Overexpression of any fibrinogen chain by Hep G2 cells specifically elevates the expression of the other two chains.

Earlier studies showed that overexpression of B beta fibrinogen chains, by transfection of Hep G2 cells with B beta cDNA, specifically enhanced the synthesis of all three fibrinogen chains (Roy, S. N., Mukhopadhyay, G., and Redman, C. M. (1990) J. Biol. Chem. 265, 6389-6393). To determine whether overexpression of any of the three component chains of fibrinogen affects the synthesis of the other two chains, we developed stable Hep G2 cell lines transfected with individual fibrinogen chain cDNAs. As a control, cells were also transfected with expression vector, which did not contain fibrinogen cDNA. Transfection with any fibrinogen cDNA increased the synthesis of all three fibrinogen chains but not of other plasma proteins. Hep G2 cells transfected with B beta cDNA produced 3-4-fold more fibrinogen than control cells, and cells transfected with A alpha or gamma cDNA made about 2-fold more fibrinogen. Northern blot analyses showed that levels of all 3 fibrinogen mRNAs were increased and were highest in Hep G2-B beta cells. Nuclear run-on transcription assays demonstrated that increased expression of the chains was due to increased transcriptional activity. These studies show that transcription of the three fibrinogen chains is tightly linked, and increased expression of any chain specifically leads to increased synthesis of the other two chains.

dimer, with each half-molecule containing a set of the three chains (for reviews, see Refs. [1][2][3]. Each of the component chains is the product of a separate gene (4-7), which are clustered together on the long arm of chromosome 4, with the Bj3 gene in opposite transcriptional orientation to the Aa and y genes (8,9). Each gene is separately transcribed and translated (lo), and the nascent chains are discharged into the lumen of the endoplasmic reticulum, where chain assembly into dimeric fibrinogen occurs in a stepwise manner (11)(12)(13).
Fibrinogen is an acute-phase protein, and in the early stages of inflammation or on treatment of hepatocytes with interleukin-6 and glucocorticoids its synthesis is increased, and this is directly related to enhanced transcription of the three genes (14)(15)(16). In these conditions of enhanced fibrinogen produc-* This work was supported by National Institutes of Health Grant HL 37457. These studies were presented in preliminary form at the 32nd annual meeting of the American Society for Cell Biology in November, 1992. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ TO whom correspondence should be addressed New York Blood Center, 310 E. 67 St., New York, NY 10021. Tel. 212-570-3059; Fax: 212-879-0243. tion both increased transcription and the half-life of each mRNA are coordinately regulated (17). The mechanisms by which the expression of the three fibrinogen genes is regulated are not fully understood. There is no single sequence in the fibrinogen gene that is involved in the regulation of all three genes, and although several nuclear factors have been described, which bind to the fibrinogen promoters, no single factor has been found that activates all three genes (18-21).
Hep G2 cells have surplus Aa and y chains, which also occur as an Aa y complex. We have suggested that Bj3 chain synthesis may be a rate-limiting step in the production of fibrinogen by Hep G2 cells (12,13). This was supported by the fact that increased Bj3 chain synthesis, elicited by transfection of Hep G2 cells with Bj3 cDNA, led to increased synthesis and secretion of fibrinogen. However, transfection with Bj3 cDNA not only increased the synthesis of Bj3 but also that of Aa and y chains, and the transfected Hep G2 cells maintained surplus amounts of Aa and y chains. This indicated that hepatocytes stringently regulate expression of the three fibrinogen genes and that over-production of Bfl chains specifically up-regulates expression of the other two genes (22).
In this study we show that enhanced expression of fibrinogen is not only regulated by Bj3 in synthesis but that transfection of Hep G2 cells with any fibrinogen chain cDNA elevates the expression of the other two genes, leading to the specific synthesis and secretion of fibrinogen.

EXPERIMENTAL PROCEDURES
Materials-The expression vectors containing full length Aa, BO, and y chain cDNA, pRSVNeo-Aa, pRSVNeo-Bo, and pRSVNeo-y have been previously described (22, 23). Human @-actin cDNA was purchased from Clontech and RNasin from Promega Corp. Other reagents used have also been previously described (22-25).
Cell Culture, Transfection, and Selection of Stable Cell Lines-Hep G2 cells were transfected with pRSVNeo-Aa, pRSVNeo-Bo, pRSVNeo-y, or as a control with pRSVNeo, which did not contain a fibrinogen cDNA insert, by the calcium phosphate method (26). methionine and immunoprecipitation of nascent fibrinogen, albumin, transferrin, al-antichymotrypsin, and C-reactive protein were performed as previously described (11,12,23). Protein radioactivity of individual proteins was determined by cutting out the radioactive areas from the SDS-PAGE' gels and counting by liquid scintillation spectrometry (12) or by measuring the optical density of the fluorograms using a scanning densitometer. Northern Blot Analyses-Poly(A+) RNA from Hep GB-Neo, Hep The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.

