Tumor necrosis factor regulation of major histocompatibility complex gene expression

Immunol Res 1991;10:141-155 �9 1991 S. Karger AG, Basel

0257-277XI91/0102-014152.75/0

Tumor Necrosis Factor Regulation of Major Histocompatibility Complex Gene Expression

David R. Johnson, Jordan S. Pober

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass., USA

Introduction

Two qualitatively different activity types of tumor necrosis factor (TNF) have been extensively characterized: (1) TNF is directly cytotoxic, independent of protein or mRNA synthesis, to some oncogenically transformed cells [1], and (2) TNF regulates the expression of proteins in normal and transformed cells, including the highly poly- morphic, immunoregulatory molecules encoded within the major histocompatibility complex (MHC). The immunological importance of the MHC arises from the fact that specific helper or killer T cells only recognize antigen efficiently when it is bound by one of two classes of MHC-encoded cell surface proteins: class-I molecules, which are ex- pressed on nearly all cell types, or class-II molecules, which are normally found only on B cells, macrophages and certain other cells. Although both class-I and class-II molecules appear to fold into similar peptide-binding structures, these molecules show important functional differences. Class-I molecules preferentially bind peptide antigens from en- dogenously synthesized proteins, e.g. viraI proteins, that are subsequently displayed on the cell surface to be recognized by cytotoxic

CD8 + T cells, which kill virus-infected cells. In contrast, class-II MHC molecules preferentially bind peptides from exogenous proteins and present antigen most efficiently to helper CD4 § T cells. The quantitative expression of class-I and class-II MHC molecules may be important in determining the efficiency of T-cell stimulation. Here we review the specific actions of TNF upon MHC molecule expression and discuss the mecha- nisms of TNF-mediated gene regulation.

TNF Effects on M H C Molecule Expression

TNF increases the cell surface expression of class-I molecules, 2- to 4-fold in 24 h [2]. This increase in surface expression reflects the increase in total cellular class-I protein, which is preceded by a rise in the steady-state levels of mRNA encoding both subunits of the class-I ctl3 heterodimer. In endothelial cells we have found that the increase in mRNA results from increased transcription, with no evidence of increased mRNA stability [3]. Since peptides bind most readily to class I as the nascent a and 13 chains fold to form the antigen-binding structure [4], this

142 Johnson/Pober

increased rate of MHC protein biosynthesis might be more relevant than the overall level of surface expression in determining the efficiency of antigen presentation.

TNF is usually found together with other cytokines at sites of inflammation. In partic- ular, interferon-13 (IFN-[3) and TNF are synthesized by virus-infected cells [5] and IFN-y and TNF are released by some activated T cells [6]. Individually, these interferons also induce MHC class-I expression (8- to 10- fold). However, TNF combines with either IFN to produce a much greater than additive (i.e. synergistic) increase in MHC class-I expression (>20-fold). In contrast, the two IFN types combine to produce a less than additive induction of class I. Although these observations were initially made in cultured vascular endothelial cells, which are of par- ticular interest because endothelium might play a role in recruiting specific T cells from the bloodstream into inflammatory sites, these observations have since been extended to several other cell types [7] (and HeLa, unpublished observation).

Class-II regulation by TNF is controver- sial, perhaps reflecting differences among the cell types in which it has been studied. TNF does not induce de novo expression of class-II mRNA in human endothelial cells [2, 8] (although contradictory results have been reported in rat endothelial cells [9], in human vascular smooth muscle cells [10]), or in normal murine macrophage, although induction occurs in malignant murine macrophage [11]. In several studies TNF inhibited the induction of class-II molecules by IFNq, in human endothelial cells [12-I4], although the effect is small in comparison to that of IFN-13, which markedly inhibits IFN- 7 induction of class-II mRNA [12, 13]. In contrast, TNF has been reported to enhance

class-II expression on virus- or IFNq,-induced astrocytes [ 15, 16], activated T-cell- or IFN-y-induced Schwann cells [17], and IFN- y-induced monocytes [18]. In human mono- cyte cell lines TNF increases constitutive class-II expression in THP-t but does not induce de novo expression in U937 [11]. Similarly, class II is induced in human pancreatic [3 cells by combined IFN-y and TNF but not by either cytokine alone [19], although it should be noted that this combination markedly impairs 13-cell function [20] and has been implicated in insulin-dependent diabetes [21]. Therefore, TNF may regulate on-going class-II synthesis but it is usually unable to initiate transcription of the class-II gene.

