0192-0561/92 $5.00 + .00 Pergamon Press pie. Internatio1"~al Society for lrnmunopharmacology.

Int. J. lmmunopharmac., Vol. 14, No. 3, pp. 401-411, 1992. Printed in Great Britain.

REVIEW: T R A N S C R I P T I O N A L A N D P O S T - T R A N S C R I P T I O N A L R E G U L A T I O N OF I N T E R L E U K I N 1 G E N E E X P R E S S I O N MATTHEW J. FENTON Department of Medicine and Evans Department of Clinical Research, Boston University Medical Center, Boston, MA 02118, U.S.A.

Interleukin la (IL-la) and/3 (IL-I/3) are proinflammatory cytokines that are encoded by distinct genes, but share most biological activities. During the past several years, intense investigation has focused on elucidating the molecular basis for the regulation of IL-la and /3 gene expression. While the overall organization of both genes is conserved in mammals, the DNA sequence homology is surprisingly limited. This supports the growing body of evidence suggesting that each gene is regulated by distinct cis- and transacting elements. Most recently, novel regulatory DNA sequence elements and several nuclear regulatory proteins have been identified, which ultimately participate in the control of IL-I/3 gene transcription. In addition to transcriptional controls, the stability of IL-1 mRNA can be selectively regulated by various inducing stimuli and other cytokines. Taken together, these transcriptional and post-transcriptional regulatory mechanisms provide stringent, yet flexible, control over expression of the IL-la and/3 genes. Abstract --

Interleukin 1 (IL-1) is a polypepetide cytokine that mediates a wide variety of inflammatory, metabolic, immunologic, and hematopoietic processes. IL-1 was described originally in the 1940s as an endogenous pyrogen, and later as a lymphocyte activating factor. Two distinct IL-! polypeptides, termed I L - l a and IL-lp, have been identified and are known to be the products of distinct genes. IL-1 belongs to a group of proinflammatory cytokines with overlapping bioactivities. This group includes tumor necrosis factor a (TNF) and interleukin 6 (IL-6). In combination, these cytokines can often act synergistically on target cells. While the biological and clinical properties of IL-1 have been well characterized [reviewed in (Dinarello, 1991)], the regulation of IL-1 production at the transcriptional and post-transcriptional levels remains poorly understood. In the following sections, recent progress in the study of the genomic organization of the IL-1 genes, the identification and characterization of cis- and trans-acting regulatory factors, and the selective regulation of IL-1 mRNA stability is reviewed. GENE

STRUCTURE

AND

ORGANIZATION

In 1984, the human IL-1/3 (Auron et al., 1984) and murine I L - l a cDNAs (Lomedico et al., 1984) were cloned and sequenced. Subsequently, additional

cDNAs were obtained from human (March et al., 1985), mouse, cow, pig, rabbit and rat cells. Earlier studies had described two different forms of IL-I based on their distinct isoelectric points (Dinarello et al., 1974); I L- l a has a pl of 5.3 and IL-1/3 has a pI of 7.2. The identification of distinct IL-1 cDNAs not only confirmed the existence of two forms of IL-1, but sequence analysis demonstrated that both forms are initially translated as 31 kd precursor proteins (prolL-l). While intracellular IL-1 exists exclusively as the 31 kd precursor, proteolytic processing by specific proteases generates a 17 kd biologically active carboxy-terminal polypeptide (mature IL-1) upon secretion. Distinct proteases appear to be responsible for cleavage of the p r o l L - l a (Kobayashi et al., 1990) and prolL-1/3 (Kostura et al., 1989) proteins, although several extracellular proteases can also cleave prolL-1 to generate biologically active molecules (Hazuda et al., 1990). While the mature forms of IL-lo and IL-1/3 can bind to both of the two known forms of the high affinity IL-1 receptor [IL-1RtI (Sims et al., 1988) and IL-1RtlI (Chizzonite et al., 1989], the human polypeptides share only 26°7o amino acid homology. A similar situation exists for TNF and lymphotoxin, which share 35°70 amino acid homology, exhibit identical biological activities, and are recognized by the same receptor (Aggarwal et al., 1986). Murine IL-1/3 is much more homologous to 401

