- Email: [email protected]
An alternate insulin-like growth factor I receptor signaling pathway for the progression of endothelial–mesenchymal transition
Bioscience Hypotheses (2008) 1, 312e318 available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/bihy
An alternate insulin-like growth factor I receptor signaling pathway for the progression of endothelialemesenchymal transition Enrique Arciniegas a,*, Daniel Candelle b a
Servicio Auto´nomo Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Apartado de correos 4043, Carmelitas, Caracas 1010, Venezuela b Universidad Nacional Experimental Francisco de Miranda, Area Ciencias de la Salud, Coro, Estado Falco´n, Venezuela Received 16 July 2008; accepted 21 July 2008
KEYWORDS Endotheliale mesenchymal transition; Cellecell contacts; Microtubules
Abstract Emerging evidence suggests that endothelial-to-mesenchymal transition (EndoMT) is an important contributor to cardiovascular diseases and to vascular development and pathologies as well as in cancer progression. As in epithelialemesenchymal transition (EMT), EndoMT may involve several regulated steps: disassembly of adherence junctions or loss of cellecell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation. Loss of cellecell contacts is a necessary and sufficient step in the progression of EndoMT. In endothelial cells, adherence junctions are composed of transmembrane adhesive proteins belonging to the cadherin family, with the VE-cadherin being the most important. This protein interacts with b-catenin, which links cadherin to the actin cytoskeleton. Tyrosine phosphorylation of both VE-cadherin and b-catenin is considered an important mechanism associated to the disassembly of adherence junctions or loss of celle cell contacts. Insulin-like growth factor receptor I (IGFIR) is a transmembrane tyrosine kinase that has been involved in the alterations of cellecell contacts and in the expression of some genes during cancer development and progression. Here, it is hypothesized that IGFIR autophosphorylation may initiate a signaling pathway that would lead to the loss of cellecell contacts or adherence junctions, remarkable remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here. ª 2008 Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: þ58 212 860 4630; fax: þ58 212 861 1258. E-mail address: [email protected] (E. Arciniegas).
Endothelial-to-mesenchymal transition (EndoMT) is a process by which certain endothelial cell (EC) subsets lose
1756-2392/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bihy.2008.07.012
Insulin-like growth factor I receptor signaling pathway endothelial characteristics and transform into mesenchymal cells or smooth muscle (SM)-like cells [1] (Fig. 1). There is emerging evidence to suggest that this process is an important contributor to cardiovascular diseases [2] and to vascular pathologies such as neointimal thickening formation observed in atherosclerosis and restenosis, and pulmonary vascular remodeling induced by hypoxia [1]. In addition to this role, several groups have provided evidence implying that EndoMT is critical in cardiogenesis and vascular development, granulation tissue formation and in cancer progression [2e4]. As in epithelialemesenchymal transition (EMT), EndoMT seems to progress through a series of important steps whose interdependence and order are not clear as yet [1]. This complex process may involve several characterized regulated steps: disassembly of the adherence junctions or loss of endothelial cellecell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation [1]. Importantly, loss of cell-to-cell contacts is consistently observed at EMT sites during development, and in cancer and fibrosis [5,6]. Regarding the endothelium, endothelial cell-to-cell contacts are maintained by adherens junctions composed of transmembrane adhesive proteins belonging to the cadherin family, with the vascular endothelial (VE)-cadherin being
313 the most important. This protein interacts with cytoplasmic proteins called catenins, in particular b-catenin, which links cadherin to the actin cytoskeleton. VE-cadherin and bcatenin functions are generally related to the maintenance of cellecell contacts and regulation of the intracellular signaling pathways that are implicated in cell growth, apoptosis, matrix, and cytoskeletal remodeling [7]. Tyrosine phosphorylation of both VE-cadherin and b-catenin is considered an important mechanism associated to the disassembly of endothelial adherence junctions [7,8]. In this respect, there are reports showing that disruption of endothelial cellecell contacts may occur in response to mechanical stimuli and/or soluble growth factors such as FGF-2, TGFb, and insulin-like growth factor II (IGFII) [9e11], and that this event is a necessary and sufficient step in the progression of EndoMT [9]. However, neither the molecular and cellular mechanisms operating to cause the endothelial transformation nor the signaling cascades controlling it have been elucidated. In this article, it is speculated that the binding of IGFII to the insulin-like growth factor receptor I (IGFIR) may initiate several signaling cascades that would lead to the loss of cellecell contacts or adherence junctions, dramatic remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here.
Figure 1 Confocal laser scanning microscope fluorescence (CLSM) images of vWf and a-SM actin expression in a monolayer of endothelial cells attached to fibronectin after 48 h in culture in complete medium. Strong immunoreactivity for vWf (red) in a granular pattern typical of ECs, and for a-SM actin (green) delineating cellular margin are observed. Double immunofluorescence of the same field shows some of the cells exhibiting immunoreactivity for both vWf and a-SM actin (merged).
314
Involvement of IGFII and IGFIR in the regulation of cellecell contacts Both IGFI and IGFII, in addition to being considered as important factors modulating wound healing, carcinogenesis, cancer progression, metastasis [12e15], as well as several cardiovascular diseases associated to intimal thickening formation [16e18], are also considered to participate during embryonic development [11,19e21]. Of particular significance, ontogenic changes in the circulating levels of both IGFI and IGFII have been reported in the chicken embryo model, suggesting that they may participate in regulating different processes during chicken embryo development [20,22]. Recently, we have suggested a probable role for IGFII and vitronectin in the regulation of endothelial cellecell contacts during vascular remodeling and the EndoMT [11]. However, studies about the effects of these growth factors in vascular development, particularly IGFII, are scarce. Because, there is evidence showing that IGFIIR has no apparent intrinsic signaling transduction potentialities and seems to act as a ‘‘sink’’ for IGFII [13,14,23], we will focus in this article mainly on the role of IGFIR activation. IGFIR is a transmembrane tyrosine kinase that contains two extracellular a-subunits and two intracellular bsubunits. The a-subunits bind IGFI, IGFII, and IGF, whereas the b-subunits transmit ligand-induced signals. Binding of ligands to the extracellular a-subunits results in autophosphorylation of its b-subunits on specific tyrosine residues followed by phosphorylation and recruitment of various cytoplasmic adaptor signaling proteins that include insulinreceptor substrate-1 (IRS-1), its major substrate [24]. Autophosphorylation of IGFIR potentially results in the initiation of some specific signaling pathways that eventually culminates in the activation of some transcription factors. One of them involves phosphorylation of IRS-1 and the subsequent phosphoinositide 3-kinase (PI3K), MAP kinases (MAPKs) and AKT/protein kinase B (AKT/PKB) activation [13,25e27]. The activated AKT then phosphorylates various target proteins, resulting in the regulation of various cellular processes including EMT [28]. Importantly, signals generated through this pathway could also collaborate in cell proliferation and differentiation control and apoptosis [25,26,29]. Another signaling pathway, in response to IGFI binding, would involve the phosphorylation of focal adhesion kinase (FAK) and some of its associated proteins such as paxillin as well as crosstalk with integrins during the cell migration [30]. An alternate signaling pathway has emerged about how IGFIR participates in the alteration of cellecell contacts during cancer development and progression. In fact, there is evidence that ligation of IGFIR by IGFI may also generate signals controlling the cellecell contacts by modulating the activities of the cadherins and catenins as well as of the small Rho GTPases, Rho, Rac-1, and Cdc42 [31]. For instance, binding of IGFI to IGFIR can induce tyrosine phosphorylation of members of the E-cadherinecatenin complex promoting epithelial cell migration [31,32], whereas binding of IGFII induces the cytoplasmic redistribution of IGFIR and E-cadherin and nuclear translocation of b-catenin, where it modulates gene activation, during normal and pathological EMT [33]. In ECs, binding of IGFII to IGFIR could induce cell separation, involving VE-cadherin cytoplasmic relocation and b-catenin nuclear translocation, during the EndoMT
E. Arciniegas, D. Candelle [11]. Others found that ligation of IGFIR by IGFI could induce migration by disruption of a multiproteic complex formed at cellecell contacts sites by IGFIR, E-cadherin, avintegrin in epithelial cells, suggesting that this disruption might involve tyrosine phosphorylation of these proteins [34]. However, previous reports have proposed that IGFIR activation may also occur in a ligand-independent way during malignant transformation [25] and during neointimal formation in vein graft induced by mechanical stretch [18]. From these and other studies, it is clear that activation of the IGFI signaling pathways is at least critical for cancer development and progression [27,29,31,35]. Less is known regarding the activation of IGFII signaling pathways. However, there is increasing experimental evidence to suggest that IGFII plays an important role in many developmental and pathological processes [15,36]. Even so, the manner by which IGFIR activation contributes to the control of various aspects of cell behavior continues being a subject of debate [37]. Nevertheless, if one assumes that IGFIR activation may be involved in the regulation of endothelial cellecell contacts and the modulation of gene transcription then one can suspect that this could initiate the generation of signaling pathways that govern the EndoMT process. Apart from its association to cancer development and progression [13,23,24,27,29,31], IGFIR expression and signaling have been associated to cardiovascular function and diabetic cardiomyopathy [17].
