1 Molecular and Cellular Biochemistry DOI 10.1007/s11010-006-9259-7
Springer 2006
Up-regulation of Metabotropic glutamate receptor 3 (mGluR3) in rat fibrosis and cirrhosis model of persistent hypoxic condition Sun Hee Do,1 Hae-Sun Yun,1 Won-Il Jeong,1 Da-Hee Jeong,1 Mi-Ran Ki,1 Jae-Yong Chung,1 Sang-Joon Park,1 Soon-Bok Kim2 and Kyu-Shik Jeong1 1
Department of Veterinary Pathology, College of Veterinary Medicine, Kyungpook National University, 702-701, #1370 Sangyeok-dong, Buk-gu, Daegu, Republic of Korea; 2Department of Veterinary Pathology, College of Veterinary Medicine, Gyeongsang National University, Jinju, 660-701, Republic of Korea Received 29 March 2006; accepted 1 June 2006
Abstract Glutamate is the major excitatory neurotransmitter in the central nervous system, and evidence for peripheral glutamatergic fibers in mammals is still lacking. However, glutamate receptors have been identified in peripheral organs, including taste buds, myenteric plexus, and pancreatic islet cell. Protection against anoxic damage could also be explained by mechanisms mediated by postsynaptic mGluR2 or mGluR3, such as the inhibition of membrane excitability resulting from a reduction of cAMP formation by a G-protein-dependent modulation of ion channels. In addition, activation of mGluR3 present in glial cells may contribute to neuroprotection by enhancing the production of death. Thus, mGluR2/3 behaves potentially as a major defensive mechanism anoxia-tolerant species. There are a few reports for the regional pattern of hypoxic damage, which was inversely related to the expression of mGluR2/3. The aim of this study was to characterize the expression of mGluR3 in hypoxic liver in experimental model of rat liver. Proteomic analysis of protein extracts from CCl4–induced cirrhotic liver revealed the presence of the mGluR3. The presence of mGluR3 in the cirrhotic liver was confirmed by immunohistochemical analysis. There were a number of macrophages expressing mGluR3 mainly in the fibrous septa. After 2 weeks recovery, however, most of mGluR3 positive macrophages disappeared with collagen fibers. These results demonstrate that mGluR3 involved in the liver in response to persistent hypoxic status such as fibrotic/cirrhotic condition, and suggest that the expression of mGluR3 may be a key role functional metabolism and viability in the liver by interacting with the glutamate receptors in vivo. Key words: cirrhosis, hypoxia, liver, macrophage, metabotropic glutamate receptor
Introduction Receptors for glutamate, are currently classified into two major categories: ionotropic glutamate receptors (N-methyl-
D-aspartic
acid (NMD), amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainite receptors), which form membrane ion channels, and metabotropic glutamate receptors (subtype mGlu1–8), which are coupled to
Address for offprints: Kyu-Shik Jeong, Department of Veterinary Pathology, College of Veterinary Medicine, Kyungpook National University, 702-701, #1370 Sangyeok-dong, Buk-gu, Daegu, Republic of Korea (E-mail:
[email protected])
GTP-binding proteins [11, 22]. They are subdivided into three main groups. Group I includes mGluR1 and mGluR5, which are coupled to phosphoinositide (PI) hydrolysis, while group II (mGluR2 and mGluR3) and group III cirrhotic (mGluR4, mGluR6, mGluR7, mGluR8) mGluRs are negatively coupled to adenylyl cyclase [33]. Evidence from experimental pain research has revealed that metabotropic glutamate receptors (mGluRs) play a pivotal role in nociceptive processing, inflammatory pain and hyperalgesia [44]. Werner et al. reported that active multiple sclerosis lesions showed high-level glutaminase expression in macrophages and microglia in close proximity to dystrophic axons, and white matter from other inflammatory neurologic diseases displayed glutaminase reactivity, whereas normal and non-inflammatory conditions showed none [55]. Moreover, there has been a report that hepatocytes express mGluR5 and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes [66]. Recent studies have demonstrated that endogenous activation of mGluR5 contributes to the development of hypoxia-induced liver cell. Hepatocytes isolated from knockout mice were less sensitive to hypoxic damage than hepatocytes from wild-type mice as assessed by lactate dehydrogenases release and formation of reactive oxygen species [77]. However, no information is available about the expression of mGluR3 in liver hepatocytes and hepatic stellate cells in fibrotic/cirrhotic diseases. In the view of these findings and need to understand of the function of mGluR3 in fibrotic and liver that underlie persistent hypoxic condition, this study aimed to identify if fibortic/cirrhotic condition in liver were associated with mGluR3 expression in cirrhotic rat liver model. To gain further insights into the potential role of mGluR3 in hepatic fibrosis/cirrhosis, we performed an immunohistochemical study involving antibodies to mGluR3 on control and fibrotic/cirrhotic rat liver tissue. Our aim was to define the possible involvement of mGluR3 in the pathophysiology of hepatic fibrosis/cirrhosis, thereby increasing the knowledge on the role of this mGluR3 on pathological conditions that lead to hepatocytes and HSC reactivities during the progression of fibrosis/cirrhosis.
