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Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain
ELSEVIER
Neuroscience Letters 213 (1996) 153-156
NEORUSCIHC[ I IIS
Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain K a z u h i k o M i s h i m a a, S h i g e k i H i g a s h i y a m a b, Y o u j i N a g a s h i m a c, Y o h e i M i y a g i c, A k i r a T a m u r a d, N o b u t a k a K a w a h a r a a, N a o y u k i T a n i g u c h i b, A k i o A s a i a,*, Y o s h i y u k i K u c h i n o e, T a k a a k i K i r i n o a ~Department of Neurosurgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan hDepartment of Biochemistry, Osaka University Medical School, Osaka, Japan CSecond Department of Pathology, Yokohama City University, Yokohama, Japan JDepartment of Neurosurgery, Teikyo University, Tokyo, Japan eNational Cancer Center Research Institute, Tokyo, Japan Received 12 June 1996; accepted 24 June 1996
Abstract
Heparin-binding epidemlal growth factor-like growth factor (HB-EGF) is a recently described member of the EGF family that binds to and stimulates phospho~ylation of the EGF receptor (EGFR). In this study, we examined the cellular localization of HB-EGF gene transcripts and protein in adult rat forebrain. In situ hybridization studies showed that neurons in various regions, including cortex, hippocampus, and deep structures, express HB-EGF mRNA. Positively labeled cells were also present in white matter, which suggests that both neurons and gl:ia express HB-EGF mRNA. Immunohistochemical studies with an antibody specific to proHB-EGF, a transmembrane form of HB-EGF, demonstrated ubiquitous immunoreactivity in neurons and glial cells in white matter. In view of the wide expression of its cognitive receptor, EGFR, in central nervous system neurons, our results suggest that HB-EGF is an endogenous ligand for EGFR in the central nervous system and may play an important role in physiological conditions.
Keywords: Heparin-binding EGF-like growth factor; In situ hybridization; Immunohistochemistry; Rat brain
Epidermal growth factor (EGF), a peptide that is mitogenic for a variety of cell types, has neurotrophic and protective effects on neurons [3,13,15,20]. EGF enhances neurite outgrowth and survival of cultured striatal, cortical, and cerebellar neurons [ 11,15] and protects hippocampal neurons in vitro against anoxia and nitric oxide exposure [ 13]. Although its in vivo expression and localization in the brain are somewhat controversial, EGF-like immunoreactivity has been demonstrated in a specific population of neurons, such as cortical, pallidal and nigral structures [5,10,16], suggesting that it has a physiological role in vivo. Several EGF-related molecules, such as transforming growth factor (TGF)-c~ [12], heparin-binding EGF-like growth factor (HB-EGF) [7], amphiregulin [17], and betacellulin [19], have been discovered, and * Corresponding author. Tel.: +81 3 38155411, ext. 3345; fax: +81 3 38118647
some are thought to act as neurotrophic factors in the brain as well. For example, TGF-ot has neurotrophic effects on cultured ventral midbrain dopaminergic neurons and dorsal root ganglion neurons [2,4], and its mRNA has been identified in neurons and glia in the adult rodent central nervous system (CNS) [18]. HB-EGF is initially synthesized as a large membrane anchored protein, proHB-EGF, and its extracellular domain consisting of 86 amino acids is cleaved and released from the cellular membrane (the secreted form) [8]. In in vitro studies using macrophage and transfected melanoma cells, both the secreted form and proHB-EGF have been shown to have biological effect by binding to and phosphorylation of EGF receptor (EGFR) [7,9]. In view of the wide expression of EGFR in adult rat neurons in vivo [6], this raises the possibility that HB-EGF is an endogenous ligand for EGFR in the brain as well. Although the expression of HB-EGF mRNA in adult rat
