Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M701570200 on March 30, 2007

J. Biol. Chem., Vol. 282, Issue 21, 15843-15850, May 25, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/21/15843    most recent
M701570200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koike, N.
Right arrow Articles by Itoh, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koike, N.
Right arrow Articles by Itoh, N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Brorin, a Novel Secreted Bone Morphogenetic Protein Antagonist, Promotes Neurogenesis in Mouse Neural Precursor Cells*

Naomi Koike, Yoshiaki Kassai, Yuya Kouta, Hiroyuki Miwa, Morichika Konishi, and Nobuyuki Itoh1

From the Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Sakyo, Kyoto 606-8501, Japan

Received for publication, February 22, 2007 , and in revised form, March 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We identified a gene encoding a novel secreted protein in mice and humans and named it Brorin. Mouse Brorin consists of 324 amino acids with a putative secreted signal sequence at its amino terminus and two cysteine-rich domains in its core region. Positions of 10 cysteine residues in the domains of Brorin are similar to those in the cysteine-rich domains of members of the Chordin family. However, the amino acid sequence of Brorin is not significantly similar to that of any other member of the Chordin family, indicating that Brorin is a unique member of the family. Mouse Brorin protein produced in cultured cells was efficiently secreted into the culture medium. The protein inhibited the activity of bone morphogenetic protein 2 (BMP2) and BMP6 in mouse preosteoblastic MC3T3-E1 cells. Mouse Brorin was predominantly expressed in neural tissues in embryos and also predominantly expressed in the adult brain. In the brain, the expression was detected in neurons, but not glial cells. The neural tissue-specific expression profile of Brorin is quite distinct from that of any other member of the Chordin family. Brorin protein promoted neurogenesis, but not astrogenesis, in mouse neural precursor cells. The present findings indicate that Brorin is a novel secreted BMP antagonist that potentially plays roles in neural development and functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone morphogenetic proteins (BMPs)2 are secreted signaling molecules belonging to the TGFbeta superfamily (1). BMPs regulate embryonic development in most tissues and organs. It has become apparent that the fine-tuning of BMP signaling is critical for a variety of their functions, for example, in neural induction, limb morphogenesis, and skeletal development (2, 3). The functions of BMPs are regulated by secreted regulators. An increasing number of secreted BMP regulators have been described. In vertebrates, such proteins include Noggin, the Chordin family, Follistatin, FSRP, and the DAN/Cerberus family (3).

BMPs are also expressed in developing neural tissues (4). BMPs alter the fate of neural precursor cells from neurogenesis to astrocytogenesis (5, 6). Additional observations that BMPs are also continuously expressed in the adult brain (7-10) led us to the notion that BMPs may be involved in adult neurogenesis.

Many secreted signaling proteins, including BMPs, FGFs, Wnts, and their regulators, play crucial roles in cell proliferation and differentiation. The identification and characterization of novel secreted signaling proteins are expected to provide new insights into the mechanism of cell proliferation and differentiation. We identified many genes encoding novel secreted proteins from mouse cDNAs of unknown function in the DNA databases. We termed one of them Brorin. Brorin has two cysteine-rich domains. The positions of cysteine in the domains are similar to those of members of the Chordin family that are secreted BMP regulators (11). We report here the identification of a novel secreted BMP antagonist, Brorin, which was predominantly expressed in the neural tissues in mouse adults and embryos and promoted neurogenesis in mouse neural precursor cells. Although its physiological role remains to be elucidated, Brorin is expected to play roles in neural development and functions.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Identification of Mouse and Human Brorin—Amino acid sequences predicted from mouse cDNAs of unknown function in nucleotide sequence databases were randomly analyzed using PSORT (Prediction of Protein Sorting Signals and Localization Sites in Amino Acid Sequences) (psort.ims.u-tokyo.ac.jp/form2.html). Many cDNAs encoding putative secreted proteins were identified. We isolated full-length cDNAs by polymerase chain reaction (PCR) with the embryonic cDNA (embryonic day 14.5, E14.5) as a template and cloned them in a vector DNA, pBluescript II SK (+) (Stratagene). We termed one of the cDNAs mouse Brorin. Human Brorin cDNA was also identified in a homology-based search of human cDNA sequences in nucleotide sequence databases with the amino acid sequence of mouse Brorin.

Forced Expression of Mouse Brorin cDNA in COS-7 Cells—The mouse Brorin cDNA with a DNA fragment encoding a Myc tag (EQKLISEEDL) and a His6 tag (HHHHHH) at the 3' terminus of the coding region was constructed between EcoRI and XbaI sites in a vector DNA, pcDNA3.1(+) (Invitrogen).

COS-7 cells were plated at ~70-80% confluency (~2 x 104 cells/cm2) in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences) on 12-well plates and were transfected with the recombinant vector using Lipofectamine 2000 (Invitrogen) at 37 °C for 16 h in a humidified atmosphere of 5% CO2 in air. After the transfection, COS-7 cells were further cultured in fresh culture medium at 37 °C for 72 h.

