RESUMO
Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment.
Assuntos
Fibroblastos , Hidrogéis , Macrófagos , Cicatrização , Animais , Hidrogéis/química , Cicatrização/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Feminino , Comunicação Celular/efeitos dos fármacos , Materiais Biocompatíveis/química , Ligante RANK/metabolismo , Camundongos Endogâmicos C57BL , Reagentes de Ligações Cruzadas/química , Gelatina/química , Inflamação/metabolismo , Inflamação/patologiaRESUMO
For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.
Assuntos
Desdiferenciação Celular , Folículo Piloso , Melanócitos , Nicho de Células-Tronco , Células-Tronco , Animais , Humanos , Camundongos , Folículo Piloso/citologia , Melanócitos/citologia , Células-Tronco/citologia , Microambiente Celular , Linhagem da Célula , Envelhecimento , Homeostase , Cor de Cabelo/fisiologiaRESUMO
BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a multi-resistant variant of prostate cancer (PCa) that has become a major challenge in clinics. Understanding the neuroendocrine differentiation (NED) process at the molecular level is therefore critical to define therapeutic strategies that can prevent multi-drug resistance. METHODS: Using RNA expression profiling and immunohistochemistry, we have identified and characterised a gene expression signature associated with the emergence of NED in a large PCa cohort, including 169 hormone-naïve PCa (HNPC) and 48 castration-resistance PCa (CRPC) patients. In vitro and preclinical in vivo NED models were used to explore the cellular mechanism and to characterise the effects of castration on PCa progression. RESULTS: We show for the first time that Neuropilin-1 (NRP1) is a key component of NED in PCa cells. NRP1 is upregulated in response to androgen deprivation therapies (ADT) and elicits cell survival through induction of the PKC pathway. Downmodulation of either NRP1 protein expression or PKC activation suppresses NED, prevents tumour evolution toward castration resistance and increases the efficacy of docetaxel-based chemotherapy in preclinical models in vivo. CONCLUSIONS: This study reveals the NRP1/PKC axis as a promising therapeutic target for the prevention of neuroendocrine castration-resistant variants of PCa and indicates NRP1 as an early transitional biomarker.
Assuntos
Neoplasias de Próstata Resistentes à Castração , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/patologia , Neuropilina-1 , Neoplasias de Próstata Resistentes à Castração/patologia , Antagonistas de Androgênios , Resistência a Medicamentos , Diferenciação Celular , Linhagem Celular TumoralRESUMO
Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.
Assuntos
Macrófagos/imunologia , Macrófagos/metabolismo , Fagocitose/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt , Cicatrização , Fibroblastos/metabolismo , Fibrose , Humanos , Proteólise , Pele/imunologia , Pele/lesões , Pele/metabolismo , Cicatrização/imunologiaRESUMO
Hair follicle stem cells may themselves regulate the niche environment for hair follicle regrowth. A recent Science paper from Elaine Fuchs and colleagues (Gur-Cohen et al., 2019) suggests that this involves regulation of the lymphatic system and may have implications in understanding tissue regeneration.
Assuntos
Folículo Piloso , Solo , Células-TroncoRESUMO
Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.
Assuntos
Carcinogênese/patologia , Melanócitos/patologia , Melanoma/patologia , Células-Tronco Neoplásicas/patologia , Animais , Carcinogênese/metabolismo , Transformação Celular Neoplásica/patologia , Derme/patologia , Modelos Animais de Doenças , Epiderme/patologia , Homeostase , Humanos , Melanócitos/metabolismo , Camundongos , Mutação/genética , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Microambiente Tumoral , Via de Sinalização WntRESUMO
Recent research shows that potentially cancerous, somatic mutations can reside in normal cells. Pineda et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201907178) report on a unique management technique by hair follicle stem cells to evade tumorigenesis.
Assuntos
Carcinogênese , Folículo Piloso , HumanosRESUMO
During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.
Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Análise de Célula Única/métodos , Pele/citologia , Células-Tronco/citologia , Animais , Western Blotting , Células Cultivadas , Feminino , Masculino , Camundongos , Análise de Sequência de RNA , Células-Tronco/metabolismo , Cicatrização/fisiologiaRESUMO
Despite numerous observations linking protracted exposure to low-dose (LD) radiation and leukemia occurrence, the effects of LD irradiation on hematopoietic stem cells (HSCs) remain poorly documented. Here, we show that adult HSCs are hypersensitive to LD irradiation. This hyper-radiosensitivity is dependent on an immediate increase in the levels of reactive oxygen species (ROS) that also promotes autophagy and activation of the Keap1/Nrf2 antioxidant pathway. Nrf2 activation initially protects HSCs from the detrimental effects of ROS, but protection is transient, and increased ROS levels return, promoting a long-term decrease in HSC self-renewal. In vivo, LD total body irradiation (TBI) does not decrease HSC numbers unless the HSC microenvironment is altered by an inflammatory insult. Paradoxically, such an insult, in the form of granulocyte colony-stimulating factor (G-CSF) preconditioning, followed by LD-TBI facilitates efficient bone marrow transplantation without myeloablation. Thus, LD irradiation has long-term detrimental effects on HSCs that may result in hematological malignancies, but LD-TBI may open avenues to facilitate autologous bone marrow transplantation.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Estresse Oxidativo/genética , Irradiação Corporal Total/métodos , Animais , Humanos , CamundongosRESUMO
Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro. Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.
Assuntos
Adipócitos/fisiologia , Reprogramação Celular , Miofibroblastos/fisiologia , Regeneração , Cicatrização , Animais , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Proteínas Morfogenéticas Ósseas/metabolismo , Células Cultivadas , Cicatriz/patologia , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/patologia , Folículo Piloso/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Wound induced hair follicle neogenesis (WIHN) describes a regenerative phenomenon in adult mammalian skin, wherein fully functional hair follicles regenerate de novo in the center of large excisional wounds. Originally described in rats, rabbits, sheep, and humans in 1940-60, the WIHN phenomenon was reinvestigated in mice only recently. The process of de novo hair regeneration largely duplicates the morphological and signaling features of normal embryonic hair development. Similar to hair development, WIHN critically depends on the activation of canonical WNT signaling. However, unlike hair development, WNT activation in WIHN is dependent on Fgf9 signaling generated by the immune system's gamma delta (γδ) T cells. The cellular bases of WIHN remain to be fully characterized, however, the available evidence leaves open the possibility for a blastema-like mechanism, wherein epidermal and/or dermal wound cells undergo epigenetic reprogramming toward a more plastic, embryonic-like state. De novo hair follicles do not regenerate from preexisting hair-fated bulge stem cells. This suggests that hair neogenesis is not driven by preexisting lineage-restricted progenitors, as is the case for amputation-induced mouse digit tip regeneration, but rather may require a blastema-like mechanism. The WIHN model is characterized by several intriguing features, which await further explanation. These include: (i) minimum wound size requirement for activating neogenesis, (ii) restriction of hair neogenesis to the wound's center, (iii) imperfect patterning outcomes, both in terms of neogenic hair positioning within the wound and in terms of their orientation. Future inquires into the WIHN process, made possible by a wide array of the available skin-specific genetic tools, will undoubtedly expand our understanding of the regeneration mechanisms in adult mammals.
RESUMO
Genetic studies suggest that the major events of human hair follicle development are similar to those in mice, but detailed analyses of this process are lacking. In mice, hair follicle placode "budding" is initiated by invagination of Wnt-induced epithelium into the underlying mesenchyme. Modification of adherens junctions (AJs) is clearly required for budding. Snail-mediated downregulation of AJ component E-cadherin is important for placode budding in mice. Beta-catenin, another AJ component, has been more difficult to study owing to its essential functions in Wnt signaling, a prerequisite for hair follicle placode induction. Here, we show that a subset of human invaginating hair placode cells expresses the stem cell marker CD133 during early morphogenesis. CD133 associates with membrane beta-catenin in early placodes, and its continued expression correlates with loss of beta-catenin and E-cadherin from the cell membrane at a time when E-cadherin transcriptional repressors Snail and Slug are not implicated. Stabilization of CD133 via anti-CD133 antibody treatment of human fetal scalp explants depresses beta-catenin and E-cadherin membrane localization. We discuss this unique correlation and suggest a hypothetical model whereby CD133 promotes morphogenesis in early hair follicle placodes through the localized removal of membrane beta-catenin proteins and subsequent AJ dissolution.
