RESUMO
Burning mouth syndrome (BMS) is a chronic pain condition that most commonly affects postmenopausal women older than 50 years of age. Xerostomia is a common complaint among BMS patients. However, previous studies showed inconsistent findings regarding saliva flow rate reduction. This study examined saliva flow rates, degree of mucosal hydration, xerostomia, and clinical characteristics in BMS patients compared with healthy controls. Unstimulated whole saliva (USWS) was collected through passive drooling; residual mucosal saliva (RMS) was collected using filter paper strips. Stimulated whole saliva (SWS) was collected while chewing on gum base. Oral exam and self-report data were collected. A total of 50 women (22 BMS cases and 28 healthy controls) aged 50 years or older were included in the analysis of this study. Mean age was 62 years for cases and 56 years for controls (P=0.05). Compared with controls, cases had significantly lower USWS flow rates (P<0.001) and had a higher prevalence of xerostomia (P=0.001), gastrointestinal disease (P<0.001), and vaginal dryness (P=0.01). These data show that oral and vaginal dryness are common among BMS patients. Further studies are needed to investigate potential pathophysiological mechanisms related to the quality of saliva and mucosal barrier status among these patients.
Assuntos
Síndrome da Ardência Bucal , Dor Crônica , Xerostomia , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , SalivaRESUMO
For decades, dental schools in the United States have endured a significant faculty shortage. Studies have determined that the top 2 sources of dental faculty are advanced education programs and private practice. Those who have completed both DDS and PhD training are considered prime candidates for dental faculty positions. However, there is no national database to track those trainees and no evidence to indicate that they entered academia upon graduation. The objective of this study was to assess outcomes of dental school-affiliated oral sciences PhD program enrollment, graduates, and placement between 1994 and 2016. Using the American Dental Association annual survey of advanced dental education programs not accredited by the Commission on Dental Accreditation and data obtained from 22 oral sciences PhD programs, we assessed student demographics, enrollment, graduation, and placement. Based on the data provided by program directors, the average new enrollment was 33, and graduation was 26 per year. A total of 605 graduated; 39 did not complete; and 168 were still in training. Among those 605 graduates, 211 were faculty in U.S. academic institutions, and 77 were faculty in foreign institutions. Given that vacant budgeted full-time faculty positions averaged 257 per year during this period, graduates from those oral sciences PhD programs who entered academia in the United States would have filled 9 (3.6%) vacant faculty positions per year. Therefore, PhD programs have consistently generated only a small pipeline of dental school faculty. Better mentoring to retain talent in academia is necessary. Stronger support and creative funding plans are essential to sustain the PhD program. Furthermore, the oral sciences PhD program database should be established and maintained by dental professional organizations to allow assessments of training models, trends of enrollment, graduation, and placement outcomes.
Assuntos
Educação de Pós-Graduação em Odontologia/estatística & dados numéricos , Humanos , Faculdades de Odontologia/estatística & dados numéricos , Inquéritos e Questionários , Estados UnidosRESUMO
Calcium and phosphorus homeostasis is achieved by interplay among hormones, including 1,25(OH)2D3 (1,25D), parathyroid hormone, and fibroblast growth factor 23 (FGF23), and their interactions with other proteins. For example, mutations in dentin matrix protein 1 (DMP-1) result in increased FGF23 and hypophosphatemic rickets. 1,25D is reported to modulate FGF23; thus, we hypothesized that 1,25D may be involved in modulating DMP-1 in an intermediary step. Murine cementoblasts (OCCM-30) and osteocyte-like cells (MLO-Y4 and MLO-A5), known to express DMP-1, were used to analyze effects of 1,25D on DMP-1 expression in vitro. DMP-1 mRNA levels decreased by 50% (p < .05) in the presence of 1,25D in all cell types, while use of a vitamin D receptor (VDR) agonist (EB1089) and antagonist (23S,25S)-DLAM-2P confirmed that VDR pathway activation was required for this response. Further analysis showed that histone deacetylase recruitment was necessary, but neither protein kinase A nor C pathways were required. In conclusion, our results support the hypothesis that 1,25D regulates DMP-1 expression through a VDR-dependent mechanism, possibly contributing to local changes in bone/tooth mineral homeostasis.
