Neuroscience Letters 569 (2014) 55–58
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Expression of the JAK/STAT3/SOCS3 signaling pathway in herniated lumbar discs Koji Osuka a,∗ , Nobuteru Usuda b , Masahiro Aoyama a , Hitoshi Yamahata a , Mikinobu Takeuchi a , Muneyoshi Yasuda a , Masakazu Takayasu a a b
Department of Neurological Surgery, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan Department of Anatomy II, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake, Toyoake, Aichi 470-1192, Japan
h i g h l i g h t s • JAK1 and STAT3 are expressed in the chondrocytes of herniated lumbar discs. • Phosphorylated STAT3 is expressed within the nuclei of chondrocytes. • JAK–STAT3 signaling pathway is activated within the chondrocytes of herniated discs.
a r t i c l e
i n f o
Article history: Received 16 February 2014 Received in revised form 17 March 2014 Accepted 19 March 2014 Keywords: Herniated disk Janus kinase Signal transducer and activator of transcription Suppressor of cytokine signaling
a b s t r a c t The inflammatory cytokine interleukin-6 (IL-6) plays an important role in causing symptoms of lumbar disk herniation. The present study clarifies the expression of the signaling pathway of IL-6 in herniated discs. Homogenates prepared from lumbar herniated discs from 10 patients were assessed. The expression of janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), phosphorylated (p)-STAT3 at Tyr705 , suppressor of cytokine signaling 3 (SOCS3) and actin was examined by Western blot analysis. The expression of JAK1, STAT3, and p-STAT3 at Tyr705 was also examined by immunostaining. JAK1, STAT3, p-STAT3 at Tyr705 and SOCS3 were detected in almost all cases. Immunoreactivity against JAK1 and STAT3 was observed mainly in chondrocytes, whereas immunoreactivity against p-STAT3 at Tyr705 was observed in the nuclei of chondrocytes. The JAK/STAT signaling pathway might be activated by IL-6 and transmit messages from the cell surface to the nucleus, and the pathway is negatively regulated by SOCS3. These JAK1, STAT3 and SOCS3 molecules might tightly regulate and play a role in the degeneration of chondrocytes within herniated discs. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Interleukin-6 (IL-6) is considered an important mediator of acute inflammatory responses. The expression of IL-6 in chondrocytes of lumbar herniated discs has been reported [11]. The infiltration of macrophages into herniated disk material may play a major role in IL-6 production [13]. In addition to IL-6, prostaglandin E2, nitric oxide, IL-1␣ and tumor necrosis factor-␣ (TNF-␣) are produced within lumbar disk materials in response to an inflammatory stimulus [5,14]. In addition, cyclooxygenase-2, the rate-limiting enzyme in prostaglandin E2 production, was observed in the
chondrocytes of lumbar herniated discs [8]. These data suggest that inflammatory reactions occur within herniated discs and play a role in disk degeneration and lumbar radiculopathy. Although numerous studies have confirmed the clinical importance of IL-6 in herniated discs, the mechanism of signal transduction of IL-6 in herniated discs still remains unknown. The present study was therefore undertaken to clarify this point through immunoblot analysis and immunohistochemistry.
2. Materials and methods 2.1. Patients
Abbreviations: JAK, janus kinase; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; IL, interleukin. ∗ Corresponding author. Tel.: +81 561 62 3311; fax: +81 561 63 2879. E-mail addresses: [email protected], k [email protected] (K. Osuka). http://dx.doi.org/10.1016/j.neulet.2014.03.045 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Ten patients (7 men, 3 women; age range: 58–79 years; mean age: 67 years) suffering from lumbar disk herniation confirmed by magnetic resonance (MR) imaging were enrolled in this study.
