RESUMO
The idea and meetings that planned this issue focused on extracellular matrix (ECM) started over 4 years ago. The invitations were sent to investigators over 2 years ago and manuscripts have been submitted, reviewed, and edited since the summer and fall of 2018. Most of the manuscripts were published in early view in 2019, and we are thrilled to share the final collection. This volume contains 6 reviews, 13 original research papers, and 4 remembrances. Marion (Emmy) Gordon and I organized the articles into seven topic areas, including ECM structure, genetics, and development; cancer; vascular structures and development; inflammation and wound healing; collagen in special structures; cornea and other ocular tissues; and extracellular vesicles. Anat Rec, 2020. © 2020 American Association for Anatomy.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Animais , Humanos , Inflamação/metabolismo , Cicatrização/fisiologiaRESUMO
Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.
Assuntos
Fármacos Dermatológicos/toxicidade , Laminina/metabolismo , Gás de Mostarda/toxicidade , Pele/metabolismo , Cicatrização/fisiologia , Animais , Movimento Celular/fisiologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Laminina/genética , Camundongos , Pele/efeitos dos fármacos , Regulação para CimaRESUMO
Purpose: Organ cultures of rabbit corneas have been used to ascertain the effectiveness of a human fibroblast growth factor (FGF)-1 derivative (TTHX1114), lacking cysteine residues, to protect against and/or repair epithelial lesions following exposure to nitrogen mustard (NM). Methods: Rabbit corneas were exposed to NM and cultured for up to 14 days, with or without drug (TTHX1114). At specified times, tissue was examined by histopathology and graded by a novel composite scale. Proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and the expression of native FGF-1 and ADAM-17 after NM exposure was determined by immunofluorescence. Results: Rabbit corneas, exposed to a single dose of NM, showed a nearly complete loss of epithelial cells by day 6 but were significantly regenerated by day 14. When treated continuously with TTHX1114 following vesicant exposure, the losses remained at day 2 levels. The loss of keratocytes in the stroma was not affected by TTHX1114. EdU incorporation over the same time course showed a steady increase in tissue that had not been treated with TTHX1114, while corneas that were treated with the drug showed a higher percent incorporation initially, which then decreased, indicating the strong proliferative response to TTHX1114. ADAM-17 was not significantly altered by TTHX1114 treatment. Corneal epithelial FGF-1 disappeared after only 1 day following exposure to NM. Conclusions: TTHX1114 is protective against NM-induced damage of the corneal epithelium, possibly by supplying an NM-resistant source of trophic support and by stimulating regeneration of new epithelial cells. These responses underscore the potential value of TTHX1114 as an anti-vesicant therapeutic.
Assuntos
Substâncias para a Guerra Química/toxicidade , Córnea/efeitos dos fármacos , Lesões da Córnea/prevenção & controle , Fator 1 de Crescimento de Fibroblastos/uso terapêutico , Mecloretamina/toxicidade , Proteína ADAM17/metabolismo , Animais , Córnea/metabolismo , Córnea/patologia , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/metabolismo , Dano ao DNA , Fator 1 de Crescimento de Fibroblastos/análogos & derivados , Fator 1 de Crescimento de Fibroblastos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Técnicas de Cultura de Órgãos , Engenharia de Proteínas , CoelhosRESUMO
Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a chemical warfare agent that generates an inflammatory response in the skin and causes severe tissue damage and blistering. In earlier studies, we identified cutaneous damage induced by SM in mouse ear skin including edema, erythema, epidermal hyperplasia and microblistering. The present work was focused on determining if SM-induced injury was associated with alterations in mRNA and protein expression of specific cytokines and chemokines in the ear skin. We found that SM caused an accumulation of macrophages and neutrophils in the tissue within one day which persisted for at least 7â¯days. This was associated with a 2-15 fold increase in expression of the proinflammatory cytokines interleukin-1ß, interleukin-6, and tumor necrosis factor α at time points up to 7â¯days post-SM exposure. Marked increases (20-1000 fold) in expression of chemokines associated with recruitment and activation of macrophages were also noted in the tissue including growth-regulated oncogene α (GROα/CXCL1), monocyte chemoattractant protein 1 (MCP-1/CCL2), granulocyte-colony stimulating factor (GCSF/CSF3), macrophage inflammatory protein 1α (MIP1α/CCL3), and IFN-γ-inducible protein 10 (IP10/CXCL10). The pattern of cytokines/chemokine expression was coordinate with expression of macrophage elastase/MMP12 and neutrophil collagenase/MMP8 suggesting that macrophages and neutrophils were, at least in part, a source of cytokines and chemokines. These data support the idea that inflammatory cell-derived mediators contribute to the pathogenesis of SM induced skin damage. Modulating the infiltration of inflammatory cells and reducing the expression of inflammatory mediators in the skin may be an important strategy for mitigating SM-induced cutaneous injury.
