RESUMO
AIMS: Abundance of connexin 43 (Cx43), a transmembrane protein that forms hemichannels (HCs) and gap junctions (GJs), is dynamically regulated in human gingival fibroblasts (GFBLs) during wound healing. This may be important for fast and scarless gingival wound healing as Cx43 is involved in key cell functions important during this process. Our aim was to uncover the factors that regulate Cx43 expression and abundance in GFBLs. We hypothesized that cytokines and growth factors released during wound healing coordinately regulate Cx43 abundance in GFBLs. RESULTS: TGF-ß1, -ß2, -ß3, PGE2 and IL-1ß significantly upregulated, while TNF-α and IFN-γ downregulated Cx43 in cultured GFBLs. TGF-ß1, -ß2, -ß3, IL-1ß and IFN-γ modulated Cx43 abundance at both mRNA and protein levels, while TNF-α and PGE2 regulated only Cx43 protein abundance, suggesting involvement of distinct transcriptional/post-transcriptional and translational/post-translational mechanisms, respectively. TGF-ß1-induced upregulation of Cx43 was mediated by TGFßRI (ALK5) and SMAD2/3 signaling, and this was potently suppressed by PGE2, IL-1ß, TNF-α and IFN-γ that inhibited SMAD2/3 phosphorylation. CONCLUSION: Regulation of Cx43 abundance in GFBLs involves transcriptional/post-transcriptional and translational/post-translational mechanisms that are distinctly modulated by an interplay between TGF-ß isoforms and PGE2, IL-1ß, TNF-α and IFN-γ.
Assuntos
Conexina 43/genética , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Fator de Crescimento Transformador beta1/farmacologia , Adolescente , Adulto , Idoso , Bioensaio , Conexina 43/metabolismo , Dinoprostona/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Gengiva/efeitos dos fármacos , Gengiva/metabolismo , Humanos , Interferon gama/farmacologia , Masculino , Cultura Primária de Células , Isoformas de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Neural crest (NC)-derived stem cells (NCSC) have an exceptionally wide differentiation potential, but their use in regenerative therapy has been hampered by their scarcity in adult tissues and complex isolation protocols. Human oral mucosal gingiva may provide an attractive source of these cells as it contains NC-derived cells, the tissue is easily accessible and wound healing is fast and scarless with very little morbidity. To this end, we first investigated whether NC-derived cells are retained in adult gingiva by examining 8-months-old NC-reporter Wnt1-Cre/R26RYFP mice. We then hypothesised that gingival cell NC-like phenotype can be further enhanced by floating neurosphere cultures generated from standard human gingival fibroblast (GF) and pooled CFU-F (GSC) cultures. Findings showed that NC-derived cells are retained in the gingival connective tissue of aged mice. Human GFs and GSCs expressed NC-related genes nestin, Snai1, Twist1, Pax3, Sox9 and FoxD3, and generated neurospheres. This was mediated via calcium- and connexin 43-dependent cell communication, which is similar to neurospheres formed by neural progenitors. Cells in the spheres showed significantly increased expression of NC-related genes, and down regulation of fibroblast-related type I collagen. Structurally, the neurospheres were polarised with nestin positive cells located on the outer layers underlined with an extracellular matrix rich in molecules typical to embryonic NC. Sphere-derived cells expressed significantly elevated levels of neural markers, and differentiated into Tau, neurofilament-M and GFAP-positive cells suggesting neural differentiation potential. Thus, human GF and GSC cultures may provide an efficient source of NC-derived cells via enrichment by floating sphere cultures.