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human IL-1/3 (67°7o) than to either murine IL-la (22%) or human IL-la (16%) (Gray et al., 1986). The entire genomic sequences for human IL-I~ (Furutani et al., 1986), human IL-I/3 (Clark et al., 1986), and murine IL-1/3 (Telford et al., 1986) have been reported. The human 1L-la gene is approximately 10 kb in length, whereas the human and murine IL-1/3 genes are approximately 7 kb long. The sequence of the routine IL-I~ gene has not yet been reported. The human and routine IL-1 genes are located on the same region of chromosome 2 in both species (position 2q13 in humans), although the linkage seen in the mouse does not seem to be conserved in man (D'Eustachio et al., 1987; Lafage et al., 1989; Webb et al., 1986). In spite of significant exon sequence divergence, the human IL-la and ILl/3 genes share an almost perfectly conserved i n t r o n - e x o n structure (Clark et al., 1986), suggesting that discrete structural domains are encoded by individual exons (Gilbert, 1978). The existence of two IL-1 genes in the rabbit places the date of a putative gene duplication event prior to human rabbit lineage divergence over 270 million years ago. In addition to the similar structural organization, lmman and murine IL-1/3 genes show significant sequence homology within the intron regions. The considerable conservation of the intron sequences contrasts with what is found for other highly conserved genes. For example, the large intron of the rabbit and murine /3-globin genes are not homologous, suggesting that a divergence of over 70 million years is sufficient to completely randomize the nucleotide sequences (Van Ooyen et al., 1979). The persistence of sequence homology between the introns of the human and murine IL-I genes suggests a functional role for these regions in the regulation of IL-1 gene expression. Furthermore, the IL-la gene appears to be evolving more rapidly than the IL-1/3 gene, although the regions which are most highly conserved differ between each gene (Young & Sylvester, 1989). Both IL-1 genes are comprised of seven exons, the first of which contains the majority of the 5' untranslated mRNA leader sequences. A highly conserved homopurine tract is also located within the first intron of the human and murine I D l p genes. Additional sequences within the first intron of the human IL-I/3 gene appear to possess both positive (Bensi et al., 1990) and negative (Clark et al., 1988) regulatory activities in transient transfection assays. The fourth intron of the human IL-la gene, and the third intron of the human IL-1/3 gene, contain members of the A l u family of repetitive sequence elements. Intron 6 of the human IL-la gene and

intron 5 of the human IL-1/3 gene contain sequences that are homologous to the glucocorticoid response element consensus sequence (TGTYCT), although the role of these sequences in glucocorticoidmediated suppression of IL-1 transcription has not been demonstrated. The IL-la gene is unusual in the extent of the restriction fragment length polymorphism associated with a site within the sixth intron (Haugen et al., 1989); six alleles could be distinguished among eight inbred strains of mice surveyed (D'Eustachio et aL, 1987). In comparison, only one variant IL-lp allele was observed. The seventh exon of both genes contains the 3' untranslated sequences (3' UTR) and the carboxy-terminal sequences of the coding region. Tile 3'UTR of the IL-I~ and IL-1/~ genes contains AU-rich sequences, including several copies of the ATTTA motif, that have been implicated in the selective destabilization of cytokine and cellular proto-oncogene mRNAs (Caput et al., 1986; Shaw & Kamen, 1986).

