Protease-activated receptor 2: activation, signalling and function

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR(2), which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR(2). Proteases cleave PAR(2) to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR(2) signalling is attenuated by beta-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR(2). beta-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR(2)-deficient mice support a role for PAR(2) in inflammation, and many of the effects of PAR(2) activators promote inflammation. Inflammation is mediated in part by activation of PAR(2) in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

Hypoxia and neurodegeneration

Periods of chronic hypoxia, which can arise from numerous cardiorespiratory disorders, predispose individuals to the development of dementias, particularly Alzheimer's disease (AD). AD is characterized in part by the increased production of amyloid beta peptide (Abeta), which forms the extracellular plaques by which the disease can be identified post mortem. Numerous studies have now shown that hypoxia, even in vitro, can increase production of Abeta in different cell types. Evidence has been produced to indicate hypoxia alters both expression of the Abeta precursor, APP, and also the expression of the secretase enzymes, which cleave Abeta from APP. Other studies implicate reduced Abeta degradation as a possible means by which hypoxia increases Abeta levels. Such variability may be attributable to cell-specific responses to hypoxia. Further evidence indicates that some, but not all of the cellular adaptations to chronic hypoxia (including alteration of Ca(2+) homeostasis) require Abeta formation. However, other aspects of hypoxic remodeling of cell function appear to occur independently of this process. The molecular and cellular responses to hypoxia contribute to our understanding of the clinical association of hypoxia and increased incidence of AD. However, it remains to be determined whether inhibition of one or more of the effects of hypoxia may be of benefit in arresting the development of this neurodegenerative disease.

Are transgenic mice the 'alkahest' to understanding myocardial hypertrophy and failure?

Murine transgenesis using cardioselective promoters has become increasingly common in studies of cardiac hypertrophy and heart failure, with expression mediated by pronuclear microinjection being the commonest format. Without wishing to decry their usefulness, in our view, such studies are not necessarily as unambiguous as sometimes portrayed and clarity is not always their consequence. We describe broadly the types of approach undertaken in the heart and point out some of the drawbacks. We provide three arbitrarily-chosen examples where, in spite of a number of often-independent studies, no consensus has yet been achieved. These include glycogen synthase kinase 3, the extracellular signal-regulated kinase pathway and the ryanodine receptor 2. We believe that the transgenic approach should not be viewed in an empyreal light and, depending on the questions asked, we suggest that other experimental systems provide equal (or even more) valuable outcomes.

MICAL-1 is a negative regulator of MST-NDR kinase signaling and apoptosis.

MICALs (molecules interacting with CasL) are atypical multidomain flavoenzymes with diverse cellular functions. The molecular pathways employed by MICAL proteins to exert their cellular effects remain largely uncharacterized. Via an unbiased proteomics approach, we identify MICAL-1 as a binding partner of NDR (nuclear Dbf2-related) kinases. NDR1/2 kinases are known to mediate apoptosis downstream of the mammalian Ste-20-like kinase MST1, and ablation of NDR1 in mice predisposes the mice to cancer as a result of compromised apoptosis. MST1 phosphorylates NDR1/2 kinases at their hydrophobic motif, thereby facilitating full NDR kinase activity and function. However, if and how this key phosphorylation event is regulated are unknown. Here we show that MICAL-1 interacts with the hydrophobic motif of NDR1/2 and that overexpression or knockdown of MICAL-1 reduces or augments NDR kinase activation or activity, respectively. Surprisingly, MICAL-1 is a phosphoprotein but not an NDR or MST1 substrate. Rather, MICAL-1 competes with MST1 for NDR binding and thereby antagonizes MST1-induced NDR activation. In line with this inhibitory effect, overexpression or knockdown of MICAL-1 inhibits or enhances, respectively, NDR-dependent proapoptotic signaling induced by extrinsic stimuli. Our findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling.

Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Cortical hot spots and labyrinths: why cortical neuromodulation for episodic migraine with aura should be personalized

Stimulation protocols for medical devices should be rationally designed. For episodic migraine with aura we outline model-based design strategies toward preventive and acute therapies using stereotactic cortical neuromodulation. To this end, we regard a localized spreading depression (SD) wave segment as a central element in migraine pathophysiology. To describe nucleation and propagation features of the SD wave segment, we define the new concepts of cortical hot spots and labyrinths, respectively. In particular, we firstly focus exclusively on curvature-induced dynamical properties by studying a generic reaction-diffusion model of SD on the folded cortical surface. This surface is described with increasing level of details, including finally personalized simulations using patient's magnetic resonance imaging (MRI) scanner readings. At this stage, the only relevant factor that can modulate nucleation and propagation paths is the Gaussian curvature, which has the advantage of being rather readily accessible by MRI. We conclude with discussing further anatomical factors, such as areal, laminar, and cellular heterogeneity, that in addition to and in relation to Gaussian curvature determine the generalized concept of cortical hot spots and labyrinths as target structures for neuromodulation. Our numerical simulations suggest that these target structures are like fingerprints, they are individual features of each migraine sufferer. The goal in the future will be to provide individualized neural tissue simulations. These simulations should predict the clinical data and therefore can also serve as a test bed for exploring stereotactic cortical neuromodulation.

Regulation of phospholipases C and D in rat ventricular myocytes: stimulation by endothelin-1, bradykinin and phenylephrine.

The physiological activator of protein kinase C (PKC), diacylglycerol, is formed by hydrolysis of phosphoinositides (PI) by phospholipase C (PLC) or phosphatidylcholine by phospholipase D (PLD). We have measured activation of these phospholipases by endothelin-1 (ET-1), bradykinin (BK), or phenylephrine (PE) in ventricular myocytes cultured from neonatal rat. The stimulation of PI hydrolysis after 10 min by 0.1 microM ET-1 (about 12-fold) was much greater than for BK or PE (each about four-fold), and did not correlate with translocation of nPKC delta or nPKC epsilon (Clerk A. Bogoyevitch MA. Andersson MB. Sugden PH, 1994. J Biol Chem 269: 32848-32857: Clerk A, Gillespie-Brown J, Fuller SJ, Sugden PH, 1996. Biochem J 317: 109-118). However, ET-1 and BK stimulated a similar rapid increase in [3H]InsP, formation (< 30 s), which was much greater than that seen with PE. This early phase correlated with PKC translocation. Acute or chronic exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) or treatment with Ro-31-8220 showed that the stimulation of PI hydrolysis by PE, but not ET-1 or BK, was inhibited by activation of PKC. Furthermore, ET-1 and BK heterologously desensitized the stimulation of PI hydrolysis by PE, ET-1 or BK homologously uncoupled their own receptors from [3H]InsP3 formation, but there was no evidence of heterologous desensitization with these two agonists. Anomalously, chronic exposure to TPA increased the stimulation of PI hydrolysis by BK, but this probably resulted from an increase in BK receptor density. PLD was also rapidly activated by TPA. ET-1, BK or PE. Experiments with Ro-31-8220 showed that the stimulation of PLD by ET-1 and BK was mediated through activation of PKC. We discuss the characteristics of the activation of PI hydrolysis and PLD by ET-1, BK, and PE with respect to the translocation of PKC.

Activation of c-Jun N-terminal kinases and p38-mitogen-activated protein kinases in human heart failure secondary to ischaemic heart disease.

Three well-characterized mitogen-activated protein kinase (MAPK) subfamilies are expressed in rodent and rabbit hearts, and are activated by pathophysiological stimuli. We have determined and compared the expression and activation of these MAPKs in donor and failing human hearts. The amount and activation of MAPKs was assessed in samples from the left ventricles of 4 unused donor hearts and 12 explanted hearts from patients with heart failure secondary to ischaemic heart disease. Total MAPKs or dually phosphorylated (activated) MAPKs were detected by Western blotting and MAPK activities were measured by in gel kinase assays. As in rat heart, c-Jun N-terminal kinases (JNKs) were detected in human hearts as bands corresponding to 46 and 54 kDa; p38-MAPK(s) was detected as a band corresponding to approximately 40 kDa, and extracellularly regulated kinases, ERK1 and ERK2, were detected as 44- and 42-kDa bands respectively. The total amounts of 54 kDa JNK, p38-MAPK and ERK2 were similar in all samples, although 46-kDa JNK was reduced in the failing hearts. However, the mean activities of JNKs and p38-MAPK(s) were significantly higher in failing heart samples than in those from donor hearts (P<0.05). There was no significant difference in phosphorylated (activated) ERKs between the two groups. In conclusion, JNKs, p38-MAPK(s) and ERKs are expressed in the human heart and the activities of JNKs and p38-MAPK(s) were increased in heart failure secondary to ischaemic heart disease. These data indicate that JNKs and p38-MAPKs may be important in human cardiac pathology.

