- Email: [email protected]
PDE4D-deficient mice knock the breath out of asthma
HIGHLIGHTS
Pass the protease: new interactions between GPCRs When is a G-protein-coupled receptor (GPCR) not a GPCR? When, according to the report by Nakanishi-Matsui et al.1, it instead acts as a non-signalling cofactor, aiding the activation of another GPCR. Such novel cooperativity appears to occur during thrombin activation of platelets in the mouse. Thrombin, a serine protease and the main enzyme of the blood coagulation cascade, acts directly on platelets via protease-activated receptors (PARs). By slicing off the N-terminal portion of the PAR protein, thrombin releases ‘tethered’ ligand sequences that auto-activate the receptor and its associated G protein. Mouse platelets express subtypes PAR3 and PAR4, PAR3 having greater affinity for thrombin. But surprisingly, when expressed alone, PAR3 is unresponsive to thrombin, whereas PAR4 responds only to unnaturally
high thrombin concentrations. Might PAR3 and PAR4 therefore act together to mediate thrombin’s effects? Nakanishi-Matsui et al. showed that expressing the genes encoding PAR3 and PAR4 together drastically lowered the threshold for thrombin activation of phosphoinositide by increasing the rate of PAR4 cleavage. To test the theory that PAR3 might be contributing its bound thrombin to cleave a neighbouring PAR4, chimaeric PAR molecules were constructed with the thrombin-binding region of PAR3 inserted into the N-terminal domain of PAR4. On expression, these too displayed low thresholds for thrombin activation. A final convincing piece of evidence, this time from actual mouse platelets, was that desensitization of PAR4 by a PAR4-specific agonist abolished thrombin signalling.
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
per se. The authors also reported that ovalbumin (OA)-sensitized PDE4D2/2 mice do not exhibit airways hyperreactivity (AHR) to MCh unlike their PDE4D1/1 counterparts. The inability of MCh to contract the airways of PDE4D2/2 mice is not associated with a downregulation of muscarinic receptor number but to defective receptor signalling. Of the five muscarinic receptors known, the M2 subtype, which accounts for 60–80% of the total number of muscarinic receptors in the airways of a variety of species, is negatively coupled to adenylate cyclase and reduces cAMP in response to appropriate agonists. However, this response is abolished in lung from PDE4D2/2 mice, which suggests that the M2 receptor cannot inhibit adenylate cyclase. Intriguingly, the abnormality in M2 receptor function is absent in the heart where the extent of MCh-induced bradycardia is identical
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
1 Nakanishi-Matsui, M. et al. (2000) PAR3 is a cofactor for PAR4 activation by thrombin. Nature 404, 609–613
Adam Smith E-mail: [email protected]
PII: S0165-6147(00)01512-1
PDE4D-deficient mice knock the breath out of asthma Much speculation surrounds the functional role of phosphodiesterase 4 (PDE4) and why multiple isoforms derived from four human PDE4 genes (PDE4A–D) are coexpressed in cells to perform the same function (i.e. to metabolize cAMP). Selective inhibitors of PDE4 are being developed for the treatment of asthma. To understand further the functional significance of PDE4 multiplicity in this context, Hansen and colleagues1 generated mice with targeted disruption of PDE4D and have uncovered a completely unexpected association with muscarinic acetylcholine receptor function. Thus, methacholine (MCh) fails to evoke bronchoconstriction in homozygous PDE4D2/2 null mice when compared with wild-type animals, whereas the same response evoked by 5-HT was unaffected. This indicates that disruption of PDE4D does not impair airway smooth muscle function
The authors propose a mechanism, novel for GPCRs, whereby PAR3, PAR4 and thrombin form a ternary complex. Thrombin first binds to and cleaves PAR3, which then unselfishly donates its captured thrombin to PAR4 without itself becoming activated. The role of PAR3 as a cofactor for PAR4 activation might serve to enhance the specificity of thrombin’s actions. Although this mechanism appears to offer an additional stage for pharmacological regulation of coagulation, cooperativity appears to be absent from human platelets, which express PAR1 and PAR4, but not PAR3. One straightforward verification of the cofactor model will be a complete absence of thrombin signalling in platelets derived from the awaited mPAR4 knockout mice.
