of IrBa is upregulated by NFKB, and thus a feedback loop is generated. Like AP-1, functional NFKB is a homo- or heterodimer. These dimers are made up of members of the rel family of proto-oncogenes (c-Rel, RelA, RelB, ~50, ~52) that share a 300 amino acid region termed the Rel-homology domain. I&a phosphorylation, degradation and NFKB nuclear translocation occur within 5 minutes of IL-l stimulation in numerous cell types, and the cytoplasmic domain of the IL-l RI is required for this activation (126,127). Additionally, c-Rel and RelA components of NFKB are themselves rapidly phosphoxylated following IL-I stimulation in murine LBRM and IL-lRI-transfected human Jurkat T cell lines (Bankers-Fulbright et al., in preparation). Although the in viva function of this phosphorylation remains unknown, it has been reported that phosphorylation of RelA increases the DNA-binding affinity of NFKB in vifro (128). In addition to activating NFKB by inducing nuclear translocation, IL-I also has been reported to activate pre-existing nuclear NFKB in an immature T cell line (129) and appears to upregulate expression of certain NFKB components (McKean, unpublished observations). Reactive oxygen intermediates (ROI) also have been reported to be associated with IL-I induction of NFltB (130,13 1). Activation by ROI was originally thought to be common to many activators of NFKB, including IL-l in 7OZ/3 cells and TNFa or PMA in Jurkat T and 7OZ/3 cells (130). The activation of NFltB by these cytokines could be mimicked by hydrogen peroxide and inhibited by the oxygen radical scavengers N-ace@ cysteine (NAC) and a pyrrolidone derivative of dithiocarbamate (PDTC). However, a later report suggested that ROI involvement in NFKB activation was not universal, especially in regard to IL-l-mediated induction (13 1). This study showed that NFKB activation was not inhibited by NAC in IL-l-stimulated EL4 cells, and could not be stimulated in these cells by hydrogen peroxide. In addition, Anderson and colleagues (132) have demonstrated that, although only one of the two Jurkat T cell lines they examined activated NFKB in response to oxidants, both activated NFKB in response to TNFa or PMA stimulation. NFKB activation in both of these Jurkat T cell lines was inhibited by herbimycin A and tyrophostin 47, inhibitors of protein tyrosine kinases, suggesting that tyrosine phosphorylation is requisite for NFKB activation. Activation of NFKB by IL-l has been reported to be sensitive to tyrosine kinase inhibitors in EL4 cells, 7OZ/3 cells and human melanoma cells (133-135). However, caution must be used when interpreting these results in light of the report that the tyrosine kinase inhibitor herbimycin A can directly modify NFKB and prevent it from binding to DNA in vitro (136). The model of IL-l induced NFKB activation by release from IKB is paralleled in the Drosophila dorsoventral signaling system (137-140). The Drosophila gene products Toll, cactus, and dorsal are homologous to the mammalian IL- 1RI, IKB and NFKB, respectively. Residues conserved in the cytoplasmic domain of Toll and IL-lR1 have been shown to be critical for signaling in both systems (141). However, unlike the mammalian system, NFKB activation in Drosophila has been reported to

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involve phosphorylation of only NFKB, not IKB.Two proteins involved in the Drosophila signaling cascade have been identified. Tube and pelle have been determined to be downstream from Toll in the dorsoventral signaling pathway that activates dorsal. Although the function of tube is still unknown, pelle has been identified as a serine/threonine protein kinase and may be directly activated by tube (142). Alternatively, there is some evidence that tube may function as a chaperon or transcriptional coactivator with dorsal (143). It is possible that these Drosophila proteins have mammalian counterparts that may be directly involved in the activation of NFrcB by IL- 1. Interestingly, a recent report has suggested that IL-l-induced NFKB activation is dependent upon a kinase activity, IL-I receptor associated kinase (IRAK), that co-precipitates with ligated IL- 1RI (144). Identification of these proteins will not only aid in the understanding of IL-l signal transduction, but may give insight into the receptor-proximal events of NFKB activation.