Assuntos
Gengiva/citologia , Crista Neural/citologia , Células-Tronco Neurais/citologia , Esferoides Celulares/citologia , Adolescente , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Fibroblastos/citologia , Humanos , Masculino , Camundongos , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genéticaRESUMO
Early development of the urodele amphibian Pleurodeles waltl is accompanied by a process of progressive fibronectin (FN) fibrillogenesis. FN begins to assemble into fibrils on the inner surface of the blastocoele roof at the early blastula stage and progressively forms a complex extracellular matrix. We have analyzed the mechanisms of FN-fibril formation under normal and experimental conditions in vivo with the following probes: iodinated FN, fluorescein-labeled FN, synthetic peptides containing the Arg-Gly-Asp (RGD) cell surface recognition sequence of FN, and polyclonal antibodies against both beta 1 subunit of the amphibian FN receptor and the cytoplasmic domain of beta 1 subunit. We report that in living embryos, exogenous labeled mammalian FN injected into the amphibian blastocoele undergoes FN-fibril formation in spatiotemporal patterns similar to those of endogenous FN. This indicates regulation of fibrillogenesis by the cell surface rather than by changes in the type of FN. Fibrillogenesis is inhibited in a dose-dependent manner both by the GRGDS peptide and monospecific antibodies to amphibian integrin beta 1 subunit. Furthermore, when injected intracellularly into uncleaved embryos or into selected blastomeres, antibodies to the cytoplasmic domain of integrin beta 1 subunit produce a reversible inhibition of FN-fibril formation that follows early cell lineages and cause delays in development. Together, these data indicate that in vivo, the integrin beta 1 subunit and the RGD recognition signal are essential for the proper assembly of FN fibrils in early amphibian development.
Assuntos
Fibronectinas/metabolismo , Integrinas/fisiologia , Pleurodeles/embriologia , Salamandridae/embriologia , Sequência de Aminoácidos , Animais , Anticorpos , Blastômeros/metabolismo , Matriz Extracelular/metabolismo , Imunofluorescência , Substâncias Macromoleculares , Dados de Sequência Molecular , Peptídeos , Pleurodeles/metabolismo , Relação Estrutura-AtividadeRESUMO
We have examined the expression, localization, and function of beta 1 integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits. The beta 1 polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were alpha 2 and alpha 3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor alpha 5 subunit showed no such accumulations in regions of cell-cell contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the beta 1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore, beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaCl2 also resulted in the loss of organization of intracellular F-actin filaments and beta 1 integrins, even when the anti-beta 1 monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that beta 1 integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cell-substrate adhesion.
Assuntos
Adesão Celular , Integrinas/fisiologia , Queratinócitos/fisiologia , Actinas/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Células Cultivadas , Fibroblastos/fisiologia , Fibronectinas/fisiologia , Imunofluorescência , Glicoproteínas/fisiologia , Humanos , Integrinas/biossíntese , Integrinas/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , VitronectinaRESUMO
Extracellular matrix proteins and their cellular receptors, integrins, play a fundamental role in keratinocyte adhesion and migration. During wound healing, keratinocytes detach, migrate until the two epithelial sheets confront, and then regenerate the basement membrane. We examined the expression of different integrins and their putative ligands in keratinocytes during human mucosal wound healing. Migrating keratinocytes continuously expressed kalinin but not the other typical components of the basement membrane zone: type IV collagen, laminin, and type VII collagen. When the epithelial sheets confronted each other, these missing basement membrane components started to appear gradually through the entire wound area. The expression of integrin beta 1 subunit was increased in keratinocytes during migration. The beta 1-associated alpha 2 and alpha 3 subunits were expressed constantly by wound keratinocytes whereas the alpha 5 subunit was present only in keratinocytes during reepithelialization. Furthermore, migrating cells started to express alpha v-integrins which were not present in the nonaffected epithelium. All keratinocytes also expressed the alpha 6 beta 4 integrin during migration. In the migrating cells, the distribution of integrins was altered. In normal mucosa, beta 1-integrins were located mainly on the lateral plasma membrane and alpha 6 beta 4 at the basal surface of basal keratinocytes in the nonaffected tissue. In wounds, integrins were found in filopodia of migrating keratinocytes, and also surrounding cells in several cell layers of the migrating sheet. The results indicate that migrating keratinocytes, in deep human wounds enlarge their integrin repertoire. The changes in integrin expression take place concomitantly with changes in the basement membrane composition, suggesting a close interplay of these two groups of molecules during wound healing.
Assuntos
Membrana Basal/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Integrinas/metabolismo , Queratinócitos/metabolismo , Cicatrização , Anticorpos Monoclonais , Membrana Basal/citologia , Movimento Celular , Imunofluorescência , HumanosRESUMO
VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.
