- Email: [email protected]
Altered expression of Smad family members in injured motor neurons of rat
BR A IN RE S EA RCH 1 1 32 ( 20 0 7 ) 3 6 –41
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Altered expression of Smad family members in injured motor neurons of rat Noriko Okuyama, Sumiko Kiryu-Seo, Hiroshi Kiyama⁎ Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka 545-8585, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
We examined changes in the expression of Smad family members, which transduce signals
Accepted 6 November 2006
from TGF-β superfamily ligands, following hypoglossal nerve injury. RT–PCR and in situ
Available online 12 December 2006
hybridization revealed that Smad1, 2, 3 and 4 mRNAs were significantly up-regulated in injured side, whereas Smad8 mRNA was down-regulated. Immunohistochemistry and
Keywords:
Western blotting analysis confirmed the alterations of Smad1, 2 and 4 in injured neurons.
TGF-β
These results suggest that the Smad signaling may be important for nerve regeneration.
BMP
© 2006 Elsevier B.V. All rights reserved.
Smad Regeneration Nerve injury
Injured neurons in the peripheral nervous system (PNS) are able to survive and regenerate, but those in central nervous system (CNS) are not. In response to peripheral nerve injury, numerous molecules are expressed in neurons, and most of these are assumed to be implicated in regeneration process. Amongst those that have been identified, neurotrophins and cytokines have crucial roles in cell survival and nerve regeneration (Moran and Graeber, 2004; Makwana and Raivich, 2005). It is very intriguing that the receptors for neurotrophins and cytokines, such as TrkA, TrkB, cRet, GFRα1, LIFR and CNTFR-α, are simultaneously induced after nerve injury and contribute effectively to transferring signal transduction (Honma et al., 2002; Makwana and Raivich, 2005). Intracellular signaling molecules such as Shc, ERK, PI3K, Akt, JAKs, Tyk and STAT3, which act downstream of those receptors, are also upregulated after nerve injury (Kiryu et al., 1995a,b; Yao et al., 1997; Tanabe et al., 1998; Namikawa et al., 2000; Snider et al.,
2002; Makwana and Raivich, 2005). Therefore, the orchestrated inductions of both receptors and their downstream signaling molecules are perhaps important for proper regeneration. In addition to those protective molecules, TGF-β superfamily members, including transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP) and activin, are putative protective factors (Kiefer et al., 1993; Jiang et al., 2000). TGF-β family members bind to type I and type II serine/ threonine kinase receptors and activate intracellular Smad proteins, which are key mediators for TGF-β family signaling (Miyazawa et al., 2002; Ten Dijke and Hill, 2004). Smads are classified into three subclasses based on their structure and function. The first class, called receptor-regulated Smads (RSmads), includes Smads 1, 2, 3, 5 and 8. This class can be further divided into two categories: Smad2 and Smad3 respond to TGF-β and activins, whereas Smads 1, 5 and 8 function in BMP signaling pathway. R-Smads are directly
⁎ Corresponding author. Fax: +81 666 45 3702. E-mail address: [email protected] (H. Kiyama). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.11.019
BR A IN RE S E A RCH 1 1 32 ( 20 0 7 ) 3 6 –4 1
phosphorylated and activated by the type I serine/threonine kinase receptor. The second class is the common partner Smad (Co-Smad), which contains only Smad4. R-Smads oligomerize with Co-Smad to form a heterodimeric complex that is translocated into the nucleus where it modulates transcriptional responses. The third class, called inhibitory Smads (I-Smads), includes Smads 6 and 7. They act as inhibitors of Smad-mediated signal transduction by interacting with the type I receptor and preventing activation of RSmads (Miyazawa et al., 2002). It is therefore critical to understand whether Smad-mediated signaling is involved in nerve regeneration. However, little is known of the alterations of Smad proteins in response to nerve injury. In this study, we examined the mRNA expression and protein levels of Smad family members using a hypoglossal nerve injury model of rat. Male Wistar rats (7 weeks old, 42 rats) were anesthetized with pentobarbital (45 mg/kg, i.p.) and placed in a supine position. Their right hypoglossal nerve was cut just proximal to the bifurcation of the nerve. For reverse transcriptase polymerase chain reaction (RT–PCR), five rats were killed by decapitation 7 days after hypoglassal nerve injury. Control and injured hypoglossal nuclei were dissected under a microscope and quickly dipped into liquid nitrogen. Total RNA was obtained from the control and injured hypoglossal nuclei, and reverse-transcribed with oligo dT using SuperScript II (Invitrogen) according to the manufacturer's protocol. RT–PCR was performed using following specific primers for Smads 1–8: Smad1 sense 5′-TTGTTTAGAAATGAATGGGTT-3′, Smad1 antisense 5′-ACAGTTAGAGGAATTAACCAGCTG-3′, Smad2 sense 5′-CAGCTTCTCTGAACAAACCAGG-3′, Smad2 antisense 5′-TACTCTGTGGCTCAATTCCTGCTG-3′, Smad3 sense 5′-TGACAGTGCTATTTTCGTCCAGTCT-3′, Smad3 antisense 5′-CGATCCCTTTACTCCCAGTGTCT-3′, Smad4 sense 5′-TGTCTCACCTGGAATTGATCTCTCAG-3′, Smad4 antisense 5′-AATCCATTCTGCTGCTGTCCTGGCTG-3′, Smad5 sense 5′CAGATGGGCTCTCCGCTGAACC-3′, Smad5 antisense 5′-TCGT