Assuntos
Calcitriol/farmacologia , Cemento Dentário/efeitos dos fármacos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Osteócitos/efeitos dos fármacos , Vitaminas/farmacologia , Animais , Calcitriol/análogos & derivados , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação para Baixo , Estrenos/farmacologia , Proteínas da Matriz Extracelular/efeitos dos fármacos , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/farmacologia , Flavonoides/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Maleimidas/farmacologia , Camundongos , Proteína Quinase C/antagonistas & inibidores , Pirrolidinonas/farmacologia , Receptores de Calcitriol/agonistas , Receptores de Calcitriol/antagonistas & inibidores , Fosfolipases Tipo C/antagonistas & inibidores , VorinostatRESUMO
Extracellular inorganic phosphate (ePi) is a key regulator of cementoblast behavior, both in vivo and in vitro, and results in a marked increase in osteopontin expression in vitro. To examine the molecular mechanisms involved in ePi induction of osteopontin gene expression, we transfected a series of osteopontin promoter-luciferase constructs into OCCM-30 cementoblasts. Our results demonstrate that ePi can directly induce osteopontin gene transcription. The region responsive to ePi signaling was localized to a 53-bp region of the promoter between -1454 and -1401 that contains a glucocorticoid response element (GRE). Mutation of the GRE abolished the ePi response, suggesting that glucocorticoid receptor (GR) signaling is required for ePi-mediated transcription. In addition, treatment of cells with the GR antagonist RU-486 (Mifepristone) prevented promoter activation by ePi. The results presented support a model demonstrating that inorganic phosphate regulates OPN gene transcription in cementoblasts through a pathway that requires a functional GR.
Assuntos
Regulação Bacteriana da Expressão Gênica/genética , Osteopontina/genética , Fósforo/farmacologia , Transcrição Gênica/genética , Animais , Pareamento de Bases/genética , Células Cultivadas , Cemento Dentário/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Antagonistas de Hormônios/farmacologia , Camundongos , Mifepristona/farmacologia , Mutação/genética , Fosfatos/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Transdução de Sinais/genética , Transfecção/métodos , Regulação para Cima/genéticaRESUMO
Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/fisiologia , Junções Aderentes/metabolismo , Animais , Antibacterianos/farmacologia , Apoptose/fisiologia , Caderinas/metabolismo , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Tamanho Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Desmogleínas , Desmoplaquinas , Desmossomos/metabolismo , Células Epidérmicas , Proteínas Filagrinas , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Técnicas In Vitro , Proteínas de Filamentos Intermediários/análise , Filamentos Intermediários/metabolismo , Queratinócitos/química , Queratinócitos/citologia , Queratinas/metabolismo , Microscopia Imunoeletrônica , Ratos , Tetraciclina/farmacologia , Vimentina/genética , Vimentina/metabolismo , gama CateninaRESUMO
Vitamin A and other retinoids profoundly inhibit both morphological and biochemical aspects of epidermal differentiation in vitro. Profilaggrin, like most other markers of keratinocyte differentiation, is negatively regulated by retinoic acid in vitro, both at the level of mRNA synthesis and by inhibiting the activity of endoproteases that convert profilaggrin to filaggrin. Profilaggrin is an abundant component of keratohyalin granules and forms the precursor of filaggrin, the keratin associated protein of the stratum corneum. In this report, we identify a region of the human profilaggrin promoter that is involved in the transcriptional regulation of expression by retinoic acid (RA). A series of promoter deletions linked to the chloramphenicol acetyl transferase (CAT) reporter gene were prepared and analyzed by transfection into Hela cells and keratinocytes. We also cotransfected vectors expressing retinoic acid receptor and cultured the transfected cells in the presence and absence of ligand. The region responsive to retinoic acid was localized to a 53 bp sequence between -1109 and -1056 (relative to the mRNA start site at +1) that contains a cluster of five retinoic acid response elements with variable spacing and orientation. In vitro gel shift analysis demonstrated that nuclear retinoid receptors do not bind directly to the identified sequence, suggesting that the mode of regulation by RA may be indirect or that binding requires another cofactor in addition to retinoid receptors. Whereas in keratin genes retinoic acid and glucocorticoid responsive sequences frequently coincide, the glucocorticoid response element in the profilaggrin promoter was located downstream of the RARE cluster between -965 and -951. These studies demonstrate that RA and glucocorticoids regulate profilaggrin expression at least in part by transcriptional mechanisms, via a region of the promoter that contains both retinoid and glucocorticoid responsive elements.
