Keratin Intermediate Filament Proteins Research Articles

Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory‐Denk body formation

Mallory-Denk bodies (MDBs) are hepatocyte inclusions that are associated with poor liver disease prognosis. The intermediate filament protein keratin 8 (K8) and its cross-linking by transglutaminase-2 (TG2) are essential for MDB formation. K8 hyperphosphorylation occurs in association with liver injury and MDB formation, but the link between keratin phosphorylation and MDB formation is unknown. We used a mutational approach to identify K8 Q70 as a residue that is important for K8 cross-linking to itself and other liver proteins. K8 cross-linking is markedly enhanced on treating cells with a phosphatase inhibitor and decreases dramatically on K8 S74A or Q70N mutation in the presence of phosphatase inhibition. K8 Q70 cross-linking, in the context of synthetic peptides or intact proteins transfected into cells, is promoted by phosphorylation at K8 S74 or by an S74D substitution and is inhibited by S74A mutation. Transgenic mice that express K8 S74A or a K8 G62C liver disease variant that inhibits K8 S74 phosphorylation have a markedly reduced ability to form MDBs. Our findings support a model in which the stress-triggered phosphorylation of K8 S74 induces K8 cross-linking by TG2, leading to MDB formation. These findings may extend to neuropathies and myopathies that are characterized by intermediate filament-containing inclusions.

Keratin function and regulation in tissue homeostasis and pathogenesis.

Abstract Epithelial tissues act as hubs in metabolism and communication and protect the organism against dehydration, infections, pharmacological and physical stress. Keratin intermediate filament proteins are well established as major cytoskeletal players in maintaining epithelial integrity. More recently, an involvement of keratins in growth control and organelle functions has emerged. Disruption of the keratin cytoskeleton by mutations or its reorganization following posttranslational modifications can render epithelia susceptible to tissue damage and various stresses, while loss of keratin expression is a hallmark of epithelial-mesenchymal transition (EMT). To understand the molecular mechanisms by which keratins perform their functions remains a formidable challenge. Based on selected examples, we will discuss how cell-specific expression of keratin isotypes affects cytoarchitecture and cell behavior. Further, we ask how posttranslational modifications alter keratin organization and interactions during signaling. Next, we discuss pathomechanisms of epidermal keratin disorders in the light of novel data. Finally, we raise open questions and point out future directives.

Placental Vasculogenesis Is Regulated by Keratin-Mediated Hyperoxia in Murine Decidual Tissues

The mammalian placenta represents the interface between maternal and embryonic tissues and provides nutrients and gas exchange during embryo growth. Recently, keratin intermediate filament proteins were found to regulate embryo growth upstream of the mammalian target of rapamycin pathway through glucose transporter relocalization and to contribute to yolk sac vasculogenesis through altered bone morphogenetic protein 4 signaling. Whether keratins have vital functions in extraembryonic tissues is not well understood. Here, we report that keratins are essential for placental function. In the absence of keratins, we find hyperoxia in the decidual tissue directly adjacent to the placenta, because of an increased maternal vasculature. Hyperoxia causes impaired vasculogenesis through defective hypoxia-inducible factor 1α and vascular endothelial growth factor signaling, resulting in invagination defects of fetal blood vessels into the chorion. In turn, the reduced labyrinth, together with impaired gas exchange between maternal and embryonic blood, led to increased hypoxia in keratin-deficient embryos. We provide evidence that keratin-positive trophoblast secretion of prolactin-like protein a (Prlpa) and placental growth factor (PlGF) during decidualization are altered in the absence of keratins, leading to increased infiltration of uterine natural killer cells into placental vicinity and increased vascularization of the maternal decidua. Our findings suggest that keratin mutations might mediate conditions leading to early pregnancy loss due to hyperoxia in the decidua.

