regulation of complement factor h in a human liver cell line by interferon-γ

Regulation of Complement Factor H in a Human Liver CellLine by Interferon-g

W. LUO & D. P. VIK

Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, USA

(Received 31 July 1998; Accepted in revised form 8 December 1998)

Luo W, Vik DP. Regulation of Complement Factor H in a Human Liver Cell Line by Interferon-g. Scand JImmunol 1999;49:487–494

Factor H is a regulatory protein of the alternative pathway of complement activation. The liver is the majorsite of synthesis. We have used the Hep3b human liver cell line as a model for examining its regulation byinterferon-g (IFN-g). The maximal response was achieved at 50 U/ml of IFN-g. An increase in H mRNA wasobserved as early as 2 h after addition of IFN-g; the response peaked at 24 h. The half-life of H mRNA in thepresence of IFN-g was 3.86 0.8 h. The increase in H mRNA by IFN-g was partly dependent on proteinsynthesis, as cycloheximide (CHX) reduced the response by 40% and the level of H mRNA decreased in adose-dependent manner with increasing concentrations of CHX. Phosphorylation events were also importantin this induction because the kinase inhibitors staurosporine and genistein inhibited the induction of H mRNAby 88% and 68%, respectively. The induction could be inhibited completely when Hep3b cells were treatedwith CHX and staurosporine. Thus induction of factor H by IFN-g apparently involves two factors. One islikely to be Stat1a and the other is a CHX-sensitive protein.

Dennis P. Vik, Department of Molecular Genetics and Microbiology, University of New Mexico School ofMedicine, Albuquerque, NM 87131–5276, USA

INTRODUCTION

The immune system defends the host against infection. Thecomplement system is part of the innate immunity of an organ-ism and serves as the first line of defence against bacteria. Thereare two pathways of complement activation, the classical comple-ment pathway, which is activated by immunoglobulin (Ig)G- orIgM-containing immune complexes, and the alternative comple-ment pathway, which is an antibody-independent pathway. Thelatter initiates directly on pathogen surfaces. Each pathwaygenerates a C3/C5 convertase by a different route. Complementactivation can opsonize pathogens for uptake by phagocytes,recruit phagocytes to sites of infection, and directly destroypathogens by creating membrane pores in their surfaces.Although the complement system usually plays a protectiverole, there is a number of instances in which complementactivation plays a role in the destruction of normal tissue.Tissue injury due to complement activation has been shown tooccur during the ischaemia/reperfusion of myocardial tissue [1–3], skeletal muscle [4, 5] and gut [6], in graft rejection [7], lunginjury [8] and multiple sclerosis [9]. Thus it is imperative that thecomplement system be strictly regulated. Complement activation

is regulated by a series of proteins that serve to protect host cellsfrom accidental damage. Factor H is a 150 kDa plasma proteinthat regulates the alternative pathway of complement activation[10]. The functions of factor H include: (1) blocking the access offactor B to C3b [11]; (2) accelerating the dissociation of Bb fromthe active C3b–Bb complex [12]; and (3) modifying C3b so thatit becomes susceptible to cleavage by factor I, i.e. it serves as acofactor for the cleavage of C3b by factor I [13, 14].

The liver is a main site of factor H synthesis in humans androdents [15–17], but other cell types also produce factor H.These include human umbilical vein endothelial cells [18–21],primary skin fibroblasts [20–22], glomerular mesangial cells[23], peripheral blood monocytes [21, 24], and glioma [25] andepithelial [26] cell lines. These extrahepatic sites of synthesismay play a role during localised inflammatory responses inminimising damage to tissue from complement activation.

Most complement proteins are synthesised by the liver and areacute-phase proteins (APP), i.e. proteins of hepatic origin whoseplasma concentration increases following tissue injury andinflammation. The major cytokine to affect production ofacute-phase proteins in the liver is interleukin (IL)-6 [27, 28],although glucocorticoids, interferon-g (IFN-g), IL-1 and tumour

Scand. J. Immunol.49, 487–494, 1999

q 1999 Blackwell Science Ltd

necrosis factora (TNF-a ) also play a role [28]. The plasmalevels of some of the complement proteins are increased duringthe acute phase, including C3, C4 and factor B [29]. Interferon-g,which is not generally considered a major inducer of APP, is themain cytokine inducing up-regulation of C4 gene expression; italso stimulates synthesis of factor B, C1 inhibitor, factor H andC4 bp. Thus IFN-g appears to be a major acute-phase inducer ofcomplement proteins [30], and has been shown to up-regulatefactor H expression in cell lines of both murine and human origin[16, 18, 21, 22, 25].