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brain has been confirmed by Northern blot analysis [ 1], the pattern and site of its mRNA and protein expression at the cellular level has not been well studied. This information is crucial for understanding the role of HB-EGF in the brain. In the present study, the cellular localization of HB-EGF mRNA and protein in adult rat forebrain was evaluated by in situ hybridization with an HB-EGF cRNA probe and by immunohistochemical staining with an antibody specific for proHB-EGF. Partial cDNA of rat HB-EGF corresponding to nucleotides 20 to 613 [1] was obtained by reverse transcriptase polymerase chain reaction with poly(A) + RNA of adult rat brain and was used for probe preparation. The amplified fragment was subcloned into pTZ19R plasmid vector, and the nucleotide sequence of the cloned cDNA was confirmed by the dideoxy chain termination method. The cloned cDNA was cut out by digestion with EcoRI and Bam HI and subcloned into a pGEM-4Zf(+) vector. The resultant plasmid was linearized by digestion with EcoRI (sense) or BamHI (antisense), and each cRNA probe was synthesized by in vitro transcription with SP6 or T7 RNA polymerase using digoxigenin-labeled UTP according to the manufacturer's protocol (Boehringer Mannheim Biochemicals, Indianapolis, IN). The labeling of the cRNA probe was confirmed by filter hybridization. Normal brains were obtained from male Sprague-Dawley rats 8-9 weeks old that were deeply anesthetized with pentobarbital (i.p.) and perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative overnight, embedded in paraffin, and cut into 5-/~m-thick sections. After dewaxing in xylene and rehydration in decreasing concentrations of ethanol, the sections were submerged in 0.3% Triton X-100 in phosphate-buffered saline for 5 min and then in 0.2 N HCI for 20 min, digested with 20 mg/ml of proteinase K in 50 mM Tris-HCl and 2 mM CaC12 (pH 7.5) for 5 min at room temperature, postfixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline, neutralized twice in 0.2% glycine for 30 min each, and prehybridized in 2 x SSC containing 50% formamide for 10 min. Hybridization was carried out at 50°C overnight in hybridization buffer containing 50% formamide, 5 x SSC, 1 x Denhardt's solution, 0.1 mg/ml of salmon sperm DNA, 0.1 mg/ml of heparin sulfate, 0.2 mg/ml of E. c o l i tRNA, 10% dextran sulfate, and 1 mg/mi of digoxigenin-labeled RNA probe. After in situ hybridization, the sections were washed in 4 x SSC at 40°C for 1 h, incubated with 20 mg/ml of RNase A at 37°C for 1 h, and washed twice in0.1 × SSC at 50°C for 1 h each. Hybridizedprobes were detected by anti-digoxigenin antibody, which was conjugated to alkaline phosphatase (Nucleic Acid Detection Kit, Boehringer) according to the manufacturer's protocol. For the immunohistochemical studies, fresh frozen sections were used. Rats were decapitated under deep
anesthesia with pentobarbital (i.p.), and the brains were immediately removed, cut into coronal sections, embedded in OCT compound (Miles Laboratory, Elkhart, IN), quickly frozen, and stored at -80°C until use. The frozen tissues were cut into 4-/~m-thick sections and fixed in 4% paraformaldehyde at 4°C for 5 min. After inactivation of intrinsic peroxidase with 1% hydrogen peroxide, the sections were incubated with anti-human HBEGF antisera H1 (diluted 500-fold) at 4°C overnight, and the labeled antigens were visualized with the streptavidinbiotin method (Histo-Fine Kit, Nichirei, Tokyo, Japan). The HB-EGF antibody HI (rabbit polyclonal antibody) recognizes proHB-EGF, but it does not cross-react with mature HB-EGF [14] and does not detect EGF, TGF-c~, or amphiregulin. The sections were counterstained with methyl green. In situ hybridization studies with the antisense probe demonstrated positively labeled neuronal cells throughout the forebrain, including neocortex, piriform cortex, hippocampus, thalamus, hypothalamus, striatum, and basal ganglia. In the cerebral neocortex, the hybridizing cells were distributed in all cortical layers (Fig. 1A,B). Expression of HB-EGF mRNA was especially conspicuous in the piriform