The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane domains. Numerous transport pathways warrant the unique structural identity and protein/lipid composition of the brush border membrane. Annexin II (Ca 2+-dependent lipid-binding protein) is an important component of one of the apical protein transport machineries, which involves detergent-resistant membranes and the actin cytoskeleton. Here, we investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This reduction is accompanied by a redistribution of these proteins to intracellular compartments and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are characterized by an almost flat apical membrane with sparse and short microvilli. Concomitant with this alteration is the redistribution of the intermediate filament protein keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19 interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and polarization of intestinal cells.

The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation--causes, mechanisms, and consequences--will allow for a greater understanding of epithelial cell biology and lung pathology alike.

The ubiquitin ligase CHIP/STUB1 targets mutant keratins for degradation

Keratin (K) intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which leads to a large number of human disorders. KRT5 or KRT14 mutations cause epidermolysis bullosa simplex (EBS). The considerable intra- and interfamilial variability in EBS suggests modifying loci, most of which are unknown. In many human disorders, chaperones and the ubiquitin-proteasome system have been found to modify disease severity, thereby providing novel therapy targets. Here, we demonstrate upregulation of stress-induced Hsp70 and Hsp90 in two EBS models, namely, in neonatal K5(-/-) mice and upon proteasome inhibition in cells that stably express the disease-causing mutation K14-p.Arg125Cys, both harboring keratin aggregates. Furthermore, proteasome inhibition caused nuclear translocation of pHSF-1 and an increase in K14-p.Arg125Cys-positive aggregates in cells. Overexpression of the chaperone-associated ubiquitin ligase CHIP/STUB1 strongly reduced keratin aggregates through increased degradation of mutant K14. Using CHIP-p.Met1_Ala142del (DeltaTPR-CHIP), we demonstrated the involvement of Hsc70 and Hsp70 in mutant keratin degradation. Our data uncover common principles between EBS and other protein misfolding disorders, revealing that aggregation-prone keratins are targeted by components of the chaperone machinery. Thus, modulation of the chaperone machinery using small molecules may represent a novel therapeutic strategy for dominant EBS, allowing reformation of an intact keratin cytoskeleton.

Keratins regulate yolk sac hematopoiesis and vasculogenesis through reduced BMP-4 signaling

Keratin intermediate filament proteins form the major cytoskeleton in all embryonic and adult epithelia. Increasing evidence suggests that keratins, besides their primary cytoskeletal function, can act as scaffolds which locally regulate cell growth and survival in epithelial cells. Many of these functions, however, are not understood in full, owing to keratin redundancy. We have recently created mice which lack all keratins and found that keratins act upstream of mTOR signaling to regulate protein biosynthesis via GLUT localization. Here, we report that keratins are necessary to maintain adhesion between endodermal and mesodermal cell layers of the yolk sac. As a consequence, keratin -/- embryos suffer from reduced yolk sac hematopoiesis and vasculogenesis. Pathway analysis revealed a reduction of the hedgehog target Foxf1 in yolk sac mesoderm of keratin -/- embryos, and subsequent reduction of BMP-4 and P-p38 MAPK. These defects may be caused by the overall reduction in protein biosynthesis and diminished adhesion. Our data show for the first time that keratins are necessary for the differentiation of a non-epithelial cell lineage through a combination of mechanical and signaling mechanisms.

Emerging issues with the current keratin-associated protein nomenclature

Keratin associated proteins (KAPs) are a class of proteins that associate with keratin intermediate filament proteins through disulphide linkages to give fibres such as hair and wool their unique properties. Up to 90 proteins from some 25 families have been identified and this does not include polymorphic variants of individual proteins within these families. The existence of this diverse group of proteins has been known for some 75 years but, despite this, there is still no universally accepted nomenclature for them. This paper sets out the case for revising the current system to deal with this nomenclature issue.

Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

BackgroundOncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα) activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear.ResultsYeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP) identified the cytoplasmic intermediate filament protein keratin 18 (K18) as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression.ConclusionsK18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability of LRP16 to ERα and promotes the proliferation of ERα-positive breast tumor cells. K18 plays an important functional role in regulating the ERα signaling pathway.

