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781.Full.Pdf Research Article 781 Characterization of human epiplakin: RNAi-mediated epiplakin depletion leads to the disruption of keratin and vimentin IF networks Shyh-Ing Jang*,‡,§, Alexandr Kalinin*, Kaoruko Takahashi, Lyuben N. Marekov and Peter M. Steinert¶ Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, MD 20892-8023, USA *These authors contributed equally to this work ‡Present address: Human Craniofacial Genetics Section, CRC, NIDCR, NIH, DHHS, Building 10, Room 5N102, 10 Center Drive, Bethesda, MD 20892-1423, USA ¶This paper is dedicated to the memory of Peter Steinert who initiated and provided great inspiration on this study §Author for correspondence (e-mail: [email protected]) Accepted 16 November 2004 Journal of Cell Science 118, 781-793 Published by The Company of Biologists 2005 doi:10.1242/jcs.01647 Summary Epiplakin is a member of the plakin family with multiple networks in transient knockdown; however, it could only copies of the plakin repeat domain (PRD). We studied partially restore the keratin but not the vimentin IF the subcellular distribution and interactions of human network in stably knocked down HeLa cells. We suggest epiplakin by immunostaining, overlay assays and RNAi that epiplakin is a cytolinker involved in maintaining knockdown. Epiplakin decorated the keratin intermediate the integrity of IF networks in simple epithelial cells. filaments (IF) network and partially that of vimentin. In Furthermore, we observed an increase of epiplakin the binding assays, the repeat unit (PRD plus linker) expression in keratinocytes after the calcium switch, showed strong binding and preferentially associated suggesting the involvement of epiplakin in the process of with assembled IF over keratin monomers. Epiplakin keratinocyte differentiation. knockdown revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF Key words: epiplakin, intermediate filaments, keratinocyte Introduction fragility such as that displayed in muscular dystrophy and the Journal of Cell Science Epiplakin was first discovered as an autoantigen in a patient skin disease epidemolysis bullosa simplex (Fuchs and with subepidermal blistering skin disease (Fujiwara et al., Cleveland, 1998). 1996). Later it was found in a variety of tissues, such as liver, Most plakins are very large proteins that share certain small intestine, stomach, salivary gland, esophagus and skin functional domains such as the actin-binding domain (ABD), (Fujiwara et al., 2001). Based on its deduced protein sequence, plakin domain, coiled-coil rod domain and plakin-repeat it was classified as a member of the plakin family with the domain (PRD). These proteins contribute to the maintenance presence of plakin-repeat domain (PRD) homologous to the of tissue integrity by interacting with cytoskeletal filaments and B subdomain of desmoplakin (Green et al., 1990). Plakin anchoring them to each other and to membrane junction family members, including desmoplakin, plectin, envoplakin, complexes (reviewed by Ruhrberg and Watt, 1997; Fuchs periplakin, bullous pemphigoid antigen 1 and microtubule- and Karakesisoglou, 2001; Leung et al., 2001; Leung et al., actin crosslinking factor, have been characterized as 2002). For instance, desmoplakin interacts with intermediate cytolinkers involved in the organization of the cytoskeleton filaments (IFs) and anchors them to desmosomes (Bornslaeger (reviewed by Leung et al., 2001; Leung et al., 2002). Plakins et al., 2001). Plectin, the most versatile plakin cytolinker with are expressed mainly in tissues that are subject to mechanical five copies of B subdomains, decorates IF networks in the stress such as epithelia and muscle. In addition, it has been cytoplasm and co-localizes with cortical actin at focal adhesion reported that some plakins participate in the development and contracts (Wiche et al., 1983; Seifert et al., 1992; Wiche et al., maintenance of cytoskeletal architecture in neurons (Guo et al., 1993). Plectin also plays a major role in linking the IF networks 1995; Lee et al., 2000). Many plakins are present in multiple to hemidesmosomes (Borradori and Sonnenberg, 1996; Green isoforms and differentially express through alternative splicing and Jones, 1996; Burgeson and Christiano, 1997). It has been in a tissue-specific expression pattern (Brown et al., 1995; shown that the PRD of desmoplakin and plectin plays a critical Bernier et al., 1996; Elliott et al., 1997). Depending on the type role in the direct binding of IFs (Stappenbeck and Green, 1992; of tissue, each isoform has a distinct interaction with the Steinbock et. al., 2000). In keratinocytes, envoplakin and cytoskeletal networks (Yang et al., 1996; Yang et al., 1999). periplakin, together with