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Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma
Tissue & Cell, 2000 32 (5) 399–404 © 2000 Harcourt Publishers Ltd doi: 10.1054/tice.2000.0128, available online at http://www.idealibrary.com
Tissue&Cell
Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma A. Bajo1, I. Carrero2, R.L. Hrïstov1, P. Valenzuela3, P. Martínez4, J. Cortés3, J. C. Prieto1, L. G. Guijarro1
Abstract. The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of αs , αi1/2, αi3 and αi0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in αi3 and αio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues. © 2000 Harcourt Publishers Ltd
Keywords. Guanine nucleotide-binding regulatory protein, G-protein, adenylate cyclase, signal transduction, leiomyoma
Introduction Uterine leiomyoma is the most common benign neoplasia in women, one of the most frequent causes of infertility in
1 Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, 2Departamento de Bioquímica y Biología Molecular, Universidad de Valladolid, Soria, 3Servicio de Obstetricia y Ginecología, Hospital Príncipe de Asturias, Alcalá de Henares, 4Servicio de Anatomía Patológica, Hospital Príncipe de Asturias, Alcalá de Henares, Spain
Received 10 March 2000 Accepted 21 July 2000 Correspondence to: Juan C. Prieto, Unidad de Neuroendocrinología Molecular, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain. Tel.: +34 918854527; Fax: +34 918854585; E-mail: [email protected]
reproductive years, and the leading cause for hysterectomy. Most of the available information about the growth of leiomyomas in vivo points to the central role of estrogen and progestin (Koutsilieris, 1992). However, the mechanism underlying the growth of the tumour remains unknown. The patients with uterine leiomyomas have not increased circulating progesterone or estradiol (E2) levels (Brandon et al., 1993). This suggests that tumour growth results from increased end-organ sensitivity to these steroid hormones. In fact, it has been described recently the overexpression of functional estradiol receptors in human uterine leiomyomata that results in an increased sensitivity to the hormone (Brandon et al., 1995). The mediators of estradiol in the regulation of tumour growth are local factors such as insulin-like growth factor-I (IGF-I), epidermal growth 399
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factor (EGF), or platelet-derived growth factor (PDGF) (Tommola et al., 1989; Fayed et al., 1989; Sourla & Koutsilieris, 1995) that stimulate DNA synthesis in human myometrial cells in culture (Rossi et al., 1992) by a mitogen-activating protein kinase (MAP-kinase) dependent mechanism (Ohmichi et al., 1995). However, some reports suggest that cAMP plays also a role in the growth of uterine cells. Human endometrial fibroblast proliferation is inhibited by cAMP (Matsunami et al., 1991), and analogues of this cyclic nucleotide or effectors of the adenylate cyclase system (such as forskolin or ritodrine) inhibit the MAP-kinase cascade in human myometrial cells (Ohmichi et al., 1995; Dodge & Sanborn, 1998) by a protein kinase A dependent mechanism. The cross-talk between both regulatory pathways could have important consequences in the control of uterine leiomyomas. However, the status of the adenylate cyclase system in this very frequent pathology has not been described at present. In this report we studied the general characteristics of adenylate cyclase activity in basal conditions as well as by stimulation with forskolin, guanosine 5′-[βγ-imido] triphosphate (Gpp[NH]p) and pituitary adenylate cyclaseactivating polypeptide (PACAP-27) in human leiomyoma and adjacent myometrium. We also determined the levels of α subunits (αs, αi1/2, αi3 and αi0) of guanyl nucleotidebinding regulatory proteins (G-proteins) in both tissues.
Materials and methods Reagents The antisera specific for G α-protein subunits were supplied by Du Point New England Nuclear (Boston, MA, USA). 125 I-labelled donkey antibody to rabbit IgG was from Amersham (UK). All other reagents were from Sigma (Alcobendas-Madrid, Spain) and had the highest purity available. Preparation and source of membranes Menstruating women (37–47 years old) underwent total hysterectomy for leiomyomas. Informed consent was obtained from each subject and from the Review Board of the University Hospital Príncipe de Asturias. The endometrium was removed from the myometrium, and the same was done with the leiomyoma. The different tissues were finely minced and homogenized with a Polytron (Kinematica, Luzern, Switzerland) (setting 9, 1 min) in 0.01 M triethanolamine-HCl buffer (pH 7.5) containing 0.25 M sucrose, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF) and 0.1 mg/ml bacitracin. The mixture was filtered through two layers of medical gauze and centrifuged at 1,300 × g for 10 min at 4°C. The pellet was discarded and the supernatant was again centrifuged at 45,000×g for 45 min at 4°C. The final pellet was washed twice in 50 mM Tris-HCI buffer (pH 7.5), 0.1 mM PMSF and 0.1 mg/ml bacitracin, and then immediately frozen at –80°C until use.
