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Cell Biology International (2006) 30, 533–540 (Printed in Great Britain)
Adenylyl cyclase signaling mechanisms of relaxin and insulin action: Similarities and differences
Marianna Pertsevaa*, Alexander Shpakova, Ludmila Kuznetsovaa, Svetlana Plesnevaa and Evgeniya Omeljaniukb
aSechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Thorez pr. 44, 194223, St. Petersburg, Russia
bOtt Institute of Obstetrics and Gynecology, Russian Academy of Medical Sciences, Mendeleev line 3, 199034, St. Petersburg, Russia Abstract The adenylyl cyclase signaling mechanism (ACSM) of relaxin H2 action was discovered and deciphered in mammalian muscles. A study of signaling blocks involved in ACSM of relaxin in comparison with that of insulin previously detected showed a close similarity throughout the post-receptor signaling chain of both hormones. The inhibitory action of tyrosine kinase blockers on the hormone AC activating effect indicates that the relaxin receptor involved in ACSM is likely to be of the tyrosine kinase type. However, a recent discovery of a relaxin receptor with serpentine architecture leaves open the question concerning the existence of receptor of the tyrosine kinase type. The structural-functional organization of the ACSM due to the action of relaxin—shown here for the first time—can be presented as the following signaling sequence: relaxin receptor Keywords: Adenylyl cyclase signaling, Relaxin, Insulin, Human, Rat. *Corresponding author. Fax: +7 (812) 552 3012. 1 Introduction In the last decade, interest in relaxin has continued to grow, probably because of the following three findings. First, in addition to the regulatory function in the reproductive system, this hormone has a variety of roles in the cardiovascular, renal, central nervous and other systems (Ivell and Einspanier, 2002). Second, new hormones of the relaxin family (relaxin-3, relaxin-like peptides and others) were discovered. And finally, some relaxin receptors of the serpentine type have been cloned (Hsu et al., 2000, 2002; Liu et al., 2003a,b; Scott et al., 2004). Relaxin is one of the regulatory peptides of the insulin superfamily, which includes insulin, other insulin-like hormones, and growth factors of vertebrates and invertebrates. These peptides have a structural-functional similarity consistent with a common evolutionary origin (Chan et al., 1992; Murray-Rust et al., 1992). Relaxin is a pleiotropic endocrine and paracrine factor capable of regulating many cell processes, such as growth, differentiation, apoptosis and metabolism (Ivell and Einspanier, 2002; Lee et al., 2004; Schwabe and Bullesbach, 1994). However, the molecular mechanisms of the relaxin effects remain obscure. Second messengers, such as cyclic nucleotides, nitric oxide, phosphoinositides and calcium, are assumed to be involved in relaxin action (Bani, 1997; Nguyen et al., 2003; Nistri and Bani, 2003). The study of molecular mechanisms involved in the regulatory effects of peptides of the insulin superfamily carried out in our laboratory for many years led to the discovery of the adenylyl cyclase signaling mechanism (ACSM) of insulin action in muscle of vertebrates and invertebrates that hitherto has been unknown (Pertseva et al., 1995, 1996, 2003; Plesneva et al., 2001). This novel mechanism of insulin action involves the following signaling chain: receptor-tyrosine kinase 2 Materials and methods 2.1 Experimental subjects For the experiments, male rats Rattus norvegicus (weight 150–200 2.2 Chemicals and radiochemicals Creatine phosphate, creatine phosphokinase, GTP, GTPγS, Gpp[NH]p, Tris–HCl, alumina for column chromatography, ATP, cAMP, NAD, NADP, thymidine, DTT, EDTA, imidazole were obtained from Sigma (USA). [α- 2.3 Hormones Human relaxin 2 was kindly provided by Dr J Wade (Howard Florey Institute, University of Melbourne, Australia). Porcine insulin was obtained from Sigma (USA). 2.4 Inhibitors Genistein is a specific inhibitor of tyrosine kinase activity. Wortmannin is a potent, selective and irreversible inhibitor of PI3K (Yano et al., 1993). These agents were obtained from Sigma (USA). The effects of hormones and inhibitors were studied in vitro. The agents were added to the sample for determination of AC activity. Relaxin and insulin were dissolved in 0.01 2.5 Antibodies For identification of PKCζ in relaxin signaling, specific antibodies against the mammalian (rabbit) enzyme (synthetic peptides corresponding to C-terminal region 577–592 of PKCζ) from Sigma (USA) were used. 2.6 Bacterial toxins Pertussis (PT) and cholera toxins (ChT) were obtained from Sigma (USA). PT, an exotoxin isolated from the culture of Bordetella pertussis, has been an invaluable tool in the characterization of G 2.7 ADP ribosylation procedure Samples of the membrane fraction (concentration of membrane protein was 0.95–1.0 2.8 Membrane preparation Sarcolemma membrane fractions were isolated from the leg skeletal muscle of the rat Rattus norvegicus (for each fraction, 4–6 rats were used) according to a modified method of Kidwai et al. (1973). To obtain a crude membrane fraction of human myometrium, tissue pieces were homogenized and centrifuged at 1000 2.9 Adenylyl cyclase assay AC (EC 4.6.1.1) activity was determined by a modified method of Salomon et al. (1974). The reaction mixture (final volume 50 2.10 Protein assay The protein content was determined according to the method of Lowry et al. (1951), using bovine serum albumin as a standard. 