Transcriptional regulation of endothelial cell adhesion molecules: NF ...

REVIEW

Transcriptional

regulation

molecules:

NF-icB

of endothelial

cell adhesion

and cytokine-inducible

enhancers

TUCKER COLLINS,” MARGARET A. READ,* ANDREW S. NEISH,* MARYANN Z. WIIITLEY, DIMITRIS THANOS,t52 AND TOM MANIATISt *Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA; and tDepjment University, Cambridge, Massachusetts 02138, USA

Transcription of endothelial-leukocyte adhesion molecule-i (E-selectin or ELAM- 1), vascular cell adhesion molecule-i (VCAM- 1), and intercellular adhesion molecule-i (ICAM-1) is induced by the inflammatory cytokines interleukin-i3 (IL-in) and tumor necrosis factor-a (TNFa). The positive regulatory domains required for maximal levels of cytokine induction have been defined in the promoters of all three genes. DNA binding studies reveal a requirement for nuclear factor-icB (NF-KB) and a small group of other transcriptional activators. The organization of the cytokine-inducible element in the E-selectin promoter is remarkably similar to that of the virus-inducible promoter of the human interferon-[ gene in that both promoters require NF-icB, activating transcription factor-2 (ATF..2), and high mobility group protein 1(Y) for induction. Based on this structural similarity, a model has been proposed for the cytokine-induced E-selectin enhancer that is similar to the stereospecific complex proposed for the interferongene promoter. In these models, multiple DNA bending proteins facilitate the assembly of higher order complexes of transcriptional activators that interact as a unit with the basal transcriptional machinery. The assembly of unique enhancer complexes from similar sets of transcriptional factors may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules.-Collins, T., Read, M. A., Neish, A. S., Whitley, M. Z., Thanos, D., Maniatis, T. Transcriptional regulation of endothelial cell adhesion molecules: NF-icB and cytokine-inducible enhancers. FA.SEBJ. 9,899-909 (1995) ABSTRACT

of Molecular and Cellular

end othelium

.

cytokinc

.

g

expression

enhancer

OF LEUKOCYTES FROM the circulation into the extravascular space is critical for inflammatory responses and repair of tissue injury. The process of leukocyte emigration involves several steps (reviewed in refs i and 2). The initial interaction between leukocytes and endothelium appears to be transient, resulting in the rolling of leukocytes along the vessel wall. The rolling leukocytes then THE RECRUITMENT

0892-6638/95/0009-0899/$01

.50. © FASEB

Harvard

become activated by local factors generated by the endothehum, resulting in their arrest and firm adhesion to the vessel wall. Finally, the leukocyte transmigrates the endothehium. These complex processes are regulated in part by specifIc endothelial-leukocyte adhesion molecules. The initial rolling interactions are mediated by the selectins, whereas firm adhesion and diapedesis appear to be mediated by the interaction of integrins on the surface of leukocytes with immunoglobulin gene superfamily members expressed by endothelial cells. Expression of some of the endothelial-leukocyte adhesion molecules is dynamically regulated at sites of leukocyte recruitment. For example, endothelial expression of E-selectin and vascular cell adhesion molecule-i (VCAM-i)3 is dramatically induced, and expression of intercellular adhesion molecule-i (ICAM-i) is substantially increased at sites of inflammation. These dynamic changes in surface proteins provide the endothelial cell with a mechanism of regulating cell-cell interactions. The three endothehial cell-surface proteins have different patterns of expression, and are structurally and functionally distinct. Transcription of all three of these genes is substantially increased when endothelial cells are exposed to cytokines. In this review, the common feature of these endothelial proteins, namely, transcriptional induction of the corresponding genes by the inflammatory cytokines, will be explored. The regulatory elements involved in cytokine-induced expression of these three leukocyte adhesion molecule genes and the transcription factors that bind to these elements will be described. We will summarize evidence that suggests cytokine-induced gene expression involves the synergistic interaction of a small group of transcription factors, which results in the

1T whom correspondence

Key Words:

Biology,

and reprint

requests

should

be addressed,

at:

Vascular Research Division, Department of Pathology, Brigham and Women’s Hopital, 221 Longwood Ave., Boston, MA 02115, USA. 2Present address: Department of Biochemistry and Moleular Biophysics Columbia University, New York, NY 10032, USA. Abbreviations: VCAM-1, vascular cell adhesion molecule-i; ICAM-1, intercellular adhesion molecule-i; NF-KB, nuclear factor-KB; IL-1J3, interleukin-1; LPS, lipopolysaccharide; TNFa, tumor necrosis factor-a; PRD, positive regulatory domains; HMG, high mobility group; IRF, interferon regulatory factor; MHC, major histocompatibility complex; IFN, interferon -; ATF, activating transcription factor; GAS, i-activated sequences.

899

REVIEW assembly stimulate

NUCLEAR

of unique transcription factor the basal transcription apparatus.

FACTOR-KB:

complexes

that

Signals

AN OVERVIEW

The transcription factor nuclear factor-KB (NF-KB) is a pleiotropic regulator of many genes involved in immune and inflammatory responses, including the leukocyte adhesion molecules (reviewed in ref 3). This family of dimeric transcription factor complexes consists of p5O (NF-KB1) and p52 (NF-icB2), which are both generated by proteolytic kBa processing of precursor molecules plO5 and plOO. The other members of this family, p65 (Re1A), c-Rel, and ReIB, have potent transactivation domains. All belong to the Rel family, which also includes the v-rel oncogene, c-rel protooncogene, and the Drosophila regulatory factors dif and dorsal. In resting cells, NF-KB binding proteins are in an inactive cytosohic form and are complexed to members of a family of inhibitory proteins referred to as 1KB. Several 1KB proteins have been identified including 1KB-a, IKB-y, bcl3, plOO, and p105 (reviewed in ref 4), as well as the recently cloned IKB-f3 (5). All forms of 1KB identified to date contain multiple copies of a protein motif known as an ankyrin repeat. NF-KB is sequestered in the cytoplasm by at least two mechanisms (Fig. 1): First, one of the inhibitors, 1KB-a, binds to the p65 subunit of NF-KB via the Figure 1. The proteasome and the NF-1CB/IKB--a autoregulatory system. ankyrin repeats present in the inhibitor. The bound 1KB-a Inactive NF-KB is maintained in the cytoplasm either by association with masks the NF-KB nuclear localization signal and thereby 1KB-CC or by association of p65 with p105. Multiple extracellular signals activate signal transduction pathways that lead to phosphozylation of inhibits its nuclear transport. Second, the p65 component IicB-a or plO5. IicB-a is degraded by the proteasome, which results in of NF-KB is bound to the piOS precursor of p50 or the p100 the appearance of functional pSO-p65. In parallel, the COOH terminus of precursor of p52. Like IKB-a, these bound precursors piO5 is degraded by the proteasome to generate the p50 subunit of the function as inhibitors by masking the nuclear localization p50-p65 heterodimer. Heterodimeric NF-icB enters and accumulates in signal (reviewed in ref 6). NF-KB can be activated by many the nucleus and activates the expression of many genes relevant to endothelial pathophysiology, including the gene for the inhibitor 1KB-a. diverse agents such as the inflammatory mediators, viruses, Increased expression of IKB-a replenishes the cytoplasmic levels of the and physical forces (reviewed in refs 3, 4, 6). Upon activainhibitor and returns the the endothelial cell to quiescence. This diagram tion of the cell, 1KB-a is phosphorylated and subsequently was modified and redrawn from a similar figure in Palombella et al. (9). degraded (reviewed in ref 4). Phosphorylation apparently does not result in the dissociation of the inactive NF-KBlicE-a complex, but rather in the targeting of 1KB-a for degradation (7-10). Degradation of IicB-a and processing of the piO5 NF-KB precursor into the active p50 subunit vascular pathophysiology. Functional NF-KB elements can involve the proteasome proteolytic pathway (9, 10). The be found in many genes whose expression is increased in release of NF-KB from 1KB-a, or processing of plO5 to vascular cells at sites of inflammatory responses (reviewed in refs 15, 16). The existence of multiple Rel family mempSO. allows the active p5O-p65 dimer to translocate and accumulate in the nucleus, bind to its recognition DNA bers and 1KB components emphasizes the need to examine element, and participate in the activation of transcription. the structure and regulation of this transcription factor sysNF-KB and 1KB-a interact in an autoregulatory mechatem in vascular cells. Although surprisingly little is known nism. NF-KB mediates activation of the 1KB-a gene, resultabout the expression of Rel family members in vascular ing in replenishment of the cytoplasmic pool of its own smooth muscle cells, the NF-KB system has been characinhibitor (11-14). Restored expression of 1KB-a decreases terized in cultured endothelial cells. Endothelial cells express both protein and transcript for a variety of Rel family NF-KB activation and diminishes expression of KB-dependent genes. members, including plOS/pSO, piOO/p52, p65, c-Rel, and Re1B (17; M. A. Read and T. Collins, unpublished results). In quiescent cultured endothehial cells, both p5O and p65 THE NF-KB/IKB-a SYSTEM IN ENDOTHEUAL CELLS protein can be detected in the cytoplasm and small amounts Evidence is accumulating that the transcription factor of p50 can be detected in the nucleus. Endothehial cells NF-icB and its inhibitors may play a key role in regulating also express the inhibitors 1KB-a, IKB-, bcl-3, p105, and