TTACAATACTTTTGAAAG-3′, Smad6 sense 5′-ATGACCAGGCTGTCAGCATCTTCTA-3′, Smad6 antisense 5′ATCTGTGGTTGTTGAGGAGGATCT-3′, Smad7 sense 5′-TCAGATTCCCAACTTCTTCTGGAGCC-3′, Smad7 antisense 5′-TGT GAAGATGACCTCCAGCCAGCAC-3′, Smad8 sense 5′AGCACCCCCTGCTGGAT-3′ and Smad8 antisense 5′-AAGTAGGTAGCACAGAAC-3′. RT–PCR was performed by 23 to 32 cycles of PCR depending on the target genes, with annealing temperatures 60 °C. Products were separated on an agarose gel and visualized using ethidium bromide. For in situ hybridization, brains were removed quickly 7 days after axotomy and frozen in powdered dry ice. Sections were cut at a thickness of 15 μm using a cryostat. All procedures for in situ hybridization were performed as described previously (Kiryu et al., 1995a,b). Data are representative of three independent experiments using at least seven animals. For immunohistochemistry, brains (n = 20) were removed 7 days after hypoglossal nerve injury and divided into two groups; one for fresh–frozen brain preparation and the other for perfusion fixation with Zamboni's fixative (picric acid/paraformaldehyde). 15 μm sections were cut using a cryostat, thawmounted onto 3-aminopropyltriethoxysilane-coated slides. For Smad1 immunoreactivity, fresh-frozen sections were fixed in 2% paraformaldehyde, permealized for 10 min in 1%
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Triton X-100 in TBS (20 mM Tris–HCl, 136 mM NaCl), blocked with 1% bovine serum albumin and incubated with antiSmad1 antibody (sc-7965, Santa Cruz). For Smad2/3 immunoreaction, Zamboni-fixed section was used. The sections were permealized for 10 min in 1% Triton X-100 in TBS (20 mM Tris–HCl, 136 mM NaCl), blocked with 1% bovine serum albumin and incubated with anti-Smad2/3 antibody (sc-6033, Santa Cruz). For Smad4 immunoreaction, freshfrozen sections were fixed. To retrieve antigen, sections were boiled in a microwave in 10 mM citrate buffer for 20 min, permealized for 10 min in 1% Triton X-100 in TBS (20 mM Tris–HCl, 136 mM NaCl), blocked with 1% bovine serum albumin and then incubated with anti-Smad4 antibody (sc7966, Santa Cruz). After incubation with the antibodies at 4 °C overnight, sections were incubated in secondary antibodies conjugated to Alexa Fluor 488 (Molecular Probes) for 1 h at room temperature. For Western blotting, samples collected from control and injured hypoglossal nuclei of five rats 7 days after axotomy were homogenized in lysis buffer (8 M Urea, 2% CHAPS, 40 mM Tris, 1 mM Na3VO4, 10 mM NaF). The homogenate was centrifuged at 4 °C for 10 min at 14,000 rpm. 20 μg of supernatant was loaded and immunoblotted with antiSmad1 (sc-7965, Santa Cruz), anti-Smad2/3 (sc-6033, Santa Cruz), anti-Smad4 (sc-7966, Santa Cruz) and anti-GAPDH (#4300, Ambion), visualized with horseradish peroxidase-conjugated secondary antibodies using electrochemiluminescence (Perkin-Elmer). To clarify whether Smad family members are involved in nerve regeneration, we examined the expression profiles of Smad members such as Smads 1, 2, 3, 4, 5, 6, 7 and 8 after hypoglossal nerve injury. We initially performed RT– PCR analysis using mRNAs isolated from control and injured hypoglossal nuclei. Among the various Smad family members, mRNAs for Smad1–4 were significantly increased after axotomy, whereas that for Smad8 was decreased. No significant changes in mRNA expression were observed in Smads 5, 6, and 7 (Fig. 1). To confirm these alterations and further identify the cell types expressing those members, in situ hybridization was carried out. In situ hybridization showed that the expression of Smad1, 2, 3 and 4 mRNAs was markedly up-regulated on the injured side 7 days after hypoglossal nerve transection, whereas that of Smad8 mRNA was down-regulated (Fig. 2A). The expression levels of Smad5, 6 and 7 mRNAs were very low and the apparent alterations of mRNAs were not detected. To semi-quantify the fold increase of mRNA expression after axotomy, we measured the signal intensity on the film-autoradiogram shown in Fig. 2A. Among the Smad family members examined, mRNAs for Smads 1–4 were significantly upregulated by 3- to 7-fold following nerve injury. In contrast, Smad8 mRNA was down-regulated by 4-fold after axotomy (Fig. 2B). These results were consistent with those of RT– PCR. To clarify the cell types expressing Smad mRNAs, an emulsion autoradiogram was observed under bright-field illumination after Nissl staining. The hybridization signals for Smads 1, 2 and 4 were accumulated on large neurons but not in the surrounding glial cells (Fig. 2C arrows), whereas that of Smad3 was not seen on large neurons suggesting that it was expressed in non-neuronal cells (Fig. 2C arrow heads). Smad8 mRNA signal was also observed in
38
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Fig. 1 – Expression of Smad family members 7 days after hypoglossal nerve injury. (A) RT–PCR analysis of Smad family members in control and injured hypoglossal nuclei of rats. cont, control side; inj, injured side. (B) The relative mRNA signal intensity in the injured side compared with that in control side was calculated. Bars represent means ± SD. *Significant difference compared with the control side, when analysed by Student's t-test (p < 0.01). Equivalent amounts of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are amplified, verifying standardization of conditions.