Nuclear Run-on Transcription
Assay-Nuclear transcription ass a y were performed according to Groudine et al. (28). The nuclei from about 1 X lo7 Hep Ga-Neo, Hep G2-Aa, Hep GP-BB, or Hep G2-7 cells were isolated and resuspended in reaction buffer (150 mM KCl, 10 mM Tris-HC1, pH 8.0, 5 mM MgC12, 1 mM MnClZ, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 80 units of RNasin, 5 mM dithiothreitol, 1 mM each ATP, GTP, and CTP, and 250 pCi of [mP]UTP (800 Ci/mmol, 10 pCi/pl)) and incubated at 25 "C for 45 min. Transcripts were deproteinized, DNase I-treated, and further purified by chromatography on a G-50 Sephadex spin column. Hybridizations were performed in slots on nitrocellulose membranes containing 0.5 or 5.0 pg of alkaline-denatured Aa, BB, or y cDNA at 42 'C for 3 days. Radioactivity was detected by fluorography and quantitated by measuring the density of the film in a scanning densitometer.

Synthesis and Secretion of Fibrinogen by Hep G2 Cells
Transfected with Individual C h i n cDNAs-Transfection with any fibrinogen chain cDNA increased the synthesis of fibrinogen, as measured intracellularly in a 5-min pulse period. As previously described (22) Hep G2-Bj3 cells synthesized 2.5-3-fold more fibrinogen in a 5-min pulse period than the control Hep G2-Neo cells. Hep G2-Aa and Hep G2-7, however, also synthesized more fibrinogen (40-80%) than control Hep G2-Neo cells (Fig. 1).
Although the rate of secretion of all cells was similar, Hep G2-Aa, Hep GZ-BB, and Hep G2-y secreted more fibrinogen than Hep G2-Neo cells. Most of the fibrinogen synthesized by Hep G2-Aa, Hep G2-Bj3, and Hep G2-y cells were secreted into the medium during the first 45 min of the chase period. Only a small amount, less than 20%, of the pulse-labeled fibrinogen was retained intracellularly after 2 h of chase incubation ( Fig. 1). About 80% of the pulse-labeled fibrinogen was recovered.
Increased Synthesis of Individual Fibrinogen Chains-To determine the initial rate of synthesis of individual fibrinogen chains, the intracellular radioactive fibrinogen chains synthesized at the end of a 5-min pulse incubation with L-[~%] methionine were immunoprecipitated, separated in reducing conditions on SDS-PAGE, and the radioactivities of the individual chains measured. The amount of radioactivity in each of the chains was corrected to account for different amounts of methionine in each of the chains.
Transfection of Hep G2 cells with cDNA for individual fibrinogen chains caused an increase in synthesis not only of a single chain but also of the other two component chains of fibrinogen (Fig. 2). Hep G2-Aa, Hep G2-Bj3, and Hep G2-y synthesized increased amounts of all three chains as compared with Hep G2-Neo. The increases varied from 1.7-4.2-fold ( Table I). Hep G2-Bj3 cells synthesized more of each of the fibrinogen chains than Hep G2-Aa or Hep-G2-y ( Fig. 2 and Table I).
Synthesis of Variant Aa Chin-In addition to the normal Aa chain (M, = 65,000) commonly present in fibrinogen, a variant Aa chain, containing a C-terminal extension, due to the additional splicing of a sixth exon, is also present in fibrinogen. This variant Aa chain that contains 847 amino acids is termed (YE and is synthesized and secreted by Hep G2 cells (29).

TABLE I Fold increase over Hep G2-Ne0 cells
Transcription and mRNA levels were measured as described in Figs. 6 and 7. The "protein" column indicates synthesis of individual fibrinogen chains following a 5-min pulse incubation with L -[~~S ] methionine (Fig. 3). In all cases the values represent increases in Hep GB-Act, Hep G2-BB, and Hep G2-7 as opposed to Her, G2-Neo cells. trypsin, and C-reactive protein were determined. Transfection of Hep G2 cells with expression vectors containing any one fibrinogen chain cDNA caused an increase in synthesis and secretion of fibrinogen but did not affect albumin, transferrin, al-antichymotrypsin, and C-reactive protein (Fig. 4). The control Hep G2-Neo cells produced more radioactive albumin and transferrin than fibrinogen and less alantichymotrypsin and C-reactive protein. However, the cells transfected with the fibrinogen cDNAs (Hep G2-Aa, Hep G2-BP, and Hep G2-7) synthesized and secreted more fibrinogen than the other proteins measured. This demonstrates that overexpression of any one fibrinogen chain specifically increases the synthesis and secretion of fibrinogen but does not affect other plasma proteins, including al-antichymotrypsin and C-reactive protein, which like fibrinogen are acute-phase proteins.
Fibrinogen mRNA Levels-Hep G2-Aa, Hep G2-BB, and Hep G2-y cells contained elevated levels of mRNA of all three fibrinogen chains when compared with Hep G2-Neo cells. Fig.  6 shows Northern blot analyses. Densitometric readings of the specific hybridized bands showed that in all cases the mRNA levels of the three fibrinogen chains in Hep G2-Aa, Hep GZ-BP, and Hep G2-y cells were greater than that of control Hep G2-Neo. The mRNA levels in Hep G2-Aa and Hep G2-y were nearly double that of Hep G2-Neo and the levels in Hep GP-BP were 3-5-fold higher than the control levels. In the three cell lines overproducing fibrinogen, the BB mRNA levels were greater than that of Aa and y (Fig. 5 and Table I). As a control &actin mRNA levels were also determined and found not to differ.
Nuclear Run-on Transcription-To determine whether the increased synthesis of fibrinogen and the elevated levels of the three-fibrinogen chain mRNAs were due to a stimulation of gene expression, nuclear run-on transcription assays were performed on isolated nuclei of Hep G2-Neo, Hep G2-Aa, Hep GB-BP, and Hep G2-y cells. Slots on nitrocellulose membranes containing 0.5 or 5 pg of alkaline-denatured Aa, BB, and y cDNA were hybridized separately with 32P-labeled nuclear RNA from the various stable-transfected Hep G2  Fig. 6 shows an autoradiogram and the lower panel presents the intensities of the bands in the slots containing 5 pg of cDNAs.