The IGFIR, VE-cadherin, and b-catenin complex and the regulation of gene expression Cadherins are calcium-dependent proteins which, through their extracellular domain, promote homotypic cellecell interactions and modulate cell migration by affecting adhesion, cytoskeletal organization, and cell polarity [38e 40]. Classical cadherins include epithelial- (E-), vascular endothelial- (VE-) and neural- (N-) cadherins. Several cytoplasmic proteins, such as the catenins, interact with classical cadherins linking it to the actin-associated cytoskeletal proteins and intracellular signaling pathways [7,38]. The architecture of this complex may be disrupted by tyrosine phosphorylation of cadherin and catenin, involving specific tyrosine kinases. In epithelial cells, where b-catenin is bound to E-cadherin, tyrosine phosphorylation of these proteins by tyrosine kinases such as IGFIR, not only provokes the dissociation of b-catenin from E-cadherin affecting celle cell contacts, but also allows its relocation to the cytoplasm and the nucleus where it may activate the transcription of target genes [32,41]. Interestingly NF-kB, a transcriptional factor that regulates the expression of hundred genes and whose activation proceeds rapidly and depends on the type and intensity of the stimulus, has been shown to be activated by IGFII stimulation during skeletal muscle differentiation [42] and in human keratinocytes [43]. With respect to the potential participation of NF-kB in the EMT process, studies in epithelial cells have indicated that constitutively active IGFIR causes EMT associated with increased NF-kB activity [44]. Significantly, recent reports have also proposed NF-kB as a mediator of the EMT process [45,46]. In ECs, recent data suggest that the VE-cadherinebcatenin complex in addition to being an important site for,
Insulin-like growth factor I receptor signaling pathway recent data suggest that the VE-cadherineb-catenin complex in addition to being an important site for mechanotransduction and mechanosensing is also involved in the activation of some cell signaling pathways regulating endothelial cell behavior [7]. Like in epithelial cells, disruption of VE-cadherineb-catenin complex may also cause the translocation of b-catenin to the nucleus where it influences gene transcription [7]. Recently, we have shown that IGFII stimulation of embryonic endothelial cells results in the loss of cellecell contacts by the removal of VE-cadherin and redistributions of b-catenin probably conducing to the activation and translocation of NF-kB with the consequent regulation of a-SM actin expression [47]. How these signals generated locally after the activation of IGFIR and disruption of celle cell contacts could be influencing not only the epithelial gene expression but also the endothelial gene expression, is still matter of discussion. It is possible that alterations in the cytoskeleton organization could be contributing to the expression of some genes considering that cadherins in addition to being related to the actin cytoskeleton, also seem to be associated physically with the microtubules (MTs) regulating thus their organization [48].
Microtubules in the dynamics of cellecell contacts The cytoskeleton is considered as a complex network of actin filaments, intermediate filaments, and microtubules (MTs) [49,50] that links the surface of the cell to the nucleus and participates in the formation of adherens junctions and intracellular signal transduction [48,51]. Various lines of evidence indicate that these cytoskeletal components in response to external stimuli can be used as linear tracks to move organelles, secretory vesicles, and protein complexes, within the cell and provide transient docking sites for proteins and lipids. Here, we will discuss some aspects about the MTs and their possible role in the formation or disruption of cellecell contacts as well as the possible repercussion of these events on intracellular signaling, using both the epithelial and endothelial cells as reference models. MTs are highly dynamic polymers of a/b-tubulin heterodimers that appear oriented with their minus ends clustered around the microtubule-organizing center (MTOC) located close to the nucleus and their plus ends oriented towards the cell periphery where they participate in the conformation of the multiproteic complex. In addition to its known role as cell scaffolding and track for the directed delivery of proteins, vesicles and other factors from the cell center to the cell periphery (anterograde transport), recent studies suggest that MTs may also regulate the dynamics of cellecell contacts by the formation or disruption of cadherin-mediated cell contacts which result in alteration of the b-catenin distribution [48]. A notable characteristic of the MTs behavior is its dynamic instability, in which their plus ends undergo frequent periods of slow growth and rapid shrinkage by the addition or loss of tubulin [52e54]. The control of MT dynamics is regulated spatially and temporally by proteins that associate physically with them. In general, every protein that associates with MTs can be considered to be a microtubule-associated protein (MAP). Among this class of proteins are the so called structural MAPs which bind to the MT plus or minus ends, MAPKs also known as extracellular-signal
315 regulated kinases (ERKs), other kinases such as IkB kinase (IKK) and PI3K, MT motor proteins such as cytoplasmic dynein and kinesins, and adaptor proteins. There is evidence that MAP phosphorylation leads to their detachment from the MTs, thereby contributing to the MTs instability [53,55,56]. This phosphorylation seems to be provoked by specific MAPKs that are bound to the MTs via ATPase MT motor proteins, and occurs in response to cell stimulation, thus regulating the MT dynamics and influencing cell signaling. Among the MT motor proteins, one of the most studied is cytoplasmic dynein [57,58]. This protein is actually considered as part of the multiproteic adherens junction complex and as responsible not only for tethering of MTs at sites of epithelial cellecell contacts [59], but also in the anterograde transport of proteins for the assembly of junctions such as b-catenin, acatenin, and p120-catenin [60]. Nonetheless, a possible role for dynein in the retrograde transport of proteins from the cell periphery to the cell center that includes tyrosine phosphorylated b-catenin, during the disruption of cellecell contacts and signal transmission has also been proposed [60].
Microtubules in the regulation of gene expression Current knowledge indicates that regulation of MT dynamics by a variety of signals, including mechanical stimuli, kinase inhibitors, and soluble growth factors would have an important repercussion on the behavior of cancer and endothelial cells [53]. Little is known about how extracellular stimuli regulate the MT dynamics in these cells. However, punctual studies have proposed that modulation of MT dynamics is also crucial for the nuclear or retrograde transport of activated NF-kB and control of gene expression [53]. For instance, disruption of MTs in epithelial cells activates NF-kB and induces the expression of several genes that are regulated by this transcriptional factor. This activation probably requires either direct degradation of inhibitor kB-alpha (IkB-a), which would be linked to MTs via dynein, or degradation of or degradation of this inhibitor through an activated specific MAPK or kinase that would also be an activated specific MAPK or kinase that would also be anchored to MTs, via dynein [61e64]. By contrast, in neuronal cells, a functional cytoskeleton seems to be required for the retrograde transport of activated NF-kB mediated by dynein, after phosphorylation and degradation of IkB-a which is located at the cytoplasm instead of being anchored to the MTs [65]. From these evidences it is clear that the normal nuclear NF-kB transport seems to depend on the MT dynamics, considering that the disruption of MTs affects both the retrograde transport of NF-kB and the NFkB-dependent transcriptional activity and that the alterations in the cytoskeleton, in part occasioned by the disruption of cellecell contacts also contribute with the upregulation of the NF-kB activity [66]. As already indicated, separate studies by different investigators using different systems concerning ECs, have emphasized that vascular remodeling and neointimal formation might be the results of blood pressure alterations and that such alterations might generate a range of responses involving the disturbance of MT dynamics [51,67] as well as the activation and translocation of transcriptional factors such as NF-kB, leading to gene expression
316 modulation [68]. However, a link among these different responses as well as a link between them and the EndoMT process have not been explored. Therefore, more studies are obviously necessary to gain insights into the mechanisms regulating the MT dynamics during the transition of endothelial cells towards a mesenchymal phenotype.