Detection of Recombinant Mouse Brorin Protein by Western Blotting—The culture medium and lysate of the transfected cells were separated by SDS-polyacrylamide gel (12.5%) electrophoresis under reducing conditions and transferred onto a nitrocellulose membrane (Hybond-ECL; Amersham Biosciences). The protein with the Myc tag on the membrane was detected using mouse monoclonal anti-Myc tag antibody (Cell Signaling) (1:500) as primary antibody in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (ZYMED) (1:1,000) as secondary antibody in PBS containing 0.1% Tween 20. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (PerkinElmer Life Sciences) as described (12). Prestained Protein Marker Broad Range (New England Biolabs) was used as molecular mass standard proteins.

Production of Recombinant Mouse Brorin in Insect Cells—The coding region (without the amino-terminal secreted signal sequence of 27 amino acids) of mouse Brorin cDNA with a DNA fragment encoding a Myc tag and a His6 tag at the 3' terminus of the coding region was constructed in a transfer vector DNA, pAcGP67A. Recombinant baculovirus containing the cDNA was obtained by cotransfection of Sf9 cells with the recombinant pAcGP67A and a Bsu36I-digested expression vector, BacPak6.

High Five cells (~2 x 106 cells/ml) infected with the recombinant baculovirus were cultured at 27 °C for 72 h in serum-free medium EX-CELL 400 (JRH Bioscience). Recombinant mouse Brorin was purified from the culture medium by affinity chromatography using Ni-NTA agarose and desalted by gel filtration chromatography using Bio-Gel P-6 DG in PBS containing 100 µg/ml of bovine serum albumin as a carrier. Purified recombinant mouse Brorin was separated by SDS-polyacrylamide gel (12.5%) electrophoresis under reducing conditions followed by protein staining with Coomassie Brilliant Blue R-250. Purified recombinant mouse Brorin was also analyzed by Western blotting as described above.

Reverse Transcription-PCR—RNA was extracted from mouse tissue using an RNeasy Mini kit (Qiagen). Mouse cDNA was transcribed from the RNA as a template with Molony murine leukemia virus reverse transcriptase. Brorin cDNA was amplified from the cDNA with Taq DNA polymerase and primers specific for mouse Brorin (5'-acgtggtaggggatgcctagctc-3', 5'-gtctacatttgtctgcactcatg-3'). Gapdh cDNA was amplified with primers specific for mouse Gapdh (5'-tgaccacagtccatgccatc-3',5'-tccaccaccctgttgctgta-3') as a control. The amplified DNA was analyzed by agarose gel (1.5%) electrophoresis. Thereafter, the gel was stained with ethidium bromide. The expected sizes of Brorin cDNA and Gapdh cDNA are 988 and 454 base pairs, respectively.

In Situ Hybridization—For in situ hybridization, embryos and mouse brain (postnatal day 56, P56) were frozen in powdered dry ice, and sections were cut at 16 µm with a cryostat, thaw-mounted onto poly-L-lysine-coated slides, and stored at -85 °C prior to use. A 35S-labeled mouse sense or antisense Brorin RNA probe was transcribed from SpeI or HindIII-digested mouse brorin cDNA cloned in pBlueScript II SK (+) (Stratagene) using T7 or T3 RNA polymerase with uridine 5'-[{alpha}-35S]thiotriphosphate (~30 TBq/mmol) (Amersham Biosciences), respectively. The sections were examined by in situ hybridization with the labeled probe, followed by exposure to x-ray film (BioMax MR; Kodak) for 10 days or dipping in liquid emulsion (Kodak NTB3) diluted 1:1 and exposure for 3 weeks as described (13). The sections of mouse embryos and brain were counterstained with hematoxylin-eosin and cresyl-violet (Nissl staining), respectively. Silver grains were visualized by microscopy.

Alkaline Phosphatase Activity in MC3T3-E1 Cells—Mouse preosteoblastic MC3T3-E1 cells were maintained and subcultured at 37 °C for 3 or 4 days in a humidified atmosphere of 5% CO2 in air in {alpha}-modified minimum essential medium (MEM) containing 10% FBS, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin in a humidified CO2 incubator.

For the determination of alkaline phosphatase activity, MC3T3-E1 cells were plated at a density of 1 x 105 cells/well in 48-well plates for 48 h. Once confluent, the cells were cultured in {alpha}-MEM containing 10% FBS, 100 units/ml of penicillin G, 100 µg/ml of streptomycin, 10 mM beta-glycerophosphate, and 50 µg/ml of ascorbic acid for 72 h. The cells were then cultured in {alpha}-MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin for 24 h and further cultured in {alpha}-MEM containing 0.3% FBS, 100 units/ml of penicillin G, 100 µg/ml of streptomycin, recombinant human BMP2 or BMP6 (0 or 10 ng/ml) (R&D Systems), recombinant mouse Noggin (0 or 100 ng/ml) (R&D systems), and recombinant mouse Brorin protein (0-100 ng/ml) for 72 h. The cells were washed twice with ice-cold PBS and scraped in 10 mM Tris-HCl containing 2 mM MgCl2 and 0.05% Triton X-100, pH 8.2. The cell suspensions were sonicated on ice. Aliquots of supernatants were assayed for protein concentration and alkaline phosphatase activity as described (14). For the alkaline phosphatase activity, the assay mixture contained 10 mM p-nitrophenyl phosphate in 0.1 M sodium carbonate buffer, pH 10, supplemented with 1 mM MgCl2, and was incubated at 37 °C for 30 min. After 0.1 M NaOH was added, the amount of p-nitrophenol liberated was measured by using a spectrophotometer.