Assuntos
Carcinoma Basocelular/análise , Carcinoma de Células Escamosas/análise , Receptores de Superfície Celular/análise , Receptores Imunológicos/análise , Neoplasias Cutâneas/análise , Pele/análise , Adulto , Membrana Basal/análise , Epiderme/análise , Feto , Imunofluorescência , Humanos , Recém-Nascido , Receptores de Colágeno , Receptores de Fibronectina , Receptores de Laminina , Pele/embriologia , Distribuição TecidualRESUMO
Toll-like-receptors (TLRs) play a significant role in the generation of a specific innate immune response against invading pathogens. TLR2 and TLR4 signaling contributes to infection-induced inflammation in periodontal disease (PD) and atherosclerosis. Observational studies point towards a relationship between PD and atherosclerosis, but the role of TLR2 and TLR4 in the recognition of multiple oral pathogens and their modulation of host response leading to atherosclerosis are not clear. We evaluated the role of TLR2 and TLR4 signaling in the induction of both PD and atherosclerosis in TLR2-/- and TLR4-/- mice to polymicrobial infection with periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Polybacterial infections have established gingival colonization in TLR2-/- and TLR4-/- mice and induction of a pathogen-specific immunoglobulin G immune response. But TLR deficiency dampened accelerated alveolar bone resorption and intrabony defects, indicating a central role in infection-induced PD. Periodontal bacteria disseminated from gingival tissue to the heart and aorta through intravascular dissemination; however, there was no increase in atherosclerosis progression in the aortic arch. Polybacterial infection does not alter levels of serum risk factors such as oxidized low-density lipoprotein, nitric oxide, and lipid fractions in both mice. Polymicrobial-infected TLR2-/- mice demonstrated significant levels (P < 0.05 to P < 0.01) of T helper type 2 [transforming growth factor-ß1 , macrophage inflammatory protein-3α, interleukin-13 (IL-13)] and T helper type 17 (IL-17, IL-21, IL-22, IL-23) splenic T-cell cytokine responses. Increased heat-shock protein expression, hspa1a for Hsp 70, was observed for both TLR2-/- and TLR4-/- mice. This study supports a role for TLR2 and TLR4 in PD and atherosclerosis, corroborating an intricate association between two inflammatory diseases.
Assuntos
Aterosclerose/fisiopatologia , Reabsorção Óssea/fisiopatologia , Coinfecção/imunologia , Inflamação/fisiopatologia , Receptor 2 Toll-Like/deficiência , Receptor 4 Toll-Like/deficiência , Animais , Aterosclerose/etiologia , Aterosclerose/imunologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/imunologia , Coinfecção/microbiologia , Citocinas/sangue , Fusobacterium nucleatum/imunologia , Proteínas de Choque Térmico/sangue , Inflamação/etiologia , Inflamação/imunologia , Lipoproteínas LDL/sangue , Camundongos , Óxido Nítrico/sangue , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/imunologia , Tannerella forsythia/imunologia , Células Th2/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Treponema denticola/imunologiaRESUMO
We examined the biosynthesis and localization of the fibronectin receptor integrin from normal and transformed cultured human cells. Normal cells required a minimum of 20 h for the biosynthesis of completely mature fibronectin-receptor beta-subunit, while transformed cells required only 6-8 h. There was a correspondingly major decrease in the amount of the intracellular beta-chain precursor in the transformants. Immunostaining of normal fibroblastic cells with monoclonal antibodies indicated that both alpha- and beta-polypeptides of the fibronectin receptor are localized in cell surface streaks and focal contact areas. In contrast, both subunits lacked this clustering and had a more diffuse distribution on the surfaces of transformed cells, even though quantitative immunofluorescence experiments indicated that similar or larger amounts of each subunit were present on a per cell basis. Both immunostaining and biochemical analyses also indicated the presence of a relatively large intracellular pool of beta-polypeptides in normal fibroblasts that is not present in transformed cells. There was no major transformation-dependent change in total quantities of mature fibronectin receptor subunit expressed and inserted into the plasma membrane, when normalized to total protein synthesis. Our results indicate that malignant transformation of cultured human cells results in altered localization and processing of the fibronectin receptor. Such changes involving pathways of crucial cell surface molecules may contribute to alterations in their interactions with extracellular macromolecules, including during the process of cellular invasion.