Materials and methods Animals and treatment Male Wistar rats (n = 72) weighing 200–220 g were housed in a room at 20 ± 2 C and a 12 h light–dark cycle and were given rat chow and water ad libitum. The animal experiments were performed in accordance with the NIH guidelines for the care and use of laboratory animals. Liver fiborsis/cirrhosis was induced by intraperitoneal injection of
1.0 ml/kg body weight of 10% CCl4 in olive oil three times a week for 14 weeks. Following the necropsy schedule, eight rats were sacrificed at week 0, 2, 4, 6, 8, 10, 12, and 14 weeks, respectively. The rest of the animals (n = 8) is recovery group with CCl4 treatment for 12 weeks and was allowed to recover during the last 2 weeks. These eight rats were also sacrificed at week 14.
Histology and Immunohistochemistry Liver pieces from each rat were rapidly removed and fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections of 4 lm thickness were made. The sections were stained with hematoxylin and eosin (H–E) and specially stained with Azan for collagen fibers. To quantify hepatic fibrosis, we used grade 0–4 [88]. Grade 0; none, Grade 1; short collagenous septa extended from central veins, Grade 2; slender septa link the central veins or portal veins, but lobular architecture is preserved. Grade 3; pseudolobules are formed by thin septa. Grade 4; parenchyma is subdivided into smaller pseudolobules by thin septa. For immunohistochemistry, sections of liver were deparaffinized in xylene, rehydrated in graded alcohol series, incubated in a solution of 3% hydrogen peroxide in methanol for 30 min. Tissue sections were washed with PBS, and then immunostained with primary antibody for monocytes/ macrophages, at dilution of 1:600 (ED1, Chemicon International, Temecula, CA, USA), and glutamate receptor 3 (Zymed Laboratories Inc., San Francisco, CA, USA) at dilution of 1:100. The antigen–antibody complex was visualized by an avidin– biotin peroxidase complex solution using an ABC kit (Vector Laboratories, Burlingame, CA, USA) with 3,3-diamino benzidine (Zymed Laboratories Inc., San Francisco, CA, USA). Tissue sections were then rinsed in distilled water and counterstained with Mayer’s hematoxylin. For negative control, the primary antibody was replaced by PBS.
Sample preparation, 2-DE and staining Frozen liver tissue (200 mg) was homogenized in 3 ml lysis buffer (40 Mm Tris, 8 M urea, 4% CHAPS, 1 mM EDTA, 10 mM ditioerythritol) containing a protease inhibitor cocktail tablet (Roche, Mannheim, Germany). The mixture was again ground to a fine powder in liquid nitrogen and left to thaw at room temperature. The lysate was placed on ice for 1 h and subjected to centrifugation for 1 h at 15,000 rpm. The supernatant was recovered and its protein concentration determined using a commercial 2-D Quant kit (Amersham Biosciences, Uppsala, Sweden) [99]. 2-DE was performed based on previous studies [1010] except for the
voltages used for the IEF step: (i) 500 V, 1 h; (ii) 1000 V, 1 h; (iii) 1000–8000 V, 2000 Vh; (iv) 8000 V, 32,000 Vh. All samples were run in triplicate to ensure reproducibility. At the end of the run, CBB staining of the 2-D gels was performed. Electronic images of the gels were obtained by PowerLook 1120 (UMAX System GmbH, Willich, Germany), recorded, and quantified using the Image Master 2 D software (Amersham Biosciences).
