Clinical and Experimental Allergy. 1992. Volume 22. pages 907-915
REVIEW
The proliferative response of airway smooth muscle S. J. HIRST and C. H. C. TWORT Respiratory Research Laboraiorics. Smooth Muscle Research Group. UMDS Division of Medicine, St Thomas' Hospital. London. U.K. The inability to reverse completely airway narrowing in many cases oCchronic severe uslhma suggests a persistent component of the long-term pathological process, which may involve remodelling or architectural changes in the lung. Indeed one of the most striking and characteristic pathological features of chronic asthma is an increase in [he smooth muscle mass of the airways [1-4]. Both hyperplasia (increase in cell number) and hypertrophy (increase in cell size) seem to contribute to this increase and its associated reduction in airway luminal diameter. For this reason characterization and elucidation of the cellular and biochemical events which regulate growth and division of airway smooth muscle cells to various stimuli is likeiy to be of great importance in designing novel therapies for asthma, particularly for the development of disease-modifying drugs. Investigation of the mechanisms controlling smooth muscle growth in airways has lagged far behind that of vascular research, and consequently current theories of airway smooth muscle (ASM) proliferation rely heavily on the results of studies on vascular smooth muscle. In this review we discuss some of the growth factors controlling smooth muscle proliferation, their associated signal transduction pathways for both stimulation and inhibition of the mitogenic response and the extent to which they operate in ASM. The cell cycle Cell growth and division processes are regulated predominantly by the response to appropriate growth factors and the expression of their receptors. Proliferation in mammalian cells involves a programmed series of complex genetic and biochemical events which fall into four distinct phases (outlined in Fig. 1). The first stage, G| or •gap", is characterized by a lack of DNA synthesis, but there is elevated synthesis of RN A and protein. The second stage or 'S-phase' is a period of increased DNA synthesis during which elevated RNA and protein synthesis are maintained. In the third stage or G^ phase. DNA synthesis ceases while RNA and protein synthesis continue. This phase often results in cytoplasmic volume expansion. Correspondence: Dr S. J. Hirst. Respiratory RL-scitrch Laboratories, Smooth MusL-le Reseiireh Group. LJMDS Division of Medicine. St Thomas' Hospital. Lambeth Palaee Road, London SEl 7EH. U.K.
Collectively, G|, Sand G;comprise the'interphase" which is distinct from the actual cell division (i.e. mitosis). Mitosis is the shortest phase of the cell cycle, usually occurring within 60 minutes, in which little or no DNA, RNA or protein synthesis occurs, A fifth phase called Go has been proposed in which cells have come to rest and remain viable, but do not traverse the cell cycle. Under conditions when the growth arrest is not irreversible, polypeptidc growth faetors will induce cells in Gd to reenter the cycle and proliferate. Incubation of cultured smooth muscle cells for periods of 24-72 hr in serum-free culture media supplemented with insulin, ascorbate and transferrin will maintain the cells in such a viable but nondividing state [5] and has the experimental advantage of synchronizing the cell cycle kinetics. Whether this growth-arrested state is positively controlled by the expression of certain inhibitory factors has not been established. The events which control the ability of a growtharrested cell to re-enter the cell cycle are far from understood, particularly in the ASM cell. What is clear, however, is the central role of growth factors which, acting through specific plasma membrane receptors, initiate inlracellular signal transduction events which lead to phosphorylation of a large number of target intraceilular regulatory proteins [see 6], These regulatory proteins are believed to activate the intracellular processes including altered gene expression underlying cell growth and division [7]. In I9H1 Smith and Stiles [8] classified growth factors into two major groups, competence factors and progression factors. Growth factors affecting early Gu/G| events (i.e. the preparedness to enter the cell cycle) commit the cell to growth and are termed competence factors; while those which provide a committed cell with a series of necessary external stimuli rquired for it to continue through G|. S, Gi and on to mitotic division, are termed progression factors. These regulatory factors mediate their ejects through the expression of genes and the transcription of competence proteins and progression proteins. The identity of these proteins is unclear but they are likely to include elements of the signal transduction cascade and regulators of DNA synthesis and of further (late cycle) gene transcription [9]. Most growth factors do not possess both competence and progression activity. 907
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Cell division
DNA synthesis
or Quiescent phose
Fig. I. The cell cycle of a typical mammalian cell, showing the phases leading to division. The durations of each stage have not been indicated since they vary considerably between different cell types. Consequently, cellular proliferation normally requires exposure ofeells to multiple growth factors in sequence, with each of the growth faetors and progression factors selectively activating particular events iti the proliferation signalling cascade [10]. Understanding the role of regulatory genes governing cell cycle kinetics is still in its infancy, but at least two classes of genes have been identified. These are called "cell cycle-dependent' genes and 'early response' genes [10]. The expression of cell cycle-dependent genes is modulated throughout the cell cycle: these genes encode for a variety of cellular proteins including major histone proteins, calmodulin, actin and enzymes such as thymidine kinase and dihydrofolate reductase [see 10]. "Early response" genes include the cellular proto-oncogenes., c-myc, c-jun and C'fos (induction ofc-fos expression being one of the earlier markers of proliferation and differentiation) [11]. The products of c-/»/; and c-/ftv are known lo dimerize. translocate to the nucleus and bind to activator protein-1 (AP-I) promoter regions on the DNA. increasing expression of the long-term regulated genes considered to be a prerequisite to mitosis [see 12 for review]. The use of cultured airway smooth muscle cells in assessing the proliferative response To date, assessment of ASM proliferation has relied heavily upon morphometric analysis of histological sections of the bronchi of asthmatics which reveal marked thickening of the smooth muscle layers [1-4, 13-17]. A correlation between an increase in the number of cell nuclei and muscle thickening is observed, suggesting thai the increase in tnuscle mass in asthma results more from hyperplasia rather than from hypertrophy [2]. Ceil culture, unlike morphometric analysis, can pro-
vide information about the effects of mitogenic stimuH on the cell cycle and cellular proliferation. In contrast to the widespread use of cultured vascular smooth muscle in the investigation of smooth muscle proliferation [see 18,19], experience in culturing ASM has until recently been more limited. However, with therecent resurgence of interest in the thickening of the airway wail in asthma, various groups have now succeeded in culturing cells from canine [20-22], bovine [23], rabbit [24,25] and human ASM [2630]. Cultured smooth muscle cells of tracheal or bronchial origin have a similar appearance to the differentiated contractile smooth muscle cells in the intact tissue, appearing flattened and ribbon- or spindle-shaped with central oval nuclei and prominent nucleoli [20,21,25,28]. Confluent cultured ASM cells, like those of vascular origin, align themselves in parallel and have a highly contoured architecture with the formation of ridges and nodules giving a characteristic 'hill and valley' appearance [18-21,28]. They stain positively for both smooth muscle actin and myosin [20,21], features not shared by contaminating fibroblasts or other non-muscle cells [20]. These cells maintain also their physiological responsiveness to agonists implicated in inflammatory airway diseases [26 28] and display functional coupling of /?adrenergic receptors [28,31], localized to the //2-adrenoceptor [30]. Growth factors and regulation of smooth muscle growth and division In chronic asthma, in which there is persisting inflammatory cell recruitment [32], the ASM cell is likely to be exposed to a large number of growth factors and inflammatory mediators. Many of the cells present in the inflamed airway synthesize growth factors which have the potential to stimulate hypertrophic and/or hyperplastic changes in the airway wall smooth muscle. Of particular interest is the alveolar macrophage, capable of releasing a plethora of growth factors and mediators, many of which are known to mediate fibroblast proliferation [reviewed in 33]. The macrophage is a major cell source of epidermal growth factor (EGF) and its homologue transforming growth factor-a (TGFa), fibroblast growth factors (FGF), insuiin-like growth factor (IGF-i), platelet-derived growth factor (PDGF), colony-stimulating factors such as granulocyte-colony-stimulating factor (G-CSF) and members of the interleukin family, IL-lx and IL-6 [see 34]. The platelet may also contribute since it contains large amounts of PDGF and TGF^ [33]. Curiously, the eosinophil appears to have escaped attention as a source of growth factors, despite its proposed role in the late asthmatic response and associated airway hyperresponsi-
Proliferative response of airway smooth muscle
veness [35.36]. Other inflammatory cells such as mast cells. T cells and other monocytes are also recognized sources ofa variety of different growth factors., CSFs and interleukins. Despite the presence of cells in asthmatic bronchi capable of producing a variety of growth factors, almost nothing is known about the effects of any of these factors on the ASM cell either in culture or in vivo. Our current knowledge depends heavily on studies of the effects of growth factors on vascular smooth muscle cell growth and division [37]. To date, histamine is the only mediator studied in any detail for effects on the proliferative response of cultured ASM cells. In cultured canine and human ASM cells, histamine not only induces the expression of the protooncogene c-fos^ whose expression is one of the earliest markers ofcells re-entering the cell cycle [12], but also acts as a mitogen in the absence of any other stimulus in cells rendered quiescent [22,38,39]. This suggests that histamine not only induces competence in these cells but also behaves as a progression factor. The only other reports describing the actions of growth factors on cultured ASM cells concern the expression of c-/fj.v induced by PDGF in canine cells [22,38]. EGF, however, stimulates DNA synthesis in cultured human ASM cells [31 ]. but in canine cells it failed to stimulate expression of c-fos above control levels [22]. The reasons for this apparent anomaly are not clear, but may lie in the use of [^H]-thymidine uptake (DNA synthesis) as a proliferation assay. Several criticisms have been levelled at the use of this method [see 40]. Of particular concern with this method is the absence of actual proliferation in cells which continue to incorporate thymidine suggesting a block in the S or G: phases of the cell cycle {see earlier). For this reason alternative proliferation assays such as cleavage of the tetrazolium salt, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dephenyl tetrazolium bromide (MTT) by mitochondrial succinyl dehydrogenase and assay of cellular protein by specific binding to protein of the dye. Coomassie Brilliant blue have been developed, which in ASM cultures have been found to correlate closely with actual cell counts determined by haemocytometry [41].
