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International Immunology, Vol. 14, No. 10, pp. 1193-1201, October 2002
© 2002 Japanese Society for Immunology

Functional characterization of human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2 promoters, and comparison with the C1s promoter

Yuichi Endo1, Minoru Takahashi1, Mikio Kuraya1, Misao Matsushita1, Cordula M. Stover2, Wilhelm J. Schwaeble2 and Teizo Fujita1

1 Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikarigaoka, Fukushima 960-1295, Japan 2 Department of Microbiology and Immunology, University of Leicester, Leicester LE1 9HN, UK

Correspondence to: Y. Endo; E-mail: yendo{at}fmu.ac.jp
Transmitting editor: K. Sugamura


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and discussion
 References
 
The 5'-flanking regions of the genes encoding human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2, key enzymes in the lectin complement pathway, were isolated and characterized. The features of their promoters were compared with those of the human gene for C1s, the effector component of the classical pathway. The sequences upstream from the transcription start sites of the three genes contained the elements essential for transcription and liver-specific expression. Transient expression of constructs of these genes fused to the luciferase reporter gene confirmed their liver-specific expression and showed that the MASP promoters were slightly up-regulated by the presence of IL-1ß. The stimulatory effects of IL-1ß on MASP1/3 and MASP2 gene expression were abolished by the simultaneous presence of IL-6. MASP-1/3 promoter activity was also down-regulated by IFN-{gamma}. In contrast, C1s promoter activity was strongly up-regulated by IL-6, IL-1ß and IFN-{gamma}. These results indicate that IL-6 and IFN-{gamma} affect the expression of the MASP genes in a different fashion from that of the C1s gene, implying differential regulatory effects of these cytokines on the biosynthesis of lectin pathway-specific serine proteases and classical pathway-specific serine proteases.

Keywords: C1s, classical pathway, complement, cytokine, lectin complement pathway, luciferase reporter assay, mannose-binding lectin-associated serine protease, promoter, serine protease


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and discussion
 References
 
Three pathways of mammalian complement activation have been reported: the antibody-dependent classical pathway, the antibody-independent alternative pathway and the recently described lectin pathway. Activation of each pathway involves formation of serine protease complexes, which results in activation of the central complement component C3. In the lectin pathway, mannose-binding lectin (MBL)-associated serine proteases (MASP) (1–5) form complexes with polymeric lectin molecules which are involved in pattern recognition (6). Upon binding of the recognition molecules to carbohydrates on the surface of microorganisms, MASP are converted to their active forms and initiate complement activation. To date, three pattern recognition molecules, the well-characterized MBL (7) of the collectin family (8), and ficolin L/P35 (9,10) and ficolin H/Hakata antigen (11,12) of the ficolin family (13), have been shown to form complexes with human MASP. Three types of human MASP have been reported, MASP-1 (1–4), MASP-2 (5) and the recently identified MASP-3 which is produced from the MASP1/3 gene by alternative splicing (14). Human MASP-1 cleaves C3 and C2 (15), whereas human MASP-2 cleaves C4 and C2 (15,16). The enzymatic substrate of human MASP-3 is still unknown. As is the case with the lectin pathway, the initiation of the classical pathway also involves a recognition molecule. The hexaoligomeric molecule C1q binds to immune complexes, resulting in the activation of C1r, one of the C1q-associated serine proteases. Activated C1r, in turn, activates another C1q-associated serine protease, C1s. Activated C1s then cleaves C4 and C2 to generate C3 convertase. Thus, in both the lectin and classical pathways, MASP-1/MASP-2 and C1s respectively are key enzymes which directly cleave early complement components. Since the overall structures of these MASP and C1r/C1s are closely related (2,4,5,14), they form the so-called MASP/C1r/C1s family (17).