In summary, the inflammatory cytokines TNF, IFN-[3 and IFN-y, each induce class-I MHC expression; however, TNF combines with either IFN type to increase synergistically the transcription of MHC class-I genes. In general TNF does not induce class-II molecule expression de novo and its interaction with IFNs may be cell-type dependent.

TNF Receptor Signal Transduction

All TNF effects are believed to be ini- tiated by the binding of TNF to cell surface receptors. The signal generated by TNF binding is transduced by activation of one or more second messenger pathways. Second messenger pathways may be complex, with single agents activating multiple, cell-specific enzyme systems. Therefore, to unam- biguously identify a second messenger pathway as the mediator of a cytokine signal, three criteria must be met: (1) the cytokine must activate the second messenger pathway in the cell of interest; (2) activators of the

TNF Regulation of MHC Gene Expression 143

second messenger must mimic the cytokine, and (3) inhibitors of the second messenger must inhibit the cytokine. To date, these criteria have not been met for the TNF regulation of MHC genes. Several second messengers are activated by TNF in a variety of experimental systems. We will review the interaction of TNF with its receptor and discuss TNF-act ivated second messengers as they may relate to the induction of transcription factors that regulate MHC genes.

TNF Receptor Biochemistry The detailed structure of the TNF recep-

tor (TNF-R) is a yet unknown. Human endothelial cells [22] and HeLa cells [23] have a single class of high affinity receptors (K~ about 10-1~ although a second class of lower affinity receptors may exist, or co-exist (SK-CO-1) [24] on myeloid cells [25]. The TNF-R affinity has been shown to change with cell density in cultured bovine endothelial cells [26]. Biochemical analyses suggest that the two classes of TNF-Rs are each com- posed of at least two, possibly distinct [25], TNF-binding subunits that combine to form one or more TNF-binding sites and that TNF binding stabilizes this receptor complex [25, 27].

Lymphotoxin (LT), also known as TNF- ~, is a close functional homologue of TNF that competes with TNF for binding to TNF- Rs. TNF and LT have essentially identical activities and potencies on MHC genes [12, 28], although potencies may differ for some activities [24].

Interleukin-1 (IL-1) does not stimulate MHC gene expression [28], although IL-1 and TNF share many other activities [29]. Therefore, second messenger pathways activated by both TNF and IL-1 are less likely to be directly involved in MHC regulation.

However, IL-1 may regulate the response of MHC genes to other cytokines; IL-let has been reported to antagonize TN F synergy with IFN-7 inducing class-II expression on rat endothelium [30].

TNF Receptor Regulation The cytotoxic, antiviral [31] and MHC-

inducing [12] activities of T N F are enhanced by IFN-3,, which was thought to result from an IFN-7-induced increase in TNF-receptor expression on the cell surface [32]. However, this view is challenged by the observation that in some cells IFN-ct and IFN-[3 also potentiate TN F cytotoxicity though they do not increase TNF-R expression [33]. Simi- larly, synergy between IFNs and TNF in inducing MHC class-I expression by endothelial cells does not appear to require increased TNF-R expression, since IFN-y does not significantly alter the number of TNF receptors or their affinity on these cells [3].

TNF-R expression is increased up to 7- fold in some human tumor cells and normal peripheral blood monocytes by direct activators of the cAMP-dependent protein ki- naseA (PKA), dibutyric-cAMP and 8-bro- too-cAMP, and by inhibitors of phosphodiesterase, which stimulate PKA indirectly by elevating cytosolic cAMP levels. This PKA activity is blocked on these cells by activators of protein kinase C (PKC), which act by reducing the affinity of the TNF-R [34]. TNF-R expression is also downregu- lated by PKC activators on human fibroblast [35] and cell lines [36, 37]. In contrast, PKC activators reverse TNF-R downregulation induced by IFN-et or IFN-y on primary or transformed murine macrophages, which is surprising since IFN-y has been shown to stimulate PKC activity in the macrophage cell lines THP-I and U937 [38] (and endo-

144 Johnson/Pober

thelial cells [39]). These reports of changes in TNF-R number have been interpreted as implicating PKC and PKA in regulating TNF signal transduction in several cell types. However, this interpretation is open to ques- tion since both growth- and MHC-regulating activities of TNF appear to be independent of TNF-R modulation.

