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1.
Nat Commun ; 12(1): 448, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469008

RESUMO

In self-renewing somatic tissue such as skin epidermis, terminal differentiation genes must be suppressed in progenitors to sustain regenerative capacity. Here we show that hundreds of intronic polyadenylation (IpA) sites are differentially used during keratinocyte differentiation, which is accompanied by downregulation of the Cleavage and Polyadenylation Specificity Factor (CPSF) complex. Sustained CPSF expression in undifferentiated keratinocytes requires the contribution from the transcription factor MYC. In keratinocytes cultured in undifferentiation condition, CSPF knockdown induces premature differentiation and partially affects dynamically used IpA sites. These sites include an IpA site located in the first intron of the differentiation activator GRHL3. CRISPR knockout of GRHL3 IpA increased full-length GRHL3 mRNA expression. Using a targeted genetic screen, we identify that HNRNPA3 interacts with CPSF and enhances GRHL3 IpA. Our data suggest a model where the interaction between CPSF and RNA-binding proteins, such as HNRNPA3, promotes site-specific IpA and suppresses premature differentiation in progenitors.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Queratinócitos/fisiologia , Reepitelização/genética , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Autorrenovação Celular/genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Íntrons/genética , Poliadenilação/genética , Cultura Primária de Células , Fatores de Transcrição/genética
2.
Mol Immunol ; 130: 104-112, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33309306

RESUMO

Atopic dermatitis is a severe, chronic relapsing inflammatory disease of the skin with family clustering. It is characterized into acute phase, which is dominated by T helper 2-type immune responses, and chronic phase, which is dominated by T helper 1-type immune responses. Studies have shown that 3,3'-diindolylmethane not only has antitumor effects but also can relieve symptoms of inflammatory diseases by inhibiting the nuclear factor-κB signaling pathway and regulating T cell differentiation. To study the effect of 3,3'-diindolylmethane on atopic dermatitis and the underlying mechanism, a mouse model of acute atopic dermatitis was established using 2,4-dinitrofluorobenzene. After intraperitoneal injection of 3,3'-diindolylmethane, skin erythema and edema in mice were significantly alleviated. Furthermore, 3,3'-diindolylmethane reduced immune activation, probably by inhibiting the secretion of thymic stromal lymphopoietin by keratinocytes. 3,3'-Diindolylmethane also promoted the differentiation of regulatory T cells and inhibited the activation of T helper 2 and T helper 17 cells to reduce atopic dermatitis-related immune responses. However, it showed no significant effect on the differentiation of T helper 1 cells. These results indicate that 3,3'-diindolylmethane has a significant inhibitory effect on T helper 2 cells in the acute phase of atopic dermatitis. Our findings may provide not only more insights into the pathological mechanism of AD, but also a new candidate medicine for it.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Dermatite Atópica/tratamento farmacológico , Indóis/uso terapêutico , Linfócitos T/efeitos dos fármacos , Doença Aguda , Adulto , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Dermatite Atópica/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Indóis/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Pele/efeitos dos fármacos , Pele/imunologia , Linfócitos T/fisiologia
3.
Proc Natl Acad Sci U S A ; 117(36): 22173-22182, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32843345

RESUMO

While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these-ceramides and glucosylceramides-induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.


Assuntos
Diferenciação Celular/fisiologia , Queratinócitos/fisiologia , Células-Tronco/fisiologia , Células Cultivadas , Epiderme , Humanos , Metabolismo dos Lipídeos
5.
J Surg Res ; 254: 102-109, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32422429