900

Vol. 9

July, 1995

The FASEB Journal

COLLINS El AL

REVIEW p100. In resting cultured endothehial cells, IKB-a is localized primarily in the cytoplasm and bcl-3 is localized in the nucleus (M. A. Read, M. E. Gerritsen, and T. Collins, unpublished observations). NF-KB can be activated by a variety of signals relevant to endothelial pathophysiology. This list of activating agents includes the inflammatory cytokines interleukin-1 (IL-l ) and tumor necrosis factor-a (TNFa), lipopolysaccharide (LPS), the viral homolog, poly(I:C), as well as oxidative and fluid mechanical stress (reviewed in refs 15, 18). Similarly, this regulatory system can be suppressed by antioxidants (reviewed in ref 19) and proteasome inhibitors (20, 21). The pattern of Rel family members and 1KB proteins expressed by endothelial cells is dynamic. Activation of NF-KB in response to TNFa is rapid and occurs in parallel with degradation of 1KB-a (17). A phosphorylated form of 1KB-a can be detected in endothehial cells after cytokine treatment (17, 20, and unpublished data) and may target the inhibitor for degradation. The nature and regulation of the kinase phosphorylating 1KB-a is not clear. TNFa exposure did not effect levels of plO5 (20), which also retains Rel family members in the cytoplasm, similar to findings in other cell types (22). Cytokine-induced activation of cultured endothehial cells is associated with dramatic nuclear accumulation of both pSO and p65 (17; M. A. Read, M. E. Gerritsen, and T. Collins, unpublished observations). Although cultured endothehial cells express a variety of Rel family members, p5O and p65 are the predominant species found in the nucleus upon cytokine activation. Once translocated to the nucleus, pSO/p6S binds to the KB sites in a variety of genes, including elements in the E-selectin (17, 23-25), VCAM-1 (26), and ICAM-1 (27, 28) promoters. Because the Rel family members can form combinations of homo- and heterodimers each with potentially different binding and activation specificities, it is important to examine the structure of the transcription factor associating with each functional KB element in the regulatory region of a relevant gene. For example, p65 c-Rel may play an important role in TNFa-induced transcriptional activation of the ICAM-1 (28) and tissue factor genes (29, 30). These findings illustrate the point that different combinations of Rel family proteins bind to distinct KB sites to regulate transcription. The proteasome plays an important role in the activation of the NF-KB system and cytokine-induced gene expression in endothelial cells. The proteasome is a multicatalytic 26S complex present in both the cytoplasm and nucleus (reviewed in refs 31, 32). This nonlysosomal pathway of protein degradation is responsible for the turnover of both abnormal and biologically active proteins in intact cells (33). Inhibitors of the proteasome blocked 1KB-a degradation and activation of NF-KB in response to TNFa in MG63 and HeLa cells (9). Recently, we demonstrated that inhibitors that block the proteolytic activity of the proteasome decrease nuclear accumulation of NF-KB and abrogate TNFa-induced cell-surface expression of E-selectin, VCAM-1, and ICAM-1 in endothehial cells (20). These inhibitors exhibit profound functional effects by blocking .

CYrOKINE-INDUCED ENDOTHELIALGENE EXPRESSION

static adherence transendothelial These findings

of leukocytes, as well as attachment and migration under defined flow conditions. are consistent with the concept that the NF-KB/llcB--a system may play a pivotal role in regulating leukocyte adhesion and transmigration in the vessel wall. Moreover, these findings demonstrate that rational manipulation of the activation of the NF-KB/IKB-a system may regulate sets of genes controlling complex cellular events, such as those involved in leukocyte recruitment. NF-KB and 1KB-a interact in an autoregulatory mechanism in endothehial cells (Fig. 1). 1KB-a transcript is transiently up-regulated in endothelial cells responding to TNFa (17). NF-KB mediates activation of the 1KB-a gene, resulting in replenishment of the cytoplasmic pooi of its own inhibitor (14, 34). Increased expression of 1KB-a decreases cytoplasmic NF-KB activation and diminishes expression of KB-dependent genes. Once translocated to the nucleus, NF-icB may be under additional regulatory control of 1KB molecules. In vitro, 1KB-a is capable of specifically displacing heterodimeric p5O/p6S from the E-selectin and VCAM-1 KB elements (21; M. A. Read, A. Neish, M. E. Gerritsen, and T. Collins, manuscript submitted). This displacement process may play a role in postinduction repression of the leukocyte adhesion molecules and help prevent inappropriate expression of these proteins. Thus, the endothelial NF-KB/IKB-a autoregulatory system may ensure that the induction of NF-KB is transient and that the activated endothehial cell returns to a quiescent state. This dynamic balance may be offset by the diverse agents associated with the onset of vascular disease. Although the endothehial NF-KB transcription factor system is necessary, it is not sufficient for cytokine-induced endothehial-leukocyte adhesion molecule expression. A relatively small number of other transcription factors must assemble with NF-KB to generate unique transcriptional activating complexes. In subsequent sections, the functional elements composing the cytokine-inducible enhancers of the three leukocyte adhesion molecules will be characterized and a model for the assembly of a cytokineinduced enhancer generated.

REGULATORY SEQUENCES AND TRANSCRIPTIONAL ACTIVATORS CONTROLLING CYTOKINE-INDUCED E-SELECTIN GENE EXPRESSION E-selectin is a member of the selectin family of proteins, which consists of three members: E-selectin, P-selectin, and L-selectin (reviewed in ref 35). E-selectin has an NH2terminal lectin domain, an epidermal growth factor-like domain, six complement regulatory protein-like domains, a single transmembrane, and a short cytoplasmic domain (36). The protein recognizes specific proteins or glycolipids that have sialyl Lewis X or related carbohydrates found on neutrophils. E-selectin appears to play a role in leukocyte trafficking in both acute and chronic inflammatory responses and its expression is both cell type-specific and inducible.

901

REVIEW E-selectin gene expression is normally not detected in resting endothehium, but is strongly and rapidly induced by the inflammatory cytokines and lipopolysaccharide. E-selectin appears on the endothehial cell surface within 1 to 2 h of cytokine treatment, is expressed maximally at 4 to 6 h, and then rapidly declines even in the continuous presence of cytokine. To understand the mechanism regulating the cytokineinduced expression, the architecture of the gene for E-selectin was determined. The human E-selectin gene contains 14 exons and spans approximately 13 kb of DNA (37; Fig. 2). A DNase I-hypersensitive site was identified in the 5’ proximal promoter region of the E-selectin gene in human umbilical vein endothehial cells only after TNFa treatment, suggesting the presence of a cytokine-inducible regulatory element close to the transcriptional start site (24). The regulatory elements required for cytokine activation have been localized to the first 160 base pairs immediately upstream of the start site of transcription (38). This region can function as a cytokine-inducible transcriptional enhancer in both endothehial and nonendothelial cells. Cell type-specific expression of E-selectin has been associated with hypomethylation of the cytokine response region of the E-selectin promoter (39). The organization of the regulatory elements required for cytokine-induced expression of the E-selectin gene has been defined. Four positive regulatory domains (PDs) were defined in the E-selectin promoter (Fig. 3) (25). A consensus KB element and an ATF-hike site were designated PDI and PDII, respectively. Two more recently described novel elements were designated PDIII and PDIV (Fig. 3). These domains have been mapped by deletion and point mutation

LTIN

COMPlEMENT

REPEATS CTO-

I

EM M I GENE

I NF,B

SOT

501-2

IMMUNOGLOBUUN

VCAM I GENE

TATA

II

I

...,JJJ_J..._. I,

III

IV

V

VII

u’

TM. CYTO. SOT

IMMUNOGLOBUIJN

ICAM I GENE

VI

liii

NFB

5UT

DOMAINS

I

TATA

I

-r-9]

DOMAINS

I II IDII

III IV V

NF,,B 5_J

SP

TM. CYTO.SUT

Figure 2. Structural organization of the E-selectin, VCAM-i, and ICAM1 genes. In each gene, the positions of the exon-intron boundaries correlates with the domain structure of the protein (37, 50, 57). In all three genes, exons are indicated by boxes. Introns as well as 5’ and 3’ flanking sequences are represented by lines. The 5’ and 3’ untranslated regions are represented by open boxes. The locations of putative domains are designated. The positions of the consensus transcriptional elements TATAA and NF-icB are indicated. SP, signal peptide; TM, transmembrane region; CYTO, cytoplasmic domain; and UT, untranslated region, In the E-selectin gene, EGF, epidennal growth factor-like domain. In the VCAM-i and ICAM-l genes, the immunoglobulin-like domains are indicated with Roman numerals.