NF-IL6 NF-IL6 (UEBP-p or LAP) was fust described in 1990 as an IL-l-inducible factor that could upregulate transcription of the IL-6 gene in the human glioblastoma line SK-MG4 and mouse L cells (145). NF-IL6 has also been reported to be activated by IL-6, TNF, LPS, (146) and activators of PKA (146,147) and PKC (147). Subsequent cloning indicated that NF-IL6 was highly homologous to the C-terminal region of the liver- and adipose tissue-specific transcriptional factor CYEBP, and contained a potential leucine zipper motif suggesting that NF-IL6 functioned as a dimer (148). NF-IL6 has subsequently been reported to utilize its leucine zipper domain to interact with other members of the C/EBP family (149), as well as Jun and Fos (150) and NFKB subunits (15 1,152). NF-IL6 and C/EBP both bind avidly to CCAAT and viral enhancer core DNA sequences (reviewed in 153). In addition, NF-IL6 homodimers can bind to AP-1 responsive elements (150). The transactivating ability of NF-IL6, like AP- 1, appears to be regulated at both the transcriptional and post-translational levels. Expression of NF-IL6 has been reported to be negligible in most normal mouse tissues, but can be rapidly and strongly induced by IL- 1, IL-6 and LPS. However, stimulation of the glioblastoma SK-MG-4 with IL-l resulted in the rapid activation of NF-IL6 binding activity without a concomitant increase in NF-IL6 mRNA (148). Recent reports have suggested that phosphorylation may play a critical role in NF-IL6 activation. There are reports of NF-IL6 phosphorylation resulting from PKA and PKC activation (57,146,147). Metz and Ziff (146) reported that forskolin treatment of the PC12 cell line resulted in the phosphorylation and translocation of pre-existing NF-IL6 from the cytosol to the nucleus. Subsequent studies in vivo (57) and in vitro (147) have indicated that phosphorylation of NF-IL6 within its DNA binding domain by PKC resulted in a marked decrease in DNA binding. On the other hand, phosphorylation of Ser-105, located in the transactivating domain of NF-IM, did not affect DNA binding but augmented NF-IL6 transcriptional efticiency. In addition, Nakajima and colleagues (I 54) have reported increased NF-IL6 phosphorylation and transactivating activity in NIH-3T3 cells when NF-IL6 was coexpressed with oncogenic p2 1m. This same group also found that both purified ERK and oncogenic p2 1” could phosphorylate NF-IL6 in vitro. Since MAPK pathways have been implicated in IL-l signal transduction, they could be components of the mechanism of IL- 1-induced NF-IL6 activation. However, studies to date have not used cytokines to induce phosphorylation of NF-IL6, so the relevance of the modifications in IL-l-stimulated NF-IL6 induction remains unclear.

Transcrbtional

Redation

AP-1, NFKB, and NF-IL6 are critically involved in the regulation of genes expressed in response to IL- 1. Almost every gene reported to be transcriptionally activated by IL- 1 has at least one of these

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TABLE 1 IL-I SIGNAL TRANSDUCTION PATHWAYS GENE

FACTORS’

INVOLVED”

NOT INVOLVED”

NF-IL6

AGP angioteluiaogea

NFxB

Cl

NFa AP-I

complementFactor B

NFcB

f0s G-CSF

PKA. PKC

-

AP-I

-.

NF-IL6

PKC -

AP-1. NF.B

-

167,168.212 170

-

ERK2. PKCa. F’TK. ROI. Shfase

NFrB. NF-116

GM-CSF

164

PKC

AP-I. NFxB

.8~adoQhin

163

.-

165.166

coll1gcnase

cyclmrygcnaac

Rf FERENCES

CAMP CAMP

7,.17L-174 43 49.146.175 176.177 178.182

gro

NFls

PI-K

-

134.183.184

HIV LTR

NF.B

PKC

ca’+

18J.186

IrBo

NFrB

-

IL.10

NFrB. NF-IL6

MAF’K. PKC. PTK. S/l-K

CAMP. PKC

188.193

IL-2

AP-I. NFIB

PTx substrate.ROI

cMIP. G-pmkt~. PKC

IL-2Ro

AP-1. NF.B

187

c,UP.

-

PKC

IL-4

none

Cs’.

IL-5

n0r.c

CAMP. PKA. PKC

PKA. PKC

PKC -

26.28.122.lS6.194-19 31.122.19S.197 I95 36.49.198

fLd

AP-I. NFI.

NF-IL6

MAPK. PM*.

CAMP. PKA. PKC

4S.49.33.36(3.19%203

R-8

AP-I. NF.6. NF-IL5

-

PKC. nd-SMa.w

77.202.204.206

ICM-I

NF.S

PKC

PKC. ROI

207.209

P

AP-I

PKC

49.123.l81.210

CAMP

37.211

‘ lighhr Cham

NFIB

myb

tI0r.e

mYc

NFxB

NGF

AF-I

INOS

AP-I. NFxB

CPLAI SAA

A&,.

NFI;B. NF-IL6

NFeB. NF-X.6 AP-I

ShlXLUlySln sv40

AP-I,NFrB. NF-IL6

TNFo

AP-I. NFxB. NF-U.6

TSG-6

AP-I. NF-IL6

VCAM

.

..

PL$.

PKC. PTK

PTK

CAMP CAMP. PM.