Human Hep3b cells, derived from normal liver tissue, do notexpress factor H constitutively, but when they were incubated withIFN-g, factor H mRNA transcription was induced [31]. In thisstudy, we used Hep3b cells to extend our previous studies on theeffects of IFN-g on factor H expression in an attempt to under-stand the molecular mechanism of its action and to compare theeffects of IFN-g in a human liver cell line to the previouslycharacterised effects in a mouse liver cell line [32].

MATERIALS AND METHODS

Cell culture. The human hepatoma cell line Hep3b cells (American TypeCulture Collection, Rockville, MD, USA) were maintainedon 100× 20 mm tissue culture dishes in an atmosphere of 10% carbondioxide at 378C, and fed with 10 ml of medium containing 0.45%glucose (w/v), 1% penicillin/streptomycin/Fungizone, 1%L-glutamine,and 10% fetal calf serum (BioWhittaker Inc., Walkersville, MD, USA),in a solution of Dulbecco’s Modified Eagle’s Medium. The completemedium was exchanged every 48 h.

RNA isolation, RT-PCR and Southern blot analysis.Total RNA wasisolated from Hep3b cells by the guanidinium isothiocyanate method. Atotal of 1mg of RNA from cellular extracts was placed in each assay tubefor simultaneous reverse transcriptase-polymerase chain reaction (RT-PCR). Oligonucleotide primers of human factor H (50-CAGTGAAA-GAGGAGATGCTG-30, huH PCR for) and (50-CTGGAAAG-TATGGTCTACGC-30, huH PCR rev) were used in thissemiquantitative RT-PCR assay. The RT-PCR amplification of thehuman actin mRNA was performed with the primers (50-CGTCGCCCTGGACTTCGAGCAAG-30, hu Actin PCR for) and (50-GGTGGTGCCGCCAGACAGCACT-30, hu Actin PCR rev). Reactionswere performed using the Access RT-PCR* kit (Promega, Madison, WI,USA) following the manufacturer’s protocol for 24 rounds of amplifica-tion. A total of 2.5ml RT-PCR products were electrophoresced on 2%agarose gels, and analysed by Southern blots with factor H and actin. Thefactor H cDNA probe used was a 0.7-kb fragment representing positions750–1425 of published cDNA sequence, which identified a 4.5-kbmRNA [15]. The actin probe was a cDNA fragment of chicken actin[33]. After probing, the blots were exposed to X-ray film for autoradio-graphy. The amount of radioactivity in each band was subsequentlyquantified using a phosphorimager (Molecular Dynamics, Sunnyvale,CA, USA).

Determination of factor H mRNA half-life in Hep3b cells.The half-lifeof mRNA in IFN-g-treated cells was measured following the inhibitionof transcription with actinomycin D (5mg/ml: Sigma, St Louis, MO,USA) [34], which binds to DNA and blocks the movement of RNApolymerase, preventing RNA synthesis. The cells were incubated withIFN-g for 24 h before the addition of actinomycin D, and total cellular

RNA was prepared at timed intervals after the addition of actinomycinD, analysed by RT-PCR.

Inhibition of protein synthesis by cycloheximide.Cycloheximide(CHX), which can stop protein synthesis, was purchased from Sigma.First, Hep3b cells were incubated for 24 h in medium alone or mediumcontaining either 50 U/ml IFN-g, 5mg/ml CHX or both. Second, Hep3bcells were incubated with 50 U/ml IFN-g and different concentrations ofCHX (0, 0.5, 1, 5, 10 and 15mg/ml) for 24 h. The RNA was isolated andanalysed by semiquantitative RT-PCR.

Inhibitors of protein kinases.We incubated Hep3b cells with variousinhibitors of protein kinases. H7 (Sigma) and H8 (Calbiochem, SanDiego, CA, USA) are Thr/Ser protein kinase inhibitors that inhibitprotein kinase C (PKC). We incubated Hep3b cells for 1 h in thepresence of 50mM H7 and H8, and then 100 U/ml IFN-g was addedand the incubation continued for 24 h. As a control, cells were alsoincubated with the kinase inhibitors without the addition of IFN-g. TheRNA was then isolated and analysed by semiquantitative RT-PCR forfactor H mRNA levels. Herbimycin A (Calbiochem) and genistein(Sigma) are tyrosine kinase inhibitors. We incubated Hep3b cells with250 ng/ml herbimycin A for 18 h or 50mg/ml genistein for 1 h: then100 U/ml IFN-g was added to the cultures and the incubation continuedfor 24 h. The RNA was isolated and analysed by semiquantitative RT-PCR. Staurosporine is a potent inhibitor of phospholipid/calcium-dependent protein kinase. We incubated Hep3b cells with 250 nM

staurosporine for 1 h, after which 100 U/ml IFN-g was added and theincubation continued for 24 h. The RNA was then isolated and analysedby semiquantitative RT-PCR.