Assuntos
Glucocorticoides/fisiologia , Células HeLa/fisiologia , Proteínas de Filamentos Intermediários/genética , Queratinócitos/fisiologia , Regiões Promotoras Genéticas/fisiologia , Precursores de Proteínas/genética , Tretinoína/fisiologia , Sequência de Bases/genética , Núcleo Celular/metabolismo , Dexametasona/farmacologia , Células Epiteliais/fisiologia , Proteínas Filagrinas , Glucocorticoides/farmacologia , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Precursores de Proteínas/metabolismo , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta/genética , Transfecção , Tretinoína/farmacologiaAssuntos
Envelhecimento/fisiologia , Processamento Alternativo/genética , Caspases/genética , Embrião de Mamíferos/embriologia , Epiderme/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Queratinócitos/metabolismo , RNA Mensageiro/metabolismo , Animais , Caspase 14 , Diferenciação Celular/fisiologia , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Epiderme/crescimento & desenvolvimento , Epiderme/metabolismo , Proteínas de Fluorescência Verde , Indicadores e Reagentes/farmacocinética , Queratinócitos/citologia , Proteínas Luminescentes/farmacocinética , CamundongosRESUMO
Epidermolysis bullosa simplex are dominant disorders of skin fragility characterized by intraepidermal blistering upon mild mechanical trauma. Skin fragility is caused by expression of either an abnormal keratin 5 or an abnormal keratin 14 protein, which compromises the structure and function of the keratin cytoskeleton of basal cells. We report an epidermolysis bullosa simplex patient with a novel single base substitution (A-->T1414) that changes the lysine residue at amino acid 472 to a non-sense codon (K472X). This change predicts the synthesis of a truncated keratin 5, missing 119 amino acids, including the entire tail domain and the highly conserved KLLEGE motif at the carboxy terminus of the 2B domain of the central rod. Expression of an altered keratin 5, of predicted mass and pI for the product of the K472X allele, was documented by one- and two-dimensional western blots of protein extracts from patient skin. Ultrastructural analysis of the patient's nonhyperkeratotic skin was remarkable for basal keratinocytes with dense and irregular keratin filaments proximal to the basement membrane. Keratinocytes, transfected with a cDNA carrying the A-->T1414 non-sense mutation, overexpressed a truncated keratin 5, and showed a disorganized and collapsed keratin filament cytoskeleton. This is the second epidermolysis bullosa simplex patient reported with a premature termination mutation in the KLLEGE motif. The remarkable occurrence of severe palmar--plantar hyperkeratosis in both patients suggests that the keratin 5 tail domain may have unrecognized, but important, normal functions in palmar-plantar tissues.
Assuntos
Epidermólise Bolhosa Simples/genética , Queratinas/genética , Ceratodermia Palmar e Plantar/etiologia , Mutação , Sequência de Aminoácidos , Epidermólise Bolhosa Simples/patologia , Humanos , Queratinas/química , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Pele/ultraestruturaRESUMO
Specific proteolysis plays an important role in the terminal differentiation of keratinocytes in the epidermis and several types of proteases have been implicated in this process. The proprotein convertases (PCs) are a family of Ca2+-dependent serine proteases involved in processing and activation of several types of substrates. In this study we examined the expression and some potential substrates of PCs in epidermis. Four PCs are expressed in epidermis: furin, PACE4, PC5/6 and PC7/8. Furin is detected in two forms, either with or without the transmembrane domain, suggesting occurrence of post-translational cleavage to produce a soluble enzyme. In addition the furin active site has differential accessibility in the granular layer of the epidermis relative to the basal layer, whereas antibodies to the transmembrane domain stain both layers. These findings suggest that furin has access to different types of substrates in granular cells as opposed to basal cells. PC7/8, in contrast, is detected throughout the epidermis with antibodies to both the transmembrane and active site and no soluble form observed. A peptide PC inhibitor (dec-RVKR-CMK) inhibits cleavage of Notch-1, a receptor important in cell fate determination that is found throughout the epidermis. Profilaggrin, found in the granular layer, is specifically cleaved by furin and PACE4 in vitro at a site between the amino terminus and the first filaggrin repeat. This work suggests that the PCs play multiple roles during epidermal differentiation.