902

Vol.9

July, 1995

analysis, as well as by systematic site-directed mutagenesis (23-25). The inducibihity of the E-selectin gene requires NF-KB binding to PDI, PDIII, and PDIV. Because these regulatory domains bind similar factors, the elements will not be discussed separately. Two observations indicate that NF-KB may play an important role in cytokine induction of the E-selectin gene. First, mutations that decrease the binding of NF-KB to any one of the NF-KB binding sites in vitro result in diminished cytokine-induced E-selectin expression. Second, Rel family proteins found in nuclear extracts from cytokine-activated endothelial cells specifically interact with these elements. Supershift and UV cross-linking studies demonstrate that heterodimeric pSO/p6S is the NF-KB species binding to these elements (17, 23-25). The presence of three closely spaced functionally important NF-KB sites raised the question of whether one site is preferentially occupied. DNase I footprinting revealed that when exposed to increasing amounts of pSO/p65 heterodimer, PDI, and PDIII were occupied whereas PDIV was not (24, 25). It appears that NF-KB binding to PDIII sterically interferes with binding at PDIV. Two models may resolve these findings. First, other members of the Rel family may bind PDIV. We have noted that recombinant c-Rel homodimers can simultaneously bind to all three KB elements in the cytokine response region of the intact promoter. Second, other factors may interact with the E-selectin promoter that alter the conformation of the protein-DNA complex, allowing NF-KB to interact with PDIV. The high mobility group (11MG) protein 1(Y) is required for cytokine-induced E-selectin expression, binds to both PDIII and PDIV, and enhances the binding of N F-KB to these elements (23, 25). As discussed in subsequent sections, HMG 1(Y) may function as an architectural element in the assembly of the cytokineinduced enhancer complex. The sequence of PDII is similar to the DNA sequences recognized by the ATF family of proteins. This family of transcriptional regulators share a basic leucine zipper motif and can selectively heterodimerize with members of the c-fos and c-jun groups of factors (reviewed in ref 40). Several observations suggest that this ATF-like element is important for cytokine-induced expression of the E-selectin gene. First, deletion and site-directed mutagenesis studies identify the CRE/ATF site as a functional element for maximal cytokine-induced expression of the gene (25, 41). Mutations that decrease the binding of nuclear proteins to the CRE/ATF element in PDII abolish cytokine-induced E-selectin expression. Conservation of the DNA sequence of this element across species is consistent with a functional role for this site in the regulation of E-selectin expression (42). Second, the E-selectin site has been shown to be bound by recombinant ATF-a, ATF-2, ATF-3, c-Jun (41), and CREB (43), and changes in the level of induced E-selectin expression can be correlated with alterations in the composition of the proteins binding to this element (43). Third, members of the ATF family can physically interact with both NF-icB and HMG 1(Y) (41, 44). Fourth, mice lacking ATF-2 show defective E-selectin induction (A. M. Reimold et al., un-

The FASEB Journal

COLLINS

FT AL.

REVIEW Interferon-3

p501p65

ATF-2

Jlfl

l

CFAAAATGrMATGACATAGGAAA,.CTGMAGGGAGAAcrGR.AA0TGGGAM2TcCTC GMracM?rACurATccrTGACTcCCYTcAe1rTcAcCCTlTA,cGAD

v-’

__1

PAD IV

PRD III

PRDI

PAD II

E-Select in p6 5/ p5 0 p50/p65

ATF-2

__

p50/p65

llM1\

HMG I

‘#{176}#{176}

DrcrGACApcAT1cTMT-rTTAsGcATcErcGApaDSCcccGoAAAGT1’rTrcGATcCcATruccGATT1ccTC

_____

IrJy\th

MGACTGTAGTAACATTMAADTCGTAOCACCTATAAGGGCCCTTICAAAAACCTACOGTAACCCCTAAAGGRG

4

_________

POll

PDIV

_________ P0111

PDI

VCAM-1 p501p65 fl\ /IIIIIVII I

-76

ffl

I

p50/p65 (f\

tional

Spi ,

IIIIIIVII V

\

r-i

ccCccrrrcccxOAAcGGATrrCCCTCCDCClTTGCAACAAGACCCNTAPAAAGCACAGACTTrCTADr2TACrC CDOACCCMAGQGGAACTTCCCTAMCGGADOCGGAGACDTITCT000AAAThTcItTGAMGATAAACTGAO

1Y\J

‘1W

published results). Despite the potential for overlap in function among the members of the ATF family, ATF-2 is probably essential in cytokine-induced expression of the E-selectin gene. ATF-2 may be a target of signal transduction pathways that activate E-selectin induction. Treatment of cells with inflammatory cytokine or UV radiation causes activation of the c-Jun NH2-terminal protein kinase (JNK1/2) (45). Both ATF-2 and c-Jun are targets of this kinase, and phosphorylation of these factors is required for transcriptional activity (45). Activation of ATF-2 and/or c-Jun by iNK in endothehal cells may be an important signal transduction pathway parallel to that of NF-KB activation required for cytokineinduced E-selectin expression. Analysis of the E-selectin promoter revealed a small cytokine response region with multiple NF-KB elements and an CRE/ATF element that must interact cooperatively to induce transcription. Similar findings from the analysis of other the structurally distinct gene inducible promoters suggested that synergy between a small set of transcrip-

..

activators

may

be

a common

theme

in

cytokine-in-

duced gene expression. these cytokine-induced

As discussed in subsequent sections, enhancers may involve specific spatial arrangements of transcription factors, in association with other protein components that function as architectural elements, to generate unique higher order complexes.

ICAM-1 /EBP -212

,

c.Rel/p65

,4lTllifl\ -1T2

p91/p91

AcccccGcccGArrac-rrraccrrrncAAATT1ccGGAGc

1/

CGMSGrrTCCGGGAAAGCNSCACCG

rsGccGcGcccrAAcGAAATCGMACCrrrMAQGCCDCG

//

GCTCCAAAGGCCCIITCGTCGTGGC

Figure 3. Comparison of the organization of the human promoters for the IFN-f, E-selectin, VCAM-l, and ICAM-1 genes. The double-stranded DNA sequences are shown of the IFNpromoter from -110 to -53, the E-selectin from -156 to -83, the VCAM-l from -76 to + 1, and selected regions of the the ICAM-i promoter from -212 to -57 relative to the transcriptional start sites. The inducible enhancers in IFNand E-selectin contain four regulatory domains, designated PRDI-PRDIV in the human lFNgene and PDI-IV in the E-selectin gene (25). Transcription factors that bind to each of the elements are shown for both genes. Those for the IFN-13 gene are as follows (reviewed in ref 61): NF-KB binds to PRDII, interferon regulatory factor-i (IRF-1) binds to PRDI and III, and the activating transcription factor-2 (ATF-2) binds to PRDIV. Binding sites for the high mobility group protein (11MG 1(Y)) are found in PRDs II and IV and are indicated. Transcription factors that bind to the E-selectin cytokine response region are as follows: NF-icB binds to PDI, PDIII, and PDIV (23-25, 38); ATF-2 or ATF-a homodimers or the corresponding c-jun heterodimers bind to PDII (41). HMG 1(Y) binds to a region within PDII, as well as to the AT-rich regions in the KB sites contained in PDI, PDIII, and PDIV (23, 25). The organization of the known regulatory elements required for cytokine-induced expression of the VCAM-1 gene are shown. Two binding sites for NF-icB and a single IRF-1 element have been identified (26, 51, 52). NF-icB interacts with Spi to activate gene expression from the VCAM-1 cytokine enhancer (A. Neish, V. Baichwal, and T. Collins, manuscript submitted). The organization of the regulatory elements required for cytokine-induced expression of the ICAM-l gene are shown. The TNFa response element is composite, consisting of binding sites for NF-KB and C/EBP. The IFN-y response element consists of a single binding site for p91 (27, 28, 58).