in wild-type and PDE4D knockout mice. To explain this discrepancy the authors propose either that little PDE4D is expressed in murine myocardium or that other PDE4 isoforms compensate for the loss in PDE4D activity – although the latter idea seems more unlikely as this might have also been expected in the lung. Another unanticipated and equally intriguing finding is the development of pulmonary inflammation (characterized by increased numbers of eosinophils, lymphocytes and neutrophils in the bronchoalveolar lavage fluid) in OAchallenged, sensitized PDE4D2/2 mice. Thus, inhibition of PDE4 isoforms other than PDE4D must play a central role in abrogating pulmonary inflammation. Several conclusions and additional questions arise from these studies. Importantly, it is clear that PDE4D plays an essential and previously unappreciated role in M2 receptor signalling although the mechanistic basis for this altered state is unknown. However, given that the M2 receptor subtype is thought to play, at most, a supportive
PII: S0165-6147(00)01510-8
TiPS – August 2000 (Vol. 21)
291
HIGHLIGHTS role in MCh-induced contraction of the airways, it will be instructive to learn whether the defect in muscarinic receptor signalling also extends to the contractile M3 receptor subtype. Equally, it will be important to establish that the defect in M2 receptor signalling is present in the relevant tissue (i.e. airways smooth muscle cells). One concern that has been voiced in the literature is that significant PDE4 redundancy might
exist such that inhibition of a specific subtype will have little long-lasting impact as a result of the induction of an alternative isoenzyme. However, the results of this study unequivocally refute this theory and demonstrate that PDE4D plays a non-redundant role in regulating cAMP homeostasis and serves unique and non-overlapping functions. This latter conclusion has important therapeutic implications for the design
Retinoids offer hope for the treatment of emphysema Is cessation of cigarette smoking the only effective ‘treatment’ for emphysema? Potentially no, according to a recent paper by Massaro and Massaro1, who found that all-trans retinoic acid rescued alveoli formation in rats, in which septation was blocked pharmacologically with the glucocorticoid dexamethasone, and in mice genetically predisposed to emphysema. In mammals, alveoli are formed, in part, by the developmentally regulated subdivision (septation) of the saccules that comprise the gas-exchange region of the architectually immature lung. There is a critical period in the last month of pregnancy or shortly after birth (depending on the species) during which septation must occur. If septation is prevented or fails to occur at the appropriate time, alveoli will not form and there is no current means of
inducing post hoc septation. In a previous study, the authors found that all-trans retinoic acid, a fat-soluble metabolite of vitamin A that exerts its effects by interacting with specific cytosolic receptors that act as ligand-dependent transcription factors, abrogated elastase-induced pulmonary emphysema in adult rats through the re-formation of alveoli. Now Massaro and Massaro have extended those original observations with all-trans retinoic acid to include a rat model in which failure of septation was induced by dexamethasone and in ‘tight-skin’ (Tsk) mice, in which a tandem duplication within the fibrillin-1 gene genetically predisposes the animals to emphysema. In both models of failed septation, all-trans retinoic acid increased the number of alveoli without affecting lung volume and, in the mice, also increased alveolar surface area. To
of PDE4 subtype inhibitors because compounds that inhibit PDE4A or PDE4B should be effective in the resolution of asthmatic inflammation. 1 Hansen, G. et al. (2000) Absence of muscarinic cholinergic airway responses in mice deficient in the cyclic nucleotide phosphodiesterase PDE4D. Proc. Natl. Acad. Sci. U. S. A. 97, 6751–6756
Mark Giembycz E-mail: [email protected]
