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G. W. Laurie, N. Wang, R. W. Raab, R. L. McKown; Initial Identification of a Second Mitogenic Site in Lacritin for Human Corneal Epithelial Cell Renewal. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2726.
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© ARVO (1962-2015); The Authors (2016-present)
Lacritin is a prosecretory mitogen apically released from human lacrimal acinar cells. Lacritin is also expressed by basal proliferative cells of the human corneal epithelium. Prior C-terminal deletions have identified a SDC1-binding/mitogenic site between amino acids 100 - 109 of mature lacritin (Wang et al, J. Cell Biol. 174:689-700, 2006; Ma et al, J. Cell Biol. 174:1097-1106, 2006). Since lacritin signaling is more complex than that known to be mediated by SDC1 alone, we hypothesized that SDC1 might serve as a co-receptor. If true, the hypothetical signaling receptor might bind elsewhere on a SDC1-conformationally altered lacritin. Here we tested several new lacritin N-terminal deletions. We also advanced our understanding of lacritin-stimulated MUC16 production.
Recombinant human lacritin N-deletions (N-35, N-45, N-55, N-65, N-71) were generated from the pLAC vector in E.coli and purified. Positive controls were recombinant lacritin, N-24 and EGF. Negative controls were inactive C-25 lacking the 100 - 109 site, or no treatment. For proliferation assays, HCE-T cells were treated with 10 nM of lacritin or with each deletion construct in serum-free medium. Proliferation was determined by 3H-thymidine uptake. MUC16 mRNA was monitored by real time PCR.
A) Lacritin, N-24, N-45, N-55 and N-65 are all mitogenic, but not N-71. Thus, activity is lost when the amino acids KSIVEK are removed from N-65. Interestingly, the alternative splice form of lacritin known as lacritin-b lacks the sequence SIVEKSILLTE. This suggests that lacritin-b, if expressed, may be inactive. Similarly, the lacritin-c form has an entirely different C-terminus and also should be inactive. Prior data demonstrated that C-25 does not inhibit lacritin binding of SDC1. Thus an alternative single-site model seems unlikely in which the SDC1 site extends more N-terminal in a more complex interaction with a signaling receptor. B) Although lacritin rapidly stimulates MUC16 protein production by HCE-T cells, MUC16 mRNA is unchanged, suggesting post-transcriptional regulation that lends itself to a rapid lacritin response. In contrast, serum upregulates MUC16 mRNA and protein.
Two lacritin sites may be required for HCE-T cell proliferation. Both are subject to alternative splicing and raise the question as to whether alternative splice forms are more prevalent in dry eye. Lacritin, but not serum, regulates MUC16 post-transcriptionally.
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