How IGFIR activation contributes to the transformation of endothelium derived mesenchymal cells into a smooth muscle-like phenotype As already mentioned, phosphorylation or activation of IGFIR is involved in the alteration of cellecell contacts and in the expression of some genes, but how this activation collaborates in the transformation of epithelium or endothelium derived mesenchymal cells into a SM-like phenotype still remains to be determined. It is possible that the binding of IGFII to IGFIR also may modulate, through lateral interactions, the activation or phosphorylation of endoglin, a receptor that is expressed on the surface of vascular endothelial cells and is concentrated at endothelial cellecell contacts mediating the functions of TGFb superfamily members [69,70]. This inference would be based on the fact that endoglin, apart from its close association with other TGFb receptors such as TbRII, is also able to
E. Arciniegas, D. Candelle interact with a member of the dynein family regulating not only its own retrograde transport but also the activated TGFb signal transduction pathway [71]. This is of great interest since the presence of activated TGFb isoforms has been associated with alterations in cadherin expression and bcatenin redistribution and the modulation of NF-kB activity not only during EMT but also in EndoMT [1,10,45,72]. Moreover, activated TGFb-1 has been shown to promote the conversion of endothelial cells into SM-like cells [9,73,74]. Thus, it is possible to speculate that activated IGFIR, in functional cooperation with endoglin, may participate in the transformation of endothelial cells into mesenchymal cells or SM-like cells (defined by the expression of a-SM actin). It is also possible that endoglin activation provokes the activation of IGFIR, considering that the activation of this receptor may also occur in a ligand-independent manner.
The hypothesis In conclusion, a complex cascade of signals that may be generated after the binding of IGFII to IGFIR may result in: (a) IGFIR tyrosine autophosphorylation, (b) VE-cadherin and b-catenin tyrosine phosphorylation, (c) perturbation of microtubule (MT) dynamics involving phosphorylation of MT-associated proteins (MAPs), MAP kinases (MAPKs), kinases and phosphatases as well as alteration of actin filaments and intermediate filaments organization, (d)
Figure 2 Schematic representation of some of the events that may occur after the binding of IGFII to IGFIR (1). Retrograde transport along MTs of b-catenin and NF-kB (2). Anterograde transport of intermediate filaments (3). Regulation of target genes (4) and functional cooperation of endoglin/TbIIR complex (5).
Insulin-like growth factor I receptor signaling pathway retrograde transport along MTs of molecules such as phospho-b-catenin and NF-kB and anterograde transport of intermediate filaments, both assisted by MT motor proteins that include cytoplasmic dynein, (e) regulation of target genes, and (f) functional cooperation of endoglin during the transformation of endothelial cells into mesenchymal or SM-like cells (Fig. 2). The relevance of knowing which are the signaling pathways generated when IGFIR is activated and how these signals lead to the endothelial cell transformation resides in the possibility of explaining several of the cellular events occurring not only during embryonic heart development but also in the context of the pathogenesis of various cardiovascular diseases occurring not solely in adult life but also throughout childhood and adolescence.
Acknowledgments This work was supported by the Fondo Nacional de Investigaciones Cientı´ficas y Tecnolo ´gicas (FONACIT) (grant n G2005000405).
References [1] Arciniegas E, Frid MG, Douglas IS, Stenmark KR. Perspectives on endothelial-to-mesenchymal transition: potential contribution to vascular remodeling in chronic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2007;293:L1e8. [2] Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman A, McMullen J, Gustafsson E, et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 2007;13:952e61. [3] Mironov V, Visconti RP, Markwald RR. On the role of shear stress in cardiogenesis. Endothelium 2005;12:259e61. [4] Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R. Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Cancer Res 2007;67:10123e81. [5] Thiery JP, Sleeman JP. Complex networks orchestrate epithelialemesenchymal transitions. Nat Rev Mol Cell Biol 2006; 7:131e42. [6] Baum B, Settleman J, Quinlan MP. Transitions between epithelial and mesenchymal states in development and disease. Semin Cell Dev Biol 2008;19:294e308. [7] Liebner S, Cavallaro U, Dejana E. The multiple languages of endothelial cell-to-cell communication. Arterioscler Thromb Vasc Biol 2006;26:1431e8. [8] van Buul JD, Anthony EC, Fernandez-Borja M, Burridge K, Hordijk PL. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cellecell adhesion by regulating b-catenin tyrosine phosphorylation. J Biol Chem 2005;280:21129e36. [9] Frid MG, Kale VA, Stenmark KR. Mature vascular endothelium can give rise to smooth muscle cells via endothelial mesenchymal transdifferentiation: in vitro analysis. Circ Res 2002; 90:1189e96. [10] Liebner S, Catellino A, Gallini R, Rudini N, Iurlaro M, Piccolo S, et al. b-catenin is required for endothelialemesenchymal transformation during heart cushion development in the mouse. J Cell Biol 2004;166:359e67. [11] Arciniegas E, Neves YC, Carrillo LM. Potential role for insulin-like growth factor II and vitronectin in the endothelialemesenchymal transition process. Differentiation 2006;74:277e92. [12] Le Roith D, Roberts Jr CT. The insulin-like growth factor system and cancer. Cancer Lett 2003;195:127e37. [13] Pollak MN, Schernhammer ES, Hankinson SE. Insulin-like growth factors and neoplasia. Cancer 2004;4:507e18.
317 [14] Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine Growth Factor Rev 2005;16:421e39. [15] Chao W, D’Amore PA. IGF2: epigenetic regulation and role in development and disease. Cytokine Growth Factor Rev 2008; 19:111e20. [16] Zaina S, Nilsson J. Insulin-like growth factor II and its receptors in atherosclerosis and in conditions predisposing to atherosclerosis. Curr Opin Lipidol 2003;14:483e9. [17] Opgaard OS, Wang PH. IGF-I is matter of heart. Growth Horm IGF Res 2005;15:89e94. [18] Cheng J, Du J. Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulinlike growth factor I receptor. Arterioscler Thromb Vasc Biol 2007;27:1744e51. [19] Louvi A, Accili D, Efstratiadis A. Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev Biol 1997;189:33e48. [20] Scanes CG, Thommes RC, Radecki SV, Buonomo FC, Woods JE. Ontogenic changes in the circulating concentrations of insulin-like growth factor (IGF)-I, IGF-II, and IGFbinding proteins in the chicken embryo. Gen Comp Endocrinol 1997;106:265e70. [21] Allan GJ, Zannoni A, McKinnell I, Otto W, Holzenberger M, Flint DJ, et al. Major components of the insulin-like growth factor axis are expressed early in chicken embryogenesis, with IGF binding protein (IGFBP) expression subject to regulation by sonic hedgehog. Anat Embryol 2003;207:73e84. [22] McMurtry JP, Rosebrough RW, Brocht DM, Francis GL, Upton Z, Phelps P. Assessment of developmental changes in chicken and turkey insulin-like growth factor-II by homologous radioimmunoassay. J Endocrinol 1998;157:463e73. [23] Yee D. Targeting insulin-like growth factor pathways. Br J Cancer 2006;94:465e8. [24] Baserga R, Peruzzi F, Reiss K. The IGF-I receptor in cancer biology. Int J Cancer 2003;107:873e7. [25] Valentinis B, Baserga R. IGF-I receptor signalling in transformation and differentiation. J Clin Pathol Mol Pathol 2001; 54:133e7. [26] Wu Y, Karas M, Dupont J, Zhao H, Toyoshima Y, Le Roith D. Multiple signaling pathways are involved in the regulation of IGF-I receptor inhibition of PTEN-enhanced apoptosis. Growth Horm IGF Res 2004;14:52e8. [27] Riedemann J, Macaulay VM. IGFIR signalling and its inhibition. Endocr Relat Cancer 2006;13:S33e43. [28] Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, Van Roy F, et al. Activation of NF-kappaB by Akt upregulates snail expression and induces epithelium mesenchyme transition. Oncogene 2007;26:7445e56. [29] Ryan PD, Goss PD. The emerging role of the insulin-like growth factor pathway as a therapeutic target in cancer. Oncologist 2008;13:16e24. [30] Shen MR, Hsu YM, Hsu KF, Chen YF, Tang MJ, Chou CY. Insulinlike growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by alphavbeta3 integrin signaling. Carcinogenesis 2006;27:962e71. [31] Mauro L, Surmacz E. IGF-I receptor, cellecell adhesion, tumour development and progression. J Mol Histol 2004;35:247e53. [32] Mauro L, Salerno M, Morelli C, Boterberg T, Bracke ME, Surmacz E. Role of the IGF-I receptor in the regulation of cellecell adhesion: implications in cancer development and progression. J Cell Physiol 2002;194:108e16. [33] Morali OG, Delmas V, Moore R, Jeanney C, Thiery JP, Larue L. IGF-II induces rapid b-catenin relocation to the nucleus during epithelium to mesenchyme transition. Oncogene 2001;20: 4942e50. [34] Canonici A, Steelent W, Rigot V, Khomitch-Baud A, BoutaghouCherid, Bruyneel E, et al. Insulin-like growth factor I receptor,