account for the discrepancy between mice and rats, the authors proposed that all-trans retinoic acid regulates the eruption of septa and their spacing, whereas another factor determines the surface area. If a recent Phase II pilot trial of all-trans retinoic acid in patients with emphysema by Mao et al.2 proves beneficial, then this pharmacological approach could provide general remedial action in humans with respiratory diseases that are characterized by an inadequate number of alveoli for adequate gas exchange, such as emphysema, interstitial fibrosis and bronchopulmonary dysplasia. 1 Massaro, G.D. and Massaro, D. (2000) Retinoic acid treatment partially rescues failed septation in rats and in mice. Am. J. Physiol. 278, 955–960 2 Mao, J.T. et al. (2000) A phase II pilot study of all-trans retinoic acid for the treatment of human emphysema. Am. J. Respir. Crit. Care Med. 161, 583
Mark Giembycz E-mail: [email protected]
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
How hot is chilli? The recently cloned capsaicin (vanilloid) receptor (VR1), normally expressed by small-size dorsal root ganglion (DRG) neurones, is a cation channel that confers to transfected cells the sensitivity to both heat and acid pH values, as well as to capsaicin and pharmacologically related substances. By using low or high doses of capsaicin or analogues to stimulate or block DRG neurones that express VR1, respectively, a role in thermal nociception was suggested for these
292
TiPS – August 2000 (Vol. 21)
capsaicin-sensitive DRG neurones. Now, two studies offer the opportunity to identify the differences in electrophysiological properties of DRGs and in nociceptive behaviour between mice lacking VR1 receptors (VR12/2) and their normal counterparts (VR11/1)1,2. Both the development and gross behaviour of VR12/2 mice was normal so the observed changes in the behavioural reactions to nociceptive stimuli are considered to be specific. The results
PII: S0165-6147(00)01509-1
from the studies of both Caterina and colleagues1 and Davis and colleagues2 largely agree; a loss of a heat (.40 °C)evoked inward current in DRG neurones of VR12/2 mice was reported by both groups. However, the conclusions about the behavioural response to heat stimulation (hot-plate and plantar test) in these mice differ between the two studies. Caterina et al. found a reduced thermal nociception in VR12/2 mice whereas Davis et al. could not demonstrate this effect. This discrepancy could be more apparent than real; Caterina et al. found that the withdrawal latency of
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0165-6147(00)01511-X
Pass the protease: new interactions between GPCRs When is a G-protein-coupled receptor (GPCR) not a GPCR? When, according to the report by Nakanishi-Matsui et al.1, it instead acts as a non-signalling cofactor, aiding the activation of another GPCR. Such novel cooperativity appears to occur during thrombin activation of platelets in the mouse. Thrombin, a serine protease and the main enzyme of the blood coagulation cascade, acts directly on platelets via protease-activated receptors (PARs). By slicing off the N-terminal portion of the PAR protein, thrombin releases ‘tethered’ ligand sequences that auto-activate the receptor and its associated G protein. Mouse platelets express subtypes PAR3 and PAR4, PAR3 having greater affinity for thrombin. But surprisingly, when expressed alone, PAR3 is unresponsive to thrombin, whereas PAR4 responds only to unnaturally
high thrombin concentrations. Might PAR3 and PAR4 therefore act together to mediate thrombin’s effects? Nakanishi-Matsui et al. showed that expressing the genes encoding PAR3 and PAR4 together drastically lowered the threshold for thrombin activation of phosphoinositide by increasing the rate of PAR4 cleavage. To test the theory that PAR3 might be contributing its bound thrombin to cleave a neighbouring PAR4, chimaeric PAR molecules were constructed with the thrombin-binding region of PAR3 inserted into the N-terminal domain of PAR4. On expression, these too displayed low thresholds for thrombin activation. A final convincing piece of evidence, this time from actual mouse platelets, was that desensitization of PAR4 by a PAR4-specific agonist abolished thrombin signalling.