IL-I PROMOTER A N D UPSTREAM ELEMENTS

The IL-1 genes are transcriptionally silent in unstimulated peripheral blood monocytes (PBMs), vascular endothelial (Warner et al., 1987b), fibroblasts (Yamato et al., 1989), and vascular smooth muscle cells (Libby et al., 1986), but can be rapidly induced by a variety of agents (see below). In contrast, these genes are constitutively expressed in epithelial cells (Ansel et al., 1988; Kupper et al., 1986), large granular lymphocytes (Galli et al., 1990), Kupffer cells (Fox et al., 1991), and some transformed cells (Griffin et al., 1987; Wano et al., 1987). Pretreatment of lipopolysaccharide-nonresponsive U937 histiocytic lymphoma cells with 5-azacytidine has been shown to render these cells responsive to lipopolysaccharide (LPS), suggesting that demethylation of the IL-1 genes themselves (or genes that code for components of the LPS signal pathway) can influence subsequent IL-I expression (Kovacs et al., 1987). To date, few reports have described the upstream elements that control basal and inducible expression of the IL-1 genes. Transient transfection studies, using human IL-1/3 upstream sequences fused to the chloramphenicol acetyl transferase (CAT) reporter gene, demonstrated that CAT expression could be detected in transfected THP-1 human monocytic leukemia cells, but not in transfected HeLa cells (Clark et al., 1988). In contrast, CAT constructs that included the entire IL-I/3 first intron could be expressed in transfected

lnterleukin 1 Gene Expression HeLa cells (Bensi et al., 1990). Furthermore, when these intron sequences were present, only 132 bp of DNA located immediately upstream of the transcriptional start site are sufficient to promote constitutive expression of the gene, albeit at low levels. The activity of this 132 bp minimal promoter element can be substantially increased by the immediate early gene products of human cytomegalovirus (G. Hunninghake and M. Fenton, unpublished observations). Taken together, these data suggest that the IL-1 promoter is expressed in a cell type-specific manner that is mediated by distinct sequence elements and trans-acting factors. Recently Beusi et al. (1990) identified an enhancer sequence within the human IL-lp gene that mediates the induction of transcription by phorbol 12-myristate-13-acetate (PMA). This enhancer is located between positions - 2982 and -- 2795 upstream from the transcriptional start site. One copy of this enhancer sequence inserted upstream from an enhancerless SV40 promoter allows the expression of the CAT gene in PMA-stimulated THP-1 cells, whereas PMA-stimulated HeLa cells require the presence of two copies of the enhancer in order to express CAT activity. This enhancer could also confer PMA-responsiveness on CAT constructs containing a portion of the human IL-la gene ( - 4 2 0 0 to +723). Sequence analysis of this enhancer region revealed the presence of DNA motifs similar to the AP-1 binding site of the collagenase gene (Angel et al., 1987) and the positive regulatory domain I binding site in the human /3 interferon gene (Goodburn et al., 1986). Although these two DNA motifs may play a role in enhancer function, deletion of additional DNA upsteam of these sequences dramatically decreases CAT activity. While a LPS-responsive element has yet to be reported, preliminary evidence suggests that it overlaps and extends upstream of the PMA-responsive element (P. Auron, personal communication).

GENE EXPRESSION AND TRANSCRIPTIONAL

REGULATION

It is now recognized that a wide variety of cells have the capacity to synthesize IL-1, including blood monocytes, tissue macrophages, blood neutrophils, B- and T-lymphocytes, keratinocytes, fibroblasts, endothelial cells, smooth muscle cells, astrocytes, microglia, dermal dendritic cells, glomerular mesangial cells, and Kupffer cells. Transcription of