Protein concentration was determined according to the method of Bradford (1976) using bovine serum albumin as a standard. Adenylate cyclase assay Adenylate cyclase activity was measured as described previously (Houslay et al., 1976) with some modifications. Membranes (0.05 mg protein/ml) were incubated in 0.1 ml of total volume with an ATP-regenerating system (7.5 mg/ml phosphocreatine and 1 mg/ml creatine phosphokinase) dissolved in 0.25 M triethanolamine-HCI buffer (pH 7.4) in the presence of 1.5 mM ATP, 5 mM MgSO4, 1 mM 3-isobutyl-1-methylxanthine, 1 mM EDTA, 1 mg/ml bacitracin and test substances. The incubation was carried out for 30 min at 30°C and the reaction was stopped by heating the mixture for 3 min. After addition of 0.2 ml of an alumina slurry (0.75 mg/ml in triethanolamine-HCl buffer, pH 7.4) and centrifugation, the supernatant was taken for cAMP measurement (Gilman, 1970). Immunodetection of αs and αi subunits of G-proteins Membrane proteins (30 mg) were solubilized in 20 mM Tris-HCl buffer (pH 8.0) containing 10% (v/v) glycerol, 0.05% (w/v) Lubrol-PX, 1% (w/v) sodium dodecylsulfate (SDS), 1 mM EDTA and 1 mM dithiothreitol (DTT), and boiled for 5 min. N-ethylmaleimide was added to a final concentration of 33 mM and samples were incubated for 15 min at room temperature. Proteins were resolved on a 10% polyacrylamide gel and then transferred to nitrocellulose sheets (Hoeffer, San Francisco, CA, USA). Immunodetection of different a-subunits was carried out with the corresponding antisera: RM/1 (αs), AS/7 (αi1/2), EC/2 (αi3/0) and GC/2 (α0) as described (Mumby et al., 1986). Briefly, the nitrocellulose sheet was sliced and soaked in 50 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.2% (v/v) Nonidet-P40, 0.2% (w/v) NaN3 and 5% (w/v) non-fat dry milk. The different antisera were diluted in the same buffer (1/1,000 dilution) and the incubation took 1 h at room temperature. Immunoreactive proteins were revealed using 125I-labelled donkey antibody against rabbit IgG. Immunoblots were dried and exposed at –80°C after extensive washing. Data were obtained under conditions giving a linear relationship between the amount of protein loaded and the intensity of the radioactive signal from the immunoblots. For each sample, immunoblots were repeated at least three times with similar results. Autoradiograms were analysed using an Ultroscan XL Enhanced Laser Densitometer (Amersham Pharmacia Biotech, Uppsala, Sweden). Data analysis Results were calculated as the means ± S.E.M. of the number of experiments indicated. Statistical analysis was carried out using the nonparametric test of Wilcoxon for paired values with two-tails. The level of significance was set as p<0.05.
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Fig. 1 Comparison of basal levels of adenylate cyclase activity in uterine leiomyoma (hatched bars) and adjacent myometrium (open bars) (n = 12). The enzyme activity was measured as described in Methods.
Results As shown in Fig. 1, basal adenylate cyclase activity decreased in membranes from human leiomyomas as compared with the adjacent myometrium. From 12 patients studied, 67% presented a marked decrease in the basal adenylate cyclase activity in the tumour whereas 33% did not present any changes. When comparing mean values from the 12 patients, a 43% decrease in the enzyme activity was observed in leiomyoma with respect to the adjacent myometrium (Table 1). Forskolin- and Gpp[NH]p-stimulated adenylate cyclase activity were also decreased in the uterine tumour in the same population studied (Table 1). The observed changes seem to correspond to alterations in
Table 1
the enzyme content and not in the affinity for forskolin because the potency of the diterpene did not change in leiomyoma with respect to the myometrium (Table 1 and Fig. 2 left). When adenylate cyclase was stimulated with Gpp[NH]p, a non-hydrolysable guanyl nucleotide analogue that acts primarily through G-proteins, a decrease in efficiency without changes in the potency of the nucleotide was observed in the leiomyoma (Table 1 and Fig. 2, right). The alterations observed could be related with a decreased content of some G-protein subunits. To test this hypothesis, we carried out the Western analysis of αs, αi1+2, αi3 and α0 subunits in the same population (12 patients; a representative patient is shown in Fig. 3). The videodensitometric analysis of the corresponding bands gave the results
Adenylate cyclase activity in human leiomyoma and the adjacent myometrium.
Basal Forskolin (1 mM) Gpp[NH]p (1 mM) PACAP-27 (1 µM) + GTP (1 µM) Forskolin (ED50)a Gpp[NH] (ED50)a
Myometrium
Leiomyoma
Significance
21.9 ± 5.3 233.1 ± 43.0 62.5 ± 8.9 60.0 ± 15.0 14.0 ± 5.7 0.50 ± 0.20
12.5 ± 3.0 167.0 ± 32.6 45.7 ± 6.4 20.4 ± 3.0 19.3 ± 8.1 0.40 ± 0.06
p < 0.01 p < 0.005 p < 0.001 p < 0.05 N.S. N.S.
Basal and stimulated adenylate activity is expressed in pmol cAMP/min/mg protein. aThe ED50 values for forskolin and Gpp[NH]p are expressed in µM. Data correspond to the mean ± S.E.M. for 12 patients. N.S. = not significant.