RESUMO

BACKGROUND: Wound healing is a complex process aiming at repairing the damaged skin. MiR-23b has been reported to be upregulated during wound healing. In this study, we intended to explore the working mechanism of miR-23b during wound healing. METHODS: Quantitative real-time polymerase chain reaction was performed to detect the enrichment of miR-23b and tissue inhibitor of metalloproteinase-3 (TIMP3) in HaCaT cells. Scratch wound assay was carried out to measure the migration of HaCaT cells. The target of miR-23b was predicted by microT-CDS software, and the combination was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay. The abundance of TIMP3 protein was detected by Western blot assay. RESULTS: The abundance of miR-23b was positively related to the concentration and time of transforming growth factor ß1 treatment in HaCaT cells. MiR-23b promoted the migration of keratinocytes. TIMP3 was a direct target of miR-23b and was negatively regulated by miR-23b. TIMP3 inhibited the migration of keratinocytes. MiR-23b accelerated the migration of keratinocytes by downregulating the abundance of TIMP3. CONCLUSIONS: MiR-23b promoted the migration of keratinocytes partly through reducing the enrichment of TIMP3. MiR-23b might be a promising target for the treatment of wound healing-associated diseases.


Assuntos
Movimento Celular/genética , Regulação para Baixo/genética , Queratinócitos/fisiologia , MicroRNAs/fisiologia , Inibidor Tecidual de Metaloproteinase-3/genética , Linhagem Celular , Movimento Celular/fisiologia , Queratinócitos/química , MicroRNAs/análise , Inibidor Tecidual de Metaloproteinase-3/análise , Transfecção , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/genética
6.
J Biomed Sci ; 27(1): 56, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32312260

RESUMO

BACKGROUND: Human keratinocytes and derived products are crucial for skin repair and regeneration. Despite substantial advances in engineered skin equivalents, their poor availability and immunorejection remain major challenges in skin grafting. METHODS: Induced keratinocyte-like cells (iKCs) were directly reprogrammed from human urine cells by retroviral transduction of two lineage-specific transcription factors BMI1 and △NP63α (BN). Expression of keratinocyte stem cell or their differentiation markers were assessed by PCR, immunofluorescence and RNA-Sequencing. Regeneration capacity of iKCs were assessed by reconstitution of a human skin equivalent under air-interface condition. RESULTS: BN-driven iKCs were similar to primary keratinocytes (pKCs) in terms of their morphology, protein expression, differentiation potential, and global gene expression. Moreover, BN-iKCs self-assembled to form stratified skin equivalents in vitro. CONCLUSIONS: This study demonstrated an approach to generate human iKCs that could be directly reprogrammed from human somatic cells and extensively expanded in serum- and feeder cell-free systems, which will facilitate their broad applicability in an efficient and patient-specific manner.


Assuntos
Reprogramação Celular/fisiologia , Queratinócitos/fisiologia , Células Cultivadas/fisiologia , Técnicas de Reprogramação Celular , Feminino , Humanos , Técnicas In Vitro , Masculino , Fenômenos Fisiológicos da Pele
7.
Hum Cell ; 33(3): 459-469, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32306195

RESUMO

Studies of human genetic disorders and animal models indicate that matriptase plays essential roles in proteolytic processes associated with profilaggrin processing and desquamation at late stages of epidermal differentiation. The tissue distribution profile and zymogen activation status in human skin, however, suggests that matriptase physiological function in the skin more likely lies in the proliferating and differentiating keratinocytes in the basal and spinous layers. Marked acanthosis with expanded spinous layer and lack of significant changes in intensity and expression pattern for several terminal differentiation markers in the skin of ARIH patients support matriptase's role in earlier rather than the later stages of differentiation. In addition to the tissue distribution, differential subcellular localization further limits the ability of extracellular matriptase proteolytic activity to access the cytosolic non-membrane-bound keratohyalin granules, in which profilaggrin processing occurs. The short lifespan of active matriptase, which results from tightly controlled zymogen activation, rapid inhibition by HAI-1, and shedding from cell surface, indicates that active matriptase likely performs physiological functions via limited proteolysis on its substrates, as needed, rather than via a continuous bulk process. We, here, review these spatiotemporal controls of matriptase proteolytic activity at the biochemical, cellular, and tissue level. Based on this in-depth understanding of how matriptase activity is regulated, we argue that there is no direct involvement of matriptase proteolytic activity in profilaggrin processing and desquamation. The defects in epidermal terminal differentiation associated with matriptase deficiency are likely secondary and are due to putative disruption at earlier stages of differentiation.


Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Proteólise , Serina Endopeptidases/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Precursores Enzimáticos/metabolismo , Células Epidérmicas/fisiologia , Humanos , Queratinócitos/fisiologia , Camundongos , Mutação , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
8.
PLoS One ; 15(4): e0231174, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267880

RESUMO

As the outermost organ, the skin can be damaged following injuries such as wounds and bacterial or viral infections, and such damage should be rapidly restored to defend the body against physical, chemical, and microbial assaults. However, the wound healing process can be delayed or prolonged by health conditions, including diabetes mellitus, venous stasis disease, ischemia, and even stress. In this study, we developed a vibrational cell culture model and investigated the effects of mechanical vibrations on human keratinocytes. The HaCaT cells were exposed to vibrations at a frequency of 45 Hz with accelerations of 0.8g for 2 h per day. The applied mechanical vibration did not affect cell viability or cell proliferation. Cell migratory activity did increase following exposure to vibration, but the change was not statistically significant. The results of immunostaining (F-actin), western blot (ERK1/2), and RT-qPCR (FGF-2, PDGF-B, HB-EGF, TGF-ß1, EGFR, and KGFR) analyses demonstrated that the applied vibration resulted in rearrangement of the cytoskeleton, leading to activation of ERK1/2, one of the MAPK signaling pathways, and upregulation of the gene expression levels of HB-EGF and EGFR. The results suggest that mechanical vibration may have wound healing potential and could be used as a mechanical energy-based treatment for enhancing wound healing efficiency.


Assuntos
Queratinócitos/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Estresse Fisiológico , Vibração/efeitos adversos , Cicatrização/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Técnicas de Cultura de Células/métodos , Linhagem Celular , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Receptores ErbB/metabolismo , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Regulação para Cima
9.
Science ; 367(6483)2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165560

RESUMO

At the body surface, skin's stratified squamous epithelium is challenged by environmental extremes. The surface of the skin is composed of enucleated, flattened surface squames. They derive from underlying, transcriptionally active keratinocytes that display filaggrin-containing keratohyalin granules (KGs) whose function is unclear. Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. The dynamics of phase separation governed terminal differentiation and were disrupted by human skin barrier disease-associated mutations. We used fluorescent sensors to investigate endogenous phase behavior in mice. Phase transitions during epidermal stratification crowded cellular spaces with liquid-like KGs whose coalescence was restricted by keratin filament bundles. We imaged cells as they neared the skin surface and found that environmentally regulated KG phase dynamics drive squame formation. Thus, epidermal structure and function are driven by phase-separation dynamics.


Assuntos
Epiderme/fisiologia , Transição de Fase , Animais , Citoplasma/metabolismo , Humanos , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Queratinas/metabolismo , Camundongos
10.
J Gen Virol ; 101(5): 523-532, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32182205

RESUMO

The infectious life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Evidence suggests a sophisticated interplay between host gene regulation and virus replication. Alternative splicing is an essential process for host and viral gene expression, and is generally upregulated by serine arginine-rich splicing factors (SRSFs). SRSF activity can be positively or negatively controlled by cycles of phosphorylation/dephosphorylation. Here we show that HPV16 infection leads to accumulation of the paradigm SRSF protein, SRSF1, in the cytoplasm in a keratinocyte differentiation-specific manner. Moreover, HPV16 infection leads to increased levels of cytoplasmic and nuclear phosphorylated SRSF1. SR protein kinase 1 (SRPK1) phosphorylates SRSF1. Similar to HPV upregulation of SRSF1, we demonstrate HPV upregulation of SRPK1 via the viral E2 protein. SRPK1 depletion or drug inhibition of SRPK1 kinase activity resulted in reduced levels of SRSF1, suggesting that phosphorylation stabilizes the protein in differentiated HPV-infected keratinocytes. Together, these data indicate HPV infection stimulates the SRPK1-SRSF axis in keratinocytes.


