- Email: [email protected]
Matrix-degrading podosomes in smooth muscle cells
ARTICLE IN PRESS
European Journal of Cell Biology 85 (2006) 183–189 www.elsevier.de/ejcb
REVIEW
Matrix-degrading podosomes in smooth muscle cells Thomas Lenera, Gerald Burgstallerb,1, Luca Crimaldic, Sibylle Lachc, Mario Gimonac, a
Division of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria Department of Cell Biology, Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstr. 11, A-5020 Salzburg, Austria c Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8a, I-66030 Santa Maria Imbaro, Italy b
Abstract Activation of protein kinase C by phorbol esters triggers the remodelling of the actin cytoskeleton and the formation of podosomes in smooth muscle cells (SMCs). Regional control of actin dynamics at specialised microdomains results in a local reduction in contractile forces. The molecular basis for this local inhibition of contractility includes the clustering of cortactin during podosome formation (which precedes the rapid, local dispersion of myosin, tropomyosin and h1 calponin), and the specific recruitment of 110-kDa actin filament-associated protein (AFAP-110) and 190-kDa Rho-specific GTPase-activating protein (p190RhoGAP) to the microdomains. Podosome formation also correlates with cell polarisation, the induction of cell motility, and local degradation of the extracellular matrix. These findings may provide explanations for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis, and support the concept that matrix degradation and the concomitant engagement of the molecular machinery initiating actin-based cell motility drive tissue invasion in smooth muscle. r 2005 Elsevier GmbH. All rights reserved. Keywords: Podosomes; Cell motility; Smooth muscle; Matrix resorption; Cytoskeleton; Phenotypic plasticity
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Podosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Podosomes in vascular and visceral smooth muscle cells . . . . . . . The microdomain – where podosome formation begins . . . . . . . . Inhibition of podosome formation by increased actin stabilisation Smooth muscle cell polarisation requires flexible substrates . . . . . Matrix degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions, hypotheses and future challenges . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
184 184 184 184 186 186 187 187 187 188
Abbreviations: AFAP-110, 110-kDa actin filament-associated protein; Arp2/3, Protein complex containing actin-related proteins 2 and 3; ECM, Extracellular matrix; h1 CaP, High-molecular-weight calponin variant 1; p190RhoGAP, 190-kDa Rho-specific GTPase-activating protein; SMC, Smooth muscle cell; WASp, Wiskott Aldrich syndrome protein Corresponding author. Tel.: +39 0872 570 293; fax: +39 0872 570 412. E-mail address: [email protected] (M. Gimona). 1 Present address: Department of Molecular Cell Biology, Campus Vienna Biocenter 4, Dr. Bohr Gasse 9, A-1030 Vienna, Austria. 0171-9335/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2005.08.001
ARTICLE IN PRESS 184
T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
Introduction Directed cell migration and tissue invasion of vascular smooth muscle cells (SMCs) are key elements in the development and progression of vascular diseases. Two necessary prerequisites for the transmigration of vascular SMCs from the medial to the intimal layer of the artery are seen in the phenotypic modulation from the contractile (differentiated) to the synthetic (proliferative) state (Campbell and Chamley-Campbell, 1981), and in the proteolytic processes mediating extracellular matrix (ECM) degradation (Pauly et al., 1994; Newby and Zaltsman, 1999; Goodall et al., 2002; Aguilera et al., 2003). Albeit both in vitro cell motility and the production of matrix metalloproteinases (MMPs) have been described independently, the unifying molecular mechanisms that would support such a scenario have only recently been unravelled. SMCs form podosomes in response to phorbol-12,13dibutyrate (PDBu) (Hai et al., 2002; Webb et al., 2005). These dynamic organelles are critical for cell adhesion and substrate degradation, and are typically found in cells that can cross tissue boundaries. Podosomes and the structurally related invadopodia (Buccione et al., 2004) both have elementary roles in many different aspects of cell invasion of tissues. In particular, substrate degradation by tumour cells is confined to punctate areas that correspond in both size and location to the appearance of podosomes (Chen et al., 1984; Mizutani et al., 2002; Baldassarre et al., 2003). Thus, the ability of SMCs to form podosomes in response to activation of protein kinase C (PKC) has fuelled recent research activities that are aimed at deciphering the potential diacylglycerolmediated mechanisms for cytoskeleton remodelling and basement membrane rearrangements (Hai et al., 2002; Burgstaller and Gimona, 2004, 2005; Webb et al., 2005). In this review, we highlight the emerging concepts of podosome formation in SMCs, and discuss the potential pathways and molecular components that are involved in their regulation, stabilisation and turnover, as well as the potential roles that podosomes have in smooth muscle physiology and pathology.
Podosomes In addition to stress-fibre-anchoring focal adhesions, only a few cell types can form the transient adhesive structures that are known as podosomes (Tarone et al., 1985; Marchisio et al., 1988). These structures appear as rings of variable sizes that are initially identified as dynamic sites of actin polymerisation activity, and that can fuse into larger ribbons and rings with a wide inner cavity (Fig. 1).
While these specialised adhesions have long been confined to cells derived from the monocytic lineage (primarily osteoclasts and macrophages) and to cells transformed by the Rous sarcoma virus, recent advances have also identified similar structures in other nontransformed cell types, such as endothelial cells (Moreau et al., 2003; Osiak et al., 2005), epithelial cells (Spinardi et al., 2004) and SMCs (Hai et al., 2002). In the latter case, the formation of podosomes is elicited by contraction-inducing phorbol esters, such as PDBu and 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulate the activation of the PKCa and d isoforms (Brandt et al., 2002, 2003; Yang and Kazanietz, 2003).
Podosomes in vascular and visceral smooth muscle cells To date, there are only a few SMC lines that express critical markers of smooth muscle differentiation, such as h1 calponin (h1 CaP) and SM22. The A7r5 rat vascular SMC line (Kimes and Brandt, 1976) has proven to be a reliable model to investigate podosome formation in vitro. Studies on this cell line have revealed the characteristic structure of podosomes using electron microscopy, confirmed the co-localisation of established podosome marker proteins, and demonstrated the pathways of signalling events via the PAK-PIX route (Webb et al., 2005). Notably, podosome formation is not confined to vascular SMCs, and we have verified the potential of podosome formation also in a visceral SMC line, namely the human intestinal smooth muscle (HISM) line (M. Gimona, unpublished; Fig. 2). As with the rat vascular SMCs, human visceral SMCs respond to phorbol esters with the formation of podosome-like structures. HISM cells in culture retain a higher degree of bipolarity and display an elongated rather than rounded phenotype. In contrast to vascular SMCs, podosomes are rarely seen in the periphery of these cells, and here the podosomes commonly resemble the rosette-type arrangements that have also been described for epithelial cells.