cortex and amygdala. In the hippocampal formation, HB-EGF mRNA expression was stronger in large pyramidal cells of the CA1-4 regions than in the granular cells of the dentate gyrus (Fig. 1D). Positive cells were also present in several regions of white matter, including the corpus callosum (Fig. 1C), anterior commissure, and the external and internal capsules; because of their location, these cells are likely to be glial cells. In other parts of the brain, the ependymal cells and choroid plexus showed positive hybridization signals (Fig. 1E). In this experiment, no signal above background was observed in controls in which the sense cRNA probe was used (Fig. IF). Immunohistochemical studies demonstrated proHBEGF immunoreactivity in the cytosol and principal processes, but not in the nuclei, of neurons throughout many areas of the rat forebrain, including the neocortex, piriform cortex, hippocampus, thalamus, hypothalamus, striatum, and basal ganglia. In the neocortex, proHB-EGF immunoreactivity was relatively strong, particularly in the pyramidal cells of layers 3 and 5 (Fig. 2A,B). ProHB-EGF expression was especially strong in the amygdala, hippocampus, and piriform cortex. In the hippocampus, immunostaining was more intense in CA1-4 neurons than in the granular cells of the dentate gyrus, which is in accordance with thein situ hybridization findings (Fig. 2D). Immunoreactive cells were also present in several regions of white matter, including the corpus callosum (Fig. 2C), anterior commissure, and the external and internal capsules. Ependymal cells lining the ventricular system and choroid plexus were also stained with proHB-EGF antibody (Fig. 2E). No immunoreactivity was found in sections reacted with antiserum pre-absorbed by recombinant human HB-EGF or in control sections without primary antibody (Fig. 2F).
K. Mishima et al. / Neuroscience Letters 213 (1996) 153-156
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Fig. 1. Expression of HB-EGF transcripts in the adult rat forebrain determined by in situ hybridization with digoxygenin-labeled antisense (A-E) and sense HB-EGF probes (F). (A,B) The positive signals were localized in neurons and intense signals were observed especially in the pyramidal ceils of layers 3 and 5. (B) High-power view of the cortical layer. (C) The hybridization signals were localized in scattered glial cells in the corpus callosum. (D) Expression of HB-EGF mRNA in adult rat hippocampal formation. The HB-EGF mRNA is more abundantly expressed in the CAI--4 areas than in the dentate gyros. (E) The ependymal cells lining the lateral ventricle and the choroid plexus show hybridization signals. (F) No positive signal was observed in control cortical section hybridized with sense probe. Scale bar: (A,F) 400 #m; (B,C,E) 100/~m; (D) 200 #m.
This study demonstrates that HB-EGF mRNA and proHB-EGF protein are broadly expressed in neurons of adult rat forebrain. However, little is known about the biological effect of HB-EGF on neuronal cells. Recently,
we found that recombinant HB-EGF (rHB-EGF) stimulates neurite outgrowth of PC12 cells in vitro and prevents apoptotic cell death of nerve growth factor-deprived sympathetic neuron-like PC12 cells, thereby promoting cell
Fig. 2. lmmunohistochemical staining for proHB-EGF in adult rat forebrain. (A,B) ProHB-EGF immunoreactivity in cortex was observed in neurons in their cytoplasm and thelr principal processes. The pyramidal cells of layers 3 and 5 showed intense staining for proHB-EGF. (B) High-power view of cortical layer. (C) Positive staining was also observed in scattered glial cells in the corpus callosum. (D) ProHB-EGF immunoreactivity in the hippocampal formation is more intense in the C A I - 4 areas than in the dentate gyrus. (E) The ependymal cells lining the lateral ventricle and the choroid plexus show intense immunoreactivity for proHB-EGF. (F) No immunoreactivity was present in control cortical sections incubated with preabsorbed antiserum. Scale bar: (A,F) 400 ~m; (B,C,E) 100 ~tm; (D) 200/~m.