2.11 Data analysis The data are presented as the mean 3 Results The first stage of the work consisted in demonstration of the ability of human relaxin 2, compared to mammalian insulin, to exercise the AC activating effect in the muscle tissues of the objects under study. Relaxin (10−8 Table 1. The influence of relaxin (10−8 M), insulin (10−8 M) and the hormone plus GTPγS (10−5 M) in vitro on the adenylyl cyclase activity in the human myometrium and the rat skeletal muscles
The second stage was aimed at further deciphering of the AC activating mechanism of relaxin on the basis of functional organization of ACSM of insulin action discovered in previous studies (Pertseva et al., 2003; Plesneva et al., 2001). 3.1 Receptor tyrosine kinase: action of genistein For the elucidation of the type of relaxin receptor involved in the putative ACSM of hormone action, experiments were carried out using genistein, a specific tyrosine kinase blocker. Genistein in a dose-dependent manner (1.25–10.0 
Fig. 1 The influence of genistein on the AC activating action of relaxin (10−8 In the case of genistein, the AC activating effect of relaxin was decreased more than with insulin. In the presence of Gpp[NH]p, the blocking action of genistein was also evident. As a result, the potentiating effect of guanine nucleotide on the AC activating action of both peptides vanished. A higher sensitivity of relaxin AC effect to genistein was denoted by an IC The data is in accord with our earlier findings of the inhibitory action of tyrphostin 47, another tyrosine kinase blocker, on the AC activating effect of relaxin in human myometrium (Kuznetsova et al., 1999), and of insulin and IGF-1 in rat skeletal muscles (Pertseva et al., 1996; Plesneva et al., 2001). 3.2 G proteins: action of bacterial toxins 3.2.1 Pertussis toxin (PT) PT treatment of human myometrium and rat muscle membranes (Table 2) led to an increase of AC activity in the controls, and to a significant decrease in AC stimulating effect of relaxin and insulin, as well as with the hormones combined with GTPγS. According to the mechanism of PT action (Milligan, 1988; Reisine, 1990), the toxic effect is due to ADP-ribosylation of G The influence of pertussis toxin on the relaxin- and insulin-stimulated AC activity in the human myometrium and the rat skeletal muscles 3.2.2 Cholera toxin (ChT) ChT treatment of human myometrium and rat muscle membranes led to an increase of GTPγS-stimulated AC-activity compared without ChT (Fig. 2), which has been ascribed to stabilization of the GTP-binding form of G Fig. 2 The influence of relaxin and insulin on GTPγS-stimulated AC activity in cholera toxin-treated membranes of the human myometrium (A, B) and the rat skeletal muscles (C, D). (A, C) Without toxin; (B, D) ChT-treated membranes in the presence of GTPγS. 1, without hormone; 2, relaxin, 10−8 Thus, the ADP-ribosylation technique (i) shows the involvement of G proteins of the inhibitory and stimulatory types in the AC stimulating action of relaxin, and (ii) confirms the involvement of both G proteins in the ACSM of insulin action discovered earlier (Pertseva et al., 2003; Plesneva et al., 2001). 3.3 Phosphatidylinositol 3-kinase: effect of wortmannin The influence of wortmannin (10−10–10−7 M) on AC-activating effects of the peptides in human myometrium and rat muscles was a strong inhibition of the AC stimulating effect of relaxin and insulin in a dose-dependent manner (Fig. 3A,B). The specificity of wortmannin blocking action on AC effect of peptides of the insulin superfamily was tested in the experiments using β-adrenergic agonist isoproterenol. The action of adrenergic agonists is usually due to another signaling pathway involving three molecular blocks: receptors of the serpentine type, G Fig. 3 The inhibitory effect of wortmannin on relaxin and insulin AC stimulating effects in the human myometrium (A) and in the rat skeletal muscles (B). Vertical axis, percentage of the AC stimulating effect of hormones. 1. without wortmannin; 2–5, 10−10, 10−9, 10−8 and 10−7 3.4 Protein kinase Cζ: influence of specific antibodies against PKCζ In the human myometrium (Fig. 4A) and the rat skeletal muscles (Fig. 4B), antibodies against mammalian PKCζ diluted 1:10 to 1:1000 showed a dose-dependent inhibition of the AC activating effects of relaxin and insulin. In the myometrium, a combined effect of hormones plus Gpp[NH]p was also blocked with PKCζ antibodies, leading to the disappearance of the guanine nucleotide potentiating action (data not shown). The same was shown in our earlier work devoted to the insulin AC activating action in rat skeletal muscles (Pertseva et al., 2003; Plesneva et al., 2001).