Assuntos
Precursores Enzimáticos/metabolismo , Epiderme/enzimologia , Receptores de Superfície Celular , Subtilisinas/metabolismo , Fatores de Transcrição , Células Cultivadas , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/genética , Células Epidérmicas , Proteínas Filagrinas , Furina , Humanos , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/efeitos dos fármacos , Queratinócitos/enzimologia , Proteínas de Membrana/antagonistas & inibidores , Pró-Proteína Convertases , Precursores de Proteínas/química , Precursores de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor Notch1 , Serina Endopeptidases/farmacologia , Especificidade por Substrato , Subtilisinas/antagonistas & inibidores , Subtilisinas/genética , Subtilisinas/farmacologia , Distribuição TecidualRESUMO
Flaky tail (gene symbol ft) is an autosomal recessive mutation in mice that results in a dry, flaky skin, and annular tail and paw constrictions in the neonatal period. Previous studies demonstrated that the ft mutation maps to the central region of mouse chromosome 3, in the vicinity of the epidermal differentiation complex, a gene locus that includes many nonkeratin genes expressed in epidermis. In this study we report a detailed characterization of the flaky tail mouse. Affected homozygous ft/ft mice exhibit large, disorganized scales on tail and paw skin, marked attenuation of the epidermal granular layer, mild acanthosis, and orthokeratotic hyperkeratosis. Biochemical analysis demonstrated that ft/ft mice lacked normal high molecular profilaggrin (approximately 500 kDa), and instead expressed a lower molecular weight form of profilaggrin (220 kDa) that is not proteolytically processed to profilaggrin intermediates or filaggrin. Mutant mice lacked the large, irregular F-type keratohyalin granules that contain profilaggrin, and filaggrin was absent from the cornified layers of ft/ft epidermis. The expression of epidermal keratins was unchanged, whereas the cornified envelope proteins involucrin and loricrin were increased in ft/ft epidermis. Cultured ft/ft keratinocytes also synthesized reduced amounts of profilaggrin mRNA and protein, demonstrating that the defect in profilaggrin expression is intrinsic to epidermal cells. These findings demonstrate that flaky tail mice express an abnormal profilaggrin polypeptide that does not form normal keratohyalin F-granules and is not proteolytically processed to filaggrin. We propose that the absence of filaggrin, and in particular the hygroscopic, filaggrin-derived amino acids that are thought to function in epidermal hydration, underlies the dry, scaly skin characteristic of ft/ft mice. This animal model provides a tool for understanding the role of filaggrin in normal epidermal function and may provide insight into the molecular basis of the filaggrin-deficient human skin disorder ichthyosis vulgaris. J Invest Dermatol 115:1072-1081 2000
Assuntos
Proteínas de Filamentos Intermediários/deficiência , Camundongos Mutantes/metabolismo , Precursores de Proteínas/deficiência , Animais , Modelos Animais de Doenças , Proteínas Filagrinas , Ictiose Vulgar/metabolismo , Proteínas de Filamentos Intermediários/biossíntese , Proteínas de Filamentos Intermediários/genética , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Peso Molecular , Mutação , Fenótipo , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Pele/ultraestruturaRESUMO
Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.
Assuntos
Apoptose , Proteínas de Filamentos Intermediários/biossíntese , Queratinócitos/citologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular , Cicloeximida/farmacologia , Proteínas Filagrinas , Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Proteínas de Filamentos Intermediários/genética , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Ratos , Raios Ultravioleta , beta-Galactosidase/genéticaRESUMO
Epithelial tissues function to protect the organism from physical, chemical, and microbial damage and are essential for survival. To perform this role, epithelial keratinocytes undergo a well-defined differentiation program that results in the expression of structural proteins which maintain the integrity of epithelial tissues and function as a protective barrier. This review focuses on structural proteins of the epidermis and oral mucosa. Keratin proteins comprise the predominant cytoskeletal component of these epithelia. Keratin filaments are attached to the plasma membrane via desmosomes, and together these structural components form a three-dimensional array within the cytoplasm of epithelial cells and tissues. Desmosomes contain two types of transmembrane proteins, the desmogleins and desmocollins, that are members of the cadherin family. The desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic plaque proteins, including desmoplakin and plakoglobin (gamma-catenin). Epidermal and oral keratinocytes express additional differentiation markers, including filaggrin and trichohyalin, that associate with the keratin cytoskeleton during terminal differentiation, and proteins such as loricrin, small proline-rich proteins, and involucrin, that are cross-linked into the cornified envelope by transglutaminase enzymes. The importance of these