Phosphorylation of Smad Protein in MC3T3-E1 Cells—MC3T3-E1 cells were plated in {alpha}-MEM containing 10% FBS, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin at a density of 1 x 105 cells/well in 12-well plates for 48 h. Once confluent, the cells were cultured in {alpha}-MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin for 48 h. They were then cultured in {alpha}-MEM containing 0.3% FBS, 100 units/ml of penicillin G, and 100 µg/ml of streptomycin for 24 h and further cultured in {alpha}-MEM containing 0.3% FBS, 100 units/ml of penicillin G, 100 µg/ml of streptomycin, recombinant human BMP2 or BMP6 (0 or 10 ng/ml) (R&D Systems), recombinant mouse Noggin (0 or 100 ng/ml) (R&D Systems), and recombinant mouse Brorin protein (0 or 100 ng/ml) for 45 min. The cells were lysed in TNE buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 2 µg/ml of pepstatin. Phosphorylated Smad proteins in the cell lysate were detected by Western blotting using rabbit antibody against phosphorylated Smad1/5/8 (1:1,000) (Cell Signaling) as primary antibody in PBS containing 0.1% Tween 20 and horseradish peroxidase-conjugated goat antibody against anti-rabbit antibody (1:2,000) (VECTOR) as secondary antibody in PBS containing 0.1% Tween 20. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (PerkinElmer Life Sciences) as described (12).


Figure 1
View larger version (56K):
[in this window]
[in a new window]

  		FIGURE 1.
Comparison of amino acid sequences of mouse and human Brorin. A, comparison of amino acid sequences of mouse and human Brorin. The numbers refer to amino acid positions. Asterisks indicate identical residues in the sequences. Underlines at the amino terminus indicate putative secreted signal sequences. Underlines in the core sequence indicate cysteine-rich domains. A dash indicates a gap introduced to align the sequences. B, schematic representation of Brorin. SP and CR1 and CR2 indicate a putative secreted signal sequence and cysteine-rich domains, respectively. C, comparison of amino acid sequences of cysteine-rich domains of mouse Brorin and Crossveinless-2 (CV-2). The numbers (1-10) refer to positions of 10 conserved cysteine residues. Asterisks indicate identical residues in the sequences. Dashes indicate gaps introduced to align the sequences.

 
Culture of Mouse Neural Precursor Cells—Mouse neural precursor cells were cultured as described (5, 15). In brief, mouse embryonic diencephalon at E13.5 was mechanically dissociated into single cells in Hank's Balanced Salt Solution. The dissociated cells were seeded in N2-supplemented 1:1 Dulbecco's modified Eagle's medium: Ham's F12 containing 100 units/ml of penicillin G and 100 µg/ml of streptomycin on 35-mm dishes coated with poly-L-ornithine (15 µg/ml) (Sigma) and fibronectin (1 µg/ml) (Sigma) for differentiation and cell proliferation activity assays, respectively. The cells were cultured at 37 °C in an atmosphere of 5% CO2.

Neural Differentiation Activity Assay—Mouse neural precursor cells were seeded at a density of 3 x 105 cells/cm2 in 35-mm dishes coated with poly-L-ornithine and fibronectin. After being cultured in N2-supplemented 1:1 Dulbecco's modified Eagle's medium: Ham's F12 for 2 h, the cells were further cultured under the various conditions indicated. For immunostaining, cells were fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C and washed in PBS at room temperature for 30 min three times. They were then permeabilized with 0.1% Triton X-100 in PBS at room temperature for 30 min and immunostained using primary antibodies in PBS containing 5% bovine serum albumin and 0.1% Triton X-100 and secondary antibodies in PBS. The cells were treated with primary antibodies at 4 °C overnight. After three washes for 5 min in PBS, the cells were further treated with the secondary antibodies at room temperature for 1 h. Primary antibodies used were as follows: an anti-micro-tubule-associated protein 2 (MAP2) mouse monoclonal antibody (1:400) (Sigma) and an anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal antibody (1:100) (Sigma). Secondary antibodies used were rhodamine red- or fluorescein isothiocyanate-conjugated anti-rabbit or mouse IgG antibodies (Sigma) (1:200). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Roche Applied Science). The stained cells were observed under a fluorescence microscope. Results are the means ± S.E. for six different fields (at least 200 cells/field) from four independent slides.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Mouse and Human Brorin—We identified mouse cDNAs encoding novel putative secreted proteins by randomly analyzing amino acid sequences predicted from mouse cDNAs of unknown function in the GenBankTM nucleotide sequence data base with a computer program for the prediction of protein localization sites in cells (PSORT). The full-length cDNAs were isolated by PCR with mouse embryonic cDNA as a template. One of them encodes a putative secreted protein of 324 amino acids with a putative signal sequence (27 amino acids) at its amino terminus (GenBankTM accession code AB292670 [GenBank] ) (Fig. 1A). The protein has two cysteine-rich domains in its core region (Fig. 1, A and B). Positions of 10 cysteine residues in the domains of Brorin are similar to those in the cysteine-rich domains of members of the Chordin family, although the amino acid sequence of Brorin is not significantly similar to that of any other member of the family. The cysteine-rich domains are most similar to those of Crossveinless-2 among members of the Chordin family (16) (Fig. 1C). The gene was predominantly expressed in the brain as described below. Therefore, we termed it Brorin, short for brain-specific chordin-like protein.

