Heath (School of Biosciences, University of Birmingham, Birmingham, UK) for providing us with the clones for hLIF and LIF05 expression and Michael H

Heath (School of Biosciences, University of Birmingham, Birmingham, UK) for providing us with the clones for hLIF and LIF05 expression and Michael H. also resulted in reduced preconditioning-induced protection of the retinal Neohesperidin photoreceptors. These data demonstrate that LIFR and its ligands play an essential role in endogenous neuroprotective mechanisms triggered by preconditioning-induced stress. 1992; Lavail 1992; Penn 1987). Prolonged bright light exposure can also induce oxidative damage which, when severe, kills photoreceptors (Noell 1966; Penn 1987). However, under such unfavorable conditions, retinal cells Rabbit Polyclonal to FAKD2 initiate a response to rescue photoreceptors by recruiting or secreting a variety of antioxidants, cytokines and/or neurotrophic factors (Chaum 2003; Lavail 1992; Liu 1998; Penn 1987; Steinberg 1995; Wen 1995; Wen 1998). This has been clearly demonstrated in models where exposure to subtoxic levels of stress (e.g., bright cyclic light) induced changes in retinal tissue that protect photoreceptors from a subsequent dose of lethal stress (Li 2001; Li 2003; Liu 1998). Factors that were shown to be up regulated under oxidative stress include basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor (BDNF), LIF, and CLC (Chaum 2003; Faktorovich 1992; Lavail 1992; Rattner 2008; Samardzija 2006; Zachary 2005). While these are hypothesized to play a role in preconditioning-induced endogenous neuroprotection, it has not yet been demonstrated which factors or receptors are essential for the protection. Intriguingly, among the up regulated molecules LIF, CNTF, and CLC belong to the same family and signal through heterodimerization of leukemia inhibitory factor receptor (LIFR) and glycoprotein 130 (gp130). Since these ligands and receptors are functional in the retina (Sherry 2005; Ueki 2008; Wen 1998), our hypothesis is that activation of LIFR: gp130 complex plays an essential role in preconditioning-induced endogenous protection of retinal photoreceptors. This hypothesis predicts that inhibiting the activation of these receptors during stress would make the photoreceptor cells more susceptible to oxidative damage. LIF05, a mutant LIF molecule, antagonizes LIF, CNTF, CT-1 and CLC activities by competitively binding and blocking the LIFR dimerization with gp130 (Hudson 1996; Vernallis 1997). In this study, we tested our hypothesis by delivering LIF05 during preconditioning. The data show that inhibiting LIFR activation blocks the Neohesperidin protective effects of preconditioning resulting in increased photoreceptor sensitivity to oxidative stress. Materials and Methods Recombinant protein expression and purification Expression and purification of human LIF and LIF05 was performed as described previously (Robinson Neohesperidin 1994b). Briefly, LIF and LIF05 were expressed as glutathione-S-transferase (GST) fusion proteins in strain JM109. Cultures were grown in LB plus ampicillin (100g/ml) at 37C and 300 rpm until they reached midlog phase (A600 = 0.6). Isopropyl -D-1-thyogalactopyranoside (IPTG) was then added to the culture to a final concentration of 0.1 Neohesperidin mM and induction was carried out for additional 3 h at room temperature. Intracellular fusion protein was recovered from cell extracts by affinity binding to a slurry of glutathione-Sepharose 4B beads (GE Healthcare, Uppsala, Sweden). Washes were carried out as described by the manufacturer’s protocol. Isolation of LIF or LIF05 was achieved by cleavage of the fusion protein with human thrombin (Amersham Biosciences, Piscataway, NJ) in 1X PBS (pH 7.3) overnight at room temperature. Following cleavage, the elution Neohesperidin containing hLIF or LIF05 was pooled with additional 4 batch washes (1X PBS, pH 7.3). Cleaved hLIF or LIF05 was further purified by fast protein liquid chromatography (FPLC) using a Mono-S cationic exchange column (Amersham Biosciences, Piscataway, NJ). Elution was carried out with a linear gradient of 0 C 1 M NaCl in 20 mM MES buffer (pH 6.0). Eluted fractions were analyzed using SDS-PAGE and the fractions containing enriched LIF or LIF05 were pooled and concentrated by ultrafiltration (Millipore Corporation, Billerica, MA). Purities of LIF and LIF05 were >90% as evaluated by SDS-PAGE. Purified LIF and LIF05 were tested on human retinal Mller cell cultures to determine biological activity and rule out contamination with endotoxins. Concentrations were determined using BCA assay as described by the manufacturer’s protocol (Thermo Scientific, Rockford IL) Kinetic analysis of LIFR and gp130 interaction with LIF and LIF05 Interactions of the cytokine receptor domains with immobilized LIF or LIF05 were analyzed by surface plasmon resonance (SPR) using the SensiQ system (ICX Technologies, Oklahoma City, OK) as described by the manufacturer. A carboxyl sensor, with two channels, was installed in SensiQ and allowed to thermally equilibrate for about 15 min. The sensor was cleaned with a 3 min injection of 0.1 M HCl. An activation solution of 2 mM 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 0.5 mM N-hydroxysulfosuccinimide (NHS) was prepared in deionized water immediately.