cellular structures is highlighted by the large numbers of genetic and acquired (autoimmune) human disorders that involve mutations in, or autoantibodies to, keratins and desmosomal and cornified envelope proteins. While much progress has been made in the identification of the structural proteins and enzymes involved in epithelial differentiation, regulation of this process is less clear. Both calcium and retinoids influence epithelial differentiation by altering the transcription of target genes and by regulating activity of enzymes critical in epithelial differentiation, such as transglutaminases, proteinases, and protein kinases. These studies have furthered our understanding of how epithelial tissue and cell integrity is maintained and provide a basis for the future treatment of skin and oral disorders by gene therapy and other novel therapeutics.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Desmossomos/química , Epiderme/química , Células Epiteliais/química , Mucosa Bucal/química , Animais , Diferenciação Celular , Proteínas do Citoesqueleto/genética , Proteínas da Matriz Extracelular/fisiologia , Proteínas Filagrinas , Humanos , Queratinócitos/química , Queratinas/genética , Queratinas/fisiologia , Proteínas de Membrana/fisiologia , Camundongos , Precursores de Proteínas/fisiologia , Dermatopatias Genéticas/genéticaRESUMO
Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. Profilaggrin is phosphorylated and contains multiple filaggrin repeats separated by a hydrophobic linker peptide. We have previously shown that filaggrin constructs containing the linker, when transiently transfected into epithelial cells, lead to expression of a protein that resembles keratohyalin (Dale et al. J Invest Dermatol 108:179-187 1997). To characterize further the region(s) of the linker and/or filaggrin that are necessary for granule formation, we generated several mutant constructs from Flag-FG-1, and generated fusions of filaggrin with green fluorescent protein. We also subjected profilaggrin to protein phosphatase 2A treatment and measured its subsequent solubility. We found that granular morphology is not dependent on the linker or conserved phosphorylation sites, nor is solubility affected by protein phosphatase 2A treatment. Granule morphology was abrogated only in a truncated construct, which still contains the linker. A construct consisting of 16 amino acids of filaggrin fused to green fluorescent protein led to rounded and bizarrely shaped transfected cells with compact keratin filaments, suggesting that very little filaggrin sequence is required for keratin filament interaction. Radiolabeled filaggrin-green fluorescent protein constructs specifically bound keratin in overlay assays confirming that the observed cytoskeletal collapse is due to filaggrin-keratin interaction. Our findings indicate that profilaggrin must be extensively processed before it loses both its granule forming ability as well as its insolubility, suggesting that granule formation in vivo correlates with insolubility in vitro. Further, filaggrin retains its ability to bind keratin as it is degraded to smaller peptides.
Assuntos
Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/fisiologia , Precursores de Proteínas/fisiologia , Animais , Grânulos Citoplasmáticos/ultraestrutura , Proteínas Filagrinas , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Dermatopatias/metabolismo , SolubilidadeRESUMO
The terminal differentiation of epidermal keratinocytes from the granular to enucleated cornified layer involves drastic changes both in morphology and biochemistry. Profilaggrin is a keratinocyte-specific phosphoprotein expressed in the granular layer. Although keratinization has been regarded as a specialized form of apoptosis or programmed cell death, the mechanism for this cellular transition at the molecular level is not yet well understood. In this study, we used light and electron microscopic immunohistochemistry to investigate the localization of the profilaggrin domains during this process. Antibodies specific for the amino-terminal domains of profilaggrin showed localization in keratohyalin granules in the granular cells, but stained the nucleus in transition cells. In contrast, an antibody to filaggrin domains stained the cytoplasm in the transition cells. Nuclei that were positive to amino-terminal profilaggrin contained fragmented DNA, characteristic of apoptosis. In the epidermis of patients with progressive symmetric erythrokeratoderma carrying a mutation in the loricrin gene (loricrin keratoderma), the profilaggrin amino-terminal domains were packed within apoptotic nuclei together with loricrin aggregates and this persisted up to the parakeratotic superficial layer. The present study indicates that the amino-terminal profilaggrin domains are cleaved from the filaggrin repeats and transiently localized to the apoptotic nuclei just before the formation of enucleated stratum corneum in normal epidermis. This suggests a significant role for the profilaggrin amino-terminus in nuclear events associated with keratinocyte terminal differentiation. Disrupted apoptosis found in loricrin keratoderma might explain the marked parakeratotic hyperkeratosis characteristic of this disease.
