The Trk family of neurotrophin receptors is downregulated in the lumbar spines of rats with congenital kyphoscoliosis
Daisuke Tsunoda · Haku Iizuka · Tsuyoshi Ichinose · Yoichi Iizuka · Tokue Mieda · Noriaki Shimokawa · Kenji Takagishi · Noriyuki Koibuchi
D. Tsunoda · N. Shimokawa () · N. Koibuchi
Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan
D. Tsunoda · H. Iizuka · T. Ichinose · Y. Iizuka · T. Mieda · K. Takagishi
Department of Orthopedic Surgery, Gunma University Graduate School of Medicine, Gunma, Japan
N. Shimokawa ()
Department of Nutrition, Takasaki University of Health and Welfare, Gunma, Japan
Corresponding author: Noriaki Shimokawa, Ph.D.
Department of Nutrition, Takasaki University of Health and Welfare, 37-1 Nakaorui-machi, Takasaki, Gunma 371-0033, Japan
Phone: +81-27-352-1284 Fax : +81-27-353-2055
E-mail: [email protected] (N. Shimokawa)
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research (C) (25462286) from the JSPS (to H.I.) and the Mishima Kaiun Memorial Foundation, Japan (to N.S).
Abstract
Congenital scoliosis is a condition characterized by spinal curvature beyond the physiological norm. The molecular mechanisms underlying the pathogenesis of congenital scoliosis are beginning to be clarified; however, the genes related to congenital scoliosis are still unknown.
We herein report the results of a comprehensive analysis of gene expression in the spines from a rat model of congenital kyphoscoliosis obtained using DNA microarrays. The rats (Ishibashi rats, IS) showed decreased expression levels of genes associated with bone formation, such as those associated with retinol metabolism and type I collagen. Interestingly, the flexion sites of the IS rats showed low expression levels of tropomyosin receptor kinases (Trks: TrkA, TrkB and TrkC), which belong to the neurotrophic receptor tyrosine kinase family. Moreover, this phenomenon was observed only in the flexion sites of the spine, and the expression levels of Trks in other parts of the spine in these rats were normal. The decreased expression levels of Trks were observed at both the mRNA and protein levels. We also observed that the number of Trk-immunopositive cells in the lumbar spine in the IS rats was lower than that in wild-type rats.
These findings indicate that the Trks have an important function in regulating normal bone formation, and provide a molecular explanation for the pathogenesis of congenital kyphoscoliosis.
Keywords Congenital scoliosis · Kyphoscoliosis model rats · DNA microarrays · Trk family neurotrophin receptors
Introduction
Scoliosis is a medical condition characterized by an abnormal curvature and deformity of the spinal vertebrae. There are three main types of scoliosis: congenital, idiopathic and symptomatic secondary to another condition, such as cerebral palsy or amyotrophy [1]. Scoliosis is difficult to detect early in the course of the disease, because the curvature is rarely painful at that time.
Presently, surgery is the only way to treat patients with scoliosis. A detailed analysis of the pathogenic mechanisms and the ability to detect the disease early in its course by genetic testing could lead to the prevention of disease progression, decreasing the patients’ pain and burden and possibility avoiding the need for surgery. Genetic testing for adolescent idiopathic scoliosis was started in 2009 and is still ongoing [2]. However, little is known about the genetic background and key causal genes of congenital scoliosis.
Ishibashi (IS) rats were established by Ishibashi in 1968 as an inbred strain from a male wild-type and a female Wistar rat [3]. The IS rats are characterized by kyphoscoliosis due to congenital abnormalities of the lumbar vertebrae, and are generally regarded as an animal model of human congenital vertebral malformation. The kyphoscoliosis in this rat model is associated with restricted spinal canals and compressed spinal cords. Yamada et al. [4] suggested that one or more genetic factors may be involved in the development of this phenotype. In a previous study [5], we found that the expression levels of Hox 10 and Hox 11 paralogs, which regulate the formation of the lumbar and sacral vertebrae [6], are extremely low in the lumbosacral transitional areas of IS rats compared with those of normal rats. These findings strongly suggest that the marked decrease in Hox 10 and 11 expression levels may cause a homeotic transformation to phenotypes other than kyphoscoliosis in IS rats. However, the genetic factors responsible for the kyphoscoliosis have not yet been identified.
DNA microarray analyses have recently been verified to be effective for identifying the genes involved in adolescent idiopathic scoliosis [7, 8]. However, this method has not yet been applied for the analysis of congenital scoliosis. In this study, we investigated congenital kyphoscoliosis-related genes by a DNA microarray analysis. We herein demonstrate that the
expression levels of tropomyosin receptor kinases (Trks), which belong to the neurotrophic receptor tyrosine kinase family, are significantly lower in the lumbar spines of IS rats compared to normal rats.