The lectin pathway has been thought to play a pivotal role in innate immunity during the lag period before the onset of adaptive immunity (18), while the classical pathway plays a primary role in adaptive immunity. There is little information about the transcriptional regulation of the lectin pathway. It was reported that rat P100 (identical to MASP-1) was induced in primary cultures of hepatocytes by IL-6 and/or dexamethasone, and that P100 was also up-regulated in vivo during an experimentally induced acute-phase reaction (19). Recently, using a luciferase reporter assay, the promoter of human MBL, one of the pattern recognition molecules in the lectin pathway, was reported to be down-regulated by dexamethasone and not affected by IL-6, IFN-{gamma} or tumor necrosis factor (TNF)-{alpha} (20). There are several reports about the regulation of the components in the classical pathway, and in most cases, C1r/C1s were up-regulated by inflammatory cytokines such as IL-6 and IFN-{gamma} (21–24). However, the molecular mechanisms underlying the transcriptional regulation of MASP/C1r/C1s genes remained to be elucidated. We recently reported the nucleotide sequence of the putative promoter region of the human MASP2 gene (25). To settle the question as to whether MASP are actually acute-phase proteins or how MASP are regulated by acute-phase cytokines, in this study we report the characterization of the human MASP-1/3 and MASP-2 promoters in comparison with the C1s promoter, especially the effects of cytokines on these promoters, and discuss their biological significance with respect to complement activation by the lectin pathway and the classical pathway.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and discussion
 References
 
Materials
Restriction enzymes and modifying enzymes such as T4 DNA polymerase and Klenow fragment were purchased from Toyobo (Osaka, Japan) and Nippon Roche (Tokyo, Japan). The pGEM-T easy and pGL3 vectors, and Tfx-20 reagents for transfection of eukaryotic cells were from Promega (Madison, WI). The pBluescript II SK+ and {lambda}ZAPII vectors were from Stratagene (La Jolla, CA). The ligation kit was from Takara Shuzo (Kyoto, Japan), the human liver decapped cDNA library from Nippon Gene (Toyama, Japan), HepG2 and HeLa cells from ATCC (Rockville, MD), and RPMI 1640 culture medium and FCS from Sigma (St Louis, MO) and ICN Biomedicals (Aurora, OH) respectively. The substrate solution for the luciferase assay (Pica Gene) and lysis buffer for preparation of culture cell lysate (luc/PGC-50) were from Toyo Ink (Tokyo, Japan). [{alpha}-32P]dCTP and the Megaprime DNA labeling system were from Amersham Japan (Tokyo, Japan). Cytokines such as IL-1ß, IL-6 and IFN-{gamma} were from Pepro Tech (Rocky Hill, NJ).

5' Rapid amplification of cDNA ends (5' RACE)
To determine the transcription start sites of the human MASP1/3 and C1s genes, the cDNAs including the 5' termini of transcripts were amplified by 5' RACE using a kit (Marathon; Clontech, Palo Alto, CA), and a human liver decapped cDNA library and HepG2 cell cDNA as templates. The gene-specific primers were 5'-GAGTCTGGATAACCAGGCGAC-3' (+404 to +424 in Fig. 1A), 5'-GCATAATAGAGAAGCAGCCACCTC-3' (+311 to +334) and 5'-CAGCTTGACTTGCCTGTGAGCTC-3' (+243 to +265) for MASP-1/3, and 5'-CCCATACATGGTAG GCTCAGCAT-3' (+335 to +377 in Fig. 1B) and 5'-CTG CTCCTCTGTCCACAGAGCCT-3' (+557 to +577) for C1s. The PCR products were cloned in pGEM-T easy and sequenced.



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  		Fig. 1. Nucleotide sequences of the 5'-flanking regions of the human MASP1/3 (A) and C1s (B) genes. Nucleotide number –1 is the first nucleotide before the transcription start site. Capital letters denote exonic sequences, and small letters denote 5'-flanking and intronic sequences. Bold sequence ATG with Met represents the initiation codon in the MASP1/3 gene. The initiation codon of the C1s gene is in exon II. Nucleotide sequence data will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases with the following accession numbers: AB065435 and AB065437 for 5'-flanking sequences of the human MASP1/3 and C1s genes respectively.