Signal transduction mechanisms for control of smooth muscle growth and division The response to extracellular mediators such as growth factors requires cellular signalling mechanisms to transduce the message received by plasma membrane receptors into the intracellular events associated with the control of proliferation. Evidence is steadily accumulating implicating a variety of signal transduction pathways in the control of mitogenesis in ASM ceils [22,31.39]. The following signal transduction pathways have already
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been implicated to varying extents in the control of smooth muscle growth and division and may provide important clues to the future design of more effective antiproliferative therapies. Role of prolein kinasc C Studies to date have centred on the role of a family of up to nine phosphatidylserine-dependent, lipid-activated serine and threonine protein kinases, collectively known as protein kinase C (PKC) [42,43]. Phorbol esters and synthetic diacylglycerols, which mimic the endogenous PKC activator, diacylglycerol (DAG), have long been known to stimulate DNA synthesis and cell division, often in synergy with other growth promoting factors [44 46]. PKC activates a number of early events associated with mitogenesis including Na + /H+ exchange, resulting in intracellular alkalinization, protein synthesis and the induction/expression of nuclear proto-oncogenes such as C'fos and c-myc [47.48]. In cultured smooth muscle cells isolated from bovine carotid arteries, activation of PKC may be necessary for progression from the G; to S phase of the cell cycle [49]. Not all agonists, however, which activate PKC cause proliferation in smooth muscle cells. For example in cultured rat aortic smooth muscle cells, made quiescent in a defined serum-free media, both angiotensin II [50] and arginine-vasopressin [51] fail to induce proliferation, but do cause marked increases in cellular protein synthesis with subsequent hypertrophy. Despite this failure to cause proliferation, both angiotensin II and arginine-vasopressin stimulate early signal transduction events in cultured vascular smooth muscle cells such as activation of phospholipase C (PLC). mobilization of intracellular calcium (Ca-^ + J, activation of PKC and induction of proto-oncogenes [52 55]. This suggests that in addition to PKC activation, at least one other pathway may control the ability of an agonist to induce mitogenesis. Similar findings have already been shown in cultured human ASM ceils in which histamine and bradykinin both stimulate phosphatidylinositol 4,5biphosphate (PIP:) hydrolysis [23,56]. via a receptorcoupled PLC. Ca-^, mobilization [23.26,27] and induce cJus expression, but in which only histamine increases DNA synthesis [39] and presumably mitogenesis. This suggests that as in the case of cultured vascular smooth muscle cells, regulatory pathways additional to PKC are important in the control of ASM cell proliferation [39].
Role of receptor protein tyrosine kinases One such additional intracellular pathway controlling early mitogenic signalling events includes the receptor
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protein tyrosine kinase (PTK) family, responsible for the phosphorytation of tyrosine residues in target proteins [57]. The growth factors EGF, PDGF, insulin and insulinlike growth factor and transforming growth factors (TGF) increase PTK activity to varying extents in many cell types [reviewed in 7], although no direct evidence yet exists for tyrosine phosphorylation by PTKs in response to these growth factors in ASM. In cultured human ASM cells EGF has been shown to be mitogenic [31], but in the absence of data, for example, showing inhibitory efi'ects of EGF receptor PTK inhibitors such aserbstatinand the related tyrphostins [5S] on EGF-induced mitogenesis in these cells, the role ol'this signalling pathway must remain open. The intracclluiar target substrate(s) of the tyrosine kinase activity remains unclear. One possibility is phospholipase C. the key enzyme in phosphatidylinositol (PI) turnover. The activation of this enzyme causes hydrolysis of PIP: forming the two second messengers, DAG and inositol 1.4.,5-triphosphate (Ins 1.4.5 Pi), which activate PKC and Ca-"' mobilization from the sarcoplasmic reticulum respectively. Activation by PDGF of receptors having protein tyrosine kinase activity also causes PI turnover, mediated by PLC [59], and mitogenesis in cultured rabbit vascular smooth muscle cells [60]. Recently PLC-y, an isozyme of PLC, has been shown to be phosphorylated at tyrosine residues both in growth factor (EGF)-treated cells and directly by ihe purified EGF receptor [61-63]. Observations such as these have led to the notion of "cross-talk' between the PTK and PI turnover intracellular signalling pathways (see Fig. 2). Other cellular targets for PTK phosphorylation include phosphatidyl-3'-kinasc (P3'K), the c-r(//-l proto-oncogene product (RAF-I) and GTPase activating protein (GAP), reviewed by Cantley et al. [64]. P3"K is an enzyme which phosphorylates the 3'-position of the inositol ring of PI to give a class of phospholipids which are not substrates for PLC-y. but which are important for proliferation [65]; while RAF-I is a cytoplusmic protooncogenc product whose intrinsic serine threonine kinase activity is positively regulated by PTK, and is thought to stimulate transcription mediated by certain nuclear proto-oncogenes [66]. GAP enhances the GTPase activity of the GTP-binding product of thec-rw.v proto-oncogene, thereby attenuating its activity [67]. Although each of these proteins are associated with activated growth factor receptor PTKs. not all growth factor receptors can phosphorylatc and bind with all four proteins. Whether these proteins form a multimolecular 'signal transfer particle' even before PTK activation or whether separate sites for these proteins exist on the growth factor receptor PTK is not yet clear [57].
Changes in the activity/expression of these proteins during accelerated ASM cell growth and division cither in stimulated ASM cell culture or in intact asthmatic bronchi have yet to be investigated. Role of phospholipase D The presence of phospholipase D (PLD) in a wide variety of mammalian tissues and cells has been known for many years. Only recently has evidence implicated its role in signal transduction [68]. In fibroblasts receptor-mediated activation of PLD generates phosphatidic acid from membrane phosphatidylchoHne [69], triggering DNA synthesis and proliferation [70.71]. In cultured vascular smooth muscle cells PLD activity has been demonstrated after stimulation with phorbol ester. This stimulation was blocked by staurosporine [72], an inhibitor of PKC, indicating that PLD activity was regulated via PKC. Moreover, the smooth muscle mitogen. PDGF was able to stimulate PLD activity by a mechanism which was wholly dependent on extracellular Ca-^ and activation of PKC [72]. This highlights a pos.sible role for PLD in smooth muscle proliferation, and emphasizes the extent to which 'cross-talk' between different signalling pathways probably occurs. Further evidence of such 'crosstalk' is emerging from studies reported in Thompson ci at. in which FGF-stimulated Swiss 3T3 fibroblasts showed enhanced PLD activity which was inhibited after blocking the EGF receptor PTK [73], suggesting that PLD activation in these cells is mediated by tyrosine phosphoryiation, A number of stimuli can activate PLD in a variety of cell types. The precise nature o^ receptor coupling to PLD. however, is uncertain. Several possible mechanisms for activation of PLD following agonist-receptor interactions have been presented by Thompson cf al. [73]. It is likely that this pathway and its role in proliferation will receive much attention in ihe near future, since it represents an alternative mechanism for the production of DAG from membrane phospholipids which does not directly involve mobilization of intracellular Ca" ^ stores via Ins 1,4,5-P3. Smooth muscle .spasmogens as growth promoters It is becoming increasingly clear in vascular smooth muscle that a number of vasoactive agents and polypeptide growth factors appear to stimulate common second messenger systems which include increased PI turnover. mobilization of Ca- •, intracellular alkalinization through stimulation of Na^/H+ exchange, activation of PKC [see 37] and activation of PLD [see 74]. Further, certain smooth muscle mitogens including PDGF can also
Proliferative re.spon.se of airway smooth muscle
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EGF PDGF)
Tyrosine Phosphorylatior
1,TARGET PROTEIN •( ' PHOSHORYLATION '
-
4^
PROTO-ONCOGENE EXPRESSION
(eg. Z'fos, c-myc]
4-
DNA SYNTHESIS
MITOTIC DIVISION
Fig. 2. Key stimulus-response coupling believed lo be important in ihe control orproiifcraUon in ciilliircd airway smooth muscle cells. Events stimulating niilogenesis are indicated by unbroken lines ( ); while those inhibiting mitogenesis arc shown by broken lines ( ). Where evidence exists for particular signalling events in airway smooth muscle cells, filled arrow heads have been used; while open arrow heads link signalling events occurring in vascular smooth muscle cells, but for which no evidence is yet available for their ftinction in airway smooth muscle cells. Agonists known to stimulate or inhibit mitogenesis in airway smooth muscle cells have been included; while agonists which mediate certain events in the cascade, but have not yet been demonstrated to cause mitogenesis in these cells are shown in brackets. For clarity many of the proposed 'cross-talk" events have been omitted. R ^ receptor coupled to GTP-binding protein; G^GTP-binding protein; PLCv = phosphonpase C;; PLD = phospholipase D; PTK = protein tyrosine kinase: Adencyelase = adcnylatecyelasc: Guan cyclase^guanylatecyciase; PlP; = phosphatidyl 4,5-bisphosphate; PC^phosphatidylcholine: ATP^adcnosine tripliospliatc; GTP = guanosine triphosphate; IP.i^inositoI 1,4,5-trisphosphatc; DAGs^diaeylglycerol species; Cyclic AMP = cyclic 3 .5-adenosine monophosphatc; Cyclic GMP = cyclic 3',5'-guanosine monophosphate; PKCs^ protein kinase C species; PKA = cyclic AMP-depcndcnt protein kinase: P K G = cyclic GMP-dependent protein kJnasc; Ca/ CaM kinase^calcium/calmodulin-depcndent protein kinase.
