Secondary Logo

Journal Logo

Pancreas and Liver

Inhibition of Albumin Synthesis in Chronic Diseases

Molecular Mechanisms

Chojkier, Mario MD

Author Information
Journal of Clinical Gastroenterology: April 2005 - Volume 39 - Issue 4 - p S143-S146
doi: 10.1097/01.mcg.0000155514.17715.39
  • Free

Abstract

Gene expression can be modulated at five different levels: 1) transcription of DNA into primary RNA transcripts, 2) RNA processing into mature mRNA, 3) translation of the mRNA into protein, 4) posttranslational modifications of the protein, and 5) regulation of protein degradation.14 However, liver-specific genes, such as albumin, are usually regulated at the level of transcription.21,47,72 In the regulation of transcription, it takes more than “two to tango.” This choreography calls for dynamic interaction among transcription proteins literally dancing on DNA regulatory sequences. There are general transcription factors (eg, c-Jun, NFκB, TFIID) and specialized transcription factors (C/EBP-α, HNF1, HNF4, C/EBP-β, DBP). These specialized regulatory proteins occupy “center stage” sequences within enhancer and/or promoter regions of the DNA and induce cell-specific functions. The critical DNA regulatory sequences have been identified in many liver-specific genes.17,29,32,34,35,37,42,45-49,57,58,70

The albumin promoter is composed of a TATA motif and six upstream binding sites (A-F) for nuclear proteins.37 Two of these elements, B and D, are particularly important for the efficient liver-specific in vitro transcription from the albumin promoter (Fig. 1). Substitution of either one of these cis-acting elements with unrelated DNA dramatically reduces the transcriptional activity of the albumin promoter in liver nuclear extracts.39 Element B is a high-affinity site for the well-characterized transcription factor HNF1, a protein that binds to promoter elements of many liver-specific genes.11,18,24,36 Site D can be recognized by liver-enriched factors (C/EBP-α, DBP, and C/EBP-β) that are heat-resistant and relatively basic, due to the amino acid composition of the DNA binding domain (Fig. 1). The albumin gene is expressed specifically in the liver after birth, and this expression is regulated predominantly at the level of transcription.44,59 An upstream cis-acting element of the albumin gene (−8.5 kb to −10.4 kb), the enhancer, is sufficient to promote liver-specific expression from the albumin promoter in transgenic animals.46

FIGURE 1
FIGURE 1:
Regulatory sequences of the albumin gene. Schematic representation of the critical cis-acting elements of the albumin gene enhancer and promoter. The binding sites for the liver-specific transcription factors are indicated. The arrow indicates start of transcription of the introns and exons, which produces albumin mRNA. (Modified with permission.14)

Albumin is the most abundant protein in plasma, and the colloid pressure of plasma is maintained principally by the levels of circulating albumin.67 Albumin also performs important metabolic functions in the transport of free fatty acids, bilirubin, and many drugs.14,67 In a normal individual, approximately 15 g of albumin are synthesized daily by hepatocytes to maintain the albumin plasma steady state concentration (∼4 g/100 mL).67 Therefore, decreased albumin synthesis results in hypoalbuminemia, which facilitates excessive transudation of fluids into extravascular spaces (edema and ascites).5 Hypoalbuminemia is a frequent feature of cachectic patients afflicted with chronic diseases,61 including cancer, AIDS, and inflammatory disorders, and a major contributor to their morbidity.4,28,50,64 There is strong evidence to suggest that tumor necrosis factor-α (TNF-α) is a critical mediator,13,23,71 in concert with other cytokines,28,54,56,60 of cachexia of chronic diseases. Over the last several years, some of the molecular mechanisms responsible for hypoalbuminemia of chronic diseases have been elucidated.