Tryptic in-gel digestion and MALDI-TOF MS Protein spots of interest were excised reduced and digested with 1.25 lg/100 ll of modified porcine trypsin (Promega, Madison, WI, USA) per protein spot. Mass spectrometric analyses were performed using a MALDI-TOF/MS (Voyager-DE STR, Applied Biosystems, Foster City, CA, USA) with delayed extraction and reflectron [1111]. Spectra were calibrated upon acquisition using a ProteoMass Peptide MALDI-MS Calibration Kit, MS-CAL2 (Sigma, St. Louis, USA) with angiotensin II (1046.5423), P14R (1533.8582), and ACTH fragment 18–39 (2465.1989) as internal calibration. Database searches were carried out with the MS-Fit (http://prospector.ucsf.edu).
Results Histopathology of liver tissues Normal collagen fibers in the areas of portal and central veins were observed at week 0. After 14 weeks of CCl4 treatment, there was evidence of increased fibrosis therefore cirrhotic condition was induced (Fig. 1). The mean fibrosis score of the control (week 0) was G0, while the score for the week 14 animals was G4 and cirrhotic nodules were obvious. In the liver of recovery group, there was evidence of remarkable decrease in collagen fiber and bile ducts. Thus the mean fibrosis score was G3. In immunohistochemistry for mGluR3, there are no positive cells at week 0, while a number of macrophages expressing mGluR3 in the fibrous septa were detected at week 8 and 14. On the other hand, most of mGluR3 positive macrophages disappeared in the liver of recovery group. mGluR3 was widely distributed and was highly expressed in macrophages of fibrotic liver corresponding the expression of ED1, a macrophage marker (Fig. 2).
Up-regulated proteins after treatment of CCl4 A total of 11 protein spots that were highly expressed were detected in CCl4-induced cirrhotic rat liver and recovery
group (Fig. 3). Amongst proteins that showed interesting levels of expression at different time points, week 0 and week 14, was mGluR3. It is confirmed that glutamate receptors are present not only in the CNS but also in peripheral organs. On the other hand, disulfide isomerase ER-60 (ER-60), aldehyde dehydrogenase (ALDH), calcium/ calmodulin-dependent protein kinase I (CAM kinase I), apolipoptroein E precursor and thioredoxin peroxidase 1 (PRDX 2) that were differentially expressed were detected at week 14. In recovery group, heat shock protein 70 (HSP70), inositol hexakisphosphate kinase 6 (IP6K6), and procollagen C-proteinase 3 (PCP3) were up-regulated. Proteins related to signal transduction such as serine/ threonine kinase activity, lipid kinase activity (IP6K6) and PCP3 were also increased. Amongst these proteins, procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs), which can produce collagen. Matsui et al. [1212] reported that the effect of PCPE on the syntheses of collagen and non-collagenous protein using an HSC clone was derived from cirrhotic rat liver. Apolipoprotein E precursor was up-regualted in the CCl4-induced cirrhotic rat livers. Apoprotein E (ApoE) has emerged as an important molecule in several biological processes not directly related to its lipid transport function, including Alzheimer’s disease and cognitive function, immunoregulation, and possibly even infectious diseases. ApoE is a polymorphic protein arising from three alleles at a single gene locus. The three major isoforms, apoE4, apoE3, and apoE2 differ from one another only by single amino acid substitutions, yet these changes have profound functional consequences at both the cellular and molecular levels. PRDX2 is abundantly expressed in all types of cells and plays a major role in protecting RBCs from oxidative stress in mice. ALDH were up-regulated in the cirrhotic liver samples. They are able to eliminate toxic biogenic and xenobiotic aldehydes [1313]. In the 2-DE results, ALDH 1A1 was upregulated. It belongs to the class I ALDH, which is constitutive and inducible in the liver cytoplasm of rat [1414]. In summary, the up-regulation of these aldehyde-metabolizing enzymes, as observed in the 2-DE results, is a storng indication of the presence of increased lipid peroxides. Heat shock proteins (HSP) are known to be highly conserved for stresses, including elevated temperature, exposure to toxins, or heavy metals. In the liver, a 70 kDa HSP (stress-inducible HSP70, HSP72) has a crucial cytoprotective function mediated by its function as a ’molecular chaperon’ [1515, 1616]. mGluR3 displayed a 10-fold increase in the protein expression at week 14 compared with that of week 0 and R14. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Glutamatergic
