Transcription of the Rat Serine Protease Inhibitor 2.1 Gene in Vivo: Correlation with GAGA Box Promoter Occupancy and Mechanism of Cytokine-Mediated Down-Regulation

Anne Emmanuelle Simar-Blanchet, Catherine Legraverend, Jean Paul Thissen1 and Alphonse Le Cam

Laboratoire INSERM U376 Hôpital Arnaud de Villeneuve 34295 Montpellier cedex 05, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two GH-response elements (GHREs) and a single glucocorticoid (GC)-response element were found to regulate activity of the rat serine protease inhibitor 2.1 gene (spi 2.1) promoter in vitro. To assess the physiological relevance of these observations, we have investigated the relationship existing between the level of spi 2.1 gene transcription, structural modifications of the chromatin, and in vivo nuclear protein-promoter interactions monitored by genomic footprinting, in control, hypophysectomized, and inflamed rats. We also addressed the mechanism of inflammation-mediated gene down-regulation. We found that a high level of spi 2.1 gene transcription correlates with hypersensitivity of the promoter to deoxyribonuclease I (DNase I) and maximal occupancy of the GAGA box (GHRE-I). The failure of GAGA-box binding proteins (GAGA-BPs) to interact with the GAGA box appears to result from an impairment in GH action due to its absence (i.e. hypophysectomized animals) or to the appearance of a cytokine-mediated GH-resistant state (i.e. inflamed rats) in liver. Unlike the GAGA box, signal transducer and activator of transcription (STAT) factor-binding sites included in the GHRE-II were never found to be protected against DNase I attack but displayed a differential DNase I reactivity depending on the level of gene transcription. Alterations in DNase I reactivity of the GC-response element region suggest that GC receptor-GC complexes may associate, in a transient manner, with the promoter in the actively transcribing control state. Taken together, our studies suggest a mechanism of spi 2.1 gene activation in vivo whereby the GH-dependent chromatin remodeling caused by or concomitant to the recruitment of GAGA-box binding proteins is the first compulsory and presumably predominant step.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional promoter analysis mainly relies on the introduction of plasmid constructs bearing mutated or deleted versions of any given promoter placed in front of a reporter gene, in cells supporting its activity. Concomitantly, direct in vitro binding studies [i.e. footprinting and electrophoretic mobility shift assays (EMSA)] provide interesting information on nuclear proteins that have the potential to interact with functionally relevant sites. However, whether or not specific DNA-protein complexes that assemble in cell-free systems can also be formed in vivo in the context of intact chromatin remains a major question that must be answered before ascribing any physiological significance to such macromolecular complexes. A typical example of this problem is represented by GH-mediated formation of signal transduction activator of transcription (STAT)-DNA complexes that have been shown to play a major role in GH action in vitro, in cell culture systems (1, 2, 3, 4, 5, 6). However, their in vivo function remains still to be demonstrated since direct evidence that GH also triggers their formation in living animals have not yet, to our knowledge, been obtained. Resolving this issue is not trivial and requires the use of powerful, high resolution methods such as in vivo footprinting by ligation-mediated PCR (LMPCR) (7).

During the last decade, we have extensively studied the rat serine protease inhibitor (spi) gene system, which comprises three members (8) that are differentially expressed, depending on the physiological situation. Two of them, spi 2.1 and 2.2, are maximally expressed in normal animals and are down-regulated during acute inflammation (9). In contrast, the third one, spi 2.3, is virtually silent in control animals and is transiently induced during the acute-phase response (9). Regulation of expression of the spi 2.3 gene is complex. It involves positive promoter-regulatory elements including several interleukin-6-response sites and a glucocorticoid (GC)-response element (GRE) (10), and a negative regulatory element located in the 3'-untranslated region (11), which most likely keeps the gene silent in normal rats. Two types of effectors, GH and GC, appear to be responsible for the high level of spi 2.1 (and 2.2) gene expression in normal animals (12). We (4, 13) and others (5) have characterized a distal GH-response element (GHRE-II) in the spi 2.1 promoter which, in cultured hepatocytes, accounts for approximately 50% of the GH response (4). This element contains two potential binding sites for STAT-5 (5), a protein that belongs to a class of transcription factors activated upon tyrosine phosphorylation by a GH-activated Janus kinase (Jak2) (for a review, see Refs. 14 and 15). We have recently identified the second spi 2.1 promoter GH-response element (GHRE-I). This element also referred to as the GAGA box is structurally unrelated to STAT-binding sites (16) and behaves, at least in cultured hepatocytes, as a bifunctional enhancer controlling basal transcription and responsible for part of the GH response (4, 16). In addition to these GHREs, the spi 2.1 promoter also contains a GRE, which, in vitro, binds specifically GR receptor-GC complexes (GR-GC) and entirely mediates GC response, and several CCAAT/enhancer-binding protein (C/EBP)-binding sites that contribute to basal (i.e. hormone-independent) promoter activity (4, 13).

