Small heat shock proteins and the cytoskeleton: An essential interplay for cell integrity?

The International Journal of Biochemistry & Cell Biology 44 (2012) 1680–1686

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Small heat shock proteins and the cytoskeleton: An essential interplay for cell integrity?夽 G. Wettstein a,b , P.S. Bellaye a,b , O. Micheau a,b , Ph Bonniaud a,b,c,∗ a b c

INSERM U866, University of Burgundy, Dijon 21079, France Faculty of Medicine and Pharmacy, University of Burgundy, Dijon 21079, France Service de Pneumologie, CHU Centre Hospitalo-Universitaire du Bocage, Dijon 21079, France

a r t i c l e

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Article history: Available online 7 June 2012 Keywords: Heat shock protein Cytoskeleton Fibrosis Cancer Neurological diseases

a b s t r a c t The cytoskeleton is a highly complex network of three major intracellular filaments, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs). This network plays a key role in the control of cell shape, division, functions and interactions in animal organs and tissues. Dysregulation of the network can contribute to numerous human diseases. Although small HSPs (sHSPs) and in particular HSP27 (HSPB1) or ␣B-crystallin (HSPB5) display a wide range of cellular properties, they are mostly known for their ability to protect cells under stress conditions. Mutations in some sHSPs have been found to affect their ability to interact with cytoskeleton proteins, leading to IF aggregation phenotypes that mimick diseases related to disorders in IF proteins (i.e. desmin, vimentin and neuro-filaments). The aim of this review is to discuss new findings that point towards the possible involvement of IFs in the cytoprotective functions of sHSPs, both in physiological and pathological settings, including the likelihood that sHSPs such as HSPB1 may play a role during epithelial-to-mesenchymal transition (EMT) during fibrosis or cancer progression. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.

1. Introduction The cytoskeleton is a complex network of highly ordered intracellular filaments that play a central role in the control of cell shape, division, functions and interactions in animal organs and tissues. Regulation of this network involves a variety of mechanisms, and dysregulation of the network can contribute to numerous human diseases. Heat shock proteins (HSPs), including small HSPs (sHSPs), are involved in a wide range of physiological cellular processes and are particularly known for their ability to help cells survive under stress conditions. The molecular mass of small heat shock proteins is low, ranging from 15 to 30 kDa, and they share high sequence homology within their crystallin domain (Garcia-Ranea et al., 2002). In humans, 11 crystallin-related sHSPs have been identified so far and named HSPB1 to HSPB11 (Kampinga et al., 2009). Of these, only HSP27 (HSPB1) and ␣B-crystallin (HSPB5) are induced by heat shock. However, like HSP20 (HSPB6), they can be expressed in various tissues including the lens, heart, lung,

夽 This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology. ∗ Corresponding author at: Service de pneumologie, CHU du bocage, 21079 Dijon, France. E-mail address: [email protected] (P. Bonniaud).

kidney, bladder, stomach, skeletal muscle and skin, where HSPB1 and HSPB5 have been shown to play a physiological role in nonheat shock-dependent processes, such as cell growth (Garrido et al., 1997; Spector et al., 1993), cell differentiation (Mehlen et al., 1997; Shakoori et al., 1992), apoptosis (Garrido et al., 1999, 2006), tumorigenesis (Garrido et al., 1998), signal transduction and the modulation of cytoskeleton proteins (Lavoie et al., 1993b). The physiological functions of HSPB1 and HSPB5 are tightly regulated by the phosphorylation of serine residues, allowing the formation of either large (hypo-phosphorylated) or small (hyperphosphorylated) oligomers. Consequently sHSPs are likely to be present in different flavors in the cells and thus to display any of the three well-known and interconnected properties or functions. First, a chaperone activity (Craig et al., 1994; Ellis, 1987; Ellis and van der Vies, 1991), which prevents client-protein aggregation, second, the ability to control the redox status (Federico et al., 2005; Guo et al., 2007) and third, the ability to regulate cytoskeleton dynamics (Landry and Huot, 1995; Mounier and Arrigo, 2002). The aim of this review is to provide an overview of the main cellular and molecular events involved in the control of cytoskeleton dynamics by sHSPs.

2. Cytoskeleton network: overview In eukaryotic cells (Wickstead and Gull, 2011), the cytoskeleton is a dynamic structure composed of an interconnected

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Fig. 1. Schematic representation of the intracellular organization of the 3 main components of the cytoskeleton, namely microtubules (MT), intermediary filaments (IF) and micro filaments (MF). Left panel, 3D representation. Right panel whole cell representation.