Autocrine and Paracrine Mediators of TNF Activity In human endothelial cells, TNF-me-

diated increases in class-I mRNA levels is protein synthesis-dependent [2], consistent with the possibility that TNF induces a second cytokine which mediates or augments the TNF effect. TNF induces a transient accumulation of TNF mRNA, which is enhanced by IFNq,, in murine peritoneal macrophages [40]. TNF has also been reported to induce IL-1 and IL-6 in endothelial cells [22]. However, these cytokines do not increase class-I MHC expression. Autocrine secretion of TNF by IFN-7-treated human monocytes has been reported to enhance class-II expression [18]. In contrast, exogenous TNF inhibits IFN-y-induced class-II expression by endothelial cells [ 12]. The constitutive and TNF-induced expression of classI in human fibroblasts has been reported to be mediated by IFN-~3 [41], although this mechanism is probably not oper- ating in human endothelial cells or in HeLa cells since (1) neutralizing antibodies di- rected against IFNq3 (or IFN-a) do not inhibit TNF induction of class I on endothelial cells (unpublished observations), and (2) maximal IFN-13-induced class-I expression is markedly further enhanced by exogenous TNF [12]. Moreover, TNF does not induce measurable IFN-13 mRNA or activity in these cells.

TNF-stimulated growth of confluent human FS-4 fibroblasts is enhanced by the addition of the prostglandin inhibitors, indo- methacin or acetylsalicylic acid (aspirin), suggesting that prostaglandins induced by TNF antagonize its mitogenic activity [42, 43]. Whether prostaglandins affect the expression of MHC molecules by these cells is unclear, although prostaglandins have been shown to regulate MHC expression in murine macrophages [44].

Intracelhdar Second Messenger Coupling The best described intracellular second

messenger systems are PKC (activated by Ca 2+ and diacyl glycerol or arachidonic acid), tyrosine kinase (often receptor-associated and directly activated by ligand binding) and calmodulin-dependent kinase (activated by Ca 2§ and calmodulin). There is no evidence that TNF affects intracellular calcium or can be mimicked by calcium ionophore in endothelial cells (unpublished observations), although it should be noted that a calcium ionophore has been shown to mimic TNF in inducing a transcription factor in human fibroblasts [45]. In several systems TNF is mimicked by activators of PKC, observations that initially suggested that TNF binding is transduced by PKC activation. In endothelial cells both TNF and phorbol-i 2-myristate-13-acetate (PMA), an activator of PKC, induce transient expression (4 h maximum) of the adhesion molecule ELAM-1, although protracted PMA stimulation results in PMA unresponsive- ness while leaving the TNF response largely intact [28]. Similarly, in human gingival fibroblasts, TNF and PMA induce phosphorylation and reduction in number of epidermal growth factor receptors; the activity of PMA is blocked by the PKC inhibitor staurospo-


fine [46], but TNF is not [47]. Similarly, we have found that in human endothelium, staurosporine also blocks PMA-induced but not TNF-induced expression of ELAM-I (unpublished observations).

TNF and IL- 1 cause a rapid accumulation of intracellular cAMP in human FS-4 fibroblasts, which peaks in 5 rain and returns to basal levels by 15 min [48]. Extracts of TNF- treated cells cause the phosphorylation of added histone proteins under conditions that are chosen to minimize PKC activity and phosphorylation is blocked by the PKA inhibitor H-8. The addition of an activator of PKA, forskolin, induces a similar rise in cAMP and activation of histone kinase in these cells. Furthermore, TNF, IL-1 and forskolin each strongly induce the accumulation of IL-6 mRNA by 1 h. In contrast to the abrogation of agonist-induced cAMP eleva- tion by H-8, however, only the forskolin induction of IL-6 is blocked by H-8; the induction of IL-6 by TNF or IL- 1 is only par- tially reduced. These results implicate PKA in mediating TNF (and IL- 1 ) effects but suggest the involvement of additional second messenger systems.

