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Fibrinogen and albumin synthesis are regulated at the transcriptional level during the acute phase response
450
Biochimica et Biophysica Acta, 950 (1988) 450-454
Elsevier BBA 90116
BBA Report
F i b r i n o g e n and a l b u m i n s y n t h e s i s are regulated at the transcriptional level during the acute p h a s e r e s p o n s e H a n J. M o s h a g e , B e r n a r d E . M . K l e t e r , J o s F. v a n P e l t , H e n n i e M . J . R o e l o f s , Jos A.G.M. Kleuskens and Sing Hiem Yap Division of Gastrointestinal and Liver Diseases, Department of Medicine, St. Radboud University Hospital, Nijmegen (The Netherlands)
(Received 3 May 1988)
Key words: Fibrinogensynthesis; Albumin synthesis; Transcriptionalregulation; Acute phase response; (Rat liver nucleus)
During acute inflammation or after administration of monocytic products, an enhanced transcription of the fibrinogen polypeptide genes and a reduced transcription of the albumin gene were observed. The changes in the fibrinogen polypeptide transcriptional rate were found to precede the change in albumin gene transcription. These findings indicate that the altered synthesis of fibrinogen and albumin during inflammation are regulated at the transcriptional level and are most probably mediated by monocytic products (including interleukin-l)
During the acute phase response, the plasma concentration of fibrinogen is dramatically increased, whereas the plasma concentration of albumin is decreased [1,2]. Previously, we have shown that the increased synthesis of fibrinogen and the decreased synthesis of albumin during acute inflammation or after intraperitoneal administration of monocytic products can be attributed to changes in cytoplasmic m R N A levels coding for these proteins [3,4]. Cytoplasmic m R N A levels in eukaryotic cells can be regulated at the level of transcription or by posttranscriptional mechanisms, such as differential processing of hnRNA, nucleocytoplasmic transport of processed mRNAs
Abbreviations: PMSF, phenylmethylsulfonylfluoride; DTT, dithiothreitol. Correspondence: S.H. Yap, Division of Gastrointestinal and Liver Diseases, Department of Medicine, St. Radboud University Hospital, 6500 HB Nijmegen, The Netherlands.
or changes in cytoplasmic m R N A stability (turnover rates). Determination of cytoplasmic m R N A levels alone, therefore, does not distinguish between these possibilities. To examine the possible changes in synthesis of mRNAs coding for fibrinogen polypeptides and albumin during inflammation or after administration of monocytic products, we determined the rate of transcription of the genes coding for these proteins using in vitro transcription assays of isolated rat liver nuclei. For these experiments, male Wistar rats (180-200 g) were used and maintained on Purina chow and water ad libitum. Acute inflammation was induced by turpentine injection (1.0 ml, intramuscularly). Monocytic products were prepared by incubation of activated macrophages, isolated from peritoneal exudates induced by glycogen infusion in rabbits [5] and administered intraperitoneally (3 mg). These preparations contained high levels of interleukin-1 activity in the
0167-4781/88/$03.50 © 1988 ElsevierScience Publishers B.V. (Biomedical Division)
451