network of three major filament systems, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs) (Fuchs and Weber, 1994; Lazarides, 1980; Steinert and Parry, 1985). This filament network ensures cell rigidity and anchors subcellular organelles in place. Its constant reorganization allows internal movement, including chromosome displacement and membrane deformation, thus enabling endocytosis and cell migration. MTs (≈25 nm diameter) are responsible for structural strength and cell shape. They allow organelles to move within cells. They act like rails on which kinesin and dynein can pull organelles. MFs (≈7 nm diameter), on the other hand, are responsible for cell contraction and reinforcement of the cell surface, and allow changes in cell morphology. Actin and tubulin, two highly conserved proteins among species, are the main globular proteins that form MFs and MTs, respectively. IFs (≈10 nm diameter) are ubiquitous cytoskeletal elements and are among the most insoluble and most resilient solid structures of eukaryotic cells (Nicholl and Quinlan, 1994). They are the major components of the cytoskeleton (Evans, 1998). The key function of IFs is to support the cell membrane, serving as a structural scaffold to maintain cell shape. They are fixed to the membrane through transmembrane proteins such as cadherins, a protein family involved in the formation of cell–cell tight junctions. Thus IFs are involved in the distribution of traction forces that arise in the interspace between cells and are able to protect cells against disruption. They are encoded by the largest family of genes (70 genes) in the human genome (Helfand et al., 2004), and are divided into six groups, according to their structure. These groups are: (a) type I: keratins; (b) type II: cytokeratins (Fuchs and Weber, 1994); (c) type III: are mostly mesodermal (desmin in muscle cells, vimentin-related proteins in mesenchymal, endothelial and hemopoietic cells) (Evans, 1998; Szeverenyi et al., 2008), (d) type IV: neurofilaments and related proteins (Galou et al., 1997), (e) type V: lamins, which are exclusively nuclear and occur in all tissues (Szeverenyi et al., 2008) and (f) type VI: beaded filament proteins of the eye lens (Szeverenyi et al., 2008). Though these proteins have very different amino acid sequences, the organization of the structural domain is similar (Szeverenyi et al., 2008). IF proteins have distinct tissue-specific functions, which may explain their specific pattern of expression (Hesse et al., 2001; Lazarides, 1982). For example, epithelial cells express different specific keratins that are considered almost specific markers whereas mesenchymal cells, endothelial cells and hematopoietic cells express vimentin (Eckes et al., 2000; Herrmann et al., 2007; Kim and Coulombe, 2007; Nieminen et al., 2006). These three filament structures provide a scaffold, whose composition dictates not only cell shape, but also cell integration within a given tissue, or cell functionality (Fig. 1).

3. Intermediate filament turnover and sHSPs in normal stress conditions and diseases The classical heat shock response goes through mRNA synthesis leading to HSP overexpression. Yet, even before the predictable participation of HSPs (30–45 min after the heat shock), proteins associated with the cytoskeleton and nucleoskeleton fraction of the cell including vimentin, lamin A/C, Lamin B and emerin, have been shown to be up-regulated directly from mRNAs already present in the cell, in the absence of de novo gene synthesis (Dynlacht et al., 1999; Haddad and Paulin-Levasseur, 2008; Reiter and Penman, 1983). For this reason, these proteins are called “prompt HSPs” (Reiter and Penman, 1983). However, cytoskeleton proteins should be differentiated from HSPs. During stress conditions, IFs interact with HSPs such as HSP90 (HSPC) and HSP70 (HSPA) but most of all with sHSPs, and in particular HSPB1 and HSPB5. It has been reported that large HSPs mostly bind to the microtubule network and centrosome whereas sHSPs appear to play an important role in maintaining the integrity of IFs and actin filaments (Liang and MacRae, 1997). HSPB1 and HSPB5 exist as a complex, interacting with IFs and their soluble subunits within the cell. Perng et al. (1999) demonstrated that HSPB1 could colocalize with keratin 18 and that both HSPB5 and HSPB1 could colocalize with GFAP (glial fibrillary acidic protein). Although these interactions appeared to be stronger during heat shock, these findings indicated for the first time that sHSP may play a role during IF assembly, as well as in the control of inter-filament interactions (Perng et al., 1999). HSPB1 is known to interact not only with actin (see Section 4) (Huot et al., 1998) but also with other IF proteins and thus contributes to reorganization of the intermediate filament network. Other studies have reported similar functions for HSPB5 (Iwaki et al., 1989; Nicholl and Quinlan, 1994; Wisniewski and Goldman, 1998). Like HSPB1, HSPB5 can also be phosphorylated by MAPKAP-kinase 2 (Kato et al., 1998; Rouse et al., 1994). Thus, HSPB1 and HSPB5 share similar or overlapping physiological targets and interactions with IFs. Perng et al. (1999) pointed out though that HSPB5 may even exhibit stronger affinity than HSPB1 for IF binding, and, although HSPB1 and HSPB5 are located in similar subcellular structures, it was found that these sHSPs display distinct cellular functions, which have not yet been elucidated. For example, Kegel et al. (1996) reported differences in cell survival after hypertonic stress depending on the cell type and the balance of HSPB1 or HSPB5 expression levels. Thus, though the exact role of sHSP interactions with soluble IF subunits remains unclear, sHSP/IF interaction was shown to prevent IFs from forming non-covalent filament–filament interactions (Perng et al., 1999), even under stress conditions (Nicholl and Quinlan, 1994). Nicholl and Quinlan showed that HSPB5 binds both soluble subunits of vimentin and GFAP in an ATP-independent

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Fig. 2. Schematic representation of the interaction between sHSPs and intermediary filaments. In normal conditions this interaction allows IF formation in a well-organized network or scaffold. In stress conditions, the number of sHSP/IF interactions increases thus preventing IF aggregation. In the absence of sHSPs, IFs aggregate spontaneously. Some sHSP mutations lead to IF aggregation due to altered or exaggerated sHSP/IF interactions.