RESULTS

Dose-response and kinetics of factor H mRNA induction by IFN-g

Hep3b cells do not express factor H constitutively. However,they can be induced to transcribe the factor H gene when treatedwith IFN-g [31]. The response to various doses of IFN-g wasinvestigated in order to find an optimal concentration of IFN-g

for incubation. We seeded 1 000 000 Hep3b cells per 100-mmtissue culture dish for 24 h. These cells were then incubated withvarious amounts of IFN-g (0, 5, 10, 20, 50, 100 and 200 U/ml) foranother 24 h. The negative control consisted of medium withoutIFN-g. The RNA was isolated and analysed by RT-PCR andSouthern blot. The blots were probed with either a factor HcDNA fragment or an actin cDNA fragment. The levels of factorH mRNA were normalised to the levels of actin mRNA. Theresults of three experiments are shown in Fig. 1. The levels offactor H mRNA increased with increasing concentration of IFN-g and the maximal response of induction of factor H mRNA wasachieved at a concentration of 50 U/ml of IFN-g.

The kinetics of factor H mRNA induction by IFN-g wereexamined next. We incubated Hep3b cells with 50 U/ml IFN-g

for various periods of time, from 0 to 48 h: RNA was isolated andanalysed by semiquantitative RT-PCR and Southern blot. Theresults from three experiments are shown in Fig. 2. An increasein factor H mRNA was observed as early as 2 h after addition ofIFN-g and the response peaked at 24 h.

488 W. Luo & D. P. Vik

q 1999 Blackwell Science Ltd,Scandinavian Journal of Immunology, 49, 487–494

Half-life of factor H mRNA

In other human cell lines, it has been shown that IFN-g does notaffect the half-life of factor H mRNA [21]. Since Hep3b cells donot express factor H constitutively, we could only determine thehalf-life of factor H mRNA in the presence of IFN-g. We firstincubated Hep3b cells with 50 U/ml IFN-g for 24 h, then added5mg/ml actinomycin D, which inhibits RNA transcription initia-tion. Total cellular RNA was prepared at timed intervals after theaddition of actinomycin D (0, 1, 2, 4, 6 and 8 h), and analysed bysemiquantitative RT-PCR and Southern blots. The average ofthree experiments is shown in Fig. 3. In IFN-g-treated Hep3bcells, the half-life of factor H mRNA was determined to be3.86 0.8 h.

Requirement for protein synthesis

The requirement for the duration of exposure to IFN-g necessaryto induce factor H mRNA was examined next. We incubatedHep3b cells with 50 U/ml IFN-g for various periods of time (0, 1,2, 4, 8 and 24 h); the cells were then washed and the incubationwas continued for a total of 24 h. The RNA was isolated andanalysed by semiquantitative RT-PCR. The average of threeexperiments is shown in Fig. 4. The presence of IFN-g wasrequired for at least 8 h in order to produce a maximal response.If the IFN-g was removed after incubation with the cells, theresponse was reduced.

The kinetics of induction of factor H mRNA by IFN-g wereslow, with the response reaching near-maximal levels at 24 h,and the presence of IFN-g was required for at least 8 h in order toproduce a maximal response. These data indicate that the

response to IFN-g occurs within the first few hours of incubationbut requires up to 24 h to reach maximal levels. It seemedprobable that protein synthesis might be required. In order todetermine whether this induction did indeed require new proteinsynthesis, we used CHX to inhibit translation of proteins. Thecell cultures were treated simultaneously with CHX and IFN-g.We incubated Hep3b cells for 24 h in medium alone or medium

Regulation of Factor H by IFN-g 489


Fig. 1. Dose-response of induction of factor H mRNA by IFN-g. Weincubated Hep3b cells with various amounts of IFN-g for 24 h. TheRNA was isolated and analysed by RT-PCR and Southern blots withfactor H cDNA or actin cDNA. The levels of factor H mRNA werequantified with a phosphorimager and normalized to the levels of actinmRNA. The average of three experiments6 SD is shown.