Fig. 4 Relaxin and insulin AC activating effect in PKCζ antibody-treated membranes of human myometrium (A) and rat skeletal muscles (B). Vertical axis, AC activity in percentage as compared to the control taken as 100%. (A) 1, without antibodies; 2 and 3, with antibodies diluted 1:500 and 1:100, respectively. (B) 1, without antibodies; 2, 3 and 4, with antibodies diluted 1:1000, 1:100 and 1:10, respectively. Asterisk, a statistical significance of differences from the control (p 4 Discussion The putative AC activating mechanism of relaxin action was revealed and compared with the ACSM of insulin action described in our previous investigations (Pertseva et al., 1995, 1996, 2003; Plesneva et al., 2001). Prior to our earlier findings, involvement of the AC signaling system in regulatory effects of peptides of the insulin superfamily had been ruled out. Only scant data was available to show that relaxin and insect bombyxin are capable of inducing cAMP accumulation in some tissues. Studying the AC activating action of human relaxin 2 in the muscle tissues of mammals and mollusks (Kuznetsova et al., 1999), we had found that the hormone acts via the following signaling chain: receptor-tyrosine kinase, G In the present study the following data on the architecture of relaxin ACSM have been obtained. 4.1 Characteristics of the receptor type The experiments with tyrosine kinase blocker genistein confirmed our earlier finding made with tyrphostin 47 (Kuznetsova et al., 1999; Pertseva et al., 1996), which showed inhibition of the AC activating effects of relaxin and insulin. The inhibitory action of tyrosine kinase blockers on cAMP accumulation induced by relaxin has also been observed by other authors (Bartsch et al., 2001; Sanborn et al., 2001). This led us to suggest that the relaxin receptor, like that of insulin, is of the tyrosine kinase type. The same view has been expressed by other authors (Goldsmith et al., 2001; Hollingsworth et al., 2001). Due to discovery of the receptor of relaxin and relaxin-related peptides with serpentine organization (Hsu et al., 2000, 2002; Liu et al., 2003a,b; Scott et al., 2004), the problem now concerning the nature of relaxin receptor is apparently more complicated, and the question of the types of relaxin receptors remains open. The detection in organisms of a number of relaxin-like peptides and their receptors gives reason to suppose that among the latter there may be receptors of the tyrosine kinase and serpentine types scattered in different target-tissues. This view is in agreement with the idea that the different effects of relaxin in various tissues are mediated via several signaling pathways that vary from cell to cell depending on its type (Koos and Pillai, 2001). It may also be the case that relaxin's insulin-like effects—such as growth promoting, anti-apoptotic and metabolic changes—are elicited by a receptor of the tyrosine kinase type and via ACSM, in accord with our hypothesis, as is the case with insulin (Pertseva et al., 2003; Plesneva et al., 2003). At the same time, relaxin effects that are responsible for regulation of the reproductive system are probably mediated through G protein-coupled receptors of the serpentine type structurally related to the receptors of hormones (such as LH and FSH) belonging to the group of reproductive polypeptide hormones (Hsu, 2003; Ivell et al., 2003; Kumagai et al., 2002). To solve such an intriguing dilemma concerning the type(s) of relaxin receptor requires further analysis. There is additionally a view that the non-receptor tyrosine kinase signaling cascade is involved in the relaxin regulatory effect on phosphodiesterase/AC activity (Bartsch et al., 2001). This offers an alternative explanation of the data on the interruption of relaxin signaling by inhibitors of tyrosine kinases. 