manner. In stress conditions, they observed a redistribution of HSPB5 from the detergent-soluble phase containing the IF subunits to the detergent-insoluble phase that contain IF (Nicholl and Quinlan, 1994). The association of sHSP with IF has been observed in a wide range of cell lines presenting different IF organization (Perng et al., 1999; Wisniewski and Goldman, 1998). The interactions between sHSPs and IFs occur both in stress and normal conditions. This association is known to maintain the individuality of IFs, to modulate IF interactions in their networks and to stabilize the assembly of intermediate filaments. Indeed sHSPs are able to interact with both IF monomers and whole IFs (Landsbury, 2010) (Fig. 2). The most relevant observation, confirming the importance of sHSP and IF interactions, probably comes from the phenotypic description of cells expressing sHSP mutants. Indeed in several cases, sHSP mutants led to IF aggregation phenotypes, which mimic diseases related to disorders of IF proteins (Fig. 2). Increasing evidence indicates that sHSPs could play an important role in IF (i.e. desmin, vimentin and neuro-filament) turnover, and that their dysfunction through protein mutations could lead to several human diseases. Some of them are reviewed in detail in the following sections of this article. HSPB5 is known to interact with desmin and to inhibit both its assembly and aggregation (Perng et al., 1999). This regulatory

mechanism was proposed to maintain IF homeostasis. In line with this hypothesis, it was found that a mutation in the HSPB5 gene could lead to several muscular disorders (Vicart et al., 1998), including desmin-related myopathy (DRM), an inherited disease characterized by desmin aggregation in skeletal and cardiac muscle (Goldfarb et al., 2008). This pathology results in limb, neck, trunk and facial muscle weakness and often affects cardiac muscle leading to cardiomyopathy. Several mutations in the desmin gene have been shown to be responsible for the onset of the disease (Goldfarb et al., 2008). These mutations cause an abnormal accumulation of desmin aggregates that resist normal turnover by the proteasome machinery, thus changing the mechanical properties of IFs in several cell types including cardiomyocytes. Several HSPB5 mutations, such as the point mutation R120G found in the human genome, change both HSPB5 structure and activity giving rise to an HSPB5 mutant that exhibits more affinity for desmin than does the wild type protein. The R120G mutant thus leads to desmin aggregation and causes a phenotype similar to DRM, which highlights the importance of IF-sHSP interaction in this kind of disease (Vicart et al., 1998). Another genetic disorder, Alexander disease (AxD), is caused by mutations found in an IF protein, glial fibrillary acidic protein (GFAP). AxD is an early-onset neurodegenerative disease, which results in seizures and developmental delay. In astrocytes, GFAP mutations lead to the formation of aggregates called Rosenthal

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fibers that contain GFAP, vimentin, and several sHSP such as HSPB1 and HSPB5. Studies have shown that these mutations decrease the solubility of GFAP. The severity of the disease is directly related to the site of the mutation on the GFAP sequence (Yoshida and Nakagawa, 2011). HSPB5 is known to regulate GFAP assembly and aggregation (Nicholl and Quinlan, 1994). During the disease, the accumulation of mutant GFAP impairs the ubiquitin-proteasome system, which leads to HSPB5 and HSPB1 accumulation in the brain of patients and to their sequestration in Rosenthal fibers (Iwaki et al., 1989). Thus, Rosenthal fibers in astocytes perturb the normal interaction between GFAP and HSPB5 and thereby disturb the functions of both proteins leading to AxD. The R120G HSPB5 mutation also impairs the ubiquitin-proteasome system leading to an exacerbation of HSPB5 and HSPB1 accumulation in cells and a greater degree of IF aggregation. Evidence of the key role played by HSPB5 in this disease is provided by the positive effect of HSPB5 overexpression on survival in a mouse model of AxD. Overexpression of HSPB5 was shown to induce the disaggregation GFAP filaments and to improve survival rates in a mouse model of AxD (Tang et al., 2010). IFs are also essential for the optical properties of the lens, and vimentin is one of the most abundant IFs found there. Several mutations in both human and mouse IF components have been identified as a cause of congenital cataract, but the mechanisms underlying the etiology of this disease are not yet completely understood (Song et al., 2009). Interestingly, the HSPB5 R120G point mutation can also cause cataract. In normal conditions, HSPB5 interacts with vimentin as well as with other IFs and exerts a chaperone activity towards vimentin thus enhancing its stabilization. However, mutation of this protein on residue 120 (R120G) in the lens induces the formation of vimentin aggregates, as a result of a loss of chaperone activity (Song et al., 2008). The R120G mutant was found to give rise to a misfolded protein, which exposes hydrophobic regions on its surface. In vitro studies showed that this HSPB5 mutant was prone to self-aggregation. Therefore, as large amounts of vimentin are present in eye lens and since it interacts with HSPB5, it has been suggested that the R120G mutant induces vimentin aggregation as well (Song et al., 2008), leading to lens opacity, which characterizes cataract. Charcot–Marie–Tooth (CMT) disease is one of the most common inherited neuromuscular disorders and is characterized by the degeneration of peripheral nerves resulting in weakness in the hands and feet (Houlden et al., 2008). Several mutations, including mutations in the neurofilament light (NFL) protein, that affect both IFs and HSPB1 have already been identified as causes of this syndrome (Zhai et al., 2007). Surprisingly, the corresponding HSPB1 (S135F) mutant has been found to exhibit stronger chaperone activity and affinity for client proteins than does the wild type HSPB1 (Almeida-Souza et al., 2011). This enhanced function was shown to lead to increased affinity for tubulin and microtubules (MT), resulting in better stabilization of the microtubule network in neurons isolated from a mouse model expressing the HSPB1 mutant (Almeida-Souza et al., 2011). In this mouse model, mutation of HSPB1 (S135F) led to disorders similar to those due to IF mutations and induced a CMT phenotype. Those data provide evidence of the importance of the interaction between IFs and sHSP for neuron homeostasis.

