Although light microscopy has been used to examine the early trafficking of collagen within the cell, much of our understanding of the detailed organisation of cell deposited collagen is from static electron microscopy studies. To understand the dynamics of live cell collagen deposition and fibril organisation, we generated a bright photostable mNGCol1α2 fusion protein and employed a range of microscopy techniques to follow its intracellular transport and elucidate extracellular fibril formation. Our findings reveal the dynamics of fibril growth and the dynamic nature of collagen network interactions at the cellular level. Notably we observed molecular events that build network organisation, including fibril bundling, bifurcation, directionality along existing fibrils, and looping/intertwining behaviours. Strikingly, mNGCol1α2 fluorescence intensity maxima can mark a fibril before another growing collagen fibril intersects at this location. Real-time, high-resolution imaging of collagen has enabled fibrillogenesis and organisational dynamics to be visualised together in an actively secreting cellular system. We also show that the N-terminal protease site is not an absolute requirement for collagen fibril incorporation. This approach paves the way for assessing the dynamic organisation and assembly of collagen into the extracellular matrix in skin models and other tissues during health, ageing and disease.

BACKGROUND:Retinoid signaling is an important regulator of the epidermis and skin appendages. Therefore, synthetic retinoids have been developed for therapeutic use for skin disorders such as psoriasis and acne.AIMS:In previous studies, we showed how the photostable retinoid EC23 induces neuronal differentiation in stem cell-like cell populations, and here, we aim to investigate its ability to influence epidermal and hair follicle growth.METHODS:EC23 influence on skin biology was investigated initially in cultures of monolayer keratinocytes and three-dimentional in vitro models of skin, and finally in in vivo studies of mice back skin.RESULTS:EC23 induces keratinocyte hyperproliferation in vitro and in vivo, and when applied to mouse skin increases the number of involucrin-positive suprabasal cell layers. These phenotypic changes are similar in skin treated with the natural retinoid all-trans retinoic acid (ATRA); however, EC23 is more potent; a tenfold lower dose of EC23 is sufficient to induce epidermal thickening, and resulting hyperproliferation is sustained for a longer time period after first dose. EC23 treatment resulted in a disorganized stratum corneum, reduced cell surface lipids and compromised barrier, similar to ATRA treatment. However, EC23 induces a rapid telogen to anagen transition and hair re-growth in 6-week-old mice with synchronously resting back skin follicles. The impact of EC23 on the hair cycle was surprising as similar results have not been seen with ATRA.CONCLUSIONS:These data suggest that synthetic retinoid EC23 is a useful tool in exploring the turnover and differentiation of cells and has a potent effect on skin physiology.

Skin ageing is an intricate physiological process affected by intrinsic and extrinsic factors. There is a demand to understand how the skin changes with age and photoexposure in individuals with Fitzpatrick skin types I‐III due to accelerated photoageing and the risk of cutaneous malignancies. To assess the structural impact of intrinsic and extrinsic ageing, we analysed 14 skin parameters from the photoprotected buttock and photoexposed dorsal forearm of young and ageing females with Fitzpatrick skin types II‐III (n = 20) using histomorphic techniques. Whilst the minimum viable epidermis (Emin) remained constant (Q > 0.05), the maximum viable epidermis (Emax) was decreased by both age and photoexposure (Q ≤ 0.05), which suggests that differences in epidermal thickness are attributed to changes in the dermal‐epidermal junction (DEJ). Changes in Emax were not affected by epidermal cell proliferation. For the first time, we investigated the basal keratinocyte morphology with age and photoexposure. Basal keratinocytes had an increased cell size, cellular height and a more columnar phenotype in photoexposed sites of young and ageing individuals (Q ≤ 0.05), however no significant differences were observed with age. Some of the most striking changes were observed in the DEJ, and a decrease in the interdigitation index was observed with both age and photoexposure (Q ≤ 0.001), accompanied by a decreased height of rête ridges and dermal papilla. Interestingly, young photoexposed skin was comparable to ageing skin across many parameters, and we hypothesise that this is due to accelerated photoageing. This study highlights the importance of skin care education and photoprotection from an early age.