thymocyte proliferation assay [6] and no endotoxin in a Limulus assay. Liver nuclei were isolated according to the method of Marzluff and Huang [7]. Livers were perfused in situ with a solution containing 0.14 M NaC1 (0.3 m g / m l heparin) and 10 mM Tris-HC1 (pH 8.0), homogenized in a buffer comprising 0.32 M sucrose/3.0 mM CaC12/2.0 mM magnesium acetate/0.1 mM E D T A / 0 . 1 mM P M S F / 1 . 0 mM D T T / 0 . 1 % Triton X-100/10 mM Tris-HC1 (pH 8.0), diluted with 2 vol. of a buffer comprising 2.0 M sucrose/50 mM magnesium acetate/0.1 mM E D T A / 0 . 1 mM P M S F / 1 . 0 mM D T T / 1 0 mM Tris-HC1 (pH 8.0), layered on a 2.0 M sucrose cushion in the same buffer and centrifuged for 1 h at 30000 × g. The pellet was resuspended in a buffer comprising 25% glycerol/5.0 mM magnesium a c e t a t e / 0 . 1 m M E D T A / 0 . 1 mM P M S F / 5 . 0 mM D T T / 5 0 mM Tris-HC1 (pH 8.0), centrifuged for 1 h at 30 000 × g and resuspended in the same buffer. All procedures were carried out at 4° C. In vitro transcription experiments were performed as described by Marzluff and Huang [7]. 5 0 . 1 0 6 nuclei were incubated in 800 /zl of a solution comprising 0.5 mM A T P / 0 . 5 mM C T P / 0 . 5 mM U T P / 0 . 1 2 M KCI/2.6 mM magnesium acetate/2.5 m g / m l creatine phosphate/0.6 m g / m l creatine kinase/80 #Ci guanosine 5'-[a32P]triphosphate for 30 min at 30 ° C. The reaction was stopped by addition of 10 vol. of a solution comprising 0.1% S D S / 5 0 mM EDTA (pH 7.0). R N A was extracted, precipitated and purified as described by Marzluff and Huang [7] and finally dissolved in 400 /zl 10 mM Tris-HC1 (pH 6.9). H y b o n d nylon filters, containing 3 /tg recombinant DNA per spot were prehybridized with 0.25 M sodium phosphate/7% SDS/1 mM EDTA/0.1% bovine serum albumin for 30 min at 65°C. Filters were hybridized with 32p-labeled R N A transcripts in the same solution for 48 h at 65°C. After hybridization, filters were washed four times 20 min at 7 0 ° C with 1.5 ml of a solution composed of 1% SDS, 1 mM E D T A (pH 7.0) and, respectively, 0.25 M, 0.125 M, 0.05 M and 0.025 M sodium phosphate, and hybridized material was eluted with a solution containing 98% formamide, 0.2% SDS and 10 / l g / m l E. coli tRNA and assayed for radioactivity.
pBR 322 plasmids containing cDNAs coding for the a, fl and ~, chains of rat fibrinogen were a kind gift from Dr. G.R. Crabtree (Laboratory of Pathology, NIH, Bethesda, MD) and described previously [8]. pBR 322 plasmid containing rat albumin c D N A has been described previously [9]. Plasmid DNAs were immobilized on Hybond-N nylon filters according to the manufacturer's manual. Guanosine 5'-[a-32p]triphosphate (410 C i / mmol), [6-3H]thymidine (22 C i / m m o l ) and Hybond-N nylon membranes were obtained from Amersham International, Amersham, ATP, CTP, UTP, GTP, creatine phosphate, creatine kinase, bovine serum albumin and E. coli tRNA were from Boehringer Mannheim; heparin from porcine intestinal mucosa and D T T were from Sigma. Triton X-100 was from BDH Chemicals. Formamide was purchased from J.T. Baker Chemicals. Ribonuclease-free sucrose, EDTA, salts and solvents were from E. Merck. SDS was obtained from Bio-Rad Laboratories. Using an experimental model of inflammation in rats, we have previously shown that the plasma concentration and hepatic m R N A content of fibrinogen are markedly increased [4]. In contrast, the serum concentration and hepatic m R N A content of albumin are decreased [3]. To determine whether the changes in hepatic m R N A content under these circumstances are the result of corresponding changes in transcriptional rates of these genes or the result of posttranscriptional regulatory mechanisms, we performed in vitro transcription experiments using isolated rat liver nuclei. Incorporation of radioactivity was linear up to 35-40 min incubation at 3 0 ° C (data not shown). The results, presented in Table I (and Fig. 1), demonstrate that transcription of the fibrinogen polypeptide (a, r , y) genes was dramatically increased after in vivo administration of turpentine. The maximal induction was found between 3 and 6 h, and transcriptional rates were still significantly elevated 9 h after turpentine treatment. In contrast, transcription of the albumin gene was 80% of the control value 3 h after turpentine treatment and was still further depressed 6 and 9 h (approx. 20% of control value) after turpentine treatment. Numerous studies have demonstrated that
