RESUMO
Para-hydroxycinnamic acid (pHCA) is one of the most abundant naturally occurring hydroxycinnamic acids, a class of chemistries known for their antioxidant properties. In this study, we evaluated the impact of pHCA on different parameters of skin aging in in vitro skin models after H2O2 and UV exposure. These parameters include keratinocyte senescence and differentiation, inflammation, and energy metabolism, as well as the underlying molecular mechanisms. Here we demonstrate that pHCA prevents oxidative stress-induced premature senescence of human primary keratinocytes in both 2D and 3D skin models, while improving clonogenicity in 2D. As aging is linked to inflammation, referred to as inflammaging, we analyzed the release of IL-6, IL-8, and PGE2, known to be associated with senescence. All of them were downregulated by pHCA in both normal and oxidative stress conditions. Mechanistically, DNA damage induced by oxidative stress is prevented by pHCA, while pHCA also exerts a positive effect on the mitochondrial and glycolytic functions under stress. Altogether, these results highlight the protective effects of pHCA against inflammaging, and importantly, help to elucidate its potential mechanisms of action.
Assuntos
Senescência Celular , Ácidos Cumáricos , Queratinócitos , Estresse Oxidativo , Envelhecimento da Pele , Pele , Humanos , Ácidos Cumáricos/farmacologia , Senescência Celular/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos dos fármacos , Inflamação/metabolismo , Dano ao DNA/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Raios Ultravioleta/efeitos adversos , Antioxidantes/farmacologia , Células Cultivadas , Interleucina-8/metabolismo , Interleucina-6/metabolismoRESUMO
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.
Assuntos
Envelhecimento da Pele , Dermatopatias , Feminino , Humanos , Pele/patologia , Epiderme/fisiologia , Dermatopatias/patologiaRESUMO
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.
Assuntos
Bilirrubina , Queratinócitos , Bilirrubina/metabolismo , Bilirrubina/farmacologia , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismo , Pele/metabolismo , Sacarose/análogos & derivadosRESUMO
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.
Assuntos
Colágeno/metabolismo , Derme/citologia , Estresse do Retículo Endoplasmático , Fibroblastos/metabolismo , Homeostase , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Antioxidantes/farmacologia , Ditiotreitol/farmacologia , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oxirredução , Fosforilação/efeitos dos fármacos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Pele , Engenharia Tecidual/métodos , Membrana Basal/citologia , Membrana Basal/ultraestrutura , Diferenciação Celular , Células Cultivadas , Derme/citologia , Derme/ultraestrutura , Epiderme/ultraestrutura , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro/métodos , Queratinócitos/metabolismo , Microscopia Eletrônica , Pele/anatomia & histologia , Pele/ultraestruturaRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fármacos Dermatológicos/farmacologia , Cabelo/efeitos dos fármacos , Queratinócitos/fisiologia , Tioglicolatos/farmacologia , Linhagem Celular , Feminino , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Cabelo/diagnóstico por imagem , Cabelo/ultraestrutura , Remoção de Cabelo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Inibidores de Proteínas Quinases/farmacologia , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/metabolismoRESUMO
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.
Assuntos
Colágeno/química , Derme/fisiologia , Folículo Piloso/fisiologia , Hipófise/fisiologia , Fenômenos Fisiológicos da Pele , Estresse Mecânico , Fenômenos Biomecânicos , Núcleo Celular/metabolismo , Humanos , Imageamento Tridimensional , Queratina-14/metabolismo , Precursores de Proteínas/metabolismoRESUMO
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.
Assuntos
Cabelo , Lipídeos , Oxirredução , TensoativosRESUMO
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.
Assuntos
Folículo Piloso , Retinoides , Camundongos , Animais , Retinoides/farmacologia , Epiderme , Tretinoína/farmacologia , Queratinócitos/metabolismo , Diferenciação Celular , Proliferação de CélulasRESUMO
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.
