Abstract
Purpose: :
Lacritin is a small glycoprotein secreted by human lacrimal acinar cells. Lacritin signals to mTOR and NFATC1 via rapid PKCα; dephosphorylation and PLD activation to potentially modulate differentiation, renewal and secretion of lacrimal and corneal epithelia that it preferentially targets. Recently we showed that lacritin's C–terminus bound the core protein of a form of syndecan–1 distinct from the form that binds FGF2. We report here that heparanase digestion of heparan sulfate chains is required to expose the lacritin binding site on syndecan–1 as a prerequisite for mitogenesis.
Methods: :
Human heparanase–1 was transiently transfected into HSG cells. Lysates from HEK293T cells stably transfected with syndecan–1 were sequentially affinity precipitated with FGF–2–GST or lacritin–intein fusion proteins. Heparanase was detected with antibodies directed against human heparanase followed by HRP–conjugated secondary antibody and ECL substrate. In mitogenic heparanase depletion assays, lacritin was added with [3H]–thymidine 48 hours after 1nM siRNA transfection. To rescure heparanase–depleted cells, 1 µg of heparin–enriched heparanase was added together with lacritin and [3H]–thymidine.
Results: :
By sequential affinity precipitation, it is apparent that cells make both deglycosylated and native syndecan–1 available with the latter most prevalent. Lacritin binds deglycosylated syndecan–1 and FGF–2 binds heparan–sulfate decorated syndecan–1. 3G10 failed to detect syndecan–1 pulled down by lacritin–intein, but did so after heparitinase treatment. RNAi knockdown of the endo–ß–D–glucuronidase heparanase–1 incapacitates lacritin–dependent mitogenesis without affecting their response to EGF, and can be rescued by addition of exogenous heparanase.
Conclusions: :
Mitogenic heparanase–dependent core protein binding appropriates a widely expressed cell surface proteoglycan as a cell–restrictive co–receptor. This unique coreceptor binding mechanism involves lower pH–dependent heparanase cleavage of heparan sulfate to expose a protein binding site on syndecan–1. This mechanism may offer protection against the low pH 'danger signal', one of several sudden cell stressing 'danger signals' thought to initiate primary Sjögren's syndrome. Supported by EY13143. No commercial support.
Keywords: cornea: tears/tear film/dry eye • lacrimal gland