452
TABLE I RELATIVE TRANSCRIPTIONAL RATES OF FIBRINOGEN POLYPEPTIDE AND ALBUMIN GENES IN LIVER NUCLEI ISOLATED FROM RATS AFTER TURPENTINE TREATMENT Data are means 5: S.D. of three experiments. Control values minus background (dpm) Albumin Fibrinogen a-chain Fibrinogen fl-chain Fibrinogen y-chain Background (pBR 322)
Relative transcriptional rates (% of control) Time (h): 3
639_+124 209 _+ 38 209 _+ 29 240-+ 41 47_+ 21
80_+ 12 885 _+142 856 _+104 705 -+100
m o n o c y t i c products, including interleukin-1, are i m p o r t a n t mediators for the acute phase response [2,10]. Previously, we have also shown that m o n o cytic products, containing high levels of interleukin-1 activity, increase synthesis and m R N A content of fibrinogen and decrease synthesis and m R N A content of albumin both in vivo and in vitro (Ref. 4 and unpublished observations). Table II shows that the changes in transcriptional rates of the fibrinogen polypeptide and albumin genes after administration of monocytic p r o d u c t s are similar to the changes after turpentine treatment. Furthermore, these results are in accordance with previous findings (Refs. 3, 4 and
I
1
2
3
Li
5
Fig. l. Autoradiogram of 32p-labeled transcripts of fibrinogen polypeptide and albumin genes in liver nuclei isolated from rats 9 h after turpentine treatment. 32p-labeled RNA transcripts were hybridized and washed as described in the text and exposed to Kodak XAR-2 film for 24 h at -80 o C. (1) fibrinogen a-chain; (2) fibrinogen fl-chain; (3) fibrinogen ~/chain; (4) albumin; (5) nonrecombinant pBR 322. Upper row, control; lower row, after turpentine treatment.
6
9
21_+ 8 779 _+118 769 _+124 727 -+109
18_+ 10 508 _+117 506 _+10l 453 -+ 89
unpublished observations): after turpentine treatment, fibrinogen polypeptide m R N A content of rat liver rises at 6 h and is maximally elevated at 24 h, and is still above control values at 48 h. Likewise, the fibrinogen plasma concentration increases at 12 h, reaching a m a x i m u m at 36 h and is still elevated at 72 h. In contrast, albumin m R N A content declines at 9 h and is minimal at 12 h, and is still reduced at 72 h. However, the serum albumin concentration is only moderately depressed, p r o b a b l y as a consequence of the relatively long half-life of albumin in the circulation. As shown in Tables I and II, the transcriptional rates of the different fibrinogen polypeptide genes are increased to the same extent at all times after induction. These findings indicate that a c o m m o n mechanism regulated the transcriptional rate of these three different genes, as has also been reported for the increase of fibrinogen polypeptide m R N A s , following defibrination with snake ven o m [11]. M o d u l a t i o n of protein synthesis as a consequence of changes at the transcriptional level has been observed for h o r m o n a l l y regulated, developmentally regulated and tissue-specific genes [12-15]. Transcriptional regulation of acute phase proteins has been shown for C-reactive protein, c o m p l e m e n t factor 3 and serum amyloid A protein during inflammation in rabbits [16], and also for al-major acute phase proteins, transferrin, a 2globulin, al-acid glycoprotein and a2-macroglobulin [17], although these authors do not exclude possible additional p o s t t r a n s c r i p t i o n a l mechanisms in the regulation of the latter two proteins. Regulation of protein synthesis at the