A
0 week
8 week
14 week
R14week
B
Fig. 1. (A) Distribution of collagen fibers and mGluR3-positive cells in rat liver. Azan stain (a–d) and IHC (e–h) analyses were performed at 0 (a, e), 8 (b, f), 14 (c, g), and recovery 14 week (d, h). Normal collagen fibers were shown in the areas of portal and central veins (a). Collagen fibers surround pseudolobules and bile ducts proliferate (b, c). Remarkable decreases of collagen fibers were observed during recover (d). There were no positive cells for mGluR3 (e). A number of macrophages expressing mGluR3 were mainly detected in the fibrous septa (f, g). During recovery, most of mGluR3 positive macrophages disappeared with collagen fibers (h). Original magnifications ·33. (B) Arbitrary kinetics of the number of mGluR3 positive monocytes.
neurotransmission is involved in most aspects of normal brain function and can be perturbed in many neuropathologic conditions. Imbalances in glutamatergic function have been implicated in neuronal death following
ischemia, in hypoglycemia or anoxia, in epilepsy, and in neurodegenerative disorders. However, there was few report for function of mGluR3 on fibrotic/cirrhotic liver condition.
mGluR 3
ED1
a
b
c
d
0 week
8 week
Fig. 2. Distribution of mGluR3 and ED1 in fibrotic rat liver. Original magnifications ·66.
Discussion In addition to their expression in their central nervous system, glutamate receptors have been reported to be widely expressed in peripheral, neuronal, or non-neuronal tissues, such as bone [1717], skin [1818], and pancreas [1919]. Indeed, recent studies have revealed that peripheral glutamate receptors may be involved in a variety of physiologic functions [2020–2222]. For example, glutamate receptors in the myenteric plexus of the guinea pig ileum were related to modulation of intestinal contractility [2020, 2121], and mGluRs in the rat heart were suggested to play an important role in the cardiac function [2222]. Glutamate-stimulated tumor growth and glutamate antagonist-induced suppression of cancer cell growth strongly suggest that glutamate signaling is likely to be important in non-neuronal cancer cell [2323]. Furthermore, endogenous activation of metabotropic
glutamate receptors which contributes to the development of hypoxia-induced liver cell injury [77], and pharmacological blockade of glutamate receptors which, might represent a novel target for the treatment of drug-induced liver damage [2424], were novel clues for the present of the glutamate receptor in the liver and their regulatory mechanism in the chronic lesion. This study is the report of altered mGluR3 protein expression in the liver in an experimental animal model of persistent hypoxic condition, such as fibrosis and cirrhosis. Here, we demonstrate unique alterations in mGluR3 in rat liver fibrosis that were distinct from neuronal expression of mGluR subtypes. This work demonstrates clearly that mGluR3 are differentially modulated in these different fibrogenesis condition, representative of mild inflammation, moderate to mild fibrosis, severe fibrosis, and irreversible cirrhotic condition. In the present study, we found that mGluR3 was widely distributed in macrophages
A pI MW a (kDa)
4.99
6.34
7.51
8.12
4.99
pI
7.51
6.34
8.12
pI
b
4.99
6.34
7.51
8.12
c mGluR3 HSP70 HSP70
71.1 ER-60
ER-60
IP6K6
ALDH
47.0 CAM kinase I Apolipoprotein E precursor PCP3
25.1
PRDX 2
17.3 15.9
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50 HSP70
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Apolipoprotein E precursor
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ALDH
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1200 1000
mGluR3
Relative Intensity
B