4O3

the IL-1 genes can be initiated by LPS, phorbol esters, calcium ionophores (Yamato et al., 1989), bacterial exotoxins (Knudsen et al., 1986), exposure to u.v. light (Kupper et al., 1987), complement components (Schindler et al., 1990a), autoreactive T-cells (Wasik et al., 1988), and adhesion of cells to a surface (Fuhlbrigge et al., 1987; Haskill et al., 1988) or the cross-linking of cellular adhesion molecules (Couturier et al., 1990). Human cytomegalovirus immediate early gene products have been shown to dramatically upregulate the expression of CAT constructs containing the human IL-1/3 promoter ( - 1 0 9 7 to + 14) in transient transfection assays (Iwamoto et al., 1990). Cytokines also play a central role in regulating IL-1 expression. Specifically, TNF and IL-I itself can induce IL-I expression in monocytes (Dinarello et al., 1987), vascular smooth muscle cells (Warner et al., 1987a), and vascular endothelial cells (Warner et al., 1987b). IL-2 was recently shown to induce IL-1/3 gene expression in PBMs (Kovacs et al., 1989) and T-cells (Numerof et al., 1990). Furthermore, lymphokines can augment IL-1 expression initiated by a distinct inducer. This phenomenon has been clearly demonstrated in the ability of y-interferon (Haq et al., 1985; Ucla et al., 1990), and GM-CSF (Frendl et al., 1990), and IL-3 (Frendl et al., 1990) to synergize with LPS to induce IL-1 production. Other cytokines, such as IL-4 (Essner et al., 1989; Hart et al., 1989), IL-6 (Schindler et al., 1990c), and IL-10 (Moore et al., 1990) can suppress IL-1 expression in LPS-stimulated monocytic cells. Furthermore, IL-4 was shown to block the ability of },-interferon to augment IL-1 production in LPS-stimulated PBMs (Donnelly et al., 1990a). In contrast, },-interferon was also shown to downregulate IL-l-induced IL-la and /3 production by a mechanism that does not involve prostaglandin synthesis (Ghezzi & Dinarello, 1988). Some of the best known inhibitors of I14-1 production are the glucocorticoids. Dexamethazone (DEX) has been shown to suppress IL-1 expression at both the transcriptional (Nishida et al., 1988) and post-transcriptional (Knudsen et al., 1987; Lee et al., 1988) levels. Heat and other inducers of the heat shock response can also inhibit IL-1 production (Schmidt & Abdulla, 1988). In summary, these data demonstrate that the IL-1 genes are under unique and specific transcriptional controls which are directed by the particular inducing agent and can be further modulated by several lymphokines. While transcription of the IL-1 genes can be induced by a variety of agents, the kinetics and magnitude of the response can be quite varied. As might be expected from the relatively low sequence

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homology between the IL-I a and/3 promoters, these two genes are often differentially expressed. For example, keratinocytes constitutively express two to four fold higher levels of IL-la mRNA than IL-1/3 mRNA (Kupper et al., 1986). In contrast, human CD3 large granular lymphocytes constitutively express IL-lp mRNA and little, if any, IL-la mRNA (Galli et al., 1990). Also, LPS-stimulated PBMs express predominantly IL-1/3 message (Burchett et al., 1988), whereas stimulated human T-cell clones express predominantly IL-la message (Acres et al., 1987). PMA-stimulated U937 cells have been shown to express IL-I/3 mRNA, but not IL-la mRNA (Nishida et al., 1988). IL-la can induce both IL-la and IL-1/3 expression in PBMs (Dinarello et al., 1987), but induces only IL-lp expression in vascular smooth muscle cells (Warner et al., 1987a) and vascular endothelial cells (Warner et al., 1987b). In smooth muscle and endothelial cells, IL-la expression was only observed in cells treated concomitantly with the protein synthesis inhibitor cycloheximide (CHX). In addition to these cell typespecific differences, there is also considerable variation in the relative expression of both IL-lcr and /3 within populations of cells, depending on the state of proliferation (Lovett & Larsen, 1988), differentiation (Ansel et al., 1988; Myers et al., 1989), tissue distribution (Burchett et al., 1988; Bernaudin et al., 1988), age (Sauder et al., 1989), or time in culture (Arend et al., 1989). As discussed below, these variations can result from changes in gene transcription and/or mRNA stability. We and others have studied the regulation of IL-1/3 gene expression using THP-1 cells (Fenton et al., 1987). LPS activation of these cells was found to result in the immediate and transient increase of IL-1/3 message. Similar transient kinetics for IL-lp gene expression have also been reported for PBMs (Turner et al., 1989), vascular endothelial cells (Warner et at., 1987b), and vascular smooth muscle cells (Libby et al., 1986). The presence of CHX during LPS stimulation caused a superinduction of message synthesis, but did not abolish the transient response. These results are consistent with the findings of Collart et al. (1986) who first described the superinduction of Cytokine gene transcription in the presence of CHX, and concluded that these genes were regulated by labile transcriptional repressors. The findings of Turner et al. (1988, 1989) differed from these data, and supported the hypothesis that the major effect of CHX is to stabilize IL-1 mRNA. We later used the THP-1 cell model system to demonstrate differential expression of both IL-1/3 mRNA and protein by two distinct stimuli; LPS and