Specific binding of transcription factors (i.e. C/EBP, STATs, GR-GC complexes and GAGA box-binding proteins, GAGA-BPs) to their cognate sites in the spi 2. 1 promoter has been demonstrated by EMSA and in vitro footprinting (4, 16, 17). However, the physiological role of such factors remains questionable since identical in vitro footprinting patterns were obtained with liver nuclear extracts from control, hypophysectomized (hypox), or inflamed rats (17), which correspond to high, negligible, and intermediate levels of spi 2.1 gene expression, respectively (13). To address this issue, we have used genomic footprinting to examine changes in site occupancy occurring in vivo within the proximal spi 2.1 promoter region as a function of the level of gene transcription. Concomitantly, we have analyzed deoxyribonuclease I (DNase I) sensitivity of the promoter region to evaluate structural chromatin alterations that take place during gene activation. The data suggest that transcription of the spi 2.1 gene in vivo strictly correlates with occupancy of the GAGA box presumably triggered by or associated to a GH-dependent chromatin remodeling event. In addition, our studies largely unravel the mechanism of down-regulation of the spi 2.1 (and 2.2) gene that takes place during acute inflammation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Comparison of the Level of spi 2.1 Gene Transcription in Control, Hypophysectomized, and Acutely Inflamed Rats
Chances of establishing a relationship between binding of transcription factors to specific promoter sites and gene expression in vivo are greatly enhanced by the possibility to analyze conditions featuring significantly different levels of transcriptional activity. Figure 1Go shows that such a requirement is met with the spi 2.1 gene. The high level of transcription observed in control animals, in which it exceeds that of the albumin gene, was decreased by more than 95% upon hypophysectomy, which, in contrast, did not affect transcriptional activity of albumin and ß-actin genes. In acutely inflamed rats, spi 2.1 gene transcription was also severely depressed. The 5- to 7-fold decrease in signal intensity observed in the run-on assay, in fact, underestimates the inflammation-mediated decrease in spi 2.1 gene transcription. Indeed, because of the extensive degree of sequence identity (more than 95%) between the two cDNAs in their 5' and central regions (9), the spi 2.1 probe used also recognized the spi 2.3 transcript, which was essentially undetectable in control animals but is induced during inflammation (Refs. 9 and 10; see also Fig. 7Go). As previously observed, acute inflammation negatively affected albumin gene transcription but had no effect on the expression of the ß-actin gene (10).



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Figure 1. Nuclear Run-On Assay

Nascent in vivo liver RNA transcripts contained in nuclei from control (CONTROL), hypophysectomized (HYPOX), or 18-h inflamed (INFLAMED) rats were elongated in vitro in the presence of [{alpha}-32P]UTP, purified, and hybridized to various plasmids bearing cDNA sequences from spi 2.1, albumin, or ß-actin genes immobilized on a nylon membrane. Empty vector (pUC 19) was used as a blank control. After hybridization performed at 42 C for 48 h, the membrane was treated with RNases A and T1, extensively washed, and autoradiographed at -80 C with an intensifying screen for 3 days.

 


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Figure 7. Northern Blotting Analysis of spi 2 mRNAs in Cultured Hepatocytes: Effects of GH and Cytokines

Forty eight-hour-old cultured hepatocytes were incubated for 24 h in the absence or presence of rGH (500 ng/ml), without or with 10 ng/ml of various cytokines used alone or in combination. Total RNAs were then extracted and analyzed as described in the legend to Fig. 7Go.

 
High Levels of spi 2.1 Gene Transcription Correlate with the Appearance of DNase I Hypersensitivity within the Promoter Region
Changes in level of transcription are generally associated with alterations of the chromatin structure, more particularly in regions thought to be important for transcription in vivo. In eukaryotic genomes, chromatin remodeling has been often detected as sites of DNase I hypersensitivity (for a review, see Ref.18). Figure 2Go shows that digestion of genomic DNA with BamHI generated a 2.6-kb fragment (panel A) spanning the -862/+1725 gene region (panel B). DNase I treatment of liver nuclei from normal rats led to the appearance of three minor fragments of approximately 0.8, 0.75, and 0.67 kb that were not detected in DNA isolated from hepatic nuclei of hypox animals. This revealed the presence of three DNase I-hypersensitive sites (HSS I, II, and III) that were seen only when the gene is efficiently transcribed and that map in the proximal promoter region containing the two GHREs previously identified by functional in vitro studies (4, 5, 13). This is consistent with the observation made by Yoon et al. (19), who first reported a GH-dependent induction of DNase I-hypersensitive sites in the 5'-flanking spi 2.1 gene region. A similar analysis has also been performed with liver nuclei from inflamed rats, yielding results comparable to those obtained with nuclei from hypox animals (data not shown).



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Figure 2. DNase I Sensitivity of the spi 2.1 Gene Promoter Region in Control and Hypox Rats

A, Appearance of DNase I-hypersensitive sites in the proximal 5'-flanking region of the spi 2.1 gene. Hepatic nuclei prepared from control (lanes 1–7) and hypox (lanes 8–13) rats were exposed to varying concentrations of DNase I as described in Materials and Methods. Purified DNA was then digested with BamHI and subjected to electrophoresis. On the autoradiogram, arrows indicate the position of the 2.6-kb fragment covering the -862/+1725 gene region and that of the three fragments of approximately 0.80, 0.75, and 0.67 kb, generated by the action of 6 µg/ml DNase I on control nuclei but not on nuclei from hypox animals. Size of fragments was determined using mol wt markers run alongside. B, Diagram summarizing the organization of the -862/+1725 BamHI spi 2.1 genomic fragment, the location of the probe used for Southern analysis, and the putative position of the DNase I HSS detected in control nuclei. Open rectangles indicate the position of the two GHREs (see Fig. 3Go) identified in the promoter.

