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 alpha 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.

Fig. 1. Effects of rSLURP-1 and rSLURP-2 on the Tumorigenic Activities of NNK and NNN.

The Het-1A cells were preincubated for 16 h with 2 μg/ml of either rSLURP-1 or rSLURP-2 and then exposed to 1 μM of NNK or NNN for 24 h, followed by 3 passages in culture. After that, the tumorigenic activities of experimental and control, unexposed cells were measured in vitro and in vivo as detailed in Materials and Methods. Asterisks indicate significant (p<0.05) differences compared to the effects of each nitrosamine given alone.

1. rSLURP-1 and -2 reduce numbers of colonies in soft agar produced by nitrosamine-treated Het-1A cells.

2. rSLURP-1 and 2 reduce number of tumors nodules induced by nitrosamine-treated Het-1A cells in the skin of Nu/Nu mice.

Fig. 2. Alterations of the SLURP-1 and -2 Gene Expression.

1. The qPCR analysis was used to detect alterations in the gene expression at the mRNA level in the malignant keratinocyte cell lines SCC-25 and FaDu, and in intact (1) and NNK (2)- or NNN (3)-transformed Het-1A cells. The results were normalized relative to the rates of expression of corresponding genes in the samples of normal human oral keratinocytes (OKC), taken as 1. All experimental samples contained significantly (p<0.05) less mRNA transcripts compared to OKC. Asterisks indicate changes that significantly (p<0.05) differ in nitrosamine-treated Het-1A from intact Het-1A cells.

2. Visualization of the 9 kDa native SLURP-1 and -2 proteins in culture supernatants of intact Het-1A cells (lane 1) and normal human gingival keratinocytes (lane 2), and in a sample of normal human saliva (lane 3) using the mouse monoclonal antibodies 336H12-1A3 and 341F10-1F12, respectively, diluted 1:100.

3. Intact Het-1A cells were transfected with normal control shRNA (shRNA-NC) or shRNA directed to the specific sequences present in the nAChR α3, α5, α7 or α9 subunits, and used for quantitative analysis of the SLURP-1 and -2 gene expression by qPCR, as described in Materials and Methods.