Human Brorin cDNA (accession number AB292671 [GenBank] ) was also identified in a homology-based search of human cDNA sequences in GenBankTM with the amino acid sequence of mouse Brorin. The amino acid sequence of human Brorin (325 amino acids) was highly similar (~90% identity) to that of mouse Brorin (Fig. 1A). We examined the chromosomal localization of the human Brorin gene by searching the Ensembl data base with the nucleotide sequence of human Brorin cDNA. The gene was mapped to chromosome 7 at q12.2.


Figure 2
View larger version (20K):
[in this window]
[in a new window]

  		FIGURE 2.
Detection of recombinant mouse Brorin protein. A, the lysate and culture medium of COS-7 cells expressing mouse Brorin cDNA were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions. Recombinant mouse Brorin protein with a Myc tag was detected by Western blotting with anti-Myc tag antibody. B, mouse Brorin cDNA was expressed in cultured High Five insect cells by infection with a recombinant baculovirus containing the mouse Brorin cDNA with a 3'-terminal extension encoding Myc and His6 tags. Recombinant mouse Brorin protein was purified from the culture medium of High Five cells by affinity chromatography using Ni-NTA-agarose. Purified recombinant Brorin protein with bovine serum albumin as a carrier was separated by SDS-polyacrylamide gel electrophoresis under reducing conditions. The protein was detected by protein staining with Coomassie Brilliant Blue R-250 (CBB) and by Western blotting with anti-Myc tag antibody (Western). Prestained protein markers were used as molecular mass standard proteins of 62.0, 47.5, and 32.5 kDa.

 


Figure 3
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 3.
Expression of Brorin in mouse adult tissues and brains at different developmental stages. A, the expression of Brorin in mouse adult tissues (P56) was examined by RT-PCR followed by agarose gel (1.5%) electrophoresis. The gel was stained with ethidium bromide. The expression of Gapdh was also examined as a control. B, the expression of Brorin in mouse brain at different developmental stages (E12.5-P56) was examined by RT-PCR. The expression of Gapdh was also examined as a control.

 
Forced Expression of Mouse Brorin cDNA in Cultured Cells, COS-7 Cells—To examine whether Brorin is a secreted protein, mouse cDNA encoding Brorin with Myc and His6 tags was expressed in cultured cells, COS-7 cells. Both the culture medium and cell lysate were examined by reducing SDS-poly-acrylamide gel electrophoresis followed by Western blotting with anti-Myc tag antibody. No bands were detected in the cell lysate or culture medium of COS-7 cells transfected with the empty vector as a control (data not shown). A major band of ~49 kDa was mainly detected in the culture medium of COS-7 cells transfected with the Brorin expression vector, indicating that Brorin is a secreted protein (Fig. 2A). The observed molecular mass was larger than the calculated molecular mass of the recombinant Brorin protein (~38.1 kDa), indicating that Brorin protein might be subjected to post-translational modification.

Production of Purified Recombinant Mouse Brorin Protein—To prepare purified recombinant mouse brorin protein, mouse Brorin cDNA was expressed in cultured High Five insect cells by infection with a recombinant baculovirus containing the mouse Brorin cDNA with the 3'-terminal extension encoding Myc and His6 tags. Recombinant mouse Brorin was purified from the culture medium of High Five cells by affinity chromatography using Ni-NTA agarose. Purified recombinant mouse Brorin was separated by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by protein staining. Purified recombinant Brorin showed a single band of 48 kDa with a band of bovine serum albumin (66 kDa) as a carrier (Fig. 2B). Purified recombinant mouse Brorin was also analyzed by Western blotting. Purified recombinant Brorin also showed a single band of 48 kDa (Fig. 2B).

Expression of Brorin in Mouse Tissues and Embryos Examined by RT-PCR—The expression of Brorin in mouse tissues (P56) was examined by RT-PCR. Although the expression of Gapdh as a control was detected in all the tissues examined, the expression of Brorin was mostly detected in the brain among major tissues examined, indicating that Brorin was predominantly expressed in the brain (Fig. 3A). We also examined the expression of Brorin in the brain at different developmental stages (E12.5-P56) by RT-PCR. The expression was detected in the brain at all the stages examined. However, the expression gradually increased with development (Fig. 3B).

