Genetic organization of complement receptor-related genes in the mouse.

Using an interspecific cross, gene linkage relationships among members of the murine complement receptor-related genes, C4bp, Cfh, Mcry, and Mcr2, were analyzed by segregation of RFLP in 200 mice. The human homologues of these genes are tightly linked, composing the RCA locus, which maps to human chromosome (Chr.)1q32, within a large linkage group conserved between human Chr.1q21-32 and mouse Chr.1. RFLP associated with C4bp and Cfh map within this conserved linkage group; Cfh is located 9 cM telomeric to C4bp, which is consistent with linkage data for their human homologues. Mcry and Mcr2, while tightly linked, are located outside the conserved group, 40 cM telomeric to C4bp. These data suggest that a translocation or inversion occurred within the RCA family during the evolution of the mouse, defining a breakpoint of this large conserved linkage group.

Promoter activity of the 5' flanking region of the complement receptor type 1 (CR1) gene: basal and induced transcription.

The luciferase reporter gene system was used to assay the basal and Ara-C induced promoter activity of the human CR1 gene in K-562 erythroleukemia cells. Based on the results from clones of nested deletions, both basal and induced reporter gene activity fell to promoterless levels between constructs containing 79 bp (-79) and 41 bp (-41) upstream of the transcription start site. The -79 fragment was shifted in electrophoretic mobility assays using nuclear extracts from Ara-C induced and non-induced cells while the -41 bp fragment was not shifted. These data suggest that the 38 bp region between these constructs is necessary for the transcriptional activity of the CR1 gene and is involved in specifically binding transcription factors from the nuclei of induced and non-induced cells. Several potential transcription factor binding sites in this region were identified.

Analysis of the promoter region of the murine complement factor H gene.

We have used the luciferase system to assay basal promoter activity of the murine factor H gene. Based on the results from luciferase assays with clones of 13 nested deletions, a 242-bp region that appeared to contain an enhancer element was subcloned upstream of a heterologous promoter and was shown to enhance transcription. A 26-bp fragment from this region was shifted in electrophoretic mobility assays, and this fragment contains a consensus sequence for the adenovirus major late transcription factor/upstream stimulatory factor (MLTF/USF). This fragment had enhancing activity in a minimal factor H promoter construct, demonstrating that it is a major enhancer of the factor H gene in murine liver cells.

Complete structure of the murine p36 (annexin II) gene. Identification of mRNAs for both the murine and the human gene with alternatively spliced 5' noncoding exons.

p36 (also termed annexin II) is a 39 kDa Ca2+/phospholipid-binding, membrane-associated protein that is a protein-tyrosine kinase substrate. We report here studies of the noncoding exons of p36, which combined with our earlier studies of the coding exons, allow us to conclude that the murine p36 gene is 34 kb in length with 14 exons. Comparison of the genes coding for mouse and human p36 (annexin II) and mouse, rat and human p35 (annexin I) and pigeon cp35 (an annexin I-related protein) shows strong genomic structural conservation supporting the hypothesis that these genes had a common ancestor. Both human and murine p36 mRNAs were found to be alternatively spliced in their 5' noncoding region. In both cases exon 2 is a cassette exon, which is present in a small fraction of p36 mRNAs. In type 1 mouse p36 mRNA the first noncoding 44 base exon 1 is joined to exon 3, the first of the 12 coding exons. In type 2 mRNA a 70 base noncoding exon (exon 2) is inserted between exon 1 and exon 3. Type 1 mRNA was present in all cell types studied as revealed by Northern analysis and primer extension, whereas type 2 mRNA could only be detected by RACE or PCR, indicating that it is of very low abundance. The major transcription start site of the mouse p36 gene was mapped by primer extension to be 61 bp upstream of the AUG initiation codon, which corresponds to type 1 mRNA, The murine p36 gene enhancer/promoter region contains a putative TATA box and several other potential regulatory sequences. The two alternatively-spliced human p36 mRNAs differ by the presence or absence of a noncoding 81 base exon (exon 2) inserted after exon 1, with exon 2-containing mRNAs representing approximately 10% of total p36 mRNA. The 300 bp spanning the promoter and exons 1-3 of the human and murine p36 genes show strong sequence homology immediately before and after the major transcription start site except in the region corresponding to exon 2, where homology is more limited.

Factor H.