Assuntos
Processamento Alternativo/genética , Papillomavirus Humano 16/patogenicidade , Infecções por Papillomavirus/genética , Proteínas Serina-Treonina Quinases/genética , Células 3T3 , Animais , Células HeLa , Humanos , Queratinócitos/fisiologia , Camundongos , Fosforilação/genética , Fatores de Processamento de Serina-Arginina/genética , Regulação para Cima/genética , Proteínas Virais/genética , Replicação Viral/genética
11.
Int J Mol Sci ; 21(3)2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32033114

RESUMO

The skin is a multilayered and primary defensive organ. Intimate intercellular communication in the skin is necessary to ensure effective surveillance. Extracellular vesicles (EVs) are being explored for their involvement in intercellular skin communication. The aim of this study was to evaluate how human dermal fibroblasts (HDFs) accelerate EV production during senescence and the effects of senescence-associated EVs on epidermal homeostasis. Replicative senescent HDFs were assessed with senescence-associated ß-galactosidase staining and the expression of senescence-related markers. Isolated EVs were characterized by dynamic light scattering and EV marker expression. EVs secreted from untreated young or senescent HDFs, or from those treated with a nSMase inhibitor, antioxidant, and lysosomal activity regulators, were determined by sandwich ELISA for CD81. Human epidermal keratinocytes were treated with young- and senescent HDF-derived EVs. Compared to young HDFs, senescent HDFs produced relatively high levels of EVs due to the increased nSMase activity, oxidative stress, and altered lysosomal activity. The nSMase inhibitor, antioxidant, and agents that recovered lysosomal activity reduced EV secretion in senescent HDFs. Relative to young HDF-derived EVs, senescent HDF-derived EVs were less supportive in keratinocyte differentiation and barrier function but increased proinflammatory cytokine IL-6 levels. Our study suggests that dermis-derived EVs may regulate epidermal homeostasis by reflecting cellular status, which provides insight as to how the dermis communicates with the epidermis and influences skin senescence.


Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Derme/fisiologia , Vesículas Extracelulares/fisiologia , Fibroblastos/fisiologia , Queratinócitos/fisiologia , Adulto , Antioxidantes/metabolismo , Comunicação Celular/fisiologia , Células Cultivadas , Derme/metabolismo , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Interleucina-6/metabolismo , Queratinócitos/metabolismo , Estresse Oxidativo/fisiologia
12.
Toxicon ; 176: 1-9, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31935389

RESUMO

The present study aimed to explore the potential antioxidant molecules of the Asian hornet venom (Vespa velutina nigrithorax) responsible for radical scavenging activity and human keratinocyte protection against oxidative stress. We developed a first technical platform that combined a DPPH radical scavenging chemical assay and cytotoxicity and ROS (reactive oxygen species) production in HaCaT keratinocyte cells exposed to UVB to evaluate the antioxidant property of V. velutina venom. We further employed Thin Layer Chromatography (TLC) combined with the DPPH assay as a targeted separation approach to isolate the antioxidant compounds responsible for the free radical scavenging property of V. velutina venom. In parallel, the latter was fractionated by a HPLC-DAD non-targeted separation approach. From this experiment, nine fractions were generated which were again evaluated separately for their antioxidant properties using DPPH assays. Results showed that only one fraction exhibited significant antioxidant activity in which serotonin was identified as the major compound by a UHPLC-ESI-QTOF HRMS/MS approach. We finally demonstrated, using purified serotonin molecule that this bioactive structure is mostly responsible for the free radical scavenging property of the crude venom as evidenced by DPPH and ROS assays in HaCaT cells exposed to UVB.


