Protease-activated receptors in the musculoskeletal system

Protease-activated receptors (PARs) mediate cellular responses to a subset of extracellular proteases, including blood coagulation factors and proteases produced by inflammatory cells. Cells in bone, cartilage and muscle exhibit cell type-specific expression patterns and functional responses for the different PARs. Activators of PAR-1 include thrombin, and activators of PAR-2 include trypsin and tryptase; PARs-3 and -4 are also receptors for thrombin. Thrombin stimulates PAR-1-mediated proliferative responses in osteoblasts, chondrocytes and myoblasts, and in developing muscle, PAR-1 activation by thrombin appears to mediate activity-dependent polyneuronal synapse reduction. In bone, activation of PAR-2 leads to inhibition of osteoblast-mediated osteoclast differentiation induced by hormones or cytokines, and in muscle, PAR-2 activation leads to stimulation of myoblast proliferation. Although there is some evidence for a role for PARs expressed by cells of the musculoskeletal system at specific stages of development, their major role appears to be in protecting the tissues from the destructive effects of inflammation and promoting regeneration. This review discusses the regulation of cell function in the musculoskeletal system by receptor-mediated responses to proteases. Expression patterns of PARs, the circumstances in which PAR activators are likely to be present, functional responses of PAR activation, and responses to thrombin for which receptors have not yet been identified are considered.     

Proteinase-activated receptors in the nervous system

Recent data point to important roles for proteinases and their cognate proteinase-activated receptors (PARs) in the ontogeny and pathophysiology of the nervous system. PARs are a family of G-protein-coupled receptors that can affect neural cell proliferation, morphology and physiology. PARs also have important roles in neuroinflammatory and degenerative diseases such as human immunodeficiency virus-associated dementia, Alzheimer's disease and pain. These receptors might also influence the pathogenesis of stroke and multiple sclerosis, conditions in which the blood-brain barrier is disrupted. The diversity of effects of PARs on neural function and their widespread distribution in the nervous system make them attractive therapeutic targets for neurological disorders. Here, we review the roles of PARs in the central and peripheral nervous systems during health and disease, with a focus on neuroinflammatory and degenerative disorders.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a “regeneration cocktail.” In this review, we describe the ability of peptides derived from tenascin-C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

Promoting and directing axon outgrowth

Establishment of appropriate neuronal connections during development and regeneration requires the extension of processes that must then grow in the correct direction, find and recognize their targets, and make synapses with them. During development, embryonic neurons gradually establish central and peripheral connections in an evolving cellular environment in which neurotrophic factors are provided by supporting and target cells that promote neuronal survival, differentiation, and process outgrowth. Some cells also release neurotropic factors that direct the outgrowth of neuronal processes toward their targets. Following development the neurotrophic requirements of some adult neurons change so that, although they respond to neurotrophic factors, they no longer require exogenous neurotrophins to survive or to extend processes. Within the central nervous system (CNS), the ability of neurons to extend processes is eventually lost because of a change in their cellular environment from outgrowth permissive to inhibitory. Thus, neuronal connections that are lost in the adult CNS are rarely reestablished. In contrast, the environment of the adult peripheral nervous system fosters process outgrowth and synapse formation. This article discusses the neurotrophic requirements of embryonic and adult neurons, as well as the importance of neurotropic factors in directing the outgrowth of regenerating adult axons.     

Biochemical properties and kinetic parameters of dihydroxyphenylalanine--5-hydroxytryptophan decarboxylase in brain, liver, and adrenals of cat.

Biochemical properties and kinetic parameters of nonpurified dihydroxyphenylalanine-5-hydroxytryptophan decarboxylase extracted from brain and two peripheral organs, liver and adrenals, were studied in the cat. This study shows that decarboxylase activity in brain is lower than in peripheral organs and that 5-hydroxytryptophan can be decarboxylated without exogenous addition of pyridoxal-5'-phosphate (PLP). However, the addition of PLP substantially increases the enzyme activity. Excess of coenzyme (greater than 60 muM) induces inhibition in adrenals and liver but not in the central nervous system (CNS). The observed inhibition might be related to the presence of a tetrahydroisoquinoline derivative formed in the medium. Differentiation between mechanisms of action of decarboxylase in the CNS and peripheral organs is suggested.     

