EDITORIAL FOCUS
PARs in the stars: proteinase-activated receptors and astrocyte function. Focus on "Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways"

Morley D. Hollenberg

Diabetes/Endocrine, Mucosal Inflammation, Smooth Muscle and Cancer Biology Research Groups, Departments of Pharmacology & Therapeutics and Medicine, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1


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IN THE FIRMAMENT OF THE BRAIN, astrocytes have in the past been considered to be primarily passive, supportive bystanders in terms of maintaining an optimal environment for the function of neuronal elements. Now, however, it is accepted that astrocytes, the most numerous cell type within the central nervous system (CNS), are key multifunctional units that are in a constant dialogue with each other and with neighboring neurons and adjacent vasculature. This bidirectional communication between astrocytes and other cell types is facilitated by their processes that can enwrap synaptic terminals and also impinge on vascular arterioles. Via their interaction with capillary endothelial cells, the astrocytes are thought to play a key role in the formation and maintenance of the blood-brain barrier (16, 19, 20). Not only are astrocytes a major source of neurotrophic factors and cytokines, which in the setting of injury can affect neuronal survival and apoptosis (23, 45), but the astrocytes themselves are equipped with an abundance of receptors for neurotransmitters and neuromodulators (15, 40), including glutamate, which is believed to communicate directly via "spillover" from the neuron to the astrocyte. One common theme of the signaling pathways triggered by the astrocyte ionotropic receptors, such as those for glutamate (GluRs; see Ref. 40 for references), or by G protein-coupled receptors (GPCRs), such as those that activate Gs and cAMP-driven processes (28) or Gq and inositide-specific phospholipase Cbeta , is an elevation of intracellular Ca2+, which in turn activates a variety of intracellular signaling processes (40). Of note is the ability of the GPCR agonist endothelin (for which mRNA is detected in glia) to stimulate inositide turnover (i.e., Ca2+ signaling) and mitogenesis in astrocytes (26). Like endothelin, thrombin has long been known as a regulator of vascular contractility (i.e., for mobilizing intracellular Ca2+) and mitogenesis. Because of the likelihood of thrombin access to astrocytes in the setting of CNS vascular injury, the impact of thrombin on astrocyte function has been of interest since the mid-1980s, when it was discovered that thrombin is a potent mitogen for astrocytes (34). In addition to regulating astrocyte proliferation, thrombin can reverse astrocyte stellation via a signaling pathway involving tyrosine kinase (14), enhance astrocyte nerve growth factor production (30), and protect astrocytes from cell death caused by hypoglycemia or oxidative stress. Of note, Perraud et al. (34) showed that trypsin, like thrombin, is mitogenic for astrocytes. Although initially the circulation was considered the potential physiological source of thrombin in the CNS, mRNA for thrombin as well as its activating enzyme, factor X, along with its inhibitors, antithrombin III and protease-nexin-1, have all been detected in rat brain; all the machinery needed to generate and regulate thrombin is potentially available to expose astrocytes to thrombin action within the CNS (5, 7, 8, 36, 38). In addition to its actions on astrocytes, thrombin also has effects on neural elements (5, 10). Only recently, however, have the cellular mechanisms of action of thrombin been clarified so as to provide an infrastructure for the work described in the study by Wang et al., the current article in focus (Ref. 42, see p. C1351 in this issue).