Materials and methods Animals
The IS rats, which were used as a model of congenital kyphoscoliosis, were prepared as described in our previous work [5]. Briefly, homozygote IS rats were registered with The National Bio Resource Project for the Rat in Japan (NBRP-Rat) and were provided by the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University (Kyoto, Japan). Wistar rats (Wt; Japan SLC Inc., Shizuoka, Japan) were used as the wild-type controls and were treated in the same manner as IS rats. The animal experimental protocol used in this study was approved by the Animal Care and Experimentation Committee, Gunma University Showa Campus. All rats were housed individually under controlled light (12 h light: 12 h dark) and temperature (21 ± 1 C) conditions. Homozygotes (IS × IS) were obtained by mating. The day of delivery was designated as postnatal day (P) 0.
Homozygous newborn rats were divided into three groups (n=7/group). The first and second groups of rats were subjected to RNA and protein extraction, respectively, from samples from the spine on P4. The third group was subjected to an immunohistochemical analysis of samples from the spine on P4. In a pilot study, we found that the extraction of RNA/protein from the spine was easiest on P4 because the ossification is less-advanced. Therefore, we used the spines on P4 throughout this study. A sufficient amount of RNA for the DNA microarrays was extracted. Age-matched control rats (n=7/group) were also used for each experiment.
RNA extraction
For the DNA microarray and RT-PCR analyses, IS and Wt rats were killed on P4 by decapitation under deep anesthesia with diethyl ether. The third to fifth lumbar spine segments
(L3 – L5), where deformities appear most commonly in IS rats [4], were obtained. The 10th to 12th thoracic spine segments (Th10 – Th12), which do not show kyphoscoliosis in IS rats, were also obtained for comparison. RNA was extracted from these vertebral segments using an RNeasy kit (Qiagen, Hilden, Germany) and was treated with DNase I (Qiagen).
DNA microarray analysis
The rat gene expression (approximately 20,000 distinct genes) was analyzed using the 3D-Gene Rat Oligo chip 20k (Toray industries, Tokyo, Japan). Total RNA samples extracted from IS and Wt rats were labeled with Cy3 and Cy5, respectively, and hybridization was performed in accordance with the supplier’s protocol. Hybridization signals were scanned using a 3D gene scanner (Toray industries). The raw signal intensity of each spot was normalized by substitution with the background signal intensity. The intensities of the detected signals for each gene were normalized by a global normalization method [9].
Quantitative RT-PCR
cDNA was reverse-transcribed from total RNA using a High-Capacity RNA-to-DNA kit (Applied Biosystems, Foster City, CA). The synthesized cDNA was subjected to a quantitative RT-PCR analysis using an ABI StepOne-Real-Time PCR platform (Applied Biosystems) with TaqMan probes (Applied Biosystems) specific for rat TrkA (Rn00561634_m1), TrkB (Rn01441749_m1) and TrkC (Rn00570389_m1). The mRNA expression levels were normalized by comparison with the rat glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA expression levels (Rn01775763_q1).
Western blot analysis
The protein expression levels of the Trks in the spine were measured by a Western blot analysis.
Protein extraction from spinal samples was performed as described previously [10]. Briefly, L3 – L5 of the IS and Wt rats on P4 were removed and homogenized in lysis buffer containing
protease inhibitors, and proteins were separated by SDS-PAGE. Immunoblotting was performed using a rabbit monoclonal anti-TrkA antibody (EP1058Y, 1:1000, Abcam plc, Cambridge, UK), a rabbit monoclonal anti-TrkB antibody (80E3, 1:1000, Cell Signaling Technology, Danvers, MA) or a rabbit monoclonal anti-TrkC antibody (C44H5, 1:1000, Cell Signaling Technology), and then samples were incubated with an anti-rabbit IgG coupled to HRP (GE Healthcare, Little Chalfont, UK). The antibody-antigen complexes were detected using the ECL system (GE Healthcare). Blots were reprobed with an anti--actin antibody (#4967, 1:5000, Cell Signaling Technology) to determine the quantity of proteins. The signals were imaged (ImageQuant LAS4000, GE Healthcare) and the band intensities were determined using the ImageJ software program, version 1.41 (NIH, Bethesda, MD).
