Bernard Degryse

The double life of HMGB1 chromatin protein: architectural factor and extracellular signal

The High Mobility Group Box (HMGB) chromosomal proteins have been known and studied for a long time, but we have only recently started to understand their biological functions. They now have a clear reputation for being important architectural factors: they facilitate the assembly of site‐specific DNA binding proteins to their cognate binding sites within chromatin. Beyond this intranuclear function, they also have an extracellular function, which will be the prime focus of this short review. ### The HMGB family: structure, expression and nuclear function The HMGB family comprises the three proteins HMGB1 (previously HMG1), HMGB2 (previously HMG2) and HMGB3 (previously HMG4 or HMG2b) (Bustin, 2001). The structure of these three proteins is highly conserved (>80% amino acid identity), and their biochemical properties are so far indistinguishable. HMGBs are composed of three different domains. The two homologous DNA binding domains, HMG boxes A and B, are each ∼75 amino acids in length. The C‐terminal domain is highly negatively charged, consisting of a continuous stretch of glutamate or aspartate residues, and is longest in HMGB1 and shortest in HMGB3 (reviewed in Bustin, 1999; Bianchi and Beltrame, 2000). HMGB1 is ubiquitous and only 10 times less abundant than core histones, at ∼106 molecules per typical mammalian cell. Expression of the other two family members is more restricted: HMGB3 is only expressed to a significant amount during embryogenesis (Vaccari et al ., 1998); HMGB2 is widely expressed during embryonic development, but restricted mainly to lymphoid organs and testis in the adult mouse (Ronfani et al ., 2001). The localization of these proteins in most cells is nuclear. In their nuclear identity, HMGB1 and HMGB2 bind to the minor groove of DNA, causing a local distortion of the double helix. They have little or no sequence preference, and they are recruited to the site of action by specific DNA binding proteins. HMGB1 has …

The Low Density Lipoprotein Receptor-related Protein Is a Motogenic Receptor for Plasminogen Activator Inhibitor-1

Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

Urokinase/urokinase receptor and vitronectin/αvβ3 integrin induce chemotaxis and cytoskeleton reorganization through different signaling pathways

Vitronectin (VN) and pro-urokinase (pro-uPA) stimulated migration of rat smooth muscle cells in a dose-dependent and additive way, and induced motile-type changes in cell morphology together with a complete reorganization of the actin filaments and of the microtubules. All these effects were inhibited by pertussis toxin, or by antibodies directed against the urokinase receptor (uPAR) or against the VN receptor αvβ3 suggesting that an association between the two receptors is required to mediate both signals. Investigation of the signaling pathways showed that increasing the intracellular cAMP resulted in a selective inhibition of VN-induced cell migration. On the other hand, PD 98059, an inhibitor of MEK, differentially inhibited the pro-uPA- but not the VN-induced cell migration. Phosphorylation and nuclear translocation of Erk by pro-uPA was directly observed. We conclude that the signaling pathways of pro-uPA and VN must be at least in part different.

The nuclear protein HMGB1, a new kind of chemokine?

The chromosomal protein HMGB1 is now regarded as a proinflammatory cytokine. Importantly, HMGB1 has chemotactic activity suggesting its involvement in the early and late events of the inflammatory reaction. Therefore, HMGB1 has all the hallmarks of a chemokine (chemotactic cytokine). We propose to classify HMGB1 into a new group of proteins unrelated structurally to chemokines but having chemokine‐like functions, and to name this class CLF (chemokine‐like functions). The CLF class should include other unrelated molecules such as urokinase and its receptor, cytokines macrophage migration inhibitory factor (MIF) and interleukin (IL)‐6, anaphylatoxin C5a, ribosomal protein S19, and thioredoxin that have similar chemokine‐like activities. This innovative concept may lead to the identification of new therapeutic targets.

PAI-1 inhibits urokinase-induced chemotaxis by internalizing the urokinase receptor

PAI‐1 (plasminogen activator inhibitor‐1) binds the urokinase‐type plasminogen activator (uPA) and causes its degradation via its receptor uPAR and low‐density lipoprotein receptor‐related protein (LRP). While both uPA and PAI‐1 are chemoattractants, we find that a preformed uPA–PAI‐1 complex has no chemotactic activity and that PAI‐1 inhibits uPA‐induced chemotaxis. The inhibitory effect of PAI‐1 on uPA‐dependent chemotaxis is reversed when uPAR internalization is inhibited by the 39 kDa receptor‐associated protein or by anti‐LRP antibodies. Under the same conditions, the uPA–PAI‐1 complex is turned into a chemoattractant causing cytoskeleton reorganization and extracellular‐regulated kinase/mitogen‐activated protein kinases activation. Thus, uPAR internalization by PAI‐1 regulates cell migration.

Vitronectin inhibits plasminogen activator inhibitor-1-induced signalling and chemotaxis by blocking plasminogen activator inhibitor-1 binding to the low-density lipoprotein receptor-related protein.

We have previously reported that the serpin plasminogen activator inhibitor-1 activates the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) signalling pathway and stimulates cell migration by binding to the low-density lipoprotein receptor-related protein. All the free forms (cleaved, latent or active) of this inhibitor were shown to be motogenic. However, the plasminogen activator inhibitor-1 can also interact with vitronectin which acts as a cofactor by increasing the half-life of the active form of the serpin. Since vitronectin influences most of the biological functions of the plasminogen activator inhibitor-1, we explored the effects of vitronectin on signalling and cell migration induced by this serpin. We found that the interaction between vitronectin and the plasminogen activator inhibitor-1 suppressed signalling and cell migration. In fact, a purified vitronectin(1-97)/plasminogen activator inhibitor-1 complex was not chemotactic. Vitronectin interaction with the plasminogen activator inhibitor-1 blocks the binding of this serpin to its motogenic receptor, the low-density lipoprotein receptor-related protein. Consequently, vitronectin inhibits the activation of the Janus kinase/signal transducer and activator of transcription signalling pathway by the plasminogen activator inhibitor-1 and subsequent cell migration. In conclusion, we have unveiled a new inhibitory role of vitronectin, which turns off the intracellular signalling and migration-promoting activity of the plasminogen activator inhibitor-1. Thus, the motogenic (cleaved, latent or active) and non-motogenic (in complex with vitronectin) forms of the plasminogen activator inhibitor-1 have different properties that may explain the rather contrasting physiological and pathological roles of this serpin.