 
In Vivo DNase I Footprinting of the spi 2.1 Promoter
The results presented in Fig. 2Go suggested that chromatin remodeling occurred in the spi 2.1 promoter region, when switching from a situation of very low level (i.e. hypox rats) to a situation of very high level (i.e. control animals) of gene transcription. However, they did not provide any information on the relationship that may exist between the degree of occupancy of putative functional sites within the promoter in vivo and the level of gene transcription. Our previous in vitro binding studies (i.e. EMSA and footprinting experiments) have not allowed us to correlate changes in the level of spi 2.1 gene transcription in vivo with changes in DNA-protein interactions more particularly in the region of the GAGA box (17) which proved to be the major spi 2.1 promoter regulatory element in isolated hepatocytes (4, 16). We thus turned to the genomic footprinting technique that allows a precise analysis of nuclear protein-DNA interactions in vivo. Figure 3Go displays the results of an analysis of the lower strand of the spi 2.1 promoter region from positions -160 to +10 relative to the cap site. This region was previously found to contain the elements required for maximal basal as well as hormone-stimulated promoter activity in cultured hepatocytes (4, 13). A comparison of patterns of bands obtained with naked DNA (in vitro) and with the three DNase I-treated nuclei-derived templates (in vivo) revealed major differences throughout the promoter. In control rats, a unique region encompassing the GAGA box and presumably extending toward the 3'-region, as suggested by the slight decrease in intensity of the -37/38 and -26/-27 bands, was strongly protected against DNase I attack. In contrast, essentially no protection of the GAGA element occurred in hypox or acutely inflamed rats. It should also be noted that hypophysectomy and, to a lesser extent, inflammation induced a strong DNase I-reactive site at positions -37/-38 (presumably a doublet) and weaker sites at positions -26/-27 and +10. Unlike what was observed with the GAGA box, no DNase I reactivity of the adjacent C/EBP site spanning the -66 to -58 region was detected, regardless of the situation analyzed. This obviously precludes drawing any conclusion on the in vivo role of this site shown to be functional in vitro (4, 13).



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Figure 3. In Vivo DNase I Footprinting of the spi 2.1 Gene Promoter

Lower strand analyses of LMPCR products revealed with the 32P-labeled oligonucleotide L3. The (G+A) lane corresponds to naked DNA chemically cleaved at purine residues and the IN VITRO lane corresponds to naked DNA treated with DNase I. The three other lanes (IN VIVO) correspond to templates obtained from DNAse I-treated liver nuclei from normal (CONT), hypophysectomized (HYPOX), or LPS-treated (INFL) rats. The major spi 2.1 promoter elements found to be functional in cultured hepatocytes and shown on the diagram at the lefthand side are: the TATA box (-32/-26), the proximal GHRE (GHRE-I/GAGA box, -45/-57), the most active C/EBP site (-58/-66), the GRE (-73/-88), and the distal GHRE (GHRE-II, -104/-149) that contains the two putative STAT-5-binding sites (-124/-132 and -139/-147). The DNase I-protected region (PR) observed only with the in vivo control template and the major reproducible changes detected between either IN VITRO and IN VIVO templates, or between the three IN VIVO templates are indicated by arrows at the righthand side of the figure.

 
In addition to the GAGA box, two other promoter regions appeared to be differentially affected by hypophysectomy and acute inflammation. The first one includes the GRE, which was previously defined as the -88/-74 motif, on the basis of its functional activity (4) and its structural similarity with the consensus GRE sequence (20). In agreement with what has been observed for interactions of other genes with GR-GC complexes (21, 22), no footprint (i.e. protection) was observed in the spi 2.1 GRE region (Fig. 3Go) which, however, displayed marked differences in DNase I reactivity as a function of the level of gene transcription. Thus, a strong reactive site occurring at position -86 within the GRE undetected with a naked template (i.e. in vitro) and a minor one flanking this motif occurring at position -92 or -93 (or possibly both) also seen in vitro became apparent in control animals but almost totally disappeared upon hypophysectomy or inflammation. In contrast, enhanced DNase I reactivity was detected at positions -90, -95, -96, and -98 (i.e. 5' to the GRE) in nuclei from hypox and, to a lesser extent, inflamed animals, compared with both in vivo controls and naked DNA. Finally, the fairly strong nuclease reactive site found at position -80 in DNase I-treated naked DNA and in DNA isolated from control nuclei digested with the enzyme was significantly attenuated in hypox and acutely inflamed rat nuclei.

The second region differentially affected in both pathophysiological models relative to the control encompasses the spi GHRE-II (4) and includes the potential STAT-5 binding sites present at positions -124 to -132 and -141 to -147. In addition to other minor differences that proved poorly reproducible, two major observations were consistently made: 1) unlike what was observed with the GAGA box, no clear protection of any of the STAT-binding sites could be detected, whatever the state of the animal, by comparing in vitro and in vivo footprint patterns; 2) in the in vivo patterns, several DNase I-hyperreactive sites (positions -126, -128, and -137) were visible with hypox and, although much less pronounced (positions -126 and -128), inflamed rat liver nuclei-derived templates but not with controls. In addition, two minor nuclease-reactive sites (positions -118 and -120) that were seen with both a naked template and DNA prepared from DNase I-digested control nuclei became barely detectable upon hypophysectomy or inflammation. Figure 4Go shows a similar analysis performed with the upper strand. The most striking observation made with naked DNA as well as with templates derived from DNase-treated nuclei was the almost complete lack of DNase I reactivity of the GAGA box region and, as already noted with the lower strand, of its 5'-flanking C/EBP site. As observed with the lower strand, no clear protection but rather significant changes in DNase I reactivity of the promoter were detected in the GRE region. Thus, a set of hyperreactive sites internal to the GRE (positions -82 and -77 to -72) that were not observed with naked DNA but could be seen with templates prepared from control nuclei, were undetectable except for two of them (positions -74 and -73), in templates prepared from nuclei of hypox or inflamed rats. In addition, two other minor changes including the appearance of a weak reactive site at position -84 and attenuation of stronger ones at positions -92 and -91 were also detected in this region, with in vivo hypox and inflamed rat-derived templates compared with controls or in vitro DNase I-treated DNA.



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Figure 4. In Vivo DNase I Footprinting of the spi 2.1 Gene Promoter

Upper strand analysis of LMPCR products revealed with the 32P-labeled oligonucleotide L*3. Legend is identical to that in Fig. 3Go.