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
per se. The authors also reported that ovalbumin (OA)-sensitized PDE4D2/2 mice do not exhibit airways hyperreactivity (AHR) to MCh unlike their PDE4D1/1 counterparts. The inability of MCh to contract the airways of PDE4D2/2 mice is not associated with a downregulation of muscarinic receptor number but to defective receptor signalling. Of the five muscarinic receptors known, the M2 subtype, which accounts for 60–80% of the total number of muscarinic receptors in the airways of a variety of species, is negatively coupled to adenylate cyclase and reduces cAMP in response to appropriate agonists. However, this response is abolished in lung from PDE4D2/2 mice, which suggests that the M2 receptor cannot inhibit adenylate cyclase. Intriguingly, the abnormality in M2 receptor function is absent in the heart where the extent of MCh-induced bradycardia is identical
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
1 Nakanishi-Matsui, M. et al. (2000) PAR3 is a cofactor for PAR4 activation by thrombin. Nature 404, 609–613
Adam Smith E-mail: [email protected]
PII: S0165-6147(00)01512-1
PDE4D-deficient mice knock the breath out of asthma Much speculation surrounds the functional role of phosphodiesterase 4 (PDE4) and why multiple isoforms derived from four human PDE4 genes (PDE4A–D) are coexpressed in cells to perform the same function (i.e. to metabolize cAMP). Selective inhibitors of PDE4 are being developed for the treatment of asthma. To understand further the functional significance of PDE4 multiplicity in this context, Hansen and colleagues1 generated mice with targeted disruption of PDE4D and have uncovered a completely unexpected association with muscarinic acetylcholine receptor function. Thus, methacholine (MCh) fails to evoke bronchoconstriction in homozygous PDE4D2/2 null mice when compared with wild-type animals, whereas the same response evoked by 5-HT was unaffected. This indicates that disruption of PDE4D does not impair airway smooth muscle function
The authors propose a mechanism, novel for GPCRs, whereby PAR3, PAR4 and thrombin form a ternary complex. Thrombin first binds to and cleaves PAR3, which then unselfishly donates its captured thrombin to PAR4 without itself becoming activated. The role of PAR3 as a cofactor for PAR4 activation might serve to enhance the specificity of thrombin’s actions. Although this mechanism appears to offer an additional stage for pharmacological regulation of coagulation, cooperativity appears to be absent from human platelets, which express PAR1 and PAR4, but not PAR3. One straightforward verification of the cofactor model will be a complete absence of thrombin signalling in platelets derived from the awaited mPAR4 knockout mice.
in wild-type and PDE4D knockout mice. To explain this discrepancy the authors propose either that little PDE4D is expressed in murine myocardium or that other PDE4 isoforms compensate for the loss in PDE4D activity – although the latter idea seems more unlikely as this might have also been expected in the lung. Another unanticipated and equally intriguing finding is the development of pulmonary inflammation (characterized by increased numbers of eosinophils, lymphocytes and neutrophils in the bronchoalveolar lavage fluid) in OAchallenged, sensitized PDE4D2/2 mice. Thus, inhibition of PDE4 isoforms other than PDE4D must play a central role in abrogating pulmonary inflammation. Several conclusions and additional questions arise from these studies. Importantly, it is clear that PDE4D plays an essential and previously unappreciated role in M2 receptor signalling although the mechanistic basis for this altered state is unknown. However, given that the M2 receptor subtype is thought to play, at most, a supportive
PII: S0165-6147(00)01510-8
TiPS – August 2000 (Vol. 21)
291
HIGHLIGHTS role in MCh-induced contraction of the airways, it will be instructive to learn whether the defect in muscarinic receptor signalling also extends to the contractile M3 receptor subtype. Equally, it will be important to establish that the defect in M2 receptor signalling is present in the relevant tissue (i.e. airways smooth muscle cells). One concern that has been voiced in the literature is that significant PDE4 redundancy might
exist such that inhibition of a specific subtype will have little long-lasting impact as a result of the induction of an alternative isoenzyme. However, the results of this study unequivocally refute this theory and demonstrate that PDE4D plays a non-redundant role in regulating cAMP homeostasis and serves unique and non-overlapping functions. This latter conclusion has important therapeutic implications for the design