PMA. In contrast to LPS, the kinetics of IL-lp message production following PMA stimulation is nontransient (Fenton el al., 1988). Furthermore, PMA-induced transcription, unlike that for LPS, was not affected by the presence of CHX. These differences, along with the ability to elicit message production by either LPS or PMA under conditions where the other inducer is inactive (Fenton el al., 1988), suggest that LPS and PMA regulate IL-lp gene expression by independent mechanisms. While these two induction pathways are essentially independent, they may share some components. This possibility was strengthened by the demonstration that IL-la and p expression in murine macrophages involved both protein kinase C (PKC) and calmodulin-dependent kinase activities (Kovacs et al., 1988). Thus, the hypothesis that LPS activates two distinct kinase pathways, while PMA activates only the PKC pathway, would be consistent with our data. In contrast, IL-2 can induce IL-1/3 expression in PBMs by a pathway that can be blocked by inhibitors of PKC, but not calmodulin-dependent kinase (Kovacs et al., 1989). The cellular second messenger pathways that mediate transcriptional activation or repression have yet to be fully understood. While glucocorticoids, such as DEX, are presumed to act via receptors that have the capacity to bind to specific DNA sequences, the location of such glucocorticoid responsive elements (GREs) has yet to be identified. As mentioned above, GRE-like sequences have been identified within the introns of both the IL-lc~ and/3 genes, greater than 5000 bp downstream of the start of transcription. DEX was capable of blocking the expression of IL-lp mRNA in IL-l-stimulated astrocytoma cells (Nishida et al., 1989) and in LPS-stimulated PBMs (Lew el al., 1988). DEX was also shown to completely block IL-1/3 mRNA expression in bacterial toxin-stimulated U937 cells (Knudsen el al., 1987). In prestimulated cells, higher concentrations of DEX induced a transient increase in intracellular cAMP levels and could block IL-I release without affecting mRNA levels. Kern el •1. (1988) later reported that LPS-induced lL-lp transcription in PBMs was unaffected by DEX treatment, although the high doses of LPS were used in these studies (10 #g/ml) may have been capable of overcoming DEX inhibition. The role of cAMP and prostaglandins (PGs) in IL-I production has also been examined. Warner el al. (1987a,b) showed that PGs could block the release of IL-1/3 bioactivity, but not transcription, in both vascular smooth muscle and endothelial cells stimulated with IL-I. In LPSstimulated PBMs, PGs were also shown to post-