 
As observed with the lower strand, no significant in vivo protection of the GHRE-II region could be observed with the upper strand, regardless of the situation analyzed. However, the enhanced DNase I reactivity of the most 3'-STAT-5-binding site region (increased signal at positions -128 and -127) described above with the lower strand (Fig. 3Go) and specifically seen with hypox and inflamed rat-derived templates was also observed. Several other nuclease-reactive sites were induced in the two pathophysiological animal models examined, both in this distal region of the spi 2.1 promoter (positions -165, -106, and -104) and in a region closer to the cap site (positions -34 to -32). It is also remarkable that the cap site region (positions +1 to -15) displayed enhanced DNase I reactivity in all in vivo conditions analyzed, compared with naked DNA.

Analysis of GAGA-BPs and STAT-5 Contents of Liver Nuclei by EMSA
The almost complete absence of detectable in vivo interactions between GAGA-BPs and the GAGA box in hypox and acutely inflamed animals revealed by genomic footprinting could be merely due to a decrease in amounts of active GAGA-BPs in nuclei preparations. To test this hypothesis, we made extracts from liver nuclei prepared in the same way as those used for DNase I treatment and analyzed by EMSA the capacity of nuclear proteins to bind to the GAGA box. Figure 5AGo shows that no significant qualitative or quantitative difference could be detected between control, hypox, and inflamed rat liver nuclear extract preparations. As already observed with control extracts (16), formation of a major (a) and a minor (b) complex, previously shown to represent specific binding of nuclear factors to the spi GAGA box, occurred with all three different sets of nuclear proteins.



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Figure 5. EMSA Analysis of GAGA-BP and STAT-5 Content of Liver Nuclei

A, Ten micrograms of liver nuclear proteins prepared from normal (CONTROL), hypophysectomized (HYPOX), or LPS-treated (INFLAMED) rats were incubated with a 32P-labeled single-copy GAGA box probe (GA-ext). Bound and free probes were separated by electrophoresis on a 5% native polyacrylamide gel and detected by autoradiography. The region of the gel containing the specific (a and b) and nonspecific (c) complexes are shown. B, Ten micrograms of liver nuclear proteins prepared from normal rats were preincubated at 4 C for 1 h with preimmune serum (PI) or antibodies (1 µg purified Ig per assay) directed against STAT-5 b ({alpha}-STAT-5 b). A labeled oligonucleotide probe representing the STAT-5 ß-casein gene promoter-binding site was then added, and incubation was allowed to proceed for 15 min at RT. Bound and free probes were separated by electrophoresis on a 4% native polyacrylamide gel and detected by autoradiography. Region of the gel containing the specific DNA-protein complexes (thin arrows) and immune complexes (IC, wide arrows) are shown.

 
An intriguing observation arising from genomic footprinting of the spi 2.1 promoter was the absence of any protection of the GHRE-II region against DNase I attack even when the gene is maximally transcribed (i.e. in control animals). This was indeed unexpected in the context of GH action since this region has been shown to bind STAT-5 in vitro and activate GH-dependent transcription ex vivo in transfected cells (5, 6). A possible explanation to this apparent lack of stable STAT-5 in vivo binding could be the absence of active forms of this transcription factor that might have leaked out or be inactivated (i.e. by dephosphorylation) during nuclei preparation. The EMSA performed with liver nuclear extracts from control rats shown in Fig. 5BGo does not support this hypothesis. Indeed, significant amounts of STAT-5 present in control nuclear extracts bind to a specific STAT-5 probe derived from the ß-casein gene promoter. In addition to STAT-5, C/EBP factors massively represented in liver nuclei have also been shown to bind the spi 2.1 GHRE-II in vitro (17). However, based on our genomic footprinting data, they do not appear to form detectable stable complexes with this promoter region in vivo.

In Vivo Effect of Lipopolysaccharides (LPS) and in Vitro Effect of Cytokines on GH-Dependent or Independent spi 2 mRNA Levels
An important observation made in the genomic footprinting experiments was that the lack of protection of the GAGA box was associated with a very strong down-regulation of the spi 2.1 gene in inflamed animals. Since GH appears to strictly control spi 2.1 GAGA box occupancy in vivo (see Discussion), a perturbation occurring in the GH transduction pathway subsequent to the development of a GH-resistant state could account for this observation. In keeping with this possibility, some of us recently showed that treatment of primary hepatocytes with interleukin (IL)-1ß or tumor necrosis factor-{alpha} (TNF{alpha}) (23), which are two major lymphokines implicated in the liver acute-phase response (for a review, see Refs. 24 and 25), or injection of inflammatory compounds (LPS) to living animals (26) strongly inhibited GH-dependent insulin-like growth factor-I (IGF-I) message synthesis. It thus became important to check whether this type of effect could represent a more general phenomon and apply to the spi 2 gene system. To this aim, we investigated the effect of LPS (in vivo) or cytokines (in vitro) on GH action on the level of spi 2 messages. As previously observed (9), acute inflammation reduced by about 50% spi 2.1 and 2.2 mRNA liver steady-state levels and, inversely, induced the spi 2.3 message (Fig. 6AGo, compare lanes 1–3 to lanes 4–6; and Fig. 6BGo). Injection of GH alone slightly increased the level of spi 2.1 and 2.2 messages and had no effect on that of spi 2.3 (Fig. 6Go, A and B). Interestingly, the inflammatory drug largely prevented GH to restore normal spi 2.1 and 2.2 mRNA levels (Fig. 6AGo, lanes 10–12; and Fig. 6BGo).