neuronal cells under normal conditions, but a hybridization signal was not identified in the nerve-injured hypoglossal nucleus (data not shown). These findings suggest that Smad1, 2 and 4 mRNAs are induced in nerve-injured motor neurons. Immunohistochemical study showed that the expression levels of Smad1, 2 and 4 proteins were increased in injured motor neurons (Fig. 3A). As Smad has been detected previously in the nucleus in some cell lines, we expected Smad-like immunoreactivity to be observed in nucleus. However, in this study, Smads 1 and 4 were observed mainly in the cytoplasm as well as the nucleus, before and after axotomy (Fig. 3A). Only Smad2 immunoreactivity was detected in the nucleus (Fig. 3A). Following nerve injury, positive staining for Smad2 protein was also seen in the cytoplasm in addition to nucleus (Fig. 3A). The increases in the immunoreactivity of Smads 1, 2/3 and 4 were detected initially in the ipsilateral side 1 day after the surgical procedure; however, the immunoreactivity markedly increased to a peak level during the following 7 days and persisted at this level for 14 days. Thereafter, the positive staining gradually decreased until control levels were reached during the following 56 days (data not shown). Finally, we quantified the levels of Smad1, Smad2/3 and Smad4 proteins in control and injured hypoglossal nuclei by Western blotting. 1.2-, 1.4- and 1.5-fold increases were identified in Smad1, Smad2/3 and Smad4, respectively (Fig. 3B). Collectively, these results suggest that Smads 1, 2/3 and 4 are transcriptionally and translationally up-regulated in injured motor neurons.
In this study, we identified an alteration of mRNA and protein levels of Smad family members in injured motor neurons. In situ hybridization revealed that the expression of Smad1, 2 and 4 mRNAs was significantly up-regulated in injured motor neurons, whereas that of Smad8 mRNA was down-regulated. The alteration of mRNAs for other Smads, Smads 5–7, was not significant. This suggests that Smad family members have specific gene regulation profiles during the nerve regeneration process. Immunohistochemical analysis further demonstrated that Smad1 and Smad4 proteins are predominantly localized to injured motor neurons in the hypoglossal nucleus. Smad2 protein, but not Smad3 protein, was also localized in motor neurons. Although the antibody used to detect Smad2 protein crossreacts with Smad3, the results of in situ hybridization supported the possibility that the signal seen in motor neurons was Smad2. Taken together, Smads 1, 2 and 4 would be the major components among Smad family members that might be implicated in the response to nerve injury. It is possible that Smad1 and Smad2 could form heterodimers with Smad4 that translocate into the nucleus and trans-activate various target genes that are associated with nerve regeneration. Further study will be needed to determine if this combination of Smad1–Smad4 and Smad2–Smad4 heterodimers really exists in injured motor neurons. Another piece of evidence in this study is that increased Smad proteins, which are demonstrated by the immunohistochemistry, were prominently accumulated in cytoplasm in addition to nucleus after nerve injury. Because some previous reports demonstrated that some
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Fig. 2 – The expression of Smad mRNAs in injured hypoglossal motor neurons. (A) The film (upper panel) and emulsion (lower panel) autoradiography of in situ hybridization showed expression of Smad family members. The expression levels of Smads 1, 2, 3 and 4 were markedly increased 7 days after hypoglossal nerve injury (black arrows), whereas expression of Smad8 mRNA was markedly decreased (white arrow). cc, central canal. Scale bar: 1.3 mm for film autoradiography, 100 μm for emulsion autoradiography. (B) The relative mRNA signal intensity in the injured side compared with the control side was measured. Bars represent means ± SD. *Significant difference from each control side, when analysed by Student's t-test (p < 0.01). (C) Bright-field illumination of emulsion-autoradiography demonstrated localization of mRNA expressing cells in injured side of hypoglossal nucleus. Smad1, 2 and 4 mRNA signals (accumulation of silver grains) were observed in large-sized motor neurons (arrows), whereas Smad3 mRNA signal was seen in small-sized non-neuronal cells (arrowheads). cont, control side. Scale bar: 50 μm.
signaling molecules such as Akt and MAP kinase inhibited nuclear translocation of Smad to prevent cell death or to adjust the level of Smad signaling (Kretzschmar et al., 1997; Song et al., 2006), the Smads in cytoplasm after nerve injury
may be accumulated in cytoplasm due to the prevention of the nuclear translocation. Alternatively Smads may have some unknown roles to protect injured neurons. To elucidate more detailed mechanism, further study will be needed.
40
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Fig. 3 – Expression of Smad proteins in the hypoglossal nucleus 7 days after axotomy. (A) Immunohistochemical images labeled for Smad1, Smad2/3 and Smad4. Higher magnification of Smads' immunoreactivity on the contralateral and injured sides. cc, central canal; cont, control side; inj, injured side. Scale bar: 100 μm, 25 μm (high power magnification). (B) Immunoblot analysis of Smad proteins after axotomy. Protein extracted from control and injured hypoglossal nuclei of rats 7 days after axotomy were subjected to Western blot analysis. Arrows indicate position of Smad2 and Smad3 proteins. Lower panel: the same membrane was stripped and re-blotted with anti-GAPDH antibody. GAPDH was used as internal control. cont, control side; inj, injured side.
Several lines of evidence have shown that motor neurons and Schwann cells induce and release TGF-β following peripheral nerve injury (Kiefer et al., 1993; Scherer et al., 1993; Rufer et al., 1994; Jiang et al., 2000). It has also been reported that spinal cord injury induces expression of several types of BMP (Setoguchi et al., 2001; Setoguchi et al., 2004). Thus, TGF-β family members are possible ligands for Smad signaling in injured motor neurons. TGF-β superfamily ligands bind to two distinct receptor types, known as type I and type II serine/threonine kinase receptors (Miyazawa et al., 2002; Ten Dijke and Hill, 2004). Following hypoglossal nerve injury, the mRNA level of type I TGF-β and/or activin receptor, named B1, was substantially suppressed in the injured motor neurons, whereas the type II TGF-β receptor (TβR-II) was not observed in the hypoglossal nucleus prior to, or after, axotomy (Morita et al., 1996). In contrast, Jiang et al. have reported that high levels of type I TGF-β receptor (TβR-I) and TβR-II proteins were observed in normal hypoglossal nerves, and TβR-II protein was accumulated at the proximal nerve after ligation (Jiang et al., 2000). These findings suggest that the expression of other type I and II receptors should be examined in injured motor neurons. Recently, Wang et al. reported that Mullerian Inhibiting Substance (MIS), a member of TGF-β superfamily, functions as a survival factor of motor neurons and that type II MIS receptor (MISRII) exist in motor neurons (Wang et al., 2005). Considering this fact, MISRII may be a responsible receptor for the downstream Smad mediated signaling. Conclusively, we have demonstrated increased expression of Smads 1, 2 and 4 in injured hypoglossal neurons, and suggested that a combination of Smad1–Smad4 and Smad2– Smad4 heterodimers effect Smad signaling in the nucleus. Although the target genes transactivated by Smads and the
ligands/receptors to activate Smads in injured motor neurons are still unclear, Smad-mediated signaling might have an important role during nerve regeneration.
