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M Massaro-Giordano, CM Marshall, BC R Marsh, PJ Jensen, NM Schechter, RM Lavker; Plasminogen Activator Inhibitor Type 2 (PAI-2) is Secreted by Cultured Human Corneal and Conjunctival Keratinocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1668.
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Purpose: PAI-2, a member of the serpin family of proteins, is capable of inhibiting plasminogen activators uPA and tPA. It is produced by cells of various tissues and has intracellular and extracellular forms, but is not a constituent of normal plasma except late in pregnancy. PAI-2 is expressed by normal human corneal and conjunctival epithelia as well as cultured human corneal and conjunctival keratinocytes. Human tear fluid has been shown to contain PAI-2; the source of this PAI-2 is unknown. In this study the secretion of PAI-2 by cultured human corneal and conjunctival keratinocytes is investigated as a possible source of tear fluid PAI-2. Methods: Conditioned medium was collected from human corneal and conjunctival keratinocytes that were maintained under low (30uM) Ca2+and then switched to high (1mM) Ca2+ culture conditions. PAI-2 antigen in the conditioned medium and in the cell lysates was quantified as a function of time for 48 hr by Elisa and immunoblotting. Results: PAI-2 was detected in the conditioned media and cell lysates from both corneal and conjunctival keratinocytes. After switching to a high Ca2+ culture medium, keratinocyte cultures showed an approximately 3 fold increase in PAI-2 to an apparent plateau after 24 hr. The amount of PAI-2 in cell lysates after 48 hrs. was approximately 0.1% of the total cellular protein. In contrast, PAI-2 in the conditioned media increased linearly (1 ng PAI-2/mg total protein/hr) over the 48 hr time course. At 48 hr, 10-20% of the total PAI-2 was present in the conditioned medium. PAI-2 in the conditioned medium had a larger mass (50kDa) than PAI-2 in the lysate (45kDa), suggestive of glycoslyation. Interestingly, PAI-2 in tears had a mass of 45kDa. Conclusion: Our findings suggest that both corneal and conjunctival keratinocytes synthesize PAI-2 at substantial levels and that this inhibitor is partitioned between an intracellular and extracellular form. The continuous secretion of the extracellular form suggests that corneal and conjunctival keratinocytes may constitutively secrete PAI-2 in vivo. However, it is not clear whether keratinocyte secretion is one of the sources of PAI-2 in tears, due to the difference in mass between PAI-2 in the conditioned medium and tears. One possibility is that once secreted, PAI-2 becomes deglycoslyated in the tears.
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