Immunohistochemistry
For the immunohistochemical analysis, L3 – L5 of IS and Wt rats on P4 were immediately removed and fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS, 0.2 M, pH 7.40) overnight at 4˚C. The fixed lumbar spines were embedded in paraffin and cut into coronal 8-µm-thick sections, which were then mounted on silane-coated glass slides. After deparaffinization and rehydration, the sections were autoclaved for antigen activation and were incubated in 0.1 M PBS containing 1.0% H2O2/methanol for 30 min to inhibit endogenous peroxidase activity. After being blocked with nonfat milk to prevent nonspecific binding, the sections were incubated with a rabbit monoclonal anti-TrkA antibody (EP1058Y, 1:100, Abcam), rabbit anti-TrkB antibody (sc-12, 1:50, Santa Cruz Biotechnology, Inc., Dallas, TX) or rabbit monoclonal anti-TrkC antibody (C44H5, 1:500, Cell Signaling Technology) overnight at 4˚C. Next, the sections were incubated with biotinylated horse anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA) for 60 min at room temperature. After being rinsed in PBS, the sections were incubated with standard ultrasensitive ABC reagent (Vector Laboratories) for 1 h and visualized by reaction with 3,3’-diaminobenzidine (0.5 mg/ml Tris-HCl containing 0.01%
H2O2). After nuclear staining with Mayer’s hematoxylin solution, the slides were dehydrated
using a graded series of ethanol followed by a series of xylene, and were coverslipped with a mounting solution.
Statistical analysis
The quantitative data were expressed as the means ± SEM of the individual rats in each experimental group. The statistical analyses were conducted using an ANOVA. Post-hoc comparisons were carried out by Duncan’s multiple range tests. A value of P < 0.05 was considered to be statistically significant.
Results
Microarray analysis of lumbar spines
In the lumbar spines, 194 genes (90 genes upregulated, Cy3/Cy5 ratio ≥8.0-fold; 104 genes downregulated, Cy3/Cy5 ratio ≤0.125) were found to show altered expression levels in the IS rats compared to the Wt rats (Fig. 1A, Supplementary Tables 1, 2). According to the results of a gene clustering analysis, the genes significantly downregulated in IS rats were divided into several functional groups. In the lumbar spines of IS rats, the genes showing altered expression levels were related to retinol metabolism, namely, Rala, Adh1, Rbp1, Crabp1 and Aldh1a2 (data not shown), and the neurotrophin receptor family, TrkA, TrkB, and TrkC (Fig. 1A). The Cy3/Cy5 ratios of the Trks to Gapdh (as a control) are shown in Fig. 1B.
Validation of the transcription in lumbar spines
Among the genes showing altered expression levels in the lumbar spine of IS rats, we focused on validating those belonging to the neurotrophin receptor family by a real-time RT-PCR analysis. In the lumbar spines of the IS rats, the mRNA expression levels of all Trks were significantly lower than those of the Wt controls (Figs. 2A-C). The expression levels of TrkA, TrkB and TrkC in the IS rats were lower by 45%, 32% and 54%, respectively, than those in Wt rats.
On the other hand, the mRNA expression levels of TrkB and TrkC in the lumbar spines (L3 – L5) were significantly lower than those in the thoracic spines (Th10 – Th12) (Figs. 2E, 2F) of the same IS rats. Although the TrkA mRNA expression levels in the lumbar spines tended to be lower than those in the thoracic spines, no significant difference was observed (Fig. 2D).
Protein expression levels of Trks in IS
Next, we analyzed the protein expression levels of Trks in the lumbar spines. The expression levels of all Trks in the spines of the IS rats were lower than those in the Wt rats (Fig. 3). The expression levels of TrkA, TrkB and TrkC in the IS rats were lower by 34%, 53% and 27%, respectively, than those in Wt rats, with the levels of TrkB and TrkC showing significant differences.
With regard to the immunohistochemical findings, we identified numerous Trk-immunopositive cells that were scattered among the poorly-differentiated cells on the bone marrow side of the hypertrophic chondrocyte layer in the lumbar vertebra of Wt rats (Figs.
4A-C, Table 1). However, in IS rats, such cells were smaller in number by 50 – 70% compared to the Wt rats (Figs. 4D-F, Table 1).
Discussion
In the present study, we showed that the expression levels of Trks are significantly decreased in the lumbar spines of IS rats. The results of the DNA microarrays were supported by data from the quantitative RT-PCR and Western blot analyses. The decrease in Trk-positive cells in the lumbar spines of IS rats was also confirmed by immunohistochemistry. Furthermore, we found that Trk-immunopositive cells were present in the vertebrae, not in the intervertebral disks.
Therefore, in immature lumbar spines, the signaling of neurotrophic factors affects the vertebrae.
