Species-specific Changes in Regulatory Elements of Mouse Haptoglobin Genes*

Although expression of the haptoglobin (HP) as an acute phase reactant is evolutionarily conserved among mammals, there are differences among species with re- gard to the hormones required for stimulation. Using primary hepatocyte cultures, we show that in Mus caroli, as in rat, IL-1 and IL-6 are stimulatory, whereas in M. domesticus, as in humans, IL-1 response is diminished. In uiuo, an acute inflammatory process increases hepatic HP expression in both mouse species up to 30-fold but minimally affects the low level HP expression in the lung. To define the species-specific differences in regulation, we isolated the hormone-responsive elements of the HP gene from the Mus species, M. domesticus, M. caroli, and M. saxicola. Functional studies in transfected hepatoma cells revealed an exceptionally strong dexamethasone response for all three murine HP gene elements. The IL-6 response was less prominent than in rat or human. A modest response to IL-1 was observed in M. caroli and M. saxicola. A mouse-specific insertion of a polypurine se- quence led to a binding site for the PEA3 transcription factor in the HP gene promoter of M. domesticus and M. saxicola, but not M. caroli. The specific regulatory effects of glucocorticoid receptor, C/EBPP, and Ets proteins were documented by co-transfection.

appears to be co-regulated with al-acid glycoprotein in the well-studied experimental model system of rat hepatoma cells (8) and shows an interesting species-specific pattern of regulation.
HP is synthesized in the liver as a proprotein (ap), is processed by proteolytic cleavage into a! and p subunits, and is released into the bloodstream as a tetrameric protein (azpz) where it acts as a binding protein for free hemoglobin (9, 10). The active HP genes from human (11) and rat (12) have been cloned, and elements governing their expression and regulation have been studied in hepatoma cells. IL-6 and the functionallyrelated cytokines, LIF, IL-11, oncostatin M, and ciliary neurotrophic factor, stimulate HP gene transcription. This stimulation is enhanced by dexamethasone (8,(12)(13)(14)(15). The principal regulatory elements for these effects have been localized to three regions (termed A, B, and C) within 180 bp of the transcriptional start site in the 5"flanking region (12,(16)(17)(18). Both regions A and C serve as recognition sites for CEBP isoforms, the binding of which mediates, in part, the activation of HP transcription (16,(18)(19)(20)(21). Since IL-6 strongly stimulates expression of CEBPP in liver (22) and hepatoma cells (231, it has been postulated that haptoglobin gene regulation by IL-6 is indirect and is a consequence of an increase in CEBP protein activity (23). Structural analysis of the rat HP gene indicated homology to the human Hp' allele (11,12,24). The expression of the rat gene differs from the human gene, in that the former is stimulated not only by IL-6 and dexamethasone, but also by IL-1 or TNFa (8). The 5'4anking region of the rat gene contains a similar arrangement of the regulatory elements A-B-C, which mediate stimulation by IL-6 and dexamethasone (12). Analysis of rat regulatory elements have thus far failed to locate the region mediating the IL-1 response of the chromosomal gene. Although similarities in promoter architecture and functions have been demonstrated between the human and rat genes, substantial sequence alterations do exist, having the potential to contribute to the species-specific differences in regulatory pattern. Since the phylogenetic distance between human and rat is too large to make any meaningful conclusions about evolution of the regulatory elements, we have decided to compare the regulation of the HP gene among rodents, specifically the more closely related species of the genus Mus (separated from rat lineage about 15-30 million years ago; Ref. 26). We selected as representative species M. saxicola, M. caroli, and M. domesticus. The M. saxicola and M. caroli lineages diverged from the lineage leading to M. domesticus about 10 and 5 million years ago, respectively (26). In the present study, we show that substantial modifications have occurred in the promoter regions of the various mouse HP genes; some of these sequence differences may have contributed to the altered patterns of hormonal regulation observed among these species. We conclude that while induction of HP expression during the acute phase response has been maintained during mammalian evolution, the 2215 molecular machinery mediating this induction has been altered. MATERIALS  Laboratory). Stimulation of acute phase plasma protein genes was induced either by two subcutaneous injections of 25 p1 of turpentine or a single intraperitoneal injection of 25 pl of saline solution containing 25 pg of lipopolysaccharides or 5 pg of dexamethasone. The animals were killed 24 h later. Serum was prepared for immunoelectrophoretic analysis of the plasma proteins, and liver, lung, and other organs were collected for isolation of RNA (27). Parts of the livers were also used to prepare DNA (28).