Assuntos
Substâncias para a Guerra Química/toxicidade , Quimiocinas/biossíntese , Citocinas/biossíntese , Gás de Mostarda/toxicidade , Pele/efeitos dos fármacos , Pele/metabolismo , Animais , Orelha Externa/efeitos dos fármacos , Orelha Externa/metabolismo , Orelha Externa/patologia , Imuno-Histoquímica , Inflamação/induzido quimicamente , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Masculino , Metaloproteinase 12 da Matriz/biossíntese , Metaloproteinase 8 da Matriz/biossíntese , Camundongos , RNA/biossíntese , RNA/genética , Pele/patologia , Dermatopatias/induzido quimicamente , Dermatopatias/metabolismoRESUMO
Mustard exposures result in epithelial-stromal separations in the cornea and epidermal-dermal separations in the skin. Large blisters often manifest in skin, while the cornea develops microblisters, and, when enough form, the epithelium sloughs. If the exposure is severe, healing can be imperfect and can result in long-term adverse consequences. For the cornea, this could manifest as recurrent corneal erosions. Since the corneal epithelial-stromal separations are in the region identified by electron microscopy as the lamina lucida, the same region affected by the blistering disease junctional epidermolysis bullosa (JEB), we postulated that the molecules that are defective in JEB would be the same ones cleaved by mustard compounds. These molecules are α6ß4 integrin and collagen XVII, which can be cleaved by matrix metalloproteinase-9 (MMP-9) and ADAM17, respectively. Therefore, our laboratory has tested MMP-9 and ADAM17 inhibitors as potential therapies to attenuate corneal mustard injury. Our results demonstrated that inhibiting MMP-9 and ADAM17 resulted in less epithelial-stromal separation in the corneas at 24 h postexposure, as compared with using only medium as a therapy.
Assuntos
Membrana Basal/efeitos dos fármacos , Membrana Basal/patologia , Córnea/efeitos dos fármacos , Córnea/patologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Proteína ADAM17/antagonistas & inibidores , Proteína ADAM17/metabolismo , Administração Cutânea , Animais , Membrana Basal/metabolismo , Guerra Química/tendências , Córnea/metabolismo , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Técnicas de Cultura de ÓrgãosRESUMO
PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.
Assuntos
Proteínas ADAM/metabolismo , Doenças da Córnea/metabolismo , Epitélio Corneano/metabolismo , Mecloretamina/toxicidade , Proteína ADAM17 , Animais , Western Blotting , Células Cultivadas , Doenças da Córnea/induzido quimicamente , Doenças da Córnea/patologia , Substância Própria/efeitos dos fármacos , Substância Própria/metabolismo , Substância Própria/patologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/patologia , Humanos , Coelhos , Tomografia de Coerência Óptica , Fator de Necrose Tumoral alfaRESUMO
Exposure to diesel exhaust particles (DEP) is associated with pulmonary and cardiovascular diseases. Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane. This allows DEPs to penetrate into the cell and capillary lumen. In addition, pro-inflammatory cytokines are up-regulated and mediate vascular permeability in response to DEP. However, the mechanisms through which these DEP-induced pro-inflammatory cytokines increase vascular permeability remain unknown. Hence, we examined the ability of DEP to induce permeability of human umbilical vein endothelial cell tube cells to investigate these mechanisms. Furthermore, supplementation with NAC reduces ROS production following exposure to DEP. HUVEC tube cells contributed to a pro-inflammatory response to DEP-induced intracellular ROS generation. Endothelial oxidative stress induced the release of TNF-α and IL-6 from tube cells, subsequently stimulating the secretion of VEGF-A independent of HO-1. Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability. Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.