453 TABLE II RELATIVE TRANSCRIPTIONAL RATES OF FIBRINOGEN POLYPEPTIDE AND ALBUMIN GENES IN ISOLATED RAT LIER NUCLEI 9 h AFTER ADMINISTRATION OF MONOCYTIC PRODUCTS (3 mg) Data are means+ S.D. of three experiments. Controlv~ues minus background (dpm) Albumin Fibrinogen a-chain Fibrinogen fl-chain Fibrinogen ~-chain Background (pBR 322)
704+ 208 + 237 + 314+ 41 +
132 42 36 49 22
posttranscriptional level has been observed for vitellogenin [18], growth hormone [19] and the liver-specific proteins, albumin and a~-antitrypsin [20]. In these examples, cytoplasmic mRNA stability is regulated by hormones. In this respect, it is interesting to note that Piccoletti et al. [21] observed an increased synthesis of pre-ribosomal RNA and pre-mRNA and an increased activity of cytoplasmic-soluble factors participating in translation of RNA during inflammation, suggesting that both transcriptional and posttranscriptional regulatory mechanisms operate in the regulation of acute phase protein synthesis. In addition, these authors also demonstrated an increased methylation and nucleocytoplasmic transport of ribosomal RNA in rats during inflammation, again indicating the importance of posttranscriptional regulatory mechanisms [22]. Our results indicate that the changes in fibrinogen polypeptide and albumin mRNA content in the liver during inflammation are predominantly regulated at the transcriptional level, although additional posttranscriptional regulatory mechanisms affecting nucleocytoplasmic transport of processed mRNA and ribosomal RNA and changes in mRNA translation and stability might also be involved [21,22]. Both magnitude and time-course of the changes in transcriptional rates are in good agreement with the observed changes in the hepatic mRNA content coding for fibrinogen polypeptides and albumin [3,4]. From these findings, we can therefore conclude that the synthesis of fibrinogen and albumin during the acute phase response are predominantly regulated at the transcriptional level.
Relative transcriptional rates (% of control) Time (h):
6
9
12
68 + 18 220 + 47 242 + 39 285 + 53
47 + 12 576 + 101 527 + 87 489 5 : 9 2
44 +_13 248 + 51 207 + 39 283 + 62
In addition, monocytic products, containing high levels of interleukin-1 activity, lead to similar changes in transcriptional rates, indicating the importance of these monocytic products in the mediation of the acute phase response. This investigation has been supported in part by the Foundation for Medical and Health Research MEDIGON (grant No. 900-523-073).
References 1 Kushner, I. (1982) Ann. NY Acad. Sci. 389, 39-48. 2 Bornstein, D.L. (1982) Ann. NY Acad. Sci. 389, 323-337. 3 Moshage, H.J., Janssen, J.A.M., Franssen, J.H., Hafkenscheid, J.C.M. and Yap, S.H. (1987) J. Clin. Invest. 79, 1635-1641. 4 Princen, H.M.G., Nieuwenhuizen, W., Mol-Backx, G.P.B.M. and Yap, S.H. (1981) Biochem. Biophys. Res. Commun. 102, 717-723. 5 Ritchie, D.G. and Fuller, G.M. (1981) Inflammation 5, 275-287. 6 Gery, I., Gershon, R.K. and Waksman, B.H. (1972) J. Exp. Med. 136, 128-142. 7 Marzluff, W.F. and Huang, R.C.C. (1984) Transcription and Translation: A Practical Approach (Hames, B.D. and Higgins, S.J., eds.), pp. 89-130, IRL Press, Oxford. 8 Crabtree, G.R. and Kant, J.A. (1981) J. Biol. Chem. 256, 9718-9723. 9 Zern, M.A., Chakraborty, P.R., Ruiz-Opazo, N., Yap, S.H. and Shafritz, D.A. (1983) Hepatology 3, 317-322. 10 Dinarello, C.A. (1984) Rev. Inf. Dis. 6, 51-95. 11 Crabtree, G.R. and Kant, J.A. (1982) J. Biol. Chem. 257, 7277-7299. 12 Kulkarni, A.B., Gubits, R.M. and Feigelson, P. (1985) Proc. Natl. Acad. Sci. USA 82, 2579-2582. 13 Mueckler, M.M., Moran, S. and Pitot, H.C. (1984) J. Biol. Chem. 259, 2302-2305.