Because the TNF-α mouse model of cachexia closely resembles human cachexia,6,43,62 it provides a valuable system to analyze the molecular mechanisms responsible for inhibition of albumin synthesis. The TNF-α serum levels in TNF-α mice are only moderately increased (100-300 pg/mL) at the onset of weight loss,8 but at the time of death, values are similar to those found in patients with trauma or infectious, parasitic, and neoplastic diseases.25,26,53,65

The presence of MDA-protein adducts in the livers of TNF-α mice, indicate activation of an oxidative pathway.12,31 These findings are in agreement with evidence that TNF-α can stimulate oxidative stress in many cells and tissues.51,69 In addition, nitric oxide synthase (NOS)2 expression was markedly induced in the livers of TNF-α mice. This effect was rescued by treating these animals with the antioxidants D-α-tocopherol or BW755c, indicating that the liver induction of NOS2 in TNF-α mice is mediated by an enhanced oxidative stress.7 In addition, in primary mouse hepatocytes, SIN-1, a nitric oxide (NO) donor, was sufficient to induce the phosphorylation of C/EBP-β on Ser239 within the nuclear localization signal (NLS), and its nuclear export.7 NO plays an important role in redox signaling by interacting with superoxide to generate peroxynitrite38,55 and by nitrosylation of mitochondrial complex I.16 NOS expression is markedly increased in both the liver of patients with chronic viral hepatitis and hepatoma cells transfected with the hepatitis B virus cDNA.40 Therefore, NO may also mediate the inhibition of albumin expression in chronic viral hepatitis. In addition, wild-type p53 and tumor-derived p53 mutants can repress C/EBPβ-mediated transactivation of the albumin promoter.33

The phosphorylation of C/EBPβ on Ser239 and its cytoplasmic localization, as well as the decreased albumin gene expression in TNF-α mice, were reversed by treating these animals with antioxidants (D-α-tocopherol or BW755c) or a NOS inhibitor (nitro-L-arginine),7 indicating that oxidative pathways and activation of NOS are critical for the phosphorylation of C/EBP-β on Ser239 and the inhibition of albumin transcription in TNF-α mice (Fig. 2).6 Neither the supplemental dose of D-α-tocopherol nor the nitro-L-arginine treatment was toxic to control or TNF-α mice8,15 for up to a period of 8 weeks. The effects of antioxidants and nitro-L-arginine were not the spurious result of decreased synthesis of TNF-α, since they affected neither the secretion of biologically active TNF-α by these cells nor the serum levels of TNF-α in TNF-α animals.8 This pathway may include activation of other cytokines such as IL-1β and IL-6,1,20,51 which in turn could contribute to the inhibition of albumin gene expression in cachexia.22,23,54,56 However, LPS administration to mice, another inducer of inflammation, resulted in the increased nuclear expression of C/EBP-β, which remained unphosphorylated on Ser239.7 LPS induces C/EBPβ mRNA, C/EBP-β protein in the nucleus, and C/EBP-β binding activities.2,3

FIGURE 2
FIGURE 2:
Cachexia induces inhibition of albumin gene expression. Induction of TNF-α, oxidative stress, and NO synthesis leads to phosphorylation of C/EBP-β on its nuclear localization signal and its nuclear export. This results in the inhibition of albumin gene expression.

