Ryan P. Hobbs

The expanding significance of keratin intermediate filaments in normal and diseased epithelia

Intermediate filaments are assembled from a diverse group of evolutionary conserved proteins and are specified in a tissue-dependent, cell type-dependent, and context-dependent fashion in the body. Genetic mutations in intermediate filament proteins account for a large number of diseases, ranging from skin fragility conditions to cardiomyopathies and premature aging. Keratins, the epithelial-specific intermediate filaments, are now recognized as multi-faceted effectors in their native context. In this review, we emphasize the recent progress made in defining the role of keratins towards the regulation of cytoarchitecture, cell growth and proliferation, apoptosis, and cell motility during embryonic development, in normal adult tissues, and in select diseases such as cancer.

Plakophilin 2: a critical scaffold for PKCα that regulates intercellular junction assembly

Plakophilins (PKPs) are armadillo family members related to the classical cadherin-associated protein p120ctn. PKPs localize to the cytoplasmic plaque of intercellular junctions and participate in linking the intermediate filament (IF)-binding protein desmoplakin (DP) to desmosomal cadherins. In response to cell–cell contact, PKP2 associates with DP in plaque precursors that form in the cytoplasm and translocate to nascent desmosomes. Here, we provide evidence that PKP2 governs DP assembly dynamics by scaffolding a DP–PKP2–protein kinase Cα (PKCα) complex, which is disrupted by PKP2 knockdown. The behavior of a phosphorylation-deficient DP mutant that associates more tightly with IF is mimicked by PKP2 and PKCα knockdown and PKC pharmacological inhibition, all of which impair junction assembly. PKP2 knockdown is accompanied by increased phosphorylation of PKC substrates, raising the possibility that global alterations in PKC signaling may contribute to pathogenesis of congenital defects caused by PKP2 deficiency.

Plakophilin 2 Couples Actomyosin Remodeling to Desmosomal Plaque Assembly via RhoA

The desmosomal armadillo protein plakophilin 2 (PKP2) regulates cell contact-initiated cortical actin remodeling through the regulation of RhoA localization and activity to couple adherens junction maturation with desmosomal plaque assembly.

Desmoplakin Regulates Desmosome Hyperadhesion

The skin is subjected to continuous physical stress. Keratinocytes resist mechanical stress by tethering the tension-bearing keratin intermediate filament cytoskeleton to sites of intercellular contact known as desmosomes (Garrod and Chidgey, 2008; Green and Simpson, 2007). The plakin protein desmoplakin (DP) is an obligate desmosomal constituent necessary for keratin anchorage at cell-cell contacts. Establishing and maintaining the DP-keratin association is essential for regulating desmosomal adhesive strength in both developing epidermis and adult stratified tissue (Huen et al., 2002; Vasioukhin et al., 2001).

The calcium ATPase SERCA2 regulates desmoplakin dynamics and intercellular adhesive strength through modulation of PKCα signaling

Dariers disease (DD) is an inherited autosomal‐dominant skin disorder characterized histologically by loss of adhesion between keratinocytes. DD is typically caused by mutations in sarcoendoplasmic reticulum Ca2+‐ATPase isoform 2 (SERCA2), a major regulator of intracellular Ca2+ homeostasis in the skin. However, a defined role for SERCA2 in regulating intercellular adhesion remains poorly understood. We found that diminution of SERCA2 function by pharmacological inhibition or siRNA silencing in multiple human epidermal‐derived cell lines was sufficient to disrupt desmosome assembly and weaken intercellular adhesive strength. Specifically, SERCA2‐deficient cells exhibited up to a 60% reduction in border translocation of desmoplakin (DP), the desmosomal cytolinker protein necessary for intermediate filament (IF) anchorage to sites of robust cell‐cell adhesion. In addition, loss of SERCA2 impaired the membrane translocation of protein kinase C α (PKCα), a known regulator of DP‐IF association and desmosome assembly, to the plasma membrane by up to 70%. Exogenous activation of PKCα in SERCA2‐deficient cells was sufficient to rescue the defective DP localization, desmosome assembly, and intercellular adhesive strength to levels comparable to controls. Our findings indicate that SERCA2‐deficiency is sufficient to impede desmosome assembly and weaken intercellular adhesive strength via a PKCα‐dependent mechanism, implicating SERCA2 as a novel regulator of PKCα signaling.—Hobbs, R. P., Amargo, E. V., Somasundaram, A., Simpson, C. L., Prakriya, M., Denning, M. F., Green, K. J. The calcium ATPase SERCA2 regulates desmoplakin dynamics and intercellular adhesive strength through modulation of PKCα signaling. FASEB J. 25, 990–1001 (2011). www.fasebj.org

Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes

Expression of the intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and in many tumors originating in stratified and pseudostratified epithelia. We report that autoimmune regulator (Aire), a transcriptional regulator, is inducibly expressed in human and mouse tumor keratinocytes in a K17-dependent manner and is required for timely onset of Gli2-induced skin tumorigenesis in mice. The induction of Aire mRNA in keratinocytes depends on a functional interaction between K17 and the heterogeneous nuclear ribonucleoprotein hnRNP K. Further, K17 colocalizes with Aire protein in the nucleus of tumor-prone keratinocytes, and each factor is bound to a specific promoter region featuring an NF-κB consensus sequence in a relevant subset of K17- and Aire-dependent proinflammatory genes. These findings provide radically new insight into keratin intermediate filament and Aire function, along with a molecular basis for the K17-dependent amplification of inflammatory and immune responses in diseased epithelia.

Insights from a Desmoplakin Mutation Identified in Lethal Acantholytic Epidermolysis Bullosa

By linking tension-bearing intermediate filaments (IFs) to sites of robust cell-cell adhesion at desmosomes, desmoplakin (DP) has an essential role in maintaining integrity of tissues that experience mechanical stress such as skin and heart (Green and Simpson, 2007). Two DP isoforms are present in epithelia, resulting from alternative splicing of one gene. The C-terminal IF-binding domain in each isoform is identical, containing three plakin-repeat domains (A, B, and C), separated by flexible linker regions, and followed by a 68-residue tail involved in regulating DP-IF specificity. These motifs cooperate to anchor IFs to desmosomes (Stappenbeck et al., 1993; Meng et al., 1997; Choi et al., 2002; Fontao et al., 2003). Over 50 mutations have been identified throughout the desmoplakin gene (DSP) associated with autosomal-dominant or -recessive disorders affecting the skin, heart, hair, and nails (http://www.arvcdatabase.info; Bolling and Jonkman, 2009). Lethal acantholytic epidermolysis bullosa (LAEB) is an autosomal-recessive disease of severe skin and mucosal fragility caused by premature DSP termination. In the first reported LAEB case, the resultant truncated protein lacked the IF-binding domain and consequently lost keratin anchorage at the desmosomal plaque (Jonkman et al., 2005). The second LAEB case reported a 5-base pair deletion causing frameshift and premature termination in exon 20. No tissue was available for immunofluorescence or biochemical analysis so the effect on protein function is unknown (Bolling et al., 2010). Here, we describe a previously unreported DSP mutation resulting in LAEB without apparent cardiac involvement. Biochemical analysis of patient keratinocytes (KCs) sheds light on possible compensatory mechanisms that may allow embryonic survival. A full-term girl, born to consanguineous parents, showed virtually total denudement of skin (Figure 1a) and mucosae, absent fingernails and toenails (Figure 1b), and total alopecia (Figure 1c). Denuded sites re-epithelialized within days without residual scarring, but blistering recurred rapidly. Cardiac echocardiograms and chest X-rays were normal, but the airway filled with extensive sloughed mucosa. Histological sections revealed suprabasal acantholysis or cell clusters in a single acantholytic basal cell layer (arrow, Figure 1d). Immunofluorescence microscopy revealed staining for all basement membrane zone markers, with plectin and cytokeratin staining a discontinuous single row of basal KCs adherent to the basement membrane. DP staining was absent (data not shown). Based on the poor prognosis, the parents withdrew support and the baby expired on day 26 from airway obstruction. Autopsy was refused. Figure 1 Clinical features of lethal acantholytic epidermolysis bullosa (LAEB) and desmoplakin (DP) schematic highlighting sites of mutations After parental institutional review board-approved informed consent in adherence to the Declaration of Helsinki Principles, mutation screening was performed on genomic DNA isolated from peripheral blood. The complete genomic DNA coding sequence of DSP was amplified and directly sequenced as described (Whittock et al., 1999). The proband showed a homozygous single nucleotide deletion (DSP:c.7248delT) in exon 24, which was heterozygous in parental genomic DNA (data not shown). Consistent with a C-terminal mutation, an anti-DP C-terminal antibody (NW6), failed to reveal the punctate pattern characteristic of desmosome staining at cell-cell interfaces in cultured patient KCs (Figure 