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Figure 6. Northern Blotting Analysis of spi 2 mRNAs in Vivo: Effects of LPS and GH

Separate groups of six rats were injected with saline, LPS, or bGH or a combination of both as described in Materials and Methods. Total liver RNAs were then purified, resolved on a 1% denaturing agarose gel, and transferred onto a nylon membrane. Blots were hybridized with a spi 2.1 cDNA probe that revealed the spi 2.3 (the 2.3-kb band) and spi 2.1 + spi 2.2 mRNA species (the 1.8-kb band). Panel A, Autoradiogram of a gel in which RNAs from three different animals per group were analyzed. Panel B, The amount of spi mRNA was quantitated by scanning the autoradiograms. Each bar represents the mean +/- SEM (n = 6).

 
Unlike what we previously observed with hepatocytes maintained in primary culture on a plastic support (13), cells cultured on matrigel in the absence of GH retained a fairly high level of expression of spi 2.1 and 2.2 messages (Fig. 7Go, lane 1). This level could be increased, however, by GH by 2- to 3-fold (lane 2), an effect that was almost completely obliterated by IL-1ß (lanes 3 and 4), TNF{alpha} (lanes 7 and 8), or a combination of both agents (lanes 11 and 12). In contrast, IL-6 did not appear to signicantly influence GH action on spi 2.1 and 2.2 messages but, as expected (10), strongly induced the spi 2.3 mRNA (lanes 5 and 6). The latter, which was already slightly expressed under unstimulated conditions, was also increased by IL-1ß (compare lanes 1 and 3) but not TNF{alpha} (compare lanes 1 and 7). This effect of IL-1ß, which was not previously observed by using a different culture system [i.e. plastic dishes and a serum-containing medium (10) instead of matrigel-coated dishes and a serum-free medium, this study], can presumably be accounted for by the presence of a nuclear factor {kappa}B (NF-{kappa}B) site within the spi 2.3 promoter (8, 10). IL-1 has indeed been shown to activate NF-{kappa}B (for a review, see Ref.27), which, in turn, stimulates transcription of specific genes such as the nitric oxide synthase gene in pancreatic ß-cells (28). To further characterize the antagonistic action of IL-1ß on the GH effect, we performed dose-dependent studies. Induction of spi 2.1 and 2.2 mRNA species by GH in cultured hepatocytes was inhibited in a dose-dependent manner by IL-1ß, with a maximal effect (nearly 100% inhibition) occurring with 10 ng/ml (Fig. 8Go, panel A). A similar relationship was also observed for induction of the spi 2.3 mRNA. Production of spi 2.1 and 2.2 messages proved highly sensitive to GH since a concentration of 100 ng/ml (~5 nM) was maximally efficient (Fig. 8Go, panel B). However, regardless of the GH concentration used, the effect was totally inhibited by IL-1ß (Fig. 8Go, panel B). Thus, data obtained by analyzing IGF-I (23, 26) and spi 2 messages (present study) strongly support the notion that acute inflammation, through the action of IL-1ß (and possibly TNF{alpha}), causes the appearance of a strong GH-resistant state in the hepatocyte.



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Figure 8. Dose-Dependent Effects of IL-1ß and GH on spi 2 mRNAs in Cultured Hepatocytes Analyzed by Northern Blotting

Panel A, Forty eight-hour-old cultured hepatocytes were incubated for 24 h in the absence or presence of rGH (500 ng/ml) with increasing concentrations of IL-1ß. Panel B, Cells were incubated in the presence of increasing concentrations of rGH, without or with IL-1ß. Total RNAs were then extracted and analyzed as described in the legend to Fig. 7Go.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The rat spi 2.1 gene represents one of the very rare examples of a GH-regulated gene for which the main features of in vivo regulation can be largely reproduced in cultured hepatocytes with promoter constructs (4, 13). However, regulatory mechanisms inferred from in vitro studies do not account for all in vivo observations. Thus, a significant level of basal (i.e. GH- and GC-independent) promoter activity accounted for by a role of C/EBP sites was detected in primary hepatocytes (4, 13) whereas in vivo, in hypox animals lacking GH and GC, the gene was shown to be almost totally silent (12). Similarly, dexamethasone proved to be as efficient as GH in activating the spi 2.1 promoter in vitro (4, 13) but failed to reinduce gene expression in hypox rats (12). These discrepancies raised questions on the role of C/EBP sites and the GRE as regulatory elements in living animals. Another major, as yet unresolved, question pertains to the role played in vivo by the two spi 2.1 promoter GHREs characterized in vitro (4, 5, 13). The data presented in this paper, which for the first time demonstrate a correlation between in vivo promoter occupancy and the rate of transcription of a GH-regulated gene, introduced major information that largely reconciles in vitro and in vivo observations and provides answers to some of the aforementioned questions.

The most striking observation arising from genomic footprinting analysis of the spi 2.1 promoter is the strict correlation existing between the stable interaction of nuclear proteins with the proximal GHRE (GHRE-I) recently identified as the GAGA box (16), and the level of gene transcription in vivo. Such a correlation fully agrees with the observation that, in cultured hepatocytes, a mutation of the GAGA box that is lethal for both basal and hormone (i.e. GH and GC)-dependent promoter activities (4) also abolishes binding of as yet unidentified GAGA-BPs (16). This also further supports the notion that the GAGA box, a purine-rich promoter motif fully conserved in all three spi 2 genes (8), is a key control element working both in vivo and in vitro as an on/off transcriptional switch.

Clearly, transcriptional activation of the spi 2.1 gene in vivo is strictly GH-dependent, since only GH can reinduce gene expression in hypox rats (12). This strongly suggests that proper assembly of GAGA-BPs-GAGA box complexes, which is mandatory for spi 2.1 gene promoter activity in vivo, requires that the signal initiated by GH binding to its cell surface receptors is normally processed to the nuclei. Thus, in any situation where a severe impairment in the GH-signaling pathway occurs, formation of GAGA-BPs-GAGA box complexes should be prevented and spi 2.1 gene transcription shut off. This is the case, as expected, in hypox rats due to the absence of GH, but also in acutely inflamed animals. Indeed, lipopolysaccharides (in vivo) and inflammatory cytokines (IL-1ß and TNF{alpha}, ex vivo) induce a strong GH-resistant state in hepatocytes, presumably leading to a blockade of GAGA box-GAGA BPs complex formation and subsequent inactivation of the spi 2.1 gene. Cytokines have generally been found to directly regulate transcription of acute-phase genes via their specific promoter response elements (24, 25). Our data reveal that cytokines can also regulate acute-phase gene expression indirectly, by disturbing the functioning of a key promoter element such as the spi GAGA box.