Tissue Culture Systems-Primary cultures of hepatocytes from M. domesticus and M. caroli were prepared and maintained as outlined previously (29). Reuber H-35 cells (clone T-7-18; 8,21,23), HepG2 cells (30). and Hepa-1 cells (31) were cultured as described. All treatments were carried out for 24 h in serum-free minimal essential medium using the following factors at the indicated concentrations: 1 p~ dexamethasone, 100 ng/ml COS cell-derived human recombinant IL6 and LIF (Genetics Institute), 0.5 ng/ml human recombinant IL-1p (Immunex Corp.). The change in synthesis and secretion of specific plasma proteins were quantitated by rocket immunoelectrophoresis (32). The areas under the precipitin peaks were integrated and used to calculate the amounts of antigen.
RNAAnalysis-Total RNA(l0 pg) or polyadenylated RNA(5 pg) was separated on 1.5% agarose gels containing formaldehyde and transferred to nitrocellulose. The membranes were hybridized with 32P-labeled cDNAprobes encoding mouse or rat HP, human PEA3 (33), mouse CIEBPP (34). PU.l (35), Ets-2 (43), and Fli-1 (36). The bound radioactivities were quantitated by a PhosphorImager (Molecular Dynamics) using the volume integration mode. The cap site of HP mRNAs from liver and lung were determined by primer extension analysis (37) using the antisense sequence representing a part of the second exon (GC-CCTGGGAGCTGTCGTCA) as primer.
CAT Reporter Gene Constructs-The promoter and 5'-flanking region of all mouse HP genes (positions -237 to +20) were inserted into the Hind111 site of pOCT, which contains the enhancer-and promoterless CAT gene inserted into the HindIII-BumHI site of pUC18 and was provided by Dr. D. R. Grayson, Dept. of Anatomy and Cell Biology, Georgetown Medical School; the resulting plasmid was denoted pHP(237)OCT. The genomic SstI fragment (-1300 to -196) of M. domesticus HP gene2 was inserted into the SstI site at nucleotide -196 of pHP(237)OCT, replacing the segment -237 to -196 (=pmHP(1300)-OCT). The subfragments containing nucleotides -237 to -105, -237 to -185, -184 to +20, and -112 to +20 of the M. domesticus HP gene were generated by polymerase chain reaction. The former two were inserted into pCT 5' to the major late adenovirus promoter and the latter two into pOCT.
Cell 7kunsfection-H-35 cells were transfected by the DEAE-dextran method (38) and HepG2 cells by the calcium phosphate method (39). Plasmid DNA mixtures were composed of the CAT reporter gene construct (5-17 pg/ml) and p I E " W ( 2 pg/ml); the latter served as a marker for transfection efficiency (40). In some experiments, expression plasmids for transcription factors (5 pg/ml) were added; these included pCD-CIEBPp (23), pRSVGR (41), pMLP.PEA3K (33; provided by Dr. J. A. Hassell, MOBI, Hamilton), pCD-Fli-1 containing cDNA of Fli-1 (36) inserted into pCD (42), and pECE-Ets2 and pECE-PU.l (provided by Dr. R. A. Maki, La Jolla Cancer Research Foundation) (35,43). After overnight recovery, the transfected cell cultures were subdivided and treated for 24 h with serum-free medium containing dexamethasone andor cytokines. The CAT activity was determined in cell extracts (38) and normalized to the level of major urinary protein (MUP) expressed by the co-transfected PIE-MUP and quantitated by immunoelectrophoresis of the culture medium (40). The values were expressed as percent conversion of chloramphenicol to acetylated products per h per ng of MUP (12,21,23,25). Changes in synthesis of endogenous plasma proteins were quantitated by immunoelectrophoresis and used as a measure for the appropriate cytokine response of the cells.