involucrin were first characterized as The importance of these cytolinkers is further highlighted by precursors of the cornified cell envelope (CE) (Simon and the impairment of plakins found in patients with autoimmune Green, 1984; Ma and Sun, 1986). Periplakin, when transfected or inherited diseases. These disorders usually lead to tissue in full length, localizes to desmosomes, interdesmosomal 782 Journal of Cell Science 118 (4) plasma membrane and IFs (DiColandrea et al., 2000). In In the present study, we have determined the subcellular addition, the binding of the C terminus of periplakin with IFs localization of human epiplakin in cultured epithelial cells by has been reported (Kazerounian et al., 2002; van den Heuvel immunofluorescence analysis. We demonstrate the association et al., 2002; Karashima and Watt, 2002). Furthermore, full of epiplakin fragments with keratins and vimentin by overlay length envoplakin is expressed in aggregates associated with binding assays. We also observed, by real-time RT-PCR, the IFs (DiColandrea et al., 2000). increase of epiplakin expression in keratinocytes as they Epiplakin is mostly found in epithelial tissues (Fujiwara et underwent differentiation. In particular, we used RNAi to al., 2001; Spazierer et al., 2003). Like other plakin members, effectively reduce the level of epiplakin and conducted rescue epiplakin is a large protein. Both human and mouse epiplakin experiments with epiplakin fragments in HeLa cells. We genes have been reported, and both feature a huge single exon provide evidence that epiplakin plays a crucial role in the (more than 20 kb) encoding 13-16 PRDs with various sizes of integrity of IF networks in simple epithelial cells. linkers in between (Takeo et al., 2003; Spazierer et al., 2003). The last five to eight repeated units [linker plus B domains; nomenclature according to Fujiwara et al. (Fujiwara et al., Materials and Methods 2001)] of epiplakin are virtually identical even at the nucleotide Generation of cDNA constructs level. However, epiplakin lacks the center coiled-coil rod A 1.8 kb cDNA encompassing the last linker (L12), B repeat (B13) domain and the amino-terminal globular domain. It was and five additional residues before the stop codon (Table 1) was predicted that epiplakin would exist as a single chain molecule obtained by RT-PCR using total RNA of HeLa cells as a template. in vivo since no dimerization domain was found (Fujiwara et The cDNA was synthesized using 5′-GCACGGAGGAAGCCCAGT- al., 2001). However, the subcellular localization, the biological CAC (reverse) paired with 5′-ACTTCGACGAGGAGATGAACCGT function and its interaction partners remain unknown. (forward) by one-step RT-PCR (Invitrogen). The PCR product was Knockout studies on some plakins have been well cloned into TA vector (Invitrogen) and verified by cycling sequencing. This construct was used as a template to generate fragments with documented. Plectin-null mice showed a phenotype similar to different combinations of B repeat and linker (Table 1). The fragments the autosomal recessive skin disease known as epidermolysis were subcloned into a pGATEV vector of NdeI/XhoI sites [a gift from bullosa simplex with muscular dystrophy (EBS-MD) (Andrä et K. Alexandrov (Kalinin et al., 2001)], allowing inducible expression of al., 1997). The knockout of desmoplakin resulted in embryonic N-terminal GST fusion proteins. To generate pEGFP-fused epiplakin lethality due to the loss of integrity of the extra-embryonic constructs, the same primers with different linkers (Table 1) were paired endoderm (Gallicano et al., 2001). After the ablation of bullous with the corresponding reverse primers for PCR to obtain B13, L12 and pemphigoid antigen 1 (BPAG-1), in mice there was sensory L12B13 epiplakin fragments. These fragments were first cloned into neuron degeneration, and they died at 4-6 weeks (Brown et al., TA vector and digested with SalI/EcoRI, gel cleaned, and cloned into 1995; Guo et al., 1995). While the absence of envoplakin XhoI/EcoRI sites of pEGFP-C3 (Clontech) to obtain pEGFP-B13, resulted in subtle phenotypes with a slight delay in the pEGFP-L12 and pEGFP-L12B13. All of the DNA sequences were verified to ensure the coding region was in-frame. formation of the skin barrier (Määttä et al., 2001), the Expression constructs of human keratins 5, 14, 8 and 18 were periplakin-null mice developed normally and did not show any constructed as follows; the cDNAs were obtained with each skin phenotype (Aho et al., 2004). However, gene ablation of corresponding pair of primers (Table 1) by one-step RT-PCR Journal of Cell Science epiplakin through a conventional knockout study has not yet (Invitrogen) using total RNA isolated from HeLa and
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