Although binding of the albumin enhancer/promoter D-site14,72 by liver nuclear proteins from TNF-α mice and cachectic patients was substantially decreased, the total hepatocyte expression of C/EBP-β, a major D-site activator protein,2,14,48,63 remained unchanged.7 In highly differentiated, quiescent primary mouse hepatocytes, TNF-α was able to stimulate phosphorylation of endogenous mouse C/EBP-β on Ser239 within its NLS,68 which inhibits C/EBP-β's characteristic nuclear localization in normal hepatocytes9,19 and its binding to cognate DNA sequences necessary for high level transcription from the albumin gene.14,19,39,63 In addition to the neutralization by phosphorylation on Ser239 of the mouse C/EBP-β NLS, C/EBP-β also has a sequence within the leucine zipper domain (L271-SRE-L-ST-L-RN-L) that closely conforms to the consensus leucine-rich nuclear export signal.41 Probably, this putative nuclear export signal becomes available for interaction with the nucleocytoplasmic transporter CREM1 (exportin 1) following the phosphorylation of C/EBP-β's NLS induced by TNF-α or NO. Cells expressing the nonphosphorylatable C/EBP-β-Ala239 mutant were refractory to the inhibitory effects of TNF-α on albumin transcription. As expected, TNF-α did not induce the cytoplasmic localization of the nonphosphorylatable mutant C/EBP-β-Ala239. Nuclear proteins from cells expressing the phosphorylation-mimic C/EBP-β-Asp239 mutant, which like phosphorylated C/EBP-β on Ser239 did not localize to the nucleus, displayed negligible binding to the D-site, and these cells did not transcribe albumin reporter chimeric genes.7 There are other examples of how modification of nucleocytoplasmic transport can regulate gene expression.30,41

Why do TNF-α mice, but not C/EBP-β−/− mice,52 have a decreased expression of albumin? There are two mechanisms that can explain the apparent discrepancy. One would expect other C/EBPs expressed in hepatocytes to substitute for C/EBP-β, but because C/EBP-β-PSer239 has a normal leucine zipper domain, it could act in hepatocytes of TNF-α mice as a dominant negative by forming homodimers and heterodimers with leucine zipper proteins, including other C/EBPs,10,19 and inducing their nuclear export. Moreover, C/EBP-α, which is expressed27 and functions as a transcription factor27,66 normally, under basal conditions, in C/EBP-β−/− mice,52 is also a target of the TNF-α signal transduction pathway, given the presence of the conserved Ser phosphoacceptor in its DNA binding domain.10 Indeed, TNF-α also stimulates the phosphorylation on Ser300 and the nuclear export of C/EBP-α in C/EBP-β−/− hepatocytes.

Preliminary findings in patients with cancer cachexia indicate that the cascade leading to decreased albumin gene expression involves also oxidative stress, NOS2 expression, phosphorylation of C/EBP-β on Ser288 (the human homologue to mouse Ser239), and impaired nuclear localization of C/EBP-β and albumin-binding activities.7 These insights into the mechanisms responsible for the decreased albumin expression in cachexia may lead to novel therapeutic approaches for patients with cancer, AIDS, and chronic inflammatory diseases.

ACKNOWLEDGMENT

The author thanks Lauren de los Santos for the preparation of this manuscript.

