Journal of Neuroscience Research 92:1078–1090 (2014)
Fibronectin Enhances Spinal Cord Astrocyte Proliferation by Elevating P2Y1 Receptor Expression Maosheng Xia* and Yue Zhu* Department of Orthopaedics, The First Hospital of China Medical University, Shengyang, People’s Republic of China
After spinal cord injury (SCI), the formation of glial scar is a complex process that is attributed primarily to astrocytic proliferation, but the mechanism of astrocytes proliferation is still unclear. Fibronectin is a large extracellular glycoprotein that helps organize the matrix protein, and its main membrane receptor is the a5b1 integrin subunit. In this study, fibronectin stimulated spinal cord astrocytic proliferation from two directions: fibronectin increased astrocytic proliferation via a5b1 integrin receptor, and fibronectin upregulated the expression of P2Y1 receptor, and adenosine triphosphate (ATP) could enhance the astrocytic proliferation and induce more release of arachidonic acid and prostaglandin E2 via P2Y1. The upregulation of P2Y1 by fibronectin required [Ca21]i and the activation of integrin link kinase (ILK) and Akt. We found that [Ca21]i stimulated by fibronectin was a5b1 integrin receptor dependent and that the phosphorylation of Akt or extracellular signal-regulated protein kinase (ERK1/2) induced by fibronectin mediated the activation of cAMP response element-binding protein (CREB) and signal transducer and activator of transcription 3 (Stat3). Our research suggests that the release of fibronectin and ATP could stimulate the spinal cord astrocytic proliferation after SCI, and the expression of P2Y1 increased by fibronectin would provide more sites for ATP, which could aggravate the proliferation and inflammation of spinal cord astrocytes. VC 2014 Wiley Periodicals, Inc. Key words: fibronectin; astrocytes; integrin; ATP
After spinal cord injury (SCI), one of the major causes leading to the regenerative failure of injured axons is the formation of a glial scar. The glial scar, consisting mainly of reactive astrocytes, forms a nonpermissive physical barrier to the regenerative axons (Silver and Miller, 2004; Hsu et al., 2008). The formation of the glial scar is a complex process attributed primarily to astrocytic proliferation (Ridet et al., 1997; Fitch et al., 1999; McGraw et al., 2001; Matyash et al., 2002; Hsu et al., 2008). In addition to the glial scar, the noncellular fibrotic scar is a main obstacle to axon regeneration (Schwab and Bartholdi, 1996; Silver and Miller, 2004; Kopp et al., 2010). After SCI, the fibrotic scar results from extracellular C 2014 Wiley Periodicals, Inc. V
matrix (ECM) deposition, mainly including collagen types I, III, and IV; laminin; fibronectin (Fn); and chondroitin sulfate proteoglycans (CSPGs; Kopp et al., 2010). Fn is a ubiquitous and abundant ECM protein, which is secreted by cells as a soluble dimer and is subsequently assembled into multimeric fibrils at the cell surface. Fn can be disintegrated by matrix metalloproteinases (MMPs) into fibronectin fragments (Fn-f) and plays crucial roles in various cellular functions, including cell adhesion, migration, proliferation, and differentiation (Hynes, 1985; Miyamoto et al., 1998). Fn is mediated by its interaction with cell surface receptors, usually through binding of the a5b1 integrin receptor (Wierzbicka-Patynowski and Schwarzbauer, 2003; Wang et al., 2008). Integrins are a family of heterodimeric transmembrane proteins that serve as receptors for extracellular matrix proteins such as Fn, laminins, and collagens. Protein kinases, including extracellular signalregulated protein kinase 1/2 (ERK1/2) and Akt are important signal transducers