cells. The upper panel in
Hep G2-Aa, Hep G2-BB, and Hep G2-y cells showed greater transcription of all three fibrinogen genes than Hep GB-Neo cells. In Hep G2-Aa and Hep G2-y the increase was nearly 4fold, and in Hep G2-BB it was greatest, about 7-fold ( Fig. 6 and Table I).

DISCUSSION
Hepatocytes of different species have unequal amounts of the three fibrinogen chains. Because in Hep G2 cells BB chains occur in limiting amounts we postulated that synthesis of BP is a rate-limiting factor in the production of fibrinogen (12,13). Indeed, increasing BP chain synthesis by transfection of Hep G2 cells with B/3 chain cDNA led to increased synthesis of fibrinogen without using the surplus pools of Aa and y chains (22). We now show that increased expression of any fibrinogen chain, elicited by transfection with vectors containing individual fibrinogen chain cDNAs, specifically upregulates the expression of the other two chains. Thus the expression of all three fibrinogen genes is tightly linked, and our results suggest that there are probably feedback mechanisms that maintain a steady intracellular proportion of unequal amounts of fibrinogen chains, which apparently are needed for fibrinogen chain assembly. It is of interest to note that the expression of the variant, C terminus-extended A a chain (29) is also up-regulated indicating that it is under similar control as normal Act chains.
The regulation of fibrinogen chain production occurs at the transcriptional level, as shown by nuclear run-on assays. Increased transcription leads to higher levels of all three fibrinogen mRNAs and to increased initial rates of synthesis for all three chains, irrespective of which fibrinogen cDNA was transfected into Hep G2 cells. However transfection with BB cDNA had a greater effect than did transfection with A a or y cDNA. This may reflect the fact that BB does not accumulate in Hep G2 cells, but A a and y chains are maintained in surplus amounts. Therefore fibrinogen expression may be more sensitive to small changes in the intracellular levels of Bg.
The mechanism by which transfection with any one fibrinogen chain cDNA up-regulates the expression of the other two is not yet understood. Transfection with any fibrinogen cDNA causes an increase in both fibrinogen mRNA levels and in the amount of fibrinogen chains synthesized. Both fibrinogen mRNA and the individual chains are present in the cell cytoplasm and presumably exert, indirectly, a feedback effect that up-regulates the expression of the other two chains. At present we do not know whether it is the increased levels of a fibrinogen chain or of RNA that triggers the events leading to the up-regulation of the three fibrinogen genes. If individual chains are responsible then events that initially occur within the lumen of the endoplasmic reticulum must trigger increased transcription of fibrinogen genes, which ultimately occur in the nucleus. There are examples of autoregulation of gene expression by proteins present in the cytoplasm. For example the under-or overexpression of some heat shock proteins regulates the expression of a family of heat shock genes (30,31).
During the acute-phase reaction the synthesis of fibrinogen increases together with a subset of other plasma proteins. Expression of acute-phase proteins is enhanced by glucocorticoids and by interleukin-6 (32-34). The upstream regions of the BP fibrinogen gene, which respond to dexamethasone and interleukin-6, have been identified (34)(35)(36). However the factor(s) that enhance fibrinogen expression in Hep G2-Act, Hep G2-B/3, and Hep G2-y cells must be different from interleukin-6 or glucocorticoids, because the expression of acutephase proteins, a1-antichymotrypsin and C-reactive protein, was not affected. There may be some unknown factor(s) that specifically regulates the three fibrinogen genes, and over expression of any fibrinogen chain may activate these fibrinogen-specific factors. Although the mechanism is unknown our results show that fibrinogen gene expression is regulated at two different levels. One mechanism enhances fibrinogen gene expression together with that of other acute-phase proteins, and at a second level the fibrinogen genes are independently regulated.