Acknowledgments We are grateful to C. Kadono and I. Jikihara for technical assistance and T. Kawai for secretarial assistance. This study was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan and the MEXT.
REFERENCES
Honma, M., Namikawa, K., Mansur, K., Iwata, T., Mori, N., Iizuka, H., Kiyama, H., 2002. Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate. J. Neurochem. 82, 961–975. Jiang, Y., McLennan, I.S., Koishi, K., Hendry, I.A., 2000. Transforming growth factor-beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury. Neuroscience 97, 735–742. Kiefer, R., Lindholm, D., Kreutzberg, G.W., 1993. Interleukin-6 and transforming growth factor-beta 1 mRNAs are induced in rat facial nucleus following motoneuron axotomy. Eur. J. Neurosci. 5, 775–781. Kiryu, S., Morita, N., Ohno, K., Maeno, H., Kiyama, H., 1995a. Regulation of mRNA expression involved in Ras and PKA signal pathways during rat hypoglossal nerve regeneration. Brain Res. Mol. Brain Res. 29, 147–156. Kiryu, S., Yao, G.L., Morita, N., Kato, H., Kiyama, H., 1995b. Nerve injury enhances rat neuronal glutamate transporter
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expression: identification by differential display PCR. J. Neurosci. 15, 7872–7878. Kretzschmar, M., Liu, F., Hata, A., Doody, J., Massague, J., 1997. The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev. 11, 984–995. Makwana, M., Raivich, G., 2005. Molecular mechanisms in successful peripheral regeneration. FEBS J. 272, 2628–2638. Miyazawa, K., Shinozaki, M., Hara, T., Furuya, T., Miyazono, K., 2002. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells 7, 1191–1204. Moran, L.B., Graeber, M.B., 2004. The facial nerve axotomy model. Brain Res. Brain Res. Rev. 44, 154–178. Morita, N., Takumi, T., Kiyama, H., 1996. Distinct localization of two serine–threonine kinase receptors for activin and TGF-beta in the rat brain and down-regulation of type I activin receptor during peripheral nerve regeneration. Brain Res. Mol. Brain Res. 42, 263–271. Namikawa, K., Honma, M., Abe, K., Takeda, M., Mansur, K., Obata, T., Miwa, A., Okado, H., Kiyama, H., 2000. Akt/protein kinase B prevents injury-induced motoneuron death and accelerates axonal regeneration. J. Neurosci. 20, 2875–2886. Rufer, M., Flanders, K., Unsicker, K., 1994. Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve. J. Neurosci. Res. 39, 412–423. Scherer, S.S., Kamholz, J., Jakowlew, S.B., 1993. Axons modulate the expression of transforming growth factor-betas in Schwann cells. Glia 8, 265–276.
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Setoguchi, T., Yone, K., Matsuoka, E., Takenouchi, H., Nakashima, K., Sakou, T., Komiya, S., Izumo, S., 2001. Traumatic injury-induced BMP7 expression in the adult rat spinal cord. Brain Res. 921, 219–225. Setoguchi, T., Nakashima, K., Takizawa, T., Yanagisawa, M., Ochiai, W., Okabe, M., Yone, K., Komiya, S., Taga, T., 2004. Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor. Exp. Neurol. 189, 33–44. Snider, W.D., Zhou, F.Q., Zhong, J., Markus, A., 2002. Signaling the pathway to regeneration. Neuron 35, 13–16. Song, K., Wang, H., Krebs, T.L., Danielpour, D., 2006. Novel roles of Akt and mTOR in suppressing TGF-beta/ALK5-mediated Smad3 activation. EMBO J. 25, 58–69. Tanabe, K., Kiryu-Seo, S., Nakamura, T., Mori, N., Tsujino, H., Ochi, T., Kiyama, H., 1998. Alternative expression of Shc family members in nerve-injured motoneurons. Brain Res. Mol. Brain Res. 53, 291–296. ten Dijke, P., Hill, C.S., 2004. New insights into TGF-beta-Smad signalling. Trends Biochem. Sci. 29, 265–273. Wang, P.Y., Koishi, K., McGeachie, A.B., Kimber, M., Maclaughlin, D.T., Donahoe, P.K., McLennan, I.S., 2005. Mullerian inhibiting substance acts as a motor neuron survival factor in vitro. Proc. Natl. Acad. Sci. U. S. A. 102, 16421–16425. Yao, G.L., Kato, H., Khalil, M., Kiryu, S., Kiyama, H., 1997. Selective upregulation of cytokine receptor subchain and their intracellular signalling molecules after peripheral nerve injury. Eur. J. Neurosci. 9, 1047–1054.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Altered expression of Smad family members in injured motor neurons of rat Noriko Okuyama, Sumiko Kiryu-Seo, Hiroshi Kiyama⁎ Department of Anatomy and Neurobiology, Osaka City University, Graduate School of Medicine, 1-4-3 Asahimachi, Abenoku, Osaka 545-8585, Japan
A R T I C LE I N FO
AB S T R A C T
Article history:
We examined changes in the expression of Smad family members, which transduce signals
Accepted 6 November 2006
from TGF-β superfamily ligands, following hypoglossal nerve injury. RT–PCR and in situ
Available online 12 December 2006
hybridization revealed that Smad1, 2, 3 and 4 mRNAs were significantly up-regulated in injured side, whereas Smad8 mRNA was down-regulated. Immunohistochemistry and
Keywords:
Western blotting analysis confirmed the alterations of Smad1, 2 and 4 in injured neurons.
