3 Glycobiology Research Group, Tokyo Metropolitan Institute of Gerontology, Foundation for Research on Aging and Promotion of Human Welfare, 35-2 Sakaecho, Itabashi-ku, Tokyo 173-0015, Japan; and 4 Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
Received on November 12, 2003; revised on November 30, 2003; accepted on December 4, 2003
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: astrocyte differentiation / brain-derived neurotrophic factor / galectin-1
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Astrocytes are a major cell type in the CNS. They are believed to act in cooperation with neurons and other glial cells and to participate in the development and maintenance of functions of the CNS. Immature astrocytes possess a polygonal shape, have no processes, and continue to proliferate, whereas mature astrocytes have a stellate cell morphology, increased glial fibrillary acidic protein (GFAP) expression, and proliferate slowly (Bovolenta et al., 1984; Hatten, 1984
). However, little is known about how astrocytes are induced to differentiate. Previously, we revealed that a lectin, Datura stramonium agglutinin (DSA), induced astrocyte differentiation from an immature polygonal shape to a matured stellate shape (Sasaki and Endo, 2000
). DSA is known to bind the Galß1-4GlcNAcß1-6Man
1 branching multiantennary complex-type or two or more linear N-acetyllactosamine repeats (Cummings and Kornfeld, 1984
; Yamashita et al., 1987
). However, DSA is a plant agglutinin and has never been found in brain. We wanted to determine whether a similar molecule with the same carbohydrate-binding specificity and same ability to induce astrocyte differentiation is present in the brain in a more biological context.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Induction of morphological changes of astrocytes by galectin-1
To examine the ability of galectin-1 to induce astrocyte differentiation, recombinant human galectin-1 was added to an immature astrocyte culture. A dose-response analysis demonstrated that galectin-1 was able to induce morphological changes in astrocytes at a concentration of 10 µM (Figure 2A). This is similar to the concentration of galectin-1 needed for apoptosis of T cells (Perillo et al., 1995). Because the dimeric form of galectin-1 was required for induction of T-cell apoptosis (Perillo et al., 1995
) and the Kd for the monomer-dimer of galectin-1 is 7 µM (Cho and Cummings, 1995
), the results indicate that galectin-1 is also acting in a dimeric form in its induction of stellation. Addition of recombinant galectin-1induced astrocyte morphological changes and an increase in the intensity of staining by anti-GFAP antibody (Figure 2B and 2C). No DNA fragmentation was observed (data not shown), indicating that galectin-1 did not induce apoptosis of astrocytes. GFAP is expressed exclusively in astrocytes, and the expression level increases during differentiation (Bovolenta et al., 1984
). Expression of GFAP was increased 50-fold following the addition of galectin-1 (Figure 2F). Differentiated astrocytes are characterized by a stellate cell morphology, increased GFAP expression, and inhibited proliferation (Bovolenta et al., 1984
; Hatten, 1984
). The appearance of all of these characteristics after the addition of galectin-1 strongly indicates that galectin-1 induced astrocyte differentiation.
|
Galectin-1 loses its carbohydrate-binding activity after oxidation (Tracey et al., 1992). The ability of lactose to specifically inhibit the morphological changes of astrocytes suggested that the carbohydrate-binding activity of galectin-1 was involved in astrocyte stellation. This hypothesis was confirmed by the use of an oxidation-resistant galectin-1, C2S, in which the second cysteine residue is changed to serine. C2S is considerably more stable than wild-type galectin-1 under nonreducing conditions (Hirabayashi and Kasai, 1991
). Although C2S induced astrocyte differentiation (Figure 2D), wild-type galectin-1 left for a day did not show any activity. These results indicated that the carbohydrate-binding activity of galectin-1 was essential for induction of astrocyte differentiation. It should be noted that induction of astrocyte stellation by galectin-1 was irreversible. After 1 h exposure to galectin-1, replacing the medium with galectin-1-free medium did not cause the cells to revert to their former polygonal state.
Effect of galectin-1 on the proliferation of cultured astrocytes
Recombinant human galectin-1 inhibited astrocyte proliferation (Figure 2G). The doubling time of astrocytes in culture was around 4.5 days. After the addition of recombinant galectin-1 or C2S, however, astrocyte proliferation was almost completely arrested.