CYrOKINE-INDUCED

ENDOTHELIAL

GENE EXPRESSION

REGULATORY SEQUENCES ACTIVATORS CONTROLLING VCAM-1 EXPRESSION

AND TRANSCRIPTIONAL CYTOKINE-INDUCED

Vascular cell adhesion molecule 1 (VCAM-1) is a 110 kDa member of the immunoglobuhin gene superfamily first described as a cytokine-inducible endothehial adhesion molecule (46). VCAM-l binds circulating monocytes and lymphocytes expressing the integrmns a4f31 and a47 (47) and may participate in the recruitment of these chronic inflammatory cells from the bloodstream to sites of tissue injury. Vascular expression of VCAM-1 is found associated with a variety of inflammatory processes and in atherosclerotic lesions (e.g., ref 48). VCAM-1 has a distinct pattern of cytokine induction in cultured endothehial cells. Naive cells do not express VCAM-1 message; however, exposure to inflammatory cytokines IL-l and TNFa, lipopolysaccharide, and to the synthetic double-stranded RNA, poly(I:C) results in rapid up-regulation (46). VCAM-1 message levels reach a sustained high level by 2-3 h, and then gradually diminish over several days. The relatively delayed and sustained VCAM-1 response to cytokine corresponds with the preferential adhesion and infiltration of mononuclear leukocytes typical of chronic inflammatory processes (reviewed in ref 49). In an effort to understand the molecular mechanisms controlling the characteristic VCAM-1 response to cytokines, the structure of the VCAM-1 gene was determined (50) and the VCAM-1 5’ flanking region was characterized (51). The human gene for VCAM-1 spans about 25 kb of DNA and

903

REVIEW 9 exons that correlate with the functional domains (Fig. 2). Nuclear run-off analysis revealed that the gene is quiescent in cultured endothelial cells and expression was induced at the transcriptional level 1 h after TNFx induction. Stimulated levels of VCAM-1 transcript are reduced in the presence of cycloheximide, indicating that protein synthesis is necessary for maximal transcriptional activation. This finding is in striking contrast to E-selectin, which is superinduced in the presence of the protein synthesis inhibitor. The organization of the regulatory elements required for cytokine-induced expression of the VCAM-1 has been partially defined. Transient transfection experiments with segments of VCAM-1 5’ flanking sequence coupled to a CAT reporter defined a small region upstream of the transcriptional start site capable of directing full cytokine-induced gene expression (51, 52). By mutational analysis, it has been found that activation of VCAM-1 requires two tandem binding sites for NF-KB located in the basal VCAM-1 promoter at positions -73 and -58, both of which are necessary for cytokine-mediated transcriptional response. Mutation of either of these elements abolishes cytokine responsiveness. These findings are consistent with results from a systematic site-directed mutagenesis (linker scan) of the proximal 100 bp, which also revealed the two KB sites and an Spi element (A. Neish, V. Baichwal, and T. Collins, manuscript submitted). Both VCAM-1 NF-KB sites are required to confer cytokine inducibihity on a minimal promoter. When the isolated VCAM-1 KB sites were placed 5’ of a basally active viral promoter and transfected into endothelial cells, the resulting constructs exhibited minimal response to cytokine. However, when both NF-KB sites were present the resulting construct showed striking inducibility, although not to the level seen with the intact VCAM-1 basal promoter. This suggests a cooperative interaction between the NF-KB dimers bound to the tandem KB elements in the VCAM-1l promoter. Furthermore, the findings demonstrate that the activity of the intact regulatory region of the VCAM-1 promoter is distinct from that of the individual elements. This is a common feature of cytokine-inducible regulatory regions. Several findings indicate that heterodimeric p5O and p65 is the NF-KB species binding to the human VCAM-1 promoter in TNFa-activated endothelial cells. First, gel shift and supershift analysis reveal that the induced nucleoprotein complexes assembled on both NF-KB sites contain pSO and p65; second, UV cross-linking studies also reveal that both the 5’ and 3’ VCAM-1 NF-KB sites bind predominately heterodimeric p50 and p65 (26). In cotransfection experiments involving overexpressed Rel proteins, p65 acts as a powerful activator of VCAM-1 promoter reporter constructs; and addition of p50, although having no transactivating potential of its own further increases the level of expression. It is interesting that higher levels of p5O result in transcriptional repression. This experimental phenomenon has been observed with other promoters (e.g., IFN-), and may reflect competition between homodimeric p50 and heterodimeric p5O/p65 for NF-KB sites. In addition, mice lacking contains in the

protein

904 Vol. 9 July, 1995

p5O show LPS-induced expression of VCAM-1 (A. Neish, B. Sha, D. Baltimore, and T. Collins, unpublished results), suggesting that p50 is not essential for induction of VCAM1 and may play a role in regulating induced expression of the adhesion molecule. The sensitivity of VCAM-1 transcript induction to protein synthesis blockade is consistent with factors other than NF-KB contributing to cytokine-induced VCAM-1 expression. Additionally, deletion analysis suggested the existence of other functionally important protein-DNA interations. Inspection of the VCAM-1 core promoter revealed the motif GAAATAGAAA, consistent with a binding site for the interferon regulatory factor (IRF) family of transcriptional activators. This motif is a functional element in the promoters of other inducible genes, such as interferon-f3 (IFN-), the inducible form of nitric oxide synthase, and major tional

histocompatibility activator observations

eral element

class-i,

and

binds

the

transcrip-

interferon regulatory factor-i (IRF-1). Sevsupport the proposal that the IRF-i

is important in cytokine-induced VCAM-1 gene (26). First, DNA/protein binding studies with endothehial nuclear extracts revealed that IRF-i is cytokine-inducible and binds specifically to the VCAM-1 IRF binding motif. Second, mutations that decrease the binding of IRF-i to the IRF binding site in vitro decreased TNFainduced VCAM-1 expression. Third, activation of the VCAM-i promoter with overexpressed p5O/p65 could be increased further by the addition of overexpressed IRF-1. This effect could be duplicated with promoter constructs bearing isolated VCAM-i NF-KB and IRF binding elements. Fourth, IRF-i was found to physically interact with p5O in vitro. Interestingly, DNase I footprint analysis revealed that binding of NF-icB to the tandem sites on the VCAM-11 promoter increased the binding affinity of IRF-1 to its cognate site. Taken together, these data implicate IRF-1 as a factor that cooperates with NF-KB to mediate cytokine induction of VCAM-1. Analysis of both the VCAM-i and E-selectin promoters revealed small cytokine response regions with multiple NF-KB elements that must interact cooperatively to induce transcription. In addition to these two leukocyte adhesion molecules, two closely spaced functional KB elements have been identified in the MAdCAM-11 (53), class I major histocompatibility complex (MHC) promoters (54) and 1KB-a (34) promoters. As discussed below, transcriptional synergy between the factors bound to the KB elements and a small set of other transcriptional activators may be a common theme in cytokine-induced gene expression. expression

REGULATORY ACTIVATORS EXPRESSION ICAM-1

SEQUENCES CONTROLLING OF ICAM-1

AND TRANSCRIPTIONAL CYTOKINE-INDUCED

facilitates

selective cellular interactions (reviewed is an inducible counter receptor for several leukocyte f2 integrins (e.g., lymphocyte function-associated antigen (LFA-1 or CD11a/CD18) and Mac-i in ref 1). ICAM-1

The FASEB Journal

COLLINS

FT AL.