Novel association of APC with intermediate filaments identified using a new versatile APC antibody

BackgroundAs a key player in suppression of colon tumorigenesis, Adenomatous Polyposis Coli (APC) has been widely studied to determine its cellular functions. However, inconsistencies of commercially available APC antibodies have limited the exploration of APC function. APC is implicated in spindle formation by direct interactions with tubulin and microtubule-binding protein EB1. APC also interacts with the actin cytoskeleton to regulate cell polarity. Until now, interaction of APC with the third cytoskeletal element, intermediate filaments, has remained unexamined.ResultsWe generated an APC antibody (APC-M2 pAb) raised against the 15 amino acid repeat region, and verified its reliability in applications including immunoprecipitation, immunoblotting, and immunofluorescence in cultured cells and tissue. Utilizing this APC-M2 pAb, we immunoprecipitated endogenous APC and its binding proteins from colon epithelial cells expressing wild-type APC. Using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), we identified 42 proteins in complex with APC, including β-catenin and intermediate filament (IF) proteins lamin B1 and keratin 81. Association of lamin B1 with APC in cultured cells and human colonic tissue was verified by co-immunoprecipitation and colocalization. APC also colocalized with keratins and remained associated with IF proteins throughout a sequential extraction procedure.ConclusionWe introduce a versatile APC antibody that is useful for cell/tissue immunostaining, immunoblotting and immunoprecipitation. We also present evidence for interactions between APC and IFs, independent of actin filaments and microtubules. Our results suggest that APC associates with all three major components of the cytoskeleton, thus expanding potential roles for APC in the regulation of cytoskeletal integrity.

Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor

Keratin intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which affects cytoarchitecture, cell growth, survival, and organelle transport. However, owing to redundancy, the global function of keratins has not been defined in full. Using a targeted gene deletion strategy, we generated transgenic mice lacking the entire keratin multiprotein family. In this study, we report that without keratins, embryonic epithelia suffer no cytolysis and maintain apical polarity but display mislocalized desmosomes. All keratin-null embryos die from severe growth retardation at embryonic day 9.5. We find that GLUT1 and -3 are mislocalized from the apical plasma membrane in embryonic epithelia, which subsequently activates the energy sensor adenosine monophosphate kinase (AMPK). Analysis of the mammalian target of rapamycin (mTOR) pathway reveals that AMPK induction activates Raptor, repressing protein biosynthesis through mTORC1's downstream targets S6 kinase and 4E-binding protein 1. Our findings demonstrate a novel keratin function upstream of mTOR signaling via GLUT localization and have implications for pathomechanisms and therapy approaches for keratin disorders and the analysis of other gene families.

Keratin 18 is over-expressed in osteoblasts derived from Pagetic lesions but is not involved in the formation of disrupted matrix

