Scientific Report 2005

Immunology

Regulation of Protease Signaling Pathways

W. Ruf, J. Ahamed, M. Belting,* A. Dorfleutner, E. Hintermann,** M. Kerver, B.M. Mueller,*** F. Niessen, T. Kurokawa, Y. Kurokawa, Y. Lee, M. Majumdar, H. Peterson, Y. Takada,**** H.H. Versteeg

* University of Lund, Lund, Sweden
** Department of Cell Biology, Scripps Research
*** La Jolla Institute for Molecular Medicine, San Diego, California
**** University of California, Davis, California

Therapeutic Intervention with Tissue Factor–Signaling Pathways

We are interested in protease systems that regulate inflammation, cancer, and angiogenesis. Activation of the coagulation pathway by tissue factor (TF) triggers cell signaling events that contribute to lethality in sepsis. Therapy with activated protein C, the natural counterbalance of TF-initiated coagulation, markedly improves survival in severe sepsis.

We recently showed that activated protein C elicits a unique cellular response in inflamed endothelial cells through protease-activated receptor 1 (PAR-1) signaling. The established gene profile is consistent with antiapoptotic and anti-inflammatory protection of the endothelium. Importantly, the pattern of gene induction is distinct from the profile of the proinflammatory protease thrombin, which signals through the same G protein–coupled receptor PAR-1. Thus, signaling specificity appears to be determined by accessory protease-binding receptors, and protease-specific effects appear to be crucial for therapeutic benefit.

How to appropriately target direct TF-initiated cell signaling pathways remains an active area of research. Inhibition of TF can improve survival in animal models of lethal hemorrhagic fever, similar to the results of earlier studies of bacterial septicemia. However, clinical trials with a recombinant form of TF pathway inhibitor, the physiologic inhibitor of TF, have been disappointing. We established that the recombinant inhibitor is a poor inhibitor of signaling of the TF initiation complex at doses that are highly effective in blocking activation of coagulation. Because bleeding complications are the major concern with TF-directed inhibitors, the ratio of anticoagulant efficiency to antisignaling potency of TF-targeted inhibitors is a consideration in using such drugs to treat inflammatory disorders or cancer.

We are identifying downstream signaling responses that predict inhibitory potency of TF-directed strategies. We are also continuing basic mechanistic studies on the specificity of the TF signaling pathway; our goal is to identify inhibitors with highly selective antisignaling activities. We identified a candidate that specifically blocks signaling by the complex consisting of TF and coagulation factor VIIa without impairing the coagulant response. Such strategies may allow intervention in TF signaling pathways while reducing the risk of impairments in hemostasis.

Regulation of Integrin Function by TF Cytoplasmic Domain Signaling

We discovered an important regulatory role of the TF cytoplasmic domain in tumor and developmental angiogenesis. In diabetic eye diseases, we showed that the cytoplasmic domain of TF is phosphorylated specifically in neovasculature associated with pathologic changes but not in normal endothelial cells or in the vessel wall. PAR-2, the target for TF-VIIa signaling, was also expressed in neovasculature. These data suggest that the TF-VIIa complex is an important regulator of vascular cell signaling in angiogenesis. Results of recent in vitro studies lend further support to the concept that the TF–PAR-2 pathway regulates cell migration in angiogenesis.

The extracellular domain of TF is involved in an interaction with integrins, but we found no evidence that the TF-integrin interaction results in competition or inhibition of binding to the extracellular matrix by these adhesive receptors. Rather, TF specifically suppressed cell migration on laminin 5 that depends on activation of integrin α₃β₁. Inhibition of promigratory α₃β₁ depended on the TF cytoplasmic domain. Mutagenesis indicated that integrin function is suppressed when the TF cytoplasmic domain is not phosphorylated. However, phosphorylation of the TF cytoplasmic domain by TF-VIIa–mediated PAR-2 signaling is sufficient to release integrin inhibition. Thus, protease-driven signaling pathways of TF regulate cell migration by targeting the cross talk between the cytoplasmic domain of TF and integrins.

Laminin 5 is a component of basement membranes, including the subendothelial matrix. In addition, α₃β₁ is targeted by antiangiogenic molecules. We are using genetic approaches to define components of the TF–VIIa–PAR-2 signaling pathway and the relationship between the pathway and integrins during angiogenesis in vivo. In vitro, we are mapping the signaling pathways downstream of the TF cytoplasmic domain and the cross talk of TF with PARs and integrins.

Publications

Ahamed, J., Belting, M., Ruf, W. Regulation of tissue factor-induced signaling by endogenous and recombinant tissue factor pathway inhibitor 1. Blood 105:2384, 2005.

Belting, M., Ahamed, J., Ruf, W. Signaling of the tissue factor coagulation pathway in angiogenesis and cancer. Arterioscler. Thromb. Vasc. Biol. 25:1545, 2005.

Dorfleutner, A., Hintermann, E., Tarui, T., Takada, Y., Ruf, W. Cross-talk of integrin α₃β₁ and tissue factor in cell migration. Mol. Biol. Cell 15:4416, 2004.

Majumdar, M., Tarui, T., Shi, B., Akakura, N., Ruf, W., Takada, Y. Plasmin-induced migration requires signaling through protease-activated receptor 1 and integrin α₉β₁. J. Biol. Chem. 279:37528, 2004.

Peerschke, E.I.B., Petrovan, R.J., Ghebrehiwet, B., Ruf, W. Tissue factor pathway inhibitor-2 (TFPI-2) recognizes the complement and kininogen binding protein gCIqR/p33 (gCIqR): implications for vascular inflammation. Thromb. Haemost. 92:811, 2004.

Riewald, M., Ruf, W. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J. Biol. Chem. 280:19808, 2005.

Ruf, W. Emerging roles of tissue factor in viral hemorrhagic fever. Trends Immunol. 25:461, 2004.

Shi, X., Gangadharan, B., Brass, L.F., Ruf, W., Mueller, B.M. Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol. Cancer Res. 2:395, 2004.