REVIEW (CDlib/CD18). Adhesion of ICAM-i to the leukocyte integrins plays an important role in a variety of cellular interactions, including leukocyte trafficking and initiation of antigen-specific immune responses. ICAM-i is a member of the immunoglobulin gene superfamily and has five extracellular immunoglobulin-like domains, a single transmembrane region, and a short cytoplasmic domain. Endothelial cells constitutively express ICAM-i, but during inflammatory responses levels of the protein can be increased. TNFa and IFN-y act synergistically to enhance ICAM-1 expression by endothelial cells (reviewed in ref 49). Nuclear run-off analysis demonstrated that TNFa activates ICAM- i gene expression at the transcriptional level (55). The molecular mechanisms controlling expression of the protein were defined by identifying the promoter of the gene (56, 57). The ICAM-i gene contains 7 exons in which each immunoglobulin domain is contained in a single exon (Fig. 2). The organization of the regulatory elements required for both basal and cytokine-induced expression of the ICAM-i have been defined (27, 28, 58). Regulatory elements implicated in basal (or cytokine-independent) ICAM-i expression were located 115, 60, and 40 bp upstream from the ICAM-i transcriptional start site. Mutations in these regions of the gene reduced but did not abolish inducibility by both TNFa and IFN-y. Regulatory elements directing TNFa and IFN-y responses were found 190 and 90 bp, respectively, upstream of the ICAM-i transcriptional start site. Analysis of mutations in these elements and DNA binding assays revealed that the TNFa response element is composite, consisting of binding sites for NF-KB and C/EBP (Fig. 3). Mutations within these sites did not affect the basal level of ICAM-1 expression. The p5O/p6S heterodimeric form of NF-KB, as well as c-Rel/p65 and p65 homodimers, bound to the ICAM-1 KB element (27-29). The C/EBP element was bound by a mixture of factors including C/EBPa homodimers and C/EBPa-C/EBP heterodimers. A similar arrangement of NF-KB and C/EBP elements has been noted in the promoter of the IL-8 gene (59). In addition, direct protein-protein associations have been demonstrated between NF-KB and C/EBP, suggesting that these proteins may bind cooperatively to the TNFa response element of the ICAM-i promoter. The IFN-’y response element mapped to a symmetrical element that is homologous to ‘y-activated sequences (GAS) found in other IFN-’y-responsive genes (reviewed in ref 60). IFN-y binds with high affinity to IFN-y surface receptor. Once stimulated by binding to IFN-’y, the receptor activates the protein tyrosine kinases JAK1 and JAK2, which in turn phosphorylate the latent cytoplasmic transcription factor STAT1. Phosphorylated STAT1 is translocated to the nucleus, where it binds to the GAS sequence motif of IFN‘y-responsive genes and increases gene expression. The GAS element in the ICAM-1 promoter bound an IFN-y--inducible activity that was composed of p9l (STAT1). Exposure of cells to both TNFa and IFN-’y led to a synergistic induction of ICAM-1 gene expression. The distinct regulatory elements within the ICAM-1 promoter mediate tran-

CYTOKINE-INDUCED ENDOTHELIAL GENE EXPRESSION

scriptonal induction in response to the inflammatory mediators. This response required both the TNFa and IFN-y response elements, although the molecular basis for this cytokine-mediated synergism is unclear. The preceding paragraphs outline how cytokine-induced expression of three leukocyte adhesion molecule genes involves the synergistic interaction of NF-KB and of a limited number of other transcription factors. These findings explain the transcriptional activation that occurs in response to cytokine, but do not provide an understanding of how specificity of gene expression is achieved in response to these extracellular signals. Two general mechanisms are in place to achieve specificity. First, variations in the linear arrangement of distinct transcription factor binding elements would allow unique initiation complexes to be assembled. However, the analysis of the regulatory elements in the three distinct leukocyte adhesion molecule genes suggests that cytokine-induced expression involves a restricted and overlapping set of transcription factors (Fig. 3). Second, activation of distinct cytokine-response enhancers could involve the assembly of specific higher order complexes. Once assembled, the entire enhancer complex would interact with targets within the nearby basal transcriptional apparatus. As illustrated in subsequent sections, this latter mechanism may be quite important in achieving the high levels of specifically induced transcription characteristic of activated endothelium.

VIRAL INDUCTION OF THE HUMAN GENE: A MECHANISTIC PARADIGM GENE EXPRESSION

INTERFERONFOR INDUCIBLE

The virus-inducible enhancer of the human interferon-3 (IFN-) gene provides an important example of combinatorial interactions among distinct regulatory proteins in the assembly of an inducible enhancer (reviewed in refs 61, 62). The human IFNgene is transiently induced by virus. The regulatory elements required for viral activation have been localized to the first 104 base pairs immediately upstream of the start site of transcription. This regulatory region consists of four overlapping positive regulatory domains (PRDI-PRDIV). This region can function as a virus-inducible transcriptional enhancer. NF-KB binds to PRDII, IRF-1 binds to PRDI and PDIII, and ATF-2 binds to PRDIV (Fig. 3). None of these regulatory elements can activate transcription in isolation, but two or more copies of any one of the sites can act as a virus-inducible enhancer. However, the synthetic enhancers display unusually high levels of basal activity and are less inducible than the authentic regulatory region. Also, the synthetic enhancers are activated by multiple agents, whereas the intact enhancer is inducible only by virus. Thus, like the cyokine enhancers in the leukocyte adhesion molecules, the activity of the intact regulatory region of the IFN-13 promoter is distinct from that of the individual elements. The high mobility group protein HMG 1(Y) is required for virus induction of the human IFNgene. The 11MG

905

REVIEW proteins are low molecular weight nonhistone chromosomal proteins that have been placed into three groups: HMG 1/2, HMG 14/17, and HMG 1(Y) (63). These proteins do not have transcriptional activating capacity. Analysis of PRDII and IV mutations on in vitro binding and virus induction reveal that HMG 1(Y) is required for virus induction of the IFNgene (44). Binding sites for 11MG 1(Y) are found in PRDs II and IV in the IFN-f gene promoter are indicated (Fig. 3). The mechanisms by which HMG 1(Y) acts on these regulatory elements appear to be similar. 11MG 1(Y) binds to an (A+T)-rich region within PRDII and to the two (A+T)-rich regions immediately flanking PRDIV. 11MG 1(Y) binds to the minor groove of DNA, induces DNA bending, and increases the affinity of both NF-KB and ATF-2 for their respective DNA binding sites. Additionally, HMG 1(Y) can directly interact with both NF-KB and ATF-2 in the absence of DNA. The interactions between 11MG 1(Y) and DNA, and with other proteins, are essential for IFNgene expression. Thus induction of the IFN-13 enhancer by virus appears to result from the specific arrangement of transcription factor binding sites and the ability of 11MG 1(Y) to function as an architectural component to assemble an enhancer complex. Models for the protein-DNA and proteinprotein associations in the complex formed on the 1FNgene promoter have been generated (62, 64). After virus induction, a heterodimer of p50 and p65 components of NF-KB, two IRF molecules and an ATF-2/c-Jun heterodimer bind to the promoter. The binding of HMG 1(Y) at multiple sites increases the binding of NF-KB and ATF-2 for their respective binding sites and bends the DNA in a manner that facilitates the formation of a high-order complex. The relative positions of the NF-KB, IRF-1, ATF-2, and HMG 1(Y) binding sites in the IFNpromoter cannot be altered without adversely affecting enhancer function (D. Thanos and T. Maniatis, unpublished results). Once assembled, the inducible enhancer would transmit activation signals to coactivators or to the basal transcription machinery, although the nature of the signal and the target (or targets) of the signal within the transcription initiation complex are not yet known.

REMARKABLE SIMILARITY BETWEEN THE CYTOKINE-INDUCIBLE ENHANCER IN THE E-SELECTIN GENE AND THE VIRAL ENHANCER THE IFN-3 GENE

OF

There is a striking similarity between the functional elenents in the cytokine-induced E-selectin promoter and those in the virally induced n-interferon gene promoter. Both genes are silent in the absence of inducing agent and are dramatically induced by activating agents at the transcriptional level of gene expression. The regulatory elements found in the promoters of both genes are- entirely contained in a small region of about 100 bp located just upstream of unique transcriptional start sites. When coupled to a reporter gene, these regulatory regions can function as inducible enhancers. Both inducible, enhancers

906

Vol.9

July, 1995

contain four regulatory domains (designated PRDI-PRDIV i the human IFNgene and PDI-IV in the E-selectin gene); the transcription factors that bind to each of the elements are shown for these genes in Fig. 3. Both regulatory regions have a CRE/ATF binding element at the 5’ boundary and a consensus NF-KB element at the 3’ boundary of the regulatory region. Located between these elements is a duplicated transcription factor binding site: the IFN-13 gene promoter has two IRF-i elements; the E-selectin promoter has two additional NF-KB sites. Induction of both genes depends on the presence of HMG 1(Y). Like the IFNgene, the high mobility group protein HMG 1(Y) is required for cytokine induction of the human E-selectin gene (25). HMG 1(Y) binds to a region within the E-selectin PDII, as well as to the AT-rich regions in the KB sites contained in PDI, PDIII, and PDIV (23, 25). Analysis of PDIII and IV mutations on in vitro binding and cytokine induction reveal that 11MG 1(Y) is required for TNFa induction of E-selectin (25). The binding sites for HMG 1(Y) found in PDs III and IV in the E-selectin gene promoter are indicated in Fig. 3. HMG 1(Y) probably functions as an important architectural component in the assembly of the cytokine-induced transcription complex on the E-selectin promoter as it does in the IFNgene promoter.