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Fig. 2 Dose-dependent effect of forskolin and Gpp[NH]p on adenylate cyclase activity in human leiomyoma and the adjacent myometrium. Membranes from human leiomyoma (● ●) or the surrounding myometrium (●) were incubated with increasing doses of forskolin (left) or Gpp[NH]p (right) as described under Methods. Data correspond to the means ± S.E.M. for 12 patients; *P < 0.05, ***P < 0.01.
summarized in Table 2. A significant reduction in αs and αi1+2 was observed in the tumoral tissue without significant changes in αi3 and α0. Present data show a reduction in the adenylate cyclase activity from human leiomyoma with a concomitant decrease in the G-regulatory proteins αs and αi1+2. The observed changes could have consequences on the sensitivity of the tumour to hormones or neurotransmitters. In this context, we observed a reduced stimulatory effect of PACAP-27 (Table 1) on adenylate cyclase from human leiomyoma as compared to that in the adjacent myometrium.
Discussion The present report shows that the adenylate cyclase signal transduction pathway is impaired in human uterine leiomyomas. It was suggested by the study of the enzymatic activity in both basal and stimulated conditions, as well as by Western-blot analysis of α-subunits of G-proteins, that points to a reduction of the cAMP generating system in human uterine leiomyoma as compared to normal myometrium. The changes observed can result in the desensitisation of the tumour to agents such as PACAP-27 (this report) or PTHrP (Weir et al., 1994), two agonists that act through receptors coupled to adenylate cyclase. The importance of present results derives from the fact that cAMP has an inhibitory role upon uterine cell proliferation (Matsunami et al., 1991) related to an inhibitory cross talk between the protein kinase A (PKA) activation and the MAP-kinase pathway (Ohmichi et al al., 1995; Dodge &
Table 2 Distribution of Ga proteins in human leiomyoma and the adjacent myometrium Antiserum
Myometrium
Leiomyoma
Significance
RM/1 (αs) AS/7 (αi1/2) EC/2 (αi3/0) GC/2 (α0)
8.2 ± 1.7 5.3 ± 1.4 4.8 ± 1.3 2.0 ± 0.3
5.8 ± 1.2 2.9 ± 0.6 3.2 ± 0.2 1.9 ± 0.5
P < 0.05 P < 0.05 N.S. N.S.
Ga proteins were immunodetected in human leiomyoma and the adjacent myometrial tissue as shown in Fig. 3. Thereafter, a photodensitometric quantitation of the autoradiograms was performed. Data are expressed in arbitrary units and correspond to the means ± S.E.M. for 12 patients.
Sandborn, 1998). Many data exist on the connexion between G-protein coupled receptors to MAP-kinase cascades which, in turn, control gene expression by phosphorylating nuclear regulatory molecules (Murga et al., 1999). The interest has been frequently focused on the mitogenic potential of growth factors involving the extracellular signal-regulated kinase (ERK) cascade and the interaction with the cAMP-raising PKA activator forskolin, which mostly attenuates MAP-kinase action (Sevetson et al., 1993). Interestingly, the involvement of the MAPkinase cascade in the proliferation of human leiomyoma cells and the stimulatory role of prolactin in this event have been shown (Nohara et al., 1997). These features together present observations deserve further investigation on the expression of different components of MAP-kinase signalling cascades in human uterine leiomyoma which may be also extended to protein kinase C (PKC) isoforms that have been shown to play distinct roles in the myometrial growth process (Tertrin-Clary et al., 1999).
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Fig. 3 Immunodetection of Gα proteins in human leiomyoma (L) and the adjacent myometrium (M). The autoradiograms correspond to a patient from 12 analysed.
Cyclic AMP analogues, forskolin or ritodrine (a β-adrenergic agonist used in tocolytic therapy) attenuated oxytocininduced MAP kinase activity and phosphorylation in myometrial cells (Ohmichi et al., 1995). In rats, the chronic treatment with ritodrine (or other β-adrenergic agonists such as salbutamol or terbutaline) significantly increases the development of uterine leiomyomas (Colbert et al., 1991), which apparently disagrees with present results. However, we must keep in mind that all these drugs desensitise the β-receptors coupled to the adenylate cyclase system, and even their own enzyme, when they are administered chronically (Lèvicrain et al., 1998), producing the same effect observed in human uterine leiomyomas. The mechanism of the observed impairment of the adenylate cyclase/G-protein system is unknown at present. It is unquestionable that estradiol and progesterone affect the growth, development and evolution of uterine leiomyomas (Sibarsku, 1978; Biro, 1986), modulating the expression of growth factors and their receptors (Sourla & Koutsilieris, 1995). Present results