Expression of Brorin in Mouse Embryos and Adult Brain Examined by in Situ Hybridization—The expression of Brorin in mouse embryos at E12.5, E16.5, and E18.5 was examined by in situ hybridization with a 35S-labeled antisense Brorin cRNA probe using sagittal sections. The expression in embryos was shown by red grains (Fig. 4A). In contrast, essentially no grains were detected in embryos with a 35S-labeled sense Brorin cRNA probe as a control (data not shown). The expression of Brorin was predominantly detected in the developing diencephalon at E12.5 and in the developing neural tissues and tongue at E16.5 and E18.5. However, essentially no Brorin expression was observed in the developing cerebral cortex at E16.5 and E18.5.

The expression of Brorin in the brain at P56 was also examined by in situ hybridization with the antisense Brorin probe using consecutive coronal sections. The expression of Brorin, shown by red grains, was widely detected in the brain (Fig. 4B, a-i). However, prominent expression of Brorin was observed in the diencephalon and medulla oblongata. In contrast, essentially no grains were detected with the sense Brorin cRNA probe as a control (Fig. 4B, j-r).


Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 4.
Localization of Brorin mRNA in mouse embryos and adult brain. A, sagittal sections of mouse embryos at E12.5, E16.5, and E18.5 were examined by in situ hybridization with a 35S-labeled antisense mouse Brorin RNA probe. The sections were counterstained with hematoxylin-eosin. Red grains superimposed upon a hematoxylin-eosin stain show the localization of Brorin mRNA. B, coronal sections of mouse brain (P56) were also examined by in situ hybridization with a 35S-labeled antisense (a-i) or sense (j-r) mouse Brorin RNA probe, followed by exposure to x-ray film. Red grains show the location of Brorin mRNA. Scale bar, 5 mm. C, the cellular localization of Brorin mRNA in the diencephalon (a) and medulla oblongata (b) of mouse brain (P56) was examined by in situ hybridization as described above. The sections of the brain were counterstained with cresyl-violet. Black grains in bright field photographs show the location of Brorin mRNA. The arrows and arrowheads indicate neurons and glial cells, respectively. Scale bar, 50 µm.

 
The cellular distribution of Brorin mRNA in the brain was examined using in situ hybridization followed by microautoradiography. With Nissl staining of brain sections, glial cells appeared as small intensely stained (dark) cells. In contrast, neurons were generally larger and less intensely stained (lighter) because of their volume (17). In the diencephalon and medulla oblongata, Brorin mRNA was detected in neurons, but not in glial cells (Fig. 4C). In other regions of the brain, Brorin mRNA was also detected in neurons (data not shown). These results indicated that Brorin in the brain was expressed in neurons but not glial cells.

Effects of Brorin on BMP Signaling in Cultured Osteoblasts, MC3T3-E1—Positions of 10 cysteine residues in the cysteine-rich domains of Brorin are similar to those in the cysteine-rich domains of members of the chordin family. Most members of this family are regulators of BMP signaling, indicating that Brorin might be also. BMPs were originally identified as proteins in bone that induce ectopic bone and cartilage to form in vivo. BMPs are signaling molecules for the stimulation of osteoblastic differentiation (18). Most are members of the dpp and 60A subgroups of the TGFbeta superfamily (4). We examined the effect of recombinant mouse Brorin on the activity of BMP2 and BMP6, as representatives of the dpp and 60A subgroups, respectively, for the differentiation of MC3T3-E1 cells by determining alkaline phosphatase activity, a marker for osteoblastic differentiation (19). We also examined the effect of recombinant mouse Noggin proteins as a control of a BMP antagonist. BMP2 and BMP6 greatly stimulated the alkaline phosphatase activity in MC3T3-E1 cells (Fig. 5A). The activity stimulated by BMP2 and BMP6 was inhibited by Brorin in a dose-dependent manner as well as by Noggin (Fig. 5A), indicating that Brorin is a BMP antagonist.


Figure 5
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 5.
Effects of recombinant mouse Brorin protein on alkaline phosphatase activity and phosphorylation of Smad in MC3T3-E1 cells induced by BMP. A, MC3T3-E1 cells were treated with recombinant human BMP2 or BMP6 proteins and mouse Brorin or Noggin proteins for 72 h. After treatment, the alkaline phosphatase activity in MC3T3-E1 cells was measured. B, MC3T3-E1 cells were treated with recombinant human BMP2 or BMP6 proteins (0 or 10 ng/ml) and mouse Brorin or Noggin proteins (0 or 100 ng/ml) for 45 min. After treatment, phosphorylated Smad1/5/8 in MC3T3-E1 cells was detected by Western blotting.

 
BMPs also induce the phosphorylation of Smad in MC3T3-E1 cells. To confirm the antagonistic activity of Brorin, we also examined effects of Brorin and Noggin as a control on the phosphorylation of Smad induced by BMPs in MC3T3-E1 cells. BMP2 and BMP6 induced the phosphorylation of Smad1/5/8 in MC3T3-E1 cells (Fig. 5B). The phosphorylation induced by BMP2 and BMP6 was inhibited by Brorin as well as Noggin (Fig. 5B), also indicating that Brorin is a BMP antagonist.