involved in cell survival (Lee and Ruoslahti, 2005; Sugimoto et al., 2011). Integrinlinked kinase (ILK), a potential candidate signaling molecule, has been shown to be capable of regulating integrinmediated signaling (Hannigan et al., 1996). ILK, which is an upstream regulator of Akt, can interact with the cytoplasmic domain of b-integrin subunits and is activated by Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: National Natural Science Foundation of China, contract grant number: 81200935; Contract grant sponsor: Doctoral Fund of Ministry of Education of China, contract grant number: 20102104120002; Contract grant sponsor: Educational Commission of Liaoning Province, contract grant number: L2011135. *Correspondence to: Maosheng Xia, Department of Orthopaedics, The First Hospital of China Medical University, No. 155 Nanjing Bei Street, Heping District, Shenyang, P.R. China 110001. E-mail: [email protected] or Yue Zhu, Department of Orthopaedics, The First Hospital of China Medical University, No. 155 Nanjing Bei Street, Heping District, Shenyang, P.R. China 110001. E-mail: [email protected] Received 14 July 2013; Revised 16 February 2014; Accepted 19 February 2014 Published online 31 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23384
Fn-Induced Astrocytic Proliferation
both integrin activation and growth factors (Wu and Dedhar, 2001; Hsu et al., 2007). Fn and integrin can promote proliferation in many kinds of cells. For example, in mouse embryonic stem (ES) cell, Fn increases phosphorylation of Akt and ERK1/2 and promotes ES cell proliferation (Park et al., 2011). In addition, integrins regulate multiple pathways that are frequently constitutively activated in cancer cells, including ERK and phosphoinositide 3-kinase (Grande-Garcıa et al., 2007; Huveneers and Danen, 2009). In vascular smooth muscle (VSM), the activation of a5b1 integrin leads to potentiation of L-type calcium channel current through involvement of the integrin-associated tyrosine kinases Src and focal adhesion kinase (Wu et al., 2002; Gui et al., 2006). Transcription factors such as cAMP response element-binding protein (CREB) and signal transducer and activator of transcription (Stat) are downstream of Akt and ERK1/2; these transcription factors could be promising candidates between activation of the upstream kinases and target protein regulation (Wang et al., 2010). Epidermal growth factor receptor (EGFR) participates in various disorders of the central nervous system (CNS), ranging from neurodegenerative diseases to tumor development. Rapid upregulation of EGFR ligands occurs following hypoxic or ischemic CNS injuries (Jin et al., 2002) or axotomy (Lisovoski et al., 1997). The upregulation of EGFR occurs in reactive astrocytes after a broad range of insults, including ischemia (Planas et al., 1998), electrolytic lesion (Junier et al., 1993), and entorhinal ablation (Nieto-Sampedro et al., 1988). CNS diseases suggest that the EGFR pathway may regulate postinjury activities of astrocytes and the subsequent pathological changes (Liu and Neufeld, 2004). For cultured spinal cord astrocytes, we previously reported that adenosine triphosphate (ATP) could stimulate the phosphorylation of ERK1/2 via the P2Y1 receptor, and the levels of arachidonic acid (AA) and prostaglandin E2 (PGE2) were increased (Xia and Zhu, 2011a). P2Y1 receptor was present in spinal cord astrocytes. This study shows that both Fn and ATP could promote spinal cord astrocytes proliferation, but discovering how Fn aggravates the function of ATP on spinal cord astrocytes was the most important goal, so we have investigated the potential mechanism of Fn stimulation of astrocytic proliferation.