 
Cloning of MASP-1/3, MASP-2 and C1s genomic DNAs
To clone the 5'-flanking sequences of the human MASP1/3, MASP2 and C1s genes, a human genomic DNA library cloned in {lambda}FIXII was screened with 32P-labeled human MASP-1/3, MASP-2 and C1s cDNAs respectively as probes. The positive clones, F419 for MASP-1/3 (17), F4-12 for MASP-2 (25) and F508 for C1s, were subjected to restriction mapping, Southern blot hybridization with 32P-labeled cDNA probes and subcloning at the internal restriction sites into pBluescript II SK+ and then sequenced.

DNA sequencing
DNA nucleotide sequences were determined by the dideoxy chain termination method (26) using a LI-COR DNA sequencer (Model 4000). The labeling reaction was carried out using a SequiTherm Excell II sequencing kit (Epicentre Technologies, Madison, WI). Sequencing primers were synthesized by Nisinbo (Tokyo, Japan). Consensus sequences of cis-acting elements were analyzed by databank searches: http://www.dna.affrc.go.jp/sigscan/signal1.pl and http://bimas.dcrt.nih.gov/molbio/signal/.

Reporter gene constructs of the 5'-flanking region of the MASP1/3 gene
A SacI fragment (–2119 to +247) of F419 containing 2.1 kbp upstream of the transcription start site of the MASP1/3 gene was cloned in pGL3 vector at the SacI site. The DNA sequence and orientation of the insert DNA in a cloned plasmid were determined by sequencing. The promoter activity of the insert was checked by comparing the luciferase activities of the constructs with the insert in the normal orientation with those in the reverse orientation, as described below. A plasmid construct in the normal orientation, termed 41928, was selected for further analysis. To prepare a series of 5' deletion constructs of the MASP1/3 promoter, construct 41928 was cut with EcoRI at the internal restriction site (–963) and with KpnI at the pGL3 vector arm, and then subjected to the Exonuclease III/Mung Bean nuclease deletion system (Stratagene). The DNA sequences of the resulting clones were determined. Similarly, to prepare the 3' deletion constructs, an ApaI–BglII fragment (–418 to +16) blunted with Klenow fragment was ligated into pGL3 at the SmaI site. The plasmid was cut with SacI and BglII at the vector arm, and subjected to the deletion system.

Reporter gene constructs of the 5'-flanking region of the MASP2 gene
A PvuII fragment (–2859 to +43) of F4-12 containing 2.9 kbp upstream of the transcription start site of the MASP2 gene was ligated into pGL3 at the SmaI site. The promoter activity of the construct was checked by the luciferase reporter assay as described below. A plasmid construct in the normal orientation, termed 412PV2, was selected for further analysis. To prepare a series of 5' deletion constructs, 412PV2 was digested with SacI and NheI at the vector arm, and subjected to the Exonuclease III/Mung Bean nuclease deletion system. To prepare another series of 5' deletion constructs with shorter inserts, a PstI fragment (–245 to +27) blunted with T4 DNA polymerase was ligated into pGL3 at the SmaI site. The plasmid was cut with SacI and NheI at the vector arm, and then subjected to the deletion system. The DNA sequences of the clones were determined.

Reporter gene constructs of the 5'-flanking region of the C1s gene
An EcoRI–SphI fragment (–2826 to +98) of F508 containing the upstream of the C1s gene was blunted with Klenow fragment and then ligated into pGL3 at the SmaI site. The promoter activity of the insert DNA was determined as described below. A plasmid construct in the normal orientation, termed 50824, was selected for further analysis. To prepare 5' deletion constructs, a fragment was recovered from plasmid 50824 by cutting at the internal KpnI site (–287) and at the XhoI site of the vector, blunted with Klenow fragment, and then ligated into pGL3 at the SmaI site. The construct was cut with KpnI and NheI at the vector sites, and then subjected to exonuclease III/Mung Bean nuclease deletion system. The DNA sequences of the clones were determined.