While the mouse and human H proteins are structurally and functionally similar, they differ in their genetics. Whereas there is no evidence in humans for more than one gene; in mice the H locus is complex. Based on cDNA sequence and hybridization analysis of genomic cosmid clones, there are at least three distinct genes, all highly related to one another. The consensus repeating unit that comprises this molecule has obviously been duplicated numerous times, since it is present in many other molecules. Thus, it is not surprising to discover that there are several genes related to H in the mouse. A similar case has been described for two other members of this family. In humans, CR1 cDNA hybridizes to two distinct genomic clusters in the CR1 locus (Wong et al. 1989), and in mice, mCRY hybridizes to two regions in the genome, one on chromosome 1 and another on chromosome 8 (Aegerter-Shaw et al. 1987). It will be of interest to see if any other members of this family display as complex a genetic locus as murine H.

Regulation of expression of the complement factor H gene in a murine liver cell line by interferon-gamma.

Factor H is a regulatory protein of the alternative pathway of complement activation that is synthesized mainly in the liver. The authors used the +/+ Li murine liver cell line as a model for examining its regulation. When +/+ Li cells were incubated with IFN-gamma, the levels of factor H mRNA increased in a dose-dependent manner, achieving a maximal response at a concentration of 50-100 units/ml. The increase in factor H mRNA levels was paralleled by an increase in factor H secretion. The kinetics of induction of factor H mRNA were slow, with the response reaching near maximal levels at 24 h. The increase in factor H mRNA by IFN-gamma was dependent on protein synthesis, as cycloheximide abolished the response. The presence of IFN-gamma was required for the entire incubation period in order to produce a maximal response. The luciferase system was used in an attempt to identify an interferon-responsive element. Luciferase constructs containing from 807 to 236 bp of upstream sequence responded to IFN-gamma with a twofold induction of luciferase activity, whereas a construct containing 83 bp of 5' sequence did not. Thus, IFN-gamma stimulates factor H mRNA transcription through a protein intermediary that interacts with the promoter between positions -83 and -236.

Regulation of complement factor H expression in L cells.

The luciferase system was used to assay basal promoter activity of the murine factor H gene in the fibroblast cell line L929 (L cells). Thirteen nested deletion constructs were tested, and a region between -811 and -344 was found to have enhancer activity in the context of a heterologous promoter. This fragment was subdivided further and each of the two resulting subfragments also had enhancer activity. These subfragments each were shifted in electrophoretic mobility shift assays and were able to cross-inhibit each other in binding to a nuclear factor. Sequence analysis of these subfragments revealed the presence of an octamer in each subfragment, and a synthetic oligomer containing this octamer sequence was able to block binding in the mobility shift assay. Thus, this octamer sequence appears to play a major role in the basal expression of the factor H gene in L cells.

Structure of the gene for the F allele of complement receptor type 1 and sequence of the coding region unique to the S allele.

The genes for human complement receptor type 1 (CR1) F and S alleles have been cloned and span a region of 133-160 kb on chromosome 1. The F allele was found to comprise 39 exons and the S allele contains an additional 8 exons. The leader sequence and 5'-untranslated region are contained in one exon. Each of the long homologous repeats (LHR), which contain seven short consensus repeats (SCR), is composed of 8 exons. Within a LHR, SCR 1, 5, and 7 are each encoded by a single exon, SCR 2 and 6 are each encoded by 2 exons, and a single exon codes for SCR 3 and 4. The transmembrane region is encoded by 2 exons and the cytoplasmic domain and the 3'-untranslated regions are coded for by separate exons. The sequences of the eight S allele-specific exons were very similar to those from LHR-A and -B, as was predicted by comparison of the genomic restriction maps. It had previously been suggested that the alleles of CR1 have arisen by a mechanism of unequal crossover. A comparison of intron sequences from LHR-A, -B, -C, and -S revealed data that support this hypothesis. The homologies of intron sequences from LHR-A, -B, and -C indicated that the crossover event between LHR-A and -C that gave rise to LHR-B probably occurred within the fourth exon of these LHR. Likewise, the crossover event between LHR-A and -B that produced LHR-S probably occurred within a 383 bp region around the sixth exon. Analysis of RNA from peripheral blood cells by the S1 nuclease assay indicated that the transcription start site is 111 bp upstream of the translation initiation codon ATG. The 5' rapid amplification of cDNA ends confirmed this position as a transcription start site and revealed another possible start site 29 bp further upstream.

Cellular distribution of complement receptor type 4 (CR4): expression on human platelets.