Assuntos
Cisteína/farmacologia , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Inflamação/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Emissões de Veículos/toxicidade , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Capilares/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/induzido quimicamente , Interleucina-6/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The cornea is highly sensitive to oxidative stress, a process that can lead to lipid peroxidation. Ultraviolet light B (UVB) and nitrogen mustard (mechlorethamine) are corneal toxicants known to induce oxidative stress. Using a rabbit air-lifted corneal organ culture model, the oxidative stress responses to these toxicants in the corneal epithelium was characterized. Treatment of the cornea with UVB (0.5 J/cm(2)) or nitrogen mustard (100 nmol) resulted in the generation of 4-hydroxynonenal (4-HNE), a reactive lipid peroxidation end product. This was associated with increased expression of the antioxidant, heme oxygenase-1 (HO-1). In human corneal epithelial cells in culture, addition of 4-HNE or 9-nitrooleic acid, a reactive nitrolipid formed during nitrosative stress, caused a time-dependent induction of HO-1 mRNA and protein; maximal responses were evident after 10h with 30 µM 4-HNE or 6h with 10 µM 9-nitrooleic acid. 4-HNE and 9-nitrooleic acid were also found to activate Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE- and 9-nitrooleic acid-induced HO-1 expression. Inhibition of Erk1/2, and to a lesser extent, JNK and PI3K/Akt, suppressed only 4-HNE-induced HO-1, while inhibition of JNK and PI3K/Akt, but not Erk1/2, partly reduced 9-nitrooleic acid-induced HO-1. These data indicate that the actions of 4-HNE and 9-nitrooleic acid on corneal epithelial cells are distinct. The sensitivity of corneal epithelial cells to oxidative stress may be an important mechanism mediating tissue injury induced by UVB or nitrogen mustard.
Assuntos
Aldeídos/metabolismo , Córnea/metabolismo , Peroxidação de Lipídeos , Peróxidos Lipídicos/metabolismo , Mecloretamina/toxicidade , Raios Ultravioleta/efeitos adversos , Aldeídos/toxicidade , Animais , Córnea/efeitos dos fármacos , Córnea/efeitos da radiação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Heme Oxigenase-1/biossíntese , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Peróxidos Lipídicos/toxicidade , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Coelhos , Fatores de TempoRESUMO
Inhalation of diesel exhaust particles (DEPs) is associated with pulmonary and cardiovascular disease. One contributor to pathogenesis is inhaled particles reaching and injuring the lung capillary endothelial cells, and possibly gaining access to the blood stream. Using in vitro capillary tubes as a simplified vascular model system for this process, it was previously shown that DEPs induce the redistribution of vascular endothelial cell-cadherin (VE-Cad) away from the plasma membrane to intracellular locations. This allowed DEPs into the cell cytoplasm and tube lumen, suggesting the tubes may have become permeable (Chao et al., 2011). Here some of the mechanisms responsible for endothelial tube changes after DEP exposure were examined. The results demonstrate that endothelial tube cells mounted an oxidative stress response to DEP exposure. Hydrogen peroxide and oxidized proteins were detected after 24h of exposure to DEPs. Particles induced relocalization of Nrf2 from the cytoplasm to the nucleus, upregulating the expression of the enzyme heme oxygenase-1 (HO-1). Surprisingly, vascular endothelial cell growth factor-A (VEGF-A), initially termed "vascular permeability factor" (VPF), was found to be up-regulated in response to the HO-1 expression induced by DEPs. Similar to DEPs, applied VEGF-A induced relocalization of VE-Cadherin from the cell membrane surface to an intracellular location, and relocalization of VE-cadherin was associated with permeability. These data suggest that the DEPs may induce or contribute to the permeability of capillary-like endothelial tube cells via induction of HO-1 and VEGF-A.