REFERENCES

1. Akira S, Hirano T, Taga T, et al. Biology of multifunctional cytokines: IL-6 and related molecules (IL and TNF). FASEB J. 1990;4:2860-2867.
2. Akira S, Isshiki H, Sugita T, et al. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990;9:1897-1906.
3. Alonzi T, Maritano D, Gorgoni B, et al. Essential role of STAT3 in the control of the acute-phase response as revealed by inducible gene activation in the liver. Mol Cell Biol. 2001;21:1621-1632.
4. Beutler B. Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine. New York: Raven Press, 1992.
5. Braunwald E. Edema. In: Isselbacher KJ, Braunwald E, Wilson JD, et al., eds. Harrison's Principles of Internal Medicine. New York: McGraw-Hill, 1994:210-214.
6. Brenner DA, Buck M, Feitelberg SP, et al. Tumor necrosis factor α inhibits albumin gene expression in a murine model of cachexia. J Clin Invest. 1990;85:248-255.
7. Buck M, Zhang L, Hunter T, et al. Nuclear export of phosphorylated C/EBPβ mediates the inhibition of albumin expression by TNFα. EMBO J. 2001;20:6712-6723.
8. Buck M, Chojkier M. Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants. EMBO J. 1996;15:1753-1765.
9. Buck M, Turler H, Chojkier M. LAP (NF-IL6), a tissue-specific transcriptional activator, is an inhibitor of hepatoma cell proliferation. EMBO J. 1994;13:851-860.
10. Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991;5:1538-1552.
11. Cereghini S, Blumenfeld M, Yaniv M. A liver-specific factor essential for albumin transcription differs between differentiated and dedifferentiated rat hepatoma cells. Genes Dev. 1988;2:957-974.
12. Chaudhary AK, Nokubo M, Reddy GR, et al. Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver. Science. 1994;265:1580-1582.
13. Cheng J, Turksen K, Yu QC, et al. Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNFα transgenic mice. Genes Dev. 1992;6:1444-1456.
14. Chojkier M. Regulation of liver-specific gene expression. In: Boyer J, Ockner R, eds. Progress in Liver Diseases. Orlando: Saunders, 1995:37-61.
15. Chojkier M, Houglum K, Lee KS, et al. Long- and short-term D-α-tocopherol supplementation inhibits liver collagen α1(I) gene expression. Am J Physiol. 1998;275:G1480-1485.
16. Clementi E, Brown G, Feelisch M, et al. Persistent inhibition of cell respiration by nitric oxide: crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc Natl Acad Sci USA. 1998;95:7631-7636.
17. Costa RH, Lai E, Grayson DR, et al. The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver- specific factor(s) at two other sites. Mol Cell Biol. 1988;8:81-90.
18. Courtois G, Baumhueter S, Crabtree GR. Purified hepatocyte nuclear factor 1 interacts with a family of hepatocyte-specific promoters. Proc Natl Acad Sci USA. 1988;85:7937-7941.
19. Descombes P, Chojkier M, Lichtsteiner S, et al. LAP, a novel member of the C/EBP gene family, encodes a liver-enriched transcriptional activator protein. Genes Dev. 1990;4:1541-1551.
20. Dinarello CA, Cannon JG, Wolff SM, et al. Tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of IL-1. J Exp Med. 1986;163:1433-1450.
21. Fausto N. Hepatic regeneration. In: Zakim D, Boyer T, eds. Hepatology: A Textbook of Liver Diseases. Philadelphia: Saunders, 1990:49-64.
22. Flores EA, Bistran BR, Pompselli JJ, et al. Infusion of tumor necrosis/cachectin promotes muscle catabolism in the rat: a synergistic effect with interleukin 1. J Clin Invest. 1989;83:1614-1622.
23. Fong Y, Moldawer LL, Marano MA, et al. Cachectin/TNF or IL-1 induces cachexia with redistribution of body proteins. Am J Physiol. 1989;256:R659-R665.
24. Frain M, Swart G, Monaci P, et al. The liver-specific transcription factor LF-B1 contains a highly diverged homeobox DNA binding domain. Cell. 1989;59:145-157.
25. Goodman JC, Robertson CS, Grossman RG, et al. Elevation of tumor necrosis factor in head injury. J Neuroimmunol. 1990;30:213-217.