TGF-β
These results suggest that the Smad signaling may be important for nerve regeneration.
BMP
© 2006 Elsevier B.V. All rights reserved.
Smad Regeneration Nerve injury
Injured neurons in the peripheral nervous system (PNS) are able to survive and regenerate, but those in central nervous system (CNS) are not. In response to peripheral nerve injury, numerous molecules are expressed in neurons, and most of these are assumed to be implicated in regeneration process. Amongst those that have been identified, neurotrophins and cytokines have crucial roles in cell survival and nerve regeneration (Moran and Graeber, 2004; Makwana and Raivich, 2005). It is very intriguing that the receptors for neurotrophins and cytokines, such as TrkA, TrkB, cRet, GFRα1, LIFR and CNTFR-α, are simultaneously induced after nerve injury and contribute effectively to transferring signal transduction (Honma et al., 2002; Makwana and Raivich, 2005). Intracellular signaling molecules such as Shc, ERK, PI3K, Akt, JAKs, Tyk and STAT3, which act downstream of those receptors, are also upregulated after nerve injury (Kiryu et al., 1995a,b; Yao et al., 1997; Tanabe et al., 1998; Namikawa et al., 2000; Snider et al.,
2002; Makwana and Raivich, 2005). Therefore, the orchestrated inductions of both receptors and their downstream signaling molecules are perhaps important for proper regeneration. In addition to those protective molecules, TGF-β superfamily members, including transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP) and activin, are putative protective factors (Kiefer et al., 1993; Jiang et al., 2000). TGF-β family members bind to type I and type II serine/ threonine kinase receptors and activate intracellular Smad proteins, which are key mediators for TGF-β family signaling (Miyazawa et al., 2002; Ten Dijke and Hill, 2004). Smads are classified into three subclasses based on their structure and function. The first class, called receptor-regulated Smads (RSmads), includes Smads 1, 2, 3, 5 and 8. This class can be further divided into two categories: Smad2 and Smad3 respond to TGF-β and activins, whereas Smads 1, 5 and 8 function in BMP signaling pathway. R-Smads are directly
⁎ Corresponding author. Fax: +81 666 45 3702. E-mail address: [email protected] (H. Kiyama). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.11.019
BR A IN RE S E A RCH 1 1 32 ( 20 0 7 ) 3 6 –4 1
phosphorylated and activated by the type I serine/threonine kinase receptor. The second class is the common partner Smad (Co-Smad), which contains only Smad4. R-Smads oligomerize with Co-Smad to form a heterodimeric complex that is translocated into the nucleus where it modulates transcriptional responses. The third class, called inhibitory Smads (I-Smads), includes Smads 6 and 7. They act as inhibitors of Smad-mediated signal transduction by interacting with the type I receptor and preventing activation of RSmads (Miyazawa et al., 2002). It is therefore critical to understand whether Smad-mediated signaling is involved in nerve regeneration. However, little is known of the alterations of Smad proteins in response to nerve injury. In this study, we examined the mRNA expression and protein levels of Smad family members using a hypoglossal nerve injury model of rat. Male Wistar rats (7 weeks old, 42 rats) were anesthetized with pentobarbital (45 mg/kg, i.p.) and placed in a supine position. Their right hypoglossal nerve was cut just proximal to the bifurcation of the nerve. For reverse transcriptase polymerase chain reaction (RT–PCR), five rats were killed by decapitation 7 days after hypoglassal nerve injury. Control and injured hypoglossal nuclei were dissected under a microscope and quickly dipped into liquid nitrogen. Total RNA was obtained from the control and injured hypoglossal nuclei, and reverse-transcribed with oligo dT using SuperScript II (Invitrogen) according to the manufacturer's protocol. RT–PCR was performed using following specific primers for Smads 1–8: Smad1 sense 5′-TTGTTTAGAAATGAATGGGTT-3′, Smad1 antisense 5′-ACAGTTAGAGGAATTAACCAGCTG-3′, Smad2 sense 5′-CAGCTTCTCTGAACAAACCAGG-3′, Smad2 antisense 5′-TACTCTGTGGCTCAATTCCTGCTG-3′, Smad3 sense 5′-TGACAGTGCTATTTTCGTCCAGTCT-3′, Smad3 antisense 5′-CGATCCCTTTACTCCCAGTGTCT-3′, Smad4 sense 5′-TGTCTCACCTGGAATTGATCTCTCAG-3′, Smad4 antisense 5′-AATCCATTCTGCTGCTGTCCTGGCTG-3′, Smad5 sense 5′CAGATGGGCTCTCCGCTGAACC-3′, Smad5 antisense 5′-TCGT TTACAATACTTTTGAAAG-3′, Smad6 sense 5′-ATGACCAGGCTGTCAGCATCTTCTA-3′, Smad6 antisense 5′ATCTGTGGTTGTTGAGGAGGATCT-3′, Smad7 sense 5′-TCAGATTCCCAACTTCTTCTGGAGCC-3′, Smad7 antisense 5′-TGT GAAGATGACCTCCAGCCAGCAC-3′, Smad8 sense 5′AGCACCCCCTGCTGGAT-3′ and Smad8 antisense 5′-AAGTAGGTAGCACAGAAC-3′. RT–PCR was performed by 23 to 32 cycles of PCR depending on the target genes, with annealing temperatures 60 °C. Products were separated on an agarose gel and visualized using ethidium bromide. For in situ hybridization, brains were removed quickly 7 days after axotomy and frozen in powdered dry ice. Sections were cut at a thickness of 15 μm using a cryostat. All procedures for in situ hybridization were performed as described previously (Kiryu et al., 1995a,b). Data are representative of three independent experiments using at least seven animals. For immunohistochemistry, brains (n = 20) were removed 7 days after hypoglossal nerve injury and divided into two groups; one for fresh–frozen brain preparation and the other for perfusion fixation with Zamboni's fixative (picric acid/paraformaldehyde). 15 μm sections were cut using a cryostat, thawmounted onto 3-aminopropyltriethoxysilane-coated slides. For Smad1 immunoreactivity, fresh-frozen sections were fixed in 2% paraformaldehyde, permealized for 10 min in 1%