2a). Surprisingly, an N-terminal antibody (NW161) only rarely detected faint DP punctae between cells (Figure 2a). While the desmosomal cadherin, desmoglein-2, was absent from intercellular borders (data not shown), E-cadherin, b-catenin, and plakoglobin staining were better preserved, consistent with a less-pronounced effect on adherens junctions (Figure 2b). Figure 2 Immunostaining and biochemical analysis of lethal acantholytic epidermolysis bullosa (LAEB) keratinocytes (KCs) DP was undetectable in patient KC lysates, regardless of antibody domain specificity, without reduction of other desmosomal components (Figure 2c). The identified point mutation predicts truncation (p.Phe2416LeufsX14) within plakin-repeat domain B (Figure 1e). Although quantitative real-time RTPCR analysis indicated reduced DSP transcript levels in patient KCs compared to normal human epidermal KCs, the difference was not statistically significant (Figure 2e). These data support the idea that reduced DP expression in patient KCs is controlled either translationally or through degradation pathways, the latter that was proposed for a recent case of cutaneous disease without evidence of heart defects resulting from virtually complete loss of plakoglobin in the skin (Cabral et al., 2010). Interestingly, LAEB KCs exhibit reduced E-cadherin and elevated N-cadherin protein levels compared to multiple normal human epidermal KCs isolates of the same passage number (Figure 2d and Supplementary Figure S1 online). In addition, the extracellular matrix glycoprotein fibronectin was elevated in LAEB lysates (Figure 2d). Furthermore, plakoglobin and, to a lesser extent, β-catenin were observed in the nucleus of LAEB KCs (Figure 2b), where they may initiate transcription events upon the loss of DP expression (Garcia-Gras et al., 2006). We propose that elevated N-cadherin and fibronectin may facilitate re-epithelialization in the patients skin (Clark, 1990; Peinado et al., 2004), and further speculate that loss of DP might trigger a “cadherin switch,” a process that occurs during epithelial–mesenchymal transition (Nishimura and Takeichi, 2009), but has not previously been linked with desmosome molecules. Although it was not possible to assess whether similar elevation of N-cadherin occurred in cardiac tissue, compensation by this intercalated disc protein might help counter any potential loss of DP in the heart. A consistent genotype-phenotype correlation linking DSP mutations to disease has not yet emerged. DP truncations and/or point mutations may variably cause cardiac and/or skin syndromes (Supplementary Table S1 online). In the cases of LAEB, severe skin fragility is observed when mutations cause complete loss of DP C-terminal expression. However, less severe cases of skin fragility/woolly hair and palmoplantar keratoderma, with or without cardiomyopathy, have been reported in patients when (1) at least one copy of full-length DP I is expressed, but with a point mutation (Whittock et al., 2002; Alcalai et al., 2003; Mahoney et al., 2010); (2) DP I expression is lost, but at least one copy of full-length DP II is expressed (Uzumcu et al., 2006; Asimaki et al., 2009); or (3) homozygous DP C-terminus truncation occurs downstream of plakin-repeat domain B (Norgett et al., 2000). Thus, it seems likely that presence of at least one full-length copy of a DP isoform, whether DP I or DP II, may keep patients from developing early lethality as seen in LAEB. In contrast, near-complete loss of DP (described herein) would be predicted to result in severe impairment of both epidermal and cardiac integrity (Gallicano et al., 2001). In utero survival and the lack of observed cardiac involvement in the patient of this study could reflect compensatory mechanisms that stabilized adhesion, re-epithelialization, and cardiac integrity.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Summary Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

Keratins Are Going Nuclear

Previously thought to reside exclusively in the cytoplasm, the cytoskeletal protein keratin 17 (K17) has been recently identified inside the nucleus of tumor epithelial cells with a direct impact on cell proliferation and gene expression. We comment on fundamental questions raised by this new finding and the associated significance.

Keratin intermediate filament proteins - novel regulators of inflammation and immunity in skin.

Inflammation is a complex biological response, protecting the host and promoting homeostasis following mechanical, chemical or microbial insults. Epithelia are strategically located at the interface between the external and internal milieu where, collectively, their ability to establish a