Assuntos
Antioxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Venenos de Vespas/farmacologia , Animais , Cromatografia em Camada Delgada , Humanos , Queratinócitos/fisiologia , Espécies Reativas de Oxigênio , Vespas
13.
Environ Pollut ; 259: 113908, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31931413

RESUMO

N6-methyladenosine (m6A), the most abundant and reversible RNA modification, plays critical a role in tumorigenesis. However, whether m6A can regulate p53, a leading antitumor protein remains poorly understood. In this study, we explored the regulatory role of m6A on p53 activation using an arsenite-transformed keratinocyte model, the HaCaT-T cell line. We created the cell line by exposing human keratinocyte HaCaT cells to 1 µM arsenite for 5 months. We found that the cells exhibited an increased m6A level along with an aberrant expression of the methyltransferases, demethylase, and readers of m6A. Moreover, the cells exhibited decreased p53 activity and reduced p53 phosphorylation, acetylation, and transactivation with a high nucleus export rate of p53. Knockdown of the m6A methyltransferase, METTL3 significantly decreased m6A level, restoring p53 activation and inhibiting cellular transformation phenotypes in the arsenite-transformed cells. Further, using both a bioinformatics analysis and experimental approaches, we demonstrated that m6A downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. We showed that m6A upregulated the expression of the negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, our study revealed the novel role of m6A in mediating arsenite-induced human keratinocyte transformation by suppressing p53 activation. This study further sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics.


Assuntos
Adenosina/análogos & derivados , Arsenitos/toxicidade , Queratinócitos/fisiologia , Adenosina/metabolismo , Arsenitos/metabolismo , Transformação Celular Neoplásica , Humanos , Metiltransferases , Proteínas de Ligação a RNA , Proteína Supressora de Tumor p53/metabolismo
14.
Arch Dermatol Res ; 312(1): 1-4, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31659432

RESUMO

Non-melanoma skin cancer primarily affects geriatric patients as evidenced by the fact that only 20% of these cancers are diagnosed in patients under the age of 60 years. Of importance, geriatric skin responds to procarcinogenic ultraviolet B radiation (UVB) in a manner that permits the establishment of tumor cells. Recent studies have indicated that wounding of geriatric skin with fractionated resurfacing lasers and dermabrasion upregulates fibroblast production of insulin-like growth factor-1 (IGF-1) and normalizes the procarcinogenic acute UVB response consisting of basal keratinocytes proliferating while still harboring unrepaired DNA damage. The present studies tested the ability of wounding with a commercially available microneedling device to upregulate IGF-1 levels and normalize the geriatric UVB response. Geriatric volunteers were treated with a microneedling device on buttock skin and 3 months later the IGF-1 levels and UVB responses tested in wounded vs control skin. Wounding via microneedling upregulated IGF-1 and resulted in lower levels of basal keratinocytes proliferating with unrepaired DNA damage. The ability of microneedling to protect against the formation of UVB-damaged proliferating keratinocytes indicates the potential of this wounding modality to reduce aging-associated non-melanoma skin cancer.


Assuntos
Envelhecimento , Pele/efeitos da radiação , Raios Ultravioleta , Idoso , Senescência Celular , Feminino , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Queratinócitos/fisiologia , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
15.
J Invest Dermatol ; 140(1): 75-84.e6, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31351086