THE CYTOKINE-INDUCED

ENHANCER

COMPLEX

The striking similarity between the inducible regulatory elements in the human E-selectin and IFN-13 gene promoters suggests that cytokine-induced gene expression may involve the formation of a higher order enhancer complex that is mechanistically similar to the stereospecific complex induced by virus. A model can be generated to represent the cytokine-induced enhancer on the E-selectin promoter (Fig. 4). After induction by cytokine, heterodimers of NF-KB and an ATF-2/c-Jun heterodimer (or an ATF-2 homodimer) bind to the promoter. The binding of HMG 1(Y) at multiple sites increase the binding affinity of NF-icB and ATF-2 for their respective sites and bends DNA in a way that facilitates the formation of a higher order complex necessary for transcriptional activation. The transcription factors could contact an as yet unidentified target (or targets), such as an endothelial-specific coactivator in the case of the E-selectin complex. Once assembled, the unique transcription factor enhancer complexes could act as a unit and make extensive protein-protein contacts with a series of coactivators, or the basal factors present in the transcriptional machinery. A number of observations are consistent with this model for a cytokine-induced enhancer complex. Because more is known about the E-selectin regulatory elements, it will be used to discuss the model. An important aspect of this model of the cytokine-induced enhancer complex is the spatial arrangement of the elements. The relative helical orientation or phasing of PDI and PDII in the E-selectin enhancer is more important than. the distance between these elements for cytokine responsiveness (65). Using a panel of promoter mutants, it was

The FASEB Journal

COLLINS Er AL.

REVIEW

HUG 1(Y)

HUG 1(Y)

HUG 1(Y) HUG 1(Y)

HUG 1(Y)

‘I,

HUG 1(Y)

HUG 1(Y)

HUG 1(Y)

4. Model of the cytokine-induced E-selectin enhancer. A) After induction by cytokine, heterodimers of NF-KB and an ATF-2 homodimer or an ATF-2/c-Jun heterodimer bind to the promoter. The binding of HMG 1(Y) at multiple sites increases the binding of NF-KB and ATF-2 for their Figure

respective sites and bends DNA in a way that facilitates the formation of a high-order complex necessary for transcriptional activation. The transcription factors could contact an unidentified target, which could be an endothelial-specific accessory factor. B) The transcription factors make extensive protein-protein contacts and the complex interacts as a unit with the basal transcriptional apparatus.

demonstrated that moving these elements 5 bp (half-helix turns) up- or downstream resulted in a strong reduction in promoter activity, whereas shifts of 10 bp (a full helix turn) in either direction minimally affected IL-i induciblity (65). This spacing dependence suggests that the transcription factors need to bind at the same face of the DNA-helix for optimal interactions. Protein-induced DNA bending also plays an important role in E-selectin gene expression, as has been suggested for the IFNgene enhancer (66; D. Thanos and T. Maniatis, manuscript submitted). Direct interactions between proteins bound to separate sites on DNA requires a minimum length of 130 base pairs for looping of the intervening DNA to occur. This suggests that interactions between the functional elements in the E-selectin promoter would require a bend or twist in the promoter. The energy required

CYrOKINE-INDUCED ENDOTHELIAL GENE EXPRESSION

for juxtaposition of adjacent functional elements can be provided by proteins that bend the DNA helix. All of the transcription factors that are known to bind the E-selectin promoter can bend DNA. Circular permutation assays, which detect protein-induced and sequence-specific deformation of the DNA helix, indicate that both ATF-2 and NF-KB can bend the corresponding E-selectin elements (65). These sequence-specific DNA bending proteins may direct deformation of the DNA helix and allow for a precise aligmnent of functional elements, thus stabilizing weak proteinprotein associations between the transcriptional activators. Another important component of the model is the presence of a protein that functions primarily as an architectural component of the complex. As noted before, we have demonstrated that 11MG 1(Y) is involved in the regulation of the E-selectin gene through both KB elements in PDIII and PDIV (25). HMG 1(Y) may also play a role in ATF-2 interactions. 11MG 1(Y) binding apparently alters the structure of DNA, thereby increasing the affinity of NF-KB and ATF2 for their respective recognition sequences. When the 11MG 1(Y) binding sites are placed on a model of a DNA double helix, they can be positioned on the same face of the helix. Thus, if 11MG 1(Y) binds to each of these sites simultaneously and induces a DNA bend in the same direction at each site, a loop containing the E-selectin enhancer would be formed. This loop would facilitate the interactions of NF-KB and ATF-2 and subsequent stabilization of the complex. Thus, the DNA bending proteins and the other architectural elements may create a scaffold that provides a supporting structure for the assembly of a higher order complex. Consistent with this model is the recent crystal structure of a large fragment of the p5O subunit bound as a homodimer to DNA. The pSO dimer envelopes about two-thirds of the cylindrical surface of the DNA helix making specific contacts along the 10 base pair KB recognition site (67, 68). Only the DNA’s minor groove was exposed. This is the site where minor groove binding proteins such as HMG 1(Y) may interact with DNA along with NF-KB (44). Understanding the structure of the individual transcriptional activators bound to DNA is the first step in generating an authenic model of how the transcription factors assemble into the cytokine-induced enhancer. The higher order structural features of the cytokine-induced enhancer would facilitate interactions between the proteins in the assembled complex. Direct protein-protein associations are important mechanisms by which transcription factors synergistically cooperate. A necessary element in these complexes, NF-KB, has been shown to physically interact with ATF-2 (64), ATF-a, c-Jun (41), TF-IID (69), and HMG 1(Y) (64). Additionally, direct protein-protein associations have been demonstrated between ATF-2 and HMG 1(Y) (70). Direct associations between NF-KB and IRF-i (26), as well as NF-KB and Spi (71), may be relevant for cytokine-induced expression of VCAM-i. Similarly, associations between NF-KB and C/EBP (59) may be important in induced expression of ICAM-1. These interactions may stabilize the cytokine-induced transcriptional activator complexes and provide a unique interface

907

REVIEW between the assembled enhancer basal factors in the transcriptional

CONCLUSIONS

and the coactivators machinery (Fig. 3).

or

AND PERSPECTIVES

Endothelial dysfunction has been implicated in the initiation of vascular pathology (reviewed in ref 72). This process disrupts a series of regulatory balances in the endothelium and results in multiple nonadaptive modifications, including rendering the endothelium hyperadhesive for circulating leukocytes. By examining the transcriptional regulation of three structurally and functionally distinct adhesion molecules, a theme has emerged about how the endothelial cell may coordinate a transcriptional response to the diverse agents associated with the onset of vascular disorders. The endothelial NF-KB/IKB-a system plays a pivotal role in regulating cytokine-induced leukocyte adhesion molecule expression. The transcription factor NF-KB can be activated in endothelial cells by the cytokines associated with increased adhesion molecule expression. Several leukocyte adhesion molecule genes have functional NF-KB elements. After cytokine exposure, cytoplasmic levels of an NF-KB inhibitor, 1KB-a, fall rapidly and profoundly. The proteasome pathway is required for these events. In parallel, there is nuclear accumulation of NF-KB. NF-KB and a limited set of other transcriptional activators participate in the assembly of unique transcription factor complexes that activate multiple endothelial genes, including the gene for the inhibitor IKB-a. Increased expression of 1KB-a decreases NF-KB activation and diminishes expression of KB dependent genes. The endothelial NF-KB/I1CB-a system may play an important role in regulating endothelial activation at sites of inflammatory responses. Furthermore, alterations in this regulatory system may correlate with the onset of vascular pathology. Stereospecific enhancer complexes may be important in coordinating cytokine-induced expression of the endothehal leukocyte adhesion molecules. These high-order nucleoprotein complexes may have common structural and mechanistic features. The importance of the spatial arrangement of functional elements suggests that the transcription factors interacting with these sites need to be oriented on the DNA helix in specific positions. In addition, some of these genes are dependent on the presence of chromatin components for induction. Architectural elements like HMG 1(Y) bind to multiple sites on the same side of the DNA helix and bend DNA, as well as interact with other proteins, to facilitate the assembly of high-order nucleoprotein complexes. Once assembled, the inducible enhancer would transmit specific activation signals to the coactivators or basal factors in the transcription machinery. The uniqueness of these higher order complexes may provide the specificity required to regulate complex patterns of gene expression and correlate with the distinct patterns of expression of the leukocyte adhesion molecules. We thank Vijay Baichwal (Tularik, Inc.) and Tom Parks (Boehringer Ingelheim Pharmaceuticals, Inc.) for helpful 908 Vol. 9 July, 1995

comments on the manuscript. Original work described in this manuscript was supported by National Institutes of Health grants Ai2064 to T. M., HLO3O11-Oi to A. S. N., HL45462 and HL35716 to T. C., as well as P0136028 and T32HL07627. T. C. is an Established Investigator of the American Heart Association.