Neutrophils have been shown to express a receptor for C3dg that is distinct from CR2 and is termed complement receptor type 4 (CR4). In the present study, other peripheral blood cell types were examined by indirect immunofluorescence and flow cytometry for the presence of C3dg binding activity. Specific uptake of C3dg occurred with neutrophils, platelets, and B lymphocytes, but not with eosinophils or T lymphocytes. Monocytes, contained within a mixed cell population of peripheral blood mononuclear cells and platelets, also bound C3dg, whereas purified monocytes did not. Binding of 125I-labeled glutaraldehyde-cross-linked C3dg to platelets was saturable, with an average of 1940 C3dg molecules bound per platelet at saturation (n = 8), ranging in number from 660 to 3930 molecules bound. Activation of platelets with thrombin did not consistently cause an increase in the expression of CR4 sites. 125I-C3dg binding to platelets was competitively inhibited equally well by unlabeled C3dg and iC3b, and approximately fourfold less well by C3b. The addition of platelets to elutriated monocytes generated C3dg binding activity on these cells by the formation of platelet-monocyte complexes. Thus, the CR4 on platelets accounted for the C3dg binding activity initially observed with partially purified monocytes. The adherent property of platelets may enable them to confer on certain other cell types the ability to localize C3dg-coated immune complexes or particles.

Characterization of the 5' flanking region of the human complement factor H gene.

The promoter region of the human factor H gene was cloned and a 3 kb Eco RI fragment was sequenced. Primer extension and S1 nuclease analysis were used to determine the transcription start site, which was found to be 10-11 nucleotides upstream of the published cDNA sequence. No canonical TATA or CCAAT boxes were found in conjunction with this site. The sequence from the human H promoter region was compared to that from the mouse gene. There was a region of 800 bp that was 62.5% identical between the two sequences. The sequences of the two promoter regions were compared to a database of transcription factor binding sites. Five elements were identified that matched the consensus sequence 100% and were identical in the two promoter sequences. Promoter assays using the luciferase reporter gene demonstrated that this region contained a functional transcription start site and putative enhancer elements. U118-MG astroglioma cells and Hep3b hepatoma cells were incubated with various cytokines to measure effects on their factor H mRNA levels. Interferon-gamma (IFN-gamma), but not interleukin-1 (IL-1), tumour necrosis factor alpha (TNF-alpha) or IL-6, was able to increase the level of H mRNA in both cell lines.

Regulation of complement factor H in a human liver cell line by interferon-gamma.

Factor H is a regulatory protein of the alternative pathway of complement activation. The liver is the major site of synthesis. We have used the Hep3b human liver cell line as a model for examining its regulation by interferon-gamma (IFN-gamma). The maximal response was achieved at 50 U/ml of IFN-gamma. An increase in H mRNA was observed as early as 2 h after addition of IFN-gamma; the response peaked at 24 h. The half-life of H mRNA in the presence of IFN-gamma was 3.8 +/- 0.8 h. The increase in H mRNA by IFN-gamma was partly dependent on protein synthesis, as cycloheximide (CHX) reduced the response by 40% and the level of H mRNA decreased in a dose-dependent manner with increasing concentrations of CHX. Phosphorylation events were also important in this induction because the kinase inhibitors staurosporine and genistein inhibited the induction of H mRNA by 88% and 68%, respectively. The induction could be inhibited completely when Hep3b cells were treated with CHX and staurosporine. Thus induction of factor H by IFN-gamma apparently involves two factors. One is likely to be Stat1alpha and the other is a CHX-sensitive protein.

Mechanism of regulation of complement receptor type 1 transcription by cytosine arabinoside in a pre-erythroid model.

Binding to erythrocyte complement receptor type 1 (CR1) clears immune complexes from blood and tissues, preventing complement-mediated pathological inflammation in disease. Previous work has demonstrated that Ara-C, a cytosine analogue, induces an 11-fold increase in CR1 mRNA expression in K-562 erythroleukaemia cells. In this work we therefore investigated whether the Ara-C/K-562 system could be used as a model for studying the pre-erythroid regulation of CR1. We demonstrated that increased CR1 expression could be induced independently of increased haemoglobin expression. Increases in CR1 mRNA levels produced by Ara-C treatment were not a function of increased stability of the message. However, Ara-C induced a protein synthesis-dependent increase in transcription initiation rate as early as 12h after treatment. Further data suggest that the effect of Ara-C on transcription is not a result of its direct DNA-damaging or DNA polymerase-inhibition activities. Induction of receptor transcription was inhibited by tyrosine kinase (TK) and protein kinase C (PKC) inhibitors. These data suggest that TK, PKC and dCTP-adducted phospholipid signalling pathways may all play a role in the mechanism of Ara-C-induced CR1 transcription.