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survival (unpublished data). These findings suggest that H B - E G F m a y have a similar neurotrophic function in vivo. That both H B - E G F and its receptor are widely expressed in n e u r o n s of the CNS suggests that H B - E G F is an e n d o g e n o u s ligand o f E G F R and has some role in physiological functions. In our study, H B - E G F m R N A and p r o H B - E G F were expressed at high levels in pyramidal n e u r o n s in C A 1 - 4 regions of the h i p p o c a m p u s but at relatively low levels in granular cells of the dentate gyrus. In contrast, Seroogy et al. [18], using in situ hybridization to localize T G F - ~ m R N A in rat forebrain, found that granular cells of the dentate gyrus and C A 3 pyramidal neurons were more densely labeled than other cellular c o m p o n e n t s in the hippocampus. This difference in the regional distrib u t i o n of m R N A s for TGF-ct and H B - E G F in mature rat brain is intriguing, as it raises the possibility that each of these E G F proteins has a different physiological role in the hippocampus. In this study, using proHB-EGF-specific antibody, which does not cross-react with the secreted form of H B - E G F , we demonstrated that p r o H B - E G F is expressed in n e u r o n s of the brain, in accordance with the distribution of m R N A . In n o n - n e u r o n a l cells, p r o H B - E G F is expressed on the cell surface and stimulates growth of cells expressing E G F R in a j u x t a c r i n e m a n n e r [9]. This finding suggests that neuronal p r o H B - E G F , as shown in the present study, functions in a similar manner. Further studies are warranted to clarify the function and gene regulatory m e c h a n i s m of H B - E G F in normal and pathological conditions in the CNS in vivo.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [1] Abraham, J.A., Damm, D., Bajardi, A., Miller, J., Klagsburn, M. and Ezekowitz, R.A., Heparin-binding EGF-like growth factor: characterization of rat and mouse eDNA clones, protein domain conservation across species, and transcript expression in tissues, Biochem. Biophys. Res. Commun., 190 (1993) 125-133. [2] Alexi, T. and Hefti, F., TGF ot selectively increases dopaminergic cell survival in ventral mesencephalic cultures, Soc. Neurosci. Abstr., 18 (1992) 1292. [3] Casper, D., Mytilineous, C. and Bium, M., EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture, J. Neurosci. Res., 30 (1991) 372-381. [4] Chalazonitis, A., Kessler, J.A., Twardzik, D.R. and Morrison, R.S., Transforming growth factor u, but not epidermal growth factor, promotes the survival of sensory neurons in vitro, J. Neurosci., 12 (1992) 583-594. [5] Fallon, J.H., Seroogy, K.B., Loughlin, S.E., Morrison, R.S., Bradshaw, R.A., Knaver, D.J. and Cunningham, D.D., Epidermal growth factor immunoreactive material in the central nervous system: location and development, Science, 224 (1984) 1107-1109. [6] Faundez, V., Krauss, R., Holuigue, L., Garrido, J. and Gonzalez,
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A., Epidermal growth factor receptor in synaptic fractions oftbe rat central nervous system, I. Biol. Chem,, 28 (1992) 20363-20370. Higashiyama, S., Abraham, J.A., Miller, J., Fidders, J.C. and Klagsbmn, M., A heparin binding growth factor secreted by macrophage-like cells that is related to EGF, Science, 251 (1991) 936939. Higashiyama, S., Lau, K., Besner, G.E., Abraham, J.A. and Klagsburn, M., Structure of heparin-binding epidermal growth factor-like growth factor: multiple forms, primary structure, and glycosylation of the mature protein, J. Biol. Chem., 267 (1992) 62056212. Higashiyama, S., Iwamoto, R, Goishi, K., Raab, G., Taniguchi, N., Klagsburn, M. and Mekada, E., The membrane protein CD9/ DRAP27 potentiates the juxtacrine growth factor activity of the membrane-anchored heparin-binding EGF-iike growth factor, J. Cell. Biol., 128 (1995) 929-938. Kato, M., Mizuguchi, M. and Takashima, S., Developmental