Luciferase reporter assay
All constructs in pGL3 for transfection were purified on a Qiagen column (Plasmid Midi kit; Qiagen GmbH, Hilden, Germany). HepG2 and HeLa cells were maintained in RPMI 1640 medium supplemented with 10% FCS. Semiconfluent cells in 24-well microtiter plates were transfected with 0.5 µg DNA/well using Tfx-20 reagent according to the manufacturer’s protocol. After 48 h, cell lysates were prepared using Luc/PGC-50 reagent as a solvent and 3.5 µl of each lysate was assayed for luciferase in 35 µl of Pica Gene. The luciferase activities are the means from more than three experiments.

Treatment of the transfectants with cytokines
Reporter gene constructs were transfected into HepG2 cells as described above. After 44 h, the cells were washed twice with serum-free RPMI 1640 and cultured in RPMI 1640 containing IL-6 (5–50 ng/ml), IL-1ß (0.5–10 ng/ml), IFN-{gamma} (10–100 ng/ml) or dexamethasone (10–9 to 10–5 M). After 4 h, the luciferase activities of cell lysates were measured as described above.


   Results and discussion
Top
 Abstract
 Introduction
 Methods
 Results and discussion
References
 
Nucleotide sequence and reporter activity of the 5'-flanking region of the human MASP1/3 gene
To identify the promoter region of the human MASP1/3 gene, we determined its transcription start site by 5' RACE. Each 5' RACE using HepG2 cDNA or decapped human liver cDNA as a template yielded a clear single band on an agarose gel electrophoresis (data not shown). The DNA sequences of the 5' RACE products revealed that the transcription start site in HepG2 was 209 bp upstream from that found in transcripts isolated from human liver (Fig. 1A). The upstream region of the MASP1/3 gene contained one TATA-like sequence (TFIID) at –68, and two consensus sequences of hepatocyte nuclear factor (HNF)-5 (TRTTTGY) (27) at –68 and –320. HNF-5 sequences may contribute to the liver-specific expression of the MASP1/3 gene. We reported previously that MASP-1 is expressed mainly in liver (2). Interestingly, the promoter contained two consensus sequences for Pit-1 (GHF-1), a pituitary-specific transcription factor (28). The expression of this gene in human brain might be controlled by these Pit-1 elements. The EST database reveals MASP-1 transcripts present in human brain (e.g. AL134380).

To confirm the promoter activity in the 5'-flanking region of the MASP1/3 gene, a pGL3 construct consisting of 2.1 kbp upstream from the transcription start site (full-length construct) was prepared and subjected to the reporter assay. We first compared the luciferase activities of the constructs with the insert DNA in a normal orientation with those in a reverse orientation, and found that the former had ~50- and 10-fold higher activities than the latter in HepG2 and HeLa cells respectively (Table 1). In addition, the luciferase activity in HepG2 cells was 2 orders of magnitude higher than that in HeLa cells under similar transfection conditions, consistent with the liver-specific expression of the MASP1/3 gene. To identify the regulatory elements, 5' and 3' deletion constructs were prepared from a full-length construct. As shown in Fig. 2(A), the luciferase activity of a 5' deletion construct lacking the sequence from –2328 to –283 was not significantly different from that of the full-length construct. A further deletion from –283 to –214 resulted in ~40% decrease in luciferase activity, indicating a positive controlling cis-acting element(s) in this region. A database search for elements in this region showed no known consensus sequence other than Pit-1. Deletions from –214 to a part of exon I resulted in a gradually decreasing luciferase activities over a range of 400 bp.


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  		Table 1. Luciferase reporter activities of pGL3 constructs of the human MASP1/3, MASP2 and C1s genes
 


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  		Fig. 2. Luciferase activities of the pGL3 constructs of the human MASP-1/3 (A and B), MASP-2 (C and D) and C1s (E) promoters in HepG2 cells. The 5' deletion (A, C, D and E) and 3' deletion (B) constructs were prepared from the full-length constructs (clone 419 for MASP-1/3 promoter, 412PV2 for MASP-2 promoter and 508 for C1s promoter). Each point represents the mean ± SD. Asterisks show statistically significant differences in the luciferase activities between two points.

