The endothelial glycocalyx and its regulated shedding are important to vascular health. Endo-β-D-glucuronidase heparanase-1 (HPSE1) is the only enzyme that can shed heparan sulfate. However, the mechanisms are not well understood. We show that HPSE1 activity aggravated Toll-like receptor 4 (TLR4)-mediated response of endothelial cells to LPS. On the contrary, overexpression of its endogenous inhibitor, heparanase-2 (HPSE2) was protective. The microfluidic chip flow model confirmed that HPSE2 prevented heparan sulfate shedding by HPSE1. Furthermore, heparan sulfate did not interfere with cluster of differentiation-14 (CD14)-dependent LPS binding, but instead reduced the presentation of the LPS to TLR4. HPSE2 reduced LPS-mediated TLR4 activation, subsequent cell signalling, and cytokine expression. HPSE2-overexpressing endothelial cells remained protected against LPS-mediated loss of cell-cell contacts. In vivo, expression of HPSE2 in plasma and kidney medullary capillaries was decreased in mouse sepsis model. We next applied purified HPSE2 in mice and observed decreases in TNFα and IL-6 plasma concentrations after intravenous LPS injections. Our data demonstrate the important role of heparan sulfate and the glycocalyx in endothelial cell activation and suggest a protective role of HPSE2 in microvascular inflammation. HPSE2 offers new options for protection against HPSE1-mediated endothelial damage and preventing microvascular disease.

Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle ␣-actin (␣-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression. (Blood. 2008;112: 100-110) IntroductionUrokinase-type plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes, including cell proliferation and migration during angiogenesis, tissue regeneration, inflammatory responses, and tumor growth/metastases. These complex processes all involve intracellular signal transduction and regulation of gene transcription in addition to proteolysis (see Alfano et al 1 for review). uPA is secreted as a single-chain protein (scuPA) that consists of an N-terminal EGF-like domain (GFD), a kringle domain (KD), and a serine protease domain. Binding of uPA to its high-affinity receptor CD87 (uPAR) is mediated by the GFD. 2 Plasmin converts scuPA into a proteolytically active 2-chain enzyme (tcuPA) 3 that is rapidly inhibited primarily by plasminogen activator inhibitor-1 (PAI-1). tcuPA-PAI-1 complexes are internalized with the aid of lipoprotein receptor-related protein (LRP) 4 by clathrin-mediated endocytosis. The tcuPA-PAl-1 complexes traffic to lysosomes and are degraded, while unoccupied uPAR and LRP recycle back to the cell surface. 5 uPA-induced signal transduction occurs via uPAR-dependent and uPAR-independent pathways (reviewed in Alfano et al 1 ; Kjoller 6 ; Blasi and Carmeliet 7 ). Among the latter, we have shown that cleavage of scuPA by plasmin releases the GFD fragment, generating a form of uPA unable to bind to uPAR, 8 but that stimulates migration of smooth muscle cells (SMCs). 9 Signal transduction by this scuPA fragment may be mediated in part by LRP 10 and certain integrins. 11 However, there is limited information as to the mechanism by which uPA modifies gene transcription, [12][13][14][15] and our previous studies have provided reason to hypothesize that cells express additional uPA-binding proteins that possess distinct signal-transducing activities involved in cell contractility, migration, an...

Janus kinases Jak1 and Tyk2 play an important role in urokinase-type plasminogen activator (uPA)-dependent signaling. We have recently demonstrated that both kinases are associated with the uPA receptor (uPAR) and mediate uPA-induced activation of signal transducers and activators of transcription (Stat1, Stat2, and Stat4) in human vascular smooth muscle cells (VSMC). Janus kinases are not only required for Stat activation but may also interfere with other intracellular signaling pathways. Here we report that in VSMC, Tyk2 interacts with a downstream signaling cascade involving phosphatidylinositol 3-kinase (PI3-K). We demonstrate that uPA induces PI3-K activation, which is abolished in VSMC expressing the dominant negative form of Tyk2. The regulatory subunit p85 of PI3-K co-immunoprecipitates with Tyk2 but not with Jak1, Jak2, or Jak3, and uPA stimulation increases the PI3-K activity in Tyk2 immunoprecipitates. Tyk2 directly binds to either of the two Src homology 2(SH2)p85 domains in a uPA-dependent fashion. We provide evidence that the Tyk2-mediated PI3-K activation in response to uPA is required for VSMC migration. Thus, two unrelated structurally distinct specific inhibitors of PI3-K, wortmannin and LY294002, prevent VSMC migration induced by uPA. No migratory effect of uPA was observed in VSMC expressing the dominant negative form of Tyk2. Our results underscore the versatile function of Tyk2 in uPA-related intracellular signaling and indicate that PI3-K plays a selective role in the regulation of VSMC migration.

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