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Triton X-100 in TBS (20 mM Tris–HCl, 136 mM NaCl), blocked with 1% bovine serum albumin and incubated with antiSmad1 antibody (sc-7965, Santa Cruz). For Smad2/3 immunoreaction, Zamboni-fixed section was used. The sections were permealized for 10 min in 1% Triton X-100 in TBS (20 mM Tris–HCl, 136 mM NaCl), blocked with 1% bovine serum albumin and incubated with anti-Smad2/3 antibody (sc-6033, Santa Cruz). For Smad4 immunoreaction, freshfrozen sections were fixed. To retrieve antigen, sections were boiled in a microwave in 10 mM citrate buffer for 20 min, permealized for 10 min in 1% Triton X-100 in TBS (20 mM Tris–HCl, 136 mM NaCl), blocked with 1% bovine serum albumin and then incubated with anti-Smad4 antibody (sc7966, Santa Cruz). After incubation with the antibodies at 4 °C overnight, sections were incubated in secondary antibodies conjugated to Alexa Fluor 488 (Molecular Probes) for 1 h at room temperature. For Western blotting, samples collected from control and injured hypoglossal nuclei of five rats 7 days after axotomy were homogenized in lysis buffer (8 M Urea, 2% CHAPS, 40 mM Tris, 1 mM Na3VO4, 10 mM NaF). The homogenate was centrifuged at 4 °C for 10 min at 14,000 rpm. 20 μg of supernatant was loaded and immunoblotted with antiSmad1 (sc-7965, Santa Cruz), anti-Smad2/3 (sc-6033, Santa Cruz), anti-Smad4 (sc-7966, Santa Cruz) and anti-GAPDH (#4300, Ambion), visualized with horseradish peroxidase-conjugated secondary antibodies using electrochemiluminescence (Perkin-Elmer). To clarify whether Smad family members are involved in nerve regeneration, we examined the expression profiles of Smad members such as Smads 1, 2, 3, 4, 5, 6, 7 and 8 after hypoglossal nerve injury. We initially performed RT– PCR analysis using mRNAs isolated from control and injured hypoglossal nuclei. Among the various Smad family members, mRNAs for Smad1–4 were significantly increased after axotomy, whereas that for Smad8 was decreased. No significant changes in mRNA expression were observed in Smads 5, 6, and 7 (Fig. 1). To confirm these alterations and further identify the cell types expressing those members, in situ hybridization was carried out. In situ hybridization showed that the expression of Smad1, 2, 3 and 4 mRNAs was markedly up-regulated on the injured side 7 days after hypoglossal nerve transection, whereas that of Smad8 mRNA was down-regulated (Fig. 2A). The expression levels of Smad5, 6 and 7 mRNAs were very low and the apparent alterations of mRNAs were not detected. To semi-quantify the fold increase of mRNA expression after axotomy, we measured the signal intensity on the film-autoradiogram shown in Fig. 2A. Among the Smad family members examined, mRNAs for Smads 1–4 were significantly upregulated by 3- to 7-fold following nerve injury. In contrast, Smad8 mRNA was down-regulated by 4-fold after axotomy (Fig. 2B). These results were consistent with those of RT– PCR. To clarify the cell types expressing Smad mRNAs, an emulsion autoradiogram was observed under bright-field illumination after Nissl staining. The hybridization signals for Smads 1, 2 and 4 were accumulated on large neurons but not in the surrounding glial cells (Fig. 2C arrows), whereas that of Smad3 was not seen on large neurons suggesting that it was expressed in non-neuronal cells (Fig. 2C arrow heads). Smad8 mRNA signal was also observed in
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Fig. 1 – Expression of Smad family members 7 days after hypoglossal nerve injury. (A) RT–PCR analysis of Smad family members in control and injured hypoglossal nuclei of rats. cont, control side; inj, injured side. (B) The relative mRNA signal intensity in the injured side compared with that in control side was calculated. Bars represent means ± SD. *Significant difference compared with the control side, when analysed by Student's t-test (p < 0.01). Equivalent amounts of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are amplified, verifying standardization of conditions.
neuronal cells under normal conditions, but a hybridization signal was not identified in the nerve-injured hypoglossal nucleus (data not shown). These findings suggest that Smad1, 2 and 4 mRNAs are induced in nerve-injured motor neurons. Immunohistochemical study showed that the expression levels of Smad1, 2 and 4 proteins were increased in injured motor neurons (Fig. 3A). As Smad has been detected previously in the nucleus in some cell lines, we expected Smad-like immunoreactivity to be observed in nucleus. However, in this study, Smads 1 and 4 were observed mainly in the cytoplasm as well as the nucleus, before and after axotomy (Fig. 3A). Only Smad2 immunoreactivity was detected in the nucleus (Fig. 3A). Following nerve injury, positive staining for Smad2 protein was also seen in the cytoplasm in addition to nucleus (Fig. 3A). The increases in the immunoreactivity of Smads 1, 2/3 and 4 were detected initially in the ipsilateral side 1 day after the surgical procedure; however, the immunoreactivity markedly increased to a peak level during the following 7 days and persisted at this level for 14 days. Thereafter, the positive staining gradually decreased until control levels were reached during the following 56 days (data not shown). Finally, we quantified the levels of Smad1, Smad2/3 and Smad4 proteins in control and injured hypoglossal nuclei by Western blotting. 1.2-, 1.4- and 1.5-fold increases were identified in Smad1, Smad2/3 and Smad4, respectively (Fig. 3B). Collectively, these results suggest that Smads 1, 2/3 and 4 are transcriptionally and translationally up-regulated in injured motor neurons.