Fig. 2. Kinetics of induction of factor H mRNA by IFN-g. Weincubated Hep3b cells with IFN-g for various periods of time, theRNA isolated and analysed by RT-PCR and Southern blots with factorH cDNA or actin cDNA. The levels of factor H mRNA werenormalized to the levels of actin mRNA. The average of threeexperiments6 SD is shown.

Fig. 3. Half-life of factor H mRNA. We incubated Hep3b cells withIFN-g for 24 h before the addition of actinomycin D (5mg/ml). Totalcellular RNA was prepared at timed intervals after the addition ofactinomycin D, and analysed by RT-PCR and Southern blots withfactor H cDNA. The average of three experiments6 SD is shown.

containing either 50 U/ml IFN-g, 5mg/ml CHX or both. TheRNA was isolated and analysed by semiquantitative RT-PCRand Southern blot. The results from four experiments are shownin Fig. 5. From these data, we see that the increase in factor HmRNA by IFN-g was partly dependent on protein synthesis, asCHX reduced the response by 46%. In cells incubated with CHXalone, the level was similar to that of control cells.

In order to determine whether higher concentrations of CHXcould completely inhibit the protein synthesis that is necessaryfor induction of factor H mRNA, we then incubated Hep3b cellswith 50 U/ml IFN-g and various amount of CHX (0, 0.5, 1, 5, 10and 15mg/ml) for 24 h. The RNA was isolated and analysed bysemiquantitative RT-PCR and Southern blot. The average ofthree experiments is shown in Fig. 6. When the concentrationsof CHX increase from 0mg/ml to 10mg/ml, the levels of factorH mRNA decrease from 100% to 60%, and reach a plateau athigher concentrations of CHX. This indicates that CHX inhibitsthe protein synthesis that is involved in induction of factorH mRNA by IFN-g, but cannot completely block the induction.

Effect of various inhibitors of protein kinase on factor H mRNAregulation by IFN-g

In order to investigate further the possible role of phosphoryla-tion in this induction, Hep3b cells were incubated with variousinhibitors of protein kinases. H7 and H8 are Thr/Ser proteinkinase inhibitors that inhibit cyclic nucleotide-dependent proteinkinase and PKC. Herbimycin A and genistein are tyrosine kinaseinhibitors. Staurosporine is a potent inhibitor of phospholipid/calcium-dependent protein kinase. The Hep3b cells were incu-bated for 1 h in the presence of 50mM H7 and H8, 50mg/ml

genistein and 250 nM staurosporine, for 18 h with 250 ng/mlherbimycin A, and then 100 U/ml IFN-g was added and theincubation continued for 24 h. As a control, cells were alsoincubated with the kinase inhibitors without the addition ofIFN-g. The RNA was then isolated and analysed by semiquanti-tative RT-PCR and Southern blot. The results from three experi-ments are shown in Fig. 7. Staurosporine and genistein inhibitedthe induction of factor H mRNA by IFN-g by 88% and 68%,respectively.



Fig. 4. Effect on factor H mRNA levels with IFN-g. We incubatedHep3b cells with IFN-g for various periods of time. The cells werethen washed and the incubation was continued for a total of 24 h. TheRNA was isolated and analysed by RT-PCR and Southern blots withfactor H cDNA or actin cDNA. The average of three experiments6 SD is shown.

Fig. 5. Effect of CHX treatment on factor H mRNA induction by IFN-g. We incubated Hep3b cells for 24 h in medium alone or mediumcontaining either 50 U/ml IFN-g, 5mg/ml CHX or both. The RNA wasisolated and analysed by RT-PCR and Southern blots. The average offour experiments6 SD is shown.

Fig. 6. Dose-response of CHX treatment on induction of factor HmRNA by IFN-g. We incubated Hep3b cells with IFN-g and CHX atvarious concentrations. The RNA was isolated and analysed by RT-PCR and Southern blots. The average of three experiments6 SD isshown.

Effect of both CHX and staurosporine on factor H mRNAregulation by IFN-g

We incubated Hep3b cells with 100 U/ml IFN-g alone or withboth CHX (5mg/ml) and staurosporine (250 nM) simultaneouslyfor 24 h. The RNA was isolated and analysed by a semiquanti-tative RT-PCR. The average of three experiments is shown inFig. 8. The induction of factor H mRNA by IFN-g was inhibitedcompletely when Hep3b cells were treated with CHX andstaurosporine simultaneously.

