Adrenalectomy and Dexamethasone Treatment Alter the Patterns of Basal and Acute Phase Response-induced Expression of Acute Phase Protein Genes in Rat Liver

Hormonal requirements for full hepatic expression of ₂-macroglobulin (₂M), ₁-acid glycoprotein (AGP), haptoglobin (Hp) and γ-fibrinogen (Fb) were assessed at the level of mRNA. Prior to exposure to turpentine-induced inflammation, rats were either depleted of glucocorticoids by adrenalectomy or supplemented with an excess of dexamethasone. Adrenalectomy alone did not affect the basal level of acute phase protein (APP) expression except for ₂M mRNA, the level of which was enhanced. In contrast, dexamethasone treatment alone promoted full induction of ₂M, significant, but not maximal increase of AGP and Hp mRNAs and suppression of Fb. In adrenalectomized rats, acute phase (AP)-cytokines, released in response to inflammation, promoted full expression of Fb and Hp and increased the level of AGP mRNA whereas ₂M mRNA remained at the basal level. Inflammation in dexamethasone pretreated rats elicited changes which, in comparison to mRNA values for dexamethasone unpretreated inflamed rats, were seen as overexpression of ₂M, full expression of AGP and incomplete expression of Hp, whereas Fb mRNA remained at the basal level. These data suggest that glucocorticoids are the principal inducers of ₂M and AP-cytokines of Fb. For full induction of AGP, additive actions of glucocorticoids and AP-cytokines are required whereas expression of Hp is predominantly controlled by AP-cytokines.     

The responses of rat liver glucocorticoid receptors and genes for tyrosine aminotransferase, alpha-2-macroglobulin and gamma-fibrinogen to adrenalectomy-, dexamethasone- and inflammation-induced changes in the levels of glucocorticoids and proinflammatory cytokines

The responses of liver glucocorticoid receptor (GR) and genes coding for a glucocorticoid-inducible tyrosine aminotransferase (TAT) and two acute-phase proteins (APP) [alpha2-macroglobulin (alpha2-M) and gamma-fibrinogen (Fb)] to changes in glucocorticoid (GC) and proinflammatory (AP) cytokine contents have been examined in rats after single or combined treatments with turpentine oil, dexamethasone (Dex) and adrenalectomy. Activation of two APP genes in turpentine-induced inflammation was accompanied by an increase in the level of GR mRNA and a preferential translocation of GR-GC complexes to the nucleoplasm, while the expression of TAT remained unaltered. Dex alone caused a decrease in the levels of GR and Fb mRNAs, activation of TAT and alpha2-M genes, a decrease in the affinity of hormone binding sites and redistribution of translocated GR-Dex complexes within the nuclei. Inflammation potentiated the effect which Dex alone exerted on the GR content and the number of GR binding sites but counteracted its influence on the affinity of GR binding sites and nuclear distribution of GR-Dex complexes. Adrenalectomy promoted a fall in TAT mRNA, no changes in the GR and Fb mRNA, a decrease in the affinity of GR hormone binding sites and redistribution of GR-hormone complexes within the nuclei. The AP cytokines released in response to inflammation exerted a counteracting effect on the adrenalectomy-induced changes in the affinity of hormone binding sites and nuclear distribution of GR-hormone complexes. They potentiated a fall of TAT mRNA but promoted full expression of the Fb gene. These results argue strongly for the influence of AP cytokines on the functional state of the GR and GC signaling pathways.     

Circadian rhythm of acute phase proteins under the influence of bright/dim light during the daytime

We investigated the influence of two different light intensities, dim (100 lx) and bright (5000 lx), during the daytime on the circadian rhythms of selected acute phase proteins of C-reactive protein (CRP), alpha1-acid glycoprotein (AGP), alpha1-antichymotrypsin (ACT), transfferin (TF), alpha2-macroglobulin (alpha2-m), haptoglobin (HP), and ceruloplasmin (CP). Serum samples were collected from 7 healthy volunteers at 4 h intervals during two separate single 24 h spans during which they were exposed to the respective light intensity conditions. A circadian rhythm was detected only in ACT concentration in the bright light condition. The concentration of ACT, a positive acute phase protein (APP), increased (significantly significant differences in the ACT concentration were detected at 14:00 and 22:00 h) and AGP showed a tendency to be higher under the daytime bright compared to dim light conditions. There were no significant differences between the time point means under daytime dim and bright light conditions for alpha2-M, AGP, Tf, Cp, or Hp. The findings suggest that some, but not all, APP may be influenced by the environmental light intensity.     

The protozoan parasite, Theileria annulata, induces a distinct acute phase protein response in cattle that is associated with pathology

Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.     