 
To define the core promoter of this gene, a series of 3' deletion constructs were also subjected to reporter assay. We found that the 3' deletion from –1 to –71 resulted in almost a complete loss of luciferase activity, suggesting that the essential elements are in this region (Fig. 2B). A TATA-like sequence at –69 in this region might be one of the essential elements, although its position seems somewhat distant from the transcription start site.

Reporter activity of the 5'-flanking region of the human MASP2 gene
Promoter activity was assessed for the pGL3 construct consisting of 2.9 kbp of the 5'-flanking region upstream from the transcription start site of the MASP2 gene (full-length construct). The constructs with the insert DNA in a normal orientation had ~20-fold higher activities than those in a reverse orientation in HepG2 cells (Table 1). The luciferase activities of the same constructs in HeLa cells were 2 orders of magnitude lower than those in HepG2 cells independent of the insert orientation. We recently reported that the upstream sequence of the MASP2 gene includes two tandem repeats of GC box (SP1)-like sequences at –30 and –24, but no clear TATA box (25). It also contains several elements for the liver- or hepatocyte-specific expression such as liver factor (LF)-A1 (29) and HNF-5, which may be responsible for liver-specific expression of the MASP2 gene. As shown in Fig. 2(C), the 5' deletion constructs with insert DNA lengths of 1.5–1.9 kbp had slightly lower promoter activity, compared with that of the full-length construct. The promoter activities of the constructs with the 1.0- to 1.2-kbp constructs decreased to ~30% of that of the full-length construct. This suggests the presence of enhancer elements in the regions of –2665 to –1887 and –1518 to –1192. In the latter region, a consensus sequence of TFIID at –1221 and GC box at –1296 were found, which might function as another set of the core promoter, although we did not identify any other transcription start site of this gene in this study. A further deletion from –975 to –245 resulted in a significant increase of promoter activity, indicating a silencer element(s) in this region. A CCAAT-enhancer binding protein (C/EBP) responsive elements (30,31) at –946, nuclear factor (NF)-IL6 (32,33) at –916 and –494, and NF-{kappa}B (34) at –667 and –373 were found, which might be involved in down-regulation in this region. Using another series of 5' deletion constructs with shorter DNA inserts, we observed that the reporter activity of the constructs lacking the region from –100 to –27 decreased to ~40% of that of the full-length construct, indicating the presence of essential elements in this region (Fig. 2D). A tandem repeat of GC boxes at –30 and –24 may be one of the essential elements.

Nucleotide sequence and reporter activity of 5'-flanking region of the human C1s promoter
As shown in Fig. 1(B), we determined the transcription start site of the C1s gene in human liver. The pGL3 construct containing the 2.9 kbp upstream sequence (full-length construct) was assessed in a luciferase reporter assay. We found that the constructs containing the insert DNA in a normal orientation showed ~100- and 20-fold higher activities than those in reverse orientation in HepG2 cells and HeLa cells, respectively (Table 1). In addition, the activities in HepG2 cells were 2 orders of magnitude higher than those in HeLa cells. Two GC box-like sequences were present at –64 and –281, and two NF-{kappa}B were at –41 and –197. No clear liver-specific element was found, although two LF-A1 consensus sequences are present in exon 1. Two 5' deletion constructs lacking the regions from –2826 to –1336 and to –287 showed no significant change in the luciferase activity compared with that of the full-length construct (Fig. 2E). A further 5' deletion from –242 to –191 resulted in a significant decrease in luciferase activity, probably due to the removal of enhancer elements such as NF-IL6 at –209 and NF-{kappa}B at –197. Deletion from –105 to –60 completely abolished the promoter activity, indicating the presence of essential elements in this region. This region includes a GC box-like sequence at –64.