Polymer hollow fiber-encapsulated peripheral nerve extracts change their activity towards injured hippocampal neurites in rats

The regeneration of the adult mammalian central nervous system (CNS) requires changes of the nonpromising environment. Applying peripheral nerve grafts and their extracts are both the useful method to induce regeneration of injured CNS neurites. Our previous reports showed that degeneration of peripheral nerves enhanced their neurotrophic activity in a time-dependent manner. Electrophoretical analysis of proteins obtained from degenerating sciatic nerves revealed significant changes in fractions of low molecular mass. The aim of the present work was to examine the influence of fractionated extracts from 7-day-predegenerated and non-predegenerated peripheral nerves upon injured hippocampal neurites in adult rats. The extracts were closed in fibrin-filled connective tissue chambers (CTC) or within CTC-wrapped polymer hollow fibers (PHF) of 30 kDa cut-off. The cell bodies of regrowing fibers were labeled with FITC-HRP. The CTCs appeared to be useful tool for implantation of artificial grafts into mammalian CNS. Full-spectrum nerve extracts induced strong regeneration of injured hippocampal neurites. The number of labeled cells within hippocampus was significantly lower in PHF groups than in CTC ones, indicating that low-mass proteins present in peripheral nerve extracts are not sufficient to induce successful regeneration.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors: strategies to improve neuronal regeneration

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a "regeneration cocktail." In this review, we describe the ability of peptides derived from tenascin- C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

Proteinases as hormones: targets and mechanisms for proteolytic signaling

Proteinases, such as kallikrein-related peptidases, trypsin and thrombin, can play hormone-like 'messenger roles in vivo. They can regulate cell signaling by cleaving and activating a novel family of G-protein-coupled proteinase-activated receptors (PARs 1-4) by unmasking a tethered receptor-triggering ligand. Short synthetic PAR-derived peptide sequences (PAR-APs) can selectively activate PARs 1, 2 and 4, causing physiological responses in vitro and in vivo. Using the PAR-APs to activate the receptors in vivo, it has been found that PARs, like hormone receptors, can affect the vascular, renal, respiratory, gastrointestinal, musculoskeletal and nervous systems (central and peripheral). PARs trigger responses ranging from vasodilatation to intestinal inflammation, increased cytokine production and increased nociception. These PAR-stimulated responses have been implicated in various disease states, including cancer, atherosclerosis, asthma, arthritis, colitis and Alzheimer's disease. In addition to targeting the PARs, proteinases can also cause hormone-like effects by other signaling mechanisms that may be as important as the activation of PARs. Thus, the PARs themselves, their activating serine proteinases and their signaling pathways can be considered as attractive targets for therapeutic drug development. Further, proteinases can be considered as physiologically relevant 'hormone-like' messengers that can convey signals locally or systemically either via PARs or by other mechanisms.     

Restricted inflammatory reaction in the CNS: a key impediment to axonal regeneration?

Axons in the central nervous system (CNS) of adult mammals do not regenerate after injury. Mammalian CNS differs in this respect from other mammalian tissues, including the peripheral nervous system (PNS), and from the CNS of lower vertebrates. In most parts of the body, including the nervous system, injury triggers an inflammatory reaction involving macrophages. This reaction is needed for tissue healing; when it is delayed or insufficient, healing is incomplete. The CNS, although needing an efficient inflammatory reaction resembling that in the periphery for tissue healing, appears to have lost the ability to supply it. We suggest that restricted CNS recruitment and activation of macrophages are linked to regeneration failure and might reflect the immune privilege that characterizes the mammalian CNS. As macrophages play a critical role in tissue restoration, and because their recruitment and activation are among the most upstream of the events leading to tissue healing, overcoming the deficiencies in these steps might trigger a self-repair process leading to recovery after CNS injury.     