TNF stimulates the rapid (30 s) phosphorylation of serine and tyrosine residues of a protein and myeloid HL-60 cells [49]. Tyrosine kinase activity is correlated with mitogenic activity in products of many cellular oncogenes. Both TNF and epidermal growth factor (EGF) stimulate growth in confluent diploid human FS-4 fibroblasts [50]. In human carcinoma cell lines TNF transiently stimulates EGF-R tyrosine kinase activity 3-fold within 10 min, which correlates with increased phosphorylation of the EGF-R at sites similar to those modified by EGF but distinct from those modified by PMA [42]. IL-1 also induces phosphoryla-

tion of the EGF-R, coincident with reduction in affinity for EGF, by a PKC-independent mechanism [47]. Growth conditions may directly affect constitutive and TNF- induced MHC expression by altering the levels of inhibitory or activating protooncogene products. For example, addition of serum or growth factor to endothelial cell cultures induces both c-fos, which promotes class-I expression, and c-myc, which inhibits class-I expression [51].

The coupling of ligand binding to second messenger activation is often mediated by guanosine triphosphate (GTP)-binding proteins (G proteins) and often involves the action of phospholipases. TNF increases the binding of GTPTs, an unhydrolyzable ana- log, to several cellular G proteins, and stimulates a pertussis toxin-sensitive GTPase activity nearly 4-fold in the promyelocytic leukemia cell line HE-60 and murine L929 fibroblasts [52]. One of the pertussis toxin- binding G proteins was identified as Gi, which shortens activation by virtue of its high GTPase activity. Furthermore, the TNF-induced increase in permeability of endothelial cell monolayers is blocked by pertussis toxin [53], suggesting a G-protein in- termediary in this TNF activity. However, IL-1 also acts through pertussis toxin-sensitive G proteins [54] and cAMP [55], though a novel mechanism has been proposed in T cells wherein IL-1 stimulates PKC activation by causing hydrolysis of phosphotidylcho- line, without demonstrable hydrolysis of phosphatidylinositol [56]. Similarly, TNF acts independent of phosphatidylinositol hydrolysis by phospholipase C or phospholipase D, albeit through a pertussis toxin-in- sensitive, dibutyryl cAMP-inhibited pathway in the respiratory burst response of human neutrophils [57]. Since IL-1 does not

146 Johnson/Pober

induce MHC class-I expression, these simi- larities in G-protein activation by TNF and IL-1 suggest that this is not a major pathway of TNF-regulated MHC class-I expression. On the other hand, IL-1 and TNF have similar activities on some cells in regulating class-II expression, so G proteins may mediate this TNF activity.

Phospholipase has been implicated in the cytotoxic and mitogenic activities of TNF on dividing or quiescent cultured cells [58]. Both activities are inhibited by quinacrine, an inhibitor of phospholipase, and mimicked by melittin, an activator of phospholipase-A2. Whether this phospholipase is directly involved in regulating MHC genes is unknown. However, one of the products of phospholipid hydrolysis by phospholipase- A2, arachidonic acid, can be further metabo- lized to yield prostaglandins, which are able to directly regulate MHC expression by some cell types [44].

Transcriptional Activation of MHC Genes by TNF

MHC Genes The MHC occupies approximately 3,500

kb on chromosome 6 in man and chromosome 17 in mice. Within the MHC, class-Ict chain genes, called HLA-A, B, C in man, and class-II genes, called HLA-DR, DP, DQ in man, are found grouped by class and, in class-II genes, a and [3 genes for each molecule are proximal. The class-II3 chain, also called 132-microglobulin, is monomorphic and encoded on a separate chromosome in both man and mice. The Y-flanking region of class-I genes contains homologous cis-acting DNA sequences, which allow the coordinate transcriptional regulation of these

genes. Similarly, class-II genes share regulatory elements that allow coordinate expression of the class-II genes. However, these elements differ between class-I and class-II genes, consistent with their distinct patterns of expression.