318
[35]
[36]
[37] [38] [39]
[40] [41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49] [50] [51]
[52] [53]
[54]
E. Arciniegas, D. Candelle E-cadherin and av integrin form a dynamic complex under the control of a-catenin. Int J Cancer 2008;122:572e82. Allison AS, McIntyre MA, McArdle C, Habib FK. The insulin-like growth factor type I receptor and colorectal neoplasia: insights into invasion. Hum Pathol 2007;38:1590e602. Reik W, Constancia M, Dean W, Davies K, Bowden L, Murrel A, et al. IGF2 imprinting in development and disease. Int J Dev Biol 2000;44:145e50. Baserga R. The contradictions of the insulin-like growth factor I receptor. Oncogene 2000;19:5574e81. Takeichi M. Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 1995;7:619e27. Conacci-Sorrel M, Zhurinsky J, Ben-Ze’ev A. The cadherine catenin adhesion system in signaling and cancer. J Clin Invest 2002;109:987e91. Chen X, Gumbiner BM. Crosstalk between different adhesion molecules. Curr Opin Cell Biol 2006;18:572e8. Chen J, Wu A, Sun H, Drakas R, Garofalo C, Cascio S, et al. Functional significance of type I insulin-like growth factor mediated nuclear translocation of the insulin receptor substrate-1 and b-catenin. J Biol Chem 2005;280:29912e20. Kalliman P, Canicio J, Testar J, Palacin M, Zorzano A. Insulinlike growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kB and inducible nitric-oxide synthase define a common myogenic signaling pathway. J Biol Chem 1999;274: 17437e44. Kwon YW, Jang ER, Lee YM, Kim YS, Kwon KS, Jang HS, et al. Insulin-like growth factor-II induces interleukin-6 expression via NF-kB activation in psoriasis. Biochem Biophys Res Commun 2000;278:312e7. Kim HY, Litzenburger BC, Cui X, Delgado DA, Grabiner BC, Lin X, et al. Constitutively active type I insulin-like growth factor receptor causes transformation and xenograft growth of immortalized mammary epithelial cells and is accompanied by an epithelial-to-mesenchymal transition mediated by NFkB and snail. Mol Cell Biol 2007;27:3165e75. Huber MA, Azoitel N, Baumann B, Gru ¨nert S, Sommer A, Pehamberger H, et al. NF-kB is essential for epitheliale mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest 2004;114:569e81. Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H. NF-kB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 2007;26:711e24. Arciniegas E, Carrillo LM, De Sanctis JB, Candelle D. Possible role of NF-kB in the embryonic vascular remodeling and the endothelialemesenchymal transition process. Cell Adhesion Migration 2008;2:1e13. Stehbens SJ, Paterson AD, Crampton MS, Shewan AM, Ferguson C, Akhmanova A, et al. Dynamic microtubules regulate the local concentration of E-cadherin at cellecell contacts. J Cell Sci 2006;119:1801e11. Chang L, Goldman RD. Intermediate filaments mediate cytoskeletal crosstalk. Mol Cell Biol 2004;5:601e13. Stamenovic D. Effects of cytoskeletal prestress on cell rheological behavior. Acta Biomater 2005;1:255e62. Galbraith CG, Skalah R, Chien S. Shear stress induces spatial reorganization of the endothelial cell cytoskeleton. Cell Motil Cytoskeleton 1997;40:317e30. Mitchison T, Kirschner M. Dynamic instability of microtubule growth. Nature 1984;312:237e42. Honore S, Pasquier E, Braguer D. Understanding microtubule dynamics for improved cancer therapy. Cell Mol Life Sci 2005; 62:3039e56. Akhmanova A, Steinmetz MO. Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev 2008;9:309e22.
[55] Kapeller R, Toker A, Cantley LC, Carpenter CL. Phosphoinositide 3-kinase binds constitutively to a/b-tubulin and binds to gtubulin in response to insulin. J Biol Chem 1995;270:25985e91. [56] Reszka AA, Seger R, Diltz CD, Krebs EG, Fischer EH. Association of mitogen-activated protein kinase with the microtubule cytoskeleton. Proc Natl Acad Sci U S A 1995;92:8881e5. [57] Burgess SA, Walker ML, Sakakibara H, Knight PJ, Oiwa K. Dynein structure and power stroke. Nature 2003;421:715e8. [58] Mizuno N, Narita A, Kon T, Sutoh K, Kikkawa M. Threedimensional structure of cytoplasmic dynein bound to microtubules. Proc Natl Acad Sci U S A 2007;104:20832e7. [59] Ligon LA, Karki S, Tokito M, Holzbaur ELF. Dynein binds to bcatenin and may tether microtubules at adherens junctions. Nat Cell Biol 2001;3:913e7. [60] Ligon LA, Holzbaur ELF. Microtubules tethered at epithelial cell junctions by dynein facilitate efficient junction assembly. Traffic 2007;8:808e19. [61] Rossette C, Karin M. Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kB. J Cell Biochem 1995;128:1111e9. [62] Cre ´pieux P, Kwon H, Leclerc N, Spencer W, Richard S, Lin R, et al. IkBa physically interacts with a cytoskeleton-associated protein through its signal response domain. Mol Cell Biol 1997; 17:7375e85. [63] Bourgarel-Rey V, Vallee S, Rimet O, Champion S, Braguer D, Desobry A, et al. Involvement of nuclear factor kB in c-Myc induction by tubulin polymerization inhibitors. Mol Pharmacol 2001;59:1165e70. [64] Mistry P, Deacon K, Mistry S, Blank J, Patel R. NF-kB promotes survival during mitotic cell cycle arrest. J Biol Chem 2004;279: 1482e90. [65] Mikenberg I, Widera D, Kaus A, Kaltschmidt B, Kaltschmidt C. Transcription factor NF-kB is transported to the nucleus via cytoplasmic dynein/dynactin motor complex in hippocampal neurons. PloS ONE 2007;2:e589. [66] Kuphal S, Poser I, Jobin C, Hellerbrand C, Bosserhoff AK. Loss of E-cadherin leads to upregulation of NF-kB activity in malignant melanoma. Oncogene 2004;23:8509e19. [67] Lee T-YJ, Gotlieb AI. Microfilaments and microtubules maintain endothelial integrity. Microsc Res Tech 2003;60: 115e27. [68] Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 2005;437:426e31. [69] Yamashita H, Ichijo H, Grimsby S, More ´n A, Dijke PT, Miyazono K. Endoglin forms a heterodimeric complex with the signaling receptors for transforming growth factor-b*. J Biol Chem 1994;269:1995e2001. [70] Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 2004;84:869e901. [71] Meng QJ, Lux A, Holloschi A, Li J, Hughes J, Foerg T, et al. Identification of Tctex2b, a novel dynein light chain family member that interacts with different transforming growth factor-b receptors. J Biol Chem 2006;281:37069e80. [72] Masszi A, Fan L, Rosivall LM, McCulloch CA, Rotstein L, Mucsi I, et al. Integrity of cellecell contacts is a critical regulator of TGF-b1 induced epithelial-to-myofibroblast transition. Role for b-catenin. Am J Pathol 2004;165:1955e67. [73] Arciniegas E, Sutton AB, Allen TD, Schor AM. Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 1992;103:521e9. [74] Ishisaki A, Hayashi H, Li AJ, Imamura T. Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells. J Biol Chem 2003;278: 1303e9.
journal homepage: www.elsevier.com/locate/bihy
An alternate insulin-like growth factor I receptor signaling pathway for the progression of endothelialemesenchymal transition Enrique Arciniegas a,*, Daniel Candelle b a
Servicio Auto´nomo Instituto de Biomedicina, Facultad de Medicina, Universidad Central de Venezuela, Apartado de correos 4043, Carmelitas, Caracas 1010, Venezuela b Universidad Nacional Experimental Francisco de Miranda, Area Ciencias de la Salud, Coro, Estado Falco´n, Venezuela Received 16 July 2008; accepted 21 July 2008
KEYWORDS Endotheliale mesenchymal transition; Cellecell contacts; Microtubules
Abstract Emerging evidence suggests that endothelial-to-mesenchymal transition (EndoMT) is an important contributor to cardiovascular diseases and to vascular development and pathologies as well as in cancer progression. As in epithelialemesenchymal transition (EMT), EndoMT may involve several regulated steps: disassembly of adherence junctions or loss of cellecell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation. Loss of cellecell contacts is a necessary and sufficient step in the progression of EndoMT. In endothelial cells, adherence junctions are composed of transmembrane adhesive proteins belonging to the cadherin family, with the VE-cadherin being the most important. This protein interacts with b-catenin, which links cadherin to the actin cytoskeleton. Tyrosine phosphorylation of both VE-cadherin and b-catenin is considered an important mechanism associated to the disassembly of adherence junctions or loss of celle cell contacts. Insulin-like growth factor receptor I (IGFIR) is a transmembrane tyrosine kinase that has been involved in the alterations of cellecell contacts and in the expression of some genes during cancer development and progression. Here, it is hypothesized that IGFIR autophosphorylation may initiate a signaling pathway that would lead to the loss of cellecell contacts or adherence junctions, remarkable remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here. ª 2008 Elsevier Ltd. All rights reserved.