454 14 Grouidine, M., Peretz, M. and Weintraub, H. (1981) Mol. Cell. Biol. 1,281-288. 15 Derman, E., Krauter, K., Walling, L., Weinberger, C., Ray, M. and Darnell, J.E. (1981) Cell, 23, 731-739. 16 Goldberger, G., Bing, D.H., Sipe, J.D., Rits, M. and Colten, H.R. (1987) J. Immunol. 138, 3967-3971. 17 Birch, H.E. and Schreiber, G. (1986) J. Biol. Chem. 261, 8077-8080. 18 Brock, M.L. and Shapiro, D.J. (1983) Cell 34, 207-214.
19 Diamond, D.J. and Goodman, H.M. (1985) J. Mol. Biol. 181, 41-62. 20 Jefferson, D.M., Clayton, D.F. and Darnell, J.E., Reid, L.M. (1984) Mol. Cell. Biol. 4, 1929-1934. 21 Piccoletti, R., Aletti, M.G., Cajone, F. and Bernelli-Zazzera, A. (1984) Br. J. Exp. Pathol. 65, 419-430. 22 Piccoletti, R., Aletti, M.G. and Bernelli-Zazzera, A. (1986) Inflammation 10, 109-117.
Biochimica et Biophysica Acta, 950 (1988) 450-454
Elsevier BBA 90116
BBA Report
F i b r i n o g e n and a l b u m i n s y n t h e s i s are regulated at the transcriptional level during the acute p h a s e r e s p o n s e H a n J. M o s h a g e , B e r n a r d E . M . K l e t e r , J o s F. v a n P e l t , H e n n i e M . J . R o e l o f s , Jos A.G.M. Kleuskens and Sing Hiem Yap Division of Gastrointestinal and Liver Diseases, Department of Medicine, St. Radboud University Hospital, Nijmegen (The Netherlands)
(Received 3 May 1988)
Key words: Fibrinogensynthesis; Albumin synthesis; Transcriptionalregulation; Acute phase response; (Rat liver nucleus)
During acute inflammation or after administration of monocytic products, an enhanced transcription of the fibrinogen polypeptide genes and a reduced transcription of the albumin gene were observed. The changes in the fibrinogen polypeptide transcriptional rate were found to precede the change in albumin gene transcription. These findings indicate that the altered synthesis of fibrinogen and albumin during inflammation are regulated at the transcriptional level and are most probably mediated by monocytic products (including interleukin-l)
During the acute phase response, the plasma concentration of fibrinogen is dramatically increased, whereas the plasma concentration of albumin is decreased [1,2]. Previously, we have shown that the increased synthesis of fibrinogen and the decreased synthesis of albumin during acute inflammation or after intraperitoneal administration of monocytic products can be attributed to changes in cytoplasmic m R N A levels coding for these proteins [3,4]. Cytoplasmic m R N A levels in eukaryotic cells can be regulated at the level of transcription or by posttranscriptional mechanisms, such as differential processing of hnRNA, nucleocytoplasmic transport of processed mRNAs
Abbreviations: PMSF, phenylmethylsulfonylfluoride; DTT, dithiothreitol. Correspondence: S.H. Yap, Division of Gastrointestinal and Liver Diseases, Department of Medicine, St. Radboud University Hospital, 6500 HB Nijmegen, The Netherlands.