Comparison of promoter structures of the MASP1/3, MASP2 and C1s genes
The MASP1/3 gene seems to employ a TATA-like sequence in its core promoter, whereas the MASP2 and C1s genes have GC box-like sequences in their corresponding regions. All three genes have HNF-5 as a binding motif for liver- or hepatocyte-specific factor in common. Within 1 kbp from the transcription start site, the MASP1/3, MASP2 and C1s genes contain 2, 1 and 3 consensus sequences of HNF-5 respectively. The MASP2 and C1s genes also have LF-A1 as another tissue-specific element, although it is unclear whether LF-A1 in exon 1 of the C1s gene is functional. The Pit-1 element was observed only in the MASP1/3 promoter. With respect to essential and tissue-specific elements among the three genes, all seem to employ the same elements such as HNF-5, NF-IL6 and C/EBP. On the other hand, with respect to the usage of essential elements and several elements such as NF-{kappa}B and LF-A1, the MASP-2 promoter seems to be related more to the C1s promoter than to the MASP-1/3 promoter. The difference in element usage may be due to the phylogeny of these genes. We showed previously that the MASP/C1r/C1s family evolved in two lineages from a common ancestor. MASP-1 belongs to one of the two lineages, the TCN-type, while MASP-2 and C1s are members of the other lineage, the AGY-type (17). Although MASP-3 is a member of the AGY-type MASP, the gene encoding the protease domain of MASP-3 is considered to have been inserted into the pre-existing TCN-type gene (MASP1 gene) during evolution (35).

Effects of inflammatory cytokines on human MASP-1/3, MASP-2 and C1s promoters
To determine the effects of inflammatory cytokines such as IL-6, IL-1ß and IFN-{gamma} on the transcription of the human MASP1/3, MASP2 and C1s genes, the luciferase activities of constructs 41928, 412PV and 50824 were assayed in the presence of these cytokines. As shown in Fig. 3(A), the activity of construct 41928 containing the human MASP-1/3 promoter was slightly decreased by ~20% by >50 ng/ml of IL-6. This effect of IL-6 was diminished when a 5' deletion construct 419{Delta}–468 was used, which lacks the region from –2328 to –468 and contains no NF-IL6-responsive element (TKNNGNAAK) (Fig. 4A). In contrast, the luciferase activity of construct 41928 was slightly increased by >2.5 ng/ml of IL-1ß (Fig. 3B). As shown in Fig. 4(A), a similar stimulation was observed when using 419{Delta}–468 but not 419{Delta}–122, suggesting that IL-1ß regulated the human MASP1/3 gene through the regulatory sequence in the – 468 to –122 region. No clear IL-1ß signaling elements were found in this region. The luciferase activity of construct 41928 was decreased to ~60% on exposure to >10 ng/ml of IFN-{gamma} (Fig. 3C). A similar effect was observed with the 5' deletion constructs 419{Delta}–468 and 419{Delta}–122 (Fig. 4A). Several consensus sequences of IFN-{gamma}-responsive elements (CWKKANNY) (36) are present in the –122 to +247 region.



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  		Fig. 3. Dose-dependency of IL-6 (A), IL-1ß (B) and IFN-{gamma} (C) on luciferase activities of pGL3 constructs. The full-length constructs of 41928 for MASP-1/3 (open circles), 412PV2 for MASP-2 (closed circles) and 50824 for C1s (open squares) were transfected into HepG2 cells, and then the transformants were exposed to various concentrations of cytokines for 4 h. Luciferase activity is expressed as a percentage of the activity for each construct in the absence of cytokine. Each point represents the mean ± SD of more than three experiments.