The expression of alpha(1)-acid glycoprotein mRNA during rat development. High levels of expression in the decidua

During the acute phase response to inflammation the plasma concentration of some proteins, such as alpha(1-acid glycoprotein (AGP), increases dramatically. Since breakdown and remodeling of tissue is common to both nidation and inflammation we studied the tissue distribution and regulation of AGP mRNA levels during the embryonic development of the rat. High levels of mRNA coding for AGP were detected in the placenta during early fetal development. Expression of this mRNA was confined to the decidua and was first observed approximately 1 day after implantation when proliferation of the decidua is already well advanced. Maximum levels were attained about 5 days after implantation, after which the levels decreased rapidly. In contrast to the high levels of AGP mRNA in the decidua only very low levels were detected in fetal liver and visceral yolk sac, and there was only a small increase in the levels in maternal liver. Corticosteroid hormone responsiveness of AGP mRNA synthesis by hepatocytes appeared 3 days before birth. It is likely that the synthesis of AGP by the cells of the decidua is important in establishing the precisely controlled interaction between mother and embryo during nidation.     

Cloning and developmental regulation of alpha 1 acid glycoprotein in swine

A cDNA clone of porcine alpha 1 acid glycoprotein (alpha 1AGP) has been isolated and sequenced. Sequence homologies between porcine, human, and rat indicate that porcine alpha 1AGP is similar in structure to the rat and human proteins. RNA blots from days 40, 60, 80, and 110 fetal, newborn, and adult livers showed that alpha 1AGP mRNA is relatively abundant throughout fetal development, particularly at the later stages and in the newborn; there is a rapid decline in abundance following birth. From birth to 3 days of age, there is a three- to four-fold decline in abundance, and alpha 1AGP mRNA is approximately 100 times less abundant in the adult liver than in that of perinatal pigs. Southern blots showed that alpha 1AGP is probably a single-copy gene. The isolation of a cloned cDNA for porcine alpha 1AGP provides a tool to investigate the molecular mechanisms involved in the developmental regulation of the gene and to correlate changes in gene expression during development with fetal growth and well being.     

Seasonal rhythms of "acute phase proteins" in humans

"Acute phase proteins" comprise a group of proteins whose concentrations increase or decrease by at least 25% after a damaging stimulus (burn, trauma, tissue damage, etc.) or during inflammation. We investigated the seasonal variation in the concentrations of several acute phase proteins--alpha1-antichymotrypsin (ACT), alpha1-acid glycoprotein (AGP), transferrin (Tf), alpha2-macroglobulin (alpha2-M), ceruloplasmin (Cp), antitrypsin (AT), and haptoglobin (Hp). Blood samples were collected from 15 healthy volunteers, who were subjected to the seasonal changes in illumination, were drawn at 08:00 h every 3 months (August, November, January/February, March/April, June/July). With the exception of Hp, all acute phase proteins showed an annual rhythm (ANOVA; p < 0.01). Lowest concentrations occurred in the winter months (November through February), with the exception of Tf, which was oppositely phased.     

Developmental changes in the methylation of the rat albumin and alpha-fetoprotein genes

We have analyzed methylation of the rat albumin and alpha-fetoprotein (AFP) genes by hydridizing labeled cDNA clones to HpaII and MspI digests of DNA from different stages of development. These CCGG-cutting enzymes distinguish 5-methylcystosine in mCCGG (sensitive to HpaII) and CmCGG (sensitive to MspI). In the liver, the albumin gene is heavily methylated at 18 days gestation and uniformly demethylated in the adult. The AFP gene is also heavily methylated at 18 days gestation, and develops demethylated regions at the 3' half of the gene in the adult. These methylation changes are not observed in other embryonic or adult tissues. We also evaluated expression of these genes by measuring their corresponding mRNAs. The albumin gene is actively transcribed in 18-day fetal liver, when it is heavily methylated, as well as in adult liver, when it is unmethylated. In contrast, the AFP gene is transcribed only in fetal liver, even though it is less methylated in adult liver. These findings suggest that specific methylation changes are associated with changes in gene expression, but that this association is not adequately described by the simple hypothesis that methylation turns genes off.     

The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes

During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.     