Assuntos
Transformação Celular Neoplásica/metabolismo , Fibronectinas/metabolismo , Integrinas/biossíntese , Fibroblastos/metabolismo , Imunofluorescência , Humanos , Integrinas/análise , Células Tumorais Cultivadas/metabolismoRESUMO
Microvesicles (MVs) are extracellular vesicles secreted by various cell types that are involved in intercellular communication. We hypothesized that in human periodontal disease, the pocket epithelium releases MVs, which then modulate gene expression in the underlying fibroblasts to control periodontal inflammation. MVs were isolated from culture medium of gingival epithelial cells (GECs) treated with oral bacterial biofilm extract or left untreated. Biofilm treatment significantly increased MV release from the GECs. Mass spectrometry of GEC-MVs identified a total of 2,173 proteins, of which about 80% were detected in MVs from both control and biofilm-treated GECs. Among 80 signature genes of human gingival fibroblasts, 20 were significantly regulated (P < 0.05) by MVs from control and biofilm-treated GECs in a similar manner. Matrix metalloproteinase 1 and 3 and interleukin 6 and 8 showed the strongest regulation at the mRNA and protein levels. Several cellular signaling pathways were activated by GEC-MVs in human gingival fibroblasts, including Smad and mitogen-activated protein kinase-associated pathways ERK1/2, JNK, and p38. However, ERK1/2 signaling dominated in the MV-induced gene expression changes. The results demonstrate that GEC-MVs have a strong regulatory effect on the expression of fibroblast genes associated with inflammation and matrix degradation and that bacterial biofilm stimulates the generation of GEC-MVs. This suggests that bacterial biofilms can contribute to the initiation and progression of periodontal disease by promoting a tissue-destructive phenotype in gingival fibroblasts via the enhanced secretion of epithelial MVs.
Assuntos
Células Epiteliais/metabolismo , Vesículas Extracelulares/fisiologia , Fibroblastos/fisiologia , Gengiva/citologia , Doenças Periodontais/metabolismo , Biofilmes , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Espectrometria de Massas , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Fenótipo , Transdução de SinaisRESUMO
Collagen and hyaluronic acid syntheses were studied in skin fibroblast cultures from patients with osteogenesis imperfecta and age-matched controls by labeling the cultures either with [3H]proline and separating the collagenous proteins with DEAE- and CM-cellulose chromatographies, or double-labeling the cultures with [3H]glucosamine and [14C]glycine and separating radioactive hyaluronic acid from glycoproteins and sulphated proteoglycans by DEAE-cellulose chromatography. The activities of the cell layer hyaluronate synthesizing enzyme complex (hyaluronate synthetase) were also determined. The osteogenesis imperfecta cultures were classified into three variants on the basis of type III collagen synthesis. Type III collagen amounted to approx. 40--50% from total collagen in the first variety and approx. 25--30% in the second variety. No difference was noted in the ratio of type III collagen to total collagen in the third variety in comparison with control cultures. The radioactivities of 3H-labeled hyaluronic acid in DEAE-cellulose chromatograms were compared with those of the 14C-labeled proteins. The ratios ranged 9.2--17.3 in the cultures from the patients and 4.6--8.8 in the control cultures. Hyaluronate synthetase activities were 1.3--2.0-fold higher in the osteogenesis imperfecta cells than in their controls. Increased hyaluronic acid synthesis in skin fibroblasts correlated with the severity of the disease but not with the increase in type III collagen synthesis.
Assuntos
Colágeno/biossíntese , Glicosiltransferases , Ácido Hialurônico/biossíntese , Proteínas de Membrana , Osteogênese Imperfeita/metabolismo , Pele/metabolismo , Transferases , Proteínas de Xenopus , Adolescente , Adulto , Células Cultivadas , Criança , Feminino , Fibroblastos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Hialuronan Sintases , Lactente , Masculino , Osteogênese Imperfeita/congênito , Gravidez , Prolina/metabolismo , Pele/embriologiaRESUMO
Granulation-tissue fibroblasts express an unique phenotype distinct from normal fibroblasts. Due to the importance of the cell-matrix interactions in the regulation of cell morphology and behavior, we have compared the cell adhesion apparatus, especially integrin-type receptors, in fibroblasts cultured from healthy human periodontal connective tissues and from chronic and wound granulation tissues. The spreading of granulation-tissue cells on fibronectin, but not on type I collagen or laminin, was slower when compared with the normal fibroblasts. Cell spreading on fibronectin could be inhibited by RGD-containing peptide, suggesting integrin-mediated interaction. Both cell types expressed beta 1 integrin subunit, which associated with several integrin alpha subunits, namely alpha 1, alpha 2, alpha 3, alpha 5 and alpha v. In addition to beta 1 subunit, alpha v chain formed heterodimers with beta 3 and beta 5 subunits. Thus, these cells have multiple putative fibronectin, laminin, collagen, and vitronectin receptors. Cell spreading of both cell types on fibronectin was inhibited with anti-beta 1 and anti-alpha 5 antibodies, but antibodies against other putative FN-binding integrins (alpha 3, alpha v, and alpha v beta 3) had no effects. Furthermore, granulation-tissue fibroblasts showed delayed spreading on substrates coated with anti-beta 1 or anti-alpha 5 integrin antibodies. On substrates coated with anti-alpha 3 antibody, both cell types spread equally well. By FACS analysis, the amount of beta 1 and alpha 5 integrin subunits expressed on the cell surfaces was slightly elevated in GTFs compared with HGFs. Thus, the findings in this study indicate that the weakened interaction of granulation-tissue fibroblasts with fibronectin is regulated by altered function of alpha 5 beta 1 integrin.
