EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 5, 427-435, October 2005 Heat shock protein 27 interacts with vimentin and prevents insolubilization of vimentin subunits induced by cadmium Jae-Seon Lee1,2*, Mei-Hua Zhang1*, shock protein 27 (HSP27) was colocalized and Eun Kyung Yun1, Dongho Geum3, physically associated with vimentin in unstressed Kyungjin Kim3, Tae-Hyung Kim1, cells, the roles of HSP27 with regard to vimentin were 1 1,4 assessed. HSP27-overexpressing cells prevented Yun-Sook Lim and Jeong-Sun Seo morphological alterations of the vimentin filaments, as well as reductions of soluble vimentin, in the 1ILCHUN Molecular Medicine Institute MRC cadmium-treated cells. Moreover, HSP27 antisense Department of Biochemistry and Molecular Biology oligonucleotide augmented these cadmium-in- Seoul National University College of Medicine duced changes in vimentin. These findings indicate Seoul 110-799, Korea 2 that HSP27 prevents disruption of the vimentin Laboratory of Functional Genomics intermediate filament networks and soluble vimentin Korea Institute of Radiological & Medical Sciences disappearance, by virtue of its physical interaction Seoul 139-706, Korea 3 with vimentin in cadmium-treated SK-N-SH cells. School of Biological Sciences Seoul National University Keywords: apoptosis; cadmium chloride; heat shock Seoul 151-742, Korea protein; mitogen activated protein kinases; molecular 4Corresponding author: Tel, 82-2-740-8246; chaperones; vimentin Fax, 82-2-741-5423; E-mail, [email protected] *These authors contributed equally to this work. Introduction Accepted 26 August 2005 Vimentin is a major component of intermediate fila- Abbreviation: ERK, extracellular signal-regulated kinase; GST, gluta- ment polypeptide, and forms a highly insoluble net- thione S-transferase; HSP, heat-shock protein; JNK, Jun N-terminal work in the fibroblasts. Vimentin surrounds the nu- kinase; MAPK, mitogen-activated protein kinase cleus, extending outwards to the cell periphery. It is responsible for cell elongation, regulation of cell at- tachment, subcellular organization, and signal trans- Abstract duction from the plasma membrane to the nucleus (Owen et al., 1996). Vimentin undergoes proteolysis, Vimentin is an intermediate filament that regulates and corresponds to ubiquitinated polypeptides form- cell attachment and subcellular organization. In this ed during apoptosis (Fujita et al., 2003). study, vimentin filaments were morphologically Cadmium, a toxic heavy metal, is quite harmful to altered, and its soluble subunits were rapidly re- human beings, due primarily to its long biological duced via cadmium chloride treatment. Cadmium half-life. Cadmium is a ubiquitous environmental con- chloride stimulated three major mitogen-activated taminant, and exerts mutagenic, carcinogenic, and protein kinases (MAPKs): extracellular signal-re- teratogenic effects. Humans are exposed to cadmi- gulated kinase (ERK), c-Jun N-terminal kinase (JNK), um in its forms as an industrial pollutant, a food and p38, and led apoptotic pathway via caspase-9 and contaminant, and as a component of tobacco smoke caspase-3 activations. In order to determine whether (Li et al., 2000b). Cadmium-induced DNA damage MAPKs were involved in this cadmium-induced may be the underlying cause of carcinogenic activity, soluble vimentin disappearance, we applied MAPK- along with cadmium-related effects on oncogene specific inhibitors (PD98059, SP600125, SB203580). expression (Wang and Templeton, 1998). Cadmium These inhibitors did not abolish the cadmium-in- can induce apoptosis and stress response in a variety of tissues and cells (Galan et al., 2000). The duced soluble vimentin disappearance. Caspase nervous system is a major target for the cadmium and proteosome degradation pathway were also not toxicity (Yoshida, 2001). It has been reported that involved in soluble vimentin disappearance. When cadmium disturbs important nervous system func- we observed vimentin levels in soluble and insoluble tions (Hart et al., 1989). However, the mechanisms fractions, soluble vimentin subunits shifted to an underlying many of the activities and effects of cad- insoluble fraction. As we discovered that heat- mium remain poorly understood. 