contract smooth muscle [75.76]; while other contractile agonists such us angiotensin II [77] and adrenaline [78] stimulate growth in vascular smooth muscle cells. By analogy in ASM. inilamniatory mediators including the leukotrienes, neurokinins, bradykinin and platelcl activating factor previously considered to be primarily spasmogenic in nature may also promote mitogenesis to a greater or lesser degree, as has been demonstrated with histatnine [22.38]. Thus, two broad families of growlh factor receptors have emerged: those which have intrinsic PTK activity and those which do not (see Fig. 2). Receptors lacking intrinsic PTK activity are thought to couple GTP-binding proteins. However, with the increasing number of reports
of "cross-talk", these distinctions are becoming less obvious. The intraceilular signalling pathways which are believed to be important in the control of ASM mitogenesis are summarized in Fig. 2. Inhibition of airway smooth muscle mito^fiwsis Little is known about the mechanisms and pathways controlling inhibition of ASM proliferation, but there is increasing evidence lo support a role for cyclic adenosine 3'5'-monophosphate (cyclic AMP). Human ASM cells in culture express /i-adrenoceptors of the ^^-subtype which are functionally linked to adenylate cyclase via a GTPbinding protein-dependent mechanism [29.30]. Acti-
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S.J. Hirst ami C. H. C. Twort
vation of adenylate cyclase leads to elevated cell cyclic AMP content, which can also be accomplished by inhibition of phosphodiesterase activity using a type IV isozymc selective inhibitor (rolipram) [29]. Both receptormediated (isoprenaline) and direct activation (forskolin) of adenylate cyclase activity in cultured human ASM cells inhibit both phorbo! ester- and EGF-mediated proliferation [31]. Similar eflccts are observed witb a cell-pertiieablecyclic AMP analogue, dibutryl cyclic AMP. There is, however, a marked diffcreniial sensitivity in the ability of these cyclic AMP elevators to exert an anti-proliferative effect on the ASM cells when stimulated by the PKCdependent pathway rather than the PKC-independent pathway (EGF-mediated). This apparent differential susceptibility of the PKC pathway relative to the PTK pathway to cyclic AMP-mediated eflects may provide clues to the lack of disease-modifying activity of current therapies in chronic asthma. An additional inhibitory signal transduction pathway which may regulate mitogenesis of ASM involves increased cyclic guanosine 3'5'-monophosphate (cyclic GMP) via activation of the enzyme, guanylate cyclase. Wilh the immense current interest in the role of guanylate cyclase, activated by endothelium-dependent relaxant factors such as nitric oxide, in regulating vascular smooth muscle contractility, it is not surprising to find similar reports of a role in controlling vascular smooth muscle cell growth and division [79.80]. Elevation of inlracetlular cyclic GMP by atrial natriuretic peptide or nitro-vasodilators, including sodium nitroprusside and 8-bromocyclic GMP, inhibit serum-induced [79] angiotensin 11induced [80] proliferation of cultured vascular smooth muscle cells. The extent to which these effects are mediated by a cyclic GMP-dependent protein kinase either via cyclic GMP derived from soluble or membranebound guanylate cyclase has not yet been determined. Thus available information supports the idea that factors previously considered primarily as vasorelaxant (e.g. isoprenaline and sodium nitroprusside) may also function as inhibitors of smooth muscle proliferation. Consequently, there appears to be some relationship between the ability of a factor to contract or relax smooth muscle and its ability to stimulate or inhibit proliferation. In conclusion, very little is known about the control of mitogenesis in ASM cells, but available evidence provides tentative support of a role for both the PKC and PTK pathways in stimulating milogcnesis of cultured ASM cells, while activation of adenylate cyclase may have an important role in inhibiting mitogenesis. Future directions In vascular research, the endothelium tnay function in maintaining quiescence of ihc underlying smooth muscle
cells, based upon the finding that experimental removal of the endothelium causes proliferation of smooth muscle cells in vivo [81-83]. Thus in the normal airway it is envisaged that just as in the vascular media of blood vessels, smooth muscle cells are maintained in a 'contractile phenotype' characterized by abundance of actinomysin and low rate of proliferation [84,85]. This quiescent state could result partly from the absence of mitogens and partly from the action of inhibitory factors. The presence of a similar mechanism involving the bronchial epithelial layer, which is denuded in chronic asthma [86], may provide clues to the control of the ASM cell proliferative process. The apparent inability of many current anti-asthma therapies to reverse airway narrowing in chronic asthma suggests that irreversible changes of an architectural nature occur. If ASM thickening is important in this process, understanding of the mechanisms regulating ASM growth and division is essential to the development of suitable disease-modifying therapies. This may be especially relevant since compelling evidence has been provided to suggest that thickening of the airway wall., where ASM hyperplasia/hypertrophy is a major contributory factor, explains much of the bronchial hyperresponsiveness in asthma [17]. References 1 Dunnill MS. Masserclla GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis and in emphysema. Thorax 1969; 24;l76-79. 2 Heard BE. Hossain S. Hyperplasia of bronchial muscle in asthma. J Pathol 1973; 11O:.1I9-31. 3 Hossain S. Quantitative measurement of bronchial muscle in men with asthma. Am Rev Resp Dis 1973; 107:99 109. 4 Ebina M. Yaegashi H. Chibo R. Takahashi T. Motomiya M, Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. Am Rev Resp Dis 1990; 141:1327-32. 5 Libby P, O'Brien KV. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol 1983; 115:217-23. 6 Powis G. Signalling targets for anticancer drug development. Trends Pharmacol Sci 1991; 12:188-94. 7 Yarden Y. Ullrich A. Growth factor receptor tyrosine kinases. Ann Rev Biochem ]988;57:443-78. 8 Smith JC, Stiles CD. Cytoplasmic transfer of Ihe mitogenic response to platelet-derived growlh factor. Proc NatI Acad Sei USA 1981; 78:4363-67. 9 Dcnhardt DT, Edwards DR, Parfett LJ. Gene expression during the mammalian cell cycle. Biochim Biophys Acta 1986:865:83-125. 10 Rozengurt E. Early signals in the mitogenic response. Science 1986;234:l61-6.