Interleukin 1 Gene Expression transcriptionally inhibit the release of IL-1 bioactivity (Knudsen el al., 1986). Moreover, PGs were proposed to act in this sytem by increasing intracellular cAMP levels. This phenomenon is dependent on the particular inducing signal since cAMP agonists were shown to augment IL-1 mRNA production in PMA-stimulated PBMs (Hurme, 1990). In marked contrast to these studies, Scales et al. (1989) reported that treatment of LPS-stimulated murine peritoneal macrophages with PGE2 had no suppressive effect on IL-1 mRNA levels or bioactivity, whereas TNF mRNA levels and bioactivity were dramatically reduced. Furthermore, Ohmori el al. (1990) recently showed that both PGE2 and dibutyryl cAMP could markedly enhance IL-13 message production in LPS-stimulated murine macrophages, while these agents had no effect on the levels of IL-la mRNA in these cells. The inconsistent effects of PGs and cAMP on IL-1 mRNA and bioactivity levels in these various experiments have yet to be explained. Genetic factors also contribute to the ability of cells to correctly regulate IL-1 gene expression. Strain-specific differences in the production of IL-1 has been reported in murine macrophages (Brandwein et al., 1987). Moreover, the overproduction of IL-1 clearly plays a critical role in the pathogenesis of some inherited autoimmune and inflammatory diseases. Recently, Donnelly et al. (1990b) investigated the regulation of IL-1 expression in several autoimmune-prone strains of mice. Rather than evaluating mice in which autoreactivity and tissue damage had already developed, young animals were studied at a time substantially before the onset of detectable disease. Peritoneal and bone marrow-derived macrophages from all strains of autoimmune-prone mice tested displayed transient production of IL-1 protein, in contrast to the stable expression characteristic of control normal strains. Macrophages from autoimmune-prone mice also became progressively refractory to both induction and maintenance of IL-1, a pattern that correlated with changes in the steady-state levels of IL-la and IL-13 mRNA. Subsequent analysis of transcription rates by nuclear run-on assays showed that only 50-60°7o of the reduction in steady-state IL-1 mRNA levels observed in macrophages from autoimmune-prone mice could be accounted for by decreased transcription (D. Hartwell, M Fenton and D. Belier, unpublished observations), suggesting that post-transcriptional regulation may also contribute to the marked hypoexpression of IL-1 in these cells. Finally, it should be noted that transcription,

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translation, and release of IL-I are distinct and dissociated processes. Transcription without translation has been observed in several experimental systems under conditions where increased mRNA turnover (see below) cannot account for the absence of a translated product. For example, IL-l-stimulated lung fibroblasts were shown to express IL-1/3 mRNA, but these cells did not translate this message (Elias et al., 1989). TNF-stimulated fibroblasts did not express IL-1// message, whereas IL-1/3 mRNA was expressed and translated when these cells were stimulated with both IL-1 and TNF. The presence of redundant mechanisms that regulate IL-1 production clearly underscores the necessity of preventing inappropriate production of this potent cytokine.

TRANSCRIPTIONAL REGULATORY PROTEINS

Transcriptional control of the IL-1 genes is ultimately mediated through the action of specific nuclear regulatory proteins which serve a dual role. First, these proteins recognize and bind only to specific DNA sequences. Different genes, which are coordinately expressed in distinct cell types or during development, may share common nuclear regulatory factors which recognize similar DNA sequences in each gene. For example, identical TPA (phorbol ester) responsive elements (TREs) are contained in a number of distinct genes that are coordinately induced by phorbol esters. These TREs are the binding site of the transcriptional factor AP-1, a heterodimer composed of the cellular protooncogenes c-fos and c-jun [reviewed in Curran & Franza (1988)]. The cell-type specificity of some inducible genes is often based on the presence of a required nuclear factor only in permissive cell types. The second role of nuclear regulatory proteins is to receive the stimulatory (or inhibitory) signals transmitted through several possible cellular second messenger systems which simultaneously modulate the expression of many different genes. These second messengers include various protein kinases, calcium ions, arachidonic acid metabolites, cAMP, and internalized receptor-ligand complexes. We recently used electrophoretic mobility shift assays (EMSA) and DNA footprinting to identify and characterize transcriptional regulatory proteins that bind to the human IL-13 gene. Specific DNAbinding activity was localized to a single genomic fragment ( - 5 8 to + 11) by EMSA analysis using crude nuclear extracts prepared from both resting and stimulated PBMs. DNA-binding activity was