Assuntos
Núcleo Celular/ultraestrutura , Fragmentação do DNA , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/patologia , Ceratose/patologia , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Pele/citologia , Pele/patologia , Biópsia , Diferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Proteínas Filagrinas , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/metabolismo , Ceratose/genética , Ceratose/metabolismo , Fosfoproteínas/metabolismo , Valores de Referência , Pele/metabolismoRESUMO
Profilaggrin is a large phosphoprotein that is expressed in the granular cells of epidermis where it is localized in keratohyalin. It consists of multiple copies of single filaggrin units plus N- and C-terminal sequences that differ from filaggrin. Profilaggrin is dephosphorylated and proteolytically processed during terminal differentiation to yield filaggrin, which associates with keratin intermediate filaments to form macrofibrils in the lower layers of the stratum corneum. The N-terminal sequence of human profilaggrin comprises two distinct domains; an acidic A domain of 81 amino acids that binds Ca2+, and a cationic B domain of 212 residues. In this report, we further characterize the N-terminal domain by immunohistochemistry and immunoblot analysis using anti-peptide antibodies raised to the A and B regions. All of these antibodies (n = 4) immunostained keratohyalin in the granular layer of human epidermis and also showed some reaction with the lower stratum corneum. In immunoblot studies, the high molecular weight human profilaggrin reacted with both B domain antibodies whereas it showed a weak and variable reaction with A domain antibodies. In addition to profilaggrin, a cationic 32-kDa protein was detected with all N-terminal antibodies. A similar-sized N-terminal peptide was also produced by in vitro proteolysis of human profilaggrin with endoproteinase 1 (PEP1), a protease involved in processing of mouse profilaggrin, and in cultured rat epidermal keratinocytes transfected with a human profilaggrin cDNA construct. Evidence for at least one additional cleavage within the N-terminal domain is shown by immunoreactivity of smaller (16-20 kDa) acidic and basic proteins with A and B domain antibodies, respectively. These results demonstrate that the N-terminal domain is an integral part of profilaggrin in keratohyalin but is proteolytically cleaved from profilaggrin during the terminal differentiation of keratinocytes to yield a 32-kDa peptide.
Assuntos
Células Epidérmicas , Proteínas de Filamentos Intermediários/química , Precursores de Proteínas/química , Sequência de Aminoácidos , Diferenciação Celular , Células Cultivadas , DNA Complementar/análise , Endopeptidases/metabolismo , Proteínas Filagrinas , Expressão Gênica , Humanos , Immunoblotting , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/genética , Queratinócitos/citologia , Dados de Sequência Molecular , Mucosa Bucal/citologia , Peptídeos/metabolismo , Precursores de Proteínas/genética , Estrutura Terciária de ProteínaRESUMO
Filaggrin is an intermediate filament-associated protein (IFAP) that aggregates epidermal keratin filaments in vitro and is thought to perform a similar function during terminal differentiation in vivo. To test this function in living cells, we transiently expressed constructs encoding human filaggrin in both simple epithelial cells (COS-7) and rat keratinocytes. Scanning laser confocal microscopy showed that filaggrin-positive cells had collapsed keratin and vimentin intermediate filament (IF) networks, and that filaggrin partially co-localized with the IF networks. Filaggrin was also detected diffusely in the cytoplasm and nucleus. In contrast, when profilaggrin-like constructs, containing five filaggrin domains separated by the linker sequences, were expressed in cultured cells, immunoreactive granules formed. This finding is reminiscent of the insoluble nature of native profilaggrin that accumulates in keratohyalin granules in vivo, suggesting that the linker peptides (present in profilaggrin but not filaggrin) are important for granule formation. Cells expressing filaggrin also displayed disruption of the nucleus and the nuclear envelope; they rounded up and lost attachment to the substratum, in contrast to control cells over-expressing beta-galactosidase. This functional test of filaggrin in living cells supports its role in the reorganization and packing of keratin IF in epidermal differentiation. Moreover, the observed effects on cell morphology and nuclear integrity suggest that filaggrin may contribute to the form of apoptosis associated with terminal differentiation in epidermis.