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11 Miilicr R. Bravo R, Burckhardl .1. Curran T. Induction of c-Jos gene ;ind prolein hy growth Taclors precedes activation ofc-m.iT. Nature 1984:312:716-20. 12 McKinney JD. Heintz N. Transcriptionai regulation in the eLikaryotit cell cycle. Trends Biochem Sci 1991; I6:4.'^O 5. \? Pare PD. Wiggs BR. Hogg JC. Bosken C. The comparative mechanics and morphology of airways in asthma and in chronic obstructive pulmonary disease. Am Rev Resp Dis 1991: 14.1:1189-93. 14 James Al.. Hogg JC. Dunn LA. Pare PD. The use of internal perimeter to compare airway size and to calculate smooth muscle shortening. Am Rev Dis 1988; 139;I36-9. 15 Ebina M. Yaegashi H. Takahashi T. Motomiya M. Tanemura M. Distribution of smooth muscles along the bronchial tree Am Rev Resp Dis 1990; 141:1322 6. 16 Nagai A, Inano H. Takizawa T. Morphological changes in airways induced by recurrent exposure of acetylcholine in the guinea pig. Am Rev Resp Dis 1990; 142:172-6. 17 James AL. Pare PD. Hogg JC. The mechanics of airway narrowing in asthma. Am Rev Resp Dis 1989: 139:242-6. 18 GimbroneMA.Cotran RS. Human vascular smooth muscle in culture, growth and ultrastructure. Lab Invest I975;3.V 16-27. 19 Chamlcy-Campbell J. Campbell J. Ross R. The smooth muscle cell in culture. Physiol Rev 1979; 59:1-55, 20 Tom-Moy M. Madison JM. Jones CA. DeLaneroIie P. Brown JK. Morphological characterisation of cultured smooth muscle cells isolated Irom the iracheas of adult dog.s. Anat Rec 1987:218:313-28. 21 Avner BP. DeLongo J. Wilson S. Ladman AJ. A method lor ciilturing canine tracheal smooth muscle cells in ritra: morphological observations. Anat Rec 1981; 200:357-70. 22 Panettieri RA. Yadvish PA. Kelly AM. Rubinstein NA. KotlikolT ML Hislamine stimulates proliferation of airway smooth muscle and induces c-Jos expression. Am J Physiol 199O:259:L365 L37I. 23 Marsh KA. Hill SJ. Bradkinin-induced phosphoinositide hydrolysis and calcium mobilisation in cultured bovine tracheal smooth muscle cells. Bi J Pharmacol 1992; iO5:66P. 24 Chopra LC. Twort CHC. Cameron IR. Ward JPT. Inositol 1.4.5-trisphosphate- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca-' release in cultured airway smooth muscle. Br J Pharmacol 1991: 104:901 6. 25 Hirst SJ. Warley A. Ward JPT. Preparation of cultured rabbit tracheal smooth muscle cells for X-ray microanalysis. Proc 10th Eur Congress on Electron Microscopy. Granada. Spain. September 1992. 26 Twort CMC. van Breenien C Human airway smooth muscle in culture. Tissue and Cell 1988; 20:339 44. 27 Twort CHC. van Breemen C Human airway smooth muscle in culture: control of the intracellular calcium store. Pulm Pharmacol 1989; 2:45-53. 28 Panellieri RA. Murray RK, DePalo LR. Yadvish PA. KotlikolT Ml. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physio! 1989; 2.56:C329 C335. 29 Hall IP, Townsend P. Da>kin K. Widdop S. Control of
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tissue cyclic AMP content in primary cultures of human airway smooth muscle cells. Br J Pharmacol 1992; IO5:72P. Halt IP, Widdop S. Desensitisation of isoprenalinc-lnduced ['H]-cyclic AMP formation in primary cultures of human airway smooth muscle cells. Br J Pharmacol 1992: 105:71 P. Panettieri RA. Rubinstein NA. Feuerstein B. KotlikofTML Beta-adrenergic inhibition of airway smooth muscle proliferation. Am Rev Resp Dis 1991; 143:A608. Hogg JC. James A L. Pare PD. Evidence for inflammation in asthma. Am Rev Resp Dis 1991: 143:S39-S42. King RJ, Jones MB. Minoo P. Regulation of lung cell proliferation by polypeptide growth factors. Am J Physiol 1989: 257:L23-L38. Henderson B. Blake S. Therapeutic potential of cytokine manipulation. Trends Pharmacol Sci 1992: 13:145-51. Horn BR. Robin ED. Theodore J. Van Kessel A. Total eosinophii counts in the management of asthma. New Eng J Med 1975:292:1152-5, Taylor KJ, Luksza AR. Peripheral blood eosinophii counts and bronchial hyperresponsiveness- Thorax 1987; 42:452-6. Owens GK. Control of hypertrophic versus hyperplastic growth of vascular smooth muscle cells. Am J Physiol 1989; 257:HI755 HI765, Paneitieri RA. Rubinstein NA. Kelly AM. Kothkoff MI. Histamine induces proliferation of airway smooth muscle in culture. Am Rev Resp Dis 1989: I39:A78. Panettieri RA. Rubmstein NA. Kelly AM. KotlikotT ML The specificity of C"/('),v expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Resp Dis 1991; I43:A934. Oliver MH. Harrison NK. Bishop JE. Cole PJ, Laurent GJ. A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. J Cell Sci 1989; 92:513-18. Hirst SJ. Twort CHC. Barnes PJ. Quantifying the proliferative response in cultured human and rabbit airway smooth muscle. Am Rev Resp Dis 1992: I45:A14. Kikkawa U. Kishimolo A. Nishi?:uka Y. The protein kinase C family: its heterogeneity and its implications. Ann Rev Biochem 1989; 58:31 44. Bell RB. Burns DJ, Lipid activation of protein kinase C. J BiolChem 1991; 266:4661-4. Dicker P. Rozengurt E. S;imutaiion of DNA synthesis by tumour promoter and pure mitogenic factors. Nature 1978; 276:723-6. Dicker P. Ro/engurt E. Phorbol esters and vasopressin stimulate DNA synthesis by a common mechanism. Nature 1980: 287:607-12, Rozengurt E. Rodrigue/-Pena A. Coombs M. Sinelt-Smith J. Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells: role of Ca'*-sensitive, phospholipiddependent protein kinase, Bitichem Biophys Res Common 1984: 120:1053-9. Rozengurt E, Neuropeptides as cellular growth factors: role of multiple signalling pathways. Eur J Clin Invest 1991; 21:123-34. Mitsuka M. Berk \K\ Long-term regu!ati{Hi of Na'-H *
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exchange in vascular snioolh muscle cells: role of protein kinase C. Am J Physiol 1^91; 26():C562 C569. Ohmi K. Yamashila S, Nonomura Y. Effect of K252a. a protein kinase inliihiior. on ihc prolifcralion of vascular smooth muscle cells iiiochcm Biophys RcsCommiin 1490: 173:976-81. Geisterfer A. Peach MJ. Owens GK. Angiolcnsin II induces hypertrophy, not hyperplasia of cultured rat aortic smooth muscle cells, Circ Res 1989; 62:749-56. Gcisterfcr A. Owens GK. Arginine vasopressin induced hypertrophy of cultured rat aortic smooth muscle cells. Hypertension I9S9: 14;4I3 20. Berk BC . Aronov MS. Brock TA.Cragoe E.GImbrone MA. Alexander RW. Angiotensin ll-stimu!ated N a ' , H * exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dcpendent and -indepcndeni pathways. J Biol Chem I9SS: 262:5057-64. Griendling KK. Berk BC. CJanz P. Gimbrone MA. Alexander RW. Angiotensin II stimulation of vaseular smooth muscle phosphoinositidc melabolism. Hypertension 19X7; 9:(Suppl 3):ISI .y Taubam MB. Berk BC. l/umo S. Tsuda T. Alexander RW. Nudal-Ginard B. Angiotensin II induces c-fo.s mRNA i[i aortic smooth muscle. Role of Ca-'^ mobilization. J Biol Chem 1989:264:526-30. Berk BC. Vekshtein V. Gordon HM, Suda T. Angiotensin Il-stimulated prolein synthesis in cultured va.scular smooth muscle cells. Hypertension 1989: 13:305-14. Chilvers ER. Chopra l.C. Barnes J, Twort CHC. Brndykinin-stimulated inosito! phosphate accumulation and demonstration of inositol penla- and hexakisphosphates in cultured airway smooth muscle cells. Am Rev Resp Dis 1989: l39(.Suppl4):A77. Ullrich A, Schlcssinger J. Signal iransduction by receptors with tyrosine kinase activity. Cell 1990: 61:203-12. Levitzki A. Tyrphoslins potential antiproliferative agents and novel molecular tools. Biochem Pharmacol 1990; 40:913-18. Kawahara Y. Kariya K. Araki S. Fuku/aki H, Takai Y. Platelet-derived growth factor (PDGF)-induced phospholipase C-mediated hydrolysis of phosphonositides in vascular smooth muscle cells dilTerential sensilivily of POG!-- and angiotensin ll-induced pht)SpholipaseC reactions to protein kinase C activating phorbol esters. Biochem Biophys Res Commun 1988; 165:846 54. Araki S. Kawahara Y. Kariya K. Sunako M. Tsuda T. Kukuzaki H. Yoshimi T. Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin [l in rabbit vascular smooth muscle cells. Bioclicm Biophys Res Commun 1990; UiS:35(l 7, Nishibe S. Wahl Ml. Rhee SG. Carpenter Ci. Tyrosine phosphorylation of phosphnlipaseC-l I in lilni by epidermal growth factor receptor. J Biol Chem 1989; 264:10.3.35 8. Meisenheider J. Suh P-G. Rhee SG. Hunter T. Phospholipase C-y is a subslratc for the PDGF and EGF receptor protein tyrosine kinases in rim and in ritm. Cell 1989; 57:1109-22.