Complement receptor type 1 gene regulation: retinoic acid and cytosine arabinoside increase CR1 expression.

Complement receptor type 1 (CR1) is expressed principally on erythrocytes, monocytes, neutrophils and B cells, where it acts as a negative regulator of the complement cascade and as a clearance mechanism for immune complexes. As CR1 expression occurs in a number of lineages, its regulation may parallel other steps in haematopoiesis. Several leucocyte-cell lines (including K-562, THP-1, U-937 and HL-60) and B-cell lines, as well as peripheral blood cells (PBCs), were tested for the ability of various compounds to up-regulate their CR1 expression. While most of the compounds tested had minimal effects on CR1 message level, retinoic acid induced increases in mRNA levels in nearly all cell lines studied. Furthermore, in K-562 cells, the cytosine analogue Ara-C, an inducer of erythroid differentiation, caused the highest increases in CR1 mRNA levels as compared to untreated cells. Ara-C also induced significant increases in CR1 message in PBCs. These data suggest that induction of specific CR1 expression could be an integral part of blood cell differentiation and that the identified cell induction systems may be useful as models to study the regulation of CR1 gene expression.

Neutrophils express a receptor for iC3b, C3dg, and C3d that is distinct from CR1, CR2, and CR3.

In the present study we examined human neutrophils for the expression of a receptor capable of binding C3dg and defined the relationship of this receptor to those that have been previously described, namely CR1, CR2, and CR3. C3dg was isolated from serum depleted of plasminogen, supplemented with 20 mM Mg++, and incubated at 37 degrees C for 6 to 8 days. The purified protein was homogeneous when analyzed by polyacrylamide gel electrophoresis and exhibited an apparent m.w. of 41,000. C3dg was polymerized by treatment with dimethyl suberimidate, and the dimer was isolated by gel filtration. Binding of both monomeric and dimeric 125I-labeled C3dg to neutrophils was saturable, and the latter ligand bound to an average of 12,400 sites/cell among nine normal individuals. At 4 degrees C, bound monomeric C3dg dissociated from neutrophils with an average t1/2 of 30 min, whereas dimeric C3dg dissociated with a t1/2 in excess of 120 min. Specific binding of multimeric C3dg was cation independent and was competitively inhibited by molar concentrations of iC3b and C3d that were equivalent to the inhibitory concentrations of unlabeled C3dg; C3b was less able to compete with C3dg for binding to these sites. The capacity of this neutrophil receptor to bind iC3b, C3dg, and C3d suggested its possible identity as CR2 or CR3. However, no specific binding to neutrophils of 125I-labeled HB-5 monoclonal anti-CR2 was detected. Furthermore, uptake of 125I-labeled C3dg was not inhibited by saturating concentrations of rabbit anti-CR1, anti-Mac-1, or OKM10. Thus, a receptor resides on neutrophils that binds the C3d region of iC3b and C3dg and is distinct from CR1, CR2, and CR3.

Analysis of complement factor H mRNA expression: dexamethasone and IFN-gamma increase the level of H in L cells.

Murine complement protein H is encoded by a 100-kb gene on chromosome 1. A 3.2-kb fragment of the 5' flanking region of the H gene was sequenced, and two transcription start sites for this gene were identified by RNase protection and S1 nuclease analyses, each of which had upstream TATA and CAAT boxes. This region shares sequence homology with known regulatory elements, including the SV40 enhancer consensus, the Sp1 binding site, and two glucocorticoid-responsive core elements (GRE). Tissue and cell-line specificity has been examined by Northern analysis, and the 4.4-kb full-length H messenger RNA was identified in liver, kidney, spleen, thymus, liver cell line 1469, and L cells. IFN-gamma did not induce H mRNA expression in the macrophage cell line P388D.1 but had a positive effect on both the mRNA and protein levels of H in L cells. PMA, LPS, and vitamin D did not increase H mRNA levels in L cells. Pursuant to the discovery of two GRE in the 5' regulatory region of the H gene, we examined the effects of glucocorticoids on H mRNA expression. Dexamethasone (10(-7) M) was found to increase markedly the levels of H mRNA and protein after 24 h of incubation, and the effect on the mRNA was detectable by 30 min. The fact that H is a down-regulator of complement activation is consistent with the known immunosuppressive role of glucocorticoids. To our knowledge, this is the first time that dexamethasone has been shown to increase the levels of a complement protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene.

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.

Complete structure of the murine C4b-binding protein gene and regulation of its expression by dexamethasone.