4. Small HSPs and actin fibers from physiological processes to pathology Actin is a ubiquitous and abundant eukaryotic protein. This cytoskeletal protein is involved in many cellular functions including muscle contraction, cell motility, cell adhesion, cell division, and the maintenance of cell shape. There are six closely related

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proteins in the actin family (Vandekerckhove and Weber, 1978). All six genes reside on different chromosomes. Four of them are muscle specific isoforms and two are ubiquitous. ␣-Skeletal actin (␣-SKA), ␣-cardiac actin (␣-CAA), ␣-smooth muscle actin (␣SMA), and ␥-smooth muscle actin (␥-SMA) are predominant in adult skeletal muscles, cardiac striated muscles, vascular smooth muscles and enteric smooth muscles, respectively. The two other isoforms, namely cytoplasmic ␤-(␤-CYA) and ␥-actins (␥-CYA) are ubiquitous (Chaponnier and Gabbiani, 2004). The biological significance of these different isoforms is not really understood as they share a very high degree of sequence identity (>93%) (Jaeger et al., 2009). The primary structure of all six actin isoforms is completely conserved across species from birds to humans (Kabsch and Vandekerckhove, 1992). However, in spite of their high homology, the different isoforms are not interchangeable and seem to serve a unique purpose. Actins are sorted into different subcellular compartments (Schevzov et al., 1992) and are differentially utilized by cell machinery when expressed in transfected cells (Mounier and Arrigo, 2002). The restricted distribution of the six isoforms only exists in perfectly healthy individuals. The modulation of actin isoform expression within the same tissue is an important marker of adaptative and/or pathological changes (Chaponnier and Gabbiani, 2004). Actin filaments form a double-stranded helix, which displays both structural and functional polarity. The filament has a minus end and a plus end. When elongation starts the plus end also named the fast-growing end or barbed end grows 5–10 times faster than the minus end. At the steady state, actin subunits fix at the plus end and disassemble at the minus end at the same rate, allowing the filament to maintain its length (Chaponnier and Gabbiani, 2004). To allow actin polymerization and depolymerization, a dynamic equilibrium arises between the globular monomeric form, named G-actin which is soluble, and the polymerized form, named F-actin. A large number of proteins interact with G- and/or F-actin to regulate the dynamics of these filaments. Some of them block the polymerization, promote the depolymerization or stabilize the filaments, and thus contribute to the formation of the actin network. HSPB1 and HSPB5 are known to interact with actin fibers and their interactions have been described in several papers and reviews (Bennardini et al., 1992; Doshi et al., 2010; Pivovarova et al., 2005, 2007; Singh et al., 2007). In non-stress conditions, the cytoskeleton and principally actin fibers are involved in several cellular processes such as motility and pinocytosis. The first observable effects of stress are the disruption of the cytoskeleton and the disaggregation of actin fibers. The stabilization of not only actin fibers but also all major cytoskeleton fibers is critical for cell survival. As HSPB1 is able to cap the plus end of actin filaments, thus preventing the fixation of a new actin monomer, it was identified as an inhibitor of actin polymerization (Miron et al., 1991, 1988). This inhibitory activity of HSPB1 is, however, only effective when HSPB1 is in its non-phosphorylated monomer state. Neither phosphorylated monomers nor non-phosphorylated oligomers exhibit this inhibitory property and are thus unable to inhibit actin filament polymerization (Benndorf et al., 1994). HSPB1-mediated cell survival after heat shock or oxidative stress largely relies on the ability of HSPB1 to stabilize actin filaments and thus prevent their aggregation. Moreover, actin filament rebuilding after disruption due to a stress is accelerated in cells that over-express HSPB1 (Huot et al., 1996; Lavoie et al., 1993a,b, 1995). Inversely, inhibition of HSPB1 expression leads to actin filament disorganization (Horman et al., 1999; Mairesse et al., 1996). In contrast, transfection of a non-phosphorylatable HSPB1 mutant fails to protect cells against stress-induced actin filament disruption (Huot et al., 1996; Lavoie et al., 1993a,b, 1995). Altogether, these results indicate that non-phophorylated HSPB1 inhibits actin filament growth

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Fig. 3. Schematic representation of the interaction between sHSPs and actin. Actin fibers are dynamic. There is a dynamic equilibrium between polymerization and depolymerization allowing the fibers to grow up or not. HSPB1 under its monomer form could cap actin fibers and inhibit polymerization. Under its phophorylated form HSPB1 could prevent stress induced actin fibers aggregation.

while phosphorylated HSPB1 is involved in actin filament dynamics and cell protection (Fig. 3). Albeit to a lesser extent, HSPB5 appears to show similar properties. Likewise, and as reported in a number of publications, HSPB5 was also shown to inhibit actin fiber depolymerization induced by cytochalasin D and to prevent their aggregation induced by stress (Singh et al., 2007; Wang and Spector, 1996). Bhairab et al. demonstrated that, while HSPB5 is located in the cytoplasm where it shows no specific organization in normal conditions, it is organized in fibers that colocalized with the actin after heat shock. The interaction of HSPB5 with actin was shown to be tightly regulated by HSPB5 phosphorylation on both Ser45 and Ser59 residues. Moreover the same authors demonstrated that HSPB5 can exhibit a protective function during specific physiological molecular events such as pinocytosis (Singh et al., 2007). MacIntyre et al. (2008) showed, in protein extracts obtained from laboring or non-laboring myometria, that HSPB1 can interact with both HSPB5 and ␣-SMA. The role of these complexes remains unclear, but the authors suggest that HSPB5 may release HSPB1 during labor and thus enable cytoskeleton remodeling. Their experiments highlighted the fact that in laboring but not in nonlaboring myometria the decrease in HSPB1 and HSPB5 co-localization within the cytoplasm correlated not only with an increase in HSPB1 and ␣-SMA co-localization, but also with a change in HSPB1 phosphorylation status (MacIntyre et al., 2008). Indeed, the authors found an increase in HSPB1-Ser15 phosphorylation and a decrease in HSPB1Ser82 phosphorylation during labor. It was then found that HSPB1 phosphorylation on Ser15 allowed binding to and the stabilization of actin filaments (Lambert et al., 1999). The authors suggested that the decrease in HSPB1-Ser82 phosphorylation during labor would allow a conformational change facilitating HSPB1 interactions with the cytoskeleton. Epithelial-to-mesenchymal transition (EMT) is a physiological and pathological process that leads to massive cytoskeleton changes and reorganization. In physiological conditions, during embryologic development, this process allows the formation of the mesoderm from the epithelium during gastrulation. It is involved in