Long recognized for its role in the coagulation cascade, thrombin is now known to regulate its target cells, such as platelets and endothelial cells, in part via activation of a GPCR (3, 17, 27, 32, 35, 41). The novel mechanism whereby thrombin activates its receptors involves the proteolytic unmasking of a cryptic NH2-terminal receptor sequence that, remaining tethered, binds to and triggers receptor function. In addition, short (5 to 6 amino acids) synthetic peptides, based on the proteolytically revealed motif, can activate the thrombin receptor without the unmasking of the tethered ligand (41). Since the cloning of the first receptor for thrombin (now termed "proteinase-activated receptor-1", or PAR-1), two more family members cleaved by thrombin (PAR-3 and -4) have been cloned (18, 21, 44), along with an additional PAR triggered by trypsin, but not thrombin (now termed PAR-2) (31). Key distinguishing features of the PAR family are 1) their selective sensitivity to serine proteinases (e.g., PAR-1 and -4 can be activated by both thrombin and trypsin, whereas PAR-2 is activated by trypsin and other serine proteinases, but not by thrombin; Ref. 17); and 2) their distinct proteolytically revealed tethered activating sequences (e.g., SFFLRN for rat PAR-1, SLIGRL for rat PAR-2, and GFPGKP for rat PAR-4). The "tethered ligand" sequence of PAR-3 revealed by thrombin (SFNGGPQ and TFRGAP for murine and human PAR-3, respectively) presents a puzzle, because an in-depth study of murine PAR-3 demonstrated that on its own in an expression system, this receptor fails to generate an intracellular signal when triggered by either thrombin or the tethered ligand sequence SFNGGP (29). Although the activation of PAR-3 from species other than the mouse has yet to be studied in depth, current thinking (29) portrays PAR-3 as an accessory molecule, facilitating the activation of PAR-4 by thrombin. That said, receptor-selective activating peptides such as those used by Wang et al. (42) [SLIGRL-NH2 for activating PAR-2, A(para-fluoro)F-(cyclohexyl)A(homo)RY-NH2 for PAR-1, and GYPGKF for PAR-4] have proved of enormous utility to assess the potential effects of receptor activation in the absence of the proteinases, which could have multiple effects other than activating the PARs. This issue is particularly important in the case of thrombin, which can generate cellular signals as a consequence of mechanisms apart from PAR activation: 1) fibrin formation and clot-regulated/extracellular matrix-mediated cell signaling, 2) metalloproteinase activation (25), 3) mitogenic/chemotactic peptide sequences present in the noncatalytic domains of thrombin (1, 13), and 4) thrombin-triggered glycoprotein Ib signaling (37). Thus an important aspect of the new study reported by Wang et al. is not only the use of a PAR-1-activating peptide to define clearly the potential actions of thrombin on astrocytes that are PAR-1-mediated but also the use of a PAR-1-selective activating peptide (termed "TRag" by Wang et al.), which, unlike the PAR-1-activating peptides used in previous studies with astrocytes (e.g., SFLLRNP, Ref. 14), do not readily cross-activate PAR-2 (22). Unfortunately, at the time of the early studies with astrocytes with the so-called thrombin receptor-activating peptides (or TRAPs, such as SFLLRN or SFLLRNP), it was not realized that these peptides, at the concentrations used, would also activate PAR-2. A similar comment can be made regarding the use by Wang et al. of the PAR-4-selective agonist GYPGKF, which will not activate other PARs, although the more potent and effective PAR-4-selective agonist AYPGKF-NH2 (9) might have revealed even greater effects on astrocytes.

The Magdeburg group (see references in Wang et al., Ref. 42) has evaluated the presence and potential function in astrocytes of all four PARs, employing RT-PCR and immunohistochemical detection of the receptors, and an evaluation of astrocyte responses to thrombin and PAR-activating peptides. The new study in focus (42) goes beyond previous observations of PAR-stimulated rat astrocyte Ca2+ signaling and proliferation to examine signal transduction pathways that might regulate astrocyte responsiveness, with a focus on the activation of MAPK (ERK1/2). The primary emphasis was on PAR-1 stimulation, which, compared with that of PAR-2 and -4, caused the most robust increase in phosphorylated ERK1/2 (see Fig. 2, Ref. 42), even though PAR-1, -2, and -4 can all activate ERK signaling pathways. What is somewhat puzzling is that the human PAR-3-derived tethered ligand peptide, TFRGAP, also caused an activation of MAPK. Given that this human PAR-3-derived peptide is so distinct from the murine PAR-3 sequence but somewhat related to PAR-1-activating peptides (TRag, as above; TFLLR-NH2, Ref. 22), it is possible that TFRGAP may have cross-activated rat PAR-1, rather than PAR-3. As mentioned above, murine PAR-3 fails to signal. Although preliminary data suggest signaling by human PAR-3 (4, 18), the potential signaling property of rat PAR-3 remains to be determined. The study clearly shows that signaling by thrombin to activate ERK1/2 and to affect proliferation in astrocytes can be accounted for primarily by activation of PAR-1 and not by the other potential thrombin signaling mechanisms described above. The finding that both Gq- and Gi-regulated processes can be triggered by PAR-1 may account for the very diverse effects thrombin may have on astrocyte function.