or changes in cytoplasmic m R N A stability (turnover rates). Determination of cytoplasmic m R N A levels alone, therefore, does not distinguish between these possibilities. To examine the possible changes in synthesis of mRNAs coding for fibrinogen polypeptides and albumin during inflammation or after administration of monocytic products, we determined the rate of transcription of the genes coding for these proteins using in vitro transcription assays of isolated rat liver nuclei. For these experiments, male Wistar rats (180-200 g) were used and maintained on Purina chow and water ad libitum. Acute inflammation was induced by turpentine injection (1.0 ml, intramuscularly). Monocytic products were prepared by incubation of activated macrophages, isolated from peritoneal exudates induced by glycogen infusion in rabbits [5] and administered intraperitoneally (3 mg). These preparations contained high levels of interleukin-1 activity in the
0167-4781/88/$03.50 © 1988 ElsevierScience Publishers B.V. (Biomedical Division)
451
thymocyte proliferation assay [6] and no endotoxin in a Limulus assay. Liver nuclei were isolated according to the method of Marzluff and Huang [7]. Livers were perfused in situ with a solution containing 0.14 M NaC1 (0.3 m g / m l heparin) and 10 mM Tris-HC1 (pH 8.0), homogenized in a buffer comprising 0.32 M sucrose/3.0 mM CaC12/2.0 mM magnesium acetate/0.1 mM E D T A / 0 . 1 mM P M S F / 1 . 0 mM D T T / 0 . 1 % Triton X-100/10 mM Tris-HC1 (pH 8.0), diluted with 2 vol. of a buffer comprising 2.0 M sucrose/50 mM magnesium acetate/0.1 mM E D T A / 0 . 1 mM P M S F / 1 . 0 mM D T T / 1 0 mM Tris-HC1 (pH 8.0), layered on a 2.0 M sucrose cushion in the same buffer and centrifuged for 1 h at 30000 × g. The pellet was resuspended in a buffer comprising 25% glycerol/5.0 mM magnesium a c e t a t e / 0 . 1 m M E D T A / 0 . 1 mM P M S F / 5 . 0 mM D T T / 5 0 mM Tris-HC1 (pH 8.0), centrifuged for 1 h at 30 000 × g and resuspended in the same buffer. All procedures were carried out at 4° C. In vitro transcription experiments were performed as described by Marzluff and Huang [7]. 5 0 . 1 0 6 nuclei were incubated in 800 /zl of a solution comprising 0.5 mM A T P / 0 . 5 mM C T P / 0 . 5 mM U T P / 0 . 1 2 M KCI/2.6 mM magnesium acetate/2.5 m g / m l creatine phosphate/0.6 m g / m l creatine kinase/80 #Ci guanosine 5'-[a32P]triphosphate for 30 min at 30 ° C. The reaction was stopped by addition of 10 vol. of a solution comprising 0.1% S D S / 5 0 mM EDTA (pH 7.0). R N A was extracted, precipitated and purified as described by Marzluff and Huang [7] and finally dissolved in 400 /zl 10 mM Tris-HC1 (pH 6.9). H y b o n d nylon filters, containing 3 /tg recombinant DNA per spot were prehybridized with 0.25 M sodium phosphate/7% SDS/1 mM EDTA/0.1% bovine serum albumin for 30 min at 65°C. Filters were hybridized with 32p-labeled R N A transcripts in the same solution for 48 h at 65°C. After hybridization, filters were washed four times 20 min at 7 0 ° C with 1.5 ml of a solution composed of 1% SDS, 1 mM E D T A (pH 7.0) and, respectively, 0.25 M, 0.125 M, 0.05 M and 0.025 M sodium phosphate, and hybridized material was eluted with a solution containing 98% formamide, 0.2% SDS and 10 / l g / m l E. coli tRNA and assayed for radioactivity.