suggest that they also modulate the cAMP producing system. Interestingly, it has been previously observed that estrogen reduces β-adrenoceptor-mediated cAMP production and Gs concentration in rabbit myometrium (Riemer et al., 1988). The present observation of low levels of both αs and αi1+2 (but not αi3 and α0) G-protein subunits in leiomyoma reflects a differentially abnormal expression in this pathophysiological state. Numerous disorders have been previously associated with altered G-protein expression, i.e. low αi1+2 and αi3 but normal αs and αo level have been detected in human pituitary adenomas (Ballaré et al., 1997); however, disorders frequently associated with pituitary tumours, such as hypothyroidism, hypoadrenalism and hypogonadism, may cause a reduction in αs and an increase in αi expression (Spiegel et al., 1992). Several mechanisms,
including either reduced gene expression or instability of mRNAs or their corresponding proteins may be responsible for the low expression of αs and αi1+2 in leiomyoma. This aspect deserves further experiments but, whatever the mechanism responsible, the pattern of G protein expressed results (conceivably by αs decrease) in reduced production of cAMP, which in human uterus represents an antiproliferative signal (Matsunami et al., 1991; Nilsson et al., 1998). In conclusion, present findings suggest that the decrease in the cyclic AMP producing system in human uterine leiomyomas as compared with adjacent myometrium is a factor that presumably permits the activation of MAPkinase by growth factors necessary in the development of the tumour. ACKNOWLEDGEMENTS This research was supported by grants from the Dirección General de Investigación Científica y Técnica (PB93/0492, PB94/0360, PM97/0069). REFERENCES Ballaré, E., Mantovani, S., Basetti, M., Lania, A. and Spada, A. 1997. Immunodetection of G proteins in human pituitary adenomas: evidence for a low expression of proteins of the Gi subfamily. Eur. J. Endocrinol., 137, 482–489. Biro, J.C. 1986. Estrogen-induced proteins: a new class of regulatory substances. Med. Hyphoteses, 19, 199–228. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254. Brandon, D.D., Bethea, C.L., Strawn, E.Y., Novy, M.J., Burry, K.A., Harrington, M.S., Erickson, T.E., Warner, C., Keenan, E.J. and Clinton, G.M. 1993. Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas. Am. J. Obstet. Gynecol., 169, 78–85. Brandon, D.D., Erickson, T.E., Keenan, E.J., Strawn, E.Y., Novy, M.J., Burry, K.A., Warner, C. and Clinton, G.M. 1995. Estrogen receptor
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gene expression in human uterine leiomyomata. J. Clin. Endocrinol. Metab., 80, 1876–1881. Colbert, W.E., Wilson, B.F., Williams, P.D. and Williams, G.D. 1991. Relationship between in vitro relaxation of the costo-uterine smooth muscle and mesovarial leiomyoma formation in vivo by β-receptor agonists. Arch. Toxicol., 7, 575–579. Dodge, K.L. and Sanborn, B.M. 1998. Evidence for inhibition by protein kinase A of receptor/Gaq/Phospholipase C (PLC) coupling by a mechanism not involving PLCa2. Endocrinology, 139, 2265–2271. Fayed, Y.M., Tsibris, G.C.M., Langenberg, P.W. and Robertson, A.L. 1989. Human uterine leiomyoma cells: binding and growth responses to epidermal growth factor, platelet-derived growth factor and insulin. Lab. Invest., 60, 30–37. Gilman, A.G. 1970. A protein binding assay for adenosine 3′,5′-cyclic monophosphate. Proc. Natl. Acad. Sci. USA, 67, 305–312. Houslay, M.D., Metcalfe, J.C., Warren, G.B., Hesketh, T.P. and Smith, G.A. 1976. The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it. Biochim. Biophys. Acta, 436, 489–494. Koutsilieris, M. 1992. Pathophysiology of uterine leiomyomas. Biochem. Cell. Biol., 70, 273–278. Lèvicrain J.L., Cohen-Tannoudji JC, Robin MT, Coudouel N, Legrand C, Maltier JP. Molecular mechanisms of adenylyl cyclase desensitization in pregnant rat myometrium following in vivo administration of the β-adrenergic agonist, isoproterenol. 1998. Biol. Reprod., 59, 45–52. Matsunami, K., Imai, A., Ohno, T. and Tamaya, T. 1991. Suppression of growth-promoting activity in extract from human uterine cancer by cyclic AMP-mediated mechanism. Res. Commun. Chem. Pathol. Pharmacol., 73, 371–374. Mumby, S.M., Kahn, R.A., Manning, D.R. and Gilman, A.G. 1986. Antisera of designed specificity for subunits of guanine nucleotidebinding regulatory proteins. Proc. Natl. Acad. Sci. USA, 83, 265–269. Murga, C., Fukuhara, S. and Gutkind, J.S. 1999. Novel molecular mechanisms in the pathway connecting G-protein-coupled receptors to MAP kinases cascades. Trends Endocrinol. Metab., 10, 122–127. Nilsson, U.K., Grenegard, M., Berg, G. and Svensson, S.P.S. 1998. Different proliferative responses of Gi/o-protein-coupled receptors in human myometrial smooth muscle cells. J. Mol. Neurosci., 11, 11–21.