63 Wahl MI. Olashaw NE, Nishibe S. Rhee SG. Pledger WJ, Carpenter G. Plalclet-derived growth factor induces rapid and sustained tyrosine phosphorylation ofphospholipaseCV in quiescent BAl.B/c 3T3 cells. Mol Cell Biol 1989:9:293443. 64 Cantley L C Auger KR. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Oncogenes and signal transduetion. Cell 1991; 64:281-302. 65 Escobedo. JA. Williams LT. A PDGF receptor domain essential furmitogenesis bul nol for many other responses to PDGF. Nature 1988; 335:85 7. 66 Druker BJ. Mammon H. Roberts TJ. Oncogenes. growth factors, and signal transduclion, N Eng J Med 1990; 321:1383-9!. 67 Kazlauskas A. Ellis C. Pawson T. Cooper JA. Binding of GAP to activated PDGF receptors. Science 1990; 247; 157880. 68 Biilah MM. Anthes JC. Mullmann TJ. Receptor-coupled ph
REVIEW
The proliferative response of airway smooth muscle S. J. HIRST and C. H. C. TWORT Respiratory Research Laboraiorics. Smooth Muscle Research Group. UMDS Division of Medicine, St Thomas' Hospital. London. U.K. The inability to reverse completely airway narrowing in many cases oCchronic severe uslhma suggests a persistent component of the long-term pathological process, which may involve remodelling or architectural changes in the lung. Indeed one of the most striking and characteristic pathological features of chronic asthma is an increase in [he smooth muscle mass of the airways [1-4]. Both hyperplasia (increase in cell number) and hypertrophy (increase in cell size) seem to contribute to this increase and its associated reduction in airway luminal diameter. For this reason characterization and elucidation of the cellular and biochemical events which regulate growth and division of airway smooth muscle cells to various stimuli is likeiy to be of great importance in designing novel therapies for asthma, particularly for the development of disease-modifying drugs. Investigation of the mechanisms controlling smooth muscle growth in airways has lagged far behind that of vascular research, and consequently current theories of airway smooth muscle (ASM) proliferation rely heavily on the results of studies on vascular smooth muscle. In this review we discuss some of the growth factors controlling smooth muscle proliferation, their associated signal transduction pathways for both stimulation and inhibition of the mitogenic response and the extent to which they operate in ASM. The cell cycle Cell growth and division processes are regulated predominantly by the response to appropriate growth factors and the expression of their receptors. Proliferation in mammalian cells involves a programmed series of complex genetic and biochemical events which fall into four distinct phases (outlined in Fig. 1). The first stage, G| or •gap", is characterized by a lack of DNA synthesis, but there is elevated synthesis of RN A and protein. The second stage or 'S-phase' is a period of increased DNA synthesis during which elevated RNA and protein synthesis are maintained. In the third stage or G^ phase. DNA synthesis ceases while RNA and protein synthesis continue. This phase often results in cytoplasmic volume expansion. Correspondence: Dr S. J. Hirst. Respiratory RL-scitrch Laboratories, Smooth MusL-le Reseiireh Group. LJMDS Division of Medicine. St Thomas' Hospital. Lambeth Palaee Road, London SEl 7EH. U.K.
Collectively, G|, Sand G;comprise the'interphase" which is distinct from the actual cell division (i.e. mitosis). Mitosis is the shortest phase of the cell cycle, usually occurring within 60 minutes, in which little or no DNA, RNA or protein synthesis occurs, A fifth phase called Go has been proposed in which cells have come to rest and remain viable, but do not traverse the cell cycle. Under conditions when the growth arrest is not irreversible, polypeptidc growth faetors will induce cells in Gd to reenter the cycle and proliferate. Incubation of cultured smooth muscle cells for periods of 24-72 hr in serum-free culture media supplemented with insulin, ascorbate and transferrin will maintain the cells in such a viable but nondividing state [5] and has the experimental advantage of synchronizing the cell cycle kinetics. Whether this growth-arrested state is positively controlled by the expression of certain inhibitory factors has not been established. The events which control the ability of a growtharrested cell to re-enter the cell cycle are far from understood, particularly in the ASM cell. What is clear, however, is the central role of growth factors which, acting through specific plasma membrane receptors, initiate inlracellular signal transduction events which lead to phosphorylation of a large number of target intraceilular regulatory proteins [see 6], These regulatory proteins are believed to activate the intracellular processes including altered gene expression underlying cell growth and division [7]. In I9H1 Smith and Stiles [8] classified growth factors into two major groups, competence factors and progression factors. Growth factors affecting early Gu/G| events (i.e. the preparedness to enter the cell cycle) commit the cell to growth and are termed competence factors; while those which provide a committed cell with a series of necessary external stimuli rquired for it to continue through G|. S, Gi and on to mitotic division, are termed progression factors. These regulatory factors mediate their ejects through the expression of genes and the transcription of competence proteins and progression proteins. The identity of these proteins is unclear but they are likely to include elements of the signal transduction cascade and regulators of DNA synthesis and of further (late cycle) gene transcription [9]. Most growth factors do not possess both competence and progression activity. 907
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Cell division
DNA synthesis
or Quiescent phose
Fig. I. The cell cycle of a typical mammalian cell, showing the phases leading to division. The durations of each stage have not been indicated since they vary considerably between different cell types. Consequently, cellular proliferation normally requires exposure ofeells to multiple growth factors in sequence, with each of the growth faetors and progression factors selectively activating particular events iti the proliferation signalling cascade [10]. Understanding the role of regulatory genes governing cell cycle kinetics is still in its infancy, but at least two classes of genes have been identified. These are called "cell cycle-dependent' genes and 'early response' genes [10]. The expression of cell cycle-dependent genes is modulated throughout the cell cycle: these genes encode for a variety of cellular proteins including major histone proteins, calmodulin, actin and enzymes such as thymidine kinase and dihydrofolate reductase [see 10]. "Early response" genes include the cellular proto-oncogenes., c-myc, c-jun and C'fos (induction ofc-fos expression being one of the earlier markers of proliferation and differentiation) [11]. The products of c-/»/; and c-/ftv are known lo dimerize. translocate to the nucleus and bind to activator protein-1 (AP-I) promoter regions on the DNA. increasing expression of the long-term regulated genes considered to be a prerequisite to mitosis [see 12 for review]. The use of cultured airway smooth muscle cells in assessing the proliferative response To date, assessment of ASM proliferation has relied heavily upon morphometric analysis of histological sections of the bronchi of asthmatics which reveal marked thickening of the smooth muscle layers [1-4, 13-17]. A correlation between an increase in the number of cell nuclei and muscle thickening is observed, suggesting thai the increase in tnuscle mass in asthma results more from hyperplasia rather than from hypertrophy [2]. Ceil culture, unlike morphometric analysis, can pro-
vide information about the effects of mitogenic stimuH on the cell cycle and cellular proliferation. In contrast to the widespread use of cultured vascular smooth muscle in the investigation of smooth muscle proliferation [see 18,19], experience in culturing ASM has until recently been more limited. However, with therecent resurgence of interest in the thickening of the airway wail in asthma, various groups have now succeeded in culturing cells from canine [20-22], bovine [23], rabbit [24,25] and human ASM [2630]. Cultured smooth muscle cells of tracheal or bronchial origin have a similar appearance to the differentiated contractile smooth muscle cells in the intact tissue, appearing flattened and ribbon- or spindle-shaped with central oval nuclei and prominent nucleoli [20,21,25,28]. Confluent cultured ASM cells, like those of vascular origin, align themselves in parallel and have a highly contoured architecture with the formation of ridges and nodules giving a characteristic 'hill and valley' appearance [18-21,28]. They stain positively for both smooth muscle actin and myosin [20,21], features not shared by contaminating fibroblasts or other non-muscle cells [20]. These cells maintain also their physiological responsiveness to agonists implicated in inflammatory airway diseases [26 28] and display functional coupling of /?adrenergic receptors [28,31], localized to the //2-adrenoceptor [30]. Growth factors and regulation of smooth muscle growth and division In chronic asthma, in which there is persisting inflammatory cell recruitment [32], the ASM cell is likely to be exposed to a large number of growth factors and inflammatory mediators. Many of the cells present in the inflamed airway synthesize growth factors which have the potential to stimulate hypertrophic and/or hyperplastic changes in the airway wall smooth muscle. Of particular interest is the alveolar macrophage, capable of releasing a plethora of growth factors and mediators, many of which are known to mediate fibroblast proliferation [reviewed in 33]. The macrophage is a major cell source of epidermal growth factor (EGF) and its homologue transforming growth factor-a (TGFa), fibroblast growth factors (FGF), insuiin-like growth factor (IGF-i), platelet-derived growth factor (PDGF), colony-stimulating factors such as granulocyte-colony-stimulating factor (G-CSF) and members of the interleukin family, IL-lx and IL-6 [see 34]. The platelet may also contribute since it contains large amounts of PDGF and TGF^ [33]. Curiously, the eosinophil appears to have escaped attention as a source of growth factors, despite its proposed role in the late asthmatic response and associated airway hyperresponsi-
Proliferative response of airway smooth muscle
veness [35.36]. Other inflammatory cells such as mast cells. T cells and other monocytes are also recognized sources ofa variety of different growth factors., CSFs and interleukins. Despite the presence of cells in asthmatic bronchi capable of producing a variety of growth factors, almost nothing is known about the effects of any of these factors on the ASM cell either in culture or in vivo. Our current knowledge depends heavily on studies of the effects of growth factors on vascular smooth muscle cell growth and division [37]. To date, histamine is the only mediator studied in any detail for effects on the proliferative response of cultured ASM cells. In cultured canine and human ASM cells, histamine not only induces the expression of the protooncogene c-fos^ whose expression is one of the earliest markers ofcells re-entering the cell cycle [12], but also acts as a mitogen in the absence of any other stimulus in cells rendered quiescent [22,38,39]. This suggests that histamine not only induces competence in these cells but also behaves as a progression factor. The only other reports describing the actions of growth factors on cultured ASM cells concern the expression of c-/fj.v induced by PDGF in canine cells [22,38]. EGF, however, stimulates DNA synthesis in cultured human ASM cells [31 ]. but in canine cells it failed to stimulate expression of c-fos above control levels [22]. The reasons for this apparent anomaly are not clear, but may lie in the use of [^H]-thymidine uptake (DNA synthesis) as a proliferation assay. Several criticisms have been levelled at the use of this method [see 40]. Of particular concern with this method is the absence of actual proliferation in cells which continue to incorporate thymidine suggesting a block in the S or G: phases of the cell cycle {see earlier). For this reason alternative proliferation assays such as cleavage of the tetrazolium salt, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-dephenyl tetrazolium bromide (MTT) by mitochondrial succinyl dehydrogenase and assay of cellular protein by specific binding to protein of the dye. Coomassie Brilliant blue have been developed, which in ASM cultures have been found to correlate closely with actual cell counts determined by haemocytometry [41].