1079
phe- nylthio]butadiene (U0126) was from Promega (Madison, WI). N-[(2R)22-(hydroxamidocarbonymethyl)-4-methylpentanoyl]-Ltryptophan methylamide (AG1478) was obtained from Calbiochem (La Jolla, CA). Primary antibodies anti-p-ERK1/2, antiphosphorylated Akt, anti-ERK1/2, and anti-Akt and the secondary antibodies TRITC-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies anti-p-CREB, antiphosphorylated Stat3, anti-CREB, and antitotal Stat3 were from Cell Signaling Technology (Danvers, MA). Real-time PCR kit, random hexamer, and Taq-polymerase were purchased from TaKaRa Biotechnology (Dalian, China). Astrocyte Culture Primary dissociated cultures of dorsal spinal cord were prepared from neonatal Sprague-Dawley rats (
Fibronectin Enhances Spinal Cord Astrocyte Proliferation by Elevating P2Y1 Receptor Expression Maosheng Xia* and Yue Zhu* Department of Orthopaedics, The First Hospital of China Medical University, Shengyang, People’s Republic of China
After spinal cord injury (SCI), the formation of glial scar is a complex process that is attributed primarily to astrocytic proliferation, but the mechanism of astrocytes proliferation is still unclear. Fibronectin is a large extracellular glycoprotein that helps organize the matrix protein, and its main membrane receptor is the a5b1 integrin subunit. In this study, fibronectin stimulated spinal cord astrocytic proliferation from two directions: fibronectin increased astrocytic proliferation via a5b1 integrin receptor, and fibronectin upregulated the expression of P2Y1 receptor, and adenosine triphosphate (ATP) could enhance the astrocytic proliferation and induce more release of arachidonic acid and prostaglandin E2 via P2Y1. The upregulation of P2Y1 by fibronectin required [Ca21]i and the activation of integrin link kinase (ILK) and Akt. We found that [Ca21]i stimulated by fibronectin was a5b1 integrin receptor dependent and that the phosphorylation of Akt or extracellular signal-regulated protein kinase (ERK1/2) induced by fibronectin mediated the activation of cAMP response element-binding protein (CREB) and signal transducer and activator of transcription 3 (Stat3). Our research suggests that the release of fibronectin and ATP could stimulate the spinal cord astrocytic proliferation after SCI, and the expression of P2Y1 increased by fibronectin would provide more sites for ATP, which could aggravate the proliferation and inflammation of spinal cord astrocytes. VC 2014 Wiley Periodicals, Inc. Key words: fibronectin; astrocytes; integrin; ATP
After spinal cord injury (SCI), one of the major causes leading to the regenerative failure of injured axons is the formation of a glial scar. The glial scar, consisting mainly of reactive astrocytes, forms a nonpermissive physical barrier to the regenerative axons (Silver and Miller, 2004; Hsu et al., 2008). The formation of the glial scar is a complex process attributed primarily to astrocytic proliferation (Ridet et al., 1997; Fitch et al., 1999; McGraw et al., 2001; Matyash et al., 2002; Hsu et al., 2008). In addition to the glial scar, the noncellular fibrotic scar is a main obstacle to axon regeneration (Schwab and Bartholdi, 1996; Silver and Miller, 2004; Kopp et al., 2010). After SCI, the fibrotic scar results from extracellular C 2014 Wiley Periodicals, Inc. V
matrix (ECM) deposition, mainly including collagen types I, III, and IV; laminin; fibronectin (Fn); and chondroitin sulfate proteoglycans (CSPGs; Kopp et al., 2010). Fn is a ubiquitous and abundant ECM protein, which is secreted by cells as a soluble dimer and is subsequently assembled into multimeric fibrils at the cell surface. Fn can be disintegrated by matrix metalloproteinases (MMPs) into fibronectin fragments (Fn-f) and plays crucial roles in various cellular functions, including cell adhesion, migration, proliferation, and differentiation (Hynes, 1985; Miyamoto et al., 1998). Fn is mediated by its interaction with cell surface receptors, usually through binding of the a5b1 integrin receptor (Wierzbicka-Patynowski and Schwarzbauer, 2003; Wang et al., 2008). Integrins are a family of heterodimeric transmembrane proteins that serve as receptors for extracellular matrix proteins such as Fn, laminins, and collagens. Protein kinases, including extracellular signalregulated protein kinase 1/2 (ERK1/2) and Akt are important signal transducers involved in cell survival (Lee and Ruoslahti, 2005; Sugimoto