Effect of various inhibitors of signal transduction on astrocyte stellation induced by galectin-1
To elucidate the signal transduction events that occur after galectin-1 binding, we examined the effects of various signal transduction inhibitors (Table I). PKA (protein kinase A) is thought to be involved in astrocyte stellation (Goldman and Chiu, 1984), but KT5720, an inhibitor of PKA, did not inhibit galectin-1-induced stellation, indicating that PKA is not involved in astrocyte stellation induced by galectin-1. In addition, genistein, a tyrosine kinase inhibitor, was not effective at inhibiting galectin-1-induced astrocyte stellation. However, orthovanadate, an inhibitor of protein tyrosine phosphatase (PTP) almost completely inhibited the stellation of astrocytes. These results indicate that PTP is involved in the induction of astrocyte differentiation by galectin-1. On the contrary, in the case of apoptosis of activated T cells by galectin-1, the PTP activity is down-regulated (Hernandez and Baum, 2002
; Lowe, 2001
). These observations suggest that galectin-1 binding modulates PTP activity positively or negatively depending on the cell type.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Induction of astrocyte differentiation is a new function of galectin-1. Galectin-1 has previously been associated with many cellular functions, including development, differentiation, immunity, and apoptosis (Cooper, 2002; Hirabayashi et al., 2002
; Lowe, 2001
). Galectin-1 is present in the CNS (Caron et al., 1987
; Hynes et al., 1990
; Joubert et al., 1989
; Kuchler et al., 1989
). Immunochemistry showed that galectin-1 was expressed in both neuronal cells and nonneuronal cells, including astrocytes in rat brain (Joubert et al., 1989
; Kuchler et al., 1989
). Those cells might be the source of galectin-1 when the brain was injured. In galectin-1-null mice, neurite outgrowth and targeting of olfactory neurons was altered, demonstrating a role for galectin-1 in neural development (Puche et al., 1996
). However, the effect of brain injury in galectin-1-null mice was not studied. On the other hand, in the peripheral nervous system, only the oxidized form of galectin-1 promoted axonal regeneration (Horie et al., 1999
). Because the reduced form did not show such activity, the carbohydrate-binding activity of galectin-1 was not necessary for axonal regeneration. On the contrary, as shown in this study, the carbohydrate-binding activity of galectin-1 was essential for astrocyte differentiation and BDNF production. Together these results indicate that galectin-1 is a bifunctional protein and plays different roles depending on whether it is in the oxidized or reduced form.
Probable substrates of PTP that are believed to be involved in the signal transduction after stimulation by galectin-1 remain to be determined. Immunoblotting with anti-phosphotyrosine antibody revealed that the staining intensity of several protein bands was markedly decreased after the addition of galectin-1 (Figure 3). Especially, the band around 38 kDa and several bands around 85 kDa were immediately dephosphorylated after the addition of galectin-1. These proteins remain to be identified. Tyrosine residues of proteins are phosphorylated and dephosphorylated by the action of tyrosine kinase and PTP, respectively. Galectin-1-induced astrocyte stellation was not affected by genistein but significantly blocked by the tyrosine phosphatase inhibitor orthovanadate (Table I). These results suggest that PTP activity is necessary for the induction of astrocyte stellation by galectin-1 and that galectin-1-triggered astrocyte differentiation is predominantly through a tyrosine dephosphorylation pathway.
Our results show that galectin-1 triggers differentiation, and then the differentiated astrocytes greatly increase their production of BDNF. Galectin-1 induces these phenomena through its carbohydrate-binding activity. This novel role of galectin-1 in brain raises the possibility that it might eventually have applications in the prevention of neuronal loss, such as what occurs in CNS injuries.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Asialofetuin-binding proteins from rat brain
Asialofetuin was coupled to an AF-Tresyl-Toyopearl gel according to the instructions of the manufacturer (Tosoh, Tokyo) using 50 mg asialofetuin and 1 g AF-Tresyl-Toyopearl gel resin, and more than 90% of the protein had coupled to the gel.