Protein Factors/DNA Elements Mediating TNF Effects TNF activates at least two families of

transcription factors: AP-1 and NF-~B. In human fibroblasts TNF causes transcriptional activation of e-jun and c-los [59], the genes encoding the major subunits of AP-1, which in turn promotes the transcription of genes containing a so-called TPA (12-0- tetradecanoylphorbol-13-acetate, PMA) response element (TRE) in their 5'-noncoding regions (GTGAGTA/cA) [60-62]. These pro- tooncogenes regulate their own transcription through TREs and their transcription is also regulated through serum response elements (SREs) and cAMP response elements (CREs) [63]. In T cells TNF and PMA activate the transcription factor NK-KB, which promotes transcription of genes containing a 5'-KB element (ACGGGGACTTTCCG) [64]. NF-~B is found ubiquitously associated with an inhibitory subunit, I-KB, in the cytoplasm of unstimulated cells. TNF- or PMA-activated NF-~:B dissociates from the inhibitory subunit, perhaps as a result of the phosphorylation of I-~:B, and is then translocated to the nucleus where, in T cells, it promotes ~:B- mediated transcription of IL-2 and IL-2 receptor genes [65, 66], although a require- ment for additional regulatory elements in TNF-induced IL-2 receptor expression has been suggested [67]. However, in T cells stimulated with IL-1 and antigen the transcription ofjun andros is also activated, suggesting a role for AP-1 in mediating IL-2


5' Sequence 3' Identity position #

TCCTAGAATG AGCGCCGGTG TCCCAAGCT- GGGGCGCGCA CCCCAGATCC GGAGGGCGCC ~2m (human) -201

GCTGTGTAAG GATTGGGGAG TCCCAGCCT/ -GGGATTCCC CAACTCCGCA GTTTCTTTTC HLA-A3 -148 GCGGTGTATG GATTGGGGAG TCCCAGCCTT GGGGATTCCC CAACTCCGCA GTTTCTTTTC HLA*A2 -143 GCCTTGTCTG CATTGGGGAG GCGCACAGTT GGGGATTCCC CACTCCCACG AGTTTCACTT HLA'~27 "141

GGTGAGGTCAG GGGTGGGGAA GCCCAGGGCT GGGGATTCCC CATCTCCT H-2L- -167

�9 �9 �9 CRE I I , I ( CRE I I I

H-2K b -166 GTGAGGTCAG GGGTGGGG~ GCCCAGGGCT GGGGATTCCC CATCTCCAC �9 ( ) ~ - -

ACAA AGGGACTTTC CCATITTCAGTT

A GGGGACTTTC CGAGAG

............ C ....

KB-(ike repeats a , b ' . a , b

gB-Like repeats b J . ' d

~2m (murine) -166

Ig~ ~8, NF-KB-binding

mutant K8, H2TF1/KBF1-binding

Fig. 1. Class-I enhancer sequences compared with known transcription factor binding sites. Sequences have been aligned for maximum homology, gaps are indicated by -. Sequences are from GenBank, except for H-2L d [72], for which the orientations of the class- I regulatory, elements (CRE) are indicated with arrows and H2-K b [74], for which the orientation of the

repeated elements are indicated with arrows. Position numbers indicate distance (bp) from base indicated with a # to the TATA-like TCTA sequence, except for H-2L d and H-2K b (relative to cap) and 132m, which has a TATA sequence. Underlined sequences are DNase footprints with NF-~B [96, 97]. The cAMP response-like element is double underlined.

gene induction [68]. The activity of KB-like

enhancer mediating the lipopolysaccharide (LPS)-stimulated transcription of the TNF gene itself in primary macrophages [69] suggests that this element may also mediate autocrine expression of TNF.

Recently, the transcription factor interferon regulatory factor-1 (IRF-I) has been

shown to be strongly induced by TNF in both human fibroblasts and murine L929 cells [45]. IRF-1 binds to a portion of the

interferon response element in the 5' region of MHC class-I genes [70] and the same sequence binds a second, putative negative regulatory factor IRF-2. Whether IRF-2 is modulated by TNF is unknown.