the formation of the heart and palate closure (Hay, 1995; Willis and Borok, 2007). EMT is also involved in several pathological conditions such as cancer and fibrosis. In cancer, EMT enables malignant cells to acquire a migratory phenotype and is thus associated with tumor invasiveness. In organ fibrosis and fibrogenesis, as observed in the lungs, kidneys, skin or liver, EMT refers to the formation of myofibroblasts, key cells in the synthesis and accumulation of collagen. EMT could be seen as the acquisition of extreme plasticity by epithelial cells. It is characterized by the loss of polarity, the loss of epithelial markers, reorganization of the cytoskeleton and the acquisition of mesenchymal markers (Willis and Borok, 2007). One of the main characteristics of EMT, and in particular in fibrotic processes, is also the increase in ␣-SMA expression. This form of actin is abundantly present in smooth muscle cells but also in myofibroblasts. ␣-SMA is generally considered a marker of myofibrobasts in fibrotic tissue, but its functions in the EMT process are not yet fully understood. The EMT process is linked to an increase in ␣-SMA fibers, but also to a lack of cell polarity leading to the loss of cell–cell interaction. Moreover, EMT is also associated with a concomitant decrease in cytokeratins, expressed in epithelial cells, and to an increase in vimentin and desmin, usually expressed by mesenchymal cells. The growth factor TGF-␤1 has been shown to play a key role during EMT acquisition in various fibrosis and cancer models. In a rat model of kidney fibrosis with EMT, HSPB1 and ␣-SMA were shown to co-localize in fibrotic areas. In this study, the phosphorylated form of HSPB1 was shown to interact with the cytoskeleton and was thus thought to contribute to the EMT process. In line with this hypothesis, the authors were able to show that the non-phosphorylated form of HSPB1 was able to inhibit not only the loss of E-cadherin expression but also EMT (Vidyasagar et al., 2008). Using an in vitro peritoneal mesothelial cell model, Vargha et al. (2008) showed that EMT, induced by rTGF-␤1, correlated with a concomitant increase in HSPB1 and ␣-SMA expression levels. This non-stress related upregulation of HSP-27 resulted in sufficiently high levels of HSP to attenuate stress-induced HSP expression and to protect cells moderately exposed to peritoneal dialysis fluid, which is known to induce peritoneal fibrosis.

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Interestingly, Wei et al. recently highlighted in breast cancer stem cells (BCSCs) that HSPB1 could play a role in EMT and that inactivation of HSPB1 expression decreased the EMT signature known to be associated with metastasis potency (Wei et al., 2011). Thus, the strong overexpression of HSPB1 around fibroblast foci (consisting of accumulations and aggregations of myofibroblast) in idiopathic pulmonary fibrosis suggests that HSPB1 may be a good target for some lung diseases (Chilosi et al., 2006; Korfei et al., 2011). Alternatively, the presence of HSPB1 in these areas could represent a marker of recent EMT undergone by the actual myofibroblasts. Myofibroblasts display an extremely migratory and invasive phenotype thanks to their highly contractile cytoskeleton. It is thus conceivable that sHSPs may contribute to the maintenance of the contractile phenotype of these cells and, in particular, HSPB1 may be required for EMT owing to its ability to regulate cytoskeleton maintenance and dynamics (Fig. 2). 5. Microtubules Whereas interactions between sHSPs and actin filaments or intermediate filaments are now well documented, data on the relationship between sHSPs and microtubules remain scarce. HSPB5 may nonetheless be involved in microtubule protection since microtubule depolymerization drugs such as colchicines, colcemid, vinblastine and nocodazole induce HSPB5 but not HSPB1 overexpression (Kato et al., 1996). Moreover, Arai et al. demonstrated that HSPB5 could function as a molecular chaperone to prevent tubulin aggregation. HSPB5 was found to recognize and bind to denatured tubulin at the onset of its denaturation process, thereby preventing tubulin self-aggregation (Arai and Atomi, 1997). Houck and Clark (2010) highlighted the fact that HSPB5 can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules: low molar ratios of HSPB5/tubulin are favorable for microtubule assembly, whereas high molar ratios of HSPB5/tubulin are unfavorable. The role of HSPB1 with regard to microtubules remains unclear. It has been suggested that HSPB1 may play a role in the progression of mitosis, a process that involves microtubules (Hino et al., 2000). It was found that HSPB1 only interacted with taxol-stabilized microtubules, but not denatured tubulin. However, an HSPB1 mutant was recently found to exhibit strong binding to tubulin and was associated with Charcot–Marie–Tooth disease (Almeida-Souza et al., 2011). 6. Conclusion A growing body of evidence indicates that sHSPs confer stress protection by preventing cytoskeleton aggregation. How these small proteins afford protection at the molecular level still remains uncertain. There is evidence, however, that their cytoprotective functions may be due to their ability to interact with IF polymers and IF subunits. In support of this hypothesis, mutations that affect the ability of various sHSPs to interact with cytoskeleton proteins have been found to lead to similar pathological disorders, and phenotypes, as those due to mutations in a number of cytoskeleton components. Abnormal cytoskeleton assembly may also involve sHSPs and cytoskeleton components in other pathological conditions, including the EMT process found in fibrosis and cancer, opening the way for novel therapeutic opportunities. Acknowledgments G. Wettstein is supported by the EU 7th Framework Programme (2007–2013 agreement number HEALTH-F2-2007202224 eurIPFnet.) and “Fonds de dotation Recherche en Santé Respiratoire” et la Société de Pneumologie de Langue Franc¸aise.