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None of the patients had a history of previous herniated disk therapy. All patients underwent herniectomy and discectomy at Aichi Medical University Hospital. The Ethics Committee of Aichi Medical University approved this clinical experiment. Informed consent for specimen collection was obtained from each patient. 2.2. Western blot analysis Herniated discs were homogenized using a homogenizer in 250 l of homogenization buffer containing 50 mmol/L Tris base/HCl (pH 7.5), 0.1 mmol/L dithiothreitol, 0.2 mmol/L ethylene-diaminetetraacetate (EDTA), 0.2 mmol/L ethyleneglycol bis aminoethylether tetraacetate (EGTA), 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1.25 g/mL pepstatin A, 0.2 g/mL aprotinin, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, 2 mmol/L sodium pyrophosphate and 1% Nonidet P-40. The homogenates were later centrifuged at 12,000 × g at 4 ◦ C for 10 min. The protein concentrations of the supernatants were determined by the Bradford assay using bovine serum albumin as the standard. Crude samples (25 g of each protein) were subjected to 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary polyclonal antibodies against actin (Sigma, St. Louis, MO), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) at Tyr705 (Cell Signaling Technology, Danvers, MA), suppressor of cytokine signaling 3 (␣-SOCS3, Santa Cruz technology, Dallas, TX), and primary monoclonal antibody against janus kinase 1 (JAK1, BD Transduction Lab., Lexington, KY) at a dilution of 1:500 overnight at 4 ◦ C. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology) at a dilution of 1:3000 for 30 min at room temperature. The reactions were developed with ECL or ECL plus (GE Healthcare, Buckinghamshire, U.K.). Phosphorylated STAT3 at Tyr705 immunoblots were stripped from PVDF membranes and reblotted with primary polyclonal Stat3 (Cell Signaling Technology) at a dilution of 1:500 overnight at 4 ◦ C. Finally, the membranes were developed with ECL plus. 2.3. Histological examination To study the cellular localization of JAK1, STAT3 and p-STAT3 at Tyr705 , immunohistochemical staining was performed according to the avidin-biotinylated peroxidase complex (ABC) technique at room temperature (n = 3). Herniated discs were preserved in 10 mL of ice-cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 3 h. Serial axial cryostat sections (10 m) were placed on slides for staining. These sections were first stained with hematoxylin and eosin (H&E, n = 3), and then non-specific immunoreactivity was blocked with goat serum for 30 min. Samples were treated with primary antibody against STAT3, p-STAT3 at Tyr705 (Cell Signaling Technology) and JAK1 (BD Transduction Lab.) at a dilution of 1:50 overnight at 4 ◦ C. After washing, biotinylated anti-rabbit or mouse IgG was added for 1 h, followed by a 1-h incubation with ABC. Sera for the blocking step, biotinylated antibodies and ABC were purchased from Vector Laboratories (Burlingame, CA). Reaction products were developed by incubation in 0.05% 3,3 diaminobendizine tetrachloride and 0.01% H2 O2 in 50 mM Tris–HCl (pH 7.5) for 10 min. Herniated discs that were stained without primary antibody were used as negative controls. 3. Results Nearly constant levels of actin were detected in all cases, suggesting equal levels of protein applied to SDS gels. JAK1, STAT3 and
Fig. 1. Herniated lumbar discs from 10 patients were subjected to western blotting with anti-actin (␣-actin), anti-janus kinase 1 (␣-JAK1), anti-signal transducer and activator of transcription 3 (␣-STAT3), anti-phosphorylated STAT3 at Tyr705 (␣-pSTAT3 at Tyr705 ) and anti-suppressor of cytokine signaling 3 (␣-SOCS3) antibodies. Note that JAK1, STAT3 and p-STAT3 at Tyr705 molecules could be detected in almost all cases of herniated lumbar discs. SOCS3, the negative regulator of the JAK/STAT3 signaling pathway, was also expressed in all cases.