Kallikreins and proteinase-mediated signaling: proteinase-activated receptors (PARs) and the pathophysiology of inflammatory diseases and cancer

Proteinases such as thrombin and trypsin can affect tissues by activating a novel family of G protein-coupled proteinase-activated receptors (PARs 1-4) by exposing a 'tethered' receptor-triggering ligand (TL). Work with synthetic TL-derived PAR peptide sequences (PAR-APs) that stimulate PARs 1, 2 and 4 has shown that PAR activation can play a role in many tissues, including the gastrointestinal tract, kidney, muscle, nerve, lung and the central and peripheral nervous systems, and can promote tumor growth and invasion. PARs may play roles in many settings, including cancer, arthritis, asthma, inflammatory bowel disease, neurodegeneration and cardiovascular disease, as well as in pathogen-induced inflammation. In addition to activating or disarming PARs, proteinases can also cause hormone-like effects via PAR-independent mechanisms, such as activation of the insulin receptor. In addition to proteinases of the coagulation cascade, recent data suggest that members of the family of kallikrein-related peptidases (KLKs) represent endogenous PAR regulators. In summary: (1) proteinases are like hormones, signaling in a paracrine and endocrine manner via PARs or other mechanisms; (2) KLKs must now be seen as potential hormone-like PAR regulators in vivo; and (3) PAR-regulating proteinases, their target PARs, and their associated signaling pathways appear to be novel therapeutic targets.     

Extrinsic cellular and molecular mediators of peripheral axonal regeneration

The ability of injured peripheral nerves to regenerate and reinnervate their original targets is a characteristic feature of the peripheral nervous system (PNS). On the other hand, neurons of the central nervous system (CNS), including retinal ganglion cell (RGC) axons, are incapable of spontaneous regeneration. In the adult PNS, axonal regeneration after injury depends on well-orchestrated cellular and molecular processes that comprise a highly reproducible series of degenerative reactions distal to the site of injury. During this fine-tuned process, named Wallerian degeneration, a remodeling of the distal nerve fragment prepares a permissive microenvironment that permits successful axonal regrowth originating from the proximal nerve fragment. Therefore, a multitude of adjusted intrinsic and extrinsic factors are important for surviving neurons, Schwann cells, macrophages and fibroblasts as well as endothelial cells in order to achieve successful regeneration. The aim of this review is to summarize relevant extrinsic cellular and molecular determinants of successful axonal regeneration in rodents that contribute to the regenerative microenvironment of the PNS.     

Effects of growth, degeneration and regeneration on the sensory motor system

Evidence was presented for regeneration and growth in the central nervous system of experimental animals and in the peripheral nervous system of man. Neurophysiological, neurohistological and neurochemical findings from the cerebellothalamocortical and the nigrostriatal projection systems as well as the motoneurons of the anterior horn and facial nucleus were presented and their possible functional implications discussed. Single unit recordings from regenerated primary afferents in human peripheral nerves were also shown and discussed in the light of their clinical relevance. The conclusions were reached that the findings of positive growth in the CNS following traumatic injury or in response to brain implants left little doubt that the CNS possesses the intrinsic capability of regeneration. The functional and clinical implications of this inherent capacity awaits further research. Recording the response behavior of regenerated primary afferents in man, on the other hand, provided more direct insight into functional deficits and their clinical correlates.     