RESUMO

Rac signaling affects numerous downstream targets in vitro; however, few studies have established in vivo levels. We generated mice with a single knockout (KO) of Rac1 (Keratin5(K5)-Cre;Rac1flox/flox, Rac1-KO) and double KO of Rac1 and Rac3 (K5-Cre;Rac1flox/flox;Rac3-/-, Rac1/Rac3-DKO) in keratinocytes. The hairless phenotype in Rac1-KO mice was markedly exacerbated in Rac1/Rac3-DKO mice. Strikingly, Rac1-KO mice exhibited thinner dermal white adipose tissue, which was considerably further reduced in Rac1/Rac3-DKO mice. DNA microarray using primary keratinocytes from Rac1/Rac3-DKO mice exhibited decreased mRNA levels of Bmp2, Bmp5, Fgf20, Fgf21, Fgfbp1, and Pdgfα. Combinational treatment with bone morphogenetic protein (BMP) 2 and fibroblast growth factor (FGF) 21 in culture medium, but not individual purified recombinant proteins, could differentiate 3T3-L1 fibroblasts into adipocytes, as could culture media from primary keratinocytes. Conversely, addition of anti-BMP2 or anti-FGF21 antibodies into the culture medium inhibited fibroblast differentiation. In addition, BMP2 and FGF21 treatment promoted adipocyte differentiation only of rat primary white adipocyte precursors but not rat primary brown adipocyte precursors. Furthermore, BMP2 and FGF21 treatment enhanced adipogenesis of normal human dermal fibroblasts. Notably, brown adipogenesis promoted by FGF21 was inhibited by BMP2. Thus, we propose a complex paracrine pathway from keratinocytes to intradermal pre-adipocytes, which functions as a Rac-dependent modulator of both white and brown adipogenesis.


Assuntos
Adipócitos/fisiologia , Tecido Adiposo Branco/fisiologia , Derme/patologia , Queratina-5/genética , Queratinócitos/fisiologia , Proteínas rac de Ligação ao GTP/genética , Animais , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular , Regulação para Baixo , Fatores de Crescimento de Fibroblastos/genética , Camundongos , Camundongos Knockout , Células NIH 3T3 , Comunicação Parácrina , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Análise Serial de Tecidos
16.
J Invest Dermatol ; 140(1): 66-74.e4, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31260673

RESUMO

CYLD is a deubiquitylase with tumor suppressor functions, first identified in patients with familial cylindromatosis. Despite many molecular mechanisms in which a function of CYLD was reported, affected patients only develop skin appendage tumors, and their precise pathogenesis remains enigmatic. To elucidate how CYLD contributes to tumor formation, we aimed to identify molecular partners in keratinocytes. By using yeast two-hybrid, coprecipitation, and proximity ligation experiments, we identified CENPV as a CYLD-interacting partner. CENPV, a constituent of mitotic chromosomes associating with cytoplasmic microtubules, interacts with CYLD through the region between the third cytoskeleton-associated protein-glycine domain and the active site. CENPV is deubiquitylated by CYLD and localizes in interphase to primary cilia where it increases the ciliary levels of acetylated α-tubulin. CENPV is overexpressed in basal cell carcinoma. Our results support the notion that centromeric proteins have functions in ciliogenesis.


Assuntos
Carcinoma Basocelular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cílios/metabolismo , Citoesqueleto/metabolismo , Enzima Desubiquitinante CYLD/genética , Queratinócitos/fisiologia , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Cutâneas/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Carcinogênese , Carcinoma Basocelular/genética , Proteínas Cromossômicas não Histona/genética , Clonagem Molecular , Enzima Desubiquitinante CYLD/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Neoplasias Cutâneas/genética , Ubiquitinação
17.
J Invest Dermatol ; 140(1): 103-112.e8, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276679

RESUMO

IL-17A is abundant in scleroderma but its role in fibrogenesis is controversial. We interrogated the role of IL-17A in extracellular matrix deposition and inflammation by investigating its effects on keratinocytes and fibroblasts cross-talk and in organotypic skin cultures. Keratinocyte-conditioned media of resting, IL-17A-, and/or transforming growth factor-ß-primed primary keratinocytes were used to stimulate healthy donors and scleroderma fibroblasts. Alternatively, organotypic cultures of full human skin were challenged with these cytokines. Keratinocyte-conditioned media tilted the balance of col-I to matrix metalloproteinase-1 production by fibroblasts in favor of matrix metalloproteinase-1, significantly more so in healthy donors than in scleroderma, resulting in enhanced extracellular matrix turnover, further increased by IL-17A. In organotypic skin, transforming growth factor-ß induced an extensive pro-fibrotic gene signature, including the enhanced expression of several collagen genes associated with Wnt signaling. IL-17A strongly promoted the expression of pro-inflammatory genes, with no direct effects on collagen genes, and attenuated Wnt signaling induced by transforming growth factor-ß. In this model, at the protein level, IL-17A significantly decreased col-I production. Our data strongly support a pro-inflammatory and antifibrogenic activity of IL-17A in the context of keratinocyte-fibroblast interaction and in full skin. These data help in directing and interpreting targeted therapeutic approaches in scleroderma.