Effects of Brorin on Neuronal and Astrocytic Differentiation in Cultured Neural Precursor Cells—The temporal and regional expression profiles of Brorin indicated its potential roles in neural differentiation. As the prominent expression of Brorin was observed in mouse embryonic diencephalons, we examined the effect of recombinant mouse Brorin protein on the neural differentiation of mouse diencephalic neural precursor cells. As FBS promotes neural differentiation in neural precursor cells (20), we also examined the effect of FBS (10%) on the differentiation as a positive control. After the neural precursor cells had been cultured in the presence of FBS or Brorin protein for 3 days, the differentiation was examined by double immunostaining using anti-MAP2 and GFAP antibodies for neurons and astrocytes, respectively (Fig. 6A). Green and red signals indicate MAP2-positive and GFAP-positive cells, respectively. Blue signals indicate cell nuclei counterstained with DAPI. The effects of FBS and Brorin on the total number of cells and neural differentiation were quantified by counting DAPI-positive cell nuclei and MAP2-positive and GFAP-positive cells (Fig. 6, B-D). FBS significantly increased the total number of cells. FBS also significantly increased the proportion of MAP2-positive cells and generated GFAP-positive cells among the neural precursor cells. In addition, FBS greatly stimulated the out-growth of neurites in MAP2-positive cells. As Brorin slightly increased the total number of cells, it may promote the proliferation and/or survival of the neural precursor cells. In addition, Brorin increased the proportion of MAP2-positive cells among the neural precursor cells and significantly stimulated their neurite outgrowth. However, treatment with Brorin did not produce GFAP-positive cells. These results indicate that Brorin induced neurogenesis, but not astrogenesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Brorin Is a Novel Secreted BMP Antagonist of the Chordin Family with a Unique Structure—BMPs play important roles in many organogenic processes, although they were originally identified as factors promoting the ectopic formation of cartilage and bone. The activity of BMP is controlled in part by secreted BMP regulators, including Noggin, members of the Chordin family, and members of the Dan/Cerberus family (3). The Chordin family is a group of proteins with cysteine-rich domains that consist of 10 cysteine residues. The family comprises Chordin, Chordin-like (CHL)/Neuralin, CHL2 (11), Neurogenesin-1 (21, 22), Crossveinless-2 (16, 23), and Kielin/chordin-like protein (24). These members have different numbers of cysteine-rich domains and no structural similarity to each other. CHL, CHL2, and Neurogenesin-1 have three cysteine-rich domains. Chordin, Crossveinless, and Kielin/chordin-like protein have four, eighteen, and five cysteine-rich domains, respectively. They are secreted regulators of BMP signaling. Chordin, CHL/Neuralin, CHL2, and Neurogenesin-1 are BMP antagonists. In contrast, Crossveinless-2 functions as a BMP antagonist and a pro-BMP factor. Kielin/chordin-like protein is a positive regulator of BMP signaling.


Figure 6
View larger version (53K):
[in this window]
[in a new window]

  		FIGURE 6.
Effects of recombinant mouse Brorin protein on neuronal and astrocytic differentiation in mouse neural precursor cells. A, mouse neural precursor cells were cultured in the presence of recombinant mouse Brorin protein (100 ng/ml) or FBS (10%). After being cultured for 3 days, the neural precursor cells were examined by double immunochemistry using anti-MAP2 and GFAP antibodies. Green and red signals indicate MAP2-positive and GFAP-positive cells, respectively. Blue signals indicate cell nuclei counter-stained with DAPI. Scale bar, 200 µm. B, the effect of Brorin protein on the total number of cells was quantified by counting cell nuclei counter-stained with DAPI. C, the effect of Brorin protein on neuronal differentiation was quantified by counting MAP2-positive cells. D, the effect of Brorin protein on astrocytic differentiation was quantified by counting GFAP-positive cells. Results are means ± S.E. of four different fields from four independent slides. *, p <0.005.

 
Brorin, a novel secreted BMP antagonist, has only two cysteine-rich domains, although the other members of the Chordin family have three to eighteen cysteine-rich domains as described above. In addition, the amino acid sequence of Brorin has no structural similarity to other members of the family, although the positions of cysteine residues in the domains are similar to those of other members. These results indicate that Brorin is a unique member of the Chordin family.

Brorin Is Preferentially Expressed in Adult and Embryonic Neural Tissues—Mouse Chordin is expressed at relatively high levels in mouse embryos at E7 and at much decreased levels later in development and in adult tissues (25). Mouse CHL/Neuralin is expressed in the neural plate (26). Later during development, Neuralin/CHL is expressed in neural tissue derivatives and in many mesenchymes, including the forming axial skeleton (26, 27). Mouse CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs (28). Neurogenesin-1 is expressed in the hippocampus and around the lateral ventricle in the adult brain and in the primordial cells of the skeleton (21, 22). Crossveineless-2 is expressed dynamically during mouse development, in particular in regions of strong BMP signaling such as the posterior primitive streak, ventral tail bud, and prevertebral cartilages (16). In mouse embryos, Kielin/chordin-like protein expression was prominent in two areas, the limb buds and the developing kidney, with diffuse staining in the central nervous system (24).