At least two other families of transcription factors might be involved in TNF-

induced gene activation, in addition to AP- 1, NF-~:B and IRF-1/2. The cAMP-response

element (CRE) differs from the TRE by the insertion of a single C (GTGAC__GTA/cA/6) [62] and a sequence highly homologous to the CRE is found within the class-I enhancer

(fig. 1). Several genes encoding CRE-binding proteins (CREBs) have been isolated. Since TNF elevates cAMP levels [48], it is possible

that the CREB family of transcription factors is involved in TNF regulation of MHC expression.

The recently-described transcription factor H-2RIIBP also binds to region II of the class I regulatory element [71] (fig. 1). How- ever, the nuclear factors that bind to this region have been previously shown to be

present in various issues irrespective of the

148 Johnson/Pober

A 10 20 30 40 50 60 70 80 90

I i i i i i i

AAG~TTA~T~TcT~A~AAAcT~cATG~GATGAT~TTT~TC~TA~AAGAGT~CA~GT~AcA~TAA~GA~T~G~A~T~A~GA~TC~AGTT~A~ 100

i i i | I i i

GGACAGAGATTACGGGATAA~AGGAGAGGGACGGGG~CCATGCCGAGGG~TTCTCCCTTGTTTCTCAGACAG~TCTTGGGCCAAGACTCAGGG 200 i i i r i i i

AGACATTGAGACAGAGC•CTTGGCA•AGAAGCAGAGGGGTCAGGGCGAAGTCCAGGGCCC•AGGCGTTGGCTCTCAGGGTCT•AGGCC••GAAGGCGGTG 300

i I i i i i

TATGGATTGGGGAGTCCCAGCCTTGGGGATTCCCCAAcTCCGCAGTTTCTTTTCTCCCTCTCCCAACCTATGTAGGGTCCTTCTTCCTGGATACTCACGA 400

I I I I I | I

CG~GGAC~CAGTTC~C}CCATTGGGTGT~GGGTTT~CAGAGAAG~AAT~AGTGT~GT~GCGGTCG~GGTT~TAAAGTCCGEACGCAC~CA~EGGGA 500

CTCAGATTCTCCCCAGACGCCGAGGA~GCCGTCATGGCGCCCCGAA

B C

TGAGTCA consensus TRE [60] ~ H-2Kb [70] ,..,..T HLA-A2 190" ...... HLA-A2 122" .... A.. 207 ..... C 415

Fig. 2. A Analysis of the 5' flanking region of the human class-I MHC gene HLA-A2. Sequence is from GenBank, locus HUMMHA2, accession number K02883. CAAT box and variant TATA box (TTCTAAA) are underlined, translation initiation co- don is indicated with an arrow. TPA response elements (TRE) homologies are underlined, interferon

regulatory factor-l-binding site homologies are boxed. The NF-KB/H2TFI/KBFI-binding site homology is indicated by a dotted line over the sequence [76]. B. TRE homologies are listed and compared with the consensus TRE [60]. C. IRF-l-binding site homologies are listed and compared with that of the murine class-I binding site [70].

class-I expression [72] and little is known about the regulation of this transcription factor.

Class-I Regulation The 5' regions of MHC class-I a and [3

chain genes contain a sequence homologous

to a ~:B site and several sequences with homologies to the TRE (fig. 2). In addition, a CRE is located within the class-I regulatory.

element (CRE) region I [72]. Whether these sites mediate TNF activation ofclass-I genes remains unclear.

Several groups have noted an enhancer activity in a region 5' of class-I genes [72]. Zachow and Orr [73] have shown that the

amount of enhancer binding activity corre-

lates with the level of constitutive class-I expression in human leukemic T-cell lines.

Israel et al. [74] have suggested that TNF induces transcription of the murine class-I gene (H-2K b) by activating an NK-~:B-like

transcription factor, which displaces constitutive binding proteins (AP-2 and

KBF1/H2TF1) from two imperfect ~B sites and stimulates transcription. They show that either site is unresponsive while multimers

of either site are TNF-responsive. However, the human class-I ct chain genes HLA-A2, A3

and B27, which have only a single full repeat, and human [32-microglobulin, which has only a partial element 5' of the gene, respond

to TNF [2, 3] (fig. I). In addition, in functional studies with transfected reporter genes


these investigators showed that a promoter construct lacking most of the principal ~cB site responds even better to TNF than does a promoter with the entire KB site, weakening the argument that this nB site mediates the T N F response of class-I genes.

