Alternative signaling pathways triggered by different mechanisms of serpin endocytosis

Protease Nexin-1 (PN-1), a 43 KDa glycoprotein, is known as a serpin (serine protease inhibitor) regulating extracellular proteolytic activity. It strongly inhibits the activity of several serine proteases such as thrombin, tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), trypsin and plasmin. Consequently it contributes to tissue homeostasis by inhibiting serine proteases upon formation of high molecular weight complexes that are actively removed from the extracellular space. The internalization of the PN-1 protease complexes is mediated by low-density lipoprotein receptor related protein1 (LRP1) and LRP1’s co-receptor heparan sulfate proteoglycan (HSPG). In this thesis, the mechanism and the consequence of free PN-1 internalization were examined. In cortical primary neuronal cultures prepared from PN-1 reporter mice, endogenous PN-1 was taken up by the neurons that did not expressed PN-1. Internalization of exogenous PN-1 was also studied in both wild type and LRP1-/- mouse embryonic fibroblasts (MEF). It displayed concentration- and time-dependence, and the kinetics of PN-1 uptake in LRP1-/- MEF cells was slower than that of wild type MEF cells. Receptor associated protein (RAP) interfered with PN-1 uptake in wild type but not in LRP1 -/- MEF cells. These data suggested that an alternative receptor mediates PN-1 uptake in the absence of LRP1. We identified syndecan-1, a member of HSPG family to be the receptor mediating PN-1 uptake in LRP1 -/- MEF cells. The following experimental evidences supported this conclusion. First, PN-1 uptake was sensitive to Genistein and β-cyclodextrin, both known to block syndecan-1 mediated endocytosis. Second, PN-1 uptake was increased by over-expression of full-length syndecan-1 and decreased by RNA interference targeting this proteoglycan. Furthermore, overexpression of truncated syndecan-1 lacking its intracellular domain did not influence PN1 uptake in LRP1 -/- MEF cells. These results demonstrated that syndecan-1 especially the intracellular domain of its core protein was required for syndecan-1-mediated PN-1 internalization in the absence of LRP1. We also explored the role of PN-1 in signaling transduction and cell migration. PN-1 activated PKA by binding to LRP1. More importantly, in the absence of LRP1, PN-1 stimulated Ras-Raf-MEK-ERK signaling pathway, and enhanced cell migration. The involvement of ERK signaling in PN-1 induced migration was substantiated by the fact that MEK inhibitor U0126 inhibited this migration. As downstream effector of ERK signaling, Rac1 was activated by PN-1, resulting in lamellipodia formation and increased
migration in LRP1-/- MEF cell. We further demonstrated that PN-1’s function on cell migration is coupled to syndecan-1, because anti-syndecan-1 antibody inhibited cell migration induced by PN-1. Moreover, an enhanced interaction between PN-1 and syndecan-1, by over-expression of either PN-1 or syndecan-1 in LRP1-/- MEF cells, increased cell migration.
We further identified the upstream of this signaling pathway. We found that both anti-integrin β3 and anti-uPA receptor (uPAR) antibodies inhibited PN-1 enhanced migration in LRP1-/- MEF cell. We were also able to co-immunoprecipitate PN-1 and syndecan-1 with integrin β3. Since it is know that both uPAR and syndecan-1 are the upstream of integrin αvβ3 signaling, taken all these together, we concluded that PN-1 stimulated ERK signaling influencing cell migration went through integrin via interaction either with syndecan-1 or uPAR.