Sallow and/or dull skin appearance is greatly attributable to the yellow components of skin tone. Bilirubin is a yellow chromophore known to be made in the liver and/or spleen and is transported throughout the body via the blood stream. Recent publications suggest bilirubin may be synthesized in other cells/organs, including the skin. We found human keratinocytes express the transcripts involved in bilirubin biosynthesis. In parallel, we also found human keratinocytes could indeed synthesize bilirubin in monolayer keratinocytes and in a 3D human skin-equivalent model. The synthesized amount was substantial enough to contribute to skin yellowness. In addition, oxidative stress enhanced bilirubin production. Using UnaG, a protein that forms a fluorescent species upon binding to bilirubin, we also visualized the intracellular expression of bilirubin in keratinocytes. Finally, we screened a compound library and discovered that the sucrose laurate/dilaurate (SDL) combination significantly reduced bilirubin levels, as well as bilirubin-mediated yellowness. In conclusion, bilirubin is indeed synthesized in epidermal keratinocytes and can be upregulated by oxidative stress, which could contribute to chronic or transient yellow skin tone appearance. Application of SDL diminishes bilirubin generation and may be a potential solution to mitigate yellowish and/or dull skin appearance.

Collagen is the main component of the skin extracellular matrix and plays an integral role in supporting and maintaining the structure and biomechanical properties of the skin. There is increased interest to understand how collagen quality is controlled by the skin's fibroblast cells, particularly in aging, and to quantify how product formulations improve the appearance of aging skin. In this work, we have developed a new in vitro tool to visualize collagen in skin fibroblasts at high speed and high resolution using live cell imaging and super resolution microscopy. We have developed a bright mNeonGreen-collagen reporter that is efficiently expressed in fibroblasts and other cell types. The reporter molecule assembles into higher order structures, and is transported through the secretory pathway. We have used the system to track and calculate the in vitro movement of collagen through the cell. mNeonGreen-collagen can be harnessed to quantify the in vitro effects of peptides (eg, palmitoyl pentapeptide 4 as a well-established signaling peptide) and other antiaging materials on collagen quality control and secretion. The new tool will underpin our further understanding of the cell biology of collagen and the extracellular matrix in the skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.

Thioglycolate is a potent depilatory agent. In addition, it has been proposed to be useful as a penetration enhancer for transepidermal drug delivery. However, the effects on hair structure and stress responses it elicits in epidermal keratinocytes have not been fully characterised. We have used label-free confocal and fluorescence lifetime imaging supported by electron microscopy to demonstrate how thioglycolate damages hair cuticle cells by generating breakages along the endocuticle and leading to swelling of cortex cells. Maleimide staining of free SH-groups and a decrease in the average fluorescence lifetime of endogenous fluorophores demonstrate a specific change in protein structure in both hair cuticle and cortex. We found that the thioglycolate damages cornified envelopes isolated from the stratum corneum of the epidermis. However, thioglycolate-treated epidermal equivalent cultures recover within 48 hours, which highlights the reversibility of the damage. HaCaT keratinocytes respond to thioglycolate by increased proliferation, onset of differentiation and expression of the chaperone protein Hsp 70, but not Hsp 27. Up-regulation of involucrin can be blocked by an application of c-Jun N-terminal kinase (JNK) inhibitor, but the up-regulation of Hsp 70 takes place regardless of the presence of the JNK inhibitor.

The biomechanical properties of the collagenous dermal matrix are well described but responses to mechanical force by the hair follicles have not been characterised so far. We applied a pulling force on hair follicles to visualise and quantify changes in the keratin-14 and involucrin-positive cell layers of the follicles using nuclear dimensions as an indicator of tissue deformation. Moreover, we used second-harmonic generation imaging to visualise changes in the dermal collagen. We report how the anatomical regions of the follicle respond to the force. Nuclei of the isthmus region were most affected. The nuclei in both K14-positive outer root sheath cells and in involucrin-positive cells were significantly compressed, whereas the response in the infundibulum and suprabulbar regions was more variable. The deformation of the nuclei did not correlate with lamin A/C expression. The changes in the collagenous matrix were distinct at different depths of the dermis as collagen fibrils were compressed closer to each other in the region adjacent to upper suprabulbar follicle and pulled apart near the infundibulum. Thus, the responses to the force are locally defined and the cells in the permanent and cycling parts of the follicle behave differently.