changes of epidermal growth factor-like immunoreactivity in the human fetal brain, J. Neurosci. Res., 42 (1995) 486-492. Kornblum, HT, Raymond, H.K., Morrison, R.S., Cavanaugh, K.P., Bradshaw, R.A. and Leslie, F.M., Epidermal growth factor and basic fibroblast growth factor: effects on an overlapping population of neocortical neurons in vitro, Brain Res., 535 (1990) 255-263. Massague, J., Transforming growth factor ct. A model for membrane-anchored growth factors, J. Biol. Chem., 256 (1990) 2139321396. Maiese, K., Boniece, 1., DeMeo, D. and Wagner, J.A., Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity, J. Neurosci., 13 (1993) 3034-3040. Miyagawa, J., Higashiyama, S., Kawata, S., Inui, Y., Tamura, S., Yamamoto, K., Nishida, M., Nakamura, T., Yamashita, S., Matsuzawa, Y. and Taniguchi, N., Localization of heparin-binding EGF-like growth factor in the smooth muscle cells and macrophages of human atherosclerotic plaques, J. Clin. Invest., 95 (1995) 404-411. Morrison, R.S., Komblum, HT, Leslie, F.M. and Bradshaw, R.A., Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor, Science, 238 (1987) 72-75. Schaudies, R.P., Christian, E.L. and Savage, C.R.J., Epidermal growth factor immunoreactive material in the rat brain: localization and identification of multiple species, J. Biol. Chem., 264 (1989) 10447-10450. Shoyab, M., Plowman, G.D., McDonald, V.L., Bradley, J.G. and Todaro, G.J., Structure and function of human amphiregulin: a member of the epidermal growth factor family, Science, 243 (1989) 1074-1076. Seroogy, K.B., Lundgren, K.H., Lee, D.C., Guthrie, K.H. and Gall, C.M., Cellular localization of transforming growth factor ~ mRNA in rat forebrain, J. Neurochem., 60 (1993) 1777-1782. Shing, Y., Christofori, G., Hanahan, D., Ono, Y., Sasada, R., lgarashi, K. and Folkman, J., Betacellulin: a mitogen from pancreatic beta cell tumors, Science, 259 (1993) 1604-1607. Ventreila, L.L., Effect of intracerebroventricular infusion of epidermal growth factor in rats bemitransected in the nigro-striatal pathway, J. Neurosurg. Sci., 37 (1993) 1-8.
Neuroscience Letters 213 (1996) 153-156
NEORUSCIHC[ I IIS
Regional distribution of heparin-binding epidermal growth factor-like growth factor mRNA and protein in adult rat forebrain K a z u h i k o M i s h i m a a, S h i g e k i H i g a s h i y a m a b, Y o u j i N a g a s h i m a c, Y o h e i M i y a g i c, A k i r a T a m u r a d, N o b u t a k a K a w a h a r a a, N a o y u k i T a n i g u c h i b, A k i o A s a i a,*, Y o s h i y u k i K u c h i n o e, T a k a a k i K i r i n o a ~Department of Neurosurgery, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113, Japan hDepartment of Biochemistry, Osaka University Medical School, Osaka, Japan CSecond Department of Pathology, Yokohama City University, Yokohama, Japan JDepartment of Neurosurgery, Teikyo University, Tokyo, Japan eNational Cancer Center Research Institute, Tokyo, Japan Received 12 June 1996; accepted 24 June 1996
Abstract
Heparin-binding epidemlal growth factor-like growth factor (HB-EGF) is a recently described member of the EGF family that binds to and stimulates phospho~ylation of the EGF receptor (EGFR). In this study, we examined the cellular localization of HB-EGF gene transcripts and protein in adult rat forebrain. In situ hybridization studies showed that neurons in various regions, including cortex, hippocampus, and deep structures, express HB-EGF mRNA. Positively labeled cells were also present in white matter, which suggests that both neurons and gl:ia express HB-EGF mRNA. Immunohistochemical studies with an antibody specific to proHB-EGF, a transmembrane form of HB-EGF, demonstrated ubiquitous immunoreactivity in neurons and glial cells in white matter. In view of the wide expression of its cognitive receptor, EGFR, in central nervous system neurons, our results suggest that HB-EGF is an endogenous ligand for EGFR in the central nervous system and may play an important role in physiological conditions.