Retinoids offer hope for the treatment of emphysema Is cessation of cigarette smoking the only effective ‘treatment’ for emphysema? Potentially no, according to a recent paper by Massaro and Massaro1, who found that all-trans retinoic acid rescued alveoli formation in rats, in which septation was blocked pharmacologically with the glucocorticoid dexamethasone, and in mice genetically predisposed to emphysema. In mammals, alveoli are formed, in part, by the developmentally regulated subdivision (septation) of the saccules that comprise the gas-exchange region of the architectually immature lung. There is a critical period in the last month of pregnancy or shortly after birth (depending on the species) during which septation must occur. If septation is prevented or fails to occur at the appropriate time, alveoli will not form and there is no current means of
inducing post hoc septation. In a previous study, the authors found that all-trans retinoic acid, a fat-soluble metabolite of vitamin A that exerts its effects by interacting with specific cytosolic receptors that act as ligand-dependent transcription factors, abrogated elastase-induced pulmonary emphysema in adult rats through the re-formation of alveoli. Now Massaro and Massaro have extended those original observations with all-trans retinoic acid to include a rat model in which failure of septation was induced by dexamethasone and in ‘tight-skin’ (Tsk) mice, in which a tandem duplication within the fibrillin-1 gene genetically predisposes the animals to emphysema. In both models of failed septation, all-trans retinoic acid increased the number of alveoli without affecting lung volume and, in the mice, also increased alveolar surface area. To
of PDE4 subtype inhibitors because compounds that inhibit PDE4A or PDE4B should be effective in the resolution of asthmatic inflammation. 1 Hansen, G. et al. (2000) Absence of muscarinic cholinergic airway responses in mice deficient in the cyclic nucleotide phosphodiesterase PDE4D. Proc. Natl. Acad. Sci. U. S. A. 97, 6751–6756
Mark Giembycz E-mail: [email protected]
account for the discrepancy between mice and rats, the authors proposed that all-trans retinoic acid regulates the eruption of septa and their spacing, whereas another factor determines the surface area. If a recent Phase II pilot trial of all-trans retinoic acid in patients with emphysema by Mao et al.2 proves beneficial, then this pharmacological approach could provide general remedial action in humans with respiratory diseases that are characterized by an inadequate number of alveoli for adequate gas exchange, such as emphysema, interstitial fibrosis and bronchopulmonary dysplasia. 1 Massaro, G.D. and Massaro, D. (2000) Retinoic acid treatment partially rescues failed septation in rats and in mice. Am. J. Physiol. 278, 955–960 2 Mao, J.T. et al. (2000) A phase II pilot study of all-trans retinoic acid for the treatment of human emphysema. Am. J. Respir. Crit. Care Med. 161, 583
Mark Giembycz E-mail: [email protected]
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
How hot is chilli? The recently cloned capsaicin (vanilloid) receptor (VR1), normally expressed by small-size dorsal root ganglion (DRG) neurones, is a cation channel that confers to transfected cells the sensitivity to both heat and acid pH values, as well as to capsaicin and pharmacologically related substances. By using low or high doses of capsaicin or analogues to stimulate or block DRG neurones that express VR1, respectively, a role in thermal nociception was suggested for these
292
TiPS – August 2000 (Vol. 21)
capsaicin-sensitive DRG neurones. Now, two studies offer the opportunity to identify the differences in electrophysiological properties of DRGs and in nociceptive behaviour between mice lacking VR1 receptors (VR12/2) and their normal counterparts (VR11/1)1,2. Both the development and gross behaviour of VR12/2 mice was normal so the observed changes in the behavioural reactions to nociceptive stimuli are considered to be specific. The results
PII: S0165-6147(00)01509-1
from the studies of both Caterina and colleagues1 and Davis and colleagues2 largely agree; a loss of a heat (.40 °C)evoked inward current in DRG neurones of VR12/2 mice was reported by both groups. However, the conclusions about the behavioural response to heat stimulation (hot-plate and plantar test) in these mice differ between the two studies. Caterina et al. found a reduced thermal nociception in VR12/2 mice whereas Davis et al. could not demonstrate this effect. This discrepancy could be more apparent than real; Caterina et al. found that the withdrawal latency of
0165-6147/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0165-6147(00)01511-X