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P. Bonniaud’s team is supported by “la Ligue Régionale Contre le Cancer” and ANR blanc ANR 11-BSV-011-01 Meso-IPF. O. Micheau is supported by an ANR blanc SphingoDR. Authors thank Philip Bastable for his helpful advices. References Almeida-Souza L, Asselbergh B, d‘Ydewalle C, Moonens K, Goethals S, de Winter V, et al. Small heat-shock protein HSPB1 mutants stabilize microtubules in Charcot–Marie–Tooth neuropathy. Journal of Neuroscience 2011;31:15320–8. Arai H, Atomi Y. Chaperone activity of alpha B-crystallin suppresses tubulin aggregation through complex formation. Cell Structure and Function 1997;22:539–44. Bennardini F, Wrzosek A, Chiesi M. Alpha B-crystallin in cardiac tissue. Association with actin and desmin filaments. Circulation Research 1992;71:288–94. Benndorf R, Hayess K, Ryazantsev S, Wieske M, Behlke J, Lutsch G. Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerization-inhibiting activity. Journal of Biological Chemistry 1994;269:20780–4. Chaponnier C, Gabbiani G. Pathological situations characterized by altered actin isoform expression. Journal of Pathology 2004;204:386–95. Chilosi M, Zamo A, Doglioni C, Reghellin D, Lestani M, Montagna L, et al. Migratory marker expression in fibroblast foci of idiopathic pulmonary fibrosis. Respiratory Research 2006;7:95. Craig EA, Weissman JS, Horwich AL. Heat shock proteins and molecular chaperones: mediators of protein conformation and turnover in the cell. Cell 1994;78:365–72. Doshi BM, Hightower LE, Lee J. HSPB1, actin filament dynamics, and aging cells. Annals of the New York Academy of Sciences 2010;1197:76–84. Dynlacht JR, Story MD, Zhu WG, Danner J. Lamin B is a prompt heat shock protein. Journal of Cellular Physiology 1999;178:28–34. Eckes B, Colucci-Guyon E, Smola H, Nodder S, Babinet C, Krieg T, et al. Impaired wound healing in embryonic and adult mice lacking vimentin. Journal of Cell Science 2000;113(Pt 13):2455–62. Ellis J. Proteins as molecular chaperones. Nature 1987;328:378–9. Ellis RJ, van der Vies SM. Molecular chaperones. Annual Review of Biochemistry 1991;60:321–47. Evans RM. Vimentin: the conundrum of the intermediate filament gene family. Bioessays 1998;20:79–86. Federico A, Tuccillo C, Terracciano F, D‘Alessio C, Galdiero M, Finamore E, et al. Heat shock protein 27 expression in patients with chronic liver damage. Immunobiology 2005;209:729–35. Fuchs, Weber EK. Intermediate filaments: structure, dynamics, function, and disease. Annual Review of Biochemistry 1994;63:345–82. Galou M, Gao J, Humbert J, Mericskay M, Li Z, Paulin D, et al. The importance of intermediate filaments in the adaptation of tissues to mechanical stress: evidence from gene knockout studies. Biology of the Cell 1997;89:85–97. Garcia-Ranea JA, Mirey G, Camonis J, Valencia A. p23 and HSP20/alpha-crystallin proteins define a conserved sequence domain present in other eukaryotic protein families. FEBS Letters 2002;529:162–7. Garrido C, Bruey JM, Fromentin A, Hammann A, Arrigo AP, Solary E. HSP27 inhibits cytochrome c-dependent activation of procaspase-9. FASEB Journal 1999;13:2061–70. Garrido C, Brunet M, Didelot C, Zermati Y, Schmitt E, Kroemer G. Heat shock proteins 27 and 70: anti-apoptotic proteins with tumorigenic properties. Cell Cycle 2006;5:2592–601. Garrido C, Fromentin A, Bonnotte B, Favre N, Moutet M, Arrigo AP, et al. Heat shock protein 27 enhances the tumorigenicity of immunogenic rat colon carcinoma cell clones. Cancer Research 1998;58:5495–9. Garrido C, Ottavi P, Fromentin A, Hammann A, Arrigo AP, Chauffert B, et al. HSP27 as a mediator of confluence-dependent resistance to cell death induced by anticancer drugs. Cancer Research 1997;57:2661–7. Goldfarb LG, Olive M, Vicart P, Goebel HH. Intermediate filament diseases: desminopathy. Advances in Experimental Medicine and Biology 2008;642:131–64. Guo S, Wharton W, Moseley P, Shi H. Heat shock protein 70 regulates cellular redox status by modulating glutathione-related enzyme activities. Cell Stress Chaperones 2007;12:245–54. Haddad N, Paulin-Levasseur M. Effects of heat shock on the distribution and expression levels of nuclear proteins in HeLa S3 cells. Journal of Cellular Biochemistry 2008;105:1485–500. Hay ED. An overview of epithelio-mesenchymal transformation. Acta Anatomica (Basel) 1995;154:8–20. Helfand BT, Chang L, Goldman RD. Intermediate filaments are dynamic and motile elements of cellular architecture. Journal of Cell Science 2004;117:133–41. Herrmann H, Bar H, Kreplak L, Strelkov SV, Aebi U. Intermediate filaments: from cell architecture to nanomechanics. Nature Reviews Molecular Cell Biology 2007;8:562–73. Hesse M, Magin TM, Weber K. Genes for intermediate filament proteins and the draft sequence of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. Journal of Cell Science 2001;114:2569–75. Hino M, Kurogi K, Okubo MA, Murata-Hori M, Hosoya H. Small heat shock protein 27 (HSP27) associates with tubulin/microtubules in HeLa cells. Biochemical and Biophysical Research Communications 2000;271:164–9.