Signal transduction mechanisms for control of smooth muscle growth and division The response to extracellular mediators such as growth factors requires cellular signalling mechanisms to transduce the message received by plasma membrane receptors into the intracellular events associated with the control of proliferation. Evidence is steadily accumulating implicating a variety of signal transduction pathways in the control of mitogenesis in ASM ceils [22,31.39]. The following signal transduction pathways have already
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been implicated to varying extents in the control of smooth muscle growth and division and may provide important clues to the future design of more effective antiproliferative therapies. Role of prolein kinasc C Studies to date have centred on the role of a family of up to nine phosphatidylserine-dependent, lipid-activated serine and threonine protein kinases, collectively known as protein kinase C (PKC) [42,43]. Phorbol esters and synthetic diacylglycerols, which mimic the endogenous PKC activator, diacylglycerol (DAG), have long been known to stimulate DNA synthesis and cell division, often in synergy with other growth promoting factors [44 46]. PKC activates a number of early events associated with mitogenesis including Na + /H+ exchange, resulting in intracellular alkalinization, protein synthesis and the induction/expression of nuclear proto-oncogenes such as C'fos and c-myc [47.48]. In cultured smooth muscle cells isolated from bovine carotid arteries, activation of PKC may be necessary for progression from the G; to S phase of the cell cycle [49]. Not all agonists, however, which activate PKC cause proliferation in smooth muscle cells. For example in cultured rat aortic smooth muscle cells, made quiescent in a defined serum-free media, both angiotensin II [50] and arginine-vasopressin [51] fail to induce proliferation, but do cause marked increases in cellular protein synthesis with subsequent hypertrophy. Despite this failure to cause proliferation, both angiotensin II and arginine-vasopressin stimulate early signal transduction events in cultured vascular smooth muscle cells such as activation of phospholipase C (PLC). mobilization of intracellular calcium (Ca-^ + J, activation of PKC and induction of proto-oncogenes [52 55]. This suggests that in addition to PKC activation, at least one other pathway may control the ability of an agonist to induce mitogenesis. Similar findings have already been shown in cultured human ASM ceils in which histamine and bradykinin both stimulate phosphatidylinositol 4,5biphosphate (PIP:) hydrolysis [23,56]. via a receptorcoupled PLC. Ca-^, mobilization [23.26,27] and induce cJus expression, but in which only histamine increases DNA synthesis [39] and presumably mitogenesis. This suggests that as in the case of cultured vascular smooth muscle cells, regulatory pathways additional to PKC are important in the control of ASM cell proliferation [39].
Role of receptor protein tyrosine kinases One such additional intracellular pathway controlling early mitogenic signalling events includes the receptor
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protein tyrosine kinase (PTK) family, responsible for the phosphorytation of tyrosine residues in target proteins [57]. The growth factors EGF, PDGF, insulin and insulinlike growth factor and transforming growth factors (TGF) increase PTK activity to varying extents in many cell types [reviewed in 7], although no direct evidence yet exists for tyrosine phosphorylation by PTKs in response to these growth factors in ASM. In cultured human ASM cells EGF has been shown to be mitogenic [31], but in the absence of data, for example, showing inhibitory efi'ects of EGF receptor PTK inhibitors such aserbstatinand the related tyrphostins [5S] on EGF-induced mitogenesis in these cells, the role ol'this signalling pathway must remain open. The intracclluiar target substrate(s) of the tyrosine kinase activity remains unclear. One possibility is phospholipase C. the key enzyme in phosphatidylinositol (PI) turnover. The activation of this enzyme causes hydrolysis of PIP: forming the two second messengers, DAG and inositol 1.4.,5-triphosphate (Ins 1.4.5 Pi), which activate PKC and Ca-"' mobilization from the sarcoplasmic reticulum respectively. Activation by PDGF of receptors having protein tyrosine kinase activity also causes PI turnover, mediated by PLC [59], and mitogenesis in cultured rabbit vascular smooth muscle cells [60]. Recently PLC-y, an isozyme of PLC, has been shown to be phosphorylated at tyrosine residues both in growth factor (EGF)-treated cells and directly by ihe purified EGF receptor [61-63]. Observations such as these have led to the notion of "cross-talk' between the PTK and PI turnover intracellular signalling pathways (see Fig. 2). Other cellular targets for PTK phosphorylation include phosphatidyl-3'-kinasc (P3'K), the c-r(//-l proto-oncogene product (RAF-I) and GTPase activating protein (GAP), reviewed by Cantley et al. [64]. P3"K is an enzyme which phosphorylates the 3'-position of the inositol ring of PI to give a class of phospholipids which are not substrates for PLC-y. but which are important for proliferation [65]; while RAF-I is a cytoplusmic protooncogenc product whose intrinsic serine threonine kinase activity is positively regulated by PTK, and is thought to stimulate transcription mediated by certain nuclear proto-oncogenes [66]. GAP enhances the GTPase activity of the GTP-binding product of thec-rw.v proto-oncogene, thereby attenuating its activity [67]. Although each of these proteins are associated with activated growth factor receptor PTKs. not all growth factor receptors can phosphorylatc and bind with all four proteins. Whether these proteins form a multimolecular 'signal transfer particle' even before PTK activation or whether separate sites for these proteins exist on the growth factor receptor PTK is not yet clear [57].
Changes in the activity/expression of these proteins during accelerated ASM cell growth and division cither in stimulated ASM cell culture or in intact asthmatic bronchi have yet to be investigated. Role of phospholipase D The presence of phospholipase D (PLD) in a wide variety of mammalian tissues and cells has been known for many years. Only recently has evidence implicated its role in signal transduction [68]. In fibroblasts receptor-mediated activation of PLD generates phosphatidic acid from membrane phosphatidylchoHne [69], triggering DNA synthesis and proliferation [70.71]. In cultured vascular smooth muscle cells PLD activity has been demonstrated after stimulation with phorbol ester. This stimulation was blocked by staurosporine [72], an inhibitor of PKC, indicating that PLD activity was regulated via PKC. Moreover, the smooth muscle mitogen. PDGF was able to stimulate PLD activity by a mechanism which was wholly dependent on extracellular Ca-^ and activation of PKC [72]. This highlights a pos.sible role for PLD in smooth muscle proliferation, and emphasizes the extent to which 'cross-talk' between different signalling pathways probably occurs. Further evidence of such 'crosstalk' is emerging from studies reported in Thompson ci at. in which FGF-stimulated Swiss 3T3 fibroblasts showed enhanced PLD activity which was inhibited after blocking the EGF receptor PTK [73], suggesting that PLD activation in these cells is mediated by tyrosine phosphoryiation, A number of stimuli can activate PLD in a variety of cell types. The precise nature o^ receptor coupling to PLD. however, is uncertain. Several possible mechanisms for activation of PLD following agonist-receptor interactions have been presented by Thompson cf al. [73]. It is likely that this pathway and its role in proliferation will receive much attention in ihe near future, since it represents an alternative mechanism for the production of DAG from membrane phospholipids which does not directly involve mobilization of intracellular Ca" ^ stores via Ins 1,4,5-P3. Smooth muscle .spasmogens as growth promoters It is becoming increasingly clear in vascular smooth muscle that a number of vasoactive agents and polypeptide growth factors appear to stimulate common second messenger systems which include increased PI turnover. mobilization of Ca- •, intracellular alkalinization through stimulation of Na^/H+ exchange, activation of PKC [see 37] and activation of PLD [see 74]. Further, certain smooth muscle mitogens including PDGF can also
Proliferative re.spon.se of airway smooth muscle
911
EGF PDGF)
Tyrosine Phosphorylatior
1,TARGET PROTEIN •( ' PHOSHORYLATION '
-
4^
PROTO-ONCOGENE EXPRESSION
(eg. Z'fos, c-myc]
4-
DNA SYNTHESIS
MITOTIC DIVISION
Fig. 2. Key stimulus-response coupling believed lo be important in ihe control orproiifcraUon in ciilliircd airway smooth muscle cells. Events stimulating niilogenesis are indicated by unbroken lines ( ); while those inhibiting mitogenesis arc shown by broken lines ( ). Where evidence exists for particular signalling events in airway smooth muscle cells, filled arrow heads have been used; while open arrow heads link signalling events occurring in vascular smooth muscle cells, but for which no evidence is yet available for their ftinction in airway smooth muscle cells. Agonists known to stimulate or inhibit mitogenesis in airway smooth muscle cells have been included; while agonists which mediate certain events in the cascade, but have not yet been demonstrated to cause mitogenesis in these cells are shown in brackets. For clarity many of the proposed 'cross-talk" events have been omitted. R ^ receptor coupled to GTP-binding protein; G^GTP-binding protein; PLCv = phosphonpase C;; PLD = phospholipase D; PTK = protein tyrosine kinase: Adencyelase = adcnylatecyelasc: Guan cyclase^guanylatecyciase; PlP; = phosphatidyl 4,5-bisphosphate; PC^phosphatidylcholine: ATP^adcnosine tripliospliatc; GTP = guanosine triphosphate; IP.i^inositoI 1,4,5-trisphosphatc; DAGs^diaeylglycerol species; Cyclic AMP = cyclic 3 .5-adenosine monophosphatc; Cyclic GMP = cyclic 3',5'-guanosine monophosphate; PKCs^ protein kinase C species; PKA = cyclic AMP-depcndcnt protein kinase: P K G = cyclic GMP-dependent protein kJnasc; Ca/ CaM kinase^calcium/calmodulin-depcndent protein kinase.