Footprint Analysis-Nuclear extracts were prepared from COS cells and HepG2 cells according to the protocol described by Won (Control), and a second group was treated with turpentine (Acute Phase). After 24 h, RNA were prepared from the lung and liver of each animal and analyzed by Northern blot hybridization. The autoradiograms were exposed for 24 h. The band representing the cytoplasmic HP mRNA form is shown. The ethidium bromide staining pattern of the liver RNA is reproduced to illustrate equal RNA loading. Equal amounts of serum (0.4 pl) were analyzed for HP concentrations by rocket immunoelectrophoresis.
mann (44). Forty-eight h prior to isolation, COS cells were transiently transfected with 20 pg/ml expression plasmids pCD-CIEBPP (23) or pMLP PEA3K (33). HepG2 cells were treated for 12 h with serum-free medium containing dexamethasone and IL6. End-labeled DNA fragments containing the HP promoter from position -239 to +20 from M. domesticus and from position -264 to +20 from M. caroli or M. saxicola served binding substrates. The binding reaction was performed for 30 min on ice. Following digestion with DNase I, the products were separated on an 8% denaturing polyacrylamide gel and observed by autoradiography.

RESULTS
Acute Phase Regulation of HP in Mouse Species-In mice, an experimentally induced inflammation reaction elicited a massive increase in the hepatic HP mRNA concentration in the liver and a proportional increase in HP levels in the plasma (example for M. domesticus is shown in Fig. 1). Depending upon whether lipopolysaccharide or turpentine was used as inducer, the magnitude of stimulation ranged between 9and 30-fold ( Table I ) . Acute phase induction of HP expression in M. caroli and M. saxicola was of a similar magnitude, although basal mRNA expression appeared to be severalfold higher in these two species.
An organ survey revealed that the liver is the major site of HP expression in all species. Lung exhibited 20% of the basal liver value for M. domesticus and 1% for M. caroli (data not shown) and M. saxicola (Table I ) . Expression in lung was minimally affected by an acute phase, regardless of whether it had been elicited by tissue damage or endotoxin reaction ( Table I).
Only trace amounts of HP mRNA were noted in the submaxillary gland, and none was detected in kidney, spleen, heart, pancreas, small intestine, and brain (data not shown).
HP expression in the liver was stimulated roughly 2-fold by  In both species, maximal HP expression was observed with the combination of IL-1, IL-6, and dexamethasone. Thus, there is a distinct species difference in IL-1 regulation of HP. The overall cytokine response pattern for the M. domesticus HP gene resembled that in HepG2 cells (14) and human hepatocytes (13), whereas the pattern for M. caroli was more like rat (see upper panels in Fig. 4, below). Both mouse species showed a stronger response to dexamethasone alone than seen in rat or human.
HP Gene Promoter-Southern blot analysis of liver DNA from M. domesticus, M. caroli, and M. saxicola revealed a simple pattern of restriction enzyme fragments that hybridized to HP cDNA, suggesting the presence of a single copy gene per haploid genome (data not shown).2 The existence of a single HP gene was confirmed for M. domesticus by isolation of the entire HP structural gene including flanking regions. Since the available A libraries for genomic DNA for M. caroli and M. saxicola did not contain HP genes, we isolated the promoter regions from these species by polymerase chain reaction. We assumed that the region between positions -260 and -240, that was found to be identical between M. domesticus and rat, was also identical in M. caroli and M. saxicola; therefore, we used an oligonucleotide corresponding to this region as the upstream primer. The first exon sequence, the downstream primer, was already known from cDNA analyses.2 The transcription start sites (+1) were determined by primer extension. An identical start site was observed for the M. caroli and M. saxicola HP mRNAs.
The comparison of promoter sequences ( Fig. 3) (note that all sequence information is given relative to the M. domesticus sequence) revealed several interesting features. All HP genes contain segments of homology that coincide with the regulatory elements A and B (-210 to -140). The relative location of element C (-120 to -140) appeared to be much less conserved.
Element C of M. saxicola differs from the other rodents in that a thymidine is inserted into the C/EBP binding site between -127 and -128. However, a C/EBP consensus sequence unique to the M. saxicola gene and overlapping with a PEA3 recognition sequence appeared a t position -110.