The microdomain – where podosome formation begins Cultured A7r5 SMCs exhibit a well-defined and robust actin cytoskeleton. These features allow the study of podosome formation and dynamics on the background of highly organised actin filaments. Due to the unusually large size of rat vascular SMCs in culture (with diameters of up to 180 mm), a specialised zone that is termed the microdomain has been described
ARTICLE IN PRESS T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
185
Fig. 1. Stabilised, mature podosomes in cultured vascular SMCs form larger ribbon or ring structures (arrows in A). Individual podosomes are still discernible by individual clusters of cortactin (arrowheads in B).
Fig. 2. Large podosome rosettes in vascular and visceral SMCs.
ARTICLE IN PRESS 186
T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
(Kaverina et al., 2003; Burgstaller and Gimona, 2004). These microdomains were initially characterised as a region close to where actin bundles terminate in focal adhesions sites, but which are not decorated by phalloidin. Subsequent live-cell imaging has revealed that podosome formation in SMCs originates at these sites, which are also marked by the presence of clusters of the actin-binding and protein complex containing actin-related proteins 2 and 3 (Arp2/3)-activating protein cortactin. Translocation and clustering of the Arp2/3 complex to the sites of SMC podosome formation is an early event. The presence of cortactinenriched foci within the specialised microdomains at the focal adhesion-stress fibre interface in unstimulated SMCs suggests that these sites are designated areas for cytoskeletal remodelling that recruit, stabilise and activate the actin polymerisation machinery following the appropriate stimuli. Thus, the sites of podosome formation provide a second subcellular location for intense Arp2/3-dependent actin polymerisation at the inner face of focal contacts. However, the formation of podosomes cannot correspond only to the rapid synthesis of new actin filaments, but must be preceded by local rearrangements of the existing actin cytoskeleton and the tension-generating anchorage sites. Focal adhesions indeed disassemble gradually during the dynamic formation of podosomes, and turn over in spite of a stable and contractile central cytoskeleton. Actin severing and depolymerisation may generate short nucleation sites for the newly forming, branched actin filaments that are used by the Arp2/3 machinery to rapidly generate the podosome core. Podosome formation is characterised by a concentrated burst of actin polymerisation in the podosome core, and this is preceded by the removal of actin-binding and -stabilising components, such as myosin II, tropomyosin and CaP, and the depolymerisation of the existing uncoated actin filaments. Consistent with the local dispersion of myosin and tropomyosin, podosome formation includes the rapid recruitment and accumulation of the Src activator 110-kDa actin filamentassociated protein (AFAP-110) and of 190-kDa Rhospecific GTPase-activating protein (p190RhoGAP) (Burgstaller and Gimona, 2004). AFAP-110 is involved in the spatial organisation of the sites of actin polymerisation via the Arp2/3 complex (Weed et al., 2000), and consequently in podosome formation (Gatesman et al., 2004), while p190RhoGAP appears to mediate the spatially restricted inactivation of RhoA (Nakahara et al., 1998). Thus, processes governing actin filament depolymerisation and the subsequent Arp2/3dependent filament polymerisation at the microdomains result in a local reduction in the contractile forces that appears sufficient to destabilise focal adhesions. The signalling cascade(s) initiated by PKC activation (Yang and Kazanietz, 2003) and Src phosphorylation (Hai et
al., 2002; Brandt et al., 2002) that converge at the actin cytoskeleton thus act asynchronously.
Inhibition of podosome formation by increased actin stabilisation The dramatic increase in actin polymerisation in the podosome core argues for a highly dynamic actin turnover at the microdomains. The phenotypic plasticity of SMCs combined with the rapid and transient formation of podosomes supports the concept that the engagement of the molecular machinery that initiates actin-based cell motility drives cell migration and tissue invasion in this cell type. Strikingly, phenotypic plasticity, SMC migration and podosome formation are suppressed by high expression levels of the actinbinding protein h1 CaP via the ability of CaP to regulate actin filament turnover (Gimona et al., 2003). High levels of high-molecular-weight calponin variant 1 (h1 CaP) expression are known to be required for the maintenance of the normal SMC phenotype (Horiuchi et al., 1998, 1999; Meehan et al., 2002), while downregulation of h1 CaP is generally accompanied by SMC de-differentiation (Gimona et al., 1990; Horiuchi et al., 1999) and malignant alterations in smooth muscle tissue (Takeoka et al., 2002). The general role of h1 CaP as a potent regulator of actin stability and an inhibitor of actin turnover has also been documented by its ability to inhibit metastatic cell motility in vitro (Lener et al., 2004).
Smooth muscle cell polarisation requires flexible substrates Tissue rigidity has an important role in a number of normal and pathological processes involving cell locomotion (Lo et al., 2000). On a rigid glass surface, A7r5 cells display a round shape with prominent actin stress fibres and focal adhesions, irrespective of a coating of cross-linked fibronectin or collagen I (Burgstaller and Gimona, 2005). In contrast, when grown on flexible polyacrylamide substrates (Pelham and Wang, 1997) that are coated with either fibronectin or collagen-1, A7r5 cells adopt an elongated appearance. This morphology is retained after PDBu stimulation, when the cells form a pronounced leading edge with an active, polarised lamellipodium, and podosomes are seen to be confined to the region of active actin cytoskeleton remodelling in the lamellipodium. On matrix-coated flexible substrates, vascular SMCs not only develop podosomes, but they also display increased cell polarisation and motility, indicating that the differentiated SMC phenotype is not solely influenced by the type of
ARTICLE IN PRESS T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
ECM, but also by the ability to exert forces and to undergo contraction. Consequently, when vascular SMCs are embedded in a reconstituted basement membrane matrix (MatrigelTM), the actin filaments become aligned along the longitudinal axis in the periphery of the cells. Activation of the PKC cascade(s) by PDBu then causes these spindle-shaped cells to develop numerous podosome-like complexes at the cell periphery, suggesting that podosomes in vascular SMCs are physiological structures that can be stabilised in a three-dimensional environment (Burgstaller and Gimona, 2005).