Assuntos
Capilares/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Emissões de Veículos/toxicidade , Capilares/citologia , Capilares/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Células Cultivadas , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Regulação da Expressão Gênica/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Half mustard (CEES) and nitrogen mustard (NM) are commonly used surrogates and vesicant analogs of the chemical warfare agent sulfur mustard. In the current study, in situ forming poly(ethylene glycol) (PEG)-based doxycycline hydrogels are developed and evaluated for their wound healing efficacy in CEES and NM-exposed rabbit corneas in organ culture. The hydrogels, characterized by UV-Vis spectrophotometry, rheometry, and swelling kinetics, showed that the hydrogels are optically transparent, have good mechanical strength and a relatively low degree of swelling (<7%). In vitro doxycycline release from the hydrogel disks (0.25% w/v) was found to be biphasic with release half times of approximately 12 and 72h, respectively, with 80-100% released over a 7-day period. Permeation of doxycycline through vesicant wounded corneas was found to be 2.5 to 3.4 fold higher than non-wounded corneas. Histology and immunofluorescence studies showed a significant reduction of matrix metalloproteinase-9 (MMP-9) and improved healing of vesicant-exposed corneas by doxycycline hydrogels compared to a similar dose of doxycycline delivered in phosphate buffered saline (PBS, pH 7.4). In conclusion, the current studies demonstrate that the doxycycline-PEG hydrogels accelerate corneal wound healing after vesicant injury offering a therapeutic option for ocular mustard injuries.
Assuntos
Doxiciclina/administração & dosagem , Doxiciclina/química , Portadores de Fármacos/química , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/tratamento farmacológico , Compostos de Mostarda Nitrogenada/intoxicação , Polietilenoglicóis/química , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Hidrogéis/química , Irritantes/intoxicação , Teste de Materiais , Coelhos , Resultado do TratamentoRESUMO
Epithelial cell migration during wound healing is regulated in part by enzymatic processing of laminin-332 (formerly LN-5), a heterodimer formed from alpha, beta, and gamma polypeptide chains. Under static conditions, laminin-332 is secreted into the extracellular matrix as a proform and has two chains processed to smaller forms, allowing it to anchor epithelial cells to the basement membrane of the dermis. During incisional wounding, laminin gamma2 chains in particular are processed to smaller sizes and function to promote epithelial sheet migration over the wound bed. The present study examines whether this same function occurs following chemical injury. The mouse ear vesicant model (MEVM) was used to follow the pathology in the ear and test whether processed laminin-332 enhances epithelial cell migration. Skin biopsies of sulfur mustard (SM) exposed ears for several time points were analyzed by histology, immunohistochemistry, real-time PCR, and Western blot analysis. SM exposure greatly increased mRNA levels for laminin-gamma2 in comparison to the other two chains. Protein production of laminin-gamma2 was upregulated, and there was an increase in the processed forms. Protein production was in excess of the amount required to form heterotrimeric laminin-332 and was associated with the migrating epithelial sheet, suggesting a potential role in wound healing for monomeric laminin-gamma2.
Assuntos
Moléculas de Adesão Celular/biossíntese , Movimento Celular , Células Epiteliais/metabolismo , Laminina/biossíntese , Regulação para Cima , Cicatrização , Ferimentos e Lesões/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Orelha/patologia , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Laminina/metabolismo , Camundongos , RNA Mensageiro/biossíntese , Ferimentos e Lesões/patologia , CalininaRESUMO
Sulfur mustard (HD, SM), is a chemical warfare agent that within hours causes extensive blistering at the dermal-epidermal junction of skin. To better understand the progression of SM-induced blistering, gene expression profiling for mouse skin was performed after a single high dose of SM exposure. Punch biopsies of mouse ears were collected at both early and late time periods following SM exposure (previous studies only considered early time periods). The biopsies were examined for pathological disturbances and the samples further assayed for gene expression profiling using the Affymetrix microarray analysis system. Principal component analysis and hierarchical cluster analysis of the differently expressed genes, performed with ArrayTrack showed clear separation of the various groups. Pathway analysis employing the KEGG library and Ingenuity Pathway Analysis (IPA) indicated that cytokine-cytokine receptor interaction, cell adhesion molecules (CAMs), and hematopoietic cell lineage are common pathways affected at different time points. Gene ontology analysis identified the most significantly altered biological processes as the immune response, inflammatory response, and chemotaxis; these findings are consistent with other reported results for shorter time periods. Selected genes were chosen for RT-PCR verification and showed correlations in the general trends for the microarrays. Interleukin 1 beta was checked for biological analysis to confirm the presence of protein correlated to the corresponding microarray data. The impact of a matrix metalloproteinase inhibitor, MMP-2/MMP-9 inhibitor I, against SM exposure was assessed. These results can help in understanding the molecular mechanism of SM-induced blistering, as well as to test the efficacy of different inhibitors.