et al., 2011). Integrinlinked kinase (ILK), a potential candidate signaling molecule, has been shown to be capable of regulating integrinmediated signaling (Hannigan et al., 1996). ILK, which is an upstream regulator of Akt, can interact with the cytoplasmic domain of b-integrin subunits and is activated by Additional Supporting Information may be found in the online version of this article. Contract grant sponsor: National Natural Science Foundation of China, contract grant number: 81200935; Contract grant sponsor: Doctoral Fund of Ministry of Education of China, contract grant number: 20102104120002; Contract grant sponsor: Educational Commission of Liaoning Province, contract grant number: L2011135. *Correspondence to: Maosheng Xia, Department of Orthopaedics, The First Hospital of China Medical University, No. 155 Nanjing Bei Street, Heping District, Shenyang, P.R. China 110001. E-mail: [email protected] or Yue Zhu, Department of Orthopaedics, The First Hospital of China Medical University, No. 155 Nanjing Bei Street, Heping District, Shenyang, P.R. China 110001. E-mail: [email protected] Received 14 July 2013; Revised 16 February 2014; Accepted 19 February 2014 Published online 31 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23384
Fn-Induced Astrocytic Proliferation
both integrin activation and growth factors (Wu and Dedhar, 2001; Hsu et al., 2007). Fn and integrin can promote proliferation in many kinds of cells. For example, in mouse embryonic stem (ES) cell, Fn increases phosphorylation of Akt and ERK1/2 and promotes ES cell proliferation (Park et al., 2011). In addition, integrins regulate multiple pathways that are frequently constitutively activated in cancer cells, including ERK and phosphoinositide 3-kinase (Grande-Garcıa et al., 2007; Huveneers and Danen, 2009). In vascular smooth muscle (VSM), the activation of a5b1 integrin leads to potentiation of L-type calcium channel current through involvement of the integrin-associated tyrosine kinases Src and focal adhesion kinase (Wu et al., 2002; Gui et al., 2006). Transcription factors such as cAMP response element-binding protein (CREB) and signal transducer and activator of transcription (Stat) are downstream of Akt and ERK1/2; these transcription factors could be promising candidates between activation of the upstream kinases and target protein regulation (Wang et al., 2010). Epidermal growth factor receptor (EGFR) participates in various disorders of the central nervous system (CNS), ranging from neurodegenerative diseases to tumor development. Rapid upregulation of EGFR ligands occurs following hypoxic or ischemic CNS injuries (Jin et al., 2002) or axotomy (Lisovoski et al., 1997). The upregulation of EGFR occurs in reactive astrocytes after a broad range of insults, including ischemia (Planas et al., 1998), electrolytic lesion (Junier et al., 1993), and entorhinal ablation (Nieto-Sampedro et al., 1988). CNS diseases suggest that the EGFR pathway may regulate postinjury activities of astrocytes and the subsequent pathological changes (Liu and Neufeld, 2004). For cultured spinal cord astrocytes, we previously reported that adenosine triphosphate (ATP) could stimulate the phosphorylation of ERK1/2 via the P2Y1 receptor, and the levels of arachidonic acid (AA) and prostaglandin E2 (PGE2) were increased (Xia and Zhu, 2011a). P2Y1 receptor was present in spinal cord astrocytes. This study shows that both Fn and ATP could promote spinal cord astrocytes proliferation, but discovering how Fn aggravates the function of ATP on spinal cord astrocytes was the most important goal, so we have investigated the potential mechanism of Fn stimulation of astrocytic proliferation.
1079
phe- nylthio]butadiene (U0126) was from Promega (Madison, WI). N-[(2R)22-(hydroxamidocarbonymethyl)-4-methylpentanoyl]-Ltryptophan methylamide (AG1478) was obtained from Calbiochem (La Jolla, CA). Primary antibodies anti-p-ERK1/2, antiphosphorylated Akt, anti-ERK1/2, and anti-Akt and the secondary antibodies TRITC-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies anti-p-CREB, antiphosphorylated Stat3, anti-CREB, and antitotal Stat3 were from Cell Signaling Technology (Danvers, MA). Real-time PCR kit, random hexamer, and Taq-polymerase were purchased from TaKaRa Biotechnology (Dalian, China). Astrocyte Culture Primary dissociated cultures of dorsal spinal cord were prepared from neonatal Sprague-Dawley rats (