SOCS3 were also detected in almost all cases. In addition, p-STAT3 at Tyr705 was detected (Fig. 1). H&E staining showed that chondrocytes were well developed between collagenous fibers (Fig. 2A). Immunoreactivity against JAK1 and STAT3 was observed mainly in chondrocytes (Fig. 2B and C, respectively). Immunoreactivity of p-TAT3 at Tyr705 was detected in the nuclei of chondrocytes (Fig. 2D). Immunohistochemical staining of herniated discs without primary antibodies was consistently negative (data not shown). 4. Discussion In this study, we confirmed that JAK1, STAT3, p-STAT3 at Tyr705 and SOCS3 are expressed in lumbar herniated discs. These molecules are dominantly expressed in the chondrocytes of herniated discs. JAK/STAT proteins are transcriptional factors that are responsible for transmitting messages from the cell surface to the nucleus, thereby modulating gene expression. This signaling pathway is activated by many cytokines and growth factors, one of which is a well-known inflammatory cytokine, IL-6 [4]. IL-6 signaling occurs through binding to an IL-6 receptor and coupling to two glycoprotein 130 (gp-130), which are transmembrane proteins [4]. JAKs are then activated and phosphorylate STAT proteins at tyrosine residues. The STAT proteins dimerize and translocate into the nucleus followed by binding to specific consensus sequences of target-gene promoters [4]. One previous study showed that not only IL-6, but also IL-6R was detected in herniated discs [11]. Our data suggest that this JAK/STAT signaling pathway is activated in lumbar herniated discs and plays a role in inflammatory response. The constitutive activation of STAT signaling has also been shown to participate in the development and progression of human tumors. The activation of JAK and STAT has been demonstrated in various human blood malignancies, including lymphoma and leukemia [7,15]. IL-6 plays a critical role in the proliferation of human multiple myeloma cells and induces STAT3 activation. Activated STAT3 results in the upregulation of anti-apoptotic protein Bcl-xL and increased myeloma cell survival [6]. In human breast and lung carcinomas, cell activation of STAT3 has been detected and plays a role in tumor progression [2,9]. These data suggest that activated STAT signaling participates in oncogenesis by stimulating cell proliferation. SOCS protein is one of the key factors involved in the negative regulation of JAK/STAT signaling [1,12]. SOCS genes are transcriptionally activated in response to STAT-mediated mechanisms. SOCS proteins interact with the catalytic domains of JAK proteins and inhibit JAK kinase activity, resulting in the inhibition of STAT activation [17]. This negative feedback pathway tightly regulates
K. Osuka et al. / Neuroscience Letters 569 (2014) 55–58
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Fig. 2. Hematoxylin and eosin staining demonstrated that chondrocytes are located between collagenous fibers (A). Ten-micrometer slices were immunostained with a monoclonal antibody recognizing janus kinase 1 (JAK1, B) and with polyclonal antibodies recognizing signal transducer and activator of transcription 3 (STAT3, C) and phosphorylated (p) STAT3 at Tyr705 (D) by the ABC method. Note that both JAK1 and STAT3 molecules were expressed in chondrocytes, and p-STAT3 at Tyr705 was in the nuclei of chondrocytes (D). Scale bars = 100 m (A–D).
cytokine-induced activation of STATs. The JAK/STAT signaling pathway contributes to the development of rheumatoid arthritis. Periarticular injection of SOCS3 adenovirus drastically reduced the severity of arthritis and joint swelling in experimental treatments for arthritis [10]. SOCS3 directly modulates stress-induced gp130 cytokine receptor signaling, serving as the key molecular regulator for a negative feedback circuit in myocyte hypertrophy [16]. Working in concert, these cytokines must exert endocrine, autocrine and paracrine effects and regulate local inflammation and subsequent healing within herniated discs. There are limitations to the present study that should be noted. In this in vivo study, the expression of various proteins was measured by immunoblot and immunohistochemical analyses. However, we could