Quantitative morphometric analysis of intrinsic and extrinsic skin ageing in individuals with Fitzpatrick skin types II-III.

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.

Reductive Stress Selectively Disrupts Collagen Homeostasis and Modifies Growth Factor-independent Signaling Through the MAPK/Akt Pathway in Human Dermal Fibroblasts.

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.

Functional Analysis of Periplakin and Envoplakin, Cytoskeletal Linkers, and Cornified Envelope Precursor Proteins.

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.

Annexin A9 is a periplakin interacting partner in membrane-targeted cytoskeletal linker protein complexes.

Periplakin regulates keratin organisation and participates in the assembly of epidermal cornified envelopes. A proteomic approach identified annexin A9 as a novel interacting partner for periplakin N-terminus. The presence of annexin A9 in complexes with periplakin was confirmed by immunoblotting of proteins immunoprecipitated by anti-HA or anti-annexin A9 antibodies. Both endogenous and GFP-tagged annexin A9 co-localise with endogenous periplakin and transfected periplakin N-terminus at MCF-7 cell borders and aggregate after Okadaic acid treatment. Annexin A9 and periplakin co-localise in the epidermis and annexin A9 is up-regulated in differentiating keratinocytes, but the epidermal annexin A9 expression does not require periplakin.

Plectin regulates invasiveness of SW480 colon carcinoma cells and is targeted to podosome-like adhesions in an isoform-specific manner.

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.

Dynamic expression of Syndecan-1 during hair follicle morphogenesis.

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.

Cytolinker cross-talk: periplakin N-terminus interacts with plectin to regulate keratin organisation and epithelial migration.

Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin-periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.

Down-regulation of vimentin expression inhibits carcinoma cell migration and adhesion.

Vimentin is a type III Intermediate filament protein that is expressed frequently in epithelial carcinomas correlating with invasiveness and poor prognosis. We have analysed migration and adhesion to collagenous matrix of a panel of carcinoma cell lines. In vitro invasiveness was highest in vimentin-positive SW480 colon cancer and MDA-MB-231 breast cancer cells and the role of vimentin in these cell lines was investigated by RNA interference. Down-regulation of vimentin expression resulted in impaired migration in both scratch-wound experiments and in invasion assays through cell culture inserts coated with collagen gel. Compromised migration was observed in both cell lines, whereas cell attachment assays revealed impaired adhesion to fibrillar collagen in MDA-MB-231 cells while the adhesion of vimentin-ablated SW480 cells, that express both vimentin and keratin intermediate filaments was not affected. In conclusion, ablation of vimentin expression inhibits migration and invasion of colon and breast cancer cell lines.

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier.

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 gammadelta+ 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.

Periplakin-dependent re-organisation of keratin cytoskeleton and loss of collective migration in keratin-8-downregulated epithelial sheets.

Collective migration of epithelial sheets requires maintenance of cell-cell junctions and co-ordination of the movement of the migrating front. We have investigated the role of keratin intermediate filaments and periplakin, a cytoskeletal linker protein, in the migration of simple epithelial cells. Scratch wounding induces bundling of keratins into a cable of tightly packed filaments adjacent to the free wound edge. Keratin re-organisation is preceded by a re-distribution of periplakin away from the free wound edge. Periplakin participates with dynamic changes in the keratin cytoskeleton via its C-terminal linker domain that co-localises with okadaic-acid-treated keratin granules. Stable expression of the periplakin C-terminal domain increases keratin bundling and Ser431 keratin phosphorylation at wound edge resulting in a delay in wound closure. Ablation of periplakin by siRNA inhibits keratin cable formation and impairs wound closure. Knockdown of keratin 8 with siRNA results in (1) a loss of desmoplakin localisation at cell borders, (2) a failure of MCF-7 epithelial sheets to migrate as a collective unit and (3) accelerated wound closure in vimentin-positive HeLa and Panc-1 cell lines. Thus, keratin 8 is required for the maintenance of epithelial integrity during migration and periplakin participates in the re-organisation of keratins in migrating cells.