Assuntos
Fibronectinas/metabolismo , Tecido de Granulação/metabolismo , Integrinas/fisiologia , Sequência de Aminoácidos , Membrana Celular/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Gengiva/metabolismo , Tecido de Granulação/citologia , Humanos , Dados de Sequência Molecular , Testes de Precipitina , Receptores de Fibronectina/análiseRESUMO
The hyperimmunoglobulin E syndrome (HIES) is a multisystem disorder that affects the: (1) dentition; (2) skeleton; (3) connective tissues; and (4) immune system. Little is known about periodontal manifestations of the syndrome. The purpose of this report was to describe a 5-year-old girl with suspected autosomal-recessive HIES syndrome who revealed profusely bleeding and painful gingiva and generalized aggressive periodontitis. A polymerase chain reaction (PCR)-based microbiological examination detected Porphyromonas gingivalis, Tannerella forsythia, Prevotella nigrescens, Treponema denticola, Eikenella corrodens, and Campylobacter rectus in the deep periodontitis lesions. The extraction of all deciduous teeth due to a poor prognosis and risk of systemic infection led to resolution of the oral inflammation. Long-term follow-up is required to determine the periodontal prognosis of the erupting permanent teeth.
Assuntos
Síndrome de Job/complicações , Periodontite/etiologia , Bactérias Anaeróbias/isolamento & purificação , Pré-Escolar , Consanguinidade , Feminino , Crescimento Excessivo da Gengiva/etiologia , Humanos , Periodontite/microbiologia , Extração Dentária , Dente Decíduo/cirurgiaRESUMO
Syndecans comprise a family of integral membrane proteoglycans that presumably participate in cell-matrix interactions and the modulation of growth factor response. Expression of syndecan-1, a cell surface proteoglycan that binds basic fibroblast growth factor (bFGF) and extracellular matrix components, was studied in cultured human keratinocytes from the oral mucosa and in tissue sections derived from various epithelia and carcinomas of the head and neck. For the study, polyclonal antibodies were raised against the core protein of human syndecan-1. The affinity-purified antibody (designated anti-P117) was shown to react specifically with syndecan-1. Abundant expression of syndecan-1 was detected in frozen sections of various stratified epithelia as well as in cultured keratinocytes. Keratinocytes located in the spinous cell layers showed intense immunoreactivity for syndecan-1, while basal cells stained rather weakly, suggesting that the expression of syndecan-1 could be stimulated during the differentiation of keratinocytes. Cultured human keratinocytes were induced to terminally differentiate by increasing the extracellular calcium concentration of the medium. Parallel to the induction of involucrin expression, the mRNA levels of syndecan-1 were found to increase, suggesting that syndecan-1 is indeed induced during keratinocyte differentiation. The molecular mass and glycosaminoglycan composition of syndecan-1 did not change markedly during calcium-induced differentiation. Malignant transformation was associated with marked reduction of syndecan-1 expression, based on the immunoreactivity of anti-P117 in frozen sections from squamous cell carcinomas (SCCs) of the head and neck.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Transformação Celular Neoplásica/patologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteoglicanas/fisiologia , Sequência de Aminoácidos , Animais , Northern Blotting , Mama/química , Mama/citologia , Mama/fisiologia , Cálcio/farmacologia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/química , Fibroblastos/citologia , Fibroblastos/fisiologia , Neoplasias de Cabeça e Pescoço/química , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/fisiopatologia , Soros Imunes/imunologia , Imuno-Histoquímica , Queratinócitos/química , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/imunologia , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Precursores de Proteínas/análise , Precursores de Proteínas/imunologia , Precursores de Proteínas/fisiologia , Proteoglicanas/análise , Proteoglicanas/imunologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Sindecana-1 , Sindecanas , Células Tumorais CultivadasRESUMO