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G. Wettstein et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 1680–1686

Horman S, Fokan D, Mosselmans R, Mairesse N, Galand P. Anti-sense inhibition of small-heat-shock-protein (HSP27) expression in MCF-7 mammary-carcinoma cells induces their spontaneous acquisition of a secretory phenotype. International Journal of Cancer 1999;82:574–82. Houck SA, Clark JI. Dynamic subunit exchange and the regulation of microtubule assembly by the stress response protein human alphaB crystallin. PLoS One 2010;5:e11795. Houlden H, Laura M, Wavrant-De Vrieze F, Blake J, Wood N, Reilly MM. Mutations in the HSP27 (HSPB1) gene cause dominant, recessive, and sporadic distal HMN/CMT type 2. Neurology 2008;71:1660–8. Huot J, Houle F, Rousseau S, Deschesnes RG, Shah GM, Landry J. SAPK2/p38dependent F-actin reorganization regulates early membrane blebbing during stress-induced apoptosis. Journal of Cell Biology 1998;143:1361–73. Huot J, Houle F, Spitz DR, Landry J. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Research 1996;56:273–9. Iwaki T, Kume-Iwaki A, Liem RK, Goldman JE. Alpha B-crystallin is expressed in non-lenticular tissues and accumulates in Alexander‘s disease brain. Cell 1989;57:71–8. Jaeger MA, Sonnemann KJ, Fitzsimons DP, Prins KW, Ervasti JM. Context-dependent functional substitution of alpha-skeletal actin by gamma-cytoplasmic actin. FASEB Journal 2009;23:2205–14. Kabsch W, Vandekerckhove J. Structure and function of actin. Annual Review of Biophysics and Biomolecular Structure 1992;21:49–76. Kampinga HH, Hageman J, Vos MJ, Kubota H, Tanguay RM, Bruford EA, et al. Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 2009;14:105–11. Kato K, Ito H, Inaguma Y, Okamoto K, Saga S. Synthesis and accumulation of alphaB crystallin in C6 glioma cells is induced by agents that promote the disassembly of microtubules. Journal of Biological Chemistry 1996;271:26989–94. Kato K, Ito H, Kamei K, Inaguma Y, Iwamoto I, Saga S. Phosphorylation of alphaBcrystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation. Journal of Biological Chemistry 1998;273:28346–54. Kegel KB, Iwaki A, Iwaki T, Goldman JE. AlphaB-crystallin protects glial cells from hypertonic stress. American Journal of Physiology 1996;270:C903–9. Kim S, Coulombe PA. Intermediate filament scaffolds fulfill mechanical, organizational, and signaling functions in the cytoplasm. Genes and Development 2007;21:1581–97. Korfei M, Schmitt S, Ruppert C, Henneke I, Markart P, Loeh B, et al. Comparative proteomic analysis of lung tissue from patients with idiopathic pulmonary fibrosis (IPF) and lung transplant donor lungs. Journal of Proteome Research 2011;10:2185–205. Lambert H, Charette SJ, Bernier AF, Guimond A, Landry J. HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus. Journal of Biological Chemistry 1999;274:9378–85. Landry J, Huot J. Modulation of actin dynamics during stress and physiological stimulation by a signaling pathway involving p38 MAP kinase and heat-shock protein 27. Biochemistry and Cell Biology 1995;73:703–7. Landsbury A, Der Perng M, Pohl E, Quinlan RA. Functional symbiosis between the intremediate filament cytoskeleton and small heat shock proteins. In: Small Stress Proteins and Human Diseases. Nova Science Publishers, Inc; 2010. pp. 55–87. Lavoie JN, Gingras-Breton G, Tanguay RM, Landry J. Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization. Journal of Biological Chemistry 1993a;268:3420–9. Lavoie JN, Hickey E, Weber LA, Landry J. Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. Journal of Biological Chemistry 1993b;268:24210–4. Lavoie JN, Lambert H, Hickey E, Weber LA, Landry J. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Molecular and Cellular Biology 1995;15:505–16. Lazarides E. Intermediate filaments as mechanical integrators of cellular space. Nature 1980;283:249–56. Lazarides E. Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Annual Review of Biochemistry 1982;51:219–50. Liang P, MacRae TH. Molecular chaperones and the cytoskeleton. Journal of Cell Science 1997;110(Pt 13):1431–40. MacIntyre DA, Tyson EK, Read M, Smith R, Yeo G, Kwek K, et al. Contraction in human myometrium is associated with changes in small heat shock proteins. Endocrinology 2008;149:245–52. Mairesse N, Horman S, Mosselmans R, Galand P. Antisense inhibition of the 27 kDa heat shock protein production affects growth rate and cytoskeletal organization in MCF-7 cells. Cell Biology International 1996;20:205–12. Mehlen P, Mehlen A, Godet J, Arrigo AP. hsp27 as a switch between differentiation and apoptosis in murine embryonic stem cells. Journal of Biological Chemistry 1997;272:31657–65. Miron T, Vancompernolle K, Vandekerckhove J, Wilchek M, Geiger B. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein. Journal of Cell Biology 1991;114:255–61. Miron T, Wilchek M, Geiger B. Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle. European Journal of Biochemistry 1988;178:543–53. Mounier N, Arrigo AP. Actin cytoskeleton and small heat shock proteins: how do they interact? Cell Stress Chaperones 2002;7:167–76.

