Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1β and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.     

Regulation of protein disulfide isomerase gene expression in brain and liver during rat development

A 4-fold increase in protein disulfide isomerase (PDI) mRNA is observed in brain of 10 days-old rats and in liver of 20 days-old foetuses when compared with 20 days-old (brain) and 18 days-old (liver) foetuses respectively. During further postnatal development, the mRNA for PDI decreases in both organs to the initial values present in foetuses and remains practically unchanged in brain till the adult. By contrast in liver by 35-40 days after birth, and coincident with sexual maturation, there is a 2.5-fold increase in PDI mRNA that is maintained by 55 days (adult). These results clearly show that protein disulfide isomerase gene expression is differentially regulated in liver and brain during rat development.     

Phosphorylation pattern of liver proteins during the early stages of the acute-phase response.

Liver preparations from turpentine-treated rats show an increased capacity to autophosphorylate a protein of 32.5 kDa (p 32.5): both the kinase and the substrate protein are strongly bound to the membrane fraction, but the protein is released to the cytosol after phosphorylation, which occurs exclusively in serine residues. No known second messenger-dependent protein kinase seems to be responsible for the reaction. Phosphorylation of p 32.5 could be an early post-receptorial event after turpentine-treatment possibly caused by cytokines and involved in the pathogenesis of further events of the acute-phase response.     

Expression of C/EBP delta in rat liver during development and the acute-phase response

Using Western analysis, C/EBP delta was established in the nuclear extract and nuclear matrix throughout rat liver development and in the adult. During the acute-phase response (APR), C/EBP delta increased in the nuclear extract but remained unchanged in the nuclear matrix of fetal and postnatal rats, whereas it increased in both the nuclear extract and nuclear matrix of the adult. The solubility partitioning of gene regulatory proteins in the nucleus is important for their functioning (Uskokovi? et al. 2002). The obtained different solubility partitioning profiles of C/EBP delta suggest that its activity is regulated by different mechanisms during development and in the adult.     

Distribution of transferrin synthesis in brain and other tissues in the rat

Levels of transferrin mRNA were measured by hybridization to transferrin cDNA in extracts from various areas of rat brain and other tissues. The highest concentrations of transferrin mRNA were found in the liver and the choroid plexus of the lateral and third ventricles. Lower concentrations were observed in the medulla and thalamus, choroid plexus of the fourth ventricle, cortex, hypothalamus, cerebellum, pituitary, testis, placenta, stomach, spleen, kidney, muscle, and heart. Yolk sac, small intestine, and adrenal glands did not contain detectable transferrin mRNA levels. The size of transferrin mRNA was the same in liver, brain, and testis. Upon incubation of choroid plexus pieces with [14C]leucine in vitro, about 4% of the radioactive protein secreted into the medium was found to be transferrin. Together with previous data (Dickson, P.W., Howlett, G.J., and Schreiber, G. (1985) J. Biol. Chem. 260, 8214-8219; Dickson, P.W., Aldred, A.R., Marley, P.D., Bannister, D., and Schreiber (1986) J. Biol. Chem. 261, 3475-3478) the obtained data suggest that the choroid plexus plays a role in maintenance of homeostasis in the microenvironment of the central nervous system by synthesizing and secreting plasma proteins.     

Activity and distribution of protein kinase C in liver during the acute-phase response

Activity and subcellular distribution of protein kinase C were estimated in liver cytosol and membrane fractions of rats carrying a turpentine-induced inflammation. Protein kinase C activity increases significantly 8 h after treatment in the membrane fraction, with concurrent reduction in the cytosol; 10 h after treatment the membrane-associated activity returns to normal, without concomitant recovery of that detected in the cytosol. The specific binding of phorbol dibutyrate to the liver membrane fraction increases but overall the effect is less evident and delayed in time. The changes are associated to alterations in the phosphorylation pattern of some liver proteins. Liver protein kinase C activity and intracellular distribution seem to be affected by a treatment which is known to induce an acute-phase response in the liver cells.     

Increase of transferrin receptors in hepatocytes during rat liver regeneration

Transferrin receptor in hepatocytes was studied by iron-saturated[125I]transferrin binding. In regenerating rat liver, the receptor was increased 18 hr after partial hepatectomy and decreased at 8 days. The increase of transferrin receptor in hepatocytes may be a marker of proliferation of the cells.     

Proteoglycans and the acute-phase response in Alzheimer's disease brain

Alzheimer's disease is a dementing disorder affecting increasingly large numbers of individuals in the aging population. The characteristic neuropathologic changes of Alzheimer's disease are the deposition of extracellular is the major constituent of senile plaques. In addition to the A4 peptide, senile plaques contain a variety of molecular species, including proteoglycans and inflammatory components. The presence of proteoglycans in the amyloid deposits of Alzheimer's disease and of systemic amyloidoses suggests that these molecules play an active role in the pathogenesis of amyloidosis. However, the molecular mechanisms that lead to the codeposition of amyloid peptide with proteoglycans is still unknown. Recent evidence suggests that the metabolism of proteoglycans is altered in Alzheimer's disease patients. The acute-phase response observed in the brain of patients affected by Alzheimer's disease may be responsible for this effect. In this article, we discuss the role of proteoglycans in Alzheimer's disease, and the possible interactions between factors involved in brain inflammatory mechanisms and proteoglycans in the pathogenesis of Alzheimer's disease     

Differential expression of rat brain caspase family proteins during development and aging.

It is well recognized that caspases are essential effector molecules for carrying out apoptosis in eukaryotic cells. The expression of rat brain caspase family proteins (caspase-2, -3, -6, -7, -8, -9, 10) in development and aging was assessed using immunochemical detection. All of these caspases were expressed in the rat brain. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that cytosolic caspase-3, -7, -8, and -10 were highly expressed at E19, and decreased after birth. In contrast, cytosolic caspase-2, -6, and -9 were constitutively expressed from the early stages to 96 weeks of age. These results show that the expression of rat brain caspase family proteins is differentially regulated during the development and aging.     

Localization of the iron-regulatory proteins hemojuvelin and transferrin receptor 2 to the basolateral membrane domain of hepatocytes

The newly discovered proteins hemojuvelin (Hjv) and transferrin receptor type 2 (TfR2) are involved in iron metabolism. Mutations in the Hjv and TfR2 gene cause hemochromatosis. We investigated the expression and cellular localization of Hjv and TfR2 in rat and human liver. The expression of Hjv and TfR2 was shown on mRNA and protein level by RT–PCR and immunoblot experiments. Their cellular localization was studied by immunofluorescence with antibodies raised against Hjv and TfR2. Hjv and TfR2 are present in human and rat liver and in primary human hepatocytes. Antisera raised against Hjv identified immunoreactive proteins with an apparent size of 44 and 46 kDa in immunoblot experiments of rat and human liver extracts, which are in accordance with the putative membrane-bound and cleaved soluble forms of this protein, respectively. TfR2 was detected as a 105 kDa protein corresponding to the predicted size of glycosylated TfR2 monomers. In immunofluorescence experiments, Hjv and TfR2 were found in rat liver only in hepatocytes. At the subcellular level, both proteins were predominantly localized to the basolateral membrane domain of hepatocytes. The localization of Hjv and TfR2 at the same membrane domain renders a functional interaction of these two proteins in iron homeostasis possible. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . Kulaksiz and Stremmel contributed equally to this work.