Proteoglycans participate in the assembly of extracellular matrix, directly by interacting with other matrix components and indirectly by regulating cellular growth-factor responses. We have studied the regulation of gene expression of two small extracellular matrix chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, by dexamethasone and retinoic acid in cultured human skin fibroblasts. Dexamethasone increased decorin production, maximally 4.8-fold, and decorin mRNA levels up to 2.3-fold, but had no effect on biglycan production or mRNA levels. Dexamethasone also prevented transforming growth factor-beta-elicited down-regulation of decorin mRNA levels and production by dermal fibroblasts. In addition, dexamethasone potently inhibited enhancement of biglycan production and mRNA levels by transforming growth factor-beta. Retinoic acid dose dependently reduced decorin mRNA levels (by 51%) and production (by 72%), but had no effect on biglycan gene expression. Retinoic acid did not alter the effect of transforming growth factor-beta on decorin or biglycan production or mRNA levels. These results provide evidence that the effects of glucocorticoids and retinoids on dermal connective tissue are partially mediated via altered expression of decorin and biglycan, which both in turn regulate the activity of transforming growth factor-beta, the most potent stimulator of connective tissue deposition.
Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteoglicanas/genética , Tretinoína/farmacologia , Biglicano , Células Cultivadas , Decorina , Proteínas da Matriz Extracelular , Fibroblastos/metabolismo , Humanos , Fator de Crescimento Transformador beta/farmacologiaRESUMO
To find out whether the epithelial anchoring system shows any alterations in lichen planus, we examined the distribution of type VII collagen, alpha 6 beta 4 integrin, and kalinin in lesions of lichen planus. These molecules were chosen because they are structural components of anchoring fibrils, hemidesmosome-associated complexes, and anchoring filaments. The localization of type VII collagen in lichen planus was strikingly different from that in nonaffected mucosa or dermis or in other mucocutaneous lesions. In the normal mucosa, type VII collagen was localized only at the basement membrane zone. In lichen planus, type VII collagen was present not only in the basement membrane area but also in streaked patterns deep in the connective tissue. The hemidesmosome-associated complex, alpha 6 beta 4 integrin, was localized at the basal aspect of basal epithelial cells of nonaffected sites, but was diffuse and discontinuous in lichen planus lesions. Most of the basal keratinocytes, however, stained for this integrin. Kalinin staining was discontinuous in lichen planus lesions. Often, finger-like projections of kalinin staining were found protruding into the connective tissue stroma. Kalinin was localized at the basement membrane zone of the nonaffected tissue and other mucocutaneous lesions. These results indicate that in cutaneous and mucosal lichen planus, the epithelial anchoring system is disturbed.
Assuntos
Líquen Plano/metabolismo , Antígenos de Superfície/análise , Moléculas de Adesão Celular/análise , Colágeno/análise , Imunofluorescência , Humanos , Imuno-Histoquímica , Integrina alfa6beta4 , Integrinas/análise , Líquen Plano/patologia , CalininaRESUMO
Cell adhesion receptors of the integrin family play a major role during re-epithelialization of human wounds. We have previously documented that the expression of alpha v family integrins is induced in keratinocytes of mucosal wounds [1]. In the present investigation, we extended these studies to determine whether alpha v beta 6 integrin is expressed during wound healing in humans. Mucosal and epidermal wound sections from 1- to 7-day-old wounds were used for immunolocalization of integrins and their putative ligands. In addition, freshly isolated epidermal keratinocytes were used to study integrin expression in vitro. Expression of alpha v beta 6 integrin appeared relatively late during mucosal and dermal wound healing. Maximal expression was seen in 7-day-old wounds in which epithelial sheets had fused and granulation tissue was present. Fibronectin and tenascin, both possible ligands for alpha v beta 6 integrin, were found concentrated underneath the basal epithelial cells expressing this receptor, and the maximal expression of tenascin coincided with that of alpha v beta 6 integrin. Freshly isolated epidermal keratinocytes did not stain for alpha v beta 6 integrin but began to express this integrin after subculturing. Our results suggest that the expression of alpha v beta 6 integrin, a putative binding integrin for fibronectin and tenascin, is induced in keratinocytes when epithelial sheets fuse during wound healing.