Brorin was expressed in restricted neural regions. At E12.5, Brorin was predominantly expressed in the diencephalon. Later in development (E16.5 and E18.5), Brorin was also predominantly expressed in the brain and spinal cord. However, no Brorin expression was observed in the cerebral cortex. In addition, Brorin was predominantly expressed in the brain among adult tissues examined. The expression of Brorin was wide-spread in neurons of the brain. This neural tissue-specific expression profile of Brorin is quite distinct from that of any other member of the Chordin family, indicating that Brorin might play unique roles in developing and adult neural tissues.

Possible Roles of Brorin in the Developing and Adult Neural Tissues—The developing neural tissues express multiple BMPs, including BMPs 2, 4, 5, 6, and 7. BMPs are also expressed in the postnatal and adult brain. Transcript levels for BMPs generally peak during the perinatal period and then decline in the adult neural tissues (4). However, strong signals are still detectable in discrete structures of the mature brain. For example, the adult hippocampus expresses relatively high levels of BMP5 and BMP6; the cortex: BMP5 and BMP6; the cerebellum: BMP5; the striatum: BMP5 and BMP7; and the brainstem: BMP5 and BMP6 (7, 29, 30). In addition, BMP 2, 4, 5, and 6 are expressed in the adult cortex, hippocampus, striatum, and substantia nigra (10). These expression profiles indicate that BMP signaling plays roles in developing and adult neural tissues.

BMP2, BMP4, and BMP7 inhibit neurogenesis and concomitantly induce astrogenesis in mouse neural precursor cells (5, 6). In contrast to BMPs, Brorin significantly promoted neurogenesis, but not astrogenesis, in mouse neural precursor cells. As Brorin is a BMP antagonist, the present finding is consistent with the findings that BMPs inhibit neurogenesis (5, 6). Recently, we also identified brorin in zebrafish. Zebrafish brorin was also predominantly expressed in the neural tissues in embryos. We generated brorin knockdown zebrafish embryos. Our preliminary analysis of the phenotype shows that development of the brain was greatly impaired, indicating that brorin potentially plays a role in the embryonic development of the brain.3 Although its physiological role remains to be elucidated, Brorin is expected to act as a BMP antagonist during neurogenesis in both the adult brain and developing neural tissues. The present findings should provide new insights into the roles of BMP signaling in neural tissues.


   FOOTNOTES
 
* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan (to N. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Yoshida-Shimoadachi, Sakyo, Kyoto 606-8501, Japan. Tel.: 81-75-753-4540; Fax: 81-75-753-4600; E-mail: itohnobu{at}pharm.kyoto-u.ac.jp.

2 The abbreviations used are: BMP, bone morphogenetic protein; FBS, fetal bovine serum; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilotriacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; {alpha}-MEM, {alpha}-minimal essential medium; GFAP, glial fibrillary acidic protein; MAP, microtubule-associated protein; DAPI, 4',6-diamidino-2-phenylindole; CHL, Chordin-like; RT-PCR, reverse transcription PCR. Back

