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SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that the antiproliferative activity of SLURP-1 is related to 'metabotropic' signaling pathway through α7-nAChR, that activates intracellular signaling cascades without opening the receptor channel.
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Fig 1. Effect of rSLURP-1 on the growth of Het-1A cells.(A). Amino acid sequence alignment of human SLURP-1, SLURP-2, ws-Lynx1, and non-conventional toxin WTX from N. kaouthia. Cysteine residues are labeled in gray, and the disulfide linkages are shown; additional N-terminal Met residues are underlined. (B). Influence of rSLURP-1 (diamonds) and ws-Lynx1 (squares) on cell growth. Each point is mean ± S.E. of six independent experiments. The Hill equation (Eq 1) was fitted to rSLURP-1 data (% of control) for each of the six experiments independently. After averaging the following values for EC50, nH and A1 were obtained 4.3 ± 0.6 nM, 1.4 ± 0.2 and 59.5 ± 1.3% (mean ± S.E., n = 6). (C). Effects of rSLURP-1 (1 μM), atropine (1 μM), Mec (10 μM), α-Bgtx (1 μM), polyclonal antibodies against α7 (IgG-α7, 1 μg per 50 μl), and their co-application. Each bar is mean ± S.E. of 4–6 independent experiments. ** and *** indicate significant (p<0.01 and p<0.001, t-test) differences.
Fig 2. rSLURP-1 binds α7 nAChR subunits in the human brain.Affinity purification was performed using magnetic beads covalently coupled with rSLURP-1 or non-coupled beads (Ctrl) on human temporal cortical homogenates (n = 2). Samples were submitted to gel electrophoresis and Western blotting along with samples of the homogenate used for affinity purification (Input) and the remaining homogenate after affinity purification (Output) followed by detection of nAChR subunits.
Fig 3. Inhibition of ACh-evoked current at α7-nAChR expressed in X. laevis oocytes by rSLURP-1.(A). Electrophysiological recordings of α7 nAChR inhibition by 13 μM rSLURP-1. Currents were obtained in response to 20 seconds 100 μM ACh pulses (horizontal bars). Inhibition of current amplitude was observed after 5 min pre-incubation with 13 μM rSLURP-1. Vertical bar represents current scale (500 nA). (B). Dose-response curve of 1 mM acetylcholine-evoked current by rSLURP-1. Each point is mean ± S.E. of independent measurements on three oocytes. The Hill equation (Eq 1) was fitted to normalized data (% of control) for each of the three experiments independently. After averaging the following values for IC50, nH and A1 were obtained 1.1 ± 0.5 μM, 1.4 ± 0.3 and 31 ± 3% (mean ± S.E., n = 3). (C). Dependence of 5 μM rSLURP-1 effect at α7-nAChR on ACh concentration. Data of rSLURP-1 induced inhibition is shown by filled squares, 5 μM ws-Lynx1 effect on 1 mM ACh is shown by circle. Asterisks indicate significant (p<0.05, t-test) difference from receptor inhibition by rSLURP-1 at 1 μM ACh (6 on plot). (D). Dose-response curves of acetylcholine-evoked current amplitudes in the absence (dotted line, empty circles) and presence (solid line, filled circles) of 1 μM rSLURP-1. The calculated parameters EC50 and nH were 232 ± 25 μM and 1.9 ± 0.3 in absence of rSLURP-1, and 214 ± 60 μM and 2.2 ± 0.3 in presence of rSLURP-1 (mean ± S.E., n = 3).
Fig 4. Competition of rSLURP-1 with 125I-α-Bgtx for binding to Ls-AChBP, membrane-bound nAChR from Torpedo californica, and α7-nAChR in the GH4C1 cell line.(A). Each point is mean ± S.E. of three independent experiments. The Hill equation (Eq 1, with A1 = 0%) was fitted to normalized Ls-AChBP and T. californica data (% of control binding) for each of the three experiments independently. After averaging the following values for IC50 and nH were obtained 4.8 ± 0.9 μM and 0.92 ± 0.17 for Ls-AChBP, and 54 ± 15 μM and 1.3 ± 0.3 for T. californica nAChR (mean ± S.E., n = 3). Data for T. californica were taken from [23]. (B). Displacement of 125I-α-Bgtx by unlabeled Bgtx and rSLURP-1 from α7-nAChR in the GH4C1 cells. (C). Affinity purification of α7-nAChR subunit was performed using magnetic beads covalently coupled with rSLURP-1, rSLURP-2, and α-Bgtx or non-coupled beads (control) on GH4C1 cells overexpressing α7-nAChR (n = 2). The 40 μl samples of GH4C1 cells with final concentration of α-Bgtx-binding sites of 0.4 nM were used. The blots were analyzed by densitometry using ImageJ software (http://imagej.nih.gov/ij/).
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