Keywords: Heparin-binding EGF-like growth factor; In situ hybridization; Immunohistochemistry; Rat brain
Epidermal growth factor (EGF), a peptide that is mitogenic for a variety of cell types, has neurotrophic and protective effects on neurons [3,13,15,20]. EGF enhances neurite outgrowth and survival of cultured striatal, cortical, and cerebellar neurons [ 11,15] and protects hippocampal neurons in vitro against anoxia and nitric oxide exposure [ 13]. Although its in vivo expression and localization in the brain are somewhat controversial, EGF-like immunoreactivity has been demonstrated in a specific population of neurons, such as cortical, pallidal and nigral structures [5,10,16], suggesting that it has a physiological role in vivo. Several EGF-related molecules, such as transforming growth factor (TGF)-c~ [12], heparin-binding EGF-like growth factor (HB-EGF) [7], amphiregulin [17], and betacellulin [19], have been discovered, and * Corresponding author. Tel.: +81 3 38155411, ext. 3345; fax: +81 3 38118647
some are thought to act as neurotrophic factors in the brain as well. For example, TGF-ot has neurotrophic effects on cultured ventral midbrain dopaminergic neurons and dorsal root ganglion neurons [2,4], and its mRNA has been identified in neurons and glia in the adult rodent central nervous system (CNS) [18]. HB-EGF is initially synthesized as a large membrane anchored protein, proHB-EGF, and its extracellular domain consisting of 86 amino acids is cleaved and released from the cellular membrane (the secreted form) [8]. In in vitro studies using macrophage and transfected melanoma cells, both the secreted form and proHB-EGF have been shown to have biological effect by binding to and phosphorylation of EGF receptor (EGFR) [7,9]. In view of the wide expression of EGFR in adult rat neurons in vivo [6], this raises the possibility that HB-EGF is an endogenous ligand for EGFR in the brain as well. Although the expression of HB-EGF mRNA in adult rat
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brain has been confirmed by Northern blot analysis [ 1], the pattern and site of its mRNA and protein expression at the cellular level has not been well studied. This information is crucial for understanding the role of HB-EGF in the brain. In the present study, the cellular localization of HB-EGF mRNA and protein in adult rat forebrain was evaluated by in situ hybridization with an HB-EGF cRNA probe and by immunohistochemical staining with an antibody specific for proHB-EGF. Partial cDNA of rat HB-EGF corresponding to nucleotides 20 to 613 [1] was obtained by reverse transcriptase polymerase chain reaction with poly(A) + RNA of adult rat brain and was used for probe preparation. The amplified fragment was subcloned into pTZ19R plasmid vector, and the nucleotide sequence of the cloned cDNA was confirmed by the dideoxy chain termination method. The cloned cDNA was cut out by digestion with EcoRI and Bam HI and subcloned into a pGEM-4Zf(+) vector. The resultant plasmid was linearized by digestion with EcoRI (sense) or BamHI (antisense), and each cRNA probe was synthesized by in vitro transcription with SP6 or T7 RNA polymerase using digoxigenin-labeled UTP according to the manufacturer's protocol (Boehringer Mannheim Biochemicals, Indianapolis, IN). The labeling of the cRNA probe was confirmed by filter hybridization. Normal brains were obtained from male Sprague-Dawley rats 8-9 weeks old that were deeply anesthetized with pentobarbital (i.p.) and perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were removed, postfixed in the same fixative overnight, embedded in paraffin, and cut into 5-/~m-thick sections. After dewaxing in xylene and rehydration in decreasing concentrations of ethanol, the sections were submerged in 0.3% Triton X-100 in phosphate-buffered saline for 5 min and then in 0.2 N HCI for 20 min, digested with 20 mg/ml of proteinase K in 50 mM Tris-HCl and 2 mM CaC12 (pH 7.5) for 5 min at room