Sialyl Lewisx epitopes do not occur on acute phase proteins in mice: relationship to the absence of α3-fucosyltransferase in the liver

Mice are frequently used in models for the study of immunological processes related to inflammation. Since it is known that the degree of fucosylation of human acute phase proteins (APPs) is altered as a consequence of an inflammatory response, we have undertaken this study to gain more insight into the fucosylation of acute phase proteins as it occurs in mouse liver. Mice carrying the cluster of the three genes encoding human α1-acid glycoprotein (AGP), one of the well known APPs, were used and the fucosylation of AGP was assessed. A complete absence of fucosylation on the transgenic human AGP was found, which is in sharp contrast to AGP in human serum, of which a major proportion is normally α3-fucosylated. Remarkably, a large proportion of mouse AGP did contain fucose residues. Fucosylation was also detected on another APP, mouse protease inhibitor (PI). α3-Fucosylation of the transgenic human AGP can be achieved in vitro, using an α3/4-fucosyltransferase (α3/4-FucT) isolated from human milk, showing that the glycoprotein is not intrinsically resistant to fucosylation. Upon subsequent measurement of the activities of the possible fucosyltransferases present in liver membranes of parent and transgenic mice, only an N-linked-core α6-FucT and no α2-, α3- or α4-FucT activity was detected. This indicates that fucose residues found on the mouse serum proteins AGP and PI, which are synthesized in the liver, are most probably in α6-linkage to the core chitobiosyl unit. Interestingly, both α6- and α3-FucT activity was detectable in human liver membranes. None of the above mentioned findings were influenced by the induction of an acute phase response by administration of bacterial lipopolysaccharide. This study shows that: (a) α6-FucT is probably a protein specific-glycosyltransferase, since mouse AGP, but not human AGP, may be used as an acceptor; (b) in contrast to human liver, mouse liver does not express any α3-FucT-activity, thereby making the mouse incapable of producing the Sialyl Lewisx epitope on APPs, which is an important part of the inflammatory reaction in humans. This last finding indicates that the mouse is not suitable as a model for the study of those phenomena related to inflammation in humans, in which glycosylation of acute phase proteins could play a significant role. © 1998 Rapid Science Ltd     

Expression of the rat liver haptoglobin gene is mediated by isoforms of C/EBPalpha, -beta and -delta proteins

CCAAT/enhancer binding proteins (C/EBP) alpha, -beta and -delta play an important role in mediating I interleukin-6 (IL-6) dependent expression of acute-phase protein (APP) genes in liver during acute-phase (AP) response. Based on the presence of type IL-6 responsive element (IL-6 RE) in the rat haptoglobin (Hp) gene promoter we assumed that some C/EBPalpha, -beta and/or -delta isoforms could mediate the expression of this gene during turpentine-induced AP response. By Western immunoblot and Northern blot assays, we found that turpentine treatment of rats led to a coordinate induction of C/EBPbeta and -delta as well as repression of C/EBPalpha isoforms pool levels in rat liver nuclear extracts (NEs) which was preceded by an adequate alteration of their mRNAs expression in liver. Consequently, results of DNA affinity chromatography revealed that affinity of certain C/EBPalpha isoforms to bind the type I IL-6 RE within the rat Hp gene promoter decreased whereas affinities of certain C/EBPbeta isoforms and C/EBPdelta were increased and induced, respectively. Our data suggest that turpentine-induced alterations of C/EBPalpha, -beta and -delta pool levels and DNA-binding activities can be regarded as an integral part of activation of the Hp gene expression in the course of AP response.     

Structure and expression of the human haptoglobin locus.

Human genomic clones of the haptoglobin Hp1F and the "haptoglobin related' gene (Hpr) have been isolated. The two genes are adjacent, spanning a region of approximately 21 kb. A comparison of their coding sequences shows that Hpr differs from Hp1F at 28 codons. Northern blot and primer elongation analyses with human liver RNA show that the haptoglobin gene Hp1F appears to be transcribed some 1000-fold less in fetal than in adult liver. In adult liver the amount of Hpr mRNA is at the lower limit of detection, therefore the extent of its expression is at most less than 1000-fold that of the Hp1F gene. No Hpr mRNA can be detected in fetal liver.     

Identification of candidate growth-regulating genes that are overexpressed in late gestation fetal liver in the rat

We have shown previously that hepatocyte proliferation in the late gestation fetal rat is mediated by growth factor-independent mechanisms that are distinct from the signaling pathways that promote proliferation of adult rat hepatocytes. In the present studies, we identified six candidate growth-regulating genes that are overexpressed in fetal rat liver (embryonic day 19, 2 days pre-term) relative to adult rat liver using suppressive subtractive hybridization. These included the following: Grb10, a growth factor receptor binding protein; eps15, a growth factor receptor substrate; nuc2+, a retinoblastoma protein binding protein; cdc25B, a cell cycle tyrosine phosphatase; the peroxisome proliferator-activated receptor PPAR alpha; and a deoxyuridine triphosphatase that functions as a PPAR alpha binding partner. In every case, the ontogeny of the expression of these genes declined postnatally in a manner consistent with the transition from a fetal to an adult hepatocyte phenotype. None were found to be cell cycle-dependent, in that they did not show expression that followed perinatal changes in hepatocyte cell cycle activity. Based on our identification of these genes and previous work characterizing their role in growth regulation, we conclude that they may contribute to the mitogenic signaling phenotype of fetal rat hepatocytes.