DISCUSSION

Factor H is a regulatory protein of the alternative pathway ofcomplement activation. The liver is the major site of synthesis.During the acute-phase response, the levels of factor H mRNAincrease, and this increase is due mainly to IFN-g. In this study,the cell line Hep3b, derived from normal human liver, was usedas a model for the effect of IFN-g on hepatocytes.

While Hep3b cells did not express factor H constitutively,when they were incubated with IFN-g the levels of factor HmRNA increased in a dose-dependent manner. No othercytokines have been shown to increase factor H mRNA levels,including IL-1, IL-6 and TNF-a [31]. The characteristics of theincrease caused by IFN-g were examined in an attempt toelucidate the mechanism of its effect. The amount of IFN-g

required to obtain an observable increase was fairly low, 5 U/ml,and the levels of factor H mRNA increase with increasingconcentration of IFN-g. The maximal response was achieved ata concentration of 50 U/ml (Fig. 1). This is similar to the doseused to elicit an effect on factor H expression in other cell types,such as murineþ / þ li cell line [32], and is in the range seen forother effects induced by IFN-g, including induction of class IIgenes [34], intercellular adhesion molecule-1 [35], C1q [36],carcinoembryonic antigen, biliary glycoprotein and non-specificcross-reacting antigens [37].

In other human cell lines, it has been shown that IFN-g doesnot affect the half-life of factor H mRNA [21]. Since Hep3b cellsdo not express factor H constitutively, we could only determinethe half-life of factor H mRNA in the presence of IFN-g. In IFN-g-treated Hep3b cells, the half-life of factor H mRNA wasdetermined to be 3.86 0.8 h (Fig. 3). In other studies [21], thehalf-life of factor H mRNA was determined in human fibroblastcells and human umbilical vein endothelial cells using similarmethods. The half-life of factor H mRNA in these cells was 3.2and 3.7 h, respectively. Our results agree with these studies anddemonstrate that IFN-g does not increase the stability of thefactor H message.

The kinetics of factor H mRNA by IFN-g were slow. Anincrease in factor H mRNA was observed as early as 2 h afteraddition of IFN-g and the response peaked at 24 h (Fig. 2). This issomewhat faster than the response seen inþ / þ li cells, whichrequired nearly 8 h in order to observe an increase in factor HmRNA [32]. In the experiment of minimum treatment time to



Fig. 7. Effect of kinase inhibitors on H mRNA up-regulation by IFN-g. The Hep3b cells were incubated with various inhibitors of proteinkinases and then incubated in the presence and absence of 100 U/mlIFN-g for 24 h. The RNA was then isolated and analysed bysemiquantitative RT-PCR. An average of three experiments6 SD isshown.

Fig. 8. Effect of CHX and staurosporine on factor H mRNAregulation by IFN-g. The Hep3b cells were incubated with IFN-g

alone or with CHX (5mg/ml) and staurosporine (250 nM) for 24 h. TheRNA was isolated and analysed by semiquantitative RT-PCR. Theaverage of three experiments6 SD is shown.

reach maximal factor H mRNA level (Fig. 4), we find thepresence of IFN-g was required for at least 8 h in order toproduce a maximal response. If the IFN-g was removed afterincubation with the cells, the response was reduced. These dataindicate that the response to IFN-g occurs within the first fewhours of incubation, but requires up to 24 h to reach maximallevels and also requires the presence of IFN-g for at least 8 h.Normally, after IFN-g binds to its receptor, the signal is trans-mitted to the Jak1 and Jak2 tyrosine kinases, which phosphor-ylate Stat1a [38, 39]. This phosphoprotein then translocates tothe nucleus within minutes, binds to specific DNA sequences as adimer and causes up-regulation of various genes.