PTK

PKC

49.175

rl-K

PKA. PKC

49.175.212

.%-IX

CAMP. PKC

213.215

rlx

PKCI

174.216-219

.-

CAMP

PPua PKC PKC

47.84.86 160,220.223

PKA

168.224.22s

a’+

148.226

-

148.221-229

-

I61

-

207.230.231

No inlormuion avaihble

Signalingptiwayr exammedand show 10be involvedin or not involvedkbib~mryin IL-1 inductionof the gene;puhwayy,are not oeceuarily involvedin xix IL-I inducdoaof die vdnwriplion facton lied. Cordmulw may be requiredfor IL-I inductionof somegenes.

elements in its enhancer, and most have multiple elements. Discussion of all of the genes reported to be regulated by IL-l is beyond the scope of this review, but a majority have been summarized in Table 1. Recent studies on IL-8 and IL-2 induction by IL- 1, summarized below, give new insight into how transcription factors can work together to enhance gene expression.

responsive

IL-l induction of IL-8 synthesis has been observed in human endothelial cells, a human fibrosaromca cell line (8387 cells), a human astrocytoma cell line (U373 cells) and a human glioblastoma cell line (T98G cells) (reviewed in 155). IL-8 transcription is induced rapidly by IL-l in these cell types and cannot be blocked by cycloheximide treatment indicating that de novo protein synthesis is not required. Cloning of the IL-8 gene identified potential binding sites for AP- 1, NF-IL6, and NFKB in the 5’-flanking region. Analysis of the minimal enhancer region of IL-8 indicated that deletion of the NFKB, but not the NF-IL6 or AP-1 sites, abolished the ability of IL-l to upregulate IL-8 transcription. However, if the NF-IL6 and AP-1 sites were both deleted, the NFKB site was not sufficient to confer

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IL-l inducibility. Thus, in this system, induction of NFKB binding is necessary but not sufficient, and at least one other transcription factor must also be present to induce IL-8 gene transcription. Similarly, IL-l-mediated induction of the IL-2 gene in T cells has been shown to be dependent upon NFKB. Unlike IL-8, however, IL-2 is not inducible by IL-l alone. In T cells, IL- 1 functions as a costimulatory signal that synergistically augments antigen receptor-initiated IL-2 gene transcription. The IL-2 enhancer, like the IL-8 enhancer, contains multiple response elements that could be directly affected by IL- 1 signal transduction, including one binding site for NFKB and two for AP- 1. However, only deletion or mutation of the NFKB response element, not the AP- 1 binding sites, abolishes IL- 1 mediated IL-2 upregulation (156, Kalli et al., submitted). Previously, Muegge and colleagues (122) reported that deletion of the distal AP- 1 site in the IL-2 enhancer abrogated the IL- 1 effect in murine LBRM T cells. However, this deletion from -2 18 to -176 also encompassed the NFKB binding site. In addition to NFKB and AP- 1, the T cell antigen receptor-induced transcription factors Octamer (Ott) and NF-AT are required for maximal upregulation of the IL-2 gene. Although not activated by IL- 1 directly, both NF-AT and Ott have been reported to associate with the AP-1 component polypeptides Fos and Jun (157-159). Since IL-l stimulation of T cells can upregulate transcription of the cJun and JunB components of AP- 1 as well as augment AP- 1 transactivating functions by stimulating JNKmediated phosphorylation of c-Jun, IL-1 has the potential to affect these transcription factors as well. However, studies utilizing NF-AT and Ott reporter constructs expressed in T cell lines suggest that the effect of IL-I costimulation on the activity of these T cell antigen receptor-stimulated transcription factors is minimal (Kalli et al., submitted). Thus, NFKB appears to be the major IL-l-responsive factor in the upregulation of the IL-2 gene in T cells. IL-l can induce the transcription of a number of other genes as well (Table 1). Of particular interest are recent studies of the IL-6, serum amyloid A (SAA) and TNF-stimulated glycoprotein 6 (TSG-6) enhancers. The IL-6 and SAA enhancers have binding sites for NF-IL6 and NFKB and, in both enhancers, NF-IL6 and NFKB synergize to activate gene transcription (152,160). The TSG-6 enhancer has binding sites for NF-IL6 and AP- 1. The NF-IL6 site is required for IL- 1 activation of this gene, and binding of AP-1 to the AP-1 site enhances this response (16 1). Thus the transcription factors that are responsive to IL-1 stimulation can activate a number of genes. However, this gene activation usually involves the complicated interactions of IL-l- and non-IL-1 induced transcription factors which are generally subject to multiple levels of regulation. It will be very informative to identify how these factors interact with each other in the context of different promoters and what post-translational modifications of each are required for their activity.

