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Blood Vitronectin Is a Major Activator of LIF and IL-6 in the Brain Through Integrin–FAK and Upar Signaling Matthew P

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Blood Vitronectin Is a Major Activator of LIF and IL-6 in the Brain Through Integrin–FAK and Upar Signaling Matthew P © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs202580. doi:10.1242/jcs.202580 RESEARCH ARTICLE Blood vitronectin is a major activator of LIF and IL-6 in the brain through integrin–FAK and uPAR signaling Matthew P. Keasey1, Cuihong Jia1, Lylyan F. Pimentel1,2, Richard R. Sante1, Chiharu Lovins1 and Theo Hagg1,* ABSTRACT Microglia and astrocytes express the VTN receptors αvβ3 and α β We defined how blood-derived vitronectin (VTN) rapidly and potently v 5 integrin (Herrera-Molina et al., 2012; Kang et al., 2008; activates leukemia inhibitory factor (LIF) and pro-inflammatory Milner, 2009; Welser-Alves et al., 2011). Microglia and astrocytes, interleukin 6 (IL-6) in vitro and after vascular injury in the brain. as well as endothelial cells, are major producers of pro- α in vitro Treatment with VTN (but not fibrinogen, fibronectin, laminin-111 or inflammatory cytokines, such as IL-6 and TNF , and collagen-I) substantially increased LIF and IL-6 within 4 h in after traumatic or ischemic injury to the brain (Banner et al., 1997; C6-astroglioma cells, while VTN−/− mouse plasma was less effective Erta et al., 2012; Lau and Yu, 2001) or upon self-induction by IL-6 than that from wild-type mice. LIF and IL-6 were induced by (Van Wagoner and Benveniste, 1999). IL-6 is a major regulator of a intracerebral injection of recombinant human (rh)VTN in mice, but variety of inflammatory disorders and a target for therapies (Hunter induction seen upon intracerebral hemorrhage was less in VTN−/− and Jones, 2015). Its levels are almost non-existent in the normal mice than in wild-type littermates. In vitro, VTN effects were inhibited by brain but increase rapidly and greatly after acute injuries, such as RGD, αvβ3andαvβ5 integrin-blocking peptides and antibodies. VTN stroke (Kang et al., 2013; Suzuki et al., 2009; Van Wagoner activated focal adhesion kinase (FAK; also known as PTK2), whereas and Benveniste, 1999). The initial trigger(s) for IL-6 induction pharmacological- or siRNA-mediated inhibition of FAK, but not PYK2, in the brain remains largely unresolved (Suzuki et al., 2009), – reduced the expression of LIF and IL-6 in C6 and endothelial cells and but might include leakage of blood proteins upon blood brain after traumatic cell injury. Dominant-negative FAK (Y397F) reduced the barrier disruption, which occurs rapidly after stroke (Krueger et al., amount of injury-induced LIF and IL-6. Pharmacological inhibition or 2015). knockdown of uPAR (also known as PLAUR), which binds VTN, also LIF is a GP130 (also known as IL6ST) receptor-activating reduced cytokine expression, possibly through a common target of cytokine, and as such related to the IL-6 family of cytokines uPAR and integrins. We propose that VTN leakage into tissues (Zigmond, 2012). LIF is well known for playing a role during in vitro in promotes inflammation. Integrin–FAK signaling is therefore a novel IL-6 development and for promoting stem cell self-renewal and vivo and LIF regulation mechanism relevant to the inflammation and stem (Bauer and Patterson, 2006; Cartwright et al., 2005). LIF is also cell fields. expressed by astrocytes (Banner et al., 1997), microglia (Nakanishi et al., 2007) and endothelial cells (Mi et al., 2001). It can also be pro- KEY WORDS: FAK, IL-6, Integrin, LIF, Vitronectin, uPAR inflammatory (Kerr and Patterson, 2004; Pan et al., 2008; Suzuki et al., 2009), facilitating neutrophil activation (Borish et al., 1986) INTRODUCTION and macrophage infiltration, as demonstrated by conditioned Vitronectin (VTN) is mainly produced in the liver by hepatocytes medium experiments from LIF−/− and IL-6−/− Schwann cell and endothelial cells (Seiffert et al., 1991, 1995) and is found at high preparations from denervated mouse sciatic nerves (Tofaris et al., concentrations in blood serum (Hayman et al., 1983). VTN−/− mice 2002). LIF is expressed at very low levels throughout the body, but display no overt phenotype (Zheng et al., 1995) but exhibit delayed increases following brain injury (Banner et al., 1997) and stroke angiogenesis and poor wound healing (Jang et al., 2000), and (Kang et al., 2013). Its expression in injured peripheral nerves is defective vascular repair, causing persistent leakiness after injury decreased again after repair (Dowsing et al., 2001), perhaps (Li et al., 2012). Thus, leaked VTN may induce tissue repair coincident with re-establishment of vascular integrity. The responses. VTN has been detected in tissues in inflammatory mechanisms regulating LIF expression are not well understood, rheumatoid arthritis (Tomasini-Johansson et al., 1998) and lung, but may include stimulation by