Assuntos
Núcleo Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Células Epidérmicas , Proteínas de Filamentos Intermediários/biossíntese , Filamentos Intermediários/ultraestrutura , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular/citologia , Chlorocebus aethiops , Epiderme/química , Proteínas Filagrinas , Humanos , Proteínas de Filamentos Intermediários/fisiologia , Queratinócitos/química , Rim/citologia , Peptídeos/química , Estrutura Terciária de Proteína , RatosRESUMO
Profilaggrin is a large phosphorylated protein (approximately 400 kDa in humans) that is expressed in the granular cells of epidermis where it forms a major component of keratohyalin. It consists of multiple copies of similar filaggrin units plus amino- and carboxy-terminal domains that differ from filaggrin. Proteolytic processing of profilaggrin during terminal differentiation results in the removal of these domains and generation of monomeric filaggrin units, which associate with keratin intermediate filaments to form macrofibrils in the stratum corneum. The amino-terminal domain contains two calcium-binding motifs similar to the EF-hands found in the S-100 family of calcium-binding proteins. In this report, we expressed the 293-residue amino-terminal pro-domain of human profilaggrin as a polyhistidine fusion protein in Escherichia coli, and characterized calcium binding by a 45Ca++ binding assay and fluorescence emission spectroscopy. Fluorescence measurements indicated that the profilaggrin polypeptide undergoes conformational changes upon the removal of Ca++ with ethylenediamine tetraacetic acid, demonstrating the presence of two calcium-binding sites with affinities for calcium that differ ninefold (1.4 x 10(-4) M and 1.2 x 10(-3) M). We suggest that this functional calcium-binding domain at the amino-terminus of human profilaggrin plays a role in profilaggrin processing and in other calcium-dependent processes during terminal differentiation of the epidermis.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/metabolismo , Proteínas de Filamentos Intermediários/química , Precursores de Proteínas/química , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Escherichia coli/química , Proteínas Filagrinas , Humanos , Dados de Sequência Molecular , Fosfoproteínas/química , Conformação Proteica , Proteínas Recombinantes/química , Espectrometria de FluorescênciaRESUMO
Ichthyosis vulgaris is an autosomal dominant disorder of keratinization characterized by mild hyperkeratosis and reduced or absent keratohyalin granules in the epidermis. Profilaggrin, a major component of keratohyalin granules, is reduced or absent from the skin of individuals with ichthyosis vulgaris. In this report, we have further characterized the molecular basis of low profilaggrin expression, which occurs in this disease. In situ hybridization revealed little profilaggrin mRNA in ichthyosis vulgaris-affected epidermis. In keratinocytes cultured from the epidermis of affected individuals, the abundance of profilaggrin was reduced to less than 10% of normal controls, while the mRNA level was decreased to 30-60% of controls. Expression of K1 and loricrin, other markers of epidermal differentiation, were not affected. Nuclear run-on assays indicated that the decrease in mRNA levels was not caused by aberrant transcription. Nucleotide sequencing of 5'-upstream, 3'-non-coding, and flanking regions of the profilaggrin gene from ichthyosis vulgaris-affected individuals revealed only minor changes, probably due to genetic polymorphisms. Our results indicate that defective profilaggrin expression in ichthyosis vulgaris is a result of selectively impaired posttranscriptional control.
Assuntos
Ictiose Vulgar/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Precursores de Proteínas/metabolismo , Processamento Pós-Transcricional do RNA , Adulto , Sequência de Bases , Células Cultivadas , Proteínas Filagrinas , Humanos , Ictiose Vulgar/genética , Hibridização In Situ , Proteínas de Filamentos Intermediários/genética , Queratinócitos/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Filaggrin is an intermediate filament-associated protein which functions to aggregate keratin intermediate filaments in the stratum corneum of mammalian epidermis. It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. In this report, we describe the characterization of cosmid genomic clones containing the human profilaggrin gene coding for 11 complete filaggrin repeats of 324 amino acids each. At the amino- and carboxyl-terminal ends of human profilaggrin are leader and tail peptide sequences of 293 and 157 amino acids, respectively, which differ from filaggrin. The leader peptide is composed of two distinct domains: an 81-residue segment which shows significant homology to the S-100 family of EF hand-containing calcium-binding proteins, and a hydrophilic second domain of 212 residues. The gene is divided into three exons, with one intron (approximately 9.6 kilobase pairs) in the 5' noncoding region and a second one of 570 base pairs between the EF hands. The position of intron 2 is identical to that of other members of the S-100-like family. The presence of an S-100-like domain suggests that profilaggrin binds calcium and that the calcium binding domain is functionally significant in the formation of keratohyalin and/or the subsequent processing of profilaggrin to filaggrin, both of which may be calcium-dependent events.