How GH contributes to bringing GAGA-BPs to the promoter, however, remains an open question. Experiments performed with cell-free systems strongly suggest that binding of GAGA-BPs (see Fig. 5AGo) and subsequent transcriptional activation (16) can occur independently of GH. This seems to indicate that only the recruitment of GAGA-BPs, presumably linked to the chromatin-remodeling event shown to take place during spi 2.1 gene activation, requires GH in vivo. At least two hypotheses can be put forward: 1) GH might first activate a chromatin-remodeling system independent of GAGA-BPs that would locally alter the nucleosomal structure and permit binding of those transcription factors to the promoter; 2) GAGA-BPs themselves could bear a chromatin-remodeling activity stimulated by GH (e.g. through posttranslational modifications). In keeping with the latter possibility, trans-displacement of histones has been proposed as one mechanism for trancription factor-targeted generation of a nucleosome-free region in chromatin (29). Assessing these hypotheses must await the cloning and characterization of GAGA-BPs that should allow setting up reconstitution experiments.

Unlike what was observed with the spi 2.1 GHRE-I (i.e. the GAGA box), we never detected any protection of the STAT-5-binding sites contained in the GHRE-II in vivo. This was true in control animals, despite the fact that a significant level of active nuclear STAT-5 could be demonstrated, and also in hypox rats treated for 2 h with GH, a condition under which the nuclei content of active STAT-5 exceeds that in controls (C. Legraverend, unpublished results). This, however, does not eliminate the possibility that STAT-5 can bind to the GHRE-II in living animals. It is indeed conceivable that activation of STAT-5 by GH could lead to a transient interaction with its cognate binding sites that would not be detected by the genomic footprinting technique but that could subsequently modify the local chromatin structure and contribute to transcriptional activation. Such a structural modification appears to take place upon gene activation, at least at the level of the more proximal STAT-5-binding site, as evidenced by the decrease in local DNase I reactivity detected with control templates. However, an alternative explanation to such a change in DNase I reactivity that was also observed in other parts of the promoter when switching from a transcriptionally inactive (i.e. hypox or inflamed rats) to a transcriptionally active (control animals) state could be the displacement or the structural alteration of a nucleosome. Thus, our genomic footprinting data do not provide a clear answer to the question of whether STAT-5 can form a complex with the spi 2 GHRE-II in vivo. Whatever the answer to this question might be, however, it should be kept in mind that STAT-5 could contribute to transcriptional activation of the spi 2.1 promoter without directly contacting the DNA. This transcription factor has indeed been shown to associate with GR receptors and potentiate their effect in vitro (30).

The absence of detectable C/EBP footprints, whatever the in vivo situation analyzed, appears at first to be totally paradoxical, based on our previous in vitro footprinting assays (4, 17) and heterologous transactivation experiments (11). Unlike what is happening in vitro (see EMSA in Fig. 8Go), the results of genomic footprinting clearly indicate that there is no stable interaction of C/EBP with the -130 to -122 site internal to the GHRE-II, in vivo. As pointed out for STAT-5, however, we cannot formally rule out that transient and/or very loose binding of C/EBP might occur in the context of intact chromatin and confer some functionality to this site in vivo. Due to the lack of DNase I sensitivity of the other potentially important C/EBP-binding site immediately flanking the GAGA box (-66/-58), it is not possible to exclude this site as being functional in vivo.

Unlike what has been observed in vitro (4), no protection of the spi 2.1 promoter GRE could be observed in vivo by genomic footprinting, either with control rats having normal circulating GC levels or with inflamed animals known to overproduce GCs (24, 25). However, a differential alteration in DNase I reactivity of the GRE region was observed as a function of the level of gene transcription by the technique of in vivo footprinting. The most striking change is represented by the presence of several DNase I-hyperreactive sites mapping within the GRE or in its close vicinity in chromatin from control liver nuclei and their absence in naked DNA and in chromatin from nuclei of hypox or inflamed rats. Similar changes in reactivity to DNase I occurring upon gene activation mediated by GCs have been observed with the rat tyrosine aminotransferase promoter (21) and the mouse mammary tumor virus long terminal repeat (22). They have been interpreted as the signature of a transient association of GR-GC complexes with GREs. Thus, the possibility seems to exist that those hypersensitive sites specifically detected in the GRE region in an actively transcribing state (i.e. control animals) are, at least in part, the consequence of an interaction with GR-GC complexes.

The genomic footprinting patterns of bands generated within the GRE region by DNase I treatment of nuclei isolated from hypox and inflamed rat liver clearly differed from those obtained with control nuclei but, surprisingly, were qualitatively similar. This was rather unexpected, considering that hypophysectomy and acute inflammation represent two opposed situations with respect to endogenous levels of circulating GCs (i.e. almost total absence of hormone in hypox rats vs. enhanced levels in inflamed rats). This makes it unlikely that GR-GCs complexes mediate changes occurring in DNase I reactivity in the spi 2.1 gene GRE region in either of these animal models compared with controls. Those alterations could more likely be related to an overall modification of the nucleosomal structure occurring in the promoter upon gene activation and revealed by the appearance of DNase I-hypersensitive sites. Further studies involving in vitro reconstitution of the nucleosomal structure will be necessary, however, to assess this hypothesis.