Assuntos
Fibroblastos/fisiologia , Inflamação/imunologia , Interleucina-17/metabolismo , Queratinócitos/fisiologia , Esclerodermia Localizada/imunologia , Escleroderma Sistêmico/imunologia , Pele/patologia , Células Cultivadas , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Técnicas de Cultura de Órgãos , Pele/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Via de Sinalização Wnt
18.
J Invest Dermatol ; 140(2): 455-464.e8, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31344385

RESUMO

Re-epithelialization is a complex process during skin wound healing, and cell migration is an integral part of this phenomenon. Here we identified a role for LRG1 as a key regulator of epidermal keratinocyte migration where LRG1 acts via enhancement of HIF-1α stability. We showed that LRG1 is upregulated at murine skin wound edges and that addition of recombinant human LRG1 accelerates keratinocyte migration and skin wound healing. Furthermore, we identified transcription factor ELK3 as a downstream effector of LRG1. We confirmed that elevated ELK3 levels manipulated by LRG1 can promote cell migration through upregulation of HIF-1α stability. Because hyperglycemia complicatedly affects HIF-1α stability and activation, our findings provide insights into the molecular controls of wound-associated cell migration and identify potential therapeutic targets for the treatment of chronic diabetic wounds. In conclusion, we demonstrated that LRG1 promotes wound repair through keratinocyte migration and is important for normalization of an abnormal process of diabetic wound healing where HIF-1α stability is insufficient.


Assuntos
Glicoproteínas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Queratinócitos/fisiologia , Reepitelização/fisiologia , Animais , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Pé Diabético/sangue , Pé Diabético/tratamento farmacológico , Pé Diabético/patologia , Modelos Animais de Doenças , Feminino , Glicoproteínas/administração & dosagem , Humanos , Hiperglicemia/complicações , Queratinócitos/efeitos dos fármacos , Masculino , Camundongos , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-ets/metabolismo , RNA-Seq , Reepitelização/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Pele/efeitos dos fármacos , Pele/patologia , Ferida Cirúrgica/tratamento farmacológico , Ferida Cirúrgica/patologia , Regulação para Cima
20.
Elife ; 82019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31793877

RESUMO

Stable cell-cell contacts underpin tissue architecture and organization. Quantification of junctions of mammalian epithelia requires laborious manual measurements that are a major roadblock for mechanistic studies. We designed Junction Mapper as an open access, semi-automated software that defines the status of adhesiveness via the simultaneous measurement of pre-defined parameters at cell-cell contacts. It identifies contacting interfaces and corners with minimal user input and quantifies length, area and intensity of junction markers. Its ability to measure fragmented junctions is unique. Importantly, junctions that considerably deviate from the contiguous staining and straight contact phenotype seen in epithelia are also successfully quantified (i.e. cardiomyocytes or endothelia). Distinct phenotypes of junction disruption can be clearly differentiated among various oncogenes, depletion of actin regulators or stimulation with other agents. Junction Mapper is thus a powerful, unbiased and highly applicable software for profiling cell-cell adhesion phenotypes and facilitate studies on junction dynamics in health and disease.


Assuntos
Comunicação Celular/fisiologia , Biologia Computacional/métodos , Células Endoteliais/fisiologia , Junções Intercelulares/fisiologia , Queratinócitos/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Caderinas/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Junções Intercelulares/metabolismo , Queratinócitos/metabolismo , Microscopia Confocal , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos Sprague-Dawley , Software
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