REFERENCES 1.

2. 3.

4. 5.

6. 7.

8. 9.

10. 11.

12. 13. 14.

15 16.

17.

18. 19. 20.

21. 22. 23.

24. 25.

Springer, T. (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301-314 Carlos, 1. M., and Harlan, J. M. (1994) Leukocyte-endothelial adhesion molecules. Blood 84, 2068-2101 Grilli, M., Chiu, i-S., and Lenardo, M. (1993) NF-icB and Rel, participants in a multiform transcriptional regulatory system. In International Review of Cytology. A Survey of Cell Biology, pp. 1-62, Academic Press, San Diego, California Beg, A. A., and Baldwin, A. S. (1993) The 1KB proteins: multifunctional regulators of Rel/NFKB transcription factors. Genes Dev. 7,2064-2070 Thompson, I. E., Phillips, R. J., Erdjument-Bromage, H., Tempst, P., and Ghosh, S. (1995) IicB-f regulates the persistent response in a biphasic activation of NF-KB. Cell, 80,573-582 Thanos, D., and Maniatis, T. (1995) NF-icB: a lesson in family values. Cell, 80,529-532 Miyamoto, Sh., Maki, M., Schmitt, M. J., Hatanaka, M., and Verma, I. M. (1994) Tumor necrosis factor a-induced phosphorylation of licBa is a signal for its degradation but not dissociation from NF-icB. Proc. Nod. Acad. Sci. USA 91, 12740-12744 Finco, T. S., Beg, A. A., and Baldwin, A. S., Jr. (1994) Inducible phosphorylation of lKBa is not sufficient for its dissociation from NF-KB and is inhibited by protease inhibitors. Proc. NatI. Acad. Sci. USA 91, 11884-11888 Palombella, V. J., Rando, 0. J., Goldberg, A. L., and Maniatis, T. (1994) The ubiquitin-proteasome pathway is required for processing the NF-KB1 precursor protein and the activation of NF-icB. Cell 78, 773-785 Traenckner, E. B.-M., Wilk, S., and Baeuerle, P. A. (1994) A proteasome inhibitor prevents activation of NF-KB and stabilizes a newly phosphorylated form of 1KB-a that is still bound to NF-KB. EMBOJ. 13,5433-5441 Brown, K., Park, S., Kanno, T., Franzoso, C., and Siebenlist, U. (1993) Mutual regulation of the transcriptional activator NF-icB and its inhibitor, IicBa. Proc. NatI. Acad. Sci. USA 90,2532-2536 Sun, 5.-C., Ganchi, P., Ballard, D., and Greene, W. (1993) NF-icB controls expression of inhibitor IKBa: evidence for an inducible autoregulatory pathway. Science 259, 1912-19 15 Scott, M. L, Fujita, T., Liou, H-C., Nolan, C. P., and Baltimore, D. (1993) The p#{243}S subunit of NF-icB regulates lxB by two distinct mechanisms. Genes Des. 7, 1266-1276 de Martin, R., Vanhove, B., Cheng, Q.,Hofer, E., Csizmadia, V., Winkler, H., and Bach, F. H. (1993) Cytokine-inducible expression in endothelial cells of an IKBa-like gene is regulated by NF-KB. EMBOJ. 12,2773-2779 Collins, T. (1993) Endothelial nuclear factor-KB and the initiation of the atherosclerotic lesion. Lab. Invest. 5, 499-SOB Collins, T., Palmer, H. J., Whitley, M. Z., Neish, A. S., and Williams A. J. (1993) A common theme in endothelial activation. Trend., Cardiovasc. Med. 3,92-97 Read, M. A., Whitley, M. Z., Williams, A. J., and Collins, T. (1994) NFicB and licB-a: An inducible regulatory system in endothelial activation. I. Exp. Med. 179,503-512 Resnick, N., and Gimbrone, M. A., Jr. (1995) Hemodynamic forces are complex regulators of endothelial gene expression. FASEB J. 9,874-882 Offermann, M. K., and Medford, R.. M. (1994) Antioxidants and atherosclerosis: a molecular perspective. Heart Di.s. and Stroke 3, 52-57 Read M. A., Neish, A. S., Luscinskas, F. W., Palombella, V. J., Maniatis, T., and Collins, T. (1995) The proteasome pathway is required for cytokine-induced endothelial leukocyte adhesion molecule expression. Immunity 2, 493-506 Read, M. A., Neish, A. S., Gerritsen, M. E., and Collins, T. (1995) Nuclear 1KB-a and the post-induction transcriptional repression of the E-selectin and VCAM-1 genes. FASEB J. 9, A126 (abstr.) Rice, N. R., and Ernst, M. K. (1993) In vivo control of NF-KB and activation by licBa. EMBO). 12,4685-4695 Lewis, H., Kaszubska, W., DeLamarter, J. F., and Whelan, J. (1994) Cooperativity between two NF-1CB complexes, mediated by high-mobility-group protein 1(Y), is essential for cytokine-induced expression of the E-selectin promoter. Mol. Cell. Biol. 14, 5701-5709 Schindler, U., and Baichwal, V. R. (1994) Three NF-icB binding sites in the hwnan E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol. Cell. Biol. 14, 5820-5831 Whitley, M.Z., Thanos, D., Read, M. A.- Maniatis, T., and Collins, T. (1994) A striking similarity in the organization of the E-selectin and beta interferon gene promoters. Mol. Cell. Biol. 14,6464-6475

The FASEB Journal

COLLINS FTAL

REVIEW 26.

27. 28.

29.

30. 31. 32. 33.

34.

35. 36.

37.

38.

39.

40.

41.

42.

43.

44.

45. 46.

47.

48.