C4b-binding protein (C4BP) is involved in the fluid-phase regulation of the classical pathway of complement. A murine genomic library was screened, and five clones were selected that covered the remaining four exons in the 5'-region of the C4BP gene. Together with previous work (Barnum, S. R., Kristensen, T., Chaplin, D. D., Seldin, M. F., and Tack, B. F. (1989) Biochemistry 28, 8312-8317), the entire C4BP gene has now been shown to be 23 kilobases (kb) long and comprised of 10 exons ranging in size from 86 to 442 base pairs (bp). Primer extension analysis revealed the major transcription start site to be 46 bp upstream of the published cDNA start site. Northern blot analysis of RNA isolated from several mouse tissues demonstrated that the C4BP gene is expressed in a liver-specific manner. Several regions homologous to known response elements were identified upstream of the C4BP gene including a strong hepatocyte nuclear factor 1 binding site and four putative glucocorticoid response elements. Furthermore, dexamethasone increased C4BP mRNA and protein levels in the mouse liver cell line, NMuLi. The stimulation of C4BP gene expression was rapid and independent of protein synthesis. These results suggest dexamethasone induction of the C4BP gene is a primary response and therefore a transcriptional effect. Inhibition of the dexamethasone effect on C4BP by actinomycin D supports this theory. These studies also provide evidence that, for optimal induction of the C4BP gene, the glucocorticoid receptor complex may cooperatively interact with accessory transcription factors. It is likely that stimulation of C4BP gene expression by dexamethasone may allude to a mechanism by which glucocorticoids exert their anti-inflammatory effects.

Structural aspects of the human C5 gene. Intron/exon organization, 5'-flanking region features, and characterization of two truncated cDNA clones.

Human C5 cDNA fragments were used to identify five overlapping cosmid clones that spanned the entire C5 gene. Partial sequencing and Southern analysis of the clones were performed to identify intron/exon boundaries and to map intron size. The human C5 gene is 79 kilobases in length and is comprised of 41 exons. Comparison of C5 with the homologous family members C3 and C4 revealed striking similarities in exon size and number. Less, although significant similarities were also observed with the family member alpha 2-macroglobulin. The transcriptional start site for the C5 gene was observed as a doublet at positions 29 and 28 nucleotides upstream of the ATG start codon. The 5'-flanking region of the gene contains sequences homologous with several known responsive elements, including interferon, interleukin-6, glucocorticoid, estrogen, NF-kappa B, and HNF-1. Two previously identified truncated cDNAs, pHC5A and pHC5B, contain 21 and 16 exons, respectively. The last exon in pHC5A, designated exon 21a, is a product of alternative splicing and is not present in the major full-length transcript. Truncation of pHC5A is the result of an alternative polyadenylation signal located in exon 21a. In pHC5B, exon 16 is extended on the 3' end by additional flanking genomic sequence that also contains an alternative polyadenylation signal.

Structure of the murine complement factor H gene.

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.

Identification and sequence analysis of four complement factor H-related transcripts in mouse liver.

Mouse liver RNA analyzed by Northern blotting with a full-length complement factor H cDNA probe demonstrates the 4.4-kilobase (kb) H mRNA as well as three additional hybridizing species of 3.5, 2.8, and 1.8 kb, respectively. Further characterization of these alternative transcripts was pursued by isolation of additional cDNAs from a liver library using a full-length H probe. Twelve clones homologous to but distinct from H were isolated, analyzed by restriction mapping, and divided into four classes, A, B, C, and D, based on their sequences. Clones from classes A, B, and C all contained nearly identical 5'-untranslated regions and leader sequences that differed from H at more than 50% of their nucleotide positions. The 5'-untranslated and leader sequences of the class D clone were unrelated to the corresponding regions of H or the class A, B, or C clones. The remaining portions of the H-related cDNAs were made up of short consensus repeats, 7 in class A, 4 in class B, 13 in class C, and 5 in class D. To determine the relationship between the H-related transcripts and the cDNA clones, Northern blots of liver RNA were analyzed by hybridization with two probes, one specific for the class D cDNA and the other reacting specifically with the class A, B, and C cDNAs. The class A/B/C probe detected transcripts of 3.5, 2.8, and 1.8 kb in liver RNA, and the class D probe hybridized to a distinct 1.8-kb message. Additionally, a cosmid genomic library was screened with H cDNA, and nine H-related clones were isolated. They spanned a region of approximately 120 kb, defining at least two discrete H-related gene loci. These results identify new members of the super-family of C3b/C4b binding protein genes.