pBR 322 plasmids containing cDNAs coding for the a, fl and ~, chains of rat fibrinogen were a kind gift from Dr. G.R. Crabtree (Laboratory of Pathology, NIH, Bethesda, MD) and described previously [8]. pBR 322 plasmid containing rat albumin c D N A has been described previously [9]. Plasmid DNAs were immobilized on Hybond-N nylon filters according to the manufacturer's manual. Guanosine 5'-[a-32p]triphosphate (410 C i / mmol), [6-3H]thymidine (22 C i / m m o l ) and Hybond-N nylon membranes were obtained from Amersham International, Amersham, ATP, CTP, UTP, GTP, creatine phosphate, creatine kinase, bovine serum albumin and E. coli tRNA were from Boehringer Mannheim; heparin from porcine intestinal mucosa and D T T were from Sigma. Triton X-100 was from BDH Chemicals. Formamide was purchased from J.T. Baker Chemicals. Ribonuclease-free sucrose, EDTA, salts and solvents were from E. Merck. SDS was obtained from Bio-Rad Laboratories. Using an experimental model of inflammation in rats, we have previously shown that the plasma concentration and hepatic m R N A content of fibrinogen are markedly increased [4]. In contrast, the serum concentration and hepatic m R N A content of albumin are decreased [3]. To determine whether the changes in hepatic m R N A content under these circumstances are the result of corresponding changes in transcriptional rates of these genes or the result of posttranscriptional regulatory mechanisms, we performed in vitro transcription experiments using isolated rat liver nuclei. Incorporation of radioactivity was linear up to 35-40 min incubation at 3 0 ° C (data not shown). The results, presented in Table I (and Fig. 1), demonstrate that transcription of the fibrinogen polypeptide (a, r , y) genes was dramatically increased after in vivo administration of turpentine. The maximal induction was found between 3 and 6 h, and transcriptional rates were still significantly elevated 9 h after turpentine treatment. In contrast, transcription of the albumin gene was 80% of the control value 3 h after turpentine treatment and was still further depressed 6 and 9 h (approx. 20% of control value) after turpentine treatment. Numerous studies have demonstrated that
452
TABLE I RELATIVE TRANSCRIPTIONAL RATES OF FIBRINOGEN POLYPEPTIDE AND ALBUMIN GENES IN LIVER NUCLEI ISOLATED FROM RATS AFTER TURPENTINE TREATMENT Data are means 5: S.D. of three experiments. Control values minus background (dpm) Albumin Fibrinogen a-chain Fibrinogen fl-chain Fibrinogen y-chain Background (pBR 322)
Relative transcriptional rates (% of control) Time (h): 3
639_+124 209 _+ 38 209 _+ 29 240-+ 41 47_+ 21
80_+ 12 885 _+142 856 _+104 705 -+100
m o n o c y t i c products, including interleukin-1, are i m p o r t a n t mediators for the acute phase response [2,10]. Previously, we have also shown that m o n o cytic products, containing high levels of interleukin-1 activity, increase synthesis and m R N A content of fibrinogen and decrease synthesis and m R N A content of albumin both in vivo and in vitro (Ref. 4 and unpublished observations). Table II shows that the changes in transcriptional rates of the fibrinogen polypeptide and albumin genes after administration of monocytic p r o d u c t s are similar to the changes after turpentine treatment. Furthermore, these results are in accordance with previous findings (Refs. 3, 4 and
I
1
2
3
Li
5
Fig. l. Autoradiogram of 32p-labeled transcripts of fibrinogen polypeptide and albumin genes in liver nuclei isolated from rats 9 h after turpentine treatment. 32p-labeled RNA transcripts were hybridized and washed as described in the text and exposed to Kodak XAR-2 film for 24 h at -80 o C. (1) fibrinogen a-chain; (2) fibrinogen fl-chain; (3) fibrinogen ~/chain; (4) albumin; (5) nonrecombinant pBR 322. Upper row, control; lower row, after turpentine treatment.