Matrix degradation MMPs enhance the migratory activity of vascular SMCs, and the invadopodia of metastasising tumour cells appear to coordinate the spatial secretion of these enzymes. In agreement with the situation in invadopodia of cancer cells, the A7r5 rat vascular SMCs are able to degrade a fluorescent fibronectin matrix in the vicinity of their podosomes in response to PDBu. One could thus hypothesize that the potential for podosome formation by cultured SMCs might be important for the remodelling of the actin cytoskeleton, of the denseplaque-associated cell-matrix adhesion sites, and possibly (under malignant de-differentiation) of the surrounding ECM of SMCs in vivo (Galis et al., 1995).
Conclusions, hypotheses and future challenges Podosomes and invadopodia have elementary roles in many different aspects of cell invasion of tissues, and substrate degradation of tumour cells is confined to punctate areas that correspond in both size and location to the appearance of podosomes (Baldassarre et al., 2003). Podosome formation thus provides an elegant explanation for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis. Our current knowledge provides hypotheses for the roles of podosome formation and matrix degradation of vascular SMCs in the progression of atherosclerotic processes. Activation of one or more of the PKC cascades (both PKCa and PKCd have been ascribed roles in cytoskeleton modulation in phorbol-ester-stimulated vascular SMCs) induces cell polarisation and motility, as well as podosome formation and targeted matrix degradation; this also occurs in a three dimensional matrix environment. Temptingly, these findings may help us to resolve the long-standing controversies and uncertainties as to how SMCs escape the barrier of the tunica
187
media and invade the intima in vascular diseases (see also Raines and Ross, 1993). Endothelial and epithelial cells and SMCs have been shown to form podosomes in vitro in response to stimulation with diverse cytokines (Osiak et al., 2005) and diacylglycerol analogues (Brandt et al., 2002; Hai et al., 2002). TGF-b1-, TNF-a- and VEGF-induced podosome formation in SMCs remains, however, to be confirmed experimentally both in vivo and in vitro, and despite remarkable advances, it remains debatable as to whether SMCs actually use their potential for podosome formation under physiological conditions. Little is known about the motility of SMCs in tissues, and it is unclear if smooth muscle injury is a sufficient trigger for the induction of SMCs motility and tissue invasion. On the other hand, the SMC phenotypic plasticity includes a number of hallmarks of tumour cells, like alterations in their protein expression profile. So, there is justifiable hope that the common ability for podosome and invadopodia formation in normal and malignant cells may lead to common aspects with respect to the molecular mechanisms involved in their generation and regulation. A striking feature of these cellular adhesion structures, including podosomes, invadopodia and focal adhesions alike, is that they are composed of essentially the same components and they are controlled by similar modulators (Linder and Aepfelbacher, 2003). However, only podosomes and invadopodia also recruit the Arp2/ 3-dependent, cortactin and Wiskott Aldrich syndrome protein (WASp)-stimulated actin polymerisation machinery, and require a dynamic actin cytoskeleton. In any case, it appears a paradox that only a few cell types can engage in podosome formation. A possible explanation for this phenomenon is the hypothetical existence of an ‘‘adhesion module’’, a biochemical device or programme that creates all of the adhesion structures from the same repertoire of components, generating a different output by differently ‘‘computing’’ the input. The multiprotein complex of the adhesion module may be seen as a ‘‘functional module’’ (as described by Barabasi and Oltvai, 2004; Hartwell et al., 1999). Understanding how such a device triggers the formation of podosomes, and most importantly the more ‘‘aggressive’’ invadopodia instead of (or in addition to) focal adhesions, should further our understanding of cell adhesion and tissue invasion under physiological and pathological conditions.
Acknowledgments The Gimona laboratory is supported by the Marie Curie Excellence Grant ] MEXT-CT-2003-002573 of the European Union. The authors are grateful to Dr. R. Buccione (SMI) for helpful suggestions and discussions,
ARTICLE IN PRESS 188
T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
Dr. C.P. Berrie for editorial assistance, and Drs. J.V. Small (IMBA, Vienna), I.N. Kaverina (Vanderbilt University, Tennessee) and C.M. Hai (Brown University, Rhode Island) for outstanding collaborations.
References Aguilera, C.M., George, S.J., Johnson, J.L., Newby, A.C., 2003. Relationship between type IV collagen degradation, metalloproteinase activity and smooth muscle cell migration and proliferation in cultured human saphenous vein. Cardiovasc. Res. 58, 679–688. Baldassarre, M., Pompeo, A., Beznoussenko, G., Castaldi, C., Cortellino, S., McNiven, M.A., Luini, A., Buccione, R., 2003. Dynamin participates in focal extracellular matrix degradation by invasive cells. Mol. Biol. Cell 14, 1074–1084. Barabasi, A.L., Oltvai, Z.N., 2004. Network biology: understanding the cell’s functional organization. Nat. Rev. Genetics 5, 101–113. Brandt, D., Gimona, M., Hillmann, M., Haller, H., Mischak, H., 2002. Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway. J. Biol. Chem. 277, 20903–20910. Brandt, D.T., Goerke, A., Heuer, M., Gimona, M., Leitges, M., Kremmer, E., Lammers, R., Haller, H., Mischak, H., 2003. Protein kinase C delta induces Src kinase activity via activation of the protein tyrosine phosphatase PTP alpha. J. Biol. Chem. 278, 34073–34078. Buccione, R., Orth, J.D., McNiven, M.A., 2004. Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat. Rev. Mol. Cell Biol. 5, 647–657. Burgstaller, G., Gimona, M., 2004. Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains. J. Cell Sci. 117, 223–231. Burgstaller, G., Gimona, M., 2005. Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 288, 3001–3005. Campbell, G.R., Chamley-Campbell, J.H., 1981. Smooth muscle phenotypic modulation: role in atherogenesis. Med. Hypotheses 7, 729–735. Chen, W.T., Olden, K., Bernard, B.A., Chu, F.F., 1984. Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites. J. Cell Biol. 98, 1546–1555. Galis, Z.S., Muszynski, M., Sukhova, G.K., Simon-Morrissey, E., Libby, P., 1995. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann. NY Acad. Sci. 748, 501–507. Gatesman, A., Walker, V.G., Baisden, J.M., Weed, S.A., Flynn, D.C., 2004. Protein kinase C alpha activates c-Src and induces podosome formation via AFAP-110. Mol. Cell. Biol. 24, 7578–7597. Gimona, M., Herzog, M., Vandekerckhove, J., Small, J.V., 1990. Smooth muscle specific expression of calponin. FEBS Lett. 274, 159–163.
Gimona, M., Kaverina, I., Resch, G.P., Vignal, E., Burgstaller, G., 2003. Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol. Biol. Cell 14, 2482–2491. Goodall, S., Porter, K.E., Bell, P.R., Thompson, M.M., 2002. Enhanced invasive properties exhibited by smooth muscle cells are associated with elevated production of MMP-2 in patients with aortic aneurysms. Eur. J. Vasc. Endovasc. Surg. 24, 72–80. Hai, C.M., Hahne, P., Harrington, E.O., Gimona, M., 2002. Conventional PKC mediates phorbol dibutyrate-induced cytoskeletal remodeling in A7r5 smooth muscle cells. Exp. Cell Res. 280, 64–74. Hartwell, L.H., Hopfield, J.J., Leibler, S., Murray, A.W., 1999. From molecular to modular cell biology. Nature 402, C47–C52. Horiuchi, A., Nikaido, T., Ito, K., Zhai, Y., Orii, A., Taniguchi, S., Toki, T., Fujii, S., 1998. Reduced expression of calponin h1 in leiomyosarcoma of the uterus. Lab. Invest. 78, 839–846. Horiuchi, A., Nikaido, T., Taniguchi, S., Fujii, S., 1999. Possible role of calponin h1 as a tumor suppressor in human uterine leiomyoma. J. Natl. Cancer Inst. 91, 790–796. Kaverina, I., Stradal, T.E., Gimona, M., 2003. Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains. J. Cell Sci. 116, 4915–4924. Kimes, B.W., Brandt, B.L., 1976. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp. Cell Res. 98, 349–366. Linder, S., Aepfelbacher, M., 2003. Podosomes: adhesion hotspots of invasive cells. Trends Cell Biol. 13, 376–385. Lener, T., Burgstaller, G., Gimona, M., 2004. The role of calponin in the gene profile of metastatic cells: inhibition of metastatic cell motility by multiple calponin repeats. FEBS Lett. 556, 221–226. Lo, C.M., Wang, H.B., Dembo, M., Wang, Y.L., 2000. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152. Marchisio, P.C., D’Urso, N., Comoglio, P.M., Giancotti, F.G., Tarone, G., 1988. Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts. J. Cell. Biochem. 37, 151–159. Meehan, K.L., Holland, J.W., Dawkins, H.J., 2002. Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer. Prostate 50, 54–63. Mizutani, K., Miki, H., He, H., Maruta, H., Takenawa, T., 2002. Essential role of neural Wiskott-Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in src-transformed fibroblasts. Cancer Res. 62, 669–674. Moreau, V., Tatin, F., Varon, C., Genot, E., 2003. Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA. Mol. Cell. Biol. 23, 6809–6822. Nakahara, H., Mueller, S.C., Nomizu, M., Yamada, Y., Yeh, Y., Chen, W.T., 1998. Activation of beta1 integrin signaling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia. J. Biol. Chem. 273, 9–12.
ARTICLE IN PRESS T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
Newby, A.C., Zaltsman, A.B., 1999. Fibrous cap formation or destruction – the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation. Cardiovasc. Res. 41, 345–360. Osiak, A.E., Zenner, G., Linder, S., 2005. Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling. Exp. Cell Res. 307, 342–353. Pauly, R.R., Passaniti, A., Bilato, C., Monticone, R., Cheng, L., Papadopoulos, N., Gluzband, Y.A., Smith, L., Weinstein, C., Lakatta, E.G., 1994. Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ. Res. 75, 41–54. Pelham, R.J., Wang, Y.L., 1997. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13661–13665. Raines, E.W., Ross, R., 1993. Smooth muscle cells and the pathogenesis of the lesions of atherosclerosis. Br. Heart J. 69, S30–S37. Spinardi, L., Rietdorf, J., Nitsch, L., Bono, M., Tacchetti, C., Way, M., Marchisio, P.C., 2004. A dynamic podosome-like structure of epithelial cells. Exp. Cell Res. 295, 360–375.
189
Takeoka, M., Ehara, T., Sagara, J., Hashimoto, S., Taniguchi, S., 2002. Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells. Eur. J. Cancer 38, 436–442. Tarone, G., Cirillo, D., Giancotti, F.G., Comoglio, P.M., Marchisio, P.C., 1985. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp. Cell Res. 159, 141–157. Webb, B.A., Eves, R., Crawley, S.W., Zhou, S., Cote, G.P., Mak, A.S., 2005. PAK1 induces podosome formation in A5r7 vascular smooth muscle cells in a PIX-dependent manner. Am. J. Physiol. Cell Physiol. (Jun 8; [Epub ahead of print]). Weed, S.A., Karginov, A.V., Schafer, D.A., Weaver, A.M., Kinley, A.W., Cooper, J.A., Parsons, J.T., 2000. Cortactin localization to sites of actin assembly in lamellipodia requires interaction with F-actin and the Arp2/3 complex. J. Cell Biol. 151, 29–40. Yang, C.F., Kazanietz, M.G., 2003. Divergence and complexities in DAG signaling: looking beyond PKC. Trends Pharmacol. Sci. 24, 602–608.
European Journal of Cell Biology 85 (2006) 183–189 www.elsevier.de/ejcb
REVIEW
Matrix-degrading podosomes in smooth muscle cells Thomas Lenera, Gerald Burgstallerb,1, Luca Crimaldic, Sibylle Lachc, Mario Gimonac, a
Division of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria Department of Cell Biology, Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstr. 11, A-5020 Salzburg, Austria c Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale 8a, I-66030 Santa Maria Imbaro, Italy b
Abstract Activation of protein kinase C by phorbol esters triggers the remodelling of the actin cytoskeleton and the formation of podosomes in smooth muscle cells (SMCs). Regional control of actin dynamics at specialised microdomains results in a local reduction in contractile forces. The molecular basis for this local inhibition of contractility includes the clustering of cortactin during podosome formation (which precedes the rapid, local dispersion of myosin, tropomyosin and h1 calponin), and the specific recruitment of 110-kDa actin filament-associated protein (AFAP-110) and 190-kDa Rho-specific GTPase-activating protein (p190RhoGAP) to the microdomains. Podosome formation also correlates with cell polarisation, the induction of cell motility, and local degradation of the extracellular matrix. These findings may provide explanations for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis, and support the concept that matrix degradation and the concomitant engagement of the molecular machinery initiating actin-based cell motility drive tissue invasion in smooth muscle. r 2005 Elsevier GmbH. All rights reserved. Keywords: Podosomes; Cell motility; Smooth muscle; Matrix resorption; Cytoskeleton; Phenotypic plasticity
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Podosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Podosomes in vascular and visceral smooth muscle cells . . . . . . . The microdomain – where podosome formation begins . . . . . . . . Inhibition of podosome formation by increased actin stabilisation Smooth muscle cell polarisation requires flexible substrates . . . . . Matrix degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions, hypotheses and future challenges . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
. . . . . . . . . .
184 184 184 184 186 186 187 187 187 188
Abbreviations: AFAP-110, 110-kDa actin filament-associated protein; Arp2/3, Protein complex containing actin-related proteins 2 and 3; ECM, Extracellular matrix; h1 CaP, High-molecular-weight calponin variant 1; p190RhoGAP, 190-kDa Rho-specific GTPase-activating protein; SMC, Smooth muscle cell; WASp, Wiskott Aldrich syndrome protein Corresponding author. Tel.: +39 0872 570 293; fax: +39 0872 570 412. E-mail address: [email protected] (M. Gimona). 1 Present address: Department of Molecular Cell Biology, Campus Vienna Biocenter 4, Dr. Bohr Gasse 9, A-1030 Vienna, Austria. 0171-9335/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2005.08.001
ARTICLE IN PRESS 184
T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
Introduction Directed cell migration and tissue invasion of vascular smooth muscle cells (SMCs) are key elements in the development and progression of vascular diseases. Two necessary prerequisites for the transmigration of vascular SMCs from the medial to the intimal layer of the artery are seen in the phenotypic modulation from the contractile (differentiated) to the synthetic (proliferative) state (Campbell and Chamley-Campbell, 1981), and in the proteolytic processes mediating extracellular matrix (ECM) degradation (Pauly et al., 1994; Newby and Zaltsman, 1999; Goodall et al., 2002; Aguilera et al., 2003). Albeit both in vitro cell motility and the production of matrix metalloproteinases (MMPs) have been described independently, the unifying molecular mechanisms that would support such a scenario have only recently been unravelled. SMCs form podosomes in response to phorbol-12,13dibutyrate (PDBu) (Hai et al., 2002; Webb et al., 2005). These dynamic organelles are critical for cell adhesion and substrate degradation, and are typically found in cells that can cross tissue boundaries. Podosomes and the structurally related invadopodia (Buccione et al., 2004) both have elementary roles in many different aspects of cell invasion of tissues. In particular, substrate degradation by tumour cells is confined to punctate areas that correspond in both size and location to the appearance of podosomes (Chen et al., 1984; Mizutani et al., 2002; Baldassarre et al., 2003). Thus, the ability of SMCs to form podosomes in response to activation of protein kinase C (PKC) has fuelled recent research activities that are aimed at deciphering the potential diacylglycerolmediated mechanisms for cytoskeleton remodelling and basement membrane rearrangements (Hai et al., 2002; Burgstaller and Gimona, 2004, 2005; Webb et al., 2005). In this review, we highlight the emerging concepts of podosome formation in SMCs, and discuss the potential pathways and molecular components that are involved in their regulation, stabilisation and turnover, as well as the potential roles that podosomes have in smooth muscle physiology and pathology.
Podosomes In addition to stress-fibre-anchoring focal adhesions, only a few cell types can form the transient adhesive structures that are known as podosomes (Tarone et al., 1985; Marchisio et al., 1988). These structures appear as rings of variable sizes that are initially identified as dynamic sites of actin polymerisation activity, and that can fuse into larger ribbons and rings with a wide inner cavity (Fig. 1).
While these specialised adhesions have long been confined to cells derived from the monocytic lineage (primarily osteoclasts and macrophages) and to cells transformed by the Rous sarcoma virus, recent advances have also identified similar structures in other nontransformed cell types, such as endothelial cells (Moreau et al., 2003; Osiak et al., 2005), epithelial cells (Spinardi et al., 2004) and SMCs (Hai et al., 2002). In the latter case, the formation of podosomes is elicited by contraction-inducing phorbol esters, such as PDBu and 12-O-tetradecanoylphorbol-13-acetate (TPA), which stimulate the activation of the PKCa and d isoforms (Brandt et al., 2002, 2003; Yang and Kazanietz, 2003).
Podosomes in vascular and visceral smooth muscle cells To date, there are only a few SMC lines that express critical markers of smooth muscle differentiation, such as h1 calponin (h1 CaP) and SM22. The A7r5 rat vascular SMC line (Kimes and Brandt, 1976) has proven to be a reliable model to investigate podosome formation in vitro. Studies on this cell line have revealed the characteristic structure of podosomes using electron microscopy, confirmed the co-localisation of established podosome marker proteins, and demonstrated the pathways of signalling events via the PAK-PIX route (Webb et al., 2005). Notably, podosome formation is not confined to vascular SMCs, and we have verified the potential of podosome formation also in a visceral SMC line, namely the human intestinal smooth muscle (HISM) line (M. Gimona, unpublished; Fig. 2). As with the rat vascular SMCs, human visceral SMCs respond to phorbol esters with the formation of podosome-like structures. HISM cells in culture retain a higher degree of bipolarity and display an elongated rather than rounded phenotype. In contrast to vascular SMCs, podosomes are rarely seen in the periphery of these cells, and here the podosomes commonly resemble the rosette-type arrangements that have also been described for epithelial cells.
The microdomain – where podosome formation begins Cultured A7r5 SMCs exhibit a well-defined and robust actin cytoskeleton. These features allow the study of podosome formation and dynamics on the background of highly organised actin filaments. Due to the unusually large size of rat vascular SMCs in culture (with diameters of up to 180 mm), a specialised zone that is termed the microdomain has been described
ARTICLE IN PRESS T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
185
Fig. 1. Stabilised, mature podosomes in cultured vascular SMCs form larger ribbon or ring structures (arrows in A). Individual podosomes are still discernible by individual clusters of cortactin (arrowheads in B).
Fig. 2. Large podosome rosettes in vascular and visceral SMCs.
ARTICLE IN PRESS 186
T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
(Kaverina et al., 2003; Burgstaller and Gimona, 2004). These microdomains were initially characterised as a region close to where actin bundles terminate in focal adhesions sites, but which are not decorated by phalloidin. Subsequent live-cell imaging has revealed that podosome formation in SMCs originates at these sites, which are also marked by the presence of clusters of the actin-binding and protein complex containing actin-related proteins 2 and 3 (Arp2/3)-activating protein cortactin. Translocation and clustering of the Arp2/3 complex to the sites of SMC podosome formation is an early event. The presence of cortactinenriched foci within the specialised microdomains at the focal adhesion-stress fibre interface in unstimulated SMCs suggests that these sites are designated areas for cytoskeletal remodelling that recruit, stabilise and activate the actin polymerisation machinery following the appropriate stimuli. Thus, the sites of podosome formation provide a second subcellular location for intense Arp2/3-dependent actin polymerisation at the inner face of focal contacts. However, the formation of podosomes cannot correspond only to the rapid synthesis of new actin filaments, but must be preceded by local rearrangements of the existing actin cytoskeleton and the tension-generating anchorage sites. Focal adhesions indeed disassemble gradually during the dynamic formation of podosomes, and turn over in spite of a stable and contractile central cytoskeleton. Actin severing and depolymerisation may generate short nucleation sites for the newly forming, branched actin filaments that are used by the Arp2/3 machinery to rapidly generate the podosome core. Podosome formation is characterised by a concentrated burst of actin polymerisation in the podosome core, and this is preceded by the removal of actin-binding and -stabilising components, such as myosin II, tropomyosin and CaP, and the depolymerisation of the existing uncoated actin filaments. Consistent with the local dispersion of myosin and tropomyosin, podosome formation includes the rapid recruitment and accumulation of the Src activator 110-kDa actin filamentassociated protein (AFAP-110) and of 190-kDa Rhospecific GTPase-activating protein (p190RhoGAP) (Burgstaller and Gimona, 2004). AFAP-110 is involved in the spatial organisation of the sites of actin polymerisation via the Arp2/3 complex (Weed et al., 2000), and consequently in podosome formation (Gatesman et al., 2004), while p190RhoGAP appears to mediate the spatially restricted inactivation of RhoA (Nakahara et al., 1998). Thus, processes governing actin filament depolymerisation and the subsequent Arp2/3dependent filament polymerisation at the microdomains result in a local reduction in the contractile forces that appears sufficient to destabilise focal adhesions. The signalling cascade(s) initiated by PKC activation (Yang and Kazanietz, 2003) and Src phosphorylation (Hai et
al., 2002; Brandt et al., 2002) that converge at the actin cytoskeleton thus act asynchronously.
Inhibition of podosome formation by increased actin stabilisation The dramatic increase in actin polymerisation in the podosome core argues for a highly dynamic actin turnover at the microdomains. The phenotypic plasticity of SMCs combined with the rapid and transient formation of podosomes supports the concept that the engagement of the molecular machinery that initiates actin-based cell motility drives cell migration and tissue invasion in this cell type. Strikingly, phenotypic plasticity, SMC migration and podosome formation are suppressed by high expression levels of the actinbinding protein h1 CaP via the ability of CaP to regulate actin filament turnover (Gimona et al., 2003). High levels of high-molecular-weight calponin variant 1 (h1 CaP) expression are known to be required for the maintenance of the normal SMC phenotype (Horiuchi et al., 1998, 1999; Meehan et al., 2002), while downregulation of h1 CaP is generally accompanied by SMC de-differentiation (Gimona et al., 1990; Horiuchi et al., 1999) and malignant alterations in smooth muscle tissue (Takeoka et al., 2002). The general role of h1 CaP as a potent regulator of actin stability and an inhibitor of actin turnover has also been documented by its ability to inhibit metastatic cell motility in vitro (Lener et al., 2004).
Smooth muscle cell polarisation requires flexible substrates Tissue rigidity has an important role in a number of normal and pathological processes involving cell locomotion (Lo et al., 2000). On a rigid glass surface, A7r5 cells display a round shape with prominent actin stress fibres and focal adhesions, irrespective of a coating of cross-linked fibronectin or collagen I (Burgstaller and Gimona, 2005). In contrast, when grown on flexible polyacrylamide substrates (Pelham and Wang, 1997) that are coated with either fibronectin or collagen-1, A7r5 cells adopt an elongated appearance. This morphology is retained after PDBu stimulation, when the cells form a pronounced leading edge with an active, polarised lamellipodium, and podosomes are seen to be confined to the region of active actin cytoskeleton remodelling in the lamellipodium. On matrix-coated flexible substrates, vascular SMCs not only develop podosomes, but they also display increased cell polarisation and motility, indicating that the differentiated SMC phenotype is not solely influenced by the type of
ARTICLE IN PRESS T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
ECM, but also by the ability to exert forces and to undergo contraction. Consequently, when vascular SMCs are embedded in a reconstituted basement membrane matrix (MatrigelTM), the actin filaments become aligned along the longitudinal axis in the periphery of the cells. Activation of the PKC cascade(s) by PDBu then causes these spindle-shaped cells to develop numerous podosome-like complexes at the cell periphery, suggesting that podosomes in vascular SMCs are physiological structures that can be stabilised in a three-dimensional environment (Burgstaller and Gimona, 2005).
Matrix degradation MMPs enhance the migratory activity of vascular SMCs, and the invadopodia of metastasising tumour cells appear to coordinate the spatial secretion of these enzymes. In agreement with the situation in invadopodia of cancer cells, the A7r5 rat vascular SMCs are able to degrade a fluorescent fibronectin matrix in the vicinity of their podosomes in response to PDBu. One could thus hypothesize that the potential for podosome formation by cultured SMCs might be important for the remodelling of the actin cytoskeleton, of the denseplaque-associated cell-matrix adhesion sites, and possibly (under malignant de-differentiation) of the surrounding ECM of SMCs in vivo (Galis et al., 1995).
Conclusions, hypotheses and future challenges Podosomes and invadopodia have elementary roles in many different aspects of cell invasion of tissues, and substrate degradation of tumour cells is confined to punctate areas that correspond in both size and location to the appearance of podosomes (Baldassarre et al., 2003). Podosome formation thus provides an elegant explanation for the complex mechanisms underlying SMC invasion in the course of the development of atherosclerotic lesions and restenosis. Our current knowledge provides hypotheses for the roles of podosome formation and matrix degradation of vascular SMCs in the progression of atherosclerotic processes. Activation of one or more of the PKC cascades (both PKCa and PKCd have been ascribed roles in cytoskeleton modulation in phorbol-ester-stimulated vascular SMCs) induces cell polarisation and motility, as well as podosome formation and targeted matrix degradation; this also occurs in a three dimensional matrix environment. Temptingly, these findings may help us to resolve the long-standing controversies and uncertainties as to how SMCs escape the barrier of the tunica
187
media and invade the intima in vascular diseases (see also Raines and Ross, 1993). Endothelial and epithelial cells and SMCs have been shown to form podosomes in vitro in response to stimulation with diverse cytokines (Osiak et al., 2005) and diacylglycerol analogues (Brandt et al., 2002; Hai et al., 2002). TGF-b1-, TNF-a- and VEGF-induced podosome formation in SMCs remains, however, to be confirmed experimentally both in vivo and in vitro, and despite remarkable advances, it remains debatable as to whether SMCs actually use their potential for podosome formation under physiological conditions. Little is known about the motility of SMCs in tissues, and it is unclear if smooth muscle injury is a sufficient trigger for the induction of SMCs motility and tissue invasion. On the other hand, the SMC phenotypic plasticity includes a number of hallmarks of tumour cells, like alterations in their protein expression profile. So, there is justifiable hope that the common ability for podosome and invadopodia formation in normal and malignant cells may lead to common aspects with respect to the molecular mechanisms involved in their generation and regulation. A striking feature of these cellular adhesion structures, including podosomes, invadopodia and focal adhesions alike, is that they are composed of essentially the same components and they are controlled by similar modulators (Linder and Aepfelbacher, 2003). However, only podosomes and invadopodia also recruit the Arp2/ 3-dependent, cortactin and Wiskott Aldrich syndrome protein (WASp)-stimulated actin polymerisation machinery, and require a dynamic actin cytoskeleton. In any case, it appears a paradox that only a few cell types can engage in podosome formation. A possible explanation for this phenomenon is the hypothetical existence of an ‘‘adhesion module’’, a biochemical device or programme that creates all of the adhesion structures from the same repertoire of components, generating a different output by differently ‘‘computing’’ the input. The multiprotein complex of the adhesion module may be seen as a ‘‘functional module’’ (as described by Barabasi and Oltvai, 2004; Hartwell et al., 1999). Understanding how such a device triggers the formation of podosomes, and most importantly the more ‘‘aggressive’’ invadopodia instead of (or in addition to) focal adhesions, should further our understanding of cell adhesion and tissue invasion under physiological and pathological conditions.
Acknowledgments The Gimona laboratory is supported by the Marie Curie Excellence Grant ] MEXT-CT-2003-002573 of the European Union. The authors are grateful to Dr. R. Buccione (SMI) for helpful suggestions and discussions,
ARTICLE IN PRESS 188
T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
Dr. C.P. Berrie for editorial assistance, and Drs. J.V. Small (IMBA, Vienna), I.N. Kaverina (Vanderbilt University, Tennessee) and C.M. Hai (Brown University, Rhode Island) for outstanding collaborations.
References Aguilera, C.M., George, S.J., Johnson, J.L., Newby, A.C., 2003. Relationship between type IV collagen degradation, metalloproteinase activity and smooth muscle cell migration and proliferation in cultured human saphenous vein. Cardiovasc. Res. 58, 679–688. Baldassarre, M., Pompeo, A., Beznoussenko, G., Castaldi, C., Cortellino, S., McNiven, M.A., Luini, A., Buccione, R., 2003. Dynamin participates in focal extracellular matrix degradation by invasive cells. Mol. Biol. Cell 14, 1074–1084. Barabasi, A.L., Oltvai, Z.N., 2004. Network biology: understanding the cell’s functional organization. Nat. Rev. Genetics 5, 101–113. Brandt, D., Gimona, M., Hillmann, M., Haller, H., Mischak, H., 2002. Protein kinase C induces actin reorganization via a Src- and Rho-dependent pathway. J. Biol. Chem. 277, 20903–20910. Brandt, D.T., Goerke, A., Heuer, M., Gimona, M., Leitges, M., Kremmer, E., Lammers, R., Haller, H., Mischak, H., 2003. Protein kinase C delta induces Src kinase activity via activation of the protein tyrosine phosphatase PTP alpha. J. Biol. Chem. 278, 34073–34078. Buccione, R., Orth, J.D., McNiven, M.A., 2004. Foot and mouth: podosomes, invadopodia and circular dorsal ruffles. Nat. Rev. Mol. Cell Biol. 5, 647–657. Burgstaller, G., Gimona, M., 2004. Actin cytoskeleton remodelling via local inhibition of contractility at discrete microdomains. J. Cell Sci. 117, 223–231. Burgstaller, G., Gimona, M., 2005. Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 288, 3001–3005. Campbell, G.R., Chamley-Campbell, J.H., 1981. Smooth muscle phenotypic modulation: role in atherogenesis. Med. Hypotheses 7, 729–735. Chen, W.T., Olden, K., Bernard, B.A., Chu, F.F., 1984. Expression of transformation-associated protease(s) that degrade fibronectin at cell contact sites. J. Cell Biol. 98, 1546–1555. Galis, Z.S., Muszynski, M., Sukhova, G.K., Simon-Morrissey, E., Libby, P., 1995. Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann. NY Acad. Sci. 748, 501–507. Gatesman, A., Walker, V.G., Baisden, J.M., Weed, S.A., Flynn, D.C., 2004. Protein kinase C alpha activates c-Src and induces podosome formation via AFAP-110. Mol. Cell. Biol. 24, 7578–7597. Gimona, M., Herzog, M., Vandekerckhove, J., Small, J.V., 1990. Smooth muscle specific expression of calponin. FEBS Lett. 274, 159–163.
Gimona, M., Kaverina, I., Resch, G.P., Vignal, E., Burgstaller, G., 2003. Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol. Biol. Cell 14, 2482–2491. Goodall, S., Porter, K.E., Bell, P.R., Thompson, M.M., 2002. Enhanced invasive properties exhibited by smooth muscle cells are associated with elevated production of MMP-2 in patients with aortic aneurysms. Eur. J. Vasc. Endovasc. Surg. 24, 72–80. Hai, C.M., Hahne, P., Harrington, E.O., Gimona, M., 2002. Conventional PKC mediates phorbol dibutyrate-induced cytoskeletal remodeling in A7r5 smooth muscle cells. Exp. Cell Res. 280, 64–74. Hartwell, L.H., Hopfield, J.J., Leibler, S., Murray, A.W., 1999. From molecular to modular cell biology. Nature 402, C47–C52. Horiuchi, A., Nikaido, T., Ito, K., Zhai, Y., Orii, A., Taniguchi, S., Toki, T., Fujii, S., 1998. Reduced expression of calponin h1 in leiomyosarcoma of the uterus. Lab. Invest. 78, 839–846. Horiuchi, A., Nikaido, T., Taniguchi, S., Fujii, S., 1999. Possible role of calponin h1 as a tumor suppressor in human uterine leiomyoma. J. Natl. Cancer Inst. 91, 790–796. Kaverina, I., Stradal, T.E., Gimona, M., 2003. Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains. J. Cell Sci. 116, 4915–4924. Kimes, B.W., Brandt, B.L., 1976. Characterization of two putative smooth muscle cell lines from rat thoracic aorta. Exp. Cell Res. 98, 349–366. Linder, S., Aepfelbacher, M., 2003. Podosomes: adhesion hotspots of invasive cells. Trends Cell Biol. 13, 376–385. Lener, T., Burgstaller, G., Gimona, M., 2004. The role of calponin in the gene profile of metastatic cells: inhibition of metastatic cell motility by multiple calponin repeats. FEBS Lett. 556, 221–226. Lo, C.M., Wang, H.B., Dembo, M., Wang, Y.L., 2000. Cell movement is guided by the rigidity of the substrate. Biophys. J. 79, 144–152. Marchisio, P.C., D’Urso, N., Comoglio, P.M., Giancotti, F.G., Tarone, G., 1988. Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts. J. Cell. Biochem. 37, 151–159. Meehan, K.L., Holland, J.W., Dawkins, H.J., 2002. Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer. Prostate 50, 54–63. Mizutani, K., Miki, H., He, H., Maruta, H., Takenawa, T., 2002. Essential role of neural Wiskott-Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in src-transformed fibroblasts. Cancer Res. 62, 669–674. Moreau, V., Tatin, F., Varon, C., Genot, E., 2003. Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA. Mol. Cell. Biol. 23, 6809–6822. Nakahara, H., Mueller, S.C., Nomizu, M., Yamada, Y., Yeh, Y., Chen, W.T., 1998. Activation of beta1 integrin signaling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia. J. Biol. Chem. 273, 9–12.
ARTICLE IN PRESS T. Lener et al. / European Journal of Cell Biology 85 (2006) 183–189
Newby, A.C., Zaltsman, A.B., 1999. Fibrous cap formation or destruction – the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation. Cardiovasc. Res. 41, 345–360. Osiak, A.E., Zenner, G., Linder, S., 2005. Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling. Exp. Cell Res. 307, 342–353. Pauly, R.R., Passaniti, A., Bilato, C., Monticone, R., Cheng, L., Papadopoulos, N., Gluzband, Y.A., Smith, L., Weinstein, C., Lakatta, E.G., 1994. Migration of cultured vascular smooth muscle cells through a basement membrane barrier requires type IV collagenase activity and is inhibited by cellular differentiation. Circ. Res. 75, 41–54. Pelham, R.J., Wang, Y.L., 1997. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13661–13665. Raines, E.W., Ross, R., 1993. Smooth muscle cells and the pathogenesis of the lesions of atherosclerosis. Br. Heart J. 69, S30–S37. Spinardi, L., Rietdorf, J., Nitsch, L., Bono, M., Tacchetti, C., Way, M., Marchisio, P.C., 2004. A dynamic podosome-like structure of epithelial cells. Exp. Cell Res. 295, 360–375.
189
Takeoka, M., Ehara, T., Sagara, J., Hashimoto, S., Taniguchi, S., 2002. Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells. Eur. J. Cancer 38, 436–442. Tarone, G., Cirillo, D., Giancotti, F.G., Comoglio, P.M., Marchisio, P.C., 1985. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp. Cell Res. 159, 141–157. Webb, B.A., Eves, R., Crawley, S.W., Zhou, S., Cote, G.P., Mak, A.S., 2005. PAK1 induces podosome formation in A5r7 vascular smooth muscle cells in a PIX-dependent manner. Am. J. Physiol. Cell Physiol. (Jun 8; [Epub ahead of print]). Weed, S.A., Karginov, A.V., Schafer, D.A., Weaver, A.M., Kinley, A.W., Cooper, J.A., Parsons, J.T., 2000. Cortactin localization to sites of actin assembly in lamellipodia requires interaction with F-actin and the Arp2/3 complex. J. Cell Biol. 151, 29–40. Yang, C.F., Kazanietz, M.G., 2003. Divergence and complexities in DAG signaling: looking beyond PKC. Trends Pharmacol. Sci. 24, 602–608.