26. Grau GE, Taylor TE, Molyneux ME, et al. Tumor necrosis factor and disease severity in children with Falciparum malaria. N Engl J Med. 1989;320:1586-1591.
27. Greenbaum L, Li W, Cressman D, et al. CCAAT enhancer-binding protein β is required for normal hepatocyte proliferation in mice after partial hepatectomy. J Clin Invest. 1998;102:996-1007.
28. Grunfeld C, Feingold KR. Metabolic disturbances and wasting in the acquired immunodeficiency syndrome. N Engl J Med. 1992;327:329-337.
29. Hardon EM, Frain M, Paonessa G, et al. Two distinct factors interact with the promoter regions of several liver-specific genes. EMBO J. 1988;7:1711-1719.
30. Hogan PG, Rao A. Transcriptional regulation: modification of nuclear export? Nature. 1999;398:200-201.
31. Houglum K, Filip M, Witztum JL, et al. Malondialdehyde and 4-hydroxynonenal protein adducts in plasma and liver of rats with iron overload. J Clin Invest. 1990;86:1991-1998.
32. Huang JH, Liao WS. Induction of the mouse serum amyloid A3 gene by cytokines requires both C/EBP family proteins and a novel constitutive nuclear factor. Mol Cell Biol. 1994;14:4475-4484.
33. Kubicka S, Kuhnel F, Zender L, et al. p53 represses CAAT enhancer-binding protein (C/EBP)-dependent transcription of the albumin gene. J Biol Chem. 1999;274:32137-32144.
34. Lai E, Prezioso VR, Tao W, et al. Hepatocyte nuclear factor 3 belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head. Genes Dev. 1991;5:416-427.
35. Lavery DJ, Schibler U. Circadian transcription of the cholesterol 7 hydroxylase gene may involve the liver-enriched bZIP protein DBP. Genes Dev. 1993;7:1871-1884.
36. Lichtsteiner S, Schibler U. A glycosylated liver-specific transcription factor stimulates transcription of the albumin gene. Cell. 1989;57:1179-1187.
37. Lichtsteiner S, Wuarin J, Schibler U. The interplay of DNA-binding proteins on the promoter of the mouse albumin gene. Cell. 1987;51:963-973.
38. Lipton SA, Choi Y-B, Pan Z-H, et al. A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds. Nature. 1993;364:626-632.
39. Maire P, Wuarin J, Schibler U. The role of cis-acting promoter elements in tissue-specific albumin gene expression. Science. 1989;244:343-346.
40. Majano PL, Garciá-Monzón C, López-Cabrera M, et al. Inducible nitric oxide synthase expression in chronic viral hepatitis. J Clin Invest. 1998;101:1343-1352.
41. Mattaj I, Englmeir L. Nucleocytoplasmic transport: the soluble phase. Annu Rev Biochem. 1998;67:265-306.
42. Nitsch D, Schultz G. The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors. Mol Cell Biol. 1993;13:4494-4504.
43. Oliff A, Defeo-Jones D, Boyer M, et al. Tumors secreting human TNFα/cachectin induce cachexia in mice. Cell. 1987;50:555-563.
44. Panduro A, Shalaby F, Shafritz DA. Changing patterns of transcriptional and post-transcriptional control of liver-specific gene expression during rat development. Genes Dev. 1987;1:1172-1182.
45. Park EA, Gurney AL, Nizielski SE, et al. Relative roles of CCAAT/enhancer-binding protein and cAMP regulatory element-binding protein in controlling transcription of the gene for phosphoenolpyruvate carboxykinase (GTP). J Biol Chem. 1993;268:613-619.
46. Pinkert CA, Ornitz DM, Brinster RL, et al. An albumin enhancer located 10kb upstream functions along with its promoter to direct efficient, liver-specific expression in transgenic mice. Genes Dev. 1987;1:268-276.
47. Poli V, Ciliberto G. Transcriptional regulation of acute phase genes by IL-6 and related cytokines. In: Tronche F, Yaniv M, eds. Liver Gene Expression. Austin, TX: R.G. Landes, 1994:131-151.
48. Poli V, Mancini FP, Cortese R. IL6-DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990;63:643-653.