Assuntos
Carcinógenos/toxicidade , Substâncias para a Guerra Química/toxicidade , Perfilação da Expressão Gênica , Gás de Mostarda/toxicidade , Animais , Carcinógenos/antagonistas & inibidores , Análise por Conglomerados , Citocinas/biossíntese , Citocinas/genética , Ensaio de Imunoadsorção Enzimática , Masculino , Camundongos , NF-kappa B/biossíntese , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Collagen XXIV is an ill-characterized fibrillar collagen that is predominantly expressed in the forming skeleton of the mouse embryo. Here we report that the Col24al gene is constitutively transcribed in the trabecular bone and periosteum of the newborn mouse as well. The bone specificity of Col24al was further documented using three well-characterized cell culture models of osteoblast differentiation. These in vitro analyses indicated that Col24al transcription is activated at about the same time as that of the osteocalcin gene, and gradually increases to eventually plateau as osteoblasts begin to deposit a mineralizing matrix. These findings lend further support to the hypothesis that collagen XXIV may be implicated in the formation of a mineralization-competent bone matrix.
Assuntos
Desenvolvimento Ósseo/genética , Diferenciação Celular/genética , Colágeno/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Osteoblastos/metabolismo , Osteogênese/genética , Animais , Matriz Óssea/crescimento & desenvolvimento , Matriz Óssea/metabolismo , Calcificação Fisiológica/genética , Linhagem Celular Tumoral , Marcadores Genéticos/genética , Camundongos , Células NIH 3T3 , Osteocalcina/genética , Periósteo/crescimento & desenvolvimento , Periósteo/metabolismo , RatosRESUMO
Triggering receptor expressed on myeloid cells (TREM) regulates inflammatory responses to lipopolysaccharide (LPS). In these studies, we analyzed the expression of TREM in hepatic macrophages and endothelial cells which play a central role in LPS clearance. LPS administration to C3H/HeOuJ mice resulted in a rapid induction of TREM-1 and TREM-3, but a decrease in TREM-2 in liver macrophages and endothelial cells. The observation that TREM family members are detectable in endothelial cells is novel and demonstrates that their expression is not limited to myeloid cells. LPS-induced alterations in TREM expression were not evident in cells from C3H/HeJ TLR-4 mutant mice, indicating that the response is dependent on TLR-4. IL-1beta and TNFalpha upregulated TREM-1 and TREM-3 expression and suppressed TREM-2 expression in macrophages and endothelial cells. This activity involved PI3-kinase and p38 MAP kinase signaling. Interestingly, no significant differences were noted in TREM expression between wild-type and TNFR1-/- mice treated with LPS. Treatment of macrophages and endothelial cells with LPS upregulated expression of nitric oxide synthase-2 (NOS-2). This was blocked by TREM-1 Fc/fusion protein, indicating that TREM-1 mediates LPS-induced NOS-2 expression. These results suggest that TREM proteins are important in the inflammatory response of hepatic macrophages and endothelial cells to acute endotoxemia.
Assuntos
Células Endoteliais/metabolismo , Endotoxemia/metabolismo , Células de Kupffer/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Animais , Endotoxemia/etiologia , Endotoxemia/genética , Escherichia coli , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Lipopolissacarídeos/farmacologia , Fígado/irrigação sanguínea , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/metabolismo , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/farmacologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.