temperature, postfixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline, neutralized twice in 0.2% glycine for 30 min each, and prehybridized in 2 x SSC containing 50% formamide for 10 min. Hybridization was carried out at 50°C overnight in hybridization buffer containing 50% formamide, 5 x SSC, 1 x Denhardt's solution, 0.1 mg/ml of salmon sperm DNA, 0.1 mg/ml of heparin sulfate, 0.2 mg/ml of E. c o l i tRNA, 10% dextran sulfate, and 1 mg/mi of digoxigenin-labeled RNA probe. After in situ hybridization, the sections were washed in 4 x SSC at 40°C for 1 h, incubated with 20 mg/ml of RNase A at 37°C for 1 h, and washed twice in0.1 × SSC at 50°C for 1 h each. Hybridizedprobes were detected by anti-digoxigenin antibody, which was conjugated to alkaline phosphatase (Nucleic Acid Detection Kit, Boehringer) according to the manufacturer's protocol. For the immunohistochemical studies, fresh frozen sections were used. Rats were decapitated under deep
anesthesia with pentobarbital (i.p.), and the brains were immediately removed, cut into coronal sections, embedded in OCT compound (Miles Laboratory, Elkhart, IN), quickly frozen, and stored at -80°C until use. The frozen tissues were cut into 4-/~m-thick sections and fixed in 4% paraformaldehyde at 4°C for 5 min. After inactivation of intrinsic peroxidase with 1% hydrogen peroxide, the sections were incubated with anti-human HBEGF antisera H1 (diluted 500-fold) at 4°C overnight, and the labeled antigens were visualized with the streptavidinbiotin method (Histo-Fine Kit, Nichirei, Tokyo, Japan). The HB-EGF antibody HI (rabbit polyclonal antibody) recognizes proHB-EGF, but it does not cross-react with mature HB-EGF [14] and does not detect EGF, TGF-c~, or amphiregulin. The sections were counterstained with methyl green. In situ hybridization studies with the antisense probe demonstrated positively labeled neuronal cells throughout the forebrain, including neocortex, piriform cortex, hippocampus, thalamus, hypothalamus, striatum, and basal ganglia. In the cerebral neocortex, the hybridizing cells were distributed in all cortical layers (Fig. 1A,B). Expression of HB-EGF mRNA was especially conspicuous in the piriform cortex and amygdala. In the hippocampal formation, HB-EGF mRNA expression was stronger in large pyramidal cells of the CA1-4 regions than in the granular cells of the dentate gyrus (Fig. 1D). Positive cells were also present in several regions of white matter, including the corpus callosum (Fig. 1C), anterior commissure, and the external and internal capsules; because of their location, these cells are likely to be glial cells. In other parts of the brain, the ependymal cells and choroid plexus showed positive hybridization signals (Fig. 1E). In this experiment, no signal above background was observed in controls in which the sense cRNA probe was used (Fig. IF). Immunohistochemical studies demonstrated proHBEGF immunoreactivity in the cytosol and principal processes, but not in the nuclei, of neurons throughout many areas of the rat forebrain, including the neocortex, piriform cortex, hippocampus, thalamus, hypothalamus, striatum, and basal ganglia. In the neocortex, proHB-EGF immunoreactivity was relatively strong, particularly in the pyramidal cells of layers 3 and 5 (Fig. 2A,B). ProHB-EGF expression was especially strong in the amygdala, hippocampus, and piriform cortex. In the hippocampus, immunostaining was more intense in CA1-4 neurons than in the granular cells of the dentate gyrus, which is in accordance with thein situ hybridization findings (Fig. 2D). Immunoreactive cells were also present in several regions of white matter, including the corpus callosum (Fig. 2C), anterior commissure, and the external and internal capsules. Ependymal cells lining the ventricular system and choroid plexus were also stained with proHB-EGF antibody (Fig. 2E). No immunoreactivity was found in sections reacted with antiserum pre-absorbed by recombinant human HB-EGF or in control sections without primary antibody (Fig. 2F).