Palmoplantar keratodermas are a heterogeneous group of disorders characterized by abnormal thickening of the volar epidermis (Blaydon and Kelsell, 2014Blaydon D.C. Kelsell D.P. Defective channels lead to an impaired skin barrier.J Cell Sci. 2014; 127: 4343-4350Crossref PubMed Scopus (25) Google Scholar, Maruthappu et al., 2014Maruthappu T. Scott C.A. Kelsell D.P. Discovery in genetic skin disease: the impact of high throughput genetic technologies.Genes. 2014; 5: 615-634Crossref PubMed Scopus (9) Google Scholar). A subset of palmoplantar keratodermas are associated with syndromes linked to other cutaneous features (Betz et al., 2012Betz R.C. Cabral R.M. Christiano A.M. Sprecher E. Unveiling the roots of monogenic genodermatoses: genotrichoses as a paradigm.J Invest Dermatol. 2012; 132: 906-914Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and also noncutaneous conditions such as hearing loss, cardiomyopathy, and esophageal cancer (Blaydon Diana et al., 2012Blaydon Diana C. Etheridge Sarah L. Risk Janet M. Hennies H.-C. Gay Laura J. Carroll R. et al.RHBDF2 mutations are associated with tylosis, a familial esophageal cancer syndrome.Am J Hum Genet. 2012; 90: 340-346Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, Kelsell et al., 2001Kelsell D.P. Di W.-L. Houseman M.J. Connexin mutations in skin disease and hearing loss.Am J Hum Genet. 2001; 68: 559-568Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Palmoplantar keratodermas specifically associated with defects in hair development include the desmosomal disorders linked to phenotypes such as woolly hair and alopecia (Brooke et al., 2012Brooke M.A. Nitoiu D. Kelsell D.P. Cell-cell connectivity: desmosomes and disease.J Pathol. 2012; 226: 158-171Crossref PubMed Scopus (133) Google Scholar). Two adult siblings from a consanguineous family of Pakistani origin, whose parents were first cousins, presented with an autosomal recessively inherited palmoplantar keratoderma, leukonychia, and exuberant curly scalp hair (Figure 1a). Both affected individuals described the progressive development of yellowish thickened scaly skin affecting the palms and soles since 2 years of age, and toenail dystrophy in their teenage years. Examination revealed marked diffuse, verrucous hyperkeratosis with deep fissuring affecting the soles (Figure 1a) and to a lesser extent, the palms. There was no evidence of transgradiens. The toenails were dystrophic with onycholysis and leukonychia was also present, most evident in the finger nails. Onychomycosis was excluded by negative fungal culture. No abnormalities of teeth or sweating were identified. The siblings also described having extremely thick, rapidly growing curly scalp hair since childhood, but without excessive hair growth elsewhere. Neither parent had a similar hair or skin phenotype, and they had no other offspring. Clinical photographs were obtained, and written consent was provided by patients for their publication. Blood samples were collected after written informed consent in adherence with the Declaration of Helsinki principles and approval of the East London and City Health Authority. Whole-exome capture from both siblings was performed using SeqCap EZ Human Exome Library v2.0 (Roche NimbleGen, Madison, WI) and sequenced with 100-bp paired-end reads on the HiSeq 2000 platform (Illumina, San Diego, CA). Resulting reads were mapped to the hg18 human reference genome using the Novoalign alignment tool (Novocraft Technologies Sdn Bhd, Selangor, Malaysia). Sequence variants were called with SAMtools and annotated with ANNOVAR (Wang et al., 2010Wang K. Li M. Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data.Nucleic Acids Res. 2010; 38: e164Crossref PubMed Scopus (7948) Google Scholar). Given the history of parental relatedness, the variants were filtered for homozygous changes shared by the two affected individuals, of which 83 homozygous variants reported either as novel or with an Exome Variant Server (EVS) (NHLBI GO Exome Sequencing Project, Seattle, WA) estimated frequency of less than 0.01 were selected as potential candidates. These candidates included a homozygous C to A transversion, c.C101A, in exon 2 of FAM83G (NM_001039999), a gene reported to be mutated in hereditary footpad hyperkeratosis in Kromfohrländer and Irish terrier dog breeds, which presents with fissuring hyperkeratosis of the paws and a bushy coat (Drogemuller et al., 2014Drogemuller M. Jagannathan V. Becker D. Drogemuller C. Schelling C. Plassais J. et al.A mutation in the FAM83G gene in dogs with hereditary footpad hyperkeratosis (HFH).PLoS Genet. 2014; 10: e1004370Crossref PubMed Scopus (35) Google Scholar, Sayyab et al., 2016Sayyab S. Viluma A. Bergvall K. Brunberg E. Jagannathan V. Leeb T. et al.Whole-genome sequencing of a canine family Trio reveals a FAM83G variant associated with hereditary footpad hyperkeratosis.G3 (Bethesda, Md). 2016; 6: 521-527Crossref PubMed Scopus (16) Google Scholar). Furthermore, the bushy hair phenotype of the “woolly” mice (wly mouse) has been linked to a 995-bp deletion in fam83g (Radden, 2013Radden IInd, L.A. Child K.M. Adkins E.B. Spacek D.V. Feliciano A.M. King T.R. The wooly mutation (wly) on mouse chromosome 11 is associated with a genetic defect in Fam83g.BMC Res Notes. 