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also observed in nuclear extracts prepared from human neutrophils, several monocytic cell lines, vascular endothelial cells, and foreskin fibroblasts. This DNA fragment is of particular interest since this region is highly conserved between the human and murine IL-I/3 genes (Clark et al., 1986; Telford et al., 1986), and contains the " T A T A box" motif. Factors which bind to this fragment may interact with the transcriptional machinery, and therefore represent the penultimate step in transducing signals which activate IL-1/3 gene expression. We subsequently used methylation interference footprinting to identify the binding site for the protein present in PBM nuclear extracts. This protein, termed NFIL-I/3A (/3A), was found to bind to a sequence upstream of the TATA box ( - 4 9 to 38) (Fenton, 1990). The sequence of the /3A binding site is completely conserved between the human and routine IL-1/3 genes (100070 homology over 12 bp), as is the location of this sequence relative to the TATA box of each gene. The latter observation suggests that /3A may interact with the TATA box-binding factor TF-IID (Nakajima et al., 1988). A search of the GENBANK DNA sequence database (using the FASTA search program) revealed that the /3A binding sequence does not resemble the binding site for any previously described nuclear regulatory proteins. The human IL-I~ gene, which does not possess a TATA box, contains no promoter sequences that are homologous to the/3A binding site (Furutani et al., 1986). EMSA analysis of nuclear extracts prepared from various cells showed that multiple D N A - p r o t e i n complexes could be generated using an oligonucleotide probe that contained only the /3A-binding sequence. This suggested that either distinct nuclear proteins could specifically bind to the same DNA sequence, or that a single protein could exist in several post-translationally modified isoforms such that each isoform could generate a distinct complex. While this question has yet to be definitively answered, we have found that the relative expression of the various pA isoforms can be rapidly altered upon stimulation of the cells with LPS or PMA. Furthermore, these changes are only observed when nuclear extracts are prepared with buffers containing phosphatase inhibitors, suggesting that the /3A isoforms may be post-translationally modified by phosphorylation. It should be noted that the expression of/3A isoforms in unstimulated cells is not inconsistent with the possibility that/3A plays a role in inducible IL-I/3 gene expression. The DNAbinding and transcriptional activation domains of

transcriptional regulatory proteins often act as independent functional units. Thus, a nuclear protein could be constitutively bound to DNA in the unstimulated cell, and yet have no effect on transcription until (for example) subsequent posttranslational modification of the transcriptional activation domain has occurred following stimulation of the cell. Experiments to address the functional role of/3A in transcription of the IL-1/3 gene were performed using transient transfection assays. We found that deletion or mutation of the/3A binding site abolishes, or severely reduces, IL-I/3 promoter basal activity. Additional transfection experiments have also shown that t r a n s - a c t i v a t i o n of a minimal promoter element ( - 1 3 1 to +11) by the human cytomegalovirus immediate early gene products requires the #A site (G. Hunninghake and M. Fenton, unpublished observations), thus demonstrating that pA plays a critical role in both basal and inducible IL-1/3 promoter function. In summary, our preliminary studies have identified a novel family of nuclear regulatory proteins that can be rapidly and transiently modulated by agents that can induce IL-lp gene expression (PMA and LPS). Furthermore, these proteins appear to be required for basal, and in some cases, inducible IL-1/3 promoter function.

POST-TRANSCRIPTIONAL REGULATION The regulation of mRNA decay is now clearly recognized as an important mechanism which is used to establish steady-state message levels within the cell. The ability to simultaneously regulate gene expression at the transcriptional and posttranscriptional levels provides a means to rapidly and transiently express particular gene products, including many cytokines. The presence of AU-rich sequence elements (AREs) in many cytokine messages, including IL-la and /3, suggests that selective mRNA decay is a common feature of cytokine expression (Caput et al., 1986). Two distinct ARE-binding proteins have been recently identified, and these proteins may mediate selective mRNA decay (Bohjanen et al., 1991; Gillis & Malter, 1991). Several studies have shown that the expression of both the IL-la and/3 mRNA can be regulated at the post-transcriptional level. For example, LPSstimulated THP-1 cells predominantly express IL-1/3 mRNA and protein (Fenton et al., 1987). Turner et al. (1988) showed that while both genes were

