744 Fig. 4 MDBs formation results from a complex interplay of several contributing factors. Since the cytoplasm represents a hydrophilic mi- lieu, all exposed hydrophobic molecules (depicted by red stretches within the protein) are predisposed to aggregation. Properly folded proteins usually hide their hydrophobic stretches inside, but these get exposed in nascent protein chains or after proteins become misfolded as a consequence of oxidative stress. Chaperones bind to these hydro- phobic residues and facilitate protein refolding. Alternatively, dam- aged proteins become polyubiquitinated and degraded either by the proteasomal or autophagic system. MDB-causing agents typically gen- erate extensive amount of oxidative stress with increased protein mis- folding. In addition, chaperone levels are downregulated and/or chaperone function is compromised. Dysbalanced K8/K18 expression precedes MDB formation, likely increases keratin misfolding and pre- disposes to posttranslational modiWcations, which may interfere with keratin refolding and/or repair. Accumulated misfolded proteins are sequestered as inclusions through the action of p62. p62 also shuttles polyubiquitinated proteins to degradative machineries. However, pro- teasomal degradation might be impaired by oxidative stress, may not be able to digest highly cross-linked protein species or might simply be overwhelmed by the excessive supply. On the other hand, autophagy is upregulated during MDB formation in mice and additional stimulation of autophagy attenuates MDB formation in certain conditions. Of note, supplementation of S-adenosylmethionine or mitochondrially targeted antioxidants eVectively diminishes MDB formation, thereby pointing to a central role of oxidative stress in MDB generation bling MDBs can also be produced by transfection of K18 (Nakamichi et al. 2002; Stumptner et al. 2007). Among posttranslational modiWcations, MDB formation is associated with K8 hyperphosphorylation and transami- dation (Zatloukal et al. 1992; Stumptner et al. 2000). In a recent study, ablation of K8 S73 phosphorylation site in transgenic mice resulted in diminished MDB formation after DDC exposure (Harada et al., unpublished results). Several potential mechanisms might be responsible for this observation. K8 S73 is a well-known p38 kinase target site and p38 up-regulation induces keratin network reorganiza- tion with subsequent granule formation (Ku et al. 2002b; Wöll et al. 2007). P38 kinase-induced keratin network reorganization might be a necessary prerequisite for MDB formation, since p38 kinase inhibition prevented MDB formation in vitro (Nan et al. 2006). Alternatively, K8 123 Histochem Cell Biol (2008) 129:735–749 hyperphosphorylation may induce MDB formation through inhibition of K8 degradation, which results in increased K8/K18 ratio (Ku and Omary 2000). MDBs contain highly cross-linked keratins and the abla- tion of tissue transglutaminase eVectively inhibits DDC- induced MDB formation (Zatloukal et al. 1992; Strnad et al. 2007). Since K8 is a much better in vitro transgluta- minase substrate than K18 and highly cross-linked proteins found in MDB-forming mice contain K8, but not K18, it was suggested that excessive K8 gets preferentially tran- samidated and acts as a nucleus for MDB formation. This is supported by studies in transgenic mice, where K8 overex- pression accelerates and K18 excess inhibits not only MDB, but also cross-link formation (Strnad et al. 2007). Ad 3. MDBs were shown to contain misfolded keratins (Cadrin et al. 1991, Kachi et al. 1993). The protein misfold- ing is usually counteracted by the reparative function of chaperones (Ross and Poirier 2004; Macario and Conway de Macario 2005; Bukau et al. 2006), however, DDC feed- ing is associated with diminished chaperone expression, persistent chaperone modiWcations and impairment of chaperone function (Strnad et al. submitted). Similarly, chaperone function is impaired in a rat model of chronic alcoholic liver disease (Carbone et al. 2005). Ad 4. P62 is a stress-inducible protein with multiple functions which is a constituent of multiple inclusion bod- ies termed sequestosomes (Shin 1998; Kuusisto et al. 2001; Zatloukal et al. 2002; Moscat et al. 2007). It binds proteins polyubiquitinated at lysine 63 and shuttles them for prote- asomal or autophagic degradation (Vadlamudi et al. 1996; Bjorkoy et al. 2005; Seibenhener et al. 2004; Wooten et al. 2008). p62 has been shown to enhance aggregate formation (Donaldson et al. 2003; Wang et al. 2005; Komatsu et al. 2007; Gal et al. 2007; Nezis et al. 2008). p62 action is ben- eWcial under normal conditions, since it prevents accumula- tion of abnormal proteins (Bjorkoy et al. 2005; Ramesh Babu et al. 2008), but may become harmful when protein degradation is inhibited (Komatsu et al. 2007). There are several lines of evidence implicating p62 (con- taining the ubiquitin binding site) in MDB formation. In cell culture experiments, protein aggregates resembling MDBs formed only after p62 co-transfection, but not when K8, K18, and ubiquitin were transfected alone or in combi- nation (Stumptner et al. 2007). Furthermore, p62 was rapidly induced in DDC-fed mice with p62-containing aggregates preceding the formation of MDBs (Stumptner et al. 2002). Furthermore, p62 inhibition attenuated, whereas p62 overexpression enhanced MDB formation in DDC-primed hepatocytes (Nan et al. 2006). Ad 5. Accumulation of protein aggregates, as seen dur- ing MDB formation, is counteracted by proteasomal or autophagic degradation (Glickman and Ciechanover 2002; Williams et al. 2006) and both degradation machineries are