4.2 G proteins involved in relaxin ACSM Inhibition of relaxin signal transduction via the AC system as a result of the PT treatment of muscle membranes, seen in the present study, implies the involvement of G The unexpected finding that PT inhibits relaxin (and insulin, earlier data) stimulatory effect on AC observed in the present study and our interpretation of this fact is confirmed by the work of Marjamaki et al. (1997). Studying integration of different signals directed to several AC isoforms, they found that (i) ligand activation of α 4.3 Phosphatidylinositol 3-kinase The suppressing influence of wortmannin on the relaxin AC activating effect provides evidence for involvement of this enzyme in the putative ACSM of the hormone action. In the smooth muscles of the freshwater bivalve mollusk, Anodonta cygnea, the AC activating effect of relaxin is also blocked by wortmannin (Kuznetsova et al., 2001). Similar results were obtained in the case of the ACSM of insulin action (Plesneva et al., 2001). According to Nguyen et al. (2003), wortmannin exercises a dose-dependent inhibition of relaxin-induced increase in cAMP, which agrees with their data on stimulatory effect of relaxin on PI3K activity in the human monocyte cell line, TPH-1. The fact that PI3K is involved in relaxin and insulin AC signaling coincides with our finding of the involvement in it of G 4.4 Protein kinase C zeta (PKCζ) One of the targets of the regulatory action of insulin, and probably of relaxin mediated through PI3K, is atypical PKCζ. This was found by using specific antibodies against the mammalian (rabbit) PKCζ. The treatment of membrane fractions of the human myometrium and the rat muscles with antibodies led to interruption of relaxin signal transduction via the AC signaling system (Fig. 4). The same true of ACSM with regard to insulin action in rat skeletal muscles (Pertseva et al., 2003). The involvement of PKCζ in relaxin stimulating action on the cAMP accumulation was also demonstrated in THP-1 cells (Nguyen and Dessauer, 2005). Combining the data obtained in the present work with that of our earlier studies (Kuznetsova et al., 1999, 2001), the AC signaling cascade involved in relaxin action in the mammalian tissues can be presented as follows: receptor (tyrosine kinase or serpentine type) A comparison of the structural-functional organization of ACSM of relaxin and insulin action revealed both some similar and some different features. The difference concerns the type of receptor. As is well known, the insulin receptor belongs to receptor of tyrosine kinase types. Despite inhibitory action of tyrosine kinase blockers on relaxin AC stimulating effect, which we have already shown (Kuznetsova et al., 1999, and the present study) and other authors (Bartsch et al., 2001; Sanborn et al., 2001), the structural homology has also been revealed between some receptors of relaxin peptides and receptors of the serpentine type, which hinders identification of the nature of relaxin receptors. In the mechanism of relaxin action the possibility that the non-receptor tyrosine kinase cascade is involved cannot be excluded. This was suggested by Bartsch et al. (2001), concerning the mechanism of relaxin action on phosphodiesterase/AC activity. Thus the types of relaxin receptors remain questionable. As far as the type of heterotrimeric G protein coupled with putative relaxin receptor is concerned, one idea is based on a structural study of insulin receptor-tyrosine kinase and receptors of the serpentine type. These were found to have regions with similar amino acid sequences responsible for interaction with G It follows from the above that post-receptor components of ACSM of the two related hormones are alike. According to our hypothesis that insulin-competent ACSM mediates the hormone regulatory action on the main processes in a cell (Pertseva, 2000; Pertseva et al., 2003; Plesneva et al., 2001), it can be assumed that relaxin-competent ACSM will in all probability also interfere in the regulation of cell growth, apoptosis and anabolism. This had been experimentally shown to be so for insulin (Plesneva et al., 2003). Data is available that relaxin also stimulates cell proliferation and retards apoptosis in different tissues (Lee et al., 2004). In conclusion, we propose a novel adenylyl cyclase signaling mechanism of relaxin action, which finds confirmation in the data of Nguyen et al. (2003), Nguyen and Dessauer (2005). 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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355 Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB) |
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Abstract