The murine HP promoters were characterized by the presence of a purine-rich sequence located between -120 and -70,

M. coroli
-+ -+ -+ -+ " + + " + + " " + + + + Regulatory Elements in the BI-Flanking Region of the Murine HP Genes"T0 examine the function of the murine HP promoters, the 5"flanking regions of the three mouse HP genes homologous to the M. domesticus region from -237 to +20 were inserted into pOCT, and the resulting plasmids were introduced into the cytokine-responsive human (HepG2) and rat (H-35) hepatoma cell lines. Fig. 4 shows results example for M. domesticus HP-CAT constructs. Treatment of the transfected cells with various combinations of effectors revealed that the mouse gene elements mediated an exceptionally strong response to dexamethasone in both HepG2 (Fig. 4A) and H-35 ( Fig. 4B and Table 11) cells. The magnitude of stimulation in HepG2 cells was surprisingly high (Table IV) considering that these cells have low glucocorticoid receptor levels (45).
The gene constructs showed a minor response to IL-6 that was somewhat higher in HepG2 cells than H-35 cells. There was a cooperative effect of IL-6 and dexamethasone primarily in HepG2 cells (Fig. 4, Table 11). IL-1 or TNFa, alone or in combination with dexamethasone, proved to be ineffective on the transfected M. domesticus constructs, but was capable of activating about 2-fold the M. caroli and M. saxicola constructs ( Table 11). The minor IL-1 response of the latter two HP constructs probably contributed to the substantially increased CAT expression in cells treated with the combination of IL-1, IL-6, and dexamethasone.  ..  Table 111).
To assess the significance of the A-B-C element-containing region in the regulation by dexamethasone and IG6, various lengths of 5'-flanking regions and selected subfragments were tested in transfected H-35 cells (Table 111). Deletion from -1300 to -237 did not alter the regulatory pattern significantly, although the dexamethasone response was consistently higher.
Removal of the A element (by deletion to -184) and the B-C elements (by deletion to -112) led to a stepwise reduction in dexamethasone stimulation. The A element (-237 to -185), either in single copy or in triplicate, was ineffective as a hormone-responsive element, but in the context of the A-B-C segment (-237 to -106), a significant response to both dexamethasone and IL-6 effect was observed. This finding differs from the one reported for human analog A which has a single base difference. Human element A as a dimer or trimer was able to produce a 35-fold enhancement by IL-6 (16, 17). These data suggest that the strong dexamethasone regulation observed for the HP gene elements is dependent upon the A-B-C region as well as the promoter-proximal 106 base pairs. There was no obvious IG6 response element recognizable, even though most of the constructs yielded an approximate 2-fold stimulation by IL-6. The mouse constructs differ in this respect from rat HP gene constructs, in that the latter produced a severalfold higher IL-6 response (25). -+ -+ -+ -+ + + + + D e x " + + --+ + -" + IL-1 + + + + -+ --1L-6 " " " " + + --LIF " " " " " + + TNFa

HP construct
Relative expression

IL6
-1300 to +20 OCT 16 f 5 (2) -237 to +20 OCT 2.8 f 0.6 (2) 76 t 36 (8) 2.9 t 0.4 (4) -184 to +20 OCT -112 to +20 OCT  Table IV, both the A-B-C region (-237 to -106) and the -112 to +20 region of M. domesticus produced a roughly equal magnitude of dexamethasone stimulation that was dependent upon co-expressed steroid hormone receptor. These experiments also showed more clearly that the IL-6 regulation was independent of the glucocorticoid receptor and was primarily mediated by the A-B-C region.
A similar functional test of the M. caroli and M. saxicola HP-CAT constructs (-237 to +20) indicated a n equally prominent action of the glucocorticoid receptor, as observed for the M.
domesticus construct (Table IV). Specific Role of CIEBP&C/EBPP has been proposed as an important transacting factor acting via the A and C elements in the human (19) and rat (21,23) HP genes. The sensitivity of the murine HP constructs to CIEBPP was determined in HepG2 cells by co-transfection with a C/EBPP expression vector ( Table  V). The -237 to +20 region of all three murine H P genes was transactivated by CIEBPP. The M. domesticus and M. caroli constructs increased between 4and &fold, whereas the M. saxicola constructs increased more than 30-fold. In each case, the magnitude of stimulation by dexamethasone or IL-6 was reduced in the presence of C/EBP.