Nohara, A., Ohmichi, M., Koike, K., Jikihara, H., Kimura, A., Masuhara, K., Ikegami, H., Inoue, M., Miyake, A. and Murata, Y. 1997. Prolactin stimulates mitogen-activated protein kinase in human leiomyoma cells. Biochem. Biophys. Res. Commun., 238, 473–477. Ohmichi, M., Koike, K., Nohara, A., Kanda, Y., Sakamoto, Y., Zhang, Z.X., Hirota, K. and Miyaka, A. 1995. Oxytocin stimulates mitogenactivated protein kinase activity in cultured human puerperal uterine myometrial cells. Endocrinology, 136, 2082–2087. Riemer, R.K., Wu, Y.Y., Bottari, S.P., Jacobs, M.M., Goldfien, A., Roberts, J.M. 1988. Estrogen reduces β-adrenoceptor-mediated cAMP production and the concentration of the guanyl nucleotide-regulatory protein, Gs, in rabbit myometrium. Mol. Pharmacol., 4, 389–395. Rossi, M.J., Chegini, N. and Masterson, B.J. 1992. Presence of epidermal growth factor platelet-derived growth factor, and their receptors in human myometrial tissue and smooth muscle cells: their action in smooth muscle cells in vitro. Endocrinology, 130, 1716–1727. Sevetson, B.R., Kong, X. and Lawrence, J.C.Jr. 1993. Increasing cAMP attenuates activation of mitogen-activated protein kinase. Proc. Natl. Acad. Sci. USA, 90, 10305–10309. Sibarsku, D.A. 1978. Estrogen induction of growth factor specific for hormone-responsive mammary, pituitary, and kidney tumor cells. Proc. Natl. Acad. Sci. USA, 75, 3786–3790. Sourla, A. and Koutsilieris, M. 1995. Purification and partial sequencing of the major mitogen for human uterine smooth muscle-like cells in leiomyoma extracts. J. Clin. Invest., 96, 751–758. Spiegel, A.M., Shenker, A. and Weinstein, L.S. 1992. Receptor-effector coupling by G proteins: implications for normal and abnormal signal transduction. Endor. Rev., 13, 536–565. Tertrin-Clary, C., Eude, I., Fournier, T., Paris, B., Breuiller-Fouché, M. and Ferré, F. 1999. Contribution of protein kinase C to ET-1-induced proliferation in human myometrial cells. Am. J. Physiol., 276, E503–E511. Tommola, P., Pekonen, F. and Rutanen, E.M. 1989. Binding of epidermal growth factor and insulin-like growth factor I in human myometrium and leiomyoma. Obstet. Gynecol. 74, 658–662. Weir, E.C., Goad, D.L., Daifotis, A.G., Burtis, W.J., Dreyer, B.E. and Nowak, R.A. 1994. Relative overexpression of the parathyroid hormone-related protein gene in human leiomyomas. J. Clin. Endocrinol. Metab., 78, 784–789.
Tissue&Cell
Impairment of adenylate cyclase activity and G-proteins in human uterine leiomyoma A. Bajo1, I. Carrero2, R.L. Hrïstov1, P. Valenzuela3, P. Martínez4, J. Cortés3, J. C. Prieto1, L. G. Guijarro1
Abstract. The mechanisms responsible for the growth of uterine leiomyoma (a frequent cause of infertility in women) are largely unknown. Some data supports that cAMP plays a role in the growth of uterine cells but there are no reports on the status of the cAMP producing system in this human benign neoplasia. In this study, biopsies from leiomyoma and the adjacent myometrium were taken from menstruating women subjected to total hysterectomy for leiomyoma. Adenylate cyclase activity was determined by a protein-binding method, and the expression of αs , αi1/2, αi3 and αi0) G-protein subunits was analysed by immunoblot. The leiomyoma samples exhibited a decreased expression of as and ai1/2 with respect to the adjacent myometrial tissue. No differences were observed in αi3 and αio protein expression. The basal adenylate cyclase activity as well as the efficacy (as assessed by the maximal stimulation levels) of either forskolin or, to a lesser extent, Gpp[NH]p on stimulation the enzyme activity was significantly lower in leiomyoma than in myometrium, whereas the potency (as assessed by the ED50 values) of these two agents did not vary. Present data indicate that the human leiomyoma is associated with low levels of cAMP. It is conceivable that the loss of sensitivity of adenylate cyclase to endogenous regulatory molecules could be related to the pathogenesis of human leiomyomas given that cAMP inhibits the MAP-kinase cascade in uterine tissues. © 2000 Harcourt Publishers Ltd
Keywords. Guanine nucleotide-binding regulatory protein, G-protein, adenylate cyclase, signal transduction, leiomyoma
Introduction Uterine leiomyoma is the most common benign neoplasia in women, one of the most frequent causes of infertility in
1 Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, 2Departamento de Bioquímica y Biología Molecular, Universidad de Valladolid, Soria, 3Servicio de Obstetricia y Ginecología, Hospital Príncipe de Asturias, Alcalá de Henares, 4Servicio de Anatomía Patológica, Hospital Príncipe de Asturias, Alcalá de Henares, Spain
Received 10 March 2000 Accepted 21 July 2000 Correspondence to: Juan C. Prieto, Unidad de Neuroendocrinología Molecular, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, E-28871 Alcalá de Henares, Spain. Tel.: +34 918854527; Fax: +34 918854585; E-mail: [email protected]
reproductive years, and the leading cause for hysterectomy. Most of the available information about the growth of leiomyomas in vivo points to the central role of estrogen and progestin (Koutsilieris, 1992). However, the mechanism underlying the growth of the tumour remains unknown. The patients with uterine leiomyomas have not increased circulating progesterone or estradiol (E2) levels (Brandon et al., 1993). This suggests that tumour growth results from increased end-organ sensitivity to these steroid hormones. In fact, it has been described recently the overexpression of functional estradiol receptors in human uterine leiomyomata that results in an increased sensitivity to the hormone (Brandon et al., 1995). The mediators of estradiol in the regulation of tumour growth are local factors such as insulin-like growth factor-I (IGF-I), epidermal growth 399