In order to investigate the possible role of Stat1a in thisinduction, Hep3b cells were incubated with various inhibitorsof protein kinase. Previous studies have shown that certain kinaseinhibitors can affect the transactivation of class II MHC, CIITAand Stat1a by IFN-g [40, 41]. H7 and H8 are Thr/Ser proteinkinase inhibitors that inhibit PKC. Herbimycin A and genisteinare tyrosine kinase inhibitors. Staurosporine is a potent inhibitorof phospholipid/calcium-dependent protein kinase. We incu-bated Hep3b cells with these kinase inhibitors and then inducedthem with IFN-g. From this experiment (Fig. 7), we find thatneither H7 nor H8 had any effect on the induction of factor HmRNA. This differs with the results from a human astrocytomaline where H7 and H8 completely blocked induction of class IIMHC, CIITA and Stat1a mRNA at this concentration [42]. Inour study, genistein inhibited the induction of factor H mRNA by68%, herbimycin A inhibited H induction by 25%. These data arein contrast to another study, in which genistein and herbimycin Ainhibited IFN-g-mediated induction of class II mRNA by 84%and 100%, respectively, and of CIITA mRNA by 66% and 50%,respectively [42]. Staurosporine has been shown to inhibit thephosphorylation of Stat1a [41]. In our experiment, it inhibitedinduction of factor H mRNA by IFN-g by 88%. Clearly,phosphorylation events are important in the induction of H byIFN-g, because both kinase inhibitors staurosporine and genis-tein inhibited the phosphorylation of Stat1a and reduced theinduction of H mRNA by IFN-g. This suggests that there mightbe a Stat1a binding site in the promoter of the factor H gene.Previous experiments in our laboratory using factor H promoterconstructs and transfection assays suggest that there are noresponse elements between¹1800 andþ44 [32]. The consensussequence of the Stat1a binding site is TTXCXXXAA [43], and inthe human factor H promoter sequence, we can find three sites thatmatch downstream fromþ44. They are TTCTAGCAA at posi-tions 91–99 in reverse orientation, and TTTCTGAAA, foundtwice in the first intron at positions 21–29 and 243–251,respectively. Therefore, Stat1a as a regulatory factor mayinvolve one or more of these sites in the induction of factor HmRNA by IFN-g.

Because of the delay in reaching maximal levels of factor HmRNA and the data showing that kinase inhibitors cannotcompletely inhibit the induction by IFN-g, it is possible thatother factors besides Stat1a were acting. In theþ / þ li murineliver cell line, it has been shown that the increase in factor H

mRNA by IFN-g was dependent on protein synthesis, as CHXcompletely abolished the response [33]. The experiment in thehuman Hep3b liver cell line with CHX demonstrated that proteinsynthesis is required in the induction of factor H mRNA by IFN-g (Figs 5 and 6). The increase in factor H mRNA by IFN-g waspartly dependent on protein synthesis, as 10mg/ml CHX reducedthe response by 40%. However, further increase in CHX con-centrations did not result in greater inhibition of factor Hinduction. This contrasts with data from mouse cell lines andsuggests a different mechanism of regulation of this gene.

From the above experiments, we find that there are at least twofactors involved in the regulation of factor H gene expression byIFN-g. One is likely to be Stat1a, because after the addition ofkinase inhibitors staurosporine and genistein, which inhibit thephosphorylation of Stat1a, the levels of factor H mRNA werereduced by 88% and 68%, respectively. The other factor is apreviously unidentified protein that is synthesized during theinduction by IFN-g, because when cells were treated with CHX,the levels of factor H mRNA were reduced by 60%. These factorsappear to work somewhat independently, as inhibiting one or theother led to a partial reduction in mRNA levels.

In order to test the hypothesis that two distinct factors areinvolved in the induction of factor H mRNA expression in thehuman Hep3b liver cell line by IFN-g, we incubated Hep3b cellswith IFN-g alone or with both CHX and staurosporine for 24 h.These data indicate that the expression of factor H mRNA byIFN-g was inhibited completely when Hep3b cells were treatedwith both CHX and staurosporine (Fig. 8). On the other hand,there is a possibility that Stat1a is the only factor involved andCHX may be inhibiting thede novosynthesis of Stat1a, thusreducing the response of factor H mRNA by IFN-g. In ourexperiments, it has been shown that a phosphorylation event isinvolved in the induction of factor H mRNA by IFN-g. AlthoughCHX may inhibit synthesis of Stat1a, it does not affect kinaseactivity. Therefore, a CHX-sensitive protein distinct from Stat1a

that is synthesised in the induction of factor H mRNA by IFN-g

is likely to be involved.In summary, the mechanism of IFN-g regulation of complement

factor H in the human Hep3b liver cell line appears to involve twofactors. They are Stat1a and a CHX-sensitive protein. Each ofthem binds to the specific factor H DNA sequence and regulatesthe expression of factor H gene. The kinase inhibitors such asstaurosporine and genistein can inhibit the phosphorylation ofStat1a, and CHX can inhibit the induction of a newly-synthesisedprotein. Inhibiting either protein alone leads to a partial decrease infactor H induction by IFN-g. However, the addition of both agentssimultaneously blocks up-regulation completely, suggesting thatthe two factors are distinct.

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regulation of complement factor h in a human liver cell line by interferon-γ

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