Assuntos
Antígenos CD/genética , Caderinas/metabolismo , Glicoproteínas/genética , Folículo Piloso/citologia , Folículo Piloso/fisiologia , Peptídeos/genética , beta Catenina/metabolismo , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Transição Epitelial-Mesenquimal/fisiologia , Técnicas de Introdução de Genes , Glicoproteínas/metabolismo , Folículo Piloso/crescimento & desenvolvimento , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Mutantes , Morfogênese/fisiologia , Peptídeos/metabolismo , Couro Cabeludo/citologia , Transcriptoma , Via de Sinalização Wnt/fisiologiaRESUMO
Skin wound healing is a complex regenerative phenomenon that can result in hair follicle neogenesis. Skin regeneration requires significant contribution from the immune system and involves substantial remodelling of both epidermal and dermal compartments. In this viewpoint, we consider epigenetic regulation of reepithelialization, dermal restructuring and hair neogenesis. Because little is known about the epigenetic control of these events, we have drawn upon recent epigenetic mapping and functional studies of homeostatic skin maintenance, epithelial-mesenchymal transition in cancer, and new works on regenerative dermal cell lineages and the epigenetic events that may shape their conversion into myofibroblasts. Finally, we speculate on how these various healing components might converge for wound-induced hair follicle neogenesis.
Assuntos
Epigênese Genética , Cabelo/fisiologia , Homeostase/genética , Reepitelização/genética , Pele/lesões , Animais , Transição Epitelial-Mesenquimal/genética , Humanos , Miofibroblastos/fisiologiaRESUMO
Understanding molecular mechanisms for regeneration of hair follicles provides new opportunities for developing treatments for hair loss and other skin disorders. Here we show that fibroblast growth factor 9 (Fgf9), initially secreted by γδ T cells, modulates hair follicle regeneration after wounding the skin of adult mice. Reducing Fgf9 expression decreases this wound-induced hair neogenesis (WIHN). Conversely, overexpression of Fgf9 results in a two- to threefold increase in the number of neogenic hair follicles. We found that Fgf9 from γδ T cells triggers Wnt expression and subsequent Wnt activation in wound fibroblasts. Through a unique feedback mechanism, activated fibroblasts then express Fgf9, thus amplifying Wnt activity throughout the wound dermis during a crucial phase of skin regeneration. Notably, humans lack a robust population of resident dermal γδ T cells, potentially explaining their inability to regenerate hair after wounding. These findings highlight the essential relationship between the immune system and tissue regeneration. The importance of Fgf9 in hair follicle regeneration suggests that it could be used therapeutically in humans.
Assuntos
Fator 9 de Crescimento de Fibroblastos/metabolismo , Fator 9 de Crescimento de Fibroblastos/farmacologia , Folículo Piloso/efeitos dos fármacos , Linfócitos T/metabolismo , Cicatrização/efeitos dos fármacos , Animais , Células Cultivadas , Derme/citologia , Derme/imunologia , Derme/metabolismo , Derme/fisiologia , Retroalimentação Fisiológica/efeitos dos fármacos , Folículo Piloso/crescimento & desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Linfócitos T/fisiologia , Proteínas Wnt/metabolismo , Cicatrização/fisiologiaRESUMO
Activation of epithelial stem cells and efficient recruitment of their proliferating progeny plays a critical role in cutaneous wound healing. The reepithelialized wound epidermis has a mosaic composition consisting of progeny that can be traced back both to epidermal and several types of hair follicle stem cells. The contribution of hair follicle stem cells to wound epidermis is particularly intriguing as it involves lineage identity change from follicular to epidermal. Studies from our laboratory show that hair follicle-fated bulge stem cells commit only transient amplifying epidermal progeny that participate in the initial wound re-epithelialization, but eventually are outcompeted by other epidermal clones and largely disappear after a few months. Conversely, recently described stem cell populations residing in the isthmus portion of hair follicle contribute long-lasting progeny toward wound epidermis and, arguably, give rise to new interfollicular epidermal stem cells. The role of epithelial stem cells during wound healing is not limited to regenerating stratified epidermis. By studying regenerative response in large cutaneous wounds, our laboratory uncovered that epithelial cells in the center of the wound can acquire greater morphogenetic plasticity and, together with the underlying wound dermis, can engage in an embryonic-like process of hair follicle neogenesis. Future studies should uncover the cellular and signaling basis of this remarkable adult wound regeneration phenomenon.