Introduction * Corresponding author. Tel.: þ58 212 860 4630; fax: þ58 212 861 1258. E-mail address: [email protected] (E. Arciniegas).
Endothelial-to-mesenchymal transition (EndoMT) is a process by which certain endothelial cell (EC) subsets lose
1756-2392/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bihy.2008.07.012
Insulin-like growth factor I receptor signaling pathway endothelial characteristics and transform into mesenchymal cells or smooth muscle (SM)-like cells [1] (Fig. 1). There is emerging evidence to suggest that this process is an important contributor to cardiovascular diseases [2] and to vascular pathologies such as neointimal thickening formation observed in atherosclerosis and restenosis, and pulmonary vascular remodeling induced by hypoxia [1]. In addition to this role, several groups have provided evidence implying that EndoMT is critical in cardiogenesis and vascular development, granulation tissue formation and in cancer progression [2e4]. As in epithelialemesenchymal transition (EMT), EndoMT seems to progress through a series of important steps whose interdependence and order are not clear as yet [1]. This complex process may involve several characterized regulated steps: disassembly of the adherence junctions or loss of endothelial cellecell contacts, cytoskeletal reorganization, proteases, cytokines and growth factor synthesis and secretion, extracellular matrix remodeling, membrane receptor expression, cell detachment and cell migration and differentiation [1]. Importantly, loss of cell-to-cell contacts is consistently observed at EMT sites during development, and in cancer and fibrosis [5,6]. Regarding the endothelium, endothelial cell-to-cell contacts are maintained by adherens junctions composed of transmembrane adhesive proteins belonging to the cadherin family, with the vascular endothelial (VE)-cadherin being
313 the most important. This protein interacts with cytoplasmic proteins called catenins, in particular b-catenin, which links cadherin to the actin cytoskeleton. VE-cadherin and bcatenin functions are generally related to the maintenance of cellecell contacts and regulation of the intracellular signaling pathways that are implicated in cell growth, apoptosis, matrix, and cytoskeletal remodeling [7]. Tyrosine phosphorylation of both VE-cadherin and b-catenin is considered an important mechanism associated to the disassembly of endothelial adherence junctions [7,8]. In this respect, there are reports showing that disruption of endothelial cellecell contacts may occur in response to mechanical stimuli and/or soluble growth factors such as FGF-2, TGFb, and insulin-like growth factor II (IGFII) [9e11], and that this event is a necessary and sufficient step in the progression of EndoMT [9]. However, neither the molecular and cellular mechanisms operating to cause the endothelial transformation nor the signaling cascades controlling it have been elucidated. In this article, it is speculated that the binding of IGFII to the insulin-like growth factor receptor I (IGFIR) may initiate several signaling cascades that would lead to the loss of cellecell contacts or adherence junctions, dramatic remodeling of the cytoskeleton, increased cell motility, and finally to the progressive transition towards a mesenchymal phenotype. Data supporting this hypothesis are presented here.
Figure 1 Confocal laser scanning microscope fluorescence (CLSM) images of vWf and a-SM actin expression in a monolayer of endothelial cells attached to fibronectin after 48 h in culture in complete medium. Strong immunoreactivity for vWf (red) in a granular pattern typical of ECs, and for a-SM actin (green) delineating cellular margin are observed. Double immunofluorescence of the same field shows some of the cells exhibiting immunoreactivity for both vWf and a-SM actin (merged).
314
Involvement of IGFII and IGFIR in the regulation of cellecell contacts Both IGFI and IGFII, in addition to being considered as important factors modulating wound healing, carcinogenesis, cancer progression, metastasis [12e15], as well as several cardiovascular diseases associated to intimal thickening formation [16e18], are also considered to participate during embryonic development [11,19e21]. Of particular significance, ontogenic changes in the circulating levels of both IGFI and IGFII have been reported in the chicken embryo model, suggesting that they may participate in regulating different processes during chicken embryo development [20,22]. Recently, we have suggested a probable role for IGFII and vitronectin in the regulation of endothelial cellecell contacts during vascular remodeling and the EndoMT [11]. However, studies about the effects of these growth factors in vascular development, particularly IGFII, are scarce. Because, there is evidence showing that IGFIIR has no apparent intrinsic signaling transduction potentialities and seems to act as a ‘‘sink’’ for IGFII [13,14,23], we will focus in this article mainly on the role of IGFIR activation. IGFIR is a transmembrane tyrosine kinase that contains two extracellular a-subunits and two intracellular bsubunits. The a-subunits bind IGFI, IGFII, and IGF, whereas the b-subunits transmit ligand-induced signals. Binding of ligands to the extracellular a-subunits results in autophosphorylation of its b-subunits on specific tyrosine residues followed by phosphorylation and recruitment of various cytoplasmic adaptor signaling proteins that include insulinreceptor substrate-1 (IRS-1), its major substrate [24]. Autophosphorylation of IGFIR potentially results in the initiation of some specific signaling pathways that eventually culminates in the activation of some transcription factors. One of them involves phosphorylation of IRS-1 and the subsequent phosphoinositide 3-kinase (PI3K), MAP kinases (MAPKs) and AKT/protein kinase B (AKT/PKB) activation [13,25e27]. The activated AKT then phosphorylates various target proteins, resulting in the regulation of various cellular processes including EMT [28]. Importantly, signals generated through this pathway could also collaborate in cell proliferation and differentiation control and apoptosis [25,26,29]. Another signaling pathway, in response to IGFI binding, would involve the phosphorylation of focal adhesion kinase (FAK) and some of its associated proteins such as paxillin as well as crosstalk with integrins during the cell migration [30]. An alternate signaling pathway has emerged about how IGFIR participates in the alteration of cellecell contacts during cancer development and progression. In fact, there is evidence that ligation of IGFIR by IGFI may also generate signals controlling the cellecell contacts by modulating the activities of the cadherins and catenins as well as of the small Rho GTPases, Rho, Rac-1, and Cdc42 [31]. For instance, binding of IGFI to IGFIR can induce tyrosine phosphorylation of members of the E-cadherinecatenin complex promoting epithelial cell migration [31,32], whereas binding of IGFII induces the cytoplasmic redistribution of IGFIR and E-cadherin and nuclear translocation of b-catenin, where it modulates gene activation, during normal and pathological EMT [33]. In ECs, binding of IGFII to IGFIR could induce cell separation, involving VE-cadherin cytoplasmic relocation and b-catenin nuclear translocation, during the EndoMT
E. Arciniegas, D. Candelle [11]. Others found that ligation of IGFIR by IGFI could induce migration by disruption of a multiproteic complex formed at cellecell contacts sites by IGFIR, E-cadherin, avintegrin in epithelial cells, suggesting that this disruption might involve tyrosine phosphorylation of these proteins [34]. However, previous reports have proposed that IGFIR activation may also occur in a ligand-independent way during malignant transformation [25] and during neointimal formation in vein graft induced by mechanical stretch [18]. From these and other studies, it is clear that activation of the IGFI signaling pathways is at least critical for cancer development and progression [27,29,31,35]. Less is known regarding the activation of IGFII signaling pathways. However, there is increasing experimental evidence to suggest that IGFII plays an important role in many developmental and pathological processes [15,36]. Even so, the manner by which IGFIR activation contributes to the control of various aspects of cell behavior continues being a subject of debate [37]. Nevertheless, if one assumes that IGFIR activation may be involved in the regulation of endothelial cellecell contacts and the modulation of gene transcription then one can suspect that this could initiate the generation of signaling pathways that govern the EndoMT process. Apart from its association to cancer development and progression [13,23,24,27,29,31], IGFIR expression and signaling have been associated to cardiovascular function and diabetic cardiomyopathy [17].