Epidermal growth factor and the nervous system

Various growth factors and their receptors are present in the nervous system. This review focuses on the presence of epidermal growth factor (EGF) and its receptors in the central nervous system (CNS). Evidence indicates that EGF in the CNS is the result of local synthesis, by intrinsic and blood-derived macrophages, glial cells and neurons, and uptake from the peripheral blood through the circumventricular organs and probably also through the blood-brain barrier. Evidence is accumulating suggesting that EGF regulates a variety of CNS functions in a specific manner. EGF influences CNS growth, differentiation and maintenance (actions proposed to promote neural regeneration and cell survival following a variety of insults). EGF also induces neuromodulatory actions, affects the neuroendocrine system, and suppresses food intake and gastric acid secretion. Acute and chronic pathological processes, e.g., various cancers, stimulate the production and release of EGF in various cell systems. Monitoring of EGF by the CNS may participate in several neurological manifestations (e.g., appetite suppression, neuroendocrine alterations) frequently accompanying acute and chronic disease. EGF and transforming growth factor-alpha (TGF-alpha, a factor that binds to the EGF receptor with high affinity and induces the same biological signals as EGF) also may be involved in the promotion of malignancy in the CNS and in the neuropathogenesis of degenerative disorders. Thus evidence is accumulating concerning the neurophysiological or neuropathophysiological significance of EGF in the nervous system.     

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca²⁺) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.     

中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。     

Strategies to repair lost sensory connections to the spinal cord

Failure of injured axons to regenerate in the central nervous system (CNS) is the main obstacle for repair of stroke and traumatic injuries to the spinal cord and sensory roots. This regeneration failure is high-lighted at the dorsal root transitional zone (DRTZ), the boundary between the peripheral (PNS) and central nervous system where sensory axons enter the spinal cord. Injured sensory axons regenerate in the PNS compartment of the dorsal root but are halted as soon as they reach the DRTZ. The failure of regenerating dorsal root axons to re-enter the mature spinal cord is a reflection of the generally nonpermissive nature of the CNS environment, in contrast to the regeneration supportive properties of the PNS. The dorsal root injury paradigm is therefore an attractive model for studying mechanisms underlying CNS regeneration failure in general and how to overcome the hostile CNS environment. Here we review the main lines that have been pursued to achieve growth of injured dorsal root axons into the spinal cord: (i) modifying the inhibitory nature of the DRTZ by breaking down or blocking the effect of growth repelling molecules, (ii) stimulate elongation of injured dorsal root axons by a prior conditioning lesion or administration of specific growth factors, (iii) implantation of olfactory ensheathing cells to provide a growth supportive cellular terrain at the DRTZ, and (iv) replacing the regeneration deficient adult dorsal root ganglion neurons with embryonic neurons or neural stem cells.     

RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins

In contrast to the adult mammalian central nervous system (CNS), the neurons in the peripheral nervous system (PNS) can regenerate their axons. However, the underlying mechanism dictating the regeneration program after PNS injuries remains poorly understood. Combining chemical inhibitor screening with gain- and loss-of-function analyses, we identified p90 ribosomal S6 kinase 1 (RSK1) as a crucial regulator of axon regeneration in dorsal root ganglion (DRG) neurons after sciatic nerve injury (SNI). Mechanistically, RSK1 was found to preferentially regulate the synthesis of regeneration-related proteins using ribosomal profiling. Interestingly, RSK1 expression was up-regulated in injured DRG neurons, but not retinal ganglion cells (RGCs). Additionally, RSK1 overexpression enhanced phosphatase and tensin homolog (PTEN) deletion-induced axon regeneration in RGCs in the adult CNS. Our findings reveal a critical mechanism in inducing protein synthesis that promotes axon regeneration and further suggest RSK1 as a possible therapeutic target for neuronal injury repair.

This study shows that p90 ribosomal S6 kinase 1 (RSK1) responds differentially to nerve injury in the peripheral and central nervous systems, and identifies it as a crucial regulator of axonal regeneration; mechanistically, RSK1 preferentially induces the synthesis of regeneration-related proteins via the RSK1-eEF2K-eEF2 axis.     

Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction

Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-alpha or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-alpha-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83(+) DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.