not compare our data with data on healthy control discs. A previous study used autoptic L4-5 disk tissue collected within 24 h postmortem from subjects with no history of back pain as a control [11]. Postmortem changes in the phosphorylation state of protein were reported, suggesting that rapid processing is essential for an accurate assessment of active phosphorylated isoforms [3]. Considering these issues, comparison with healthy intervertebral discs may be difficult. This study is preliminary, and further studies are necessary to explore the exact roles of the JAK/STAT pathway in herniated discs. However, the presence of these molecules suggests the involvement of IL-6 signal transduction in intervertebral disk degeneration. The present investigation reports, for the first time, the expression of JAK1, STAT3, p-STAT3 at Tyr705 and SOCS3 molecules in human lumbar herniated discs. Of particular importance is the finding that STAT3 at Tyr705 activation occurs in the nuclei of chondrocytes, suggesting that these molecules may be involved in the degeneration of chondrocytes by IL-6. Further studies using inhibitors of the JAK/STAT3 signaling pathway may help identify
clinical targets to relieve the pain caused by nerve compression from herniated discs. Acknowledgment The authors thank Hisae Inui for her technical assistance. References [1] T.A. Endo, M. Masuhara, M. Yokouchi, R. Suzuki, H. Sakamoto, K. Mitsui, A. Matsumoto, S. Tanimura, M. Ohtsubo, H. Misawa, T. Miyazaki, N. Leonor, T. Taniguchi, T. Fujita, Y. Kanakura, S. Komiya, A. Yoshimura, A new protein containing an SH2 domain that inhibits JAK kinases, Nature 387 (1997) 921–924. [2] A. Fernandes, A.W. Hamburger, B.I. Gerwin, ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells, Int. J. Cancer 83 (1999) 564–570. [3] U. Gärtner, C. Janke, M. Holzer, E. Vanmechelen, T. Arendt, Postmortem changes in the phosphorylation state of tau-protein in the rat brain, Neurobiol. Aging 19 (1998) 535–543. [4] P.C. Heinrich, I. Behrmann, S. Haan, H.M. Hermanns, G. Muller-Newen, F. Schaper, Principles of interleukin (IL)-6-type cytokine signalling and its regulation, Biochem. J. 374 (2003) 1–20. [5] J.D. Kang, H.I. Georgescu, L. McIntyre-Larkin, M. Stefanovic-Racic, W.F. Donaldson 3rd, C.H. Evans, Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2, Spine (Phila Pa 1976) 21 (1996) 271–277. [6] T. Kishimoto, T. Taga, S. Akira, Cytokine signal transduction, Cell 76 (1994) 253–262. [7] T.S. Migone, J.X. Lin, A. Cereseto, J.C. Mulloy, J.J. O’Shea, G. Franchini, W.J. Leonard, Constitutively activated JAK–STAT pathway in T cells transformed with HTLV-I, Science 269 (1995) 79–81. [8] H. Miyamoto, R. Saura, M. Doita, M. Kurosaka, K. Mizuno, The role of cyclooxygenase-2 in lumbar disc herniation, Spine (Phila Pa 1976) 27 (2002) 2477–2483. [9] C.I. Sartor, M.L. Dziubinski, C.L. Yu, R. Jove, S.P. Ethier, Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM102PT human breast cancer cells, Cancer Res. 57 (1997) 978–987. [10] T. Shouda, T. Yoshida, T. Hanada, T. Wakioka, M. Oishi, K. Miyoshi, S. Komiya, K. Kosai, Y. Hanakawa, K. Hashimoto, K. Nagata, A. Yoshimura, Induction of
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[11]
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[13]
[14]
K. Osuka et al. / Neuroscience Letters 569 (2014) 55–58 the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis, J. Clin. Invest. 108 (2001) 1781–1788. N. Specchia, A. Pagnotta, A. Toesca, F. Greco, Cytokines and growth factors in the protruded intervertebral disc of the lumbar spine, Eur. Spine J. 11 (2002) 145–151. R. Starr, T.A. Willson, E.M. Viney, L.J. Murray, J.R. Rayner, B.J. Jenkins, T.J. Gonda, W.S. Alexander, D. Metcalf, N.A. Nicola, D.J. Hilton, A family of cytokineinducible inhibitors of signalling, Nature 387 (1997) 917–921. T. Takada, K. Nishida, M. Doita, H. Miyamoto, M. Kurosaka, Interleukin-6 production is upregulated by interaction between disc tissue and macrophages, Spine (Phila Pa 1976) 29 (2004) 1089–1092 (discussion 1093). H. Takahashi, T. Suguro, Y. Okazima, M. Motegi, Y. Okada, T. Kakiuchi, Inflammatory cytokines in the herniated disc of the lumbar spine, Spine (Phila Pa 1976) 21 (1996) 218–224.