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factor (EGF), or platelet-derived growth factor (PDGF) (Tommola et al., 1989; Fayed et al., 1989; Sourla & Koutsilieris, 1995) that stimulate DNA synthesis in human myometrial cells in culture (Rossi et al., 1992) by a mitogen-activating protein kinase (MAP-kinase) dependent mechanism (Ohmichi et al., 1995). However, some reports suggest that cAMP plays also a role in the growth of uterine cells. Human endometrial fibroblast proliferation is inhibited by cAMP (Matsunami et al., 1991), and analogues of this cyclic nucleotide or effectors of the adenylate cyclase system (such as forskolin or ritodrine) inhibit the MAP-kinase cascade in human myometrial cells (Ohmichi et al., 1995; Dodge & Sanborn, 1998) by a protein kinase A dependent mechanism. The cross-talk between both regulatory pathways could have important consequences in the control of uterine leiomyomas. However, the status of the adenylate cyclase system in this very frequent pathology has not been described at present. In this report we studied the general characteristics of adenylate cyclase activity in basal conditions as well as by stimulation with forskolin, guanosine 5′-[βγ-imido] triphosphate (Gpp[NH]p) and pituitary adenylate cyclaseactivating polypeptide (PACAP-27) in human leiomyoma and adjacent myometrium. We also determined the levels of α subunits (αs, αi1/2, αi3 and αi0) of guanyl nucleotidebinding regulatory proteins (G-proteins) in both tissues.
Materials and methods Reagents The antisera specific for G α-protein subunits were supplied by Du Point New England Nuclear (Boston, MA, USA). 125 I-labelled donkey antibody to rabbit IgG was from Amersham (UK). All other reagents were from Sigma (Alcobendas-Madrid, Spain) and had the highest purity available. Preparation and source of membranes Menstruating women (37–47 years old) underwent total hysterectomy for leiomyomas. Informed consent was obtained from each subject and from the Review Board of the University Hospital Príncipe de Asturias. The endometrium was removed from the myometrium, and the same was done with the leiomyoma. The different tissues were finely minced and homogenized with a Polytron (Kinematica, Luzern, Switzerland) (setting 9, 1 min) in 0.01 M triethanolamine-HCl buffer (pH 7.5) containing 0.25 M sucrose, 0.5 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride (PMSF) and 0.1 mg/ml bacitracin. The mixture was filtered through two layers of medical gauze and centrifuged at 1,300 × g for 10 min at 4°C. The pellet was discarded and the supernatant was again centrifuged at 45,000×g for 45 min at 4°C. The final pellet was washed twice in 50 mM Tris-HCI buffer (pH 7.5), 0.1 mM PMSF and 0.1 mg/ml bacitracin, and then immediately frozen at –80°C until use.
Protein concentration was determined according to the method of Bradford (1976) using bovine serum albumin as a standard. Adenylate cyclase assay Adenylate cyclase activity was measured as described previously (Houslay et al., 1976) with some modifications. Membranes (0.05 mg protein/ml) were incubated in 0.1 ml of total volume with an ATP-regenerating system (7.5 mg/ml phosphocreatine and 1 mg/ml creatine phosphokinase) dissolved in 0.25 M triethanolamine-HCI buffer (pH 7.4) in the presence of 1.5 mM ATP, 5 mM MgSO4, 1 mM 3-isobutyl-1-methylxanthine, 1 mM EDTA, 1 mg/ml bacitracin and test substances. The incubation was carried out for 30 min at 30°C and the reaction was stopped by heating the mixture for 3 min. After addition of 0.2 ml of an alumina slurry (0.75 mg/ml in triethanolamine-HCl buffer, pH 7.4) and centrifugation, the supernatant was taken for cAMP measurement (Gilman, 1970). Immunodetection of αs and αi subunits of G-proteins Membrane proteins (30 mg) were solubilized in 20 mM Tris-HCl buffer (pH 8.0) containing 10% (v/v) glycerol, 0.05% (w/v) Lubrol-PX, 1% (w/v) sodium dodecylsulfate (SDS), 1 mM EDTA and 1 mM dithiothreitol (DTT), and boiled for 5 min. N-ethylmaleimide was added to a final concentration of 33 mM and samples were incubated for 15 min at room temperature. Proteins were resolved on a 10% polyacrylamide gel and then transferred to nitrocellulose sheets (Hoeffer, San Francisco, CA, USA). Immunodetection of different a-subunits was carried out with the corresponding antisera: RM/1 (αs), AS/7 (αi1/2), EC/2 (αi3/0) and GC/2 (α0) as described (Mumby et al., 1986). Briefly, the nitrocellulose sheet was sliced and soaked in 50 mM Tris-HCl, pH 8.0, 2 mM CaCl2, 80 mM NaCl, 0.2% (v/v) Nonidet-P40, 0.2% (w/v) NaN3 and 5% (w/v) non-fat dry milk. The different antisera were diluted in the same buffer (1/1,000 dilution) and the incubation took 1 h at room temperature. Immunoreactive proteins were revealed using 125I-labelled donkey antibody against rabbit IgG. Immunoblots were dried and exposed at –80°C after extensive washing. Data were obtained under conditions giving a linear relationship between the amount of protein loaded and the intensity of the radioactive signal from the immunoblots. For each sample, immunoblots were repeated at least three times with similar results. Autoradiograms were analysed using an Ultroscan XL Enhanced Laser Densitometer (Amersham Pharmacia Biotech, Uppsala, Sweden). Data analysis Results were calculated as the means ± S.E.M. of the number of experiments indicated. Statistical analysis was carried out using the nonparametric test of Wilcoxon for paired values with two-tails. The level of significance was set as p<0.05.