Wang et al. (42) also attempted to define the potential role of EGF receptor transphosphorylation/activation as described for other GPCRs (6) or the stimulation by zinc of Ras (43). The highly selective and potent EGF kinase inhibitor AG1478/PD-153035 (2, 11, 12) inhibited PAR-1-mediated ERK activation by ~60% (see Fig. 10, Ref. 42), thereby implying a role for EGF receptor transactivation. Yet Wang et al. were unable to detect PAR-1-stimulated EGF receptor phosphorylation at its autophosphorylation site, PY1068, which is a hallmark of receptor kinase activation (see Fig. 11C, Ref. 42). AG1478 also did not attenuate PAR-1-triggered cell proliferation (see Fig. 9C, Ref. 42), despite its ability to inhibit markedly PAR-1-triggered ERK phosphorylation (see Fig. 9B, Ref. 42). Thus EGF receptor transactivation appears not to play a role in PAR-1-mediated astrocyte proliferation. Involvement of metalloproteinase-mediated release of heparin-binding EGF to cross-activate the EGF receptor after PAR-1 triggering, as described by Daub et al. (6), was also ruled out for PAR-1, because the metalloproteinase inhibitor maximastat had no effect on TRag or thrombin activation of astrocyte ERK. One is thus left with the possibility that although EGF receptor transactivation does not play a role in the mitogenic action of thrombin on astrocytes, EGF receptor transphosphorylation (e.g., at EGF receptor residue 845, Ref. 43) might nonetheless play a role in PAR-1-mediated ERK activation. An alternative hypothesis would be that AG1478 is acting with quite high potency (IC50 < 0.05 µM; see Fig. 10, Ref. 42) to inhibit another kinase involved in astrocyte ERK activation, but not in the proliferative response of astrocytes to thrombin and SFLLRN. The role of EGF receptor transactivation in the actions of PARs on astrocytes thus remains an open question.

Over and above the demonstration that PAR-1 activation involves a number of signaling pathways in rat astrocytes (and presumably other species), the work of Wang et al. (42) highlights the potential importance of proteinases in the CNS to regulate cell function by activating signal transduction pathways or possibly via the "processing" of peptide agonist precursors, such as proendothelin. In this regard, the observations in the late 1980s/early 1990s that proteinase inhibitors can stimulate astrocyte proliferation (33) and that proteinases other than thrombin and trypsin can regulate astrocyte function (30) take on added significance. Thus the work of Wang et al. (42) raises issues not only about the physiological roles that PARs in the CNS might subserve but also about the importance of looking for the potential sources and signaling properties of proteinases, apart from thrombin- and trypsin-related proteinases, in the CNS. In this regard, it should be noted that the PARs are unique as GPCRs in that they can have not only multiple proteinase agonists but also their own multiple endogenous "antagonists," i.e., proteinases that cleave downstream of the tethered ligand domain, thereby "silencing" the receptors (as does plasmin for PAR-1, Ref. 24). As a general guiding principle for exploring the potential role of PARs in astrocytes, it can be suggested that, akin to the clotting system, PARs may play a role as an "intrinsic defense system" participating in the processes of inflammation, tissue remodeling, repair, and embryogenesis (5, 39).

It would thus appear that the PARs can now be added to the large cadre of growth factor, ion channel, and GPCR systems present on astrocytes. The challenge for the future is to determine 1) the true physiological roles that the PARs and other astrocyte-localized receptors play in intact tissue and 2) the source and regulation of the agonists (i.e., proteinases, EGF-like ligands, or other) that are the endogenous regulators of astrocytes. Further work in this most intriguing area of research will undoubtedly reveal many interesting surprises.


    ACKNOWLEDGEMENTS

I am grateful to V. Wee Yong for invaluable discussions relating to astrocyte function and to Dr. Paul Insel for skillful editorial assistance.


    FOOTNOTES

This work is supported by funds from the Canadian Institutes of Health Research, The Canadian Heart & Stroke Foundation, The Kidney Foundation of Canada, Servier, and a Johnson & Johnson focused giving program grant.

Address for reprint requests and other correspondence: M. D. Hollenberg, Diabetes/Endocrine, Mucosal Inflammation, Smooth Muscle and Cancer Biology Research Groups, Depts. of Pharmacology & Therapeutics and Medicine, Faculty of Medicine, Univ. of Calgary, Calgary, AB, Canada T2N 4N1 (E-mail: mhollenb{at}ucalgary.ca).

10.1152/ajpcell.00304.2002


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