Adult rat brains were homogenized with 10 ml/g wet tissue of cold 4 mM 2-mercaptoethanol and 0.1 M phosphate buffered saline (pH 7.4) containing 2 mM ethylenediaminetetraacetic acid (EDTA) (buffer A) in a Potter homogenizer. The homogenate was centrifuged at 100,000 x g for 1 h at 4°C. The obtained precipitate was moved to buffer A with 100 mM lactose and stirred at 4°C for 12 h. The solution was centrifuged at 100,000 x g for 1 h at 4°C, and the supernatant was dialyzed against buffer A to remove lactose completely. Then the solution (2.2 mg protein) was applied to an asialofetuin immobilized column (5 ml volume). After extensive washing of the column with buffer A, the bound proteins were eluted with buffer A containing 100 mM lactose. Asialofetuin-nonbinding fraction (2.1 mg protein) and asialofetuin-binding fraction (5 µg protein) were obtained. Both asialofetuin-binding and -nonbinding fractions were concentrated to 4 ml. Forty microliters and 4 µl each were applied to SDSpolyacrylamide gel with silver staining and immunoblot stained with anti-galectin-1, respectively. Buffer of remaining both fractions was changed to DMEM with an Ultrafree-4 Centrifugal Filter Unit having a Biomax-10 filter (Millipore, Billerica, MA), and each fraction was added to cultured astrocytes.
Recombinant galectin-1 and mutant protein, C2S
Recombinant galectin-1 and its mutant galectin-1 (termed C2S), in which the second cysteine residue was changed to serine, were produced as described by Hirabayashi and Kasai (1991). C2S is substantially resistant to oxidative inactivation while preserving carbohydrate-binding activity.
Cell cultures of astrocytes
Pregnant Fischer rats (F344/N Slc) were obtained from Japan SLC (Shizuoka). Astrocytes were prepared from the cerebellums of postnatal day 5 rats as described previously (Sasaki and Endo, 2000). None of the cells were labeled by anti-neurofilament antibody, a specific marker of neurons, and most were labeled by the anti-GFAP antibody, a specific astrocyte marker. Cells were subcultured at a 1:4 ratio every 8 days to obtain a highly homogenous astrocyte preparation.
Changes in cell morphology were assessed under a microscope (TE300, Nikon, Tokyo) with a Hoffman Modulation Contrast module (Nikon). Cells were seeded in 12-well plates (Asahi Techno glass, Tokyo) at a density of 5x 103 cells per cm2. The cells were cultured with media containing 10% FBS. Three days before the experiments, the medium was changed to FBS-free medium. Cells having three or more processes at least twice as long as the diameter of the cell body were defined as stellate. Results are expressed as the percentages of stellate cells relative to the total cell count. A cell proliferation assay and immunostaining with anti-GFAP were performed as described (Sasaki and Endo, 2000).
SDSPAGE and immunoblotting
The cells in the culture dish were washed three times with ice-cold phosphate buffered saline, and detached and homogenized with 1% SDS in 10 mM TrisHCl buffer, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 1 µM aprotinin, 1 µM pepstatin A. and 1 mM EDTA for the anti-GFAP blotting. To homogenize the cells for the anti-phosphotyrosine blotting, 2 mM sodium orthovanadate and 10 mM sodium fluoride were added to the buffer. Conditioned media were 40 times concentrated with a Biomax-10 filter. Ten-microliter samples were also subjected to SDSPAGE. Proteins were separated by 15% SDSPAGE according to Laemmli (1970) and then electroblotted onto a polyvinyl difluoride (PVDF) membrane (Millipore) as described (Sasaki and Endo, 2000
). Protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL). GFAP, galectin-1, phosphotyrosine, or BDNF was detected with anti-GFAP antibody, anti-galectin-1 antiserum, anti-phosphotyrosine antibody, or anti-BDNF antibody, respectively, with an ECL kit (Amersham Biosciences). Band intensities were measured by densitometric scanning using a densitometer and NIH Image 1.61/ppc software.