contract smooth muscle [75.76]; while other contractile agonists such us angiotensin II [77] and adrenaline [78] stimulate growth in vascular smooth muscle cells. By analogy in ASM. inilamniatory mediators including the leukotrienes, neurokinins, bradykinin and platelcl activating factor previously considered to be primarily spasmogenic in nature may also promote mitogenesis to a greater or lesser degree, as has been demonstrated with histatnine [22.38]. Thus, two broad families of growlh factor receptors have emerged: those which have intrinsic PTK activity and those which do not (see Fig. 2). Receptors lacking intrinsic PTK activity are thought to couple GTP-binding proteins. However, with the increasing number of reports
of "cross-talk", these distinctions are becoming less obvious. The intraceilular signalling pathways which are believed to be important in the control of ASM mitogenesis are summarized in Fig. 2. Inhibition of airway smooth muscle mito^fiwsis Little is known about the mechanisms and pathways controlling inhibition of ASM proliferation, but there is increasing evidence lo support a role for cyclic adenosine 3'5'-monophosphate (cyclic AMP). Human ASM cells in culture express /i-adrenoceptors of the ^^-subtype which are functionally linked to adenylate cyclase via a GTPbinding protein-dependent mechanism [29.30]. Acti-
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S.J. Hirst ami C. H. C. Twort
vation of adenylate cyclase leads to elevated cell cyclic AMP content, which can also be accomplished by inhibition of phosphodiesterase activity using a type IV isozymc selective inhibitor (rolipram) [29]. Both receptormediated (isoprenaline) and direct activation (forskolin) of adenylate cyclase activity in cultured human ASM cells inhibit both phorbo! ester- and EGF-mediated proliferation [31]. Similar eflccts are observed witb a cell-pertiieablecyclic AMP analogue, dibutryl cyclic AMP. There is, however, a marked diffcreniial sensitivity in the ability of these cyclic AMP elevators to exert an anti-proliferative effect on the ASM cells when stimulated by the PKCdependent pathway rather than the PKC-independent pathway (EGF-mediated). This apparent differential susceptibility of the PKC pathway relative to the PTK pathway to cyclic AMP-mediated eflects may provide clues to the lack of disease-modifying activity of current therapies in chronic asthma. An additional inhibitory signal transduction pathway which may regulate mitogenesis of ASM involves increased cyclic guanosine 3'5'-monophosphate (cyclic GMP) via activation of the enzyme, guanylate cyclase. Wilh the immense current interest in the role of guanylate cyclase, activated by endothelium-dependent relaxant factors such as nitric oxide, in regulating vascular smooth muscle contractility, it is not surprising to find similar reports of a role in controlling vascular smooth muscle cell growth and division [79.80]. Elevation of inlracetlular cyclic GMP by atrial natriuretic peptide or nitro-vasodilators, including sodium nitroprusside and 8-bromocyclic GMP, inhibit serum-induced [79] angiotensin 11induced [80] proliferation of cultured vascular smooth muscle cells. The extent to which these effects are mediated by a cyclic GMP-dependent protein kinase either via cyclic GMP derived from soluble or membranebound guanylate cyclase has not yet been determined. Thus available information supports the idea that factors previously considered primarily as vasorelaxant (e.g. isoprenaline and sodium nitroprusside) may also function as inhibitors of smooth muscle proliferation. Consequently, there appears to be some relationship between the ability of a factor to contract or relax smooth muscle and its ability to stimulate or inhibit proliferation. In conclusion, very little is known about the control of mitogenesis in ASM cells, but available evidence provides tentative support of a role for both the PKC and PTK pathways in stimulating milogcnesis of cultured ASM cells, while activation of adenylate cyclase may have an important role in inhibiting mitogenesis. Future directions In vascular research, the endothelium tnay function in maintaining quiescence of ihc underlying smooth muscle
cells, based upon the finding that experimental removal of the endothelium causes proliferation of smooth muscle cells in vivo [81-83]. Thus in the normal airway it is envisaged that just as in the vascular media of blood vessels, smooth muscle cells are maintained in a 'contractile phenotype' characterized by abundance of actinomysin and low rate of proliferation [84,85]. This quiescent state could result partly from the absence of mitogens and partly from the action of inhibitory factors. The presence of a similar mechanism involving the bronchial epithelial layer, which is denuded in chronic asthma [86], may provide clues to the control of the ASM cell proliferative process. The apparent inability of many current anti-asthma therapies to reverse airway narrowing in chronic asthma suggests that irreversible changes of an architectural nature occur. If ASM thickening is important in this process, understanding of the mechanisms regulating ASM growth and division is essential to the development of suitable disease-modifying therapies. This may be especially relevant since compelling evidence has been provided to suggest that thickening of the airway wall., where ASM hyperplasia/hypertrophy is a major contributory factor, explains much of the bronchial hyperresponsiveness in asthma [17]. References 1 Dunnill MS. Masserclla GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis and in emphysema. Thorax 1969; 24;l76-79. 2 Heard BE. Hossain S. Hyperplasia of bronchial muscle in asthma. J Pathol 1973; 11O:.1I9-31. 3 Hossain S. Quantitative measurement of bronchial muscle in men with asthma. Am Rev Resp Dis 1973; 107:99 109. 4 Ebina M. Yaegashi H. Chibo R. Takahashi T. Motomiya M, Tanemura M. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. Am Rev Resp Dis 1990; 141:1327-32. 5 Libby P, O'Brien KV. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol 1983; 115:217-23. 6 Powis G. Signalling targets for anticancer drug development. Trends Pharmacol Sci 1991; 12:188-94. 7 Yarden Y. Ullrich A. Growth factor receptor tyrosine kinases. Ann Rev Biochem ]988;57:443-78. 8 Smith JC, Stiles CD. Cytoplasmic transfer of Ihe mitogenic response to platelet-derived growlh factor. Proc NatI Acad Sei USA 1981; 78:4363-67. 9 Dcnhardt DT, Edwards DR, Parfett LJ. Gene expression during the mammalian cell cycle. Biochim Biophys Acta 1986:865:83-125. 10 Rozengurt E. Early signals in the mitogenic response. Science 1986;234:l61-6.
Proliferative rcspon.sc of airway smooth muscle
11 Miilicr R. Bravo R, Burckhardl .1. Curran T. Induction of c-Jos gene ;ind prolein hy growth Taclors precedes activation ofc-m.iT. Nature 1984:312:716-20. 12 McKinney JD. Heintz N. Transcriptionai regulation in the eLikaryotit cell cycle. Trends Biochem Sci 1991; I6:4.'^O 5. \? Pare PD. Wiggs BR. Hogg JC. Bosken C. The comparative mechanics and morphology of airways in asthma and in chronic obstructive pulmonary disease. Am Rev Resp Dis 1991: 14.1:1189-93. 14 James Al.. Hogg JC. Dunn LA. Pare PD. The use of internal perimeter to compare airway size and to calculate smooth muscle shortening. Am Rev Dis 1988; 139;I36-9. 15 Ebina M. Yaegashi H. Takahashi T. Motomiya M. Tanemura M. Distribution of smooth muscles along the bronchial tree Am Rev Resp Dis 1990; 141:1322 6. 16 Nagai A, Inano H. Takizawa T. Morphological changes in airways induced by recurrent exposure of acetylcholine in the guinea pig. Am Rev Resp Dis 1990; 142:172-6. 17 James AL. Pare PD. Hogg JC. The mechanics of airway narrowing in asthma. Am Rev Resp Dis 1989: 139:242-6. 18 GimbroneMA.Cotran RS. Human vascular smooth muscle in culture, growth and ultrastructure. Lab Invest I975;3.V 16-27. 19 Chamlcy-Campbell J. Campbell J. Ross R. The smooth muscle cell in culture. Physiol Rev 1979; 59:1-55, 20 Tom-Moy M. Madison JM. Jones CA. DeLaneroIie P. Brown JK. Morphological characterisation of cultured smooth muscle cells isolated Irom the iracheas of adult dog.s. Anat Rec 1987:218:313-28. 21 Avner BP. DeLongo J. Wilson S. Ladman AJ. A method lor ciilturing canine tracheal smooth muscle cells in ritra: morphological observations. Anat Rec 1981; 200:357-70. 22 Panettieri RA. Yadvish PA. Kelly AM. Rubinstein NA. KotlikolT ML Hislamine stimulates proliferation of airway smooth muscle and induces c-Jos expression. Am J Physiol 199O:259:L365 L37I. 23 Marsh KA. Hill SJ. Bradkinin-induced phosphoinositide hydrolysis and calcium mobilisation in cultured bovine tracheal smooth muscle cells. Bi J Pharmacol 1992; iO5:66P. 24 Chopra LC. Twort CHC. Cameron IR. Ward JPT. Inositol 1.4.5-trisphosphate- and guanosine 5'-O-(3-thiotriphosphate)-induced Ca-' release in cultured airway smooth muscle. Br J Pharmacol 1991: 104:901 6. 25 Hirst SJ. Warley A. Ward JPT. Preparation of cultured rabbit tracheal smooth muscle cells for X-ray microanalysis. Proc 10th Eur Congress on Electron Microscopy. Granada. Spain. September 1992. 26 Twort CMC. van Breenien C Human airway smooth muscle in culture. Tissue and Cell 1988; 20:339 44. 27 Twort CHC. van Breemen C Human airway smooth muscle in culture: control of the intracellular calcium store. Pulm Pharmacol 1989; 2:45-53. 28 Panellieri RA. Murray RK, DePalo LR. Yadvish PA. KotlikolT Ml. A human airway smooth muscle cell line that retains physiological responsiveness. Am J Physio! 1989; 2.56:C329 C335. 29 Hall IP, Townsend P. Da>kin K. Widdop S. Control of
30
31
32 33
34 35
36 37
W
39
40
4!