Fig. 3. (A) 2-D Gel electrophoresis image analysis of up-regulated proteins in carbon tetrachloride (CCl4)-administrated rat liver. First dimension, immobilized pH gradient (IPG) strip (ready-made); separation distance, 180 mm; sample application by in-gel rehydration (300 lg protein/strip); IEF was performed on the IPG system; running condition: 500 V for 1 h, 1000 V for 1 h, 1000–8000 V for 2000 Vh and 8000 V for 32,000 Vh. Second dimension, vertical SDS-PAGE (12%); CBB staining. At 0 week (a), 14 week (b), and recovery 14 week (c) after CCl4-administrated rat liver proteins were separated on IPG 3–10. Arrows indicate over-expressed proteins. (B) Expression level of up-regulated the 11 proteins was represented on the bar graph. At 14 week, expression levels of upregulated seven proteins were compared with other two groups (a). At recovery 14 week, expression levels of up-regulated four proteins were compared with other two groups (b). Gray bar: at 0 week, closed bar: at 14 week, open bar: at recovery 14 week. Values were expressed relative to the week 0 (100%).
of fibrotic conditioned liver corresponding to the expression of ED1, a macrophage marker. In the injured tissue, macrophages are involved in proinflammatory and profibrogenic cytokine release, induction of oxidative stress, and phagocytosis. Since mGluR3 are primarily expressed by macrophages, it may be associated with fibrogenic response. It may also be related to inflammatory response and other molecular and/or cellular events in the injured liver. In persistent hypoxic condition, fibrogenesis, liver showed significant increase in mGluR3, whereas irreversible hypoxic condition, cirrhotic liver, represent reduced immunoreactivity for mGluR3. This finding suggests that mGluR3 may regulate the pathophysiology of normal and fibrotic/
cirrhotic liver induced by carbontetrachloride. In immunohistochemistry, there are no positive cells for mGluR3 at week 0. However, a number of macrophages expressing mGluR3 were mainly detected in the fibrous septa at week 14. In the fibrotic rat liver, mGluR3 was expressed in the cytoplasm of the macrophages rather than the membrane, perhaps due to receptor internalization, which is a mechanism for desensitization [2525–2727]. The physiologic role of mGluR3 is important in both neuronal [2121] and nonneuronal tissues [2828, 2929]. Decreased expression of the mGluR3 gene might be targeted for therapeutic intervention in inflammatory pain states [2828]. Our data indicate that mGluR3 expression is linked to kinetics of macrophages in
liver cirrhosis. Consistent with these findings, our previous study [3030] showed kinetic relationship among the numbers of macrophages, myofibroblasts and mast cells in liver fibrosis, but none between the former two cells and the mast cells in liver cirrhosis. Based on proteomic analysis, 11 spots on 2-D gels derived from fibrotic liver perfectly matched with masses identified by mass spectrometry and identified. Among these proteins, four proteins well corresponded to mGluR3, ER60, CAM kinase I, PRDX 2 at 14 week. The most highly induced protein, mGluR3, was approximately 10-fold overexpressed compared with that of other two groups. A major function of group II mGluRs is as autoreceptors for glutamate, depressing excitatory post-synaptic potentials through inhibition of glutamate release [3131]. Dolan and Nolan [3232] reported that crucial role for selective group I mGluR antagonists and group II mGluR agonists in acute inflammatory hyperalgesia. We put forward the hypothesis that TGF-b1 is mainly produced by myofibroblasts and macrophages at early and middle stages of fibrotic processes, but it is predominantly released by hypoxic hepatocytes in the last fibrotic stage or cirrhosis. In addition, the upregulation of mGluR3 during the process of fibrosis and cirrhosis may play a critical role in processing of hepatic fibrosis and cirrhosis, and its regulatory mechanism still remains to be clarified.
Acknowledgement The work presented in this paper was supported by the Brain Korea 21 Project in 2006.
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