428 Exp. Mol. Med. Vol. 37(5), 427-435, 2005 Heat-shock proteins (HSPs) play vital roles as pho-ERK (Thr202/yr204), and phospho-p38 (Thr180/ molecular chaperones in the folding, transport, and Tyr182) were obtained from Cell Signaling (Beverly, assembly of newly synthesized polypeptides. HSPs MA). Anti-HSP27, anti-actin, anti-caspase-3, anti-vi- are induced in response to a wide range of che- mentin, and peroxidase-conjugated secondary anti- micals and biological signals, including heat shock, bodies were purchased from Santa Cruz Biotechno- heavy metals, UV- and -radiation, oxidative stress, logy (Santa Cruz, CA). Anti-caspase-9 antibody was viral and bacterial infections, and osmotic stress obtained from BD Pharmigen (San Jose, CA). MG132, (Morimoto et al., 1990; Lee and Seo, 2002). This ALLN, PD98059, and SB203580 were purchased from suggests that HSPs may be involved in a general Calbiochem (San Diego, CA). SP600125 was obtained cellular defense mechanism. They contribute to cel- from TOCRIS (Bristol, UK). Enhanced chemilumine- lular protection via the restoration of denatured pro- scense (ECL) western blotting detection reagents teins, or via physical interaction with specific target were obtained from Amersham (Little Chalfont). proteins (Li et al., 2000a; Park et al., 2001). HSPs are highly conserved from bacteria to mammals, and Western blot analysis are generally divided into five major classes, accord- ing to molecular size: HSP27, HSP60, HSP70, After treatment of cadmium chloride, cells were HSP90, and HSP110. HSP27 exhibits an ATP-inde- washed with cold PBS three times and collected with pendent chaperone activity which prevents protein cell scraper. Protein extracts were prepared as aggregation, and function as anti-apoptotic molecule described in Lee and Seo (2002). Extracts were (Jakob et al., 1993; Ehrnsperger et al., 1997; Paul et subjected to SDS-PAGE electrophoresis and trans- al., 2002). Several mechanisms have been pro- ferred to a nitrocellulose membrane. The blot was posed to account for the anti-apoptotic activities of blocked with Tris-buffered saline (TBS) (20 mM Tris, HSP27 (Bruey et al., 2000). The cytoprotective effect pH 7.5, 137 mM NaCl) containing 5% nonfat milk for of HSP27 has been attributed to its function as a 1 h at room temperature, and incubated with primary modulator of the integrity of F-actin microfilaments antibodies (1:1,000 dilutions) in TBST (20 mM Tris, (Gerthoffer and Gunst, 2001). Small HSPs, HSP27 pH 7.5, 137 mM NaCl, 0.2% Tween 20) containing and B-crystallin, are associated specifically with 3% nonfat dry milk for 2 h at room temperature. The intermediate filaments in human pathologies involving blots were washed three times with TBST, and intermediate filament aggregates (Perng et al., 1999). incubated with horseradish peroxidase-conjugated HSP27-overexpressing cells showed resistance to cell secondary antibodies (1:5,000 dilutions) in TBST death by cadmium chloride (Wu and Welsh, 1996). A containing 3% nonfat dry milk for 2 h at room tem- potential role for HSP27 in the cellular response to perature. After washing three times with TBST, the cadmium was suggested due to cadmium-dependent blots were developed with the ECL detection sys- increase in HSP27 (Bonham et al., 2003). tem. Soluble and insoluble fractions were prepared In this study, we demonstrated that HSP27 is a as described in Yaglom et al. (2003). binding partner of vimentin, by conducting a co-im- munoprecipitation assay on human neuroblastoma Co-immunoprecipitation SK-N-SH cells. In order to ascertain the role of SK-N-SH cells were incubated with lysis buffer (50 HSP27 with regard to vimentin, we observed al- mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 150 mM terations in vimentin during cadmium-induced apop- NaCl, 0.5% sodium deoxycholate, and protease inhi- tosis, using HSP27-antisense or HSP27-overexpres- bitors). The lysates (500 µg) were precleared by sion strategies. Our results indicate that HSP27 gentle agitation with protein A/G-agarose beads at plays an important role in preventing the alteration of 4°C for 4 h. And then 2 µg anti-vimentin (V9) and vimentin filaments and vimentin subunit levels in the anti-HSP27 antibodies were added and incubated cadmium-induced apoptotic pathway. for 4 h at 4°C. The immunocomplexes were col- lected on protein A/G-agarose beads at 4°C over- night. After washing three times with lysis buffer, Materials and Methods they were analyzed by Western blotting with anti- vimentin and anti-HSP27 antibodies. In a pull-down Cell culture and reagents binding assay, SK-N-SH cell extracts (1 mg) were SK-N-SH cells were maintained at 37°C in DMEM incubated with glutathione S-transferase (GST)-vi- medium supplemented with 10% heat-inactivated mentin immobilized on glutathione-sepharose 4B fetal bovine serum (GIBCO, Carlsbad, CA) in a hu- beads at 4°C for 4 h. After centrifugation, the beads midified 95% air, 5% CO2 incubator. CdCl2 was pur- were washed three times, and then the bound chased from Sigma (St. Louis, MO). Antibodies against complex was analyzed by immunoblot using anti- JNK, ERK, p38, phospho-JNK (Thr183/Tyr185), phos- HSP27 or anti-vimentin antibodies. Physical and functional interaction between HSP27 and vimentin 429 Immunofluorescence staining with 10 µM HSP27 sense or anti-sense oligonu-