Reverse transcription polymerase chain reaction
Cultured immature astrocytes were incubated with or without 10 µM recombinant galectin-1 for 24 h. Total RNA was isolated from cells using Isogen (Nippon Gene, Toyama). First-strand cDNA was synthesized using SuperScript III RNase H, Reverse Transcriptase (Invitrogen, Carlsbad, CA) and polymerase chain reaction (PCR) was performed with KOD DNA polymerase (KOD plus, Toyobo, Osaka) according to the manufacture's instruction. The following primers were used (forward and backward, respectively): BDNF, 5'-cactccgaccctgcccgccg-3' and 5'-tccactatcttcccctttta-3' (Guo et al., 2002) and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), 5'-tgaaggtcggtgtcaacggatttggc-3' and 5'-catgtaggccatgaggtccaccac-3' (Tso et al., 1985
). Cycling was preceded by 2 min at 94°C and followed by 10 min at 72°C and performed as follows: 15 s at 94°C, 30 s at annealing temperature, 1 min at 72°C. Annealing temperature was 61°C for G3PDH and 54°C for BDNF. PCR products were calculated to a length of 983 bp (G3PDH) and 364 bp (BDNF). Five microliters of each amplification reaction were resolved on a 1% agarose gel.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
2 Present address: Glycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barde, Y.A. (1989) Trophic factors and neuronal survival. Neuron, 2, 15251534.[ISI][Medline]
Bovolenta, P., Liem, R.K., and Mason, C.A. (1984) Development of cerebellar astroglia: transitions in form and cytoskeletal content. Dev. Biol., 102, 248259.[ISI][Medline]
Bresjanac, M. and Antauer, G. (2000) Reactive astrocytes of the quinolinic acid-lesioned rat striatum express GFR1 as well as GDNF in vivo. Exp. Neurol., 164, 5359.[CrossRef][ISI][Medline]
Caron, M., Joubert, R., and Bladier, D. (1987) Purification and characterization of a ß-galactoside-binding soluble lectin from rat and bovine brain. Biochim. Biophys. Acta, 925, 290296.[ISI][Medline]
Cho, M. and Cummings, R.D. (1995) Galectin-1, a ß-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization. J. Biol. Chem., 270, 51985206.
Cooper, D.N. (2002) Galectinomics: finding themes in complexity. Biochim. Biophys. Acta, 1572, 209231.[ISI][Medline]
Cummings, R.D. and Kornfeld, S. (1984) The distribution of repeating [Galß1,4GlcNAcß1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1. J. Biol. Chem., 259, 62536260.
Dougherty, K.D., Dreyfus, C.F., and Black, I.B. (2000) Brain-derived neurotrophic factor in astrocytes, oligodendrocytes, and microglia/macrophages after spinal cord injury. Neurobiol. Dis., 7, 574585.[CrossRef][ISI][Medline]
Goldman, J.E. and Chiu, F.C. (1984) Growth kinetics, cell shape, and the cytoskeleton of primary astrocyte cultures. J. Neurochem., 42, 175184.[ISI][Medline]
Guo, H., Tang, Z., Yu, Y., Xu, L., Jin, G., and Zhou, J. (2002) Apomorphine induces trophic factors that support fetal rat mesencephalic dopaminergic neurons in cultures. Eur. J. Neurosci., 16, 18611870.[CrossRef][ISI][Medline]
Hatten, M.E. (1984) Embryonic cerebellar astroglia in vitro. Brain Res., 315, 309313.[Medline]
Hernandez, J.D. and Baum, L.G. (2002) Ah, sweet mystery of death! Galectins and control of cell fate. Glycobiology, 12, 127R136R.
Hirabayashi, J. and Kasai, K. (1991) Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa ß-galactoside-binding lectin. J. Biol. Chem., 266, 2364823653.
Hirabayashi, J., Kawasaki, H., Suzuki, K., and Kasai, K. (1987) Further characterization and structural studies on human placenta lectin. J Biochem. (Tokyo), 101, 987995.[Abstract]
Hirabayashi, J., Hashidate, T., Arata, Y., Nishi, N., Nakamura, T., Hirashima, M., Urashima, T., Oka, T., Futai, M., Muller, W.E., and others. (2002) Oligosaccharide specificity of galectins: a search by frontal affinity chromatography. Biochim. Biophys. Acta, 1572, 232254.[ISI][Medline]
Horie, H., Inagaki, Y., Sohma, Y., Nozawa, R., Okawa, K., Hasegawa, M., Muramatsu, N., Kawano, H., Horie, M., Koyama, H., and others. (1999) Galectin-1 regulates initial axonal growth in peripheral nerves after axotomy. J. Neurosci., 19, 99649974.