[15] R.M. Weber-Nordt, C. Egen, J. Wehinger, W. Ludwig, V. Gouilleux-Gruart, R. Mertelsmann, J. Finke, Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein–Barr virus (EBV)-related lymphoma cell lines, Blood 88 (1996) 809–816. [16] H. Yasukawa, M. Hoshijima, Y. Gu, T. Nakamura, S. Pradervand, T. Hanada, Y. Hanakawa, A. Yoshimura, J. Ross Jr., K.R. Chien, Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130mediated cardiac myocyte hypertrophy and survival pathways, J. Clin. Invest. 108 (2001) 1459–1467. [17] H. Yasukawa, H. Misawa, H. Sakamoto, M. Masuhara, A. Sasaki, T. Wakioka, S. Ohtsuka, T. Imaizumi, T. Matsuda, J.N. Ihle, A. Yoshimura, The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop, EMBO J. 18 (1999) 1309–1320.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Expression of the JAK/STAT3/SOCS3 signaling pathway in herniated lumbar discs Koji Osuka a,∗ , Nobuteru Usuda b , Masahiro Aoyama a , Hitoshi Yamahata a , Mikinobu Takeuchi a , Muneyoshi Yasuda a , Masakazu Takayasu a a b
Department of Neurological Surgery, Aichi Medical University, 1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan Department of Anatomy II, Fujita Health University School of Medicine, 1-98 Dengakugakubo, Kutsukake, Toyoake, Aichi 470-1192, Japan
h i g h l i g h t s • JAK1 and STAT3 are expressed in the chondrocytes of herniated lumbar discs. • Phosphorylated STAT3 is expressed within the nuclei of chondrocytes. • JAK–STAT3 signaling pathway is activated within the chondrocytes of herniated discs.
a r t i c l e
i n f o
Article history: Received 16 February 2014 Received in revised form 17 March 2014 Accepted 19 March 2014 Keywords: Herniated disk Janus kinase Signal transducer and activator of transcription Suppressor of cytokine signaling
a b s t r a c t The inflammatory cytokine interleukin-6 (IL-6) plays an important role in causing symptoms of lumbar disk herniation. The present study clarifies the expression of the signaling pathway of IL-6 in herniated discs. Homogenates prepared from lumbar herniated discs from 10 patients were assessed. The expression of janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), phosphorylated (p)-STAT3 at Tyr705 , suppressor of cytokine signaling 3 (SOCS3) and actin was examined by Western blot analysis. The expression of JAK1, STAT3, and p-STAT3 at Tyr705 was also examined by immunostaining. JAK1, STAT3, p-STAT3 at Tyr705 and SOCS3 were detected in almost all cases. Immunoreactivity against JAK1 and STAT3 was observed mainly in chondrocytes, whereas immunoreactivity against p-STAT3 at Tyr705 was observed in the nuclei of chondrocytes. The JAK/STAT signaling pathway might be activated by IL-6 and transmit messages from the cell surface to the nucleus, and the pathway is negatively regulated by SOCS3. These JAK1, STAT3 and SOCS3 molecules might tightly regulate and play a role in the degeneration of chondrocytes within herniated discs. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Interleukin-6 (IL-6) is considered an important mediator of acute inflammatory responses. The expression of IL-6 in chondrocytes of lumbar herniated discs has been reported [11]. The infiltration of macrophages into herniated disk material may play a major role in IL-6 production [13]. In addition to IL-6, prostaglandin E2, nitric oxide, IL-1␣ and tumor necrosis factor-␣ (TNF-␣) are produced within lumbar disk materials in response to an inflammatory stimulus [5,14]. In addition, cyclooxygenase-2, the rate-limiting enzyme in prostaglandin E2 production, was observed in the
chondrocytes of lumbar herniated discs [8]. These data suggest that inflammatory reactions occur within herniated discs and play a role in disk degeneration and lumbar radiculopathy. Although numerous studies have confirmed the clinical importance of IL-6 in herniated discs, the mechanism of signal transduction of IL-6 in herniated discs still remains unknown. The present study was therefore undertaken to clarify this point through immunoblot analysis and immunohistochemistry.
2. Materials and methods 2.1. Patients
Abbreviations: JAK, janus kinase; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; IL, interleukin. ∗ Corresponding author. Tel.: +81 561 62 3311; fax: +81 561 63 2879. E-mail addresses: [email protected], k [email protected] (K. Osuka). http://dx.doi.org/10.1016/j.neulet.2014.03.045 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Ten patients (7 men, 3 women; age range: 58–79 years; mean age: 67 years) suffering from lumbar disk herniation confirmed by magnetic resonance (MR) imaging were enrolled in this study.