This study is the first to apply a DNA microarray analysis as a comprehensive analysis of the gene expression in congenital kyphoscoliosis. By using this method, several genes that may
be involved in bone formation and metabolism were identified. In the most common site for malformation of the lumbar spines in IS rats, the expression levels of retinol metabolism-related genes and Trks were decreased. Retinol is one of the forms of vitamin A, and vitamin A in human blood is mostly in the form of retinol. Vitamin A-deficient rats show hypoplastic cranial bones, ectopic bones in the dorsal regions of cervical spinal nerve 1 (C1), and malformation of the sternal and pelvic regions [11]. A recent study has shown that vitamin A deficiency during the fetal period induces many skeletal malformations in the cervical, thoracic, pelvic and sacral regions, as well as the limbs [12]. Therefore, we expected that the expression levels of retinol metabolism-related genes in the spines of IS rats would be decreased.
On the other hand, we could not predict the relationship between the expression of neuronal factors and their receptors and the pathogenesis of congenital kyphoscoliosis. It has recently been reported that neuronal factors and their receptors have critical roles in bone formation. For example, semaphorin 3A, a regulator of axon/dendrite growth [13], binds to its receptor, neuropilin-1, and regulates the bone mass by inhibiting osteoblast differentiation [14].
Therefore, we focused on the up- or down-regulated genes encoding neuronal molecules and their receptors. Among the identified genes, there were significant decreases in the expression levels of all Trks (TrkA, TrkB and TrkC) in the spines of IS rats, and these were the most interesting results for us, since there has been no previous report of the relevance of Trks to the pathogenesis of scoliosis and kyphoscoliosis. Furthermore, IS rats are generally regarded as an animal model of human spinal malformation in which the deformities of the bone are restricted to the spinal vertebra. It is unlikely that the congenital kyphoscoliosis in this model is due to systemic ligands, such as growth factors and hormones. Therefore, we focused on the aberrant expression levels of the neurotrophin receptors, the Trks.
TrkA is the receptor for nerve growth factor (NGF), TrkB is the receptor for brain-derived neurotrophic factor (BDNF) and neurotrophin (NT) -4/5, and TrkC is the receptor for NT3 [15].
The Trks are also members of the family of receptor tyrosine kinases (RTKs). The binding of the neurotrophins to the Trks leads to receptor tyrosine phosphorylation [16]. Their
ligand-receptor signaling triggers neuron proliferation and is important for the development and maturation of the nervous system [17].
There is no current evidence that links the aberrant expression of TrkA with malformation of the lumbar spines. However, blocking the NGF-TrkA pathway is highly efficacious in attenuating skeletal pain, because the majority of sensory nerve fibers that control the skeleton express TrkA [18]. Recently, Ghilardi and coworkers reported that therapies blocking the Trks attenuated bone pain caused by prostate cancer [19] and after bone fracture [20]. It would be interesting to determine whether such therapies could also attenuate the bone pain in patients with scoliosis.
BDNF acts on central and peripheral neurons through binding to TrkB [21]. BDNF and TrkB are also widely expressed in non-neuronal cells, such as leukocytes, chondrocytes, fibroblasts, adipocytes and osteoblasts [21, 22]. BDNF was observed to localize in osteoblast-like cells in the ribs, and its mRNA expression level in fractured ribs increased during the process of healing [23]. TrkB mRNA is expressed in developing spines on rat embryonic day 17.5 [24]. Targeted brain deletion of BDNF expression causes excess bone mass [25]. Moreover, BDNF regulates the gene expression of bone/cementum-related molecules, such as alkaline phosphatase and osteopontin, and bone morphogenetic protein (BMP)-2 [26].
Recently, Hutchison reported that mice lacking TrkB showed dwarfism (TrkBflox/flox;Col2a1-cre), and TrkB/p38 (MAPK14) double knockout mice had altered expression levels of Runx2 and Sox9, which are required for skeletogenesis [27]. In the present study, however, the IS rats did not show dwarfism or pronounced alterations of the expression levels of Runx2 (Cy3/Cy5 ratio:
1.75) or Sox9 (Cy3/Cy5 ratio: 1.16). Clearly, BDNF-TrkB signaling plays an important role in the regulation of the normal development of bone tissue; however, a more detailed analysis of the precise molecular mechanisms involved in the development of kyphoscoliosis will be needed.