Category 6. Bone formation, cartilage and bone matrix 401 Keratin 18 is over-expressed in osteoblasts derived from Pagetic lesions but is not involved in the formation of disrupted matrix D. Naot, B.G. Matthews, R.M. Locklin, Z. Xia, P.A. Hulley, J. Cornish Department of Medicine, University of Auckland, Auckland, New Zealand Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford, United Kingdom Paget's disease is a common focal bone disorder. Pagetic lesions, which result from over-activity of osteoclasts and osteoblasts, appear lytic at early stages and later turn sclerotic, with areas of irregular, disorganised bone matrix. Pagetic osteoclasts are grossly abnormal, and have been the focus of most of the research on the cellular mechanisms of Paget's. Given the tight coupling between osteoclasts and osteoblasts, we characterised changes in the pagetic osteoblast that could contribute to the development of the disease. We compared gene expression in osteoblasts and bone marrow cells from pagetic and non-pagetic patients. Microarray analysis identified a number of differentially regulated genes, and the intermediate filament protein keratin 18 (KRT18) was one of the most highly upregulated genes in pagetic osteoblasts. Real-time RT-PCR comparing 28 pagetic samples to 49 controls confirmed that KRT18 expression is more than three times higher in pagetic cells. In the present study we investigated the effects of KRT18 over-expression on osteoblasts and mesenchymal cells. Primary human osteoblasts were transduced with a KRT18 adenoviral vector and compared to cells transduced with a control vector. Real-time RT-PCR showed that KRT18 over-expressing cells have increased levels of interleukin 1β, Dkk1, and the chemokine MCP1, genes that had previously been identified as up-regulated in pagetic osteoblasts. In order to study the possible effects of KRT18 over-expression on cell morphology and extracellular matrix formation, we cultured virally transduced human primary mesenchymal cells in 3-dimensional collagen scaffolds. The scaffolds provide an extracellular structure which resembles the bone environment and is therefore better suited for the study of cell morphology and matrix formation and mineralisation. Cells were analysed using confocal imaging. Cells were also stained for alkaline phosphatase activity and calcium deposition was measured by alizarin red staining. Cultures of KRT18 over-expressing cells were not significantly different from the controls. In conclusion, these results suggest that KRT18 plays a role in osteoblast biology, and overexpression of this gene can reproduce some of the features of pagetic osteoblasts, however, this does not appear to include the disrupted matrix formation. doi:10.1016/j.bone.2009.01.454 402 EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury A. Arthur, C. Stylianou, S.A. Koblar, K. Matsuo, S. Gronthos Bone and Cancer Laboratories, Division of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, SA, Australia The Institute of Molecular and Cell Biology, Singapore's Agency for Science, Technology and Research, Singapore, Singapore School of Molecular and Biomedical Science, School of Medicine, University of Adelaide, Adelaide, SA, Australia Department of Microbiology and Immunology, Graduate School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan The bone marrow contains mesenchymal stromal/stem cells (MSCs) that reside within a perivascular niche. This precursor population is essential for regulating skeletal tissue homeostasis, including bone formation and repair. Bone remodelling is mediated by bone forming osteoblasts derived from MSC, and by bone resorbing osteoclasts that originate from the haematopoietic lineage. However, the molecular signals that maintain multi-potential MSC populations within the stem cell niche and the mechanisms that drive their mobilization towards the bone surfaces to facilitate bone formation are not well defined. The Eph/ephrin family of receptor tyrosine kinases have been implicated in the maintenance of stem cell niches including neural, intestinal and dental tissue, and more recently in the regulation of bone homeostasis. We have characterized the gene and protein expression of EphB/ephrin-B molecules on human culture expanded MSC populations and found that the EphB1, B2 and B4 receptors and ephrin-B1/B2 ligand were the highest expressed B class Eph/ephrin molecules. Functional studies using Fc fusion proteins bound to the extracellular portion of EphB2 or ephrin-B1 showed that reverse ephrin-B signalling inhibited human MSC attachment and spreading, while forward EphB signalling promoted MSC migration. Furthermore, EphB expressing MSC were found to restrict their capacity for selfrenewal while activated ephrin-B expressing MSC promoted osteogenic differentiation. A murine femoral fracture model was used to elucidate the contribution of EphB/ephrin-B molecules to skeletal tissue repair and bone remodelling. Gene expression for 8756–3282/$ – see front matter Bone 44 (2009) S142–S161

EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury

Category 6. Bone formation, cartilage and bone matrix 401 Keratin 18 is over-expressed in osteoblasts derived from Pagetic lesions but is not involved in the formation of disrupted matrix D. Naot, B.G. Matthews, R.M. Locklin, Z. Xia, P.A. Hulley, J. Cornish Department of Medicine, University of Auckland, Auckland, New Zealand Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford, United Kingdom Paget's disease is a common focal bone disorder. Pagetic lesions, which result from over-activity of osteoclasts and osteoblasts, appear lytic at early stages and later turn sclerotic, with areas of irregular, disorganised bone matrix. Pagetic osteoclasts are grossly abnormal, and have been the focus of most of the research on the cellular mechanisms of Paget's. Given the tight coupling between osteoclasts and osteoblasts, we characterised changes in the pagetic osteoblast that could contribute to the development of the disease. We compared gene expression in osteoblasts and bone marrow cells from pagetic and non-pagetic patients. Microarray analysis identified a number of differentially regulated genes, and the intermediate filament protein keratin 18 (KRT18) was one of the most highly upregulated genes in pagetic osteoblasts. Real-time RT-PCR comparing 28 pagetic samples to 49 controls confirmed that KRT18 expression is more than three times higher in pagetic cells. In the present study we investigated the effects of KRT18 over-expression on osteoblasts and mesenchymal cells. Primary human osteoblasts were transduced with a KRT18 adenoviral vector and compared to cells transduced with a control vector. Real-time RT-PCR showed that KRT18 over-expressing cells have increased levels of interleukin 1β, Dkk1, and the chemokine MCP1, genes that had previously been identified as up-regulated in pagetic osteoblasts. In order to study the possible effects of KRT18 over-expression on cell morphology and extracellular matrix formation, we cultured virally transduced human primary mesenchymal cells in 3-dimensional collagen scaffolds. The scaffolds provide an extracellular structure which resembles the bone environment and is therefore better suited for the study of cell morphology and matrix formation and mineralisation. Cells were analysed using confocal imaging. Cells were also stained for alkaline phosphatase activity and calcium deposition was measured by alizarin red staining. Cultures of KRT18 over-expressing cells were not significantly different from the controls. In conclusion, these results suggest that KRT18 plays a role in osteoblast biology, and overexpression of this gene can reproduce some of the features of pagetic osteoblasts, however, this does not appear to include the disrupted matrix formation. doi:10.1016/j.bone.2009.01.454 402 EphB/ephrin-B interactions assist in MSC niche maintenance and contribute to bone remodelling following injury A. Arthur, C. Stylianou, S.A. Koblar, K. Matsuo, S. Gronthos Bone and Cancer Laboratories, Division of Haematology, Institute of Medical and Veterinary Science/Hanson Institute, Adelaide, SA, Australia The Institute of Molecular and Cell Biology, Singapore's Agency for Science, Technology and Research, Singapore, Singapore School of Molecular and Biomedical Science, School of Medicine, University of Adelaide, Adelaide, SA, Australia Department of Microbiology and Immunology, Graduate School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan The bone marrow contains mesenchymal stromal/stem cells (MSCs) that reside within a perivascular niche. This precursor population is essential for regulating skeletal tissue homeostasis, including bone formation and repair. Bone remodelling is mediated by bone forming osteoblasts derived from MSC, and by bone resorbing osteoclasts that originate from the haematopoietic lineage. However, the molecular signals that maintain multi-potential MSC populations within the stem cell niche and the mechanisms that drive their mobilization towards the bone surfaces to facilitate bone formation are not well defined. The Eph/ephrin family of receptor tyrosine kinases have been implicated in the maintenance of stem cell niches including neural, intestinal and dental tissue, and more recently in the regulation of bone homeostasis. We have characterized the gene and protein expression of EphB/ephrin-B molecules on human culture expanded MSC populations and found that the EphB1, B2 and B4 receptors and ephrin-B1/B2 ligand were the highest expressed B class Eph/ephrin molecules. Functional studies using Fc fusion proteins bound to the extracellular portion of EphB2 or ephrin-B1 showed that reverse ephrin-B signalling inhibited human MSC attachment and spreading, while forward EphB signalling promoted MSC migration. Furthermore, EphB expressing MSC were found to restrict their capacity for selfrenewal while activated ephrin-B expressing MSC promoted osteogenic differentiation. A murine femoral fracture model was used to elucidate the contribution of EphB/ephrin-B molecules to skeletal tissue repair and bone remodelling. Gene expression for 8756–3282/$ – see front matter Bone 44 (2009) S142–S161