The regions involved in C/EBPP transactivation were further characterized by using constructs containing subregions of the M. domesticus HP gene ( Table V). As expected, the A-B-C (-237 to -106) element was transactivated by CIEBPP. More surprisingly, the 112-bp promoter sequence was also responsive. A striking difference did exist, however, in that the A-B-C region, but not the 112-bp region, was synergistically activated by  (5 pglml), p1E"UF' (2 pg/ml), and expression vectors for the of the indicated HP-CAT constructs of M. domesticus, M. saxicola, and glucocorticoid receptor (5 pg/ml). All DNA mixtures were brought to 22 pglml with PIE. Each experimental series included the HP-CAT gene construct without expression vector (only PIE) ("No addition") and served as a reference value for the culture receiving glucocorticoid receptor (GR). Subcultures were treated with medium alone ("Control") or medium containing dexamethasone or IL-6. The specific activity in each culture was determined and expressed relative to the control of the "No addition" transfections (defined as 1.     h with the indicated fractions were analyzed by Northern blot hybridization using"2PP-labeled cDNAencoding PEA3 Fli-1, ets-2, and HP. The autoradiograms of the Ets-related mRNAs were exposed for 7 days, the HP autoradiogram for 1 day. Within the -239 to +20 region of the M. domesticus, H P gene, the two C/EBPP binding sites at position A (-213 to -198) and C (-137 to -123) were observed (Fig. 5.4 ). No obvious C/EBPP binding was detectable within the proximal 112 bp of the promoter sequence, even though this sequence was a target of transactivation by C/EBPP (Table V). Equivalent analysis of the M. caroli promoter region revealed three CEBPP binding sites, two at analogous positions as in M. domesticus (Fig. 5 B ) and one additional site upstream of element A at -246 to -230. The latter site was only observed when we used COS cellderived C/EBPP preparations. Applying nuclear extracts from control or IL-6-treated HepG2 cells, which contain a mixture of C/EBP isoforms, the protection was restricted to A and C sites (Fig. 5C). The pattern of HepG2 cell protein interaction with the M. caroli promoter was very similar to that of M. domesticus (Fig. 50). The same two C/EBP recognition sites were occupied by proteins, and the cytokine treatment did not detectably induce a binding activity interacting with other promoter regions, including the B-element. Sequence analysis (Fig. 3) indicated that the C/EBPP consensus sequence at the approximate position of -130 in the M. saxicola promoter was modified by a single nucleotide insertion and thus essentially eliminating CIERPP binding at that site. An eficient binding was, however, observed at the new site that has evolved at position -104 to -110 (Fig. 5B).
The Possible Role of Ets-related Factors in HP Gene Regulation-The promoter regions of the HP genes of M. domesticus and M. saxicola, but not M. caroli, contain a potential PEA3 recognition site (Fig. 3). PEA3, which is a member of the ets transcription factor gene family (33), was not initially considered to be relevant for HP gene control, since no detectable expression of PEA3 mRNA was found in adult mouse liver (Fig.  6). However, hepatoma cells do express PEA3 mRNA (Fig. 6). Although we failed to detect PEA3 in nuclear extracts of hepatoma cells by Western blotting using monoclonal antibodies Nuclear Extract of Cos-1

A d / A
Std.
- against PEA3 (33), the physical presence of the PEA3 protein could conceivably influence expression of transfected HP gene constructs. To demonstrate that PEA3 interacts with predicted binding sites in HP promoters, a DNase I protection analysis was performed by using nuclear extracts of COS-1 cells transfected with PEA3 expression vectors (Fig. 7). Although the experimental system did not yield as prominent a PEA3 binding as achieved with CIERPP. binding was nevertheless detected in sites containing the consensus PEA3 recognition sequence. No PEA3 binding was apparent with the M. caroli promoter (Fig.  7).