6
9
21_+ 8 779 _+118 769 _+124 727 -+109
18_+ 10 508 _+117 506 _+10l 453 -+ 89
unpublished observations): after turpentine treatment, fibrinogen polypeptide m R N A content of rat liver rises at 6 h and is maximally elevated at 24 h, and is still above control values at 48 h. Likewise, the fibrinogen plasma concentration increases at 12 h, reaching a m a x i m u m at 36 h and is still elevated at 72 h. In contrast, albumin m R N A content declines at 9 h and is minimal at 12 h, and is still reduced at 72 h. However, the serum albumin concentration is only moderately depressed, p r o b a b l y as a consequence of the relatively long half-life of albumin in the circulation. As shown in Tables I and II, the transcriptional rates of the different fibrinogen polypeptide genes are increased to the same extent at all times after induction. These findings indicate that a c o m m o n mechanism regulated the transcriptional rate of these three different genes, as has also been reported for the increase of fibrinogen polypeptide m R N A s , following defibrination with snake ven o m [11]. M o d u l a t i o n of protein synthesis as a consequence of changes at the transcriptional level has been observed for h o r m o n a l l y regulated, developmentally regulated and tissue-specific genes [12-15]. Transcriptional regulation of acute phase proteins has been shown for C-reactive protein, c o m p l e m e n t factor 3 and serum amyloid A protein during inflammation in rabbits [16], and also for al-major acute phase proteins, transferrin, a 2globulin, al-acid glycoprotein and a2-macroglobulin [17], although these authors do not exclude possible additional p o s t t r a n s c r i p t i o n a l mechanisms in the regulation of the latter two proteins. Regulation of protein synthesis at the
453 TABLE II RELATIVE TRANSCRIPTIONAL RATES OF FIBRINOGEN POLYPEPTIDE AND ALBUMIN GENES IN ISOLATED RAT LIER NUCLEI 9 h AFTER ADMINISTRATION OF MONOCYTIC PRODUCTS (3 mg) Data are means+ S.D. of three experiments. Controlv~ues minus background (dpm) Albumin Fibrinogen a-chain Fibrinogen fl-chain Fibrinogen ~-chain Background (pBR 322)
704+ 208 + 237 + 314+ 41 +
132 42 36 49 22
posttranscriptional level has been observed for vitellogenin [18], growth hormone [19] and the liver-specific proteins, albumin and a~-antitrypsin [20]. In these examples, cytoplasmic mRNA stability is regulated by hormones. In this respect, it is interesting to note that Piccoletti et al. [21] observed an increased synthesis of pre-ribosomal RNA and pre-mRNA and an increased activity of cytoplasmic-soluble factors participating in translation of RNA during inflammation, suggesting that both transcriptional and posttranscriptional regulatory mechanisms operate in the regulation of acute phase protein synthesis. In addition, these authors also demonstrated an increased methylation and nucleocytoplasmic transport of ribosomal RNA in rats during inflammation, again indicating the importance of posttranscriptional regulatory mechanisms [22]. Our results indicate that the changes in fibrinogen polypeptide and albumin mRNA content in the liver during inflammation are predominantly regulated at the transcriptional level, although additional posttranscriptional regulatory mechanisms affecting nucleocytoplasmic transport of processed mRNA and ribosomal RNA and changes in mRNA translation and stability might also be involved [21,22]. Both magnitude and time-course of the changes in transcriptional rates are in good agreement with the observed changes in the hepatic mRNA content coding for fibrinogen polypeptides and albumin [3,4]. From these findings, we can therefore conclude that the synthesis of fibrinogen and albumin during the acute phase response are predominantly regulated at the transcriptional level.
Relative transcriptional rates (% of control) Time (h):
6
9
12
68 + 18 220 + 47 242 + 39 285 + 53
47 + 12 576 + 101 527 + 87 489 5 : 9 2
44 +_13 248 + 51 207 + 39 283 + 62
In addition, monocytic products, containing high levels of interleukin-1 activity, lead to similar changes in transcriptional rates, indicating the importance of these monocytic products in the mediation of the acute phase response. This investigation has been supported in part by the Foundation for Medical and Health Research MEDIGON (grant No. 900-523-073).
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