A recently described transcription factor, PRDII-BF1, binds the ~cB-like sequence in the murine class-I gene H-2K b, and displays novel characteristics [75]. Unlike other ~cB- binding factors, PRDII-BF1 is under transcriptional regulation and is induced only slowly, over a period of hours, by serum or virus stimulation of human MG63 cells. These are the characteristics that would be expected of a transcription factor mediating TNF regulation of HLA class-I genes since they are induced slowly and since cyclohex- imide, a protein-synthesis inhibitor, has previously been shown to block TNF induction, implicating a protein factor synthesized de novo [2]. Moreover, the authors report that serum stimulation increases HLA class-I mRNA with kinetics that are slightly slower than those of PRDII-BF1. A similar or identical factor has been identified in endothelial cells [Tucker Collins, Brigham and Women's Hospital, personal commun.]. Therefore this factor is an attractive candidate for a role in mediating TNF induction of class-I genes. It should be noted that PRDII-BF1 is much larger (predicted 250-290 kD) than either NF-KB (45-50 kD and an associated 65-kD polypeptide) [76] or KBF1/H2TF1 (120 kD), so it is unlikely to be the factor observed by Israel et al. [74].

Class-H Regulation The cell-type-specific regulation of class-

II mRNA expression is reflected in the variety of nuclear factors that bind to the promoter regions of class-II genes. For example.

the 5' region of the HLA-DRct gene contains a l l0-bp sequence that binds different factors in different cell types, such as B cells, T cells, murine macrophages, and IFN-v- treated or untreated HeLa cells [77, 78]. One common factor, RF-X, appears to be re- quired for IFN-y inducibility in fibroblasts, and a second, NF-S, binds to a CRE-like element [79]. Little is known about protein factors or sequence elements that may mediate positive and negative T N F regulation of class-II expression. Although TN F strongly induces IRF-1, no binding site is apparent in the l l0-bp sequence mentioned above. Whether a cryptic site exists in this region, or can be found elsewhere, remains to be seen. A second attractive mediator for TN F regulation of class II is mXBP/CRE-BP2. It is unknown whether this factor is regulated by TNF, but some regulatory effects are likely through TNF regulation of PKA activity.

Transcription Factor Modification Protein kinases can control the activity of

transcription factors. For example, PKC is believed to phosphorylate I•B, resulting in activated NF-~cB [64]. Subunits of the transcription factor AP-1 (c-Fos,c-Jun) are also regulated by phosphorylation [80, 81]. It is unclear whether Jun-B or Jun-D, which are recently described alternative, regulatory forms ofc -Jun [82, 83], are also regulated by phosphorylation. CREB is phosphorylated by PICA [84] and is also predicted to have PKC and casein kinase-II phosphorylation sites [85]. In addition, CREB [85] and CRE- BP2 [86], like Jun and Fos, contain leucine- zipper domains, which mediate hetero- and homo-dimerizat ion of these transcription factors. The presence of PKA, PKC and casein kinase-II phosphorylation sites on

150 Johnson/Pober

CREB suggests that this transcription factor could be modified by different activated second messengers systems.

Second Messenger~Transcription Factor Cross-Talk In one cell type the response of a single

gene to either IFN- 7 or IFN-a can be blocked by different second messenger antagonists [87]. This suggests that different receptors will be found that activate distinct second messengers. However, a single transcription factor subunit (c-Jun) can combine with different subunits (mXBP/CRE-BP2 or c-Fos) to form complexes mediating responses to cAMP or PMA [86]. In addition, c-Fos but not v-Fos represses c-fos transcription through a serum response element (SRE) [88], perhaps because v-Fos is not phosphorylated under the same conditions as c-Fos [80]. In contrast, the c-fos SRE mediates transcriptional stimulation by an activated G protein, c-Ha-ras, or by an inhibitor of phosphodiesterase, dibutyl cAMP, both of which may stimulate PKA [63].