In conclusion, we would like to propose a mechanism for spi 2.1 gene activation in vivo that includes first, a GH-dependent remodeling of the chromatin in the promoter region caused by, or concomitant to, the recruitment of GAGA-BPs. This would then allow association of GR-GC complexes with the GRE, which in turn could boost GAGA-BP binding through a dynamic interplay, thus accounting for the potentiating effect of GCs on GH action. At this point, the contribution of STAT-5 to GH-mediated spi 2.1 gene activation in vivo remains to be clarified. It should be pointed out, however, that unlike activation of STAT-5, which appears to be sex-dependent (31), functioning of the GAGA box as well as expression of spi 2.1 and 2.2 genes are essentially identical in the male and the female (our unpublished observations). This strongly argues for a predominant role of the GAGA box in controlling GH-dependent regulation of spi 2 gene expression in vivo. Finally, a very important outcome of these studies is the elucidation of the indirect mechanism of cytokine-mediated down-regulation that accounts for the transcriptional shut-off of spi 2.1 and 2.2 gene expression induced by acute inflammation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[{gamma}-32P]ATP (3000 Ci/mmol) and [{alpha}-32P]UTP (800 Ci/mmol) were obtained from Du Pont NEN (Paris, France). Vent Exo- and Amplitaq DNA polymerases were purchased from New England Biolabs (Beverly, MA) and Perkin Elmer (Paris, France), respectively, and DNase I (3000 U/mg) was from Worthington (Freehold, NJ). Collagenase (0.65 U/mg) was from Boehringer Mannheim (Mannheim, Germany) and bacterial (Escherichia coli) LPS were obtained from Life Technology (Cergy-Pontoise, France). Bovine GH (bGH) was a kind gift of Mosanto (St-Louis, MO). Rat GH (rGH) (NIH-B-13, AFP-87401) was a gift from the National Institute of Diabetes and Digestive and Kidney Diseases, and recombinant murine IL-1ß, IL-6, and TNF{alpha} were purchased through R & D systems (Abingdon, U.K.). Antibodies to STAT-5b were purified Ig preparations obtained from Santa Cruz Biotechnology Co. (Santa Cruz, CA). Nylon membrane (Hybond N+) was from Amersham Life Science (Amersham, U.K.). Female Wistar rats were obtained from the Katholieke Universiteit Leuven (Heverlee, Belgium) and male Wistar rats were purchased from IFFA-CREDO (l’Arbresle, France).

Probes used for EMSA listed below (genomic sequences are in uppercase letters) include: (i) an oligonucleotide (GA-ext) (8) containing the spi GAGA box (underlined) and bearing its natural 6 and 7 nt-long 5'- and 3'-flanking sequences, respectively: 5'-tcgaTTCCTAAGAGGAGGGAGGAGCCTTTGGT; (ii) an oligonucleotide comprising the high-affinity STAT-5-binding site (underlined) present in the ß-casein gene promoter (32): 5'-gTCCCTTAATTCCAAGAAGTCC.

The following spi 2.1 promoter-derived oligonucleotides were used in genomic footprinting to amplify: (i), the upper strand:

L1 (-238/-218), 5'-GTACACTCTACTTTTGCTTTG;

L2 (-211/-187): 5'-TCCCACTTTCCTCATTGACTTTGAC;

L3 (-201/-170): 5'-CTCATTGACTTTGACCACTCAATAAATAAAAGG; and (ii), the lower strand:

L11 (+73/+56), 5'-TGATACCAGCCAGCTGCC;

L21 (+50/+28), 5'-CTGTGTTTGCTGACACCTGATGT;

L31 (+42/+18), 5'-GCTGACACCTGATGTTCAGGGTTGT.

It should be mentioned that, despite the extensive sequence conservation of the three spi 2 gene family members, more particularly in the promoter region (8), sets of oligonucleotides used in our experiments were totally specific, since they allowed exclusive amplification of the spi 2.1 sequence. The linker used was the same as that described by Mueller and Wold (7).

Nuclear Run-On Assays
Nuclei isolated from the liver (33) of normal, hypox, or LPS-treated (killed 18 h after a single ip injection of 750 µg/100 g body weight) adult male rats were used to evaluate the in vivo rate of transcription initiation. The procedure for run-on analysis, which includes incubation of the nuclei with [{alpha}-32P]UTP, purification of total cellular RNA, and hybridization to templates immobilized on a nylon membrane, has been previously described (34). The following plasmids were used as hybridization templates: pUC19 bearing 0.52 kb of the spi 2.1 cDNA 5'-region (9); pBR322 bearing 1.6 kb of the mouse ß-actin cDNA (35); pBR322 containing 1.2 kb of the rat albumin cDNA (36). The amount of labeled transcripts used for hybridization corresponded to 20 x 106 trichloroacetic acid-precipitable counts.