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Fig. 1 Comparison of basal levels of adenylate cyclase activity in uterine leiomyoma (hatched bars) and adjacent myometrium (open bars) (n = 12). The enzyme activity was measured as described in Methods.
Results As shown in Fig. 1, basal adenylate cyclase activity decreased in membranes from human leiomyomas as compared with the adjacent myometrium. From 12 patients studied, 67% presented a marked decrease in the basal adenylate cyclase activity in the tumour whereas 33% did not present any changes. When comparing mean values from the 12 patients, a 43% decrease in the enzyme activity was observed in leiomyoma with respect to the adjacent myometrium (Table 1). Forskolin- and Gpp[NH]p-stimulated adenylate cyclase activity were also decreased in the uterine tumour in the same population studied (Table 1). The observed changes seem to correspond to alterations in
Table 1
the enzyme content and not in the affinity for forskolin because the potency of the diterpene did not change in leiomyoma with respect to the myometrium (Table 1 and Fig. 2 left). When adenylate cyclase was stimulated with Gpp[NH]p, a non-hydrolysable guanyl nucleotide analogue that acts primarily through G-proteins, a decrease in efficiency without changes in the potency of the nucleotide was observed in the leiomyoma (Table 1 and Fig. 2, right). The alterations observed could be related with a decreased content of some G-protein subunits. To test this hypothesis, we carried out the Western analysis of αs, αi1+2, αi3 and α0 subunits in the same population (12 patients; a representative patient is shown in Fig. 3). The videodensitometric analysis of the corresponding bands gave the results
Adenylate cyclase activity in human leiomyoma and the adjacent myometrium.
Basal Forskolin (1 mM) Gpp[NH]p (1 mM) PACAP-27 (1 µM) + GTP (1 µM) Forskolin (ED50)a Gpp[NH] (ED50)a
Myometrium
Leiomyoma
Significance
21.9 ± 5.3 233.1 ± 43.0 62.5 ± 8.9 60.0 ± 15.0 14.0 ± 5.7 0.50 ± 0.20
12.5 ± 3.0 167.0 ± 32.6 45.7 ± 6.4 20.4 ± 3.0 19.3 ± 8.1 0.40 ± 0.06
p < 0.01 p < 0.005 p < 0.001 p < 0.05 N.S. N.S.
Basal and stimulated adenylate activity is expressed in pmol cAMP/min/mg protein. aThe ED50 values for forskolin and Gpp[NH]p are expressed in µM. Data correspond to the mean ± S.E.M. for 12 patients. N.S. = not significant.
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Fig. 2 Dose-dependent effect of forskolin and Gpp[NH]p on adenylate cyclase activity in human leiomyoma and the adjacent myometrium. Membranes from human leiomyoma (● ●) or the surrounding myometrium (●) were incubated with increasing doses of forskolin (left) or Gpp[NH]p (right) as described under Methods. Data correspond to the means ± S.E.M. for 12 patients; *P < 0.05, ***P < 0.01.
summarized in Table 2. A significant reduction in αs and αi1+2 was observed in the tumoral tissue without significant changes in αi3 and α0. Present data show a reduction in the adenylate cyclase activity from human leiomyoma with a concomitant decrease in the G-regulatory proteins αs and αi1+2. The observed changes could have consequences on the sensitivity of the tumour to hormones or neurotransmitters. In this context, we observed a reduced stimulatory effect of PACAP-27 (Table 1) on adenylate cyclase from human leiomyoma as compared to that in the adjacent myometrium.
Discussion The present report shows that the adenylate cyclase signal transduction pathway is impaired in human uterine leiomyomas. It was suggested by the study of the enzymatic activity in both basal and stimulated conditions, as well as by Western-blot analysis of α-subunits of G-proteins, that points to a reduction of the cAMP generating system in human uterine leiomyoma as compared to normal myometrium. The changes observed can result in the desensitisation of the tumour to agents such as PACAP-27 (this report) or PTHrP (Weir et al., 1994), two agonists that act through receptors coupled to adenylate cyclase. The importance of present results derives from the fact that cAMP has an inhibitory role upon uterine cell proliferation (Matsunami et al., 1991) related to an inhibitory cross talk between the protein kinase A (PKA) activation and the MAP-kinase pathway (Ohmichi et al al., 1995; Dodge &
Table 2 Distribution of Ga proteins in human leiomyoma and the adjacent myometrium Antiserum
Myometrium
Leiomyoma
Significance
RM/1 (αs) AS/7 (αi1/2) EC/2 (αi3/0) GC/2 (α0)
8.2 ± 1.7 5.3 ± 1.4 4.8 ± 1.3 2.0 ± 0.3
5.8 ± 1.2 2.9 ± 0.6 3.2 ± 0.2 1.9 ± 0.5
P < 0.05 P < 0.05 N.S. N.S.