Interleukin 1 Gene Expression transcribed at similar rates, the I L - l a message was significantly more unstable than the IL-I/3 message. IL-1 mRNA stability was subsequently shown to be modulated by distinct stimuli. In THP-1 cells, LPS initially induced an unstable IL-lp message population that became more stable with time (Fenton et al., 1988). In human PBMs, the ability of ),-interferon to enhance LPS-stimulated IL-I production was found to result from both enhanced IL-I/3 gene transcription and increased message stability (Arend et al., 1989). While ),-interferon can block IL-l-induced IL-1 production in PBMs, this molecule had no effect on IL-1 mRNA stability in this system (Schindler et al., 1990b). Furthermore, stimulation of both THP-1 cells (Fenton et al., 1988) and WI-38 fibroblasts (Yamato et al., 1989) with PMA increases both IL-lp transcription and mRNA stability. It should be noted that PMA-mediated stabilization of IL-I mRNA has also been observed for the GM-CSF message (Shaw & Kamen, 1986), and correlates inversely with the levels of a specific ARE-binding protein (Bohjanen et al., 1991). The intracellular signals that initiate changes in message stability have yet to be identified. Transfection of normal human fibroblasts with a mutant H-ras oncogene could selectively increase both transcription and stability of IL-I mRNA, suggesting that G-proteins may play a role in these processes (Demetri et al., 1990). Most recently, Ohmori et al. (1990) observed that while agents which elevated cAMP levels selectively increased IL-1/3 mRNA accumulation in murine peritoneal exudate cells, these agents did not alter IL-I~ or/3 mRNA stability. Recently, we have shown in LPS-stimulated PBMs that IL-4 can destabilize IL-1/3 mRNA, by a mechanism involving new protein synthesis, in addition to its inhibitory effects on IL-1/3 transcription (Donnelly et al., 1991). As mentioned above, DEX has also been shown to selectively inhibit the transcription of the IL-1/3 gene (Nishida et al., 1988) and decrease mRNA stability in stimulated U937 cells (Lee et al., 1988). Similar to IL-4, the destabilizing effect of DEX could also be blocked in the presence of CHX, suggesting that de n o v o

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protein synthesis was required. Since DEX treatment markedly decreased the stability of IL-lp mRNA, but not c-fos mRNA, the presence of an ARE in the 3' UTR of both messages cannot explain the selective degradation of the former. It should be noted that DEX does not promote mRNA decay in all instances. Petersen et al. (1989) showed that DEX treatment resulted in a four-fold increase in the stability of phosphoenolpyruvate carboxykinase mRNA in rat hepatoma cells (Petersen et al., 1989). Although the phosphoenolpyruvate carboxykinase 3 ' U T R contains an ARE, this element could confer DEX-dependent stabilization upon a heterologous mRNA. Thus, the 3 ' U T R can play a central, albeit highly complex, role in the regulation of mRNA stability by various agents. While several reports have demonstrated that IL-1/3 mRNA decay can be selectively regulated, the sequence elements that mediate these effects have yet to be identified.

C O N C L U D I N G REMARKS

The regulation of IL-1 production has proven to be mediated by a number of complex processes. Given the variety of cells that bear IL-1 receptors, and/or are capable of synthesizing IL-I, the biological actions of this molecule are understandably broad. The biology of IL-1 in vivo is further complicated by the existence of two distinct IL-1 receptors and a myriad of potential interactions with other cytokines, prostanoids, and a novel receptor antagonist protein. Given the potentially harmful effects of IL-1, cells have developed multiple regulatory mechanisms in order to prevent inappropriate expression of IL-I. Future efforts in understanding the molecular and biochemical basis for these mechanisms will undoubtedly focus on the intracellular second messenger pathways that transduce extracellular signals, the transcriptional regulatory proteins that are ultimately the intracellular targets of these signals, and the previously underestimated role of selective mRNA destabilization.

REFERENCES

ACRES, R. B., LARSEN, A. & CONLON, P. J. (1987). IL-1 expression in a clone of human T cells. J. Immun., 138, 2132-2136. AGGARWAL,B., EESSALU,T. & HASS, P. (1986). Characterization of receptors for human tumor necrosis factor and their regulation by gamma interferon. Nature, 318, 665- 670.

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