K. Mishima et al. / Neuroscience Letters 213 (1996) 153-156
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Fig. 1. Expression of HB-EGF transcripts in the adult rat forebrain determined by in situ hybridization with digoxygenin-labeled antisense (A-E) and sense HB-EGF probes (F). (A,B) The positive signals were localized in neurons and intense signals were observed especially in the pyramidal ceils of layers 3 and 5. (B) High-power view of the cortical layer. (C) The hybridization signals were localized in scattered glial cells in the corpus callosum. (D) Expression of HB-EGF mRNA in adult rat hippocampal formation. The HB-EGF mRNA is more abundantly expressed in the CAI--4 areas than in the dentate gyros. (E) The ependymal cells lining the lateral ventricle and the choroid plexus show hybridization signals. (F) No positive signal was observed in control cortical section hybridized with sense probe. Scale bar: (A,F) 400 #m; (B,C,E) 100/~m; (D) 200 #m.
This study demonstrates that HB-EGF mRNA and proHB-EGF protein are broadly expressed in neurons of adult rat forebrain. However, little is known about the biological effect of HB-EGF on neuronal cells. Recently,
we found that recombinant HB-EGF (rHB-EGF) stimulates neurite outgrowth of PC12 cells in vitro and prevents apoptotic cell death of nerve growth factor-deprived sympathetic neuron-like PC12 cells, thereby promoting cell
Fig. 2. lmmunohistochemical staining for proHB-EGF in adult rat forebrain. (A,B) ProHB-EGF immunoreactivity in cortex was observed in neurons in their cytoplasm and thelr principal processes. The pyramidal cells of layers 3 and 5 showed intense staining for proHB-EGF. (B) High-power view of cortical layer. (C) Positive staining was also observed in scattered glial cells in the corpus callosum. (D) ProHB-EGF immunoreactivity in the hippocampal formation is more intense in the C A I - 4 areas than in the dentate gyrus. (E) The ependymal cells lining the lateral ventricle and the choroid plexus show intense immunoreactivity for proHB-EGF. (F) No immunoreactivity was present in control cortical sections incubated with preabsorbed antiserum. Scale bar: (A,F) 400 ~m; (B,C,E) 100 ~tm; (D) 200/~m.
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survival (unpublished data). These findings suggest that H B - E G F m a y have a similar neurotrophic function in vivo. That both H B - E G F and its receptor are widely expressed in n e u r o n s of the CNS suggests that H B - E G F is an e n d o g e n o u s ligand o f E G F R and has some role in physiological functions. In our study, H B - E G F m R N A and p r o H B - E G F were expressed at high levels in pyramidal n e u r o n s in C A 1 - 4 regions of the h i p p o c a m p u s but at relatively low levels in granular cells of the dentate gyrus. In contrast, Seroogy et al. [18], using in situ hybridization to localize T G F - ~ m R N A in rat forebrain, found that granular cells of the dentate gyrus and C A 3 pyramidal neurons were more densely labeled than other cellular c o m p o n e n t s in the hippocampus. This difference in the regional distrib u t i o n of m R N A s for TGF-ct and H B - E G F in mature rat brain is intriguing, as it raises the possibility that each of these E G F proteins has a different physiological role in the hippocampus. In this study, using proHB-EGF-specific antibody, which does not cross-react with the secreted form of H B - E G F , we demonstrated that p r o H B - E G F is expressed in n e u r o n s of the brain, in accordance with the distribution of m R N A . In n o n - n e u r o n a l cells, p r o H B - E G F is expressed on the cell surface and stimulates growth of cells expressing E G F R in a j u x t a c r i n e m a n n e r [9]. This finding suggests that neuronal p r o H B - E G F , as shown in the present study, functions in a similar manner. Further studies are warranted to clarify the function and gene regulatory m e c h a n i s m of H B - E G F in normal and pathological conditions in the CNS in vivo.
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