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SC Walton, IM Hussaini, GW Laurie; Intracellular Signaling by the Lacrimal Pro-Secretory Mitogen 'Lacritin' . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3148.
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Purpose: The lacrimal/corneal axis is a fundamental regulator of ocular health. In our continued efforts to gain a molecular understanding of axis function, we discovered the novel pro-secretory mitogen 'lacritin' which appears capable of acting on all exposed cells of the lacrimal/corneal axis (Sanghi et al, J. Mol. Biol. 310: 127-139, '01). Here we begin to explore what signaling mechanisms are triggered, and whether lacritin is active outside the lacrimal/corneal or salivary axis. Methods: Patterns of tyrosine, serine/threonine and MAPK phosphorylation were studied in Western blots of serum-free human HSG cells treated with recombinant human lacritin, human EGF or serum. Calcium imaging studies utilized rabbit lacrimal acinar cells labeled with calcium green fluorescent indicator. 3H-thymidine incorporation by HSG and nine other human or mouse cell lines established confluency- and time-dependence of lacritin mitogenesis, as well as target cell specificity. Results: Approximately ten intracellular proteins are tyrosine phosphorylated upon lacritin addition. Some are also involved in EGF signaling. Most are small proteins ranging from 17 to 48 kDa whose identities are not known, although the lightly phosphorylated =21 kDa band may be p21-activated kinase 1, Ras (21 kDa), Rac (21 kDa) or Rho (24 kDa) which are all known to be EGF-activated. Candidates for the lacritin-activated proteins at 28, 32, 34, 36 and 37 kDa include: Cdk1/2 (34/33 kDa), Jun (35 - 39 kDa), and CrkL (39 kDa). No activation of MAPK Erk1/Erk2 is apparent. Lacritin promotes rapid calcium signaling in lacrimal acinar cells, and was not mitogenic for 3T3, 293, K-562, TM3, TM4, U1242MG, U251MG, WM164 or HS68 cells. Conclusion: Lacritin rapidly activates both low amplitude calcium signaling and tyrosine phosphorylation, and promotes the proliferation of ductal cells with an estimated cell binding affinity of 0.03 - 0.07 nM (versus NGF [0.01 - 1 nM], EGF [0.2 nM], PDGF [0.4 - 0.7 nM]). To date, only lacrimal, ductal and corneal epithelial cells are lacritin-responsive. We hypothesize that lacritin release regulates downstream ductal and corneal epithelial cell turnover.
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