We tested the effects of PEA3 on expression of H P CAT constructs in HepG2 cells by co-transfection (Fig. 8~. A dose-dependent trans-activation was observed with the 237-bp and 112-bp M. domesticus H P CAT constructs. PEA3 in combination with C/EBPP and glucocorticoid receptor showed the following effects ( Table V)  neutralized the IL-6 response and negatively interfered with the action of the activated glucocorticoid receptor; and 3) PEA3 cooperated with CIEBPP in transactivation. The mRNA analysis (Fig. 6) indicated that fetal and adult liver as well as hepatoma cells express mRNAs encoding members of the ets gene family. Messages for Fli-l(3.7 kb) and Ets-2 (3.2 kb), but not for PU.l, were detected in liver and in HP gene-expressing hepatoma cell lines. The expression of ets family members was not appreciably influenced by an acute phase reaction in vivo, nor was it coordinately regulated with the HP gene by hormones in the hepatoma cells. In light of the fact that ets-related genes were active in hepatic cells, we determined to what extent Fli-1, Ets-2, and PU.l could mimic the action of PEA3 on HP gene promoters ( Table V). The results indicated that these three Ets-related factors exerted a much lower stimulatory or inhibitory activity than PEA3. Taken in total, these studies suggest a regulatory role in HP gene expression for members of the ets gene family.

DISCUSSION
The major conclusions of the present study are: 1) HP is a major acute phase protein in M. domesticus, M. caroli, and M. sazicola ( Fig. 1; Table I); 2) the magnitude of stimulation in vivo is approximately 30-fold, which is 5 times higher than in rat (46); 3) the 5"flanking region (Fig. 5) contains extended segments of homology among rodents and humans; 4) the 5'flanking regions of the mouse HP genes from each of the three species mediates strong dexamethasone responses and minor IL-6 stimulation in transfected hepatoma cells (Fig. 41, accounting in part for the HP regulation seen in primary liver cells (Fig. 2); 5) a minor response to IL-1 is observed only in M. caroli and M. saxicola; and 6) during evolution of the Mus genus, a purine-rich sequence was introduced into the HP gene promoters of M. domesticus and M. saxicola, giving rise to a PEA3 binding site that is potentially regulated by members of the Ets transcription factor family.
The three mouse HP gene promoters are distinguished from those of the rat and human by their low cytokine response and their prominent stimulation by dexamethasone (12,25). (The glucocorticoid regulation of the human HP gene has yet to be demonstrated.) Of most interest is the finding that differences in HP gene regulation exist among the three mouse species. A small yet significant response to IL-1 occurs in M. caroli and M. saxicola, but not in M. domesticus (Fig. 2, Table 11). In addition, a PEA3 response element exists in M. domesticus and M. saxicola, but not in M. caroli (Table IV). These features of HP regulation are summarized in Table VI.
The structure of acute phase plasma protein genes and their response to acute inflammation are evolutionarily conserved (7). Hence, it might be assumed that the mode of regulation of these genes in the various species is similar, if not identical.
Our results show that while the hepatic expression of the HP gene is similarly increased during the inflammatory reaction in each species, the relative activity of elements mediating the reaction appears rather variable. Results summarized above indicate that IL-1 contributes to the response in M. caroli and M. saxicola, while Ets-like proteins participate in M. domesticus and M. suxicola (Table VI). The previous observation that rat and human HP genes do not display identical cytokine response patterns (12,171 has been simply accepted as a logical consequence of evolution that separated these species roughly 100 million years ago. Observing equally prominent differences in regulation among rodent species, which separated less than 20 million years ago, is unexpected. Our findings raise two critical issues that may be important in evaluating acute phase gene regulation: 1) what are the physiological implications of distinct cytokine requirements for acute phase plasma protein regulation? and 2) what are the molecular genetic mechanisms underlying the differences in the regulatory phenotypes? Acute phase reactants exert a broad array of critical functions related to homeostasis, immune regulation, and tissue repair (47,481. Therefore, the ability to appropriately regulate expression of acute phase reactants, including HP, is considered to be a process that is essential for survival of the organism in its natural habitat. To define the physiologically relevant mechanisms for regulation of the HP gene, knowledge about the acute phase-mediated changes in humoral factors is needed. Indeed, analysis of the response in human or rodents has invariably documented a concerted increase in IG1-and IL-6-type cytokines along with glucocorticoids and other endocrine hormones (49). Considering the redundancy of hormone information, the precise contribution of a given factor to the overall regulation in vivo may be difficult to delineate. The relevance of specific cytokines, in particular IL-6, on HP gene regulation in mice will be gained by the analysis of recently established ILS-deficient mice.3 Regulation of the HP gene in liver is not only dependent upon the profile of factors activated by inflammatory processes, but also upon the composition and arrangement of cis-acting regulatory sequences within the gene. Earlier comparisons of rat and human HP genes has emphasized the similarity in the IL-6 response elements (12,17,18,25) and the role of the C/EBP isoforms in controlling HP gene expression (19,21,23). The issue of glucocorticoid action on the HP gene has received little attention due to the absence of obvious glucocorticoid response element consensus sequences within the gene; in addition, the dexamethasone effect on rat and human HP gene is only apparent in the presence of IL-6 (12, 25).