Neish, A. S., Read, M. A., Thanos, D., Pine, K., Maniatis, T., and Collins, T. (1995) Endothelial IRF-1 cooperates with NF-KB as a transcriptional activator of vascular cell adhesion molecule-i. Mol. Cell. Biol. 15,2558-2569 Hou, J., Baichwal, V., and Cao, Z. (1994) Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding ICAM-1. Proc. Nail. Acad. Sci. USA 91, 1641-11645 Jahnke, A., Johnson, J. P. (1994) Synergistic activation of intercellular adhesion molecule 1 (ICAM-1) by TNFa and IFN-y is mediated by p65/pSO and p65/c-Rel and interferon-responsive factor Statlcz (p9l) that can be activated by both IFN-y and IFN-a. FEDS Leu. 354,220-226 Parry, G. C., and Mackman, N. (1994) A set of inducible genes expressed by activated human monocytic and endothelial cells contain KB-like sites that specifically bind c-Rel-p65 heterodimers. J. Biol.Chem. 269,20823-20825 Mackman, N. (1995) Regulation of the tissue factor gene. FASEB J. 9, 883-889 Peters, J. M. (1994) Proteasomes: protein degradation machines of the cell. Trends Biochem. Sci. 19,377-382 Ciechanover, A. (1994) The ubiquitin-proteasome proteolytic pathway. Cell 79, 13-21 Rock, K. L., Gramm, C., Rothstein, L., Clark, K., Stein, K., Dick, L., Hwang, D., and Goldberg, A. L. (1994) Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78, 761-771 Cheng, Q., Cant, C. A., Moll, T., Hofer-Warbinek, R., Wagner, E., Bimstiel, M. L., Bach, F. H., and de Martin, K. (1994) NF-KB subunit-specific regulation of the IKBcL promoter.). Biol. Chem. 269, 13551-13557 Tedder, T. F., Steeber, D. A., Chen, A., and Engel, P. (1995) The selectins: vascular adhesion molecules. FASEB J. 9,866-873 Bevilacqua, M. P., Stengelin, S., Gimbrone, M. A., Jr., and Seed, B. (1989) Endothelial leukooyte adhesion molecule 1: an inducible receptor for neutrophils related to complement regulatory proteins and lectins. Science 243, 1160-1165 Collins, T., Williams, A., Johnson, G. I., Kim, J., Eddy, R., Shows, T., Gimbrone, M. A., Jr., and Bevilaqua, M. P. (1991) Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1.). Biol. Chem. 266,2466-2473 Whelan, J., Ghersa, P., Hooft van Huijsduijnen, K., Gray, J., Chandra, C. Talabot, F., and DeLamarter, J. F. (1991) An NF-KB-like factor is essential hut not sufficient for cytokine induction of endothelial adhesion molecule 1 (ELAM-1) gene transcription. Nucleic Acids. Ret. 19,2645-2653 Smith, G. M., Whelan, J., Pescini R., Ghersa, P., DeLamarter, J. F., and van Huijsduijnen, R. H. (1993) DNA-methylation of the E-selectin promoter represses NF-icB transactivation. Biochem. Biophys. Re.,. Commun. 194, 215-221 Lamb, P., and McKnight, S. L., (1991) Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. Trends Biochem. Sci. 16,417-422 Kaszubska, W., Hooft van Huijsduijnen, R., Ghersa, P., DeRaemy-Schenk, Chen, B. P. C., Hai, T., DeLainarter, J. F., and Whelan,J. (1993) Cyclic AMP-independent ATF family members interact with NF-KB and function in the activation of the E-selectin promoter in response to cytokines. Mol. Cell. Biol. 13, 7180-7190 Becker-Andr#{233}, M., Hooft van Huijsduijnen, R., Losberger, C., Whelan,J., and DeLamarter, J. F. (1992) Murine endothelial leukocyte-adhesion molecule 1 is a close structural and functional homologue of the human protein. Eur. J. Biochem. 206,401-411 De Luca, L. C., Johnson, D. K., Whitley, M. Z., Collins, T., and Pober, J. S. (1994) cAMP and tumor necrosis factor competitively regulate transcriptional activation through and nuclear factor binding to the cAMP-responsive element/activating transcription factor element of the enothelial leukocyte adhesion molecule-i (E-selectin) promoter. J. Biol. Chem. 269, 19193-19196 Thanos, D., and Maniatis, T. (1992) The high mobility group protein HMG 1(Y) is required for NF-KB-dependent vinis induction of the human IFN-fl gene. Cell 71, 777-89 Gupta. S., Campbell, D., Derijard, B., Davis K. J. (1995) Transcription factor ATF2 regulation by the iNK signal transduction pathway. Science 267, 389-393 Osborn, L., Hession, C., Tizard, K., Vassallo, C., Luhowskyj, S., Chi-Rosso. C., and Lobb, R. (1989) Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-,induced endothelial protein that binds to lymphocytes. Cell 59, 1203-1211 Elices, M., Osborn, L., Takada, Y., Crouse, C., Luhowskyj, S., Hemler, M., and Lobb, R. (1990) VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 60,577-584 Cybulsky, M., and Gimbrone, M. (1991) Endothelial expression of a mononuclear leukocyte adhesion molecule during atherogenesis. Science 251, 788-791

CYFOKINE-INDUCED

ENDOTHELIAL

GENE EXPRESSION

49.

Pober, J.S., and Cotran, R. C. (1990) Cytokines and endothelial cell biology. Physiol. Rev. 70,427-451 50. Cybulsky, M., Fries, J., Williams, A., Sultan, P., Eddy, R., Byers, M., Shows, T., Gimbrone, M., and Collins, T. (1991) Gene structure, chromosomal location, and basis for alternative mRNA splicing of the human VCAM1 gene. Proc. Nail. Acad. Sci. USA 88,7859-7863 51. Neish, A. S., Williams, A. J., Palmer, H. J., Whitley, M. Z., and Collins, T. (1992) Functional analysis of the human vascular cell adhesion molecule 1 promoter.). Exp. Med. 176, 1583-1593 52. Shu, H. B., Agranoff, A. B., Nabel, E. C., Leung, K., Duckett, C. S., Neish, A. S., Collins, T., and Nabel, G. (1993) Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-icB subunits in endothehal and epithehial cells. Mol. Cell. Dial. 13,6283-6289 53. Takeuchi, M., and Baichwal, V. K. (1995) Tumor necrosis factor-alpha induction of MAdCAM-1 gene is mediated by NF-KB proteins. Proc. Nail. Acad. Sci. 92,3561-3565 54. Mansky, P., Brown, W. M., Park, J.-H., Choi, J. W., and Yang, S. Y. (1994) The second KB element, KB2, of the HLA-a class I regulatory complex is an essential part of the promoter.). Immunol. 153, 5082-5090 55. Wertheimer, S. J., Myers, C. L., Wallace, K. W., and Parks, T. P. (1992) Intercellular adhesion molecule-i gene expression in human endothelial cells.). Biol. Chem. 267, 12030-12035 56. Degitz, K., Lian-Jie, L., and Caughman, S. W. (1991) Cloning and characterization of the 5’-transcriptional regulatory region of the human intercellular adhesion molecule 1 gene.). Biol. C/tern. 266, 14024-14030 57. Voraberger, C., Schafer, R., and Stratowa, C. (1991) Cloning of the human gene for intercellular adhesion molecule 1 and analysis of its 5’-regulatory region: induction by cytokines and phorbol ester. ). Immunol. 147, 2777-2786 58. Ledebur, H. C., and Parks, T. P. (1995) Transcriptional regulation of the intercellular adhesion molecule-i gene by inflammatory cytokines in human endothelial cells.). Biol. Chem. 270,933-943 59. Stein, B., Cogswell P. C., and Baldwin, A. 5. (1993) Functional and physical assocations between NF-KB and C/EBP family members: a Rd domain-bZIP interaction. Mol. Cell. Biol. 13,3964-3974 60. Damell, J. E., Kerr, 1. M., and Stark, C. R. (1994) Jak-STAT pathways and transcriptional activtion in response to IFNs and other extracellular signaling proteins. Science 264, 1415-142 1 61. Maniatis, T., Whittemore, L. A., Du, W., Fan, C. M., Keller, A., Palombella, V., and Thanos D. (1992) Positive and negative control of human interferon-J gene expression. In Transcriptional Regulation (McKnight, S. L., and Yamamoto, K., eds) Part 2, pp. 1193-1220, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 62. Thanos, D., Du, W., and Maniatis, T. (1995) The high mobility group protein HMG 1(Y) is an essential structural component of a virus-inducible enhancer complex. Cold Spring Harbor Symposia on Quantitative Biology, Vol. LVIII, Cold Spring Harbor Laboratory Press (In press) 63. Bustin, M., Lehn, D., and Landsman, D. (1990) Structural features of HMG chromosomal proteins and their genes. Biochim. Biophys. Acta 109,231-243 64. Du, W., Thanos, D., and Maniatis, T. (1993) Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements. Cell 74, 887-898 65. Meacock, S., Pescini-Gobert, K., DeLamarter, I. F., and van Huijsduijnen, R. H. (1994) Transcription factor-induced, phased bending of the E-selectin promoter.). Biol. Chem. 269, 31756-31762 66. Tjian, R., and Maniatis, T. (1994) Transcriptional activation: a complex puzzle with few easy pieces. Cell 77, 5-8 67. Muller, C. W., Key, F. A., Sodeoka, M., Verdine, C. L., and Harrison, S. C. (1995) Structure of the NF-KB p50 homodimer bound to DNA. Nature 373, 311-317 68. Chosh C., van Duyne C., Chosh, S., and SiglerP. B. (1995) Structure of NF-KB pSO homodimer bound to a KB site. Nature 373,303-310 69. Kerr. L. D., Ransone, L. J., Wamsley, P., Schmitt, M. J., Boyer, T. C.. Zhou, Q.,Berk, A. J., and Versna, I. J. (1993) Association between proto-oncoprotein Rd and TATA-binding protein mediates transcriptional activation by NF-KB. Nature 365, 412-419 70. Du, W., and Maniatis, T. (1994) The high mobility group protein HMG 1(Y) can stimulate or inhibit the binding of distinct transcription factor ATF-2 isoforms to the interferon-n gene promoter. Proc. Nail. Acad. &i. USA 91, 11318-11322 71. Perkins, N. D., Edwards N. L, Duckett C. S., Agranoff, A. B., Schmid, R. M., and Nabel, G. J. (1993) A cooperative interaction between NF-KB and Spi is required for HIV-1 enhancer activation. EMBO J. 12,3551-3558 72. Gimbrone, M. A., Jr. (1995) Vascular endothelium in health and disease. In Molecular Cardiovascular Medicine, Scientific American Medicine, New York, In press

909