3 N. Itoh, Y. Nakayama, and A. Miyake, unpublished observation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kingsley, D. M. (1994) Genes Dev. 8, 133-146[Free Full Text]
  2. Von Bubnoff, A., and Cho, K. W. (2001) Dev. Biol. 239, 1-14[CrossRef][Medline] [Order article via Infotrieve]
  3. Balemans, W., and Van Hul, W. (2002) Dev. Biol. 250, 231-250[CrossRef][Medline] [Order article via Infotrieve]
  4. Lein, P., Drahushak, K. M., and Higgins, D. (2002) Bone Morphogenetic Proteins: from Laboratory to Clinical Practice, pp. 289-319, Birkhauser Verlag, Basel, Switzerland
  5. Nakashima, K., Takizawa, T., Ochiai, W., Yanagisawa, M., Hisatsune, T., Nakafuku, M., Miyazono, K., Kishimoto, T., Kageyama, R., and Taga, T. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 5868-5873[Abstract/Free Full Text]
  6. Yanagisawa, M., Takizawa, T., Ochiai, W., Uemura, A., Nakashima, K., and Taga, T. (2001) Neurosci. Res. 41, 391-396[CrossRef][Medline] [Order article via Infotrieve]
  7. Mehler, M. F., Mabie, P. C., Zhang, D., and Kessler, J. A. (1997) Trends Neurosci. 20, 309-317[Medline] [Order article via Infotrieve]
  8. Ebendal, T., Bengtsson, H., and Soderstrom, S. (1998) J. Neurosci. Res. 51, 139-146[CrossRef][Medline] [Order article via Infotrieve]
  9. Charytoniuk, D. A., Traiffort, E., Pinard, E., Issertial, O., Seylaz, J., and Ruat, M. (2000) Neuroscience 100, 33-43[Medline] [Order article via Infotrieve]
  10. Chen, H. L., Lein, P. J., Wang, J. Y., Gash, D., Hoffer, B. J., and Chiang, Y. H. (2003) Brain Res. 994, 81-90[CrossRef][Medline] [Order article via Infotrieve]
  11. Garcia Abreu, J., Coffinier, C., Larrain, J., Oelgeschlager, M., and De Robertis, E. M. (2002) Gene 287, 39-47[Medline] [Order article via Infotrieve]
  12. Yamashita, T., Konishi, M., Miyake, A., Inui, K., and Itoh, N. (2002) J. Biol. Chem. 277, 28265-28270[Abstract/Free Full Text]
  13. Yazaki, N., Hosoi, Y., Kawabata, K., Miyake, A., Minami, M., Satoh, M., Ohta, M., Kawasaki, T., and Itoh, N. (1994) J. Neurosci. Res. 37, 445-452[CrossRef][Medline] [Order article via Infotrieve]
  14. Takeuchi, Y., Suzuwa, M., Kikuchi, T., Nishida, E., Fujita, T., and Matsumoto, T. (1997) J. Biol. Chem. 272, 29309-29316[Abstract/Free Full Text]
  15. Kimura, I., Konishi, M., Miyake, A., Fujimoto, M., and Itoh, N. (2006) J. Neurosci. Res. 83, 1415-1424[CrossRef][Medline] [Order article via Infotrieve]
  16. Coffinier, C., Ketpura, N., Tran, U., Geissert, D., and De Robertis, E. M. (2002) Mech. Dev. 119, S179-184[CrossRef][Medline] [Order article via Infotrieve]
  17. Gerfen, C. R. (1989) Methods Neurosci. 60, 101-114
  18. Hogan, B. L. (1996) Genes Dev. 10, 1580-1594[Free Full Text]
  19. Franceschi, R. T., Iyer, B. S., and Cui, Y. (1994) J. Bone Miner. Res. 9, 843-854[Medline] [Order article via Infotrieve]
  20. Brunet, J. F., Grollimund, L., Chatton, J. Y., Lengacher, S., Magistretti, P. J., Villemure, J. G., and Pellerin, L. (2004) Glia 46, 8-17[CrossRef][Medline] [Order article via Infotrieve]
  21. Ueki, T., Tanaka, M., Yamashita, K., Mikawa, S., Qiu, Z., Maragakis, N. J., Hevner, R. F., Miura, N., Sugimura, H., and Sato, K. (2003) J. Neurosci. 23, 11732-11740[Abstract/Free Full Text]
  22. Chandra. A., Itakura, T., Yang, Z., Tamakoshi, T., Xue, X., Wang, B., Ueki, T., Sato, K., Uezato, T., and Miura, N. (2006) Biochem. Biophys. Res. Commun. 344, 786-791[CrossRef][Medline] [Order article via Infotrieve]
  23. Ikeya, M., Kawada, M., Kiyonari, H., Sasai, N., Nakao, K., Furuta, Y., and Sasai, Y. (2006) Development 133, 4463-4473[Abstract/Free Full Text]
  24. Lin, J., Patel, S. R., Cheng, X., Cho, E. A., Levitan, I., Ullenbruch, M., Phan, S. H., Park, J. M., and Dressler, G. R. (2005) Nat. Med. 11, 387-393[CrossRef][Medline] [Order article via Infotrieve]
  25. Pappano, W. N., Scott, I. C., Clark, T. G., Eddy, R. L., Shows, T. B., and Greenspan, D. S. (1998) Genomics 52, 236-239[CrossRef][Medline] [Order article via Infotrieve]
  26. Coffinier, C., Tran, U., Larrain, J., and De Robertis, E. M. (2001) Mech. Dev. 100, 119-122[CrossRef][Medline] [Order article via Infotrieve]
  27. Nakayama, N., Han, C. E., Scully, S., Nishinakamura, R., He, C., Zeni, L., Yamane, H., Chang, D., Yu, D., Yokota, T., and Wen, D. (2001) Dev. Biol. 232, 372-387[CrossRef][Medline] [Order article via Infotrieve]
  28. Nakayama, N., Han, C. Y., Cam, L., Lee, J. I., Pretorius, J., Fisher, S., Rosen-feld, R., Scully, S., Nishinakamura, R., Duryea, D., Van, G., Bolon, B., Yokota, T., and Zhang, K. (2004) Development 131, 229-240[Abstract/Free Full Text]
  29. Tomizawa, K., Matsui, H., Kondo, E., Miyamoto, M., Tokuda, M., Itano, T., Nagahata, S., Akagi, T., and Hatase, O. (1995) Brain Res. Mol. Brain Res. 28, 122-128[Medline] [Order article via Infotrieve]
  30. Strelau, J., Sullivan, A., Bottner, M., Lingor, P., Falkenstein Suter-Crazzolara, F. C., Galter, D., Jai, J., Krieglstein, K., and Unsicker, K. (2000) J. Neurosci. 20, 8597-8603[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
D. J. Mahoney, K. Mikecz, T. Ali, G. Mabilleau, D. Benayahu, A. Plaas, C. M. Milner, A. J. Day, and A. Sabokbar
TSG-6 Regulates Bone Remodeling through Inhibition of Osteoblastogenesis and Osteoclast Activation
J. Biol. Chem., September 19, 2008; 283(38): 25952 - 25962.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/21/15843    most recent
M701570200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koike, N.
Right arrow Articles by Itoh, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koike, N.
Right arrow Articles by Itoh, N.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement