Podosome regulation by Rho GTPases in myeloid cells

European Journal of Cell Biology 90 (2011) 189–197

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Review

Podosome regulation by Rho GTPases in myeloid cells Suzanne F.G. van Helden ∗ , Peter L. Hordijk Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands

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Article history: Received 29 March 2010 Received in revised form 17 May 2010 Accepted 22 May 2010 Keywords: Podosomes Rho GTPase Myeloid cell Macrophage Dendritic cell and osteoclast

a b s t r a c t Myeloid cells form a first line of defense against infections. They migrate from the circulation to the infected tissues by adhering to and subsequently crossing the vascular wall. This process requires precise control and proper regulation of these interactions with the environment is therefore crucial. Podosomes are the most prominent adhesion structures in myeloid cells. Podosomes control both the adhesive and migratory properties of myeloid cells and the regulation of podosomes is key to the proper functioning of these cells. Here we discuss the regulation of podosomes by Rho GTPases, well known regulators of adhesion and migration, focusing on myeloid cells. In addition, the regulation of podosomes by GTPase regulators such as GEFs and GAPs, as well as the effects of some Rho GTPase effector pathways, will be discussed. © 2010 Elsevier GmbH. All rights reserved.

Introduction The immune system comprises cells from the haematopoietic lineage, which can be subdivided into the lymphoid and the myeloid lineage. The latter derives from a common myeloid progenitor which gives rise to the megakaryocyte/erythrocyte progenitor and the granulocyte/macrophage progenitor (GMP). The GMP gives rise to various types of innate immune cells that form the first line of defense against infections. Neutrophils and monocytes are among the first to be recruited to sites of infection where they, as well as macrophages, phagocytose pathogens and secrete cytokines. Conversely, dendritic cells (DCs) detect and take up pathogens and present antigens to T cells, initiating an adaptive immune response. A common feature of these cells is that they have to be able to migrate efficiently, from the blood across the endothelium into the tissue and within the stroma and parenchyma. In addition, DCs subsequently have to migrate into the lymphatics. For proper migration, regulated interactions with the endothelium and with the subendothelial extracellular matrix are crucial. As in other cells, integrins play an important role in myeloid cell adhesion and migration. The main integrin-based adhesion structures found in myeloid cells are podosomes. Similar as the functionally related focal adhesions in, e.g., fibroblasts, controlled formation, turnover and loss of podosomes is critical for adhesion and migration. As a result, podosome dynamics is key to the correct functioning of

∗ Corresponding author. Tel.: +31 20 5123213; fax: +31 20 5123474. E-mail address: [email protected] (S.F.G. van Helden). 0171-9335/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2010.05.008

myeloid cells during inflammation and under static state conditions. Rho GTPases have been shown to regulate integrin function, vesicle transport and cytoskeletal dynamics in non-myeloid cells and are therefore excellent candidate regulators of podosomes in myeloid cells. Here, we review recent progress made in the understanding of podosome dynamics by Rho GTPases and their regulators. Invadosomes Invadosomes are matrix-degrading cell adhesion structures, such as podosomes and invadopodia. Podosomes and invadopodia share many structural components (Ayala et al., 2006), however, the ability to degrade matrix is more pronounced in invadopodia. It remains to be established whether podosomes and invadopodia are different structures or representations of the same structure in different cell types. Podosomes Podosomes are cell–matrix adhesion structures consisting of a bundle of actin perpendicular to the membrane surrounded by a ring-like structure containing integrins and adaptor proteins (Fig. 1 shows examples of ring and core components). Deeper within the cell, the actin bundle connects to radiating actin fibers. Podosomes are about 0.5 ␮m wide and high and are very dynamic structures with a half-life of less than 10 min. Podosomes are found in myeloid cells, endothelial cells, smooth muscle cells and transformed fibroblasts (David-Pfeuty and Singer, 1980; Kaverina et al., 2003; Marchisio et al., 1984; Moreau et al., 2003; Tarone et al.,

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Fig. 1. Localization of podosome components. Immature DCs seeded on fibronectin-coated coverslips were stained with rabbit anti-pY118paxillin (Invitrogen, Praisley, UK), mouse anti-cortactin (Millipore, Billerica, MA), rabbit anti-WASp (Cell signaling, Beverly, MA) or goat anti-WIP (Santa Cruz Biotechnology, Santa Cruz, CA) (green in merge, all secondary antibodies were derived from Invitrogen) and phalloidin-TxRed (Molecular Probes, Invitrogen) to stain F-actin (red in merge). Images were obtained by confocal microscopy using a Zeiss LSM 510-meta microscope with a Plan-Apochromatic 63 × 1.4 NA oil immersion objective (Carl Zeiss, Jena, Germany). Cortactin, WASp and WIP are examples of podosome components that localize to the F-actin containing podosome core, while paxillin, in this case phosphorylated at Y118, localizes to the podosome rings surrounding the cores.

1985). Cells locally degrade the matrix underneath podosomes and this is due to metalloprotease activity, especially MT1-MMP, MMP2 and MMP9 (Cougoule et al., 2010; Mizutani et al., 2002; Nermut et al., 1991; Tatin et al., 2006). There is scarce information on the pathways and stimuli that drive podosome formation in various cell types. However, Arp2/3 mediated actin nucleation, myosin II-controlled cell contraction and an intact microtubule system are important for podosome regulation (Burgstaller and Gimona, 2004; Clark et al., 2006; Kaverina et al., 2003; Kopp et al., 2006). Src kinase activity is sufficient for podosome formation, as illustrated by the ability of fibroblasts transformed with v-Src to form podosomes, while untransformed fibroblasts are unable to form these structures (David-Pfeuty and Singer, 1980; Tarone et al., 1985). The importance of Src is further underscored by the finding that Src−/− mice have severe osteopet-

rosis coupled to a lack of podosomes in osteoclasts (Soriano et al., 1991). Smooth muscle cells can form podosomes in response to PDBu stimulation (Kaverina et al., 2003), which are thought to function in tissue remodeling and repair. In endothelial cells, podosomes form in response to stimulation with phorbol ester (Tatin et al., 2006), TGF␤ (Varon et al., 2006), VEGF or TNF␣ (Moreau et al., 2003; Osiak et al., 2005) and are important for the degradation of basement membrane (Rottiers et al., 2009). Cells of the myeloid lineage are special as these are the only cells capable of podosome formation upon adhesion without additional stimulation or transformation (Burns et al., 2001; Linder et al., 1999; Marchisio et al., 1984). In myeloid cells, podosomes are the main adhesion structures and they are important for adhesive and migratory behavior of these cells.

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Invadopodia

Osteoclasts

Invadopodia are matrix-degrading cell adhesions found in invasive tumor cells. Invadopodia appear as irregular dots in the vicinity of the nucleus and in proximity to the Golgi complex (Albiges-Rizo et al., 2009). This is in contrast to podosomes that can be organized into rosettes or cover the entire ventral surface of cells. Invadopodia lack the strict organization of core and ring structure found in podosomes and have a longer halflife. They are often larger than podosomes and invade deeper into the matrix. Inhibition of protease activity blocks invadopodia biogenesis (Ayala et al., 2008). Invadopodia are associated with breaching tissue boundaries and they enable cancer invasion and tumor metastasis (Gimona et al., 2008). In this review, data derived from studies on invadopodia (or podosomes in nonmyeloid cells) will be discussed in relation to the situation in myeloid cells.

The remodeling of bone is regulated by the balanced activity of bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoclast activity depends on their adhesion to the bone and formation of resorption pits (Teitelbaum, 2000). The resorptive activity is sealed of from the surroundings through the sealing zone through tight adhesion to the surface. Osteoclasts undergo repetitive cycles of migration and resorption. In osteoclasts, podosomes are organized into clusters, which mature into podosome rings that further transform into a podosome belt (Ory et al., 2008). A podosome belt is thought to be a precursor structure of sealing zones, which contain densely packed podosome cores (Chabadel et al., 2007; Luxenburg et al., 2007). The podosome clusters are important for migratory phase, while podosome rings and belts mature into the sealing zone promoting bone resorption. Rho GTPases, regulators and effector pathways

Myeloid cells and podosomes Within the myeloid lineage podosomes have been observed in monocytes, macrophages, DCs, osteoclasts, neutrophils and eosinophils (Burns et al., 2001; Johansson et al., 2004; Linder et al., 1999; Marchisio et al., 1984; Szczur et al., 2006). Podosomes are important for transendothelial migration (Calle et al., 2006; Carman et al., 2007), a process essential for extravasation of monocytes and neutrophils to inflamed sites. In macrophages, DCs and osteoclasts, the myeloid cells where podosomes are best studied, podosomes are the most prominent part of the actin cytoskeleton and they often cover the entire ventral surface of the cell. In contrast, transformed fibroblasts and endothelial cells only form few podosomes per cell often organized in rosettes. Macrophages and immature DCs Macrophages or immature DCs display a relatively slow migration, similar to the migration of fibroblasts (Friedl and Wolf, 2003). This slow migration is dependent on strong interactions with the extracellular matrix. Podosomes often localize just behind the leading edge of the cells and they form a link between the extracellular matrix and the cytoskeleton through integrins and adaptor proteins. Thereby podosomes regulate the adhesive and migratory behavior of macrophages and immature DCs (Linder et al., 1999) and contribute to the movement of these cells within peripheral tissues, while patrolling for antigen and signs of inflammation. DC-maturation In neutrophils or mature DCs, a fast, amoeboid-like migration, similar to the migration of T cells (Wolf et al., 2003), is observed. This fast migration is characterized by short lived and low affinity interactions with the substrate. The migration of mature DCs is 10 times faster than the migration of immature DCs, 5 ␮m/min vs. 0.5 ␮m/min respectively (De Vries et al., 2003; van Helden et al., 2006). The efficient induction of adaptive immunity depends on the induction of fast migration, allowing the DC to travel to the lymph nodes and activate T cells. DCs need to dissolve their podosomes during maturation, because podosomes limit fast migration by their strong interactions with the extracellular matrix and thus inhibit the induction of T cell responses (van Helden et al., 2006, 2010). Therefore, the switch in the mode of migration and the formation or dissolution of podosomes needs to be tightly controlled for proper induction of adaptive immunity.

Rho GTPases form a subfamily of the Ras GTPase superfamily and are master regulators of adhesion and migration. The activity of Rho GTPases is controlled by Rho GTPase regulators: guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). The output of Rho GTPase activation is controlled through specific downstream effector pathways. The role of these Rho GTPases, their regulators and downstream effectors in podosome regulation will be discussed here. Rho GTPases Rho-like small GTPases are a family of well known regulators of the cytoskeleton that control many cellular processes including polarity, adhesion and migration. In mesenchymal cells, activation of Rac1, RhoA and Cdc42, the best studied Rho GTPases, regulates F-actin structures and the formation of cell–matrix adhesions (Hall, 1998; Schmitz et al., 2000). More specifically, Rac1 induces lamellipodia, ruffles and focal complexes, RhoA induces stress fibers and focal adhesions and Cdc42 induces filopodia. Small GTPases cycle between an inactive, GDP-bound form and an active, GTP-bound form. The GEFs regulate the activation of Rho GTPases by promoting the exchange of GDP for GTP, while GAPs promote the intrinsic GTPase activity and thus the transition back to the GDP-bound state (Moon and Zheng, 2003; Rossman et al., 2005). GDP-bound Rho GTPases are sequestered by Rho guanine nucleotide dissociation inhibitor (RhoGDI) (DerMardirossian and Bokoch, 2005) that may serve as a molecular chaperone and regulator to protect the cell from aberrant GTPase activation. The balanced action of GEFs and GAPs is crucial for proper functioning of Rho GTPases and controls the timing and localization of Rho GTPase activity. The GTP-bound forms of the Rho GTPases transduce signals by binding to effector proteins, inducing a conformational change or altered localization, which is in turn required to transmit signals further downstream. The family of Rho GTPases consists of 20 members and can be divided into 8 subfamilies, which can in turn be divided into classical and atypical Rho GTPases (Table 1). The classical Rho GTPases are cycling between the active and inactive state as described above. The Rnd proteins, RhoH and possibly RhoBTB are GTPasedeficient, due to several amino acids in the GTP domain that are not conserved, rendering them constitutively GTP-bound (Aspenstrom et al., 2007; Chardin, 2006; Dallery et al., 1995; Foster et al., 1996; Nobes et al., 1998). RhoU, as well as the Rac1 splice variant Rac1B, has elevated nucleotide-exchange ability and, as a result, the protein is likely to reside predominantly in the GTP-bound conformation (Saras et al., 2004; Shutes et al., 2004). Atypical GTPases are therefore not or only partially controlled by GEFs and GAPs,

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Table 1 Family of Rho GTPases. Rho GTPase

Type

Expression

Cdc42-related subfamily Cdc42 RhoJ/TCL RhoQ/TC10

Classical Classical Classical

Ubiquitous Heart, lung, liver Heart, skeletal muscle

Rac-related subfamily Rac1 Rac2 Rac3 RhoG

Classical Classical Classical Classical

Ubiquitous Haematopoietic Brain, heart, placenta, pancreas Ubiquitous

Rho-related subfamily RhoA RhoB RhoC

Classical Classical Classical

Ubiquitous Ubiquitous Ubiquitous

RhoF-subfamily RhoD RhoF/Rif

Classical Classical

Heart, liver, placenta, pancreas, skeletal muscle Ubiquitous

RhoH/TTF RhoH/TTF

Atypical

Haematopoietic

RhoU-subfamily RhoU/Wrch1 RhoV/Chp/Wrch2

Atypical Atypical

Brain, heart, liver, lung, placenta, skeletal muscle Brain, spleen, lung, testis

Rnd family Rnd1 Rnd2 Rnd3/RhoE

Atypical Atypical Atypical

Brain, liver Brain, testis Ubiquitous

RhoBTB RhoBTB1 RhoBTB2 RhoBTB3

Atypical Atypical No Rho GTPase

Ubiquitous Brain Ubiquitous

The family of Rho GTPases contains 20 members and can be divided into 8 subfamilies. Rho GTPases can be divided into classical and atypical. The classical GTPases cycle between a GTP-bound and GDP-bound conformation and are regulated by GEFs and GAPs. The atypical Rho GTPases are predominantly in the GTP-bound conformation due to specific amino acid differences in the GTP-binding domain. Since the GTP-binding domain of RhoBTB3 is poorly conserved and is not typical for the Rho GTPases, RhoBTB3 should not be considered a Rho-family member. The tissue expression patterns of the different GTPases are indicated in the last column.

but rather by transcriptional regulation, phosphorylation and proteasomal degradation. Below we discuss in more detail the role of the main Rho GTPase subfamilies in the regulation of podosome dynamics. The Cdc42-subfamily In PAE endothelial cells the bacterial toxin Cytotoxic Necrotizing Factor-1 (CNF-1) induces the formation of podosomes. CNF-1 activates Rho, Rac and Cdc42 through transamidation of a critical glutamine residue at position 61 (Rac1, Cdc42) or 63 (RhoA) (Schmidt et al., 1997). In addition, constitutively active Cdc42 (V12Cdc42), but not RhoA (V14RhoA) or Rac1 (V12Rac1), could also induce podosome formation, suggesting that the CNF-1 effect in these cells is mediated by activation of Cdc42 (Moreau et al., 2006; Moreau et al., 2003). Furthermore, silencing of Cdc42 in endothelial cells inhibits podosome formation upon phorbol ester or TGF␤ stimulation (Tatin et al., 2006; Varon et al., 2006). Also in human HeLa-derived HtTA-1 cells, expression of V12Cdc42, not V14RhoA or V12Rac1, induces the formation of podosomes (Dutartre et al., 1996). These findings show that Cdc42 can drive podosome formation in cells that otherwise are devoid of podosomes. Furthermore, the formation of invadopodia, matrix-degrading podosome-like structures in cancer cells, is dependent on Cdc42 and expression of V12Cdc42 promotes invadopodia-mediated matrix degradation (Nakahara et al., 2003; Yamaguchi et al., 2005). An indication for the contribution of Cdc42 to the formation of podosomes in myeloid cells comes from the finding that during monocyte differentiation towards macrophages or upon phorbol ester treatment of THP-1 or U937 cells the total levels of Cdc42 and in particular the level of membrane-bound Cdc42 is increased. This

increase is associated with cell spreading and does not occur when monocytes are differentiated into non-adherent cells (Aepfelbacher et al., 1994). Furthermore, expression of dominant negative Cdc42 (N17Cdc42) inhibits podosome formation in DCs (Burns et al., 2001; West et al., 2000), showing the importance of Cdc42 in podosome formation in myeloid cells. Expression of V12Cdc42 led to an altered podosome distribution in DCs (Burns et al., 2001) and induced podosome loss in macrophages (Linder et al., 1999). These findings suggest that an excess of Cdc42 activity interferes with podosome dynamics, however this is based on overexpression of mutants that could also affect other GTPases and could be confirmed by knock down or knock out strategies. In addition to V12Cdc42, constitutive active RhoJ and RhoQ were found to induce podosome formation in endothelial cells (Aspenstrom et al., 2004; Vignal et al., 2000) and localize to podosomes (Billottet et al., 2008; Moreau et al., 2003), suggesting that this could also be the case in myeloid cells. However, this remains to be investigated. The Rac-subfamily In HUVEC, podosome formation upon cytokine stimulation was inhibited by expression of N17Rac1, N19RhoA and N17Cdc42, but the effect is most pronounced in N17Rac1 expressing cells (Osiak et al., 2005; Wang et al., 2009a), suggesting a role for Rac1 in podosome formation. Also the formation of invadopodia, as well as their matrix-degrading capacity, is induced by the expression of V12Rac1 (Furmaniak-Kazmierczak et al., 2007; Nakahara et al., 2003). Myeloid cells express Rac1 and Rac2, but lack expression of Rac3 (Wells et al., 2004). Antibodies against Rac1 or Rac2 lead to the disruption of actin rings and decreased resorption in

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saponine-treated rat osteoclasts (Razzouk et al., 1999). In addition, the expression of N17Rac1 disrupts podosomes in DCs and osteoclasts (Burns et al., 2001; Ory et al., 2000). These findings suggest that Rac activity is needed for podosome formation. Although podosomes form normally in V12Rac1 expressing DCs, they are lost in V12Rac1 expressing osteoclasts (Burns et al., 2001; Ory et al., 2000), indicating that the effect of an excess of Rac activity on podosomes is dependent of the cell type. Macrophages from mice lacking Rac expression (Rac1/2−/− mice) are unable to form podosomes and have impaired invasion into matrigel (Wheeler et al., 2006), confirming the importance of Rac in podosome formation. Moreover, macrophages of Rac2−/− mice lack podosomes completely, while Rac1−/− macrophages display actin dots, which often are not surrounded by paxillin (Wheeler et al., 2006), suggesting that podosome formation is initiated but not properly completed. Together, this data suggests that Rac2 is essential for podosome formation and that Rac1 is involved in the subsequent maturation of podosomes. The Rho-subfamily In endothelial cells podosome formation is blocked by C3 transferase, a bacterial toxin from Clostridium botulinum which ADP-ribosylates and inactivates RhoA, RhoB and RhoC, or by silencing of RhoA expression (Tatin et al., 2006; Varon et al., 2006), indicating that RhoA is required for podosome formation. In line with this notion, active RhoA has been reported to localize to podosomes in transformed fibroblasts (Berdeaux et al., 2004). In macrophage-derived multinucleated cells, osteoclasts and DCs, C3 transferase disrupts podosomes (Burns et al., 2001; Ory et al., 2000; West et al., 2000; Zhang et al., 1995), implying that Rho is also involved in podosome regulation in myeloid cells. In addition, RhoA inhibition leads to podosome ring instead of sealing zone formation on bone (Saltel et al., 2004), suggesting that RhoA activity promotes the transition of podosome rings to sealing zones in osteoclasts. Mouse macrophages express RhoA and RhoB, but not RhoC. RhoB−/− macrophages normally form podosomes and this is inhibited by C3 transferase (Wheeler and Ridley, 2007), suggesting that in RhoB deficient cells RhoA is essential for podosome formation. V14RhoA was found to inhibit podosome formation in fibroblasts, endothelial cells, multinucleated cells and osteoclasts (Moreau et al., 2003; Ory et al., 2000; Schramp et al., 2008; Zhang et al., 1995), suggesting that excess of RhoA activity blocks podosome formation. In line with these findings, V12Cdc42 induces podosome formation by preventing RhoA activation in endothelial cells (Moreau et al., 2003). This is further supported by the finding that limiting RhoA activity locally, thereby inhibiting contraction, promotes podosome formation in fibroblasts and neuroblastoma cells (Burgstaller and Gimona, 2004; Clark et al., 2006). In osteoclasts, microtubule depolymerization induces podosome belt disruption and this is dependent on RhoA activation (Destaing et al., 2005). Furthermore, RhoA activation leading to myosin II-mediated contraction in DCs is essential for the dissolution of podosomes during DC-maturation (van Helden et al., 2008), showing that RhoA activation can induce podosome loss in myeloid cells. These findings seem in contrast with the studies showing that podosome formation is RhoA dependent. However, a basal RhoA activity, possibly in areas of the cell were the podosomes are not formed, may be needed for podosome formation, while higher levels of Rho activity inhibit podosome formation and even lead to podosome loss. The RhoU-subfamily Interestingly, RhoU localizes to podosomes in osteoclasts and c-Src expressing cells (Brazier et al., 2009; Ory et al., 2007). The expression of RhoU was found to be upregulated during osteoclast differentiation (Brazier et al., 2006) and RhoU may be involved in

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the transition from podosomes rings to podosome belts (Brazier et al., 2009). Since the transitions between rings and belts may be specific for the osteoclast system, the effects of RhoU on podosome regulation in other myeloid cells remain to be investigated. The additional Rho GTPase subfamilies The RhoF-subfamily consists of RhoF and RhoD and activation of RhoF or RhoD causes long filopodia-like structures (Aspenstrom et al., 2004; Pellegrin and Mellor, 2005). However, in contrast to Cdc42, which also induces filopodia, RhoD and RhoF do not activate WASp. Expression of constitutive active RhoD or RhoF in endothelial cells did not induce podosome formation (Billottet et al., 2008), making it less probable that they regulate podosomes. RhoH and the members of the Rnd- and RhoBTB-subfamily are atypical Rho GTPases. Together with Rac2, RhoH is the only Rho GTPase restricted to the haematopoietic lineage (Dallery et al., 1995; Didsbury et al., 1989). However, RhoH was shown to be expressed in lymphoid cells, such as T and B cells, but undetectable in myeloid cells (Brazier et al., 2006; Gu et al., 2006; Li et al., 2002), suggesting that RhoH is not involved in podosome regulation in myeloid cells. Also the RhoBTBs are probably not important, since they are not expressed, or at very low levels, in organs were many myeloid cells are found e.g. thymus, lymph nodes, bone marrow and spleen, or in HL60 cells, J774 cells or peripheral blood leukocytes (Ramos et al., 2002). Rnd1 and Rnd2, not Rnd3, are expressed in parental as well as RANKL stimulated Raw264.7 cells (Brazier et al., 2006). Expression of Rnd1 or Rnd3 induces loss of focal adhesions and the effects of Rnd proteins on the cytoskeleton are linked to inhibition of RhoAmediated contraction (Chardin, 2006). Rnd3 has been described to bind to and inhibit Rho kinase (Riento et al., 2003). A discussed above inhibition of RhoA seems to be important for podosome formation. Therefore, it is tempting to speculate that Rnd protein could be involved in podosome formation by limiting RhoA activity or RhoA effector functions. Interestingly, Rnd1 and Rnd3 are described to interact with p190RhoGAP (see below) and recruit p190RhoGAP for RhoA inhibition (Wennerberg et al., 2003). In addition, Rnd3 can increase the GAP activity of p190RhoGAP, suggesting a possible route for Rnd proteins to regulate podosomes. Rho GTPase regulators The activity of the classical Rho GTPases is controlled by their respective GEFs and GAPs. In this section, the role of Rho GTPase regulators in podosome dynamics is discussed. GEFs The activation of Rho GTPases is controlled by GEFs that stimulate the release of GDP, allowing its replacement by GTP. There are two families of GEFs, the Dbl proteins and the DOCK180related proteins. Currently, there are 69 Dbl proteins and 16 DOCK180-related proteins identified in human (Cote and Vuori, 2002; Rossman et al., 2005). Currently, it is largely unknown which GEFs are involved in podosome regulation in myeloid cells. Vav proteins activate Rho, Rac, RhoG and Cdc42. Vav2 and Vav3 are ubiquitously expressed, while expression of Vav1 is restricted to haematopoietic system. Activation of ␤2-integrins induces phosphorylation of Vav1/3 and thereby Rho GTPase activation (Gakidis et al., 2004), suggesting that Vav proteins could also be involved in podosome formation upon adhesion. Furthermore, Vav1/3 deficiency blocks Arp2/3 recruitment and actin polymerization at complement-induced phagosomes, similar to Rac1/2 deficiency (Hall et al., 2006). Interestingly, Vav3−/− mice have increased bone mass due to impaired osteoclast function and these osteoclasts display defective Rac activation and podosome belt formation (Faccio

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et al., 2005), showing the importance of Vav in podosome regulation. ArhGEF5/Tim, which displays GEF activity towards RhoA and B and weakly to RhoC and RhoG, is important for chemotaxis in macrophage cell lines. ArhGEF5 can be phosphorylated by Src leading to activation of ArhGEF5 (Yohe et al., 2007). In addition, ArhGEF5 deficiency impairs chemotaxis of immature DCs and DC migration to the lymph nodes upon skin painting. The effects of ArhGEF5 are most pronounced in immature DCs (Wang et al., 2009b), this could reflect the difference in migration between podosome-bearing, slowly migrating immature DCs and fast migrating mature DCs without podosomes (van Helden et al., 2006). Therefore exploring the effects of ArhGEF5 on podosomes warrants further research. Based on their expression pattern in myeloid cells, P-Rex1, DOCK2, DOCK5, DOCK180 and ArhGEF8/Net1 are candidate regulators of podosomes, albeit that this has not been further investigated (Akakura et al., 2004; Brazier et al., 2006; Dong et al., 2005; Fukui et al., 2001; Welch et al., 2005). GAPs During podosome formation RhoA activity needs to be inhibited or at least limited (see above), which could be regulated by GAP proteins. Activation of ␤1-integrins leads to p190RhoGAP phosphorylation and p190RhoGAP localizes to invadopodia were it is needed for matrix degradation (Nakahara et al., 1998). In smooth muscle cells, p190RhoGAP localizes to PDBu induced podosomes and is recruited to podosomes together with AFAP-110 and cortactin by Tks5. Expression of GAP-deficient p190RhoGAP or knock down of p190RhoGAP prevents podosome formation in these cells. It was proposed that p190RhoGAP mediates the formation of podosomes through local inhibition of contraction mediated by Rho (Burgstaller and Gimona, 2004; Crimaldi et al., 2009). In HUVEC, the two forms of p190RhoGAP, p190RhoGAP-A and -B, localize to podosomes and p190RhoGAP-B regulates MT1MMP expression and matrix degradation. Surprisingly, reduction of p190RhoGAP-A expression by siRNA increased podosome formation, suggesting specific roles for the different p190RhoGAP forms in podosome regulation. Furthermore, reduction of p190RhoGAP-A and -B expression by siRNA had no effect on RhoA activity levels in HUVEC (Guegan et al., 2008), suggesting that Rho inactivation is a local event. Alternatively, additional GAPs may be involved in this process, such as RhoGAP7. This is based on data showing that in Srctransformed fibroblasts, GTP-loading of RhoA is limited by ERK5 through RhoGAP7 (Schramp et al., 2008). Furthermore, podosome formation is inhibited in ERK5−/− fibroblasts, suggesting that ERK5 and RhoGAP7 inhibit RhoA-GTP thereby promoting podosome formation. Whether the same GAPs regulate podosome formation in myeloid cells is unclear at this moment. In neutrophils deficient for p50RhoGAP/Cdc42GAP, a Rhofamily GAP with a preference for Cdc42, motility is increased and directional migration is decreased. In addition, podosomelike structures observed in the leading edge of neutrophils are decreased in p50RhoGAP−/− cells (Szczur et al., 2006), suggesting a role for p50RhoGAP in podosome regulation in myeloid cells. Rho GTPases effector pathways There are multiple effectors downstream of Rho GTPases and many of them could be involved in podosome regulation. For example IQGAP1, which influences invadopodia (Sakurai-Yageta et al., 2008), or Haematopoietic cell kinase (Hck), which localizes to podosomes and promotes podosome formation (Cougoule et al., 2005). Here, the best characterized effector pathways downstream of Rho GTPases involved in podosome regulation will be discussed.

Fig. 2. Overview of the regulation of podosomes in myeloid cells by Rho GTPases. Cdc42, and possibly RhoJ and RhoQ, promote podosome formation through the WASp/WIP/Arp2/3 and PAK/PIX/GIT pathways. Rac1/2 promotes podosome formation, possibly through the PAK/PIX/GIT pathway. Rac1/2 could be activated by Vav proteins to promote podosome formation. RhoU affects podosomes and could use similar effector pathways as Cdc42. RhoA-induced myosin II-mediated contraction results in podosome loss. In addition, p190RhoGAP and RhoGAP7 limit RhoA activity to promote podosome formation. Superscript no. 1 indicates components shown to be important in myeloid cells, superscript no. 2 indicates components involved in non-myeloid cells and superscript no. 3 indicates components shown to regulate invadopodia. The RhoA activation pathway leading to podosome loss is described only in myeloid cells. → Indicates activation; —| indicates inhibition. Dotted arrows indicate that additional effector pathways can be involved.

WASp-WIP Wiskott–Aldrich syndrome (WAS) patient-derived cells lack expression of the Wiskott–Aldrich syndrome protein (WASp), an effector of Cdc42, RhoJ and RhoQ (Aspenstrom et al., 2004). Macrophages and DCs from WAS patients are unable to form podosomes (Burns et al., 2001; Linder et al., 1999). Cdc42, WASp, WASp-interacting protein (WIP) and Arp2/3 all localize to podosomes in myeloid cells (Burns et al., 2001; Chou et al., 2006; Linder et al., 1999), also see Fig. 1 lower half). Cdc42 stimulates Arp2/3-induced actin nucleation through WASp and WIP and this is essential for podosome formation (Chou et al., 2006), also see Fig. 2). Furthermore, an intact microtubule network and the linkage of WASp to microtubules via Cdc42 interacting protein 4 (CIP4) is needed for podosome formation (Linder et al., 2000). RhoU can bind to Nck1 and Grb2, two adaptor proteins involved in cell adhesion (Shutes et al., 2004), which are involved in NWASP mediated actin assembly at the surface of endomembranes (Benesch et al., 2002) and a similar mechanism involving Cdc42, RhoU or both could recruit WASp to podosomes. PAK–PIX PAKs are effectors of Cdc42 and Rac that can be regulated by members of the PAK interacting exchange factor (PIX) family, which are also GEFs for Cdc42 and Rac1 (Li et al., 2003; Rosenberger and Kutsche, 2006). Antibodies against PAK1 disrupt the actin rings in saponine-treated rat osteoclasts leading to decreased bone resorption (Razzouk et al., 1999) and suggesting a role for PAK in podosome regulation. In addition, expression of PAK1 or ␤PIX induces podosome formation in smooth muscle cells (Webb et al., 2005), supporting a role for the PAK–PIX pathway in podosome formation. In macrophages, PAK4 and ␣PIX localize to podosomes and regulate podosome dynamics (Gringel et al., 2006). Interestingly, the effects of ␣PIX are independently of its GEF activity, suggesting that the complex of PAK and PIX proteins function downstream of Rho GTPases to control podosome dynamics in myeloid cells (Fig. 2). In addition to binding PAKs, PIXs also bind to G-proteincoupled receptor kinase-interacting protein 1 (GIT1) (Premont et al., 1998). Interestingly, GIT1 localizes to podosomes in HUVEC and was shown to be required for their formation (Wang et al., 2009a). Therefore, GIT1 could also be involved in podosome regulation in myeloid cells. It remains to be determined how the relative contribution of Cdc42 and Rac1/2 is in podosome regulation through the

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PAK–PIX pathway. Finally, RhoU and RhoV can bind to PAK (Shutes et al., 2004; Wherlock and Mellor, 2002) and could therefore also regulate podosomes through the PAK–PIX pathway.

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especially in myeloid cells and thus will enhance our knowledge of myeloid cell function. References

Rho kinase and mDia RhoA has several downstream effectors that regulate myosin IImediated contraction and thereby possibly involved in podosome regulation. RhoA binds Rho kinase, leading to an increase in the phosphorylation of myosin light chain and hence contractility (Amano et al., 1996; Bishop and Hall, 2000). In addition, Rho kinase promotes LIM kinase (LIMK) phosphorylation, which inhibits cofilin by phosphorylation, thereby increasing actin polymerization (Maekawa et al., 1999). Erk5/RhoGAP7 activity promotes podosome formation through RhoA inhibition and in ERK5−/− fibroblasts, podosome formation can be rescued by inhibiting Rho kinase or myosin II-mediated contraction (Schramp et al., 2008). These findings suggest that ERK5 and RhoGAP7 inhibit a cascade of RhoA/Rho kinase activation leading to contraction, which leads to podosome loss. Moreover, RhoA-mediated podosome loss in DCs is dependent on Rho kinase activity inducing myosin II-mediated contraction (van Helden et al., 2008). These findings suggest that Rho kinase is involved in RhoA-mediated negative regulation of podosomes (Fig. 2). Another Rho target, mDia, directly nucleates actin polymerization, as well as affecting the organization of microtubules (Fukata et al., 2003). In smooth muscle cells, mDia activation inhibits PDBu induced podosome formation (Burgstaller and Gimona, 2004), suggesting that also mDia is regulating podosome dynamics. Conclusions Rho GTPases are important regulators of the cytoskeleton and they are emerging as regulators of podosomes in various cell types, including myeloid cells. In Fig. 2, an overview of the Rho GTPase pathways regulating podosome dynamics in myeloid cells is given (Fig. 2). Cdc42 has a central role in podosome regulation and is essential for podosome formation. RhoJ, RhoQ and RhoU probably also promote podosome formation. In addition, Rac1 and Rac2 are involved in podosome regulation, where Rac2 seems critical for podosome formation and Rac1 for the maturation of these structures. The WASp-Arp2/3 pathway downstream of Cdc42, and possibly RhoU, is important in podosome formation. The effects of Rac1/2 could be mediated through the PAK/PIX pathway, however Cdc42 also activates this pathway and the relative contribution of Cdc42 and Rac1/2 are unclear at the moment. Although there are several candidate GEFs, Vav is currently the only GEF that has been shown to affect podosomes. RhoA appears to be a negative regulator of podosome formation. RhoA and Rho kinase activity leading to myosin II-mediated contraction needs to be limited, by p190RhoGAP or RhoGAP7, for podosome formation. Moreover, activation of the RhoA/Rho kinase axis is indispensable for podosome loss. The use of dominant negative and constitutive active mutants in overexpression studies has yielded many interesting findings, however these can also affect other Rho GTPase, especially within a subfamily. Therefore, studies based on knock out or knock down strategies, especially in myeloid cells, are required to further elucidate the effects of Rho GTPases on podosomes. An additional challenge is to assess cross talk between Rho GTPases and how this affects podosomes. In addition, the specific GEFs and GAPs involved, as well as the different effector pathways, need to be further defined. Finally, the effects of Rho GTPases on podosome function should be further elucidated. Together, this will allow us to better understand the function and regulation of podosomes,

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