In this study, we identified an alteration of mRNA and protein levels of Smad family members in injured motor neurons. In situ hybridization revealed that the expression of Smad1, 2 and 4 mRNAs was significantly up-regulated in injured motor neurons, whereas that of Smad8 mRNA was down-regulated. The alteration of mRNAs for other Smads, Smads 5–7, was not significant. This suggests that Smad family members have specific gene regulation profiles during the nerve regeneration process. Immunohistochemical analysis further demonstrated that Smad1 and Smad4 proteins are predominantly localized to injured motor neurons in the hypoglossal nucleus. Smad2 protein, but not Smad3 protein, was also localized in motor neurons. Although the antibody used to detect Smad2 protein crossreacts with Smad3, the results of in situ hybridization supported the possibility that the signal seen in motor neurons was Smad2. Taken together, Smads 1, 2 and 4 would be the major components among Smad family members that might be implicated in the response to nerve injury. It is possible that Smad1 and Smad2 could form heterodimers with Smad4 that translocate into the nucleus and trans-activate various target genes that are associated with nerve regeneration. Further study will be needed to determine if this combination of Smad1–Smad4 and Smad2–Smad4 heterodimers really exists in injured motor neurons. Another piece of evidence in this study is that increased Smad proteins, which are demonstrated by the immunohistochemistry, were prominently accumulated in cytoplasm in addition to nucleus after nerve injury. Because some previous reports demonstrated that some
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Fig. 2 – The expression of Smad mRNAs in injured hypoglossal motor neurons. (A) The film (upper panel) and emulsion (lower panel) autoradiography of in situ hybridization showed expression of Smad family members. The expression levels of Smads 1, 2, 3 and 4 were markedly increased 7 days after hypoglossal nerve injury (black arrows), whereas expression of Smad8 mRNA was markedly decreased (white arrow). cc, central canal. Scale bar: 1.3 mm for film autoradiography, 100 μm for emulsion autoradiography. (B) The relative mRNA signal intensity in the injured side compared with the control side was measured. Bars represent means ± SD. *Significant difference from each control side, when analysed by Student's t-test (p < 0.01). (C) Bright-field illumination of emulsion-autoradiography demonstrated localization of mRNA expressing cells in injured side of hypoglossal nucleus. Smad1, 2 and 4 mRNA signals (accumulation of silver grains) were observed in large-sized motor neurons (arrows), whereas Smad3 mRNA signal was seen in small-sized non-neuronal cells (arrowheads). cont, control side. Scale bar: 50 μm.
signaling molecules such as Akt and MAP kinase inhibited nuclear translocation of Smad to prevent cell death or to adjust the level of Smad signaling (Kretzschmar et al., 1997; Song et al., 2006), the Smads in cytoplasm after nerve injury
may be accumulated in cytoplasm due to the prevention of the nuclear translocation. Alternatively Smads may have some unknown roles to protect injured neurons. To elucidate more detailed mechanism, further study will be needed.
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Fig. 3 – Expression of Smad proteins in the hypoglossal nucleus 7 days after axotomy. (A) Immunohistochemical images labeled for Smad1, Smad2/3 and Smad4. Higher magnification of Smads' immunoreactivity on the contralateral and injured sides. cc, central canal; cont, control side; inj, injured side. Scale bar: 100 μm, 25 μm (high power magnification). (B) Immunoblot analysis of Smad proteins after axotomy. Protein extracted from control and injured hypoglossal nuclei of rats 7 days after axotomy were subjected to Western blot analysis. Arrows indicate position of Smad2 and Smad3 proteins. Lower panel: the same membrane was stripped and re-blotted with anti-GAPDH antibody. GAPDH was used as internal control. cont, control side; inj, injured side.
Several lines of evidence have shown that motor neurons and Schwann cells induce and release TGF-β following peripheral nerve injury (Kiefer et al., 1993; Scherer et al., 1993; Rufer et al., 1994; Jiang et al., 2000). It has also been reported that spinal cord injury induces expression of several types of BMP (Setoguchi et al., 2001; Setoguchi et al., 2004). Thus, TGF-β family members are possible ligands for Smad signaling in injured motor neurons. TGF-β superfamily ligands bind to two distinct receptor types, known as type I and type II serine/threonine kinase receptors (Miyazawa et al., 2002; Ten Dijke and Hill, 2004). Following hypoglossal nerve injury, the mRNA level of type I TGF-β and/or activin receptor, named B1, was substantially suppressed in the injured motor neurons, whereas the type II TGF-β receptor (TβR-II) was not observed in the hypoglossal nucleus prior to, or after, axotomy (Morita et al., 1996). In contrast, Jiang et al. have reported that high levels of type I TGF-β receptor (TβR-I) and TβR-II proteins were observed in normal hypoglossal nerves, and TβR-II protein was accumulated at the proximal nerve after ligation (Jiang et al., 2000). These findings suggest that the expression of other type I and II receptors should be examined in injured motor neurons. Recently, Wang et al. reported that Mullerian Inhibiting Substance (MIS), a member of TGF-β superfamily, functions as a survival factor of motor neurons and that type II MIS receptor (MISRII) exist in motor neurons (Wang et al., 2005). Considering this fact, MISRII may be a responsible receptor for the downstream Smad mediated signaling. Conclusively, we have demonstrated increased expression of Smads 1, 2 and 4 in injured hypoglossal neurons, and suggested that a combination of Smad1–Smad4 and Smad2– Smad4 heterodimers effect Smad signaling in the nucleus. Although the target genes transactivated by Smads and the
ligands/receptors to activate Smads in injured motor neurons are still unclear, Smad-mediated signaling might have an important role during nerve regeneration.