The present study suggests a stimulatory role for glucocorticoids that is characteristic for mouse HP genes. Dexamethasone enhances expression of the chromosomal HP gene in liver (Table I) and in primary hepatocyte culture (Fig. 21, although the magnitude of this stimulation was far less than that of the transfected HP-CAT constructs (Tables 11-IV). A potential explanation for such a discrepancy is that a cis-acting repressor element has been removed during subcloning of the HP gene elements. Alternatively, the cellular environment of immortalized hepatoma cells may be different from that of liver cells. The latter explanation deserves attention because of the data on expression of ets gene family members (Fig. 6). Moreover, mouse hepatoma Hepa-1 cells display a dexamethasone regulation of the endogenous HP gene (Fig. 6, Ref. 31) that is highly reminiscent of the regulation of transfected HP-CAT constructs.
The promoter analysis (Table 11) indicates that at least two regions of the HP gene promoter are sensitive to the glucocorticoid receptor (-237 to -104 and -112 to +20); also, sequences upstream (-1200 to -238) exert an inhibitory effect. It is tempting to speculate that these elements are the ones which are also necessary for steroid response of the chromosomal genes in liver and in Hepa-1 cells. Functional characterizations of HP gene constructs, including a more detailed analysis of HP transgenes, must be done to gain further information on the mechanism underlying the steroid response. The action of the glucocorticoid receptor on the acute phase genes has invariably been found to involve cooperativity with other factors (13,45,(50)(51)(52)(53)(54)(55). A positive function could be ascribed to CIEBP isoforms, since removal of the C/EBP recognition sequence in the A element (-237 to -185) lowered the dexamethasone response by severalfold (Table 111). Furthermore, co-expressed CIEBPP enhanced dexamethasone stimulation in constructs containing the sequence from -237 to -105 (Table V). This would suggest that the transcription factor composition (including CIEBP isoforms) present in hepatoma cells and assembled on the transfected mouse HP gene elements cooperate with the glucocorticoid receptor in mediating induction by the steroid. The identity of the relevant participating factors, the location of the glucocorticoid receptor binding site, and the nature of the cooperative actions among factors are subjects of future studies.
The evolution of the Mus genus is accompanied by characteristic modifications in the HP gene promoter region (Fig. 3). Ets-related transcription factors may bind to the polypurine sequence and modulate the activities of adjacent promoter elements. The second possibility is consistent with the observation that PEA3, and to a far lesser extent Ets-2 and Fli-1, have transactivating activity (Table IV), and that these factors are expressed in hepatic cells (Fig. 6). Expression of PEA3 in hepatoma cells is not surprising, since this factor has been implicated in oncogenesis (33). Transactivation of the HP gene by Ets-related proteins would explain in part activation of the HP gene during development, fine tuned expression during acute phase, and ectopic expression in neoplastic tissues (56,57).
In this study, we attempted to associate changes in the structure of the HP gene promoter and alterations in regulation by cytokines and glucocorticoids. We cannot rule out that these species-specific sequences serve additional functions. These may include developmental activation of the HP gene and control of tissue-specific expression in liver and in extrahepatic sites. The molecular basis for these regulatory processes are still unknown in any of the studied species.