Hynes, M.A., Gitt, M., Barondes, S.H., Jessell, T.M., and Buck, L.B. (1990) Selective expression of an endogenous lactose-binding lectin gene in subsets of central and peripheral neurons. J. Neurosci., 10, 10041013.[Abstract]
Isackson, P.J., Huntsman, M.M., Murray, K.D., and Gall, C.M. (1991) BDNF mRNA expression is increased in adult rat forebrain after limbic seizures: temporal patterns of induction distinct from NGF. Neuron, 6, 937948.[ISI][Medline]
Joubert, R., Kuchler, S., Zanetta, J.P., Bladier, D., Avellana-Adalid, V., Caron, M., Doinel, C., and Vincendon, G. (1989) Immunohistochemical localization of a ß-galactoside-binding lectin in rat central nervous system. I. Light- and electron-microscopical studies on developing cerebral cortex and corpus callosum. Dev. Neurosci., 11, 397413.[ISI][Medline]
Kuchler, S., Joubert, R., Avellana-Adalid, V., Caron, M., Bladier, D., Vincendon, G., and Zanetta, J.P. (1989) Immunohistochemical localization of a ß-galactoside-binding lectin in rat central nervous system. II. Light- and electron-microscopical studies in developing cerebellum. Dev. Neurosci., 11, 414427.[ISI][Medline]
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680685.[ISI][Medline]
Lowe, J.B. (2001) Glycosylation, immunity, and autoimmunity. Cell, 104, 809812.[ISI][Medline]
Ming, G.L., Wong, S.T., Henley, J., Yuan, X.B., Song, H.J., Spitzer, N.C., and Poo, M.M. (2002) Adaptation in the chemotactic guidance of nerve growth cones. Nature, 417, 411418.[CrossRef][ISI][Medline]
Miyazaki, H., Nagashima, K., Okuma, Y., and Nomura, Y. (2001) Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats. Brain Res., 922, 165172.[ISI][Medline]
Perillo, N.L., Pace, K.E., Seilhamer, J.J., and Baum, L.G. (1995) Apoptosis of T cells mediated by galectin-1. Nature, 378, 736739.[CrossRef][ISI][Medline]
Poo, M.M. (2001) Neurotrophins as synaptic modulators. Nat. Rev. Neurosci., 2, 2432.[CrossRef][ISI][Medline]
Puche, A.C., Poirier, F., Hair, M., Bartlett, P.F., and Key, B. (1996) Role of galectin-1 in the developing mouse olfactory system. Dev. Biol., 179, 274287.[CrossRef][ISI][Medline]
Rocamora, N., Palacios, J.M., and Mengod, G. (1992) Limbic seizures induce a differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, in the rat hippocampus. Brain Res. Mol. Brain Res., 13, 2733.[ISI][Medline]
Sasaki, T. and Endo, T. (2000) Both cell-surface carbohydrates and protein tyrosine phosphatase are involved in the differentiation of astrocytes in vitro. Glia, 32, 6070.[CrossRef][ISI][Medline]
Stadelmann, C., Kerschensteiner, M., Misgeld, T., Bruck, W., Hohlfeld, R., and Lassmann, H. (2002) BDNF and gp145trkB in multiple sclerosis brain lesions: neuroprotective interactions between immune and neuronal cells? Brain, 125, 7585.
Thoenen, H. (1995) Neurotrophins and neuronal plasticity. Science, 270, 593598.[Abstract]
Tracey, B.M., Feizi, T., Abbott, W.M., Carruthers, R.A., Green, B.N., and Lawson, A.M. (1992) Subunit molecular mass assignment of 14,654 Da to the soluble ß-galactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation. J. Biol. Chem., 267, 1034210347.
Tso, J.Y., Sun, X.H., Kao, T.H., Reece, K.S., and Wu, R. (1985) Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res., 13, 24852502.[Abstract]
Yamashita, K., Totani, K., Ohkura, T., Takasaki, S., Goldstein, I.J., and Kobata, A. (1987) Carbohydrate binding properties of complex-type oligosaccharides on immobilized Datura stramonium lectin. J. Biol. Chem., 262, 16021607.
Yurek, D.M., and Fletcher-Turner, A. (2001) Differential expression of GDNF, BDNF, and NT-3 in the aging nigrostriatal system following a neurotoxic lesion. Brain Res., 891, 228235.[ISI][Medline]
|