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K. Osuka et al. / Neuroscience Letters 569 (2014) 55–58
None of the patients had a history of previous herniated disk therapy. All patients underwent herniectomy and discectomy at Aichi Medical University Hospital. The Ethics Committee of Aichi Medical University approved this clinical experiment. Informed consent for specimen collection was obtained from each patient. 2.2. Western blot analysis Herniated discs were homogenized using a homogenizer in 250 l of homogenization buffer containing 50 mmol/L Tris base/HCl (pH 7.5), 0.1 mmol/L dithiothreitol, 0.2 mmol/L ethylene-diaminetetraacetate (EDTA), 0.2 mmol/L ethyleneglycol bis aminoethylether tetraacetate (EGTA), 0.2 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1.25 g/mL pepstatin A, 0.2 g/mL aprotinin, 1 mmol/L sodium orthovanadate, 50 mmol/L sodium fluoride, 2 mmol/L sodium pyrophosphate and 1% Nonidet P-40. The homogenates were later centrifuged at 12,000 × g at 4 ◦ C for 10 min. The protein concentrations of the supernatants were determined by the Bradford assay using bovine serum albumin as the standard. Crude samples (25 g of each protein) were subjected to 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary polyclonal antibodies against actin (Sigma, St. Louis, MO), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) at Tyr705 (Cell Signaling Technology, Danvers, MA), suppressor of cytokine signaling 3 (␣-SOCS3, Santa Cruz technology, Dallas, TX), and primary monoclonal antibody against janus kinase 1 (JAK1, BD Transduction Lab., Lexington, KY) at a dilution of 1:500 overnight at 4 ◦ C. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology) at a dilution of 1:3000 for 30 min at room temperature. The reactions were developed with ECL or ECL plus (GE Healthcare, Buckinghamshire, U.K.). Phosphorylated STAT3 at Tyr705 immunoblots were stripped from PVDF membranes and reblotted with primary polyclonal Stat3 (Cell Signaling Technology) at a dilution of 1:500 overnight at 4 ◦ C. Finally, the membranes were developed with ECL plus. 2.3. Histological examination To study the cellular localization of JAK1, STAT3 and p-STAT3 at Tyr705 , immunohistochemical staining was performed according to the avidin-biotinylated peroxidase complex (ABC) technique at room temperature (n = 3). Herniated discs were preserved in 10 mL of ice-cold 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 3 h. Serial axial cryostat sections (10 m) were placed on slides for staining. These sections were first stained with hematoxylin and eosin (H&E, n = 3), and then non-specific immunoreactivity was blocked with goat serum for 30 min. Samples were treated with primary antibody against STAT3, p-STAT3 at Tyr705 (Cell Signaling Technology) and JAK1 (BD Transduction Lab.) at a dilution of 1:50 overnight at 4 ◦ C. After washing, biotinylated anti-rabbit or mouse IgG was added for 1 h, followed by a 1-h incubation with ABC. Sera for the blocking step, biotinylated antibodies and ABC were purchased from Vector Laboratories (Burlingame, CA). Reaction products were developed by incubation in 0.05% 3,3 diaminobendizine tetrachloride and 0.01% H2 O2 in 50 mM Tris–HCl (pH 7.5) for 10 min. Herniated discs that were stained without primary antibody were used as negative controls. 3. Results Nearly constant levels of actin were detected in all cases, suggesting equal levels of protein applied to SDS gels. JAK1, STAT3 and
Fig. 1. Herniated lumbar discs from 10 patients were subjected to western blotting with anti-actin (␣-actin), anti-janus kinase 1 (␣-JAK1), anti-signal transducer and activator of transcription 3 (␣-STAT3), anti-phosphorylated STAT3 at Tyr705 (␣-pSTAT3 at Tyr705 ) and anti-suppressor of cytokine signaling 3 (␣-SOCS3) antibodies. Note that JAK1, STAT3 and p-STAT3 at Tyr705 molecules could be detected in almost all cases of herniated lumbar discs. SOCS3, the negative regulator of the JAK/STAT3 signaling pathway, was also expressed in all cases.