49. Potter JJ, Mezey E, Cornelius P, et al. The first 22 base pairs of the proximal promoter of the rat class I alcohol dehydrogenase gene is bipartite and interacts with multiple DNA-binding proteins. Arch Biochem Biophys. 1992;295:360-368.
50. Roubenoff R, Roubenoff RA, Cannon JG, et al. Rheumatoid cachexia: cytokine-driven hypermetabolism accompanying reduced body cell mass in chronic inflammation. J Clin Invest. 1994;93:2379-2386.
51. Schulze-Osthoff K, Beyaert R, Vandevoorde V, et al. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 1993;12:3095-3104.
52. Screpanti I, Romani L, Musiani P, et al. Lymphoproliferative disorder and imbalanced T-helper response in C/EBPβ-deficient mice. EMBO J. 1995;14:1932-1941.
53. Scuderi P, Lam KS, Ryan KJ, et al. Raised serum levels of tumor necrosis factor in parasitic infections. Lancet. 1986;2:1364-1365.
54. Spiegelman BM, Hotamisligil GS. Through thick and thin: wasting, obesity, and TNFα. Cell. 1993;73:625-627.
55. Stamler JS. Redox signaling: Nitrosylation and related interactions of nitric oxide. Cell. 1994;78:931-936.
56. Strassman G, Fong M, Kenney JS, et al. Evidence for the involvement of interleukin 6 in experimental cancer cachexia. J Clin Invest. 1992;89:1681-1684.
57. Svensson EC, Conley PB, Paulson JC. Regulated expression of α-2,6-sialyltransferase by the liver-enriched transcription factors HNF-1, DBP, and LAP. J Biol Chem. 1992;267:3466-3472.
58. Theisen M, Behringer RR, Cadd GG, et al. A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice. Mol Cell Biol. 1993;13:7666-7676.
59. Tilghman S, Belayew A. Transcriptional control of the murine albumin/α-fetoprotein locus during development. Proc Natl Acad Sci USA. 1982;79:5254-5257.
60. Todorov P, Carluk P, McDevitt T, et al. Characterization of a cancer cachectic factor. Nature. 1996;379:739-742.
61. Tracey KJ, Cerami A. Tumor necrosis factor, other cytokines and disease. Annu Rev Cell Biol. 1993;9:317-343.
62. Tracey KJ, Morgello S, Koplin B, et al. Metabolic effects of cachectin/tumor necrosis factor are modified by site of production. J Clin Invest. 1990;86:2014-2024.
63. Trautwein C, Caelles C, van der Geer P, et al. Transactivation by NF-IL6/LAP is enhanced by phosphorylation of its activation domain. Nature. 1993;364:544-547.
64. Voth R, Rossol S, Klein K, et al. Differential gene expression of IFN-α and tumor necrosis factor-α in peripheral blood mononuclear cells from patients with AIDS related complex and AIDS. J Immunol. 1990;144:970-975.
65. Waage A, Halstensen A, Espevik T. Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease. Lancet. 1987;1:355-357.
66. Wang ND, Finegold MJ, Bradley A, et al. Impaired energy homeostasis in C/EBP alpha knockout mice. Science. 1995;269:1108-1112.
67. West JB. Physiological Basis of Medical Practice. Baltimore: Williams & Wilkins, 1990.
68. Williams SC, Angerer N, Johnson PF. C/EBP proteins contain nuclear localization signals imbedded in their basic regions. Gene Expression. 1997;6:371-385.
69. Wong GHW, Elwell JH, Oberley LW, et al. Manganous superoxide dismutase is essential for cellular resistance to cytotoxicity of tumor necrosis factor. Cell. 1989;58:923-931.
70. Yano M, Falvey E, Gonzalez FJ. Role of the liver-enriched transcription factor DBP in expression of the cytochrome P450 CYP2C6 gene. Mol Cell Biol. 1992;12:2847-2854.
71. Yoneda T, Alsina MA, Chavez JB, et al. Evidence that tumor necrosis factor plays a pathogenetic role in the paraneoplastic syndromes of cachexia, hypercalcemia, and leukocytosis in a human tumor in nude mice. J Clin Invest. 1991;87:977-985.
72. Zaret K. Genetic control of hepatocyte differentiation. In: Arias IM, Boyer JL, Fausto N, et al, eds. The Liver: Biology and Pathobiology. New York: Raven Press, 1994:53-68.
Keywords:

cachexia; albumin; C/EBP-β; NOS; TNF-α

© 2005 Lippincott Williams & Wilkins, Inc.