The IGFIR, VE-cadherin, and b-catenin complex and the regulation of gene expression Cadherins are calcium-dependent proteins which, through their extracellular domain, promote homotypic cellecell interactions and modulate cell migration by affecting adhesion, cytoskeletal organization, and cell polarity [38e 40]. Classical cadherins include epithelial- (E-), vascular endothelial- (VE-) and neural- (N-) cadherins. Several cytoplasmic proteins, such as the catenins, interact with classical cadherins linking it to the actin-associated cytoskeletal proteins and intracellular signaling pathways [7,38]. The architecture of this complex may be disrupted by tyrosine phosphorylation of cadherin and catenin, involving specific tyrosine kinases. In epithelial cells, where b-catenin is bound to E-cadherin, tyrosine phosphorylation of these proteins by tyrosine kinases such as IGFIR, not only provokes the dissociation of b-catenin from E-cadherin affecting celle cell contacts, but also allows its relocation to the cytoplasm and the nucleus where it may activate the transcription of target genes [32,41]. Interestingly NF-kB, a transcriptional factor that regulates the expression of hundred genes and whose activation proceeds rapidly and depends on the type and intensity of the stimulus, has been shown to be activated by IGFII stimulation during skeletal muscle differentiation [42] and in human keratinocytes [43]. With respect to the potential participation of NF-kB in the EMT process, studies in epithelial cells have indicated that constitutively active IGFIR causes EMT associated with increased NF-kB activity [44]. Significantly, recent reports have also proposed NF-kB as a mediator of the EMT process [45,46]. In ECs, recent data suggest that the VE-cadherinebcatenin complex in addition to being an important site for,
Insulin-like growth factor I receptor signaling pathway recent data suggest that the VE-cadherineb-catenin complex in addition to being an important site for mechanotransduction and mechanosensing is also involved in the activation of some cell signaling pathways regulating endothelial cell behavior [7]. Like in epithelial cells, disruption of VE-cadherineb-catenin complex may also cause the translocation of b-catenin to the nucleus where it influences gene transcription [7]. Recently, we have shown that IGFII stimulation of embryonic endothelial cells results in the loss of cellecell contacts by the removal of VE-cadherin and redistributions of b-catenin probably conducing to the activation and translocation of NF-kB with the consequent regulation of a-SM actin expression [47]. How these signals generated locally after the activation of IGFIR and disruption of celle cell contacts could be influencing not only the epithelial gene expression but also the endothelial gene expression, is still matter of discussion. It is possible that alterations in the cytoskeleton organization could be contributing to the expression of some genes considering that cadherins in addition to being related to the actin cytoskeleton, also seem to be associated physically with the microtubules (MTs) regulating thus their organization [48].
Microtubules in the dynamics of cellecell contacts The cytoskeleton is considered as a complex network of actin filaments, intermediate filaments, and microtubules (MTs) [49,50] that links the surface of the cell to the nucleus and participates in the formation of adherens junctions and intracellular signal transduction [48,51]. Various lines of evidence indicate that these cytoskeletal components in response to external stimuli can be used as linear tracks to move organelles, secretory vesicles, and protein complexes, within the cell and provide transient docking sites for proteins and lipids. Here, we will discuss some aspects about the MTs and their possible role in the formation or disruption of cellecell contacts as well as the possible repercussion of these events on intracellular signaling, using both the epithelial and endothelial cells as reference models. MTs are highly dynamic polymers of a/b-tubulin heterodimers that appear oriented with their minus ends clustered around the microtubule-organizing center (MTOC) located close to the nucleus and their plus ends oriented towards the cell periphery where they participate in the conformation of the multiproteic complex. In addition to its known role as cell scaffolding and track for the directed delivery of proteins, vesicles and other factors from the cell center to the cell periphery (anterograde transport), recent studies suggest that MTs may also regulate the dynamics of cellecell contacts by the formation or disruption of cadherin-mediated cell contacts which result in alteration of the b-catenin distribution [48]. A notable characteristic of the MTs behavior is its dynamic instability, in which their plus ends undergo frequent periods of slow growth and rapid shrinkage by the addition or loss of tubulin [52e54]. The control of MT dynamics is regulated spatially and temporally by proteins that associate physically with them. In general, every protein that associates with MTs can be considered to be a microtubule-associated protein (MAP). Among this class of proteins are the so called structural MAPs which bind to the MT plus or minus ends, MAPKs also known as extracellular-signal
315 regulated kinases (ERKs), other kinases such as IkB kinase (IKK) and PI3K, MT motor proteins such as cytoplasmic dynein and kinesins, and adaptor proteins. There is evidence that MAP phosphorylation leads to their detachment from the MTs, thereby contributing to the MTs instability [53,55,56]. This phosphorylation seems to be provoked by specific MAPKs that are bound to the MTs via ATPase MT motor proteins, and occurs in response to cell stimulation, thus regulating the MT dynamics and influencing cell signaling. Among the MT motor proteins, one of the most studied is cytoplasmic dynein [57,58]. This protein is actually considered as part of the multiproteic adherens junction complex and as responsible not only for tethering of MTs at sites of epithelial cellecell contacts [59], but also in the anterograde transport of proteins for the assembly of junctions such as b-catenin, acatenin, and p120-catenin [60]. Nonetheless, a possible role for dynein in the retrograde transport of proteins from the cell periphery to the cell center that includes tyrosine phosphorylated b-catenin, during the disruption of cellecell contacts and signal transmission has also been proposed [60].
Microtubules in the regulation of gene expression Current knowledge indicates that regulation of MT dynamics by a variety of signals, including mechanical stimuli, kinase inhibitors, and soluble growth factors would have an important repercussion on the behavior of cancer and endothelial cells [53]. Little is known about how extracellular stimuli regulate the MT dynamics in these cells. However, punctual studies have proposed that modulation of MT dynamics is also crucial for the nuclear or retrograde transport of activated NF-kB and control of gene expression [53]. For instance, disruption of MTs in epithelial cells activates NF-kB and induces the expression of several genes that are regulated by this transcriptional factor. This activation probably requires either direct degradation of inhibitor kB-alpha (IkB-a), which would be linked to MTs via dynein, or degradation of or degradation of this inhibitor through an activated specific MAPK or kinase that would also be an activated specific MAPK or kinase that would also be anchored to MTs, via dynein [61e64]. By contrast, in neuronal cells, a functional cytoskeleton seems to be required for the retrograde transport of activated NF-kB mediated by dynein, after phosphorylation and degradation of IkB-a which is located at the cytoplasm instead of being anchored to the MTs [65]. From these evidences it is clear that the normal nuclear NF-kB transport seems to depend on the MT dynamics, considering that the disruption of MTs affects both the retrograde transport of NF-kB and the NFkB-dependent transcriptional activity and that the alterations in the cytoskeleton, in part occasioned by the disruption of cellecell contacts also contribute with the upregulation of the NF-kB activity [66]. As already indicated, separate studies by different investigators using different systems concerning ECs, have emphasized that vascular remodeling and neointimal formation might be the results of blood pressure alterations and that such alterations might generate a range of responses involving the disturbance of MT dynamics [51,67] as well as the activation and translocation of transcriptional factors such as NF-kB, leading to gene expression
316 modulation [68]. However, a link among these different responses as well as a link between them and the EndoMT process have not been explored. Therefore, more studies are obviously necessary to gain insights into the mechanisms regulating the MT dynamics during the transition of endothelial cells towards a mesenchymal phenotype.