Contrasting Expression of Keratins in Mouse and Human Embryonic Stem Cells

RNA expression data reveals that human embryonic stem (hES) cells differ from mouse ES (mES) cells in the expression of RNAs for keratin intermediate filament proteins. These differences were confirmed at the cellular and protein level and may reflect a fundamental difference in the epithelial nature of embryonic stem cells derived from mouse and human blastocysts. Mouse ES cells express very low levels of the simple epithelial keratins K8, K18 and K19. By contrast hES cells express moderate levels of the RNAs for these intermediate filament proteins as do mouse stem cells derived from the mouse epiblast. Expression of K8 and K18 RNAs are correlated with increased c-Jun RNA expression in both mouse and human ES cell cultures. However, decreasing K8 and K18 expression associated with differentiation to neuronal progenitor cells is correlated with increasing expression of the Snai2 (Slug) transcriptional repression and not decreased Jun expression. Increasing K7 expression is correlated with increased CDX2 and decreased Oct4 RNA expression associated with the formation of trophoblast derivatives by hES cells. Our study supports the view that hES cells are more similar to mouse epiblast cells than mouse ES cells and is consistent with the epithelial nature of hES cells. Keratin intermediate filament expression in hES cells may modulate sensitivity to death receptor mediated apoptosis and stress.

Epitope Mapping of Monoclonal Antibodies to Keratin 19 Using Keratin Fragments, Synthetic Peptides and Phage Peptide Libraries

To generate tools for monitoring processing and folding in keratin intermediate filaments, a group of monoclonal antibodies reacting with the intermediate filament protein keratin 19 were studied using different approaches to define the structure and localization of their epitopes. The binding pattern to bacterially expressed human keratin 19 fragments allowed the definition of minimal amino acid sequences required for antibody binding. The screening of overlapping 15-residue peptides confirmed and further specified the epitope locations for a subset of the tested antibodies. In addition, the epitope of an antibody with apparent species-restricted specificity (LE64) was revealed by isolating and characterizing a full-length keratin 19 clone from a PtK2 cDNA library. Taken together with species cross-reactivity of individual antibodies and sequence information obtained by probing a phage display library, specific amino acid residues could be highlighted as likely to be involved in the antibody binding.

Identification of novel glucocorticoid receptor-regulated genes involved in epidermal homeostasis and hair follicle differentiation

Despite that glucocorticoids (GCs), acting through the glucocorticoid receptor (GR) exert a pivotal role in skin physiopathology, specific genes regulated by GR in this tissue are largely unknown. We have used a transgenic mouse model overexpressing GR in epidermal basal cells and outer root sheath (ORS) of the hair follicle (HF) under the control of the keratin 5 regulatory sequences (K5-GR mice) to identify GR-regulated genes in mouse skin. We analyzed the transcriptomic profile of adult K5-GR skin as compared to non-transgenic adult mice by using oligonucleotide microarrays and identified 173 genes differentially regulated by GR in this tissue. Our data were further validated by semiquantitative RT-PCR and quantitative real-time PCR. We have identified a large subset of hair keratin intermediate filament ( krt) and hair keratin-associated protein ( krtap) genes, as well as several hox genes as GC-regulated. Since dysregulation of krt, krtaps and hox genes can cause hair disorders, as it occurs in adult K5-GR mice, our findings strongly suggest a role of GR in HF morphogenesis through the coordinated regulation of these hair-specific genes. In addition, we found that GR repressed several genes related to cell growth, such as the immediate early genes fosb and c-fos, according to the antiproliferative role described for this hormone receptor. By using cultured keratinocytes treated with GR-agonists and -antagonists, we demonstrated that down-regulation of fosb is mediated by GR. Identification of novel GR-regulated genes will help us to better understand the role of GCs as physiological modulators and pharmacological agents.

Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases.

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK. There are now more than 50 proteins shown to be substrates for JNK. These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K, and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Proteome Analysis of Human Hair Shaft

The human hair proteome was investigated using two-dimensional LC-MS/MS. Among the 343 identified proteins, 70 were detected in high relative abundance, including keratin intermediate filament proteins, largely extractable with denaturants. Over 300 proteins were found to constitute the insoluble complex formed by transglutaminase cross-linking. The intracellular distribution of identified proteins is wide from cytoplasm to nucleus, mitochondria, ribosome, and plasma membrane. These results help rationalize ultrastructural features visible in the mature hair. Keratins and several substrates for transglutaminase were found to be posttranslationally modified by methylation and dimethylation. Evidence for ubiquitination of hair proteins was also obtained.

Morphogenesis of the mammary gland and the role of keratin expression

The selective expression of keratin genes is highly tissue-specific, and antibodies to keratins have long been used as markers of differentiation in cell and developmental biology and in pathology applications. Antibodies to keratins can mark the progress of normal versus abnormal differentiation, can detect early apoptotic changes and may even identify stem cell-enriched tissue populations. A better understanding of the function of keratins has come from identifying links between keratin mutations and a wide range of tissue fragility disorders, which have shown that keratin intermediate filament proteins contribute physical resilience to epithelial tissues. The tissue specificity of keratins may thus reflect different requirements for stiffer or more plastic cells in particular organ sites. We re-examined the early development of the mammary gland to ask whether differences in the plasticity of cell compartments, proposed to result from expression of different keratins, may contribute to morphogenesis in development. If so, such physical differences might also be important in cancer metastasis. During early development, mouse mammary glands, like other epidermal appendages, first appear as thickenings of the ectoderm, which then expand to form solid buds of epithelial cells protruding into the mesenchyme. These buds then undergo substantial growth and remodeling. Many well-established signaling molecules are now known to play a role in epidermal appendage development, yet the physical changes that take place within the cells at these early stages, and which ultimately enable them to develop their functional tissue morphology, are still poorly understood. Using immunohistochemistry on mouse mammary gland precursors between days 12 and 19 of gestation, we mapped the sequential changes in expression of keratins and some other key structural proteins. Compartmentalization of keratin expression was observed to divide the mammary gland rudiment into a number of different zones. The subsequent fate of these zones suggests that the specific expression of keratins and other structural proteins may indeed predict the functional capacity of cell populations in tissues.

Defining the properties of the nonhelical tail domain in type II keratin 5: insight from a bullous disease-causing mutation.

Inherited mutations in the intermediate filament (IF) proteins keratin 5 (K5) or keratin 14 (K14) cause epidermolysis bullosa simplex (EBS), in which basal layer keratinocytes rupture upon trauma to the epidermis. Most mutations are missense alleles affecting amino acids located in the central alpha-helical rod domain of K5 and K14. Here, we study the properties of an unusual EBS-causing mutation in which a nucleotide deletion (1649delG) alters the last 41 amino acids and adds 35 residues to the C terminus of K5. Relative to wild type, filaments coassembled in vitro from purified K5-1649delG and K14 proteins are shorter and exhibit weak viscoelastic properties when placed under strain. Loss of the C-terminal 41 residues contributes to these alterations. When transfected in cultured epithelial cells, K5-1649delG incorporates into preexisting keratin IFs and also forms multiple small aggregates that often colocalize with hsp70 in the cytoplasm. Aggregation is purely a function of the K5-1649delG tail domain; in contrast, the cloned 109 residue-long tail domain from wild type K5 is distributed throughout the cytoplasm and colocalizes partly with keratin IFs. These data provide a mechanistic basis for the cell fragility seen in individuals bearing the K5-1649delG allele, and point to the role of the C-terminal 41 residues in determining K5's assembly properties.