Pro-inflammatory cytokines stimulate mitogen-activated protein kinase subfamilies, increase phosphorylation of c-Jun and ATF2 and upregulate c-Jun protein in neonatal rat ventricular myocytes.

Pro-inflammatory cytokines may be important in the pathophysiological responses of the heart. We investigated the activation of the three mitogen-activated protein kinase (MAPK) subfamilies ¿c-Jun N-terminal kinases (JNKs), p38-MAPKs and extracellularly-responsive kinases (ERKs) by interleukin-1 beta (IL-1 beta) or tumour necrosis factor alpha (TNF alpha) in primary cultures of myocytes isolated from neonatal rat ventricles. Both cytokines stimulated a rapid (maximal within 10 min) increase in JNK activity. Although activation of JNKs by IL-1 beta was transient returning to control values within 1 h, the response to TNF alpha was sustained. IL-1 beta and TNF alpha also stimulated p38-MAPK phosphorylation, but the response to IL-1 beta was consistently greater than TNF alpha. Both cytokines activated ERKs, but to a lesser degree than that induced by phorbol esters. The transcription factors, c-Jun and ATF2, are phosphorylated by the MAPKs and are implicated in the upregulation of c-Jun. IL-1 beta and TNF alpha stimulated the phosphorylation of c-Jun and ATF2. However, IL-1 beta induced a greater increase in c-Jun protein. Inhibitors of protein kinase C (PKC) (Ro318220, GF109203X) and the ERK cascade (PD98059) attenuated the increase in c-Jun induced by IL-1 beta, but LY294002 (an inhibitor of phosphatidylinositol 3' kinase) and SB203580 (an inhibitor of p38-MAPK, which also inhibits certain JNK isoforms) had no effect. These data illustrate that some of the pathological effects of IL-1 beta and TNF alpha may be mediated through the MAPK cascades, and that the ERK cascade, rather than JNKs or p38-MAPKs, are implicated in the upregulation of c-Jun by IL-1 beta.

Phenylephrine promotes phosphorylation of Bad in cardiac myocytes through the extracellular signal-regulated kinases 1/2 and protein kinase A.

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Ras: the stress and the strain

The last 10–15 years have seen an expansion in the understanding of the intracellular signalling pathways activated in cardiac myocytes in response to hypertrophic or lethal stimuli. The mitogen-activated protein kinases (MAPKs) were identified as potential key mediators of cardiac myocyte responses in the early to mid-1990's, with the extracellular signal-regulated kinases 1/2 (ERK1/2) being potently activated by heterotrimeric Gq protein-coupled receptor (GqPCR) agonists, and the c-Jun N-terminal kinases (JNKs) and p38-MAPKs being potently activated by cell stresses.

Dysregulation of granulosal bone morphogenetic protein receptor 1B density is associated with reduced ovarian reserve and the age-related decline in human fertility

Reproductive ageing is linked to the depletion of ovarian primordial follicles, which causes an irreversible change to ovarian cellular function and the capacity to reproduce. The current study aimed to profile the expression of bone morphogenetic protein receptor, (BMPR1B) in 53 IVF patients exhibiting different degrees of primordial follicle depletion. The granulosa cell receptor density was measured in 403 follicles via flow cytometry. A decline in BMPR1B density occurred at the time of dominant follicle selection and during the terminal stage of folliculogenesis in the 23-30 y good ovarian reserve patients. The 40+ y poor ovarian reserve patients experienced a reversal of this pattern. The results demonstrate an association between age-induced depletion of the ovarian reserve and BMPR1B receptor density at the two critical time points of dominant follicle selection and pre-ovulatory follicle maturation. Dysregulation of BMP receptor signalling may inhibit the normal steroidogenic differentiation required for maturation in older patients.