How IGFIR activation contributes to the transformation of endothelium derived mesenchymal cells into a smooth muscle-like phenotype As already mentioned, phosphorylation or activation of IGFIR is involved in the alteration of cellecell contacts and in the expression of some genes, but how this activation collaborates in the transformation of epithelium or endothelium derived mesenchymal cells into a SM-like phenotype still remains to be determined. It is possible that the binding of IGFII to IGFIR also may modulate, through lateral interactions, the activation or phosphorylation of endoglin, a receptor that is expressed on the surface of vascular endothelial cells and is concentrated at endothelial cellecell contacts mediating the functions of TGFb superfamily members [69,70]. This inference would be based on the fact that endoglin, apart from its close association with other TGFb receptors such as TbRII, is also able to
E. Arciniegas, D. Candelle interact with a member of the dynein family regulating not only its own retrograde transport but also the activated TGFb signal transduction pathway [71]. This is of great interest since the presence of activated TGFb isoforms has been associated with alterations in cadherin expression and bcatenin redistribution and the modulation of NF-kB activity not only during EMT but also in EndoMT [1,10,45,72]. Moreover, activated TGFb-1 has been shown to promote the conversion of endothelial cells into SM-like cells [9,73,74]. Thus, it is possible to speculate that activated IGFIR, in functional cooperation with endoglin, may participate in the transformation of endothelial cells into mesenchymal cells or SM-like cells (defined by the expression of a-SM actin). It is also possible that endoglin activation provokes the activation of IGFIR, considering that the activation of this receptor may also occur in a ligand-independent manner.
The hypothesis In conclusion, a complex cascade of signals that may be generated after the binding of IGFII to IGFIR may result in: (a) IGFIR tyrosine autophosphorylation, (b) VE-cadherin and b-catenin tyrosine phosphorylation, (c) perturbation of microtubule (MT) dynamics involving phosphorylation of MT-associated proteins (MAPs), MAP kinases (MAPKs), kinases and phosphatases as well as alteration of actin filaments and intermediate filaments organization, (d)
Figure 2 Schematic representation of some of the events that may occur after the binding of IGFII to IGFIR (1). Retrograde transport along MTs of b-catenin and NF-kB (2). Anterograde transport of intermediate filaments (3). Regulation of target genes (4) and functional cooperation of endoglin/TbIIR complex (5).
Insulin-like growth factor I receptor signaling pathway retrograde transport along MTs of molecules such as phospho-b-catenin and NF-kB and anterograde transport of intermediate filaments, both assisted by MT motor proteins that include cytoplasmic dynein, (e) regulation of target genes, and (f) functional cooperation of endoglin during the transformation of endothelial cells into mesenchymal or SM-like cells (Fig. 2). The relevance of knowing which are the signaling pathways generated when IGFIR is activated and how these signals lead to the endothelial cell transformation resides in the possibility of explaining several of the cellular events occurring not only during embryonic heart development but also in the context of the pathogenesis of various cardiovascular diseases occurring not solely in adult life but also throughout childhood and adolescence.
Acknowledgments This work was supported by the Fondo Nacional de Investigaciones Cientı´ficas y Tecnolo ´gicas (FONACIT) (grant n G2005000405).
References [1] Arciniegas E, Frid MG, Douglas IS, Stenmark KR. Perspectives on endothelial-to-mesenchymal transition: potential contribution to vascular remodeling in chronic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2007;293:L1e8. [2] Zeisberg EM, Tarnavski O, Zeisberg M, Dorfman A, McMullen J, Gustafsson E, et al. Endothelial-to-mesenchymal transition contributes to cardiac fibrosis. Nat Med 2007;13:952e61. [3] Mironov V, Visconti RP, Markwald RR. On the role of shear stress in cardiogenesis. Endothelium 2005;12:259e61. [4] Zeisberg EM, Potenta S, Xie L, Zeisberg M, Kalluri R. Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts. Cancer Res 2007;67:10123e81. [5] Thiery JP, Sleeman JP. Complex networks orchestrate epithelialemesenchymal transitions. Nat Rev Mol Cell Biol 2006; 7:131e42. [6] Baum B, Settleman J, Quinlan MP. Transitions between epithelial and mesenchymal states in development and disease. Semin Cell Dev Biol 2008;19:294e308. [7] Liebner S, Cavallaro U, Dejana E. The multiple languages of endothelial cell-to-cell communication. Arterioscler Thromb Vasc Biol 2006;26:1431e8. [8] van Buul JD, Anthony EC, Fernandez-Borja M, Burridge K, Hordijk PL. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cellecell adhesion by regulating b-catenin tyrosine phosphorylation. J Biol Chem 2005;280:21129e36. [9] Frid MG, Kale VA, Stenmark KR. Mature vascular endothelium can give rise to smooth muscle cells via endothelial mesenchymal transdifferentiation: in vitro analysis. Circ Res 2002; 90:1189e96. [10] Liebner S, Catellino A, Gallini R, Rudini N, Iurlaro M, Piccolo S, et al. b-catenin is required for endothelialemesenchymal transformation during heart cushion development in the mouse. J Cell Biol 2004;166:359e67. [11] Arciniegas E, Neves YC, Carrillo LM. Potential role for insulin-like growth factor II and vitronectin in the endothelialemesenchymal transition process. Differentiation 2006;74:277e92. [12] Le Roith D, Roberts Jr CT. The insulin-like growth factor system and cancer. Cancer Lett 2003;195:127e37. [13] Pollak MN, Schernhammer ES, Hankinson SE. Insulin-like growth factors and neoplasia. Cancer 2004;4:507e18.
317 [14] Denley A, Cosgrove LJ, Booker GW, Wallace JC, Forbes BE. Molecular interactions of the IGF system. Cytokine Growth Factor Rev 2005;16:421e39. [15] Chao W, D’Amore PA. IGF2: epigenetic regulation and role in development and disease. Cytokine Growth Factor Rev 2008; 19:111e20. [16] Zaina S, Nilsson J. Insulin-like growth factor II and its receptors in atherosclerosis and in conditions predisposing to atherosclerosis. Curr Opin Lipidol 2003;14:483e9. [17] Opgaard OS, Wang PH. IGF-I is matter of heart. Growth Horm IGF Res 2005;15:89e94. [18] Cheng J, Du J. Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulinlike growth factor I receptor. Arterioscler Thromb Vasc Biol 2007;27:1744e51. [19] Louvi A, Accili D, Efstratiadis A. Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev Biol 1997;189:33e48. [20] Scanes CG, Thommes RC, Radecki SV, Buonomo FC, Woods JE. Ontogenic changes in the circulating concentrations of insulin-like growth factor (IGF)-I, IGF-II, and IGFbinding proteins in the chicken embryo. Gen Comp Endocrinol 1997;106:265e70. [21] Allan GJ, Zannoni A, McKinnell I, Otto W, Holzenberger M, Flint DJ, et al. Major components of the insulin-like growth factor axis are expressed early in chicken embryogenesis, with IGF binding protein (IGFBP) expression subject to regulation by sonic hedgehog. Anat Embryol 2003;207:73e84. [22] McMurtry JP, Rosebrough RW, Brocht DM, Francis GL, Upton Z, Phelps P. Assessment of developmental changes in chicken and turkey insulin-like growth factor-II by homologous radioimmunoassay. J Endocrinol 1998;157:463e73. [23] Yee D. Targeting insulin-like growth factor pathways. Br J Cancer 2006;94:465e8. [24] Baserga R, Peruzzi F, Reiss K. The IGF-I receptor in cancer biology. Int J Cancer 2003;107:873e7. [25] Valentinis B, Baserga R. IGF-I receptor signalling in transformation and differentiation. J Clin Pathol Mol Pathol 2001; 54:133e7. [26] Wu Y, Karas M, Dupont J, Zhao H, Toyoshima Y, Le Roith D. Multiple signaling pathways are involved in the regulation of IGF-I receptor inhibition of PTEN-enhanced apoptosis. Growth Horm IGF Res 2004;14:52e8. [27] Riedemann J, Macaulay VM. IGFIR signalling and its inhibition. Endocr Relat Cancer 