Taken together, these results suggest a mechanism by which cross-talk between second messenger systems might occur: particu- lar agonist-activated second messenger enzyme(s) may modify transcription factors that are used in common by several different transcription complexes. Transcription factors may undergo phosphorylation-regulated dimerization (or form higher multimers) mediated by interaction domains, such as leucine zippers. These complexes may be them- selved modified by phosphorylation and undergo allosteric changes such as those observed in the phosphorylation of muscle phosphorylase [89]. The initial pattern of gene expression induced by TNF would be a function of the second messenger systems

activated and the pre-existing transcription factors that are substrates of the activated second messenger enzyme systems. Subse- quent gene expression is likely to be complex due to alterations in second messenger enzyme activities over time and interactions with previously modified transcription factors.

Post-Transcriptional Regulation TN F stimulates phosphorylation of a cap-

binding protein (p28) in vivo and in vitro, suggesting a role in post-transcriptional regulation [90]. However, MHC class-I expression is regulated exclusively at the transcriptional level in endothelial cells [3]. Class II is also regulated at the transcriptional level in most cells, but post-transcription regulation is reported in some cells. TN F is itself regulated in response to LPS at the transcriptional level [69], in the stability of T N F mRNA, and at the secretory level [9 i].

Oncogenes The adenovirus E IA protein is a strong,

general repressor of many different kinds of enhancers, including the ~B element in the murine class-I gene H-2K b enhancer, the AP- 1 binding element in polyomavirus and human metallothionein enhancers and the multiple enhancer elements of SV40 [92]. This is one of the ways in which adenovirus reduces the expression of class I on the cell surface. TNF overcomes this repression in adenovirus-transformed mouse fibroblast cell lines and synergizes with IFN-7 in this activity [93]. IFN-y has been shown to increase mRNA for the class I c~ and 13 chains in such cells. It is probable, therefore, that these cytokines are circumventing E1A repression at the level of class-I transcription. The cytokine stimulation of class I appears


to be independent of the anti-tumor activi-

ties of IFN-y and TNF since Ad l2 E1A

mRNA and protein continues to accumulate in treated ceils.

Conclusion

In this review we have correlated early

events in TNF signal transduction, such as second messenger activation, with later transcriptional activation of MHC genes. This

description remains incomplete at several levels. However, we have suggested guide- lines for defining activated second messengers involved in mediating TNF effects in-

tracellularly, and we have indicated some of the ways in which transcription factors may be modified by TNF-activated second messenger enzymes to regulate gene expression. An exact model for TNF-activated gene expression can be formulated only when more

is understood about the various transcription factors and sequence elements that are involved. However, from what is already

known about these mediators it can be anti- cipated that transcriptional regulation by TNF will initially involve enzymatic modification of pre-existing transcription factors

that are then capable of forming active transcription complexes. Subsequent regulation will probably involve competition for tran-

scription factor subunits between transcription complexes and allosteroic modifications of the complexes themselves.

The importance of TNF in regulating

MHC molecule expression is underscored by the observation that TNF is most potent in vivo at causing the regression of immuno- genic tumors and is less effective in immu- nosuppressed mice [94]. In addition, a role for TNF in regulating MHC expression in

vivo is suggested by the observation that TNF enhances the T-cell dependent immune

response in mice [95]. However, TNF has many additional immunoregulatory func-

tions. The induction by TNF of cell adhe-

sion molecules, such as ELAM-1, ICAM-1, INCAM-1 10/VCAM-1, and cytokines, such as IL-1, IL-6, IL-8 and MCP-1, is also clearly

of great importance in the context of the immune response. Nevertheless, because of

the central role of MHC molecules in cell and antigen recognition by T cells, the affect of TNF on MHC expression is a major com-

ponent of these immunoregulatory activi-

ties.

References

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Dr. David R. Johnson Department of Pathology Brigham and Women's Hospital and Harvard Medical School Boston, MA 02115 (USA)

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Tumor necrosis factor regulation of major histocompatibility complex gene expression