Assuntos
Neoplasias do Colo/metabolismo , Plectina/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Regulação para Baixo/efeitos dos fármacos , Humanos , Plectina/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.
RESUMO
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.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/fisiologia , Proteínas de Membrana/fisiologia , Plaquinas/fisiologia , Precursores de Proteínas/fisiologia , Animais , Fracionamento Celular , Linhagem Celular Tumoral , Proteínas Ricas em Prolina do Estrato Córneo/isolamento & purificação , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos/metabolismo , Proteínas de Membrana/isolamento & purificação , Plaquinas/isolamento & purificação , Precursores de Proteínas/isolamento & purificação , Técnicas do Sistema de Duplo-HíbridoAssuntos
DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ceratodermia Palmar e Plantar/genética , Mutação , Adulto , Biópsia , Análise Mutacional de DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ceratodermia Palmar e Plantar/diagnóstico , Ceratodermia Palmar e Plantar/metabolismo , Linhagem , Couro Cabeludo , Pele/metabolismo , Pele/patologiaRESUMO
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.
Assuntos
Anexinas/metabolismo , Plaquinas/metabolismo , Animais , Anexinas/química , Anexinas/genética , Diferenciação Celular , Linhagem Celular , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Células MCF-7 , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ácido Okadáico/farmacologia , Plaquinas/química , Plaquinas/genética , Proteômica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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 TGFalpha, 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.
Assuntos
Proliferação de Células , Epiderme/metabolismo , Guanilato Quinases/metabolismo , Guanilato Quinases/fisiologia , Queratinócitos/fisiologia , Animais , Animais Recém-Nascidos , Membrana Basal/metabolismo , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Epiderme/crescimento & desenvolvimento , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica/fisiologia , Transporte Proteico , Cicatrização/fisiologiaRESUMO
Epidermal proliferation and differentiation can be regulated by soluble morphogens and growth factors. Heparan sulfate proteoglycans (HSPGs) modulate the action of several of these effector molecules, such as members of the fibroblast growth factor (FGF) and Wnt families. Syndecan-1 is a cell-surface proteoglycan that is expressed in differentiating keratinocytes and transiently upregulated in all layers of the epidermis upon tissue injury. To address the role of syndecan-1 in the regulation of keratinocyte proliferation and differentiation, we generated transgenic mice that overexpress syndecan-1 under K14 keratin promoter in the basal layer of the epidermis. We observed epidermal hyperproliferation in newborn transgenic mice, as evidenced by increased number of suprabasal cell layers, elevated proliferating cell nuclear antigen (PCNA) expression in both basal and suprabasal cell layers and by expression of keratin 6 in the interfollicular epidermis. Compared to both wild-type and syndecan-1-null animals, the transgene expression interfered with skin wound healing in adult mice by decreasing cell proliferation in the re-epithelialized epidermis. Thus, syndecan-1 regulates keratinocyte proliferation differently during skin development and in healing wounds.
Assuntos
Células Epidérmicas , Queratinócitos/fisiologia , Sindecana-1/fisiologia , Cicatrização , Animais , Diferenciação Celular , Proliferação de Células , Epiderme/metabolismo , Epitélio/fisiologia , Fator 2 de Crescimento de Fibroblastos/análise , Fator 7 de Crescimento de Fibroblastos/análise , Humanos , Queratina-14/genética , Camundongos , Camundongos Transgênicos , Antígeno Nuclear de Célula em Proliferação/análise , Sindecana-1/química , Sindecana-1/genéticaRESUMO
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.
Assuntos
Adesão Celular , Movimento Celular , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Vimentina/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
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.
Assuntos
Movimento Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Queratinas/metabolismo , Plaquinas/química , Plaquinas/metabolismo , Plectina/metabolismo , Linhagem Celular Tumoral , Células Clonais , Citoesqueleto/metabolismo , Humanos , Imunoprecipitação , Queratinócitos/citologia , Queratinócitos/metabolismo , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
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.