 


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  		Fig. 4. (A) Effects of IL-6, IL-1ß and IFN-{gamma} on luciferase activities of deletion constructs. The full-length and deletion constructs of the human MASP-1/3, MASP-2 and C1s promoters were transfected into HepG2 cells, and the transfectants were then exposed to IL-6 (50 ng/ml), IL-1ß (10 ng/ml) or IFN-{gamma} (10 ng/ml) for 4 h. Luciferase activity is expressed as a percentage of the activity for each construct in the absence of cytokines (100%). The single and double asterisks represent significantly lower and higher luciferase activities respectively compared with that without cytokine. Solid lines show the insert DNA in each construct and open squares show the positions of NF-IL6-like sequence. (B) Cooperative effects of IL-6 and IL-1ß on luciferase activities of pGL3 constructs. Constructs 41928 (MASP-1/3 promoter), 412PV (MASP-2 promoter) and 50824 (C1s promoter) were transfected into HepG2 cells, and the transfectants were then exposed to IL-6 (50 ng/ml) and/or IL-1ß (10 ng/ml) for 4 h.

 
Luciferase activity of construct 412PV2 containing the human MASP-2 promoter was not changed significantly on exposure to IL-6 at the concentration from 10 to 200 ng/ml, whereas it was slightly up-regulated 1.3-fold, in the presence of >2.5 ng/ml of IL-1ß (Fig. 3A and B). This effect of IL-1ß on the MASP2 gene was diminished when the 5' deletion construct 412{Delta}–987 was used, indicating the presence of an IL-1ß-responsive element in the –2871 to –987 region (Fig. 4A). An NF-IL-6-responsive element is at –2169, which is known to be an IL-1ß mediator (37). When construct 412PV was exposed to IFN-{gamma}, there was no change in the reporter activity, indicating that the effects of IFN-{gamma} are different between the MASP1/3 and MASP2 genes (Fig. 3C).

The luciferase activity of construct 50824 containing the human C1s promoter sequence increased 2-fold in the presence of either 50 ng/ml of IL-6 or 10 ng/ml of IL-1ß (Fig. 3A and B). The stimulatory effect of these cytokines was also observed when the 5' deletion construct 508{Delta}–287 was used, but not when 508{Delta}–105 was used, suggesting the presence of IL-6- and IL-1ß-responsive elements in the –287 and –105 region (Fig. 4A). An NF-IL6-responsive element at –209 may be involved in these stimulations. The reporter activity of construct 50824 was increased ~3-fold in the presence of 10 ng/ml of IFN-{gamma} (Fig. 3C). This effect of IFN-{gamma} was diminished when the 5' deletion construct 508{Delta}–105 was used, suggesting the presence of a positive IFN-{gamma}-responsive element (s) in the –287 to –105 region (Fig. 4A).

To examine the cooperativity of IL-6 and IL-1ß on the three promoters, the reporter assay was performed in the presence of both of them. As shown in Fig. 4(B), stimulatory effects of IL-1ß on constructs 41928 and 412PV were abolished in the presence of IL-6, suggesting that IL-6 down-regulates the human MASP1/3 and MASP2 genes. In contrast, the luciferase activity of construct 50824 was increased 2-fold in the presence of both IL-6 and IL-1ß, compared with the activities in the presence of either one of them alone, indicating that these cytokines have a simply additive effect on C1s promoter activity, suggesting that IL-6 and IL-1ß affect C1s promoter activity through different pathways. Thus, it appears that IL-6 down-regulates the expression of the MASP1/3 and MASP2 genes, and up-regulates the expression of the C1s gene.

Dexamethasone, which is known to up-regulate acute-phase proteins (34), had no significant effect on the 41928, 412PV2 and 50824 constructs (data not shown).