SOCS3 were also detected in almost all cases. In addition, p-STAT3 at Tyr705 was detected (Fig. 1). H&E staining showed that chondrocytes were well developed between collagenous fibers (Fig. 2A). Immunoreactivity against JAK1 and STAT3 was observed mainly in chondrocytes (Fig. 2B and C, respectively). Immunoreactivity of p-TAT3 at Tyr705 was detected in the nuclei of chondrocytes (Fig. 2D). Immunohistochemical staining of herniated discs without primary antibodies was consistently negative (data not shown). 4. Discussion In this study, we confirmed that JAK1, STAT3, p-STAT3 at Tyr705 and SOCS3 are expressed in lumbar herniated discs. These molecules are dominantly expressed in the chondrocytes of herniated discs. JAK/STAT proteins are transcriptional factors that are responsible for transmitting messages from the cell surface to the nucleus, thereby modulating gene expression. This signaling pathway is activated by many cytokines and growth factors, one of which is a well-known inflammatory cytokine, IL-6 [4]. IL-6 signaling occurs through binding to an IL-6 receptor and coupling to two glycoprotein 130 (gp-130), which are transmembrane proteins [4]. JAKs are then activated and phosphorylate STAT proteins at tyrosine residues. The STAT proteins dimerize and translocate into the nucleus followed by binding to specific consensus sequences of target-gene promoters [4]. One previous study showed that not only IL-6, but also IL-6R was detected in herniated discs [11]. Our data suggest that this JAK/STAT signaling pathway is activated in lumbar herniated discs and plays a role in inflammatory response. The constitutive activation of STAT signaling has also been shown to participate in the development and progression of human tumors. The activation of JAK and STAT has been demonstrated in various human blood malignancies, including lymphoma and leukemia [7,15]. IL-6 plays a critical role in the proliferation of human multiple myeloma cells and induces STAT3 activation. Activated STAT3 results in the upregulation of anti-apoptotic protein Bcl-xL and increased myeloma cell survival [6]. In human breast and lung carcinomas, cell activation of STAT3 has been detected and plays a role in tumor progression [2,9]. These data suggest that activated STAT signaling participates in oncogenesis by stimulating cell proliferation. SOCS protein is one of the key factors involved in the negative regulation of JAK/STAT signaling [1,12]. SOCS genes are transcriptionally activated in response to STAT-mediated mechanisms. SOCS proteins interact with the catalytic domains of JAK proteins and inhibit JAK kinase activity, resulting in the inhibition of STAT activation [17]. This negative feedback pathway tightly regulates
K. Osuka et al. / Neuroscience Letters 569 (2014) 55–58
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Fig. 2. Hematoxylin and eosin staining demonstrated that chondrocytes are located between collagenous fibers (A). Ten-micrometer slices were immunostained with a monoclonal antibody recognizing janus kinase 1 (JAK1, B) and with polyclonal antibodies recognizing signal transducer and activator of transcription 3 (STAT3, C) and phosphorylated (p) STAT3 at Tyr705 (D) by the ABC method. Note that both JAK1 and STAT3 molecules were expressed in chondrocytes, and p-STAT3 at Tyr705 was in the nuclei of chondrocytes (D). Scale bars = 100 m (A–D).
cytokine-induced activation of STATs. The JAK/STAT signaling pathway contributes to the development of rheumatoid arthritis. Periarticular injection of SOCS3 adenovirus drastically reduced the severity of arthritis and joint swelling in experimental treatments for arthritis [10]. SOCS3 directly modulates stress-induced gp130 cytokine receptor signaling, serving as the key molecular regulator for a negative feedback circuit in myocyte hypertrophy [16]. Working in concert, these cytokines must exert endocrine, autocrine and paracrine effects and regulate local inflammation and subsequent healing within herniated discs. There are limitations to the present study that should be noted. In this in vivo study, the expression of various proteins was measured by immunoblot and immunohistochemical analyses. However, we could not compare our data with data on healthy control discs. A previous study used autoptic L4-5 disk tissue collected within 24 h postmortem from subjects with no history of back pain as a control [11]. Postmortem changes in the phosphorylation state of protein were reported, suggesting that rapid processing is essential for an accurate assessment of active phosphorylated isoforms [3]. Considering these issues, comparison with healthy intervertebral discs may be difficult. This study is preliminary, and further studies are necessary to explore the exact roles of the JAK/STAT pathway in herniated discs. However, the presence of these molecules suggests the involvement of IL-6 signal transduction in intervertebral disk degeneration. The present investigation reports, for the first time, the expression of JAK1, STAT3, p-STAT3 at Tyr705 and SOCS3 molecules in human lumbar herniated discs. Of particular importance is the finding that STAT3 at Tyr705 activation occurs in the nuclei of chondrocytes, suggesting that these molecules may be involved in the degeneration of chondrocytes by IL-6. Further studies using inhibitors of the JAK/STAT3 signaling pathway may help identify
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