Nicholl ID, Quinlan RA. Chaperone activity of alpha-crystallins modulates intermediate filament assembly. EMBO Journal 1994;13:945–53. Nieminen M, Henttinen T, Merinen M, Marttila-Ichihara F, Eriksson JE, Jalkanen S. Vimentin function in lymphocyte adhesion and transcellular migration. Nature Cell Biology 2006;8:156–62. Perng MD, Cairns L, van den IP, Prescott A, Hutcheson AM, Quinlan RA. Intermediate filament interactions can be altered by HSP27 and alphaB-crystallin. Journal of Cell Science 1999;112(Pt 13):2099–112. Pivovarova AV, Chebotareva NA, Chernik IS, Gusev NB, Levitsky DI. Small heat shock protein Hsp27 prevents heat-induced aggregation of F-actin by forming soluble complexes with denatured actin. FEBS Journal 2007;274:5937–48. Pivovarova AV, Mikhailova VV, Chernik IS, Chebotareva NA, Levitsky DI, Gusev NB. Effects of small heat shock proteins on the thermal denaturation and aggregation of F-actin. Biochemical and Biophysical Research Communications 2005;331:1548–53. Reiter T, Penman S. Prompt heat shock proteins: translationally regulated synthesis of new proteins associated with the nuclear matrix-intermediate filaments as an early response to heat shock. Proceedings of the National Academy of Sciences of the United States of America 1983;80:4737–41. Rouse J, Cohen P, Trigon S, Morange M, Alonso-Llamazares A, Zamanillo D, et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 1994;78:1027–37. Schevzov G, Lloyd C, Gunning P. High level expression of transfected beta- and gamma-actin genes differentially impacts on myoblast cytoarchitecture. Journal of Cell Biology 1992;117:775–85. Shakoori AR, Oberdorf AM, Owen TA, Weber LA, Hickey E, Stein JL, et al. Expression of heat shock genes during differentiation of mammalian osteoblasts and promyelocytic leukemia cells. Journal of Cellular Biochemistry 1992;48: 277–87. Singh BN, Rao KS, Ramakrishna T, Rangaraj N, Rao Ch. M. Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo. Journal of Molecular Biology 2007;366: 756–67. Song S, Hanson MJ, Liu BF, Chylack LT, Liang JJ. Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching. Molecular Vision 2008;14:1282–7. Song S, Landsbury A, Dahm R, Liu Y, Zhang Q, Quinlan RA. Functions of the intermediate filament cytoskeleton in the eye lens. Journal of Clinical Investigation 2009;119:1837–48. Spector NL, Ryan C, Samson W, Levine H, Nadler LM, Arrigo AP. Heat shock protein is a unique marker of growth arrest during macrophage differentiation of HL-60 cells. Journal of Cellular Physiology 1993;156:619–25. Steinert PM, Parry DA. Intermediate filaments: conformity and diversity of expression and structure. Annual Review of Cell Biology 1985;1:41–65. Szeverenyi I, Cassidy AJ, Chung CW, Lee BT, Common JE, Ogg SC, et al. The human intermediate filament database: comprehensive information on a gene family involved in many human diseases. Human Mutation 2008;29: 351–60. Tang G, Perng MD, Wilk S, Quinlan R, Goldman JE. Oligomers of mutant glial fibrillary acidic protein (GFAP) inhibit the proteasome system in Alexander disease astrocytes, and the small heat shock protein alphaB-crystallin reverses the inhibition. Journal of Biological Chemistry 2010;285:10527–37. Vandekerckhove J, Weber K. At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. Journal of Molecular Biology 1978;126:783–802. Vargha R, Bender TO, Riesenhuber A, Endemann M, Kratochwill K, Aufricht C. Effects of epithelial-to-mesenchymal transition on acute stress response in human peritoneal mesothelial cells. Nephrology, Dialysis, and Transplantation 2008. Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, et al. A missense mutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nature Genetics 1998;20:92–5. Vidyasagar A, Reese S, Acun Z, Hullett D, Djamali A. HSP27 is involved in the pathogenesis of kidney tubulointerstitial fibrosis. American Journal of Physiology Renal Physiology 2008;295:F707–16. Wang K, Spector A. alpha-crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner. European Journal of Biochemistry 1996;242:56–66. Wei L, Liu TT, Wang HH, Hong HM, Yu AL, Feng HP, et al. Hsp27 participates in the maintenance of breast cancer stem cells through regulation of epithelialmesenchymal transition and nuclear factor-kappaB. Breast Cancer Research 2011;13:R101. Wickstead B, Gull K. The evolution of the cytoskeleton. Journal of Cell Biology 2011;194:513–25. Willis BC, Borok Z. TGF-beta-induced EMT: mechanisms and implications for fibrotic lung disease. American Journal of Physiology Lung Cellular and Molecular Physiology 2007;293:L525–34. Wisniewski T, Goldman JE. Alpha B-crystallin is associated with intermediate filaments in astrocytoma cells. Neurochemical Research 1998;23:385–92. Yoshida T, Nakagawa M. Clinical aspects and pathology of Alexander disease, and morphological and functional alteration of astrocytes induced by GFAP mutation. Neuropathology 2011. Zhai J, Lin H, Julien JP, Schlaepfer WW. Disruption of neurofilament network with aggregation of light neurofilament protein: a common pathway leading to motor neuron degeneration due to Charcot–Marie–Tooth disease-linked mutations in NFL and HSPB1. Human Molecular Genetics 2007;16:3103–16.