2013; 6: 189Crossref PubMed Scopus (11) Google Scholar). Therefore, the variant in FAM83G presented as an obvious candidate for further analysis. The homozygous c.C101A variant in FAM83G was confirmed by Sanger sequencing in both siblings, and both parents were heterozygous carriers (primer sequences: FAM83G-F: 5′ CCGGGCTCATCAGGTCTTT 3′ and FAM83G-2R: 5′ GAGCGGTCCGACTTCTGG 3′; Figure 1b). The c.101C>A mutation results in loss of a Cac8I restriction endonuclease consensus site and segregation of the mutation with the condition was confirmed by restriction fragment length polymorphism analysis (Figure 1c). The mutation c.C101A is predicted to change an evolutionary conserved alanine to glutamate (p.A34E) in the protein FAM83G. This missense mutation was absent from the database of Single Nucleotide Polymorphisms (dbSNP), ExAC, 1000 genomes, gnomAD, and EVS. It is predicted to be deleterious to the protein structure (Polyphen2 score 1.00, SIFT score 0.00). This residue is conserved across vertebrates (Figure 1c) as well as between members of the FAM83 family of proteins. The FAM83A-H family is characterized by the presence of a conserved N-terminal domain of unknown function: DUFI 669. Like the missense variant, p.A34E, reported in this study, the missense variant, p.R52P, underlying hereditary footpad hyperkeratosis in dogs is also located in the DUFI 669 domain. FAM83G expression is specifically enriched (>fivefold) in the skin compared with 26 other tissue types (Edqvist et al., 2015Edqvist P. Fagerberg L. Hallstrom B. Danielsson A. Edlund K. Uhlén M. et al.Expression of human skin-specific genes defined by transcriptomics and antibody-based profiling.J Histochem Cytochem. 2015; 63: 129-141Crossref PubMed Scopus (49) Google Scholar). FAM83G has been implicated as a regulator of BMP signaling (Vogt et al., 2014Vogt J. Dingwell K.S. Herhaus L. Gourlay R. Macartney T. Campbell D. et al.Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling.Open Biol. 2014; 4: 130210Crossref PubMed Scopus (28) Google Scholar). A skin biopsy obtained from one of the siblings revealed acanthosis of the epidermis (Figure 1e), whereas immunohistochemistry demonstrated a marked reduction in FAM83G (Abcam ab121750, Cambridge, UK) expression compared with control skin (Figure 2a). Upregulation of Ki67 (Abcam ab15580) was evident, and keratin 14 expression (Abcam LL001) was not restricted to the basal layer suggesting dysregulated proliferation. As keratinocyte and hair growth development has been linked to Wnt signaling, immunohistochemical staining of β-catenin (BD Transduction Labs 610153, Oxford, UK) was performed and increased levels of active β-catenin were seen within cell nuclei of the patient epidermis compared with control (Merck Millipore 05-655 Clone 8E7, Watford, UK) (Figure 2a and b). As nuclear translocation of β-catenin is induced by activation of Wnt signaling (Greco et al., 2009Greco V. Chen T. Rendl M. Schober M. Pasolli H.A. Stokes N. et al.A two-step mechanism for stem cell activation during hair regeneration.Cell Stem Cell. 2009; 4: 155-169Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar), FAM83G may be a repressor of Wnt signaling and proliferation. To investigate the hair phenotype, hair from one sibling and both parents were obtained. Confocal imaging showed membrane-bound deposits on the outer cuticle of the hair shaft present in the affected sibling but absent in both parents (Figure 2b). Transmission electron microscopy revealed consistent regions of poorly differentiated cuticle cells possibly deriving from the inner root sheath (Figure 2c). FAM83G expression within hair follicles from the back skin of control mice was investigated. FAM83G was found to be expressed during the anagen growth phase (P28) (Figure 2d) and exhibited high levels of staining intensity at the inner root sheath and in the dermal papilla. The expression of FAM83G appeared to be maintained in catagen/early telogen (P42), within the inner root sheath (Figure 2e). FAM83G has previously been identified as a differentially expressed gene in adult mouse hair bulge stem cells compared with dermal papilla cells during telogen (P56) (Greco et al., 2009Greco V. Chen T. Rendl M. Schober M. Pasolli H.A. Stokes N. et al.A two-step mechanism for stem cell activation during hair regeneration.Cell Stem Cell. 2009; 4: 155-169Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Our findings highlight a key role for FAM83G in the homeostasis of the palmoplantar epidermis and hair. Dampened Wnt/β-catenin signaling is a feature of several common forms of hair loss, including alopecia areata and androgenetic alopecia. Further studies of FAM83G, in particular regarding its role in hair biology and the hair cycle, may support targeting FAM83G and associated pathways in scalp hair disorders. David P. Kelsell: http://orcid.org/0000-0002-9910-7144 The authors state no conflict of interest. TM holds a MRC Clinical Research Training fellowship (MR/K002740/1). The study was supported, in part, by the 2016 CHANEL-CERIES research award to DPK.