Assuntos
Antígenos de Neoplasias , Integrinas/fisiologia , Queratinócitos/química , Pele/lesões , Ferimentos Penetrantes/patologia , Separação Celular , Células Cultivadas , Fibronectinas/metabolismo , Humanos , Mucosa/lesões , Mucosa/metabolismo , Mucosa/patologia , Pele/metabolismo , Pele/patologia , Tenascina/metabolismo , Distribuição TecidualRESUMO
Cell surface sialoglycoproteins of human mononuclear phagocytes in different maturation stages were labelled by the periodate/borohydride method and separated by SDS-polyacrylamide gel electrophoresis. The main surface glycoproteins of peripheral blood monocytes had molecular masses of 115 and 95 kDa. During in vitro transition into adherent macrophages, the monocyte-characteristic surface glycoproteins disappeared. Most of the changes in the surface glycoprotein pattern occurred during the first 24 h and after 96 h the changes were completed. The major sialoglycoproteins of the macrophage cell surface had molecular masses of 130 and 55 kDa. The macrophage cell surface showed further changes when cultured in the presence of synovial fluid (10%). These results may reflect the in vivo maturation of monocytes into tissue macrophages. In synovium, tissue-derived factors may also take part in differentiation.
Assuntos
Macrófagos/citologia , Monócitos/citologia , Sialoglicoproteínas/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Macrófagos/metabolismo , Peso Molecular , Monócitos/metabolismo , Líquido Sinovial/fisiologia , Fatores de TempoRESUMO
The effect of cortisol on the synthesis of glycosaminoglycans (GAGs) was studied in cultured human aortic smooth muscle cells. Cortisol, at a level slightly exceeding the physiological concentration (10(-6) M), decreased the synthesis of hyaluronic acid (HA) by 50% but had no significant effect on the synthesis of sulphated GAGs. The ratio of HA to sulphated GAGs decreased by 47%. These effects were most marked in the fraction secreted into the culture medium. Cortisol neither affected the activity of the hyaluronic acid synthesizing enzyme complex in a cell-free system nor the molecular weight distribution of hyaluronic acid. We suggest that the atherogenity of cortisol and stress may be associated with their effect on the synthesis of HA by the smooth muscle cells of the arterial wall.
Assuntos
Ácido Hialurônico/biossíntese , Hidrocortisona/farmacologia , Músculo Liso Vascular/metabolismo , Aorta/metabolismo , Arteriosclerose/metabolismo , Células Cultivadas , Glicosaminoglicanos/metabolismo , Humanos , Lactente , Masculino , Estresse Fisiológico/complicações , Tripsina/metabolismoRESUMO
Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.
Assuntos
Cadeias alfa de Integrinas , Integrinas/metabolismo , Mucosa Bucal/metabolismo , Tenascina/metabolismo , Cicatrização , Processamento Alternativo , Movimento Celular , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Integrinas/genética , Isoformas de Proteínas/metabolismo , Tenascina/genéticaRESUMO
Proteoglycans (PGs) are extracellular and cell surface-associated macromolecules that regulate cell adhesion, cell growth, matrix formation, and bind growth factors. In this work we studied the distribution of core proteins of four PGs (decorin, biglycan, a large molecular weight PG, and CD44) in human gingiva and periodontal ligament by immunohistochemical staining of frozen tissue sections with specific antibodies. Decorin, a major PG of this tissue, was localized on collagen fiber bundles in the gingival and periodontal connective tissues. Staining for decorin was most intense at the subepithelial region. Biglycan was a minor PG component of the human periodontium, showing some accumulation in connective tissue under the oral epithelium. At the immunohistochemical level, biglycan appeared to form fine filament-like structures on extracellular matrix fibers. Localization of large molecular weight PG differed from that of decorin and biglycan. It was concentrated in deep connective tissue areas of the gingiva and in the periodontal ligament, and was only weakly present at the subepithelial region. CD44 was mainly concentrated in cell-cell contact areas of basal and spinous layers of oral epithelium. In the connective tissue of gingiva and periodontal ligament, CD44 was localized on fibroblast cell surfaces. Connective tissue area under the junctional epithelium contained relatively small amounts of PGs. The results indicate that different parts of human periodontium contain a typical variety of PGs, suggesting a specific function for each PG species in the location at which they accumulate.