SLURP-1 and -2 in normal, immortalized and malignant oral keratinocytes

Abstract

The secreted mammalian Ly-6/urokinase plasminogen activator receptor-related proteins (SLURP)-1 and -2 are produced by keratinocytes comprising the mucocutaneous epithelium. They regulate in autocrine and paracrine ways cell growth and differentiation through the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. Keratinocyte nAChRs are targeted by tobacco-derived carcinogenic nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) that can induce tumorigenic transformation of Het-1A keratinocytes. In this study we asked if SLURPs could abolish tumorigenic effects of nitrosamines. Preincubation with either recombinant SLURP-1 or -2 in both cases considerably reduced the number of colonies in soft agar, and the number of tumor nodules > 0.5 cm in diameter in Nu/Nu mice produced by Het-1A cells treated with nitrosamines. The levels of SLURP-1 and -2 mRNA transcripts in nitrosamine-transformed Het-1A cells as well as in the tumor cell lines SCC-25 and FaDu were significantly (p < 0.05) less compared to normal gingival keratinocytes, which are probably the major source of the secreted SLURPs found in a sample of human saliva. The expression of SLURPs was decreased due to gene silencing of different nAChR α subunits with small hairpin RNA, suggesting that a positive feedback regulation is altered in malignant cells. Thus, SLURP-1 and -2 are efficient autocrine and paracrine ligands of keratinocyte nAChRs capable of preventing tobacco nitrosamine-induced malignant transformation of oral cells. These “proof-of-concept” preliminary results have salient clinical implications.

Introduction

Recent research has indicated that novel proteins termed SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)-1 and -2 act as cholinergic signaling molecules in the human mucocutaneous epithelia (Arredondo et al., 2005b, Arredondo et al., 2006c, Chimienti et al., 2003, Tsuji et al., 2003). We cloned both human SLURPs, produced recombinant proteins, rSLURP-1 and -2, and generated mouse monoclonal antibodies that visualized SLURP-1 and -2 in the cytoplasm of normal human epidermal and oral keratinocytes. Both SLURPs were found in culture supernatants of normal human keratinocytes and serum. Radioligand binding inhibition studies showed that rSLURP-1 ligated the conventional ligand binding site of keratinocyte nicotinic acetylcholine receptors (nAChRs), showing a higher affinity to the [³H]nicotine-, compared to the [³H]epibatidine-sensitive nAChRs. In contrast, rSLURP-2 showed a higher affinity to the [³H]epibatidine- compared to the [³H]nicotine-labeled sites. Experimental results also demonstrated that rSLURP-1 increases the activities of caspases 3 and 8, and the number of TUNEL positive cells. In contrast, rSLURP-2 upregulated growth of keratinocytes and their resistance to apoptosis. The differences in effects of SLURP-1 and -2 can be explained by their differential binding to keratinocyte nAChR subtypes. These findings revealed a novel paradigm of the physiologic regulation of the mucocutaneous epithelial cells by locally produced small hormone-like peptide molecules acting via keratinocyte nAChRs, and opened novel direction toward better understanding and treating the oral disease.

The keratinocyte nAChRs have been shown to mediate pathobiologic effects of the tobacco and nicotine derivatives on non-neuronal cells (reviewed in (Minna, 2003)). Recently, it has been demonstrated that nAChRs play an important role in mediating malignant cell transformation of epithelial cells by the tobacco-derived carcinogenic nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) (Arredondo et al., 2006a, Arredondo et al., 2006b). To elucidate signaling mechanisms, we studied transcription of the genes encoding the cell cycle, apoptosis and signal transduction regulators. The nitrosamine treated Het-1A cells showed multifold increases of the PCNA and Bcl-2 mRNAs as well as an upregulated expression of the transcription factors GATA-3, NF-κB, and STAT-1, which could be abolished in the presence of nicotinic antagonists (Arredondo et al., 2006b). These results supported novel concept of receptor-mediated action of NNK and NNN, placing cellular nAChRs in the center of the pathophysiologic loop, and suggested that nAChR ligands may serve as therapeutic and/or chemopreventive agents.

In this study, we obtained preliminary evidence that SLURP-1 and -2 can protect cells from tumorigenic transformation. We also demonstrated that malignant transformation is associated with a decrease of SLURP production, which is regulated, in part, through cellular nAChRs.