Saturated and unsaturated fatty acids make up 85% of the total hair lipid content and are found in the cuticle and cortical cell membrane complex. Although these lipids only make up 2-6% of the hair's overall weight, they play a crucial role in keeping hair healthy, influencing shine, feel, manageability, and strength. The objective of this work was to understand the mechanisms of how these lipids are lost on exposure to external stressors, such as chemical treatments, washing, and UV exposure and to understand how their loss impacts hair strength. The experimental approach was to measure these lipids and oxidation products, lipid peroxides (LPOs) and correlate their loss with fatigue strength measurements. The results show lipids are lost over time by washing, exposure to chemical treatments, such as coloring, and environmental insults, such as UV, and it was confirmed that a mechanism of degradation is via oxidation of unsaturated lipids to form LPOs. In addition, it was shown that replenishment of these lipids is possible by incorporating lipids, such as fatty alcohols (FaOHs), into a gel network with anionic surfactants to create a delivery system that can efficiently penetrate FaOHs into hair and increase internal strength as measured by fatigue.

Tightly regulated homeostatic mechanisms determine the turnover of skin and hair cells to maintain stable epidermal thickness and hair growth. Genetic skin diseases that result in altered homeostasis include the palmoplantar keratodermas (PPKs), a heterogeneous group of disorders characterised by the presence of hyperproliferation and hyperkeratosis of the palmoplantar epidermis. We report two siblings from a consanguineous Pakistani family, presenting with diffuse PPK beginning in childhood, leukonychia and abundant, curly scalp hair with rapid growth. Using whole exome sequencing we identified a homozygous missense mutation in FAM83G/PAWS1. Recessively inherited mutations in PAWS1 have been linked to “Hereditary Footpad Hyperkeratosis” in dogs, which have a bushy coat and rapidly growing nails. Skin biopsy from an affected sibling showed an acanthotic epidermis and immunohistochemistry demonstrated profoundly diminished expression of PAWS1 compared with normal skin, where it was localised in the cytoplasm. There was upregulation of the proliferation markers Ki67 and KRT14 in the basal and suprabasal layers. Confocal hair microscopy showed accumulation of poorly differentiated cells along the hiar shaft in the affected sibling. Wnt signaling plays a central role in hair, skin and nail maintenance, and as the phenotype in humans and dogs share abnormalities in all three, we examined its downstream target, B-catenin. We observed that it was upregulated and translocated to the nucleus in the patient’s skin, implicating altered Wnt signaling in the clinical phenotype. Little is known about PAWS1 and this is the first report to associate it with human disease. Our data suggest that its primary function in the epidermis is to dampen proliferation. These findings shed light on a novel player in hair and skin homeostasis and uncover a hitherto unidentified potential therapeutic target in the treatment of disorders of hair growth.

Envoplakin and periplakin are the two smallest plakin family cytoskeletal linker proteins that connect intermediate filaments to cellular junctions and other membrane locations. These two plakins have a structural role in the assembly of the cornified envelope (CE), the terminal stage of epidermal differentiation. Analysis of gene-targeted mice lacking both these plakins and the third initial CE scaffold protein, involucrin, demonstrate the importance of the structural integrity of CE for a proper epidermal barrier function. It has emerged that periplakin, which also has a wider tissue distribution than envoplakin, has additional, independent roles. Periplakin participates in the cytoskeletal organization also in other tissues and interacts with a wide range of membrane-associated proteins such as kazrin and butyrophilin BTN3A1. This review covers methods used to understand periplakin and envoplakin functions in cell culture models, including siRNA ablation of periplakin expression and the use of tagged protein domain constructs to study localization and interactions. In addition, assays that can be used to analyze CEs and epidermal barrier function in gene-targeted mice are described and discussed.