42
43 44
45
46
47
48
913
tissue cyclic AMP content in primary cultures of human airway smooth muscle cells. Br J Pharmacol 1992; IO5:72P. Halt IP, Widdop S. Desensitisation of isoprenalinc-lnduced ['H]-cyclic AMP formation in primary cultures of human airway smooth muscle cells. Br J Pharmacol 1992: 105:71 P. Panettieri RA. Rubinstein NA. Feuerstein B. KotlikofTML Beta-adrenergic inhibition of airway smooth muscle proliferation. Am Rev Resp Dis 1991; 143:A608. Hogg JC. James A L. Pare PD. Evidence for inflammation in asthma. Am Rev Resp Dis 1991: 143:S39-S42. King RJ, Jones MB. Minoo P. Regulation of lung cell proliferation by polypeptide growth factors. Am J Physiol 1989: 257:L23-L38. Henderson B. Blake S. Therapeutic potential of cytokine manipulation. Trends Pharmacol Sci 1992: 13:145-51. Horn BR. Robin ED. Theodore J. Van Kessel A. Total eosinophii counts in the management of asthma. New Eng J Med 1975:292:1152-5, Taylor KJ, Luksza AR. Peripheral blood eosinophii counts and bronchial hyperresponsiveness- Thorax 1987; 42:452-6. Owens GK. Control of hypertrophic versus hyperplastic growth of vascular smooth muscle cells. Am J Physiol 1989; 257:HI755 HI765, Paneitieri RA. Rubinstein NA. Kelly AM. Kothkoff MI. Histamine induces proliferation of airway smooth muscle in culture. Am Rev Resp Dis 1989: I39:A78. Panettieri RA. Rubmstein NA. Kelly AM. KotlikotT ML The specificity of C"/('),v expression in the induction of airway smooth muscle proliferation by contractile agonists. Am Rev Resp Dis 1991; I43:A934. Oliver MH. Harrison NK. Bishop JE. Cole PJ, Laurent GJ. A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. J Cell Sci 1989; 92:513-18. Hirst SJ. Twort CHC. Barnes PJ. Quantifying the proliferative response in cultured human and rabbit airway smooth muscle. Am Rev Resp Dis 1992: I45:A14. Kikkawa U. Kishimolo A. Nishi?:uka Y. The protein kinase C family: its heterogeneity and its implications. Ann Rev Biochem 1989; 58:31 44. Bell RB. Burns DJ, Lipid activation of protein kinase C. J BiolChem 1991; 266:4661-4. Dicker P. Rozengurt E. S;imutaiion of DNA synthesis by tumour promoter and pure mitogenic factors. Nature 1978; 276:723-6. Dicker P. Ro/engurt E. Phorbol esters and vasopressin stimulate DNA synthesis by a common mechanism. Nature 1980: 287:607-12, Rozengurt E. Rodrigue/-Pena A. Coombs M. Sinelt-Smith J. Diacylglycerol stimulates DNA synthesis and cell division in mouse 3T3 cells: role of Ca'*-sensitive, phospholipiddependent protein kinase, Bitichem Biophys Res Common 1984: 120:1053-9. Rozengurt E, Neuropeptides as cellular growth factors: role of multiple signalling pathways. Eur J Clin Invest 1991; 21:123-34. Mitsuka M. Berk \K\ Long-term regu!ati{Hi of Na'-H *
914
49
50
51
52
53
54
55
56
57 58
59
60
61
62
S. J. Hirsi ami C. H. C. Twort
exchange in vascular snioolh muscle cells: role of protein kinase C. Am J Physiol 1^91; 26():C562 C569. Ohmi K. Yamashila S, Nonomura Y. Effect of K252a. a protein kinase inliihiior. on ihc prolifcralion of vascular smooth muscle cells iiiochcm Biophys RcsCommiin 1490: 173:976-81. Geisterfer A. Peach MJ. Owens GK. Angiolcnsin II induces hypertrophy, not hyperplasia of cultured rat aortic smooth muscle cells, Circ Res 1989; 62:749-56. Gcisterfcr A. Owens GK. Arginine vasopressin induced hypertrophy of cultured rat aortic smooth muscle cells. Hypertension I9S9: 14;4I3 20. Berk BC . Aronov MS. Brock TA.Cragoe E.GImbrone MA. Alexander RW. Angiotensin ll-stimu!ated N a ' , H * exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dcpendent and -indepcndeni pathways. J Biol Chem I9SS: 262:5057-64. Griendling KK. Berk BC. CJanz P. Gimbrone MA. Alexander RW. Angiotensin II stimulation of vaseular smooth muscle phosphoinositidc melabolism. Hypertension 19X7; 9:(Suppl 3):ISI .y Taubam MB. Berk BC. l/umo S. Tsuda T. Alexander RW. Nudal-Ginard B. Angiotensin II induces c-fo.s mRNA i[i aortic smooth muscle. Role of Ca-'^ mobilization. J Biol Chem 1989:264:526-30. Berk BC. Vekshtein V. Gordon HM, Suda T. Angiotensin Il-stimulated prolein synthesis in cultured va.scular smooth muscle cells. Hypertension 1989: 13:305-14. Chilvers ER. Chopra l.C. Barnes J, Twort CHC. Brndykinin-stimulated inosito! phosphate accumulation and demonstration of inositol penla- and hexakisphosphates in cultured airway smooth muscle cells. Am Rev Resp Dis 1989: l39(.Suppl4):A77. Ullrich A, Schlcssinger J. Signal iransduction by receptors with tyrosine kinase activity. Cell 1990: 61:203-12. Levitzki A. Tyrphoslins potential antiproliferative agents and novel molecular tools. Biochem Pharmacol 1990; 40:913-18. Kawahara Y. Kariya K. Araki S. Fuku/aki H, Takai Y. Platelet-derived growth factor (PDGF)-induced phospholipase C-mediated hydrolysis of phosphonositides in vascular smooth muscle cells dilTerential sensilivily of POG!-- and angiotensin ll-induced pht)SpholipaseC reactions to protein kinase C activating phorbol esters. Biochem Biophys Res Commun 1988; 165:846 54. Araki S. Kawahara Y. Kariya K. Sunako M. Tsuda T. Kukuzaki H. Yoshimi T. Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin [l in rabbit vascular smooth muscle cells. Bioclicm Biophys Res Commun 1990; UiS:35(l 7, Nishibe S. Wahl Ml. Rhee SG. Carpenter Ci. Tyrosine phosphorylation of phosphnlipaseC-l I in lilni by epidermal growth factor receptor. J Biol Chem 1989; 264:10.3.35 8. Meisenheider J. Suh P-G. Rhee SG. Hunter T. Phospholipase C-y is a subslratc for the PDGF and EGF receptor protein tyrosine kinases in rim and in ritm. Cell 1989; 57:1109-22.
63 Wahl MI. Olashaw NE, Nishibe S. Rhee SG. Pledger WJ, Carpenter G. Plalclet-derived growth factor induces rapid and sustained tyrosine phosphorylation ofphospholipaseCV in quiescent BAl.B/c 3T3 cells. Mol Cell Biol 1989:9:293443. 64 Cantley L C Auger KR. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Oncogenes and signal transduetion. Cell 1991; 64:281-302. 65 Escobedo. JA. Williams LT. A PDGF receptor domain essential furmitogenesis bul nol for many other responses to PDGF. Nature 1988; 335:85 7. 66 Druker BJ. Mammon H. Roberts TJ. Oncogenes. growth factors, and signal transduclion, N Eng J Med 1990; 321:1383-9!. 67 Kazlauskas A. Ellis C. Pawson T. Cooper JA. Binding of GAP to activated PDGF receptors. Science 1990; 247; 157880. 68 Biilah MM. Anthes JC. Mullmann TJ. Receptor-coupled ph
