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Pancreas and Liver

Inhibition of Albumin Synthesis in Chronic Diseases

Molecular Mechanisms

Chojkier, Mario MD

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

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

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.


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


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

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