Co-ordination of cytoskeletal networks and their dynamics is an essential feature of cell migration and cancer cell invasion. Plectin is a large cytolinker protein that influences tissue integrity, organisation of actin and intermediate filaments, and cell migration. Alternatively spliced plectin isoforms are targeted to different subcellular locations. Here, we show that plectin ablation by siRNA impaired migration, invasion and adhesion of SW480 colon carcinoma cells. A previously less well characterised plectin isoform, plectin-1k, co-localised with epithelial integrins, N-WASP, cortactin, and dynamin in podosome-like adhesions in invasive SW480 colon carcinoma cells. Transfection of alternative plectin N-terminal constructs demonstrated that the first exons of isoforms 1k, 1 and 1d can target the actin-binding domain of plectin to podosome-like adhesions. Finally, Plectin-1k N-terminus rescued adhesion site formation in plectin knock-down cells. Thus, plectin participates in actin assembly and invasiveness in carcinoma cells in an isoform-specific manner.

Syndecan-1 is a cell-surface heparan-sulphate proteoglycan that is involved in growth factor regulation, cell adhesion, proliferation, differentiation, blood coagulation, lipid metabolism, as well as tumour formation. In this study, investigation of discrete LCM captured dermal cells by semi-quantitative RT-PCR revealed Syndecan-1 mRNA transcripts were expressed only in the dermal condensation (DC) within this skin compartment during murine pelage hair follicle (HF) morphogenesis. Further immunofluorescence studies showed that, during early skin development, Syndecan-1 was expressed in the epidermis while being absent from the mesenchyme. As HF morphogenesis began ( approximately E14.5) Syndecan-1 expression was lost from the epithelial compartment of the HF and activated in HF mesenchymal cells. This Syndecan-1 expression profile was consistent between different hair follicle types including primary and secondary pelage, vibrissa, and tail hair follicles. Furthermore we show by using gene targeted mice lacking Syndecan-1 expression that Syndecan-1 is not required for follicle initiation and development.

Epidermal stem cells have been classically characterized as slow-cycling, long-lived cells that reside in discrete niches in the skin. Gene expression studies of niche-resident cells have revealed a number of stem cell markers and regulators, including the Wnt/beta-catenin, Notch, p63, c-Myc and Hedgehog pathways. A new study challenges the traditional developmental paradigm of slow-cycling stem cells and rapid-cycling transit amplifying cells in some epidermal regions, and there is mounting evidence to suggest that multi-lineage epidermal progenitors can be isolated from highly proliferative, non-niche regions. Whether there is a unique microenvironment surrounding these progenitors remains to be determined. Interestingly, cancer stem cells derived from epidermal tumours exist independent of the classic skin stem cell niche, yet also have stem cell properties, including multi-lineage differentiation. This review summarizes recent studies identifying the location and regulators of mouse and human epidermal stem cells and highlights the strategies used to identify cancer stem cells, including expression of normal epidermal stem cell markers, expression of cancer stem cell markers identified in other epidermal tumours and characterization of side-population tumour cells. Copyright (C) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Ca2+/calmodulin-associated Ser/Thr kinase (CASK) binds syndecans and other cell-surface proteins through its PDZ domain and has been implicated in synaptic assembly, epithelial polarity and neuronal gene transcription. We show here that CASK regulates proliferation and adhesion of epidermal keratinocytes. CASK is localised in nuclei of basal keratinocytes in newborn rodent skin and developing hair follicles. Induction of differentiation shifts CASK to the cell membrane, whereas in keratinocytes that have been re-stimulated after serum starvation CASK localisation shifts away from membranes upon entry to S phase. Biochemical fractionation demonstrates that CASK has several subnuclear targets and is found in both nucleoplasmic and nucleoskeletal pools. Knockdown of CASK by RNA interference leads to increased proliferation in cultured keratinocytes and in organotypic skin raft cultures. Accelerated cell cycling in CASK knockdown cells is associated with upregulation of Myc and hyperphosphorylation of Rb. Moreover, CASK-knockdown cells show increased hyperproliferative response to KGF and TGFα, and accelerated attachment and spreading to the collagenous matrix. These functions are reflected in wound healing, where CASK is downregulated in migrating and proliferating wound-edge keratinocytes.

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4+ T cells and a reduction in resident γδ+ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.