Ga proteins were immunodetected in human leiomyoma and the adjacent myometrial tissue as shown in Fig. 3. Thereafter, a photodensitometric quantitation of the autoradiograms was performed. Data are expressed in arbitrary units and correspond to the means ± S.E.M. for 12 patients.
Sandborn, 1998). Many data exist on the connexion between G-protein coupled receptors to MAP-kinase cascades which, in turn, control gene expression by phosphorylating nuclear regulatory molecules (Murga et al., 1999). The interest has been frequently focused on the mitogenic potential of growth factors involving the extracellular signal-regulated kinase (ERK) cascade and the interaction with the cAMP-raising PKA activator forskolin, which mostly attenuates MAP-kinase action (Sevetson et al., 1993). Interestingly, the involvement of the MAPkinase cascade in the proliferation of human leiomyoma cells and the stimulatory role of prolactin in this event have been shown (Nohara et al., 1997). These features together present observations deserve further investigation on the expression of different components of MAP-kinase signalling cascades in human uterine leiomyoma which may be also extended to protein kinase C (PKC) isoforms that have been shown to play distinct roles in the myometrial growth process (Tertrin-Clary et al., 1999).
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Fig. 3 Immunodetection of Gα proteins in human leiomyoma (L) and the adjacent myometrium (M). The autoradiograms correspond to a patient from 12 analysed.
Cyclic AMP analogues, forskolin or ritodrine (a β-adrenergic agonist used in tocolytic therapy) attenuated oxytocininduced MAP kinase activity and phosphorylation in myometrial cells (Ohmichi et al., 1995). In rats, the chronic treatment with ritodrine (or other β-adrenergic agonists such as salbutamol or terbutaline) significantly increases the development of uterine leiomyomas (Colbert et al., 1991), which apparently disagrees with present results. However, we must keep in mind that all these drugs desensitise the β-receptors coupled to the adenylate cyclase system, and even their own enzyme, when they are administered chronically (Lèvicrain et al., 1998), producing the same effect observed in human uterine leiomyomas. The mechanism of the observed impairment of the adenylate cyclase/G-protein system is unknown at present. It is unquestionable that estradiol and progesterone affect the growth, development and evolution of uterine leiomyomas (Sibarsku, 1978; Biro, 1986), modulating the expression of growth factors and their receptors (Sourla & Koutsilieris, 1995). Present results suggest that they also modulate the cAMP producing system. Interestingly, it has been previously observed that estrogen reduces β-adrenoceptor-mediated cAMP production and Gs concentration in rabbit myometrium (Riemer et al., 1988). The present observation of low levels of both αs and αi1+2 (but not αi3 and α0) G-protein subunits in leiomyoma reflects a differentially abnormal expression in this pathophysiological state. Numerous disorders have been previously associated with altered G-protein expression, i.e. low αi1+2 and αi3 but normal αs and αo level have been detected in human pituitary adenomas (Ballaré et al., 1997); however, disorders frequently associated with pituitary tumours, such as hypothyroidism, hypoadrenalism and hypogonadism, may cause a reduction in αs and an increase in αi expression (Spiegel et al., 1992). Several mechanisms,
including either reduced gene expression or instability of mRNAs or their corresponding proteins may be responsible for the low expression of αs and αi1+2 in leiomyoma. This aspect deserves further experiments but, whatever the mechanism responsible, the pattern of G protein expressed results (conceivably by αs decrease) in reduced production of cAMP, which in human uterus represents an antiproliferative signal (Matsunami et al., 1991; Nilsson et al., 1998). In conclusion, present findings suggest that the decrease in the cyclic AMP producing system in human uterine leiomyomas as compared with adjacent myometrium is a factor that presumably permits the activation of MAPkinase by growth factors necessary in the development of the tumour. ACKNOWLEDGEMENTS This research was supported by grants from the Dirección General de Investigación Científica y Técnica (PB93/0492, PB94/0360, PM97/0069). REFERENCES Ballaré, E., Mantovani, S., Basetti, M., Lania, A. and Spada, A. 1997. Immunodetection of G proteins in human pituitary adenomas: evidence for a low expression of proteins of the Gi subfamily. Eur. J. Endocrinol., 137, 482–489. Biro, J.C. 1986. Estrogen-induced proteins: a new class of regulatory substances. Med. Hyphoteses, 19, 199–228. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254. Brandon, D.D., Bethea, C.L., Strawn, E.Y., Novy, M.J., Burry, K.A., Harrington, M.S., Erickson, T.E., Warner, C., Keenan, E.J. and Clinton, G.M. 1993. Progesterone receptor messenger ribonucleic acid and protein are overexpressed in human uterine leiomyomas. Am. J. Obstet. Gynecol., 169, 78–85. Brandon, D.D., Erickson, T.E., Keenan, E.J., Strawn, E.Y., Novy, M.J., Burry, K.A., Warner, C. and Clinton, G.M. 1995. Estrogen receptor
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