Regulation of IL-1 Signal Transduction Since IL-1 is such a potent inflammatory cytokine, it is critical that its biological effects be tightly regulated. Regulation in vivo has been postulated to be due to the activities of the IL-lra and the IL-1 RII. IL-lra binds to IL- 1RI on the cell surface, thus blocking IL- 1 binding. Alternatively, soluble and cellular forms of the IL- 1RI1 can bind to IL- 101and IL- 1p thus preventing the cytokines from binding to the functional IL-1RI. The potential problem with utilizing these regulatory methods in therapeutic intervention of IL- l-mediated disease processes is the exquisite sensitivity of the IL- 1 system. It has been reported that as few as 10 IL- 1RI ligated on a cell can induce a biological response (17). Thus, clinically relevant inhibition of IL- 1RI signaling in vivo would require enough IL- 1RIIs to quantitatively bind all available IL- 1, or the production or infusion of enough IL- lra to block all IL- 1RI on every cell capable of responding to IL-l. Although both of these options may be technically possible, it would be difficult and expensive to achieve sufficient levels to inhibit a full-scale IL- 1-mediated response.

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Our studies in T cells have suggested that an alternative regulatory pathway exists (119). We have found that stimulation of IL-lRI-transfected Jurkat (Ju.1) T cells with enough IL-1 OLto ligate only 20% of the surface receptors results in the subsequent desensitization of all cell surface IL-1RI within four hours. These desensitized cells still have approximately 4,000 unligated IL-1RI on their cell surface, and the affinity of these receptors is normal. Thus, the inability of IL-1 to stimulate these cells is not due to the downregulation of surface IL-1RI. IL-1RI desensitization affects all signaling events examined, including IKBLXphosphorylation, NFKB translocation, c-jun andjunB mRNA induction, and upregulation of anti-CD3-initiated IL-2 transcription. If IL- 1 a is removed from the culture, the desensitization effect is completely reversed within 18 hours. These data suggest a novel pathway of IL-l signaling self-regulation involving an apparent transient uncoupling of signal transducers from the lL-1RI. This allows all the IL-IRIS on a cell to be desensitized without having to ligate every receptor. This post-receptor desensitization event is an additional target for therapeutically antagonizing IL- 1 effects in vim.

Conclusions It is apparent that IL-1 has a wide variety of effects on many different cell types. Despite the tremendous effort that has gone into elucidating how IL-I induces these effects, no paradigm has been established to explain the signaling mechanism. Part of the problem results from the fact that so many different types of cells and stimulation protocols have been used to analyze IL- 1 function. Signaling events occurring in some cell types may not be available in others. Special care also should be taken when interpreting results generated in cell lines. Tumor cell model systems may selectively utilize secondary signaling pathways, and thus their responses to IL- 1 can differ depending on what subclones are studied. Obviously, addressing the effect of IL- 1 on normal, untransformed cells will be of enormous importance to identify biologically relevant signaling events, and attempts to correlate cell line results with those of primary cells should be done whenever possible. More complexity arises because IL-l has been reported to induce so many different second messengers. It is possible that these signal transducers may have overlapping activities such that inhibiting one pathway would not abolish the response. Additionally, attempts to elucidate the most receptor-proximal events following IL-1 stimulation may be difficult because of the low number of ligated receptors required to generate a biological response. If the signal amplification step lies beyond the receptor, early events may simply be too small to detect with current technology. Additionally, future studies should focus on not only which signaling pathways are activated by IL-l, but the consequences of their activation. Simply because a signaling protein is activated following IL-l stimulation does not necessarily mean that it is involved in the biological response. Finally, the potential benefits of being able to manipulate the IL- 1 signaling pathway are enormous. Not only is IL-I implicated in the pathogenesis of many autoimmune and chronicinflammatory diseases, but positive regulation of this cytokine could help modulate the immune response. So far, systemic treatment of humans with IL-I has had such high toxicity that its utility is questionable, and clinical trials using IL-lra in humans with sepsis or R4 have been inconclusive (reviewed in 162). It is hoped that these problems can be overcome as we gain understanding of how stimulation of cells with IL- 1 mediates a particular biological response. Thus, defining the pathways involved in IL-1 signal transduction, understanding how these signals are translated into a biological response, and ultimately designing ways to manipulate them remain essential and worthy goals.

References 1. W. P. AREND and J. M. DAYER. Arthritis and Rheumatism 38 151-160 (1995). 2. T. MANDRUP-POULSEN, U. ZUMSTEG, J. REIMERS, F. POCIOT, L. MORCH, S. HELQVIST, C. A. DINARELLO and J. NERUP. Cytokine 5 185-191 (1993).

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3. 4. 5. 6. 7. 8. 9. IO. II. 12. 13.

14. 1.5. 16. 17.

18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

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