Acknowledgments We are grateful to C. Kadono and I. Jikihara for technical assistance and T. Kawai for secretarial assistance. This study was supported in part by grants from the Ministry of Health, Labor and Welfare of Japan and the MEXT.
REFERENCES
Honma, M., Namikawa, K., Mansur, K., Iwata, T., Mori, N., Iizuka, H., Kiyama, H., 2002. Developmental alteration of nerve injury induced glial cell line-derived neurotrophic factor (GDNF) receptor expression is crucial for the determination of injured motoneuron fate. J. Neurochem. 82, 961–975. Jiang, Y., McLennan, I.S., Koishi, K., Hendry, I.A., 2000. Transforming growth factor-beta 2 is anterogradely and retrogradely transported in motoneurons and up-regulated after nerve injury. Neuroscience 97, 735–742. Kiefer, R., Lindholm, D., Kreutzberg, G.W., 1993. Interleukin-6 and transforming growth factor-beta 1 mRNAs are induced in rat facial nucleus following motoneuron axotomy. Eur. J. Neurosci. 5, 775–781. Kiryu, S., Morita, N., Ohno, K., Maeno, H., Kiyama, H., 1995a. Regulation of mRNA expression involved in Ras and PKA signal pathways during rat hypoglossal nerve regeneration. Brain Res. Mol. Brain Res. 29, 147–156. Kiryu, S., Yao, G.L., Morita, N., Kato, H., Kiyama, H., 1995b. Nerve injury enhances rat neuronal glutamate transporter
BR A IN RE S E A RCH 1 1 32 ( 20 0 7 ) 3 6 –4 1
expression: identification by differential display PCR. J. Neurosci. 15, 7872–7878. Kretzschmar, M., Liu, F., Hata, A., Doody, J., Massague, J., 1997. The TGF-beta family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev. 11, 984–995. Makwana, M., Raivich, G., 2005. Molecular mechanisms in successful peripheral regeneration. FEBS J. 272, 2628–2638. Miyazawa, K., Shinozaki, M., Hara, T., Furuya, T., Miyazono, K., 2002. Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells 7, 1191–1204. Moran, L.B., Graeber, M.B., 2004. The facial nerve axotomy model. Brain Res. Brain Res. Rev. 44, 154–178. Morita, N., Takumi, T., Kiyama, H., 1996. Distinct localization of two serine–threonine kinase receptors for activin and TGF-beta in the rat brain and down-regulation of type I activin receptor during peripheral nerve regeneration. Brain Res. Mol. Brain Res. 42, 263–271. Namikawa, K., Honma, M., Abe, K., Takeda, M., Mansur, K., Obata, T., Miwa, A., Okado, H., Kiyama, H., 2000. Akt/protein kinase B prevents injury-induced motoneuron death and accelerates axonal regeneration. J. Neurosci. 20, 2875–2886. Rufer, M., Flanders, K., Unsicker, K., 1994. Presence and regulation of transforming growth factor beta mRNA and protein in the normal and lesioned rat sciatic nerve. J. Neurosci. Res. 39, 412–423. Scherer, S.S., Kamholz, J., Jakowlew, S.B., 1993. Axons modulate the expression of transforming growth factor-betas in Schwann cells. Glia 8, 265–276.
41
Setoguchi, T., Yone, K., Matsuoka, E., Takenouchi, H., Nakashima, K., Sakou, T., Komiya, S., Izumo, S., 2001. Traumatic injury-induced BMP7 expression in the adult rat spinal cord. Brain Res. 921, 219–225. Setoguchi, T., Nakashima, K., Takizawa, T., Yanagisawa, M., Ochiai, W., Okabe, M., Yone, K., Komiya, S., Taga, T., 2004. Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor. Exp. Neurol. 189, 33–44. Snider, W.D., Zhou, F.Q., Zhong, J., Markus, A., 2002. Signaling the pathway to regeneration. Neuron 35, 13–16. Song, K., Wang, H., Krebs, T.L., Danielpour, D., 2006. Novel roles of Akt and mTOR in suppressing TGF-beta/ALK5-mediated Smad3 activation. EMBO J. 25, 58–69. Tanabe, K., Kiryu-Seo, S., Nakamura, T., Mori, N., Tsujino, H., Ochi, T., Kiyama, H., 1998. Alternative expression of Shc family members in nerve-injured motoneurons. Brain Res. Mol. Brain Res. 53, 291–296. ten Dijke, P., Hill, C.S., 2004. New insights into TGF-beta-Smad signalling. Trends Biochem. Sci. 29, 265–273. Wang, P.Y., Koishi, K., McGeachie, A.B., Kimber, M., Maclaughlin, D.T., Donahoe, P.K., McLennan, I.S., 2005. Mullerian inhibiting substance acts as a motor neuron survival factor in vitro. Proc. Natl. Acad. Sci. U. S. A. 102, 16421–16425. Yao, G.L., Kato, H., Khalil, M., Kiryu, S., Kiyama, H., 1997. Selective upregulation of cytokine receptor subchain and their intracellular signalling molecules after peripheral nerve injury. Eur. J. Neurosci. 9, 1047–1054.