The BMPs are members of the transforming growth factor- (TGF-) superfamily [28] and are involved in bone formation [29]. BMPs exert their bioactivity by binding to their receptors,
BMP receptor types -IA, -IB and -II [30]. BMP receptors regulate TrkC expression [31], and BMP receptor type-II binds to TrkC to suppress BMP signals [32]. Schaffer et al. [33] reported that fibrodysplasia ossifications progressiva (FOP) patients exhibit congenital spinal malformations such as large posterior elements, tall narrow vertebral bodies, and fusion of the facet joints, and the pathogenesis of spinal malformation involves the overactivity of the BMP signaling pathway. In the present study, the gene expression levels of BMPs were not altered in the lumbar spines of IS rats. For example, the Cy3/Cy5 expression ratios of Bmp-2, Bmp-3 and Bmp-4 were 0.83, 1.63, and 0.73, respectively. Therefore, the spinal malformation in IS rats
might have been caused by the relatively excessive BMP signals, because the low level of TrkC would not have been able to suppress the BMP signals.
Additionally, the p75 neurotrophin receptor (p75NTR) interacts closely with Trks [34], and regulates the proliferation and differentiation of the osteoblast cell lines, MC3T3-E1 [35] and MG63 [36], in vitro. The mRNA expression level of p75NTR was also significantly decreased (Cy3/Cy5 ratio: 0.10, Supplementary Table 2) in the lumbar spines of IS rats in this study.
In conclusion, the Trk family of neurotrophin receptors was downregulated only in the flexion sites of the spine of congenital kyphoscoliosis rats. Although the mechanisms underlying this downregulation remain to be determined, the downregulation of Trk signaling, including p75NTR, provides new insight into the pathogenesis of scoliosis and kyphoscoliosis, showing that neurotrophin receptors are important to these processes.
Conflict of interest
The authors declare that they have no conflicts of interest.
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Figure legends
Fig. 1. A, A scatter plot of the signal intensity Cy3 versus that of Cy5 after normalization. The red and blue lines represent the diagonal line (y = x) and the fluctuation range of 8 times, respectively. TrkA, TrkB and TrkC correspond to the dots in each circle. B, The mRNA expression levels of Trks in the lumbar spines of IS rats. The mRNA expression levels of Trks in the IS (Cy3) and Wistar (Cy5) rats were analyzed using DNA microarrays. The mRNA expression levels of Trks are expressed as the Cy3/Cy5 ratio. The expression ratio of the housekeeping gene, Gapdh, was mostly unchanged in the IS and Wistar rats (Cy3/Cy5: 0.9).
Fig. 2. The mRNA expression levels of Trk family members in the spines of IS rats. The mRNA expression levels of TrkA (A), TrkB (B) and TrkC (C) in the lumbar spines (L3 – L5) of Wistar (Wt, ) and IS (IS, ) rats were determined by a real-time RT-PCR analysis. B, The mRNA expression levels of TrkA (D), TrkB (E) and TrkC (F) in the thoracic (Th10-Th12, ) and lumbar spines (L3 – L5, ) of IS (IS) rats were determined by a real-time RT-PCR analysis.
The expression levels of Trk mRNAs are expressed as relative quantities, which were normalized to the corresponding expression level of GAPDH mRNA. The mRNA expression level of each gene was the mean of three independent experiments. The data are expressed as the means ± SEM (n = 7). *, P < 0.05 compared with Wt rats (A) or the thoracic spine (Th10 – Th12) (B).
Fig. 3. The protein expression levels of Trk family members in the spines of IS rats. The protein expression levels of TrkA (A), TrkB (B) and TrkC (C) in the lumbar spines (L3 – L5) of Wistar (n=7) and IS (n=7) rats were determined by a Western blot analysis. Immunoblots (left panel) and quantification (right panel) of the amounts of Trk proteins in the lumbar spines are shown, and the results are representative of three independent Western blot analyses. The positions of the standard molecular masses (kDa) are indicated on the left. The protein expression levels of
Trks in Wistar (Wt, ) and IS ( ) rats are expressed as the relative optical density (OD), which was normalized to the corresponding density of -actin. The data are expressed as the means ± SEM (n = 7). *, P < 0.05 compared with Wt rats.
Fig. 4. Detection of Trk-immunopositive cells in the lumbar spines by immunohistochemistry.
The coronal sections of the lumbar spines (L3 – L5) on P4 of Wistar (Wt, A, B, C) and IS (D, E, F) rats were stained with anti-TrkA (A, D), anti-TrkB (B, E) or anti-TrkC (C, F) antibodies. The Trk-immunopositive cells are indicated by white arrowheads. Bar, 50 µm.
Table 1.
Density of Trk-immunopositive cells in lumbar spines TrkA TrkB TrkC Wt ++++ ++ ++++
IS ++ + ++
All Trk-immunopositive cells in one section of the lumbar spines of Wistar (Wt) and IS rats were counted. Each section was classified into one of four criteria on the basis of number of Trk-immunopositive cells: +, < 25; ++, 26-50; +++; 51-75; ++++, >76.