Assuntos
Células Epidérmicas , Células Epiteliais/citologia , Folículo Piloso/citologia , Reepitelização/fisiologia , Regeneração/fisiologia , Glândulas Sebáceas/citologia , Células-Tronco/citologia , Adulto , Medula Óssea/fisiologia , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Cicatriz/prevenção & controle , Epiderme/lesões , Epiderme/fisiologia , Células Epiteliais/fisiologia , Folículo Piloso/fisiologia , Humanos , Glândulas Sebáceas/fisiologia , Células-Tronco/fisiologia , Ferimentos Penetrantes/patologia , Ferimentos Penetrantes/reabilitaçãoAssuntos
Autoanticorpos/biossíntese , Subpopulações de Linfócitos B/imunologia , Tolerância Imunológica , Receptores de Antígenos de Linfócitos B/biossíntese , Animais , Autoanticorpos/genética , Subpopulações de Linfócitos B/metabolismo , Linhagem Celular , Hibridomas , Tolerância Imunológica/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/biossíntese , Cadeias kappa de Imunoglobulina/genética , Linfopenia/genética , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos MRL lpr , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/genéticaRESUMO
Nedd4 and Itch are E3 ubiquitin ligases that ubiquitinate similar targets in vitro and thus are thought to function similarly. T cells lacking Itch show spontaneous activation and T helper type 2 polarization. To test whether loss of Nedd4 affects T cells in the same way, we generated Nedd4(+/+) and Nedd4(-/-) fetal liver chimeras. Nedd4(-/-) T cells developed normally but proliferated less, produced less interleukin 2 and provided inadequate help to B cells. Nedd4(-/-) T cells contained more of the E3 ubiquitin ligase Cbl-b, and Nedd4 was required for polyubiquitination of Cbl-b induced by CD28 costimulation. Our data demonstrate that Nedd4 promotes the conversion of naive T cells into activated T cells. We propose that Nedd4 and Itch ubiquitinate distinct target proteins in vivo.
Assuntos
Ativação Linfocitária/imunologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação/imunologia , Animais , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases Nedd4 , Proteínas Proto-Oncogênicas c-cbl/imunologia , Linfócitos T/metabolismo , Quimeras de Transplante , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Engagement of the T cell receptor (TCR) with its cognate peptide/MHC initiates a cascade of signaling events that results in T cell activation. Limiting the extent and duration of TCR signaling ensures a tightly constrained response, protecting cells from the deleterious impact of chronic activation. In order to limit the duration of activation, T cells must adjust levels of key signaling proteins. This can be accomplished by altering protein synthesis or by changing the rate of protein degradation. Ubiquitination is a process of 'tagging' a protein with ubiquitin and is one means of initiating protein degradation. This process is activated when an E3 ubiquitin ligase mediates the transfer of ubiquitin to a target protein. Accordingly, E3 ubiquitin ligases have recently emerged as key regulators of immune cell function. This review will explore how a small group of E3 ubiquitin ligases regulate T cell responses and thus direct adaptive immunity.
Assuntos
Tolerância Imunológica/imunologia , Imunidade/imunologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Ativação Linfocitária/imunologia , Modelos Imunológicos , Ubiquitina-Proteína Ligases Nedd4 , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
BACKGROUND: In previous work, the cyclooxygenase-2 inhibitor NS-398 inhibited interleukin (IL)-1beta-stimulated prostaglandin E(2) (PGE(2)) production almost completely while partially inhibiting IL-6 production in aggressive periodontitis (AgP) human gingival fibroblasts. PGE(2) and the transcription factor nuclear factor-kappa B (NF-kappaB) regulate IL-1beta-stimulated IL-6 production. Cytoplasmic NF-kappaB is bound to inhibitors (IkappaB proteins). IL-1beta initiates a cascade resulting in phosphorylation and degradation of IkappaB, allowing nuclear translocation of NF-kappaB and target gene activation. The purpose of this study was to determine whether NS-398 inhibited phosphorylation of IkappaB and NF-kappaB activation. METHODS: AgP fibroblasts (1 to 2 x 10(6)) were exposed to IL-1beta (1 x 10(11)M) with or without NS-398 (10 nM) in serum-free medium. The NF-kappaB subunit p65 and phospho-IkappaBalpha were measured in whole cell, cytoplasmic, or nuclear extracts, using colorimetric assays. Enzyme-linked immunosorbent assays were used to measure PGE(2) and IL-6 