Assuntos
Colágeno/biossíntese , Colágeno/química , Regulação da Expressão Gênica , Sequência de Aminoácidos , Animais , Galinhas , Cobaias , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which displays unique structural features of invertebrate fibrillar collagens and is expressed predominantly in bone tissue. Here we report the characterization of the proximal promoter of the mouse gene (Col24a1) and its regulation in osteoblastic cells. Using well characterized murine models of osteoblast differentiation, we found that the Col24a1 gene is activated sometime before onset of the late differentiation marker osteocalcin. Additional analyses revealed that Col24a1 produces equal amounts of two alternatively spliced products with different 5'-untranslated sequences that originate from distinct transcriptional start sites. Cell transfection experiments in combination with DNA binding assays demonstrated that Col24a1 promoter activity in ROS17/2.8 osteosarcoma cells is under the control of an upstream cis-acting element, which is shared by both transcripts and is recognized by specific combinations of c-Jun, CREB1, ATF1, and ATF2 dimers. Consistent with these results, overexpression of c-Jun, ATF1, ATF2, or CREB1 in transiently transfected osteoblastic cells stimulated transcription from reporter gene constructs driven by the Col24a1 promoter to different degrees. Moreover, chromatin immunoprecipitation experiments showed that these nuclear factors bind the same upstream sequence of the endogenous Col24a1 gene. Collectively these data provide new information about transcriptional control of collagen fibrillogenesis, in addition to implicating for the first time CREB-AP1 protein complexes in the regulation of collagen gene expression in osteoblasts.
Assuntos
Colágeno/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Osteoblastos/fisiologia , Regiões Promotoras Genéticas , Isoformas de Proteínas/metabolismo , Fator de Transcrição AP-1/metabolismo , Processamento Alternativo , Animais , Células Cultivadas , Colágeno/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Humanos , Camundongos , Dados de Sequência Molecular , Osteoblastos/citologia , Osteocalcina/metabolismo , Isoformas de Proteínas/genética , Ratos , Fator de Transcrição AP-1/genética , Transcrição GênicaRESUMO
Collagens XII and XIV are members of a subfamily of fibril-associated collagens with interrupted triple-helices (FACITs) that facilitate the interactions of adjacent collagen fibrils. Using immunohistochemistry and in situ hybridization, we analyzed the spatial and temporal expression pattern of collagens XII and XIV during bleomycin-induced pulmonary fibrosis. C57Bl mice were treated with bleomycin (1 U, i.p., every other day for 8 days) or saline (control), and lung tissue samples were analyzed 2-12 weeks later. Collagen I protein expression was increased in the lung 2 weeks post bleomycin treatment and persisted for at least 12 weeks. In contrast, collagen XII and XIV expression was low until 4 weeks after bleomycin treatment. Whereas collagen XII expression was greatest between 4 weeks and 8 weeks, expression of collagen XIV persisted from 4 to 12 weeks, which suggests that these two proteins may play distinct roles in the fibrotic process. The mRNA for lysyl oxidase (LOX), an enzyme for cross-linking of collagens, had a delayed increase in the lung after bleomycin administration. It reached a maximum after 8 weeks, and persisted throughout the 12 weeks of the study. These data support the hypothesis that fibrosis is a multistep process that involves both collagen accumulation and changes in the molecules that modulate the biomechanical properties of fibrils.
Assuntos
Colágeno Tipo I/metabolismo , Colágeno Tipo XII/metabolismo , Colágeno/metabolismo , Colágenos Associados a Fibrilas/metabolismo , Glicoproteínas/metabolismo , Pulmão/patologia , Fibrose Pulmonar/metabolismo , Animais , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Bleomicina/administração & dosagem , Bleomicina/efeitos adversos , Feminino , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína-Lisina 6-Oxidase/análise , Fibrose Pulmonar/induzido quimicamente , RNA Mensageiro/análise , Fatores de TempoRESUMO
Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.
Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Antígenos CD/genética , Doença Hepática Induzida por Substâncias e Drogas , Fígado , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais , Animais , Antígenos CD/fisiologia , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Indução Enzimática , Injeções Intraperitoneais , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/análise , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Fatores de TempoRESUMO
Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.
Assuntos
Osso e Ossos/embriologia , Colágeno/biossíntese , Colágeno/química , Córnea/embriologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Osso e Ossos/metabolismo , Clonagem Molecular , Córnea/metabolismo , DNA Complementar/metabolismo , Humanos , Hibridização In Situ , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/metabolismo , Peptídeos/química , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Distribuição TecidualRESUMO
Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-alpha, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.