Physiological implications of the effects of IL-6, IL-1ß and IFN-{gamma} on the MASP-1/3, MASP-2 and C1s promoters
A classification of the three genes based on the effects of cytokines appears to be different from that based on element usage. The effects of IL-6 and IL-1ß on MASP-1/3 promoter activity are very similar to those on MASP-2 promoter activity. The fact that the C1s promoter was strongly up-regulated by IL-6, IL-1ß and IFN-{gamma} suggests that C1s is a typical acute-phase protein. This is supported by several previous reports, in which C1s protein was up-regulated by IL-6, IL-1ß, IFN-{gamma} and TNF-{alpha} (21–24). Haptoglobin, an acute-phase protein with a C-terminal domain closely related to the protease domain of C1r/C1s (38), is also stimulated by IL-6 through an NF-IL6-responsive element (32). It is likely that the sequences in both the promoter and coding regions are conserved between the haptoglobin and C1s genes.

One of our aims was to assess whether MASP are acute-phase proteins. In this study, unlike C1s, MASP-1, MASP-2 and MASP-3 seem to be in the group of constitutively expressed proteins, although they were slightly up-regulated by IL-1ß. Our present results are different from those of Knittel et al. (19). They reported that the expression of rat MASP-1 mRNA in primary culture of hepatocytes was up-regulated by exposure to IL-6 and/or dexamethasone, and that MASP-1 mRNA expression in rat liver was also up-regulated in vivo during an experimentally induced acute-phase reaction. This difference is partly due to differences in the experimental systems. We assayed MASP-1/3 promoter activity in a human hepatoma cell line in the present study, whereas MASP-1 mRNA in rat hepatocytes was assessed by Northern blots in the study of Knittel et al. Furthermore, the period of exposure was different, being 4 h in the present study and ~20 h in the study of Knittel et al. We cannot exclude a possibility that the MASP1/3 gene is up-regulated by a longer exposure to IL-6. We limited the exposure time to <4 h to keep the viability of HepG2 cells in FCS-free medium during the assay. Recently, Naito et al. (20), using a luciferase reporter assay in HepG2 cells, reported that human MBL was down-regulated by 6 h exposure to dexamethasone and not affected by IL-6, IFN-{gamma} or TNF-{alpha}. Their results are partly in concert with our present results on human MASP.

In the present study, we described distinct effects of both IL-6 and IFN-{gamma} on MASP-1/3 and C1s promoters. The down-regulation of MASP-1/3 promoter and strong up-regulation of C1s promoter by these cytokines suggest that they differentially affect gene expressions of serine protease components of the lectin and classical pathways. At the present time, it is unclear to what extent these cytokines contribute to the regulation of each pathway. However, several previous reports and our present results show that components of the classical pathway including C1s, contrary to MASP and MBL, are up-regulated by IL-6 and IFN-{gamma}, at least at the early period of the exposure. This might suggest that both pathways are also differentially regulated by these cytokines. In this context, it may be possible that these cytokines have a role in switching the host defense system from the lectin pathway to the classical pathway. It seems likely that the lectin pathway is constitutively fully active for host defense at the early phase of infection and that the classical pathway is inducible during the course of infection or inflammation.

In the lectin pathway, another important regulation mechanism may be alternative RNA processing of the MASP genes. The MASP1/3 gene produces MASP-1 and MASP-3, and the MASP2 gene produces a full-size MASP-2, and its truncated form termed sMAP or Map19 by alternative polyadenylation and alternative splicing (14,39,40). The alteration of RNA processing of the MASP genes would result in the change of stoichiometry of constituents followed by the change of lectin pathway activity. We have no information as to how cis-active elements of the MASP genes and cytokines contribute to this RNA processing. Thus, to elucidate the nature of regulation of the lectin pathway, further studies on the function of individual constituents, their stoichiometry in a MBL–MASP complex (or in ficolin–MASP complex) and RNA processing in various conditions are specially needed.


   Acknowledgements
 
The authors thank Ms K. Kanno and Ms A. Nozawa for technical assistance.


   Abbreviations
 
C/EBP—CCAAT-enhancer binding protein

LF-A1—liver factor A1

HNF-5—hepatocyte nuclear factor-5

MASP—MBL-associated serine protease

MBL—mannose-binding lectin

NF-IL6—nuclear factor-IL6

RACE—rapid amplification of cDNA ends

TNF—tumor necrosis factor


   References
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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