2006;13:S33e43. [28] Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, Van Roy F, et al. Activation of NF-kappaB by Akt upregulates snail expression and induces epithelium mesenchyme transition. Oncogene 2007;26:7445e56. [29] Ryan PD, Goss PD. The emerging role of the insulin-like growth factor pathway as a therapeutic target in cancer. Oncologist 2008;13:16e24. [30] Shen MR, Hsu YM, Hsu KF, Chen YF, Tang MJ, Chou CY. Insulinlike growth factor 1 is a potent stimulator of cervical cancer cell invasiveness and proliferation that is modulated by alphavbeta3 integrin signaling. Carcinogenesis 2006;27:962e71. [31] Mauro L, Surmacz E. IGF-I receptor, cellecell adhesion, tumour development and progression. J Mol Histol 2004;35:247e53. [32] Mauro L, Salerno M, Morelli C, Boterberg T, Bracke ME, Surmacz E. Role of the IGF-I receptor in the regulation of cellecell adhesion: implications in cancer development and progression. J Cell Physiol 2002;194:108e16. [33] Morali OG, Delmas V, Moore R, Jeanney C, Thiery JP, Larue L. IGF-II induces rapid b-catenin relocation to the nucleus during epithelium to mesenchyme transition. Oncogene 2001;20: 4942e50. [34] Canonici A, Steelent W, Rigot V, Khomitch-Baud A, BoutaghouCherid, Bruyneel E, et al. Insulin-like growth factor I receptor,
318
[35]
[36]
[37] [38] [39]
[40] [41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49] [50] [51]
[52] [53]
[54]
E. Arciniegas, D. Candelle E-cadherin and av integrin form a dynamic complex under the control of a-catenin. Int J Cancer 2008;122:572e82. Allison AS, McIntyre MA, McArdle C, Habib FK. The insulin-like growth factor type I receptor and colorectal neoplasia: insights into invasion. Hum Pathol 2007;38:1590e602. Reik W, Constancia M, Dean W, Davies K, Bowden L, Murrel A, et al. IGF2 imprinting in development and disease. Int J Dev Biol 2000;44:145e50. Baserga R. The contradictions of the insulin-like growth factor I receptor. Oncogene 2000;19:5574e81. Takeichi M. Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 1995;7:619e27. Conacci-Sorrel M, Zhurinsky J, Ben-Ze’ev A. The cadherine catenin adhesion system in signaling and cancer. J Clin Invest 2002;109:987e91. Chen X, Gumbiner BM. Crosstalk between different adhesion molecules. Curr Opin Cell Biol 2006;18:572e8. Chen J, Wu A, Sun H, Drakas R, Garofalo C, Cascio S, et al. Functional significance of type I insulin-like growth factor mediated nuclear translocation of the insulin receptor substrate-1 and b-catenin. J Biol Chem 2005;280:29912e20. Kalliman P, Canicio J, Testar J, Palacin M, Zorzano A. Insulinlike growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kB and inducible nitric-oxide synthase define a common myogenic signaling pathway. J Biol Chem 1999;274: 17437e44. Kwon YW, Jang ER, Lee YM, Kim YS, Kwon KS, Jang HS, et al. Insulin-like growth factor-II induces interleukin-6 expression via NF-kB activation in psoriasis. Biochem Biophys Res Commun 2000;278:312e7. Kim HY, Litzenburger BC, Cui X, Delgado DA, Grabiner BC, Lin X, et al. Constitutively active type I insulin-like growth factor receptor causes transformation and xenograft growth of immortalized mammary epithelial cells and is accompanied by an epithelial-to-mesenchymal transition mediated by NFkB and snail. Mol Cell Biol 2007;27:3165e75. Huber MA, Azoitel N, Baumann B, Gru ¨nert S, Sommer A, Pehamberger H, et al. NF-kB is essential for epitheliale mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest 2004;114:569e81. Chua HL, Bhat-Nakshatri P, Clare SE, Morimiya A, Badve S, Nakshatri H. NF-kB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. Oncogene 2007;26:711e24. Arciniegas E, Carrillo LM, De Sanctis JB, Candelle D. Possible role of NF-kB in the embryonic vascular remodeling and the endothelialemesenchymal transition process. Cell Adhesion Migration 2008;2:1e13. Stehbens SJ, Paterson AD, Crampton MS, Shewan AM, Ferguson C, Akhmanova A, et al. Dynamic microtubules regulate the local concentration of E-cadherin at cellecell contacts. J Cell Sci 2006;119:1801e11. Chang L, Goldman RD. Intermediate filaments mediate cytoskeletal crosstalk. Mol Cell Biol 2004;5:601e13. Stamenovic D. Effects of cytoskeletal prestress on cell rheological behavior. Acta Biomater 2005;1:255e62. Galbraith CG, Skalah R, Chien S. Shear stress induces spatial reorganization of the endothelial cell cytoskeleton. Cell Motil Cytoskeleton 1997;40:317e30. Mitchison T, Kirschner M. Dynamic instability of microtubule growth. Nature 1984;312:237e42. Honore S, Pasquier E, Braguer D. Understanding microtubule dynamics for improved cancer therapy. Cell Mol Life Sci 2005; 62:3039e56. Akhmanova A, Steinmetz MO. Tracking the ends: a dynamic protein network controls the fate of microtubule tips. Nat Rev 2008;9:309e22.
[55] Kapeller R, Toker A, Cantley LC, Carpenter CL. Phosphoinositide 3-kinase binds constitutively to a/b-tubulin and binds to gtubulin in response to insulin. J Biol Chem 1995;270:25985e91. [56] Reszka AA, Seger R, Diltz CD, Krebs EG, Fischer EH. Association of mitogen-activated protein kinase with the microtubule cytoskeleton. Proc Natl Acad Sci U S A 1995;92:8881e5. [57] Burgess SA, Walker ML, Sakakibara H, Knight PJ, Oiwa K. Dynein structure and power stroke. Nature 2003;421:715e8. [58] Mizuno N, Narita A, Kon T, Sutoh K, Kikkawa M. Threedimensional structure of cytoplasmic dynein bound to microtubules. Proc Natl Acad Sci U S A 2007;104:20832e7. [59] Ligon LA, Karki S, Tokito M, Holzbaur ELF. Dynein binds to bcatenin and may tether microtubules at adherens junctions. Nat Cell Biol 2001;3:913e7. [60] Ligon LA, Holzbaur ELF. Microtubules tethered at epithelial cell junctions by dynein facilitate efficient junction assembly. Traffic 2007;8:808e19. [61] Rossette C, Karin M. Cytoskeletal control of gene expression: depolymerization of microtubules activates NF-kB. J Cell Biochem 1995;128:1111e9. [62] Cre ´pieux P, Kwon H, Leclerc N, Spencer W, Richard S, Lin R, et al. IkBa physically interacts with a cytoskeleton-associated protein through its signal response domain. Mol Cell Biol 1997; 17:7375e85. [63] Bourgarel-Rey V, Vallee S, Rimet O, Champion S, Braguer D, Desobry A, et al. Involvement of nuclear factor kB in c-Myc induction by tubulin polymerization inhibitors. Mol Pharmacol 2001;59:1165e70. [64] Mistry P, Deacon K, Mistry S, Blank J, Patel R. NF-kB promotes survival during mitotic cell cycle arrest. J Biol Chem 2004;279: 1482e90. [65] Mikenberg I, Widera D, Kaus A, Kaltschmidt B, Kaltschmidt C. Transcription factor NF-kB is transported to the nucleus via cytoplasmic dynein/dynactin motor complex in hippocampal neurons. PloS ONE 2007;2:e589. [66] Kuphal S, Poser I, Jobin C, Hellerbrand C, Bosserhoff AK. Loss of E-cadherin leads to upregulation of NF-kB activity in malignant melanoma. Oncogene 2004;23:8509e19. [67] Lee T-YJ, Gotlieb AI. Microfilaments and microtubules maintain endothelial integrity. Microsc Res Tech 2003;60: 115e27. [68] Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 2005;437:426e31. [69] Yamashita H, Ichijo H, Grimsby S, More ´n A, Dijke PT, Miyazono K. Endoglin forms a heterodimeric complex with the signaling receptors for transforming growth factor-b*. J Biol Chem 1994;269:1995e2001. [70] Bazzoni G, Dejana E. Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiol Rev 2004;84:869e901. [71] Meng QJ, Lux A, Holloschi A, Li J, Hughes J, Foerg T, et al. Identification of Tctex2b, a novel dynein light chain family member that interacts with different transforming growth factor-b receptors. J Biol Chem 2006;281:37069e80. [72] Masszi A, Fan L, Rosivall LM, McCulloch CA, Rotstein L, Mucsi I, et al. Integrity of cellecell contacts is a critical regulator of TGF-b1 induced epithelial-to-myofibroblast transition. Role for b-catenin. Am J Pathol 2004;165:1955e67. [73] Arciniegas E, Sutton AB, Allen TD, Schor AM. Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro. J Cell Sci 1992;103:521e9. [74] Ishisaki A, Hayashi H, Li AJ, Imamura T. Human umbilical vein endothelium-derived cells retain potential to differentiate into smooth muscle-like cells. J Biol Chem 2003;278: 1303e9.