IL-1β, possibly through mRNA liver and kidney fibrosis (Jang et al., 2000). Both VTN and stabilization (Carlson et al., 1996). fibronectin (another enriched plasma protein) leak into the brain VTN has an RGD motif (Suzuki et al., 1985) with which it binds after stroke (del Zoppo et al., 2012) and can activate microglia to the VTN receptors αvβ3 and αvβ5 integrin (Plow et al., 2000). It in vitro (Milner and Campbell, 2003; Welser-Alves et al., 2011). also interacts with several other proteins (Leavesley et al., 2013). Besides its cell adhesive properties, VTN activates integrin intracellular signaling molecules (Giancotti and Ruoslahti, 1999), 1Department of Biomedical Sciences, Quillen College of Medicine, East Tennessee including FAK (also known as PTK2), one of the major integrin State University, Johnson City, TN 37614, USA. 2Keizo Asami Laboratory (LIKA), transducers. Phosphorylation of Y397 is critical to FAK activation Universidade Federal de Pernambuco, Recife, PE, Brasil. (Liu et al., 2003) and induces a number of signaling cascades *Author for correspondence ([email protected]) (Keasey et al., 2013). Phosphorylation of FAK at Y397 is critical for TNFα-stimulated expression of IL-6 (Schlaepfer et al., 2007), M.P.K., 0000-0002-1236-3840; C.J., 0000-0002-9693-0012; L.F.P., 0000-0002- 1467-7624; R.R.S., 0000-0002-9517-0752 suggesting that it might be a signaling node for cytokine regulation. VTN is unique among extracellular matrix (ECM) molecules Received 10 February 2017; Accepted 6 December 2017 because it also binds to urokinase-type plasminogen activator (uPA) Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs202580. doi:10.1242/jcs.202580 receptor (uPAR; also known as PLAUR) (Madsen et al., 2007), a serum withdrawal. We followed up this broad approach by testing membrane-bound glycoprotein that serves as the receptor for uPA. specific ECM proteins. C6 cells were seeded onto plastic culture Here, we determined whether blood-derived proteins such as plates and maintained for 24 h and serum-deprived for a further 24 h. VTN regulate LIF and IL-6 expression through integrin–FAK and/ VTN, fibronectin, fibrinogen, laminin-111 (the isoform comprising or uPAR signaling, by using cultured astroglioma and endothelial α1, β1 and γ1 chains) or collagen-I were then added directly to the cell, and adult mouse models. culture medium. Remarkably, after 4 h, only VTN produced a significant ∼8-fold increase in LIF (Fig. 1D) and ∼4-fold increase in RESULTS IL-6 mRNA expression (Fig. 1E). VTN treatment for 24 h showed VTN uniquely increases LIF and IL-6 expression in vitro no discernable differences relative to vehicle controls (data not We had previously shown that some ECM molecules, including shown, n=2), suggesting that LIF and IL-6 are rapidly induced but VTN, inhibit CNTF through integrin signaling (Keasey et al., 2013). then return to baseline levels. This could be due to VTN endocytosis To determine whether the related cytokines LIF and IL-6 are and subsequent degradation (Memmo and McKeown-Longo, regulated by such blood proteins we first used serum, which has very 1998). VTN effects were dose dependent (Fig. 1F,G) and resulted high levels of ECM proteins such as VTN, fibronectin (FN1) and in increased LIF and IL-6 protein release from cells as confirmed fibrinogen (Hayman et al., 1985). We cultured C6 astroglioma cells with a dot blot assay of conditioned medium (Fig. 1H,I). In addition, for 24 h in 10% fetal bovine serum (FBS), then changed the medium C6 cells were grown in a medium containing 1% (v/v) plasma from to low 1% (v/v) or regular 10% serum medium for an additional VTN−/− or VTN+/+ littermate mice, but without fetal bovine serum. 24 h. In 1% serum, LIF and IL-6 mRNA levels were only ∼33 and After 4 h, LIF and IL-6 mRNA increases with VTN−/− plasma were 45% (Fig. 1A,B), respectively, of that found when cells were in 10% only ∼30% and 50%, respectively, of that seen with VTN+/+ plasma serum. CNTF was upregulated in 1% serum (Fig. 1C), consistent (Fig. 1J,K). This suggests that native mouse VTN can increase LIF with our previous study (Keasey et al., 2013), and suggesting that the and IL-6 expression, and that an additional plasma molecule(s) also decreases in LIF and IL-6 were not a general cellular response to has such effects. Fig. 1. Among ECM proteins, VTN uniquely activates LIF and IL-6 expression in C6 astroglioma cells. Serum contains a variety and various abundances of integrin-binding ECM proteins. We plated C6 cells for 24 h in 10% serum, then replaced the medium with a low serum formulation (1% v/v; LS) for 24 h, which reduced LIF (A) and IL-6 (B) mRNA expression, as measured by RT-qPCR, relative to that seen in control 10% serum (Ctrl). (C) In the same experiment as in A and B, CNTF mRNA was increased, likely due to inhibitory activity of serum ECMs, such as VTN, on CNTF expression which is negatively regulated by integrin–FAK signaling.
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