EMSA
Liver nuclear extracts were prepared from control, LPS-treated (18 h), or hypox rats, according to the method of Gorski et al. (33) as modified by Sierra (37). The following minor changes were introduced: 0.5 mM phenymethylsulfonylfluoride (PMSF), 14 µg/ml aprotinin, 1 mM Na3VO4, and 10 mM NaF were added to the homogenizing buffer; 0.1 mM PMSF, 0.1 mM Na3VO4, and 0.1 mM NaF were added to the nuclear lysis buffer; and 0.1 mM Na3VO4 and 0.1 mM NaF were added to the dialysis buffer. Double-stranded oligonucleotides were end-labeled using T4 polynucleotide kinase and [{gamma}-32P]ATP. Probes were purified on nondenaturing polyacrylamide gels before use. The incubation mixture (20 µl) contained 10 µl liver nuclear extracts (10 µg proteins diluted in dialysis buffer), 1 µl (2 µg) poly(deoxyinosinic-deoxycytidylic)acid, 1 µl (100 µg) BSA, 4 µl of 5x buffer (25 mM MgCl2, 20 mM spermidine, 4.4 mM dithiothreitol, 0.25 mM EDTA, 50% glycerol, 0.25 mM Na3VO4, 0.25 mM NaF), and 2 µl water or antibodies. After a 15-min preincubation at room temperature (RT), the labeled probe (~0.2 ng of oligonucleotide, 50–100.000 cpm) was then added in 2 µl, and the binding reaction was allowed to proceed for 15 min at RT. For supershift experiments, nuclear proteins were first incubated with preimmune or immune serum for 1 h at 4 C. Samples were electrophoresed for 3 h at RT on 5% nondenaturing polyacrylamide gels in 0.5 x TBE (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA) at 130 V.

In Vivo Footprinting of the spi 2.1 Promoter
Preparation of DNase I-Treated Templates
Nuclei isolated from the liver of control, hypox, or inflamed (i.e. LPS-treated for 18 h) rats were incubated for 10 min on ice in a mixture containing 10 mM Tris-HCl (pH 7.4), 15 mM NaCl, 60 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 0.5 mM CaCl2 and 1 mM MgCl2, in the presence of DNase I (10 µg/ml). Reactions were stopped by adding 4 vol of a lysis solution containing 1 mg/ml proteinase K, 30 mM EDTA, and 2.5% SDS, and nuclear lysates were incubated for 4 h at 45 C. Naked DNA was concomitantly treated with DNase I. Genomic DNA was purified as described by Rigaud et al. (21) by phenol extraction and was ethanol precipitated and dissolved in water.

Genomic Sequencing with the LMPCR Procedure
This was performed using a modification of the method of Mueller and Wold (7) described by Grange et al. (38), except for ligation of the linker that was carried out for 1.5 h at RT, in the presence of 15% polyethylene glycol (39). Extension of primers 1 (L1 or L11) was performed at 50 C, exponential amplification (30 cycles) with primers 2 (L2 or L21) was performed at 66 C, and extension (9 cycles) of 32P-labeled primers 3 (L3 or L31) for detection of LMPCR products was performed at 68 C. Chemical sequencing reactions were performed by the Maxam and Gilbert procedure (40). Labeled products were separated on 6% polyacrylamide-urea sequencing gels that were dried and autoradiographed without an intensifying screen for 12–24 h at RT. Autoradiographs shown in Results are raw data from a representative experiment. In in vivo footprinting, only alterations in the pattern of bands that have been reproduced with at least three independent template preparations have been considered as real.

Analysis of DNase I-Hypersensitive Sites
Conditions used to incubate nuclei isolated from the liver of control or hypox male rats with increasing concentrations of DNase I (0–20 µg/ml) and the procedure to purify genomic DNA were as described for the preparation of DNase I-treated templates for LMPCR. DNA samples (25 µg) were then digested with BamHI, and fragments were resolved on a 1% agarose gel and transferred onto a nylon membrane. The membrane was hybridized to a uniformly 32P-labeled single-stranded RNA probe corresponding to the BamHI-PstI fragment (positions -862 to -479) of the spi 2.1 gene (8) and submitted to autoradiography at -80 C for 48 h with an intensifying screen.

Animal Treatment, Hepatocyte Culture, and Northern Blotting Analysis of spi 2 mRNAs
All animal studies were conducted in accord with the principles and procedures outlined in "Guidelines for Care and Use of Experimental Animals." Four groups (n = 6/group) of 4-week-old female Wistar rats were used to analyze the effect of LPS and GH on liver spi 2 mRNA levels in vivo. The first group received one ip injection of LPS (750 µg/100 g body weight) and one sc injection of saline; the second group received one sc injection of bGH (200 µg/100 g body weight) and one ip injection of saline; the third group was administered LPS and bGH; and the last group received two injections of saline. All animals were killed 10 h after injections.

Rat hepatocytes cultured on matrigel-coated plates for 48 h in serum-free medium were exposed during 24 h to cytokines and rGH at different doses. Procedures used to culture hepatocytes and extract total RNAs from the liver of adult rats or from cultured cells were as previously described (23). For Northern blotting analysis, 20-µg samples of total RNA were resolved on 1% denaturing agarose gels and transferred onto nylon membranes (Hybond) by vacuum blotting. Blots were then hybridized under standard conditions with a random-primed 32P-labeled spi 2.1 cDNA probe (fragment 60–1800) that was previously shown to recognize all spi messages (9). The 2.3-kb band corresponds to the spi 2.3 mRNA species, whereas the 1.8-kb band represents undistinguishable, size-matched spi 2.1 and 2.2 messages which, on the basis of the abundance of corresponding proteins, appeared to be equally represented in the normal liver (12).


    ACKNOWLEDGMENTS
 
The technical assistance of Josiane Verniers for hepatocyte culture and of Dominique Defalque for in vivo experiments is acknowledged. We are deeply indebted to Thierry Grange for his help in setting up the genomic footprinting method, his advice throughout these studies, and his critical reading of the manuscript. We thank Michel Raymondjean and Claude Sardet for reading the manuscript and Laurent Charvet for art work.


    FOOTNOTES
 
Address requests for reprints to: Dr. Alphonse Le Cam, INSERM U376, Hopital Arnaud de Villeneuve, 371 Rue du Doven Gaston Giraud, Montpellier, Cedex 05 France 34295.

1 Present address: Unité de Diabétologie et Nutrition, School of Medicine, The University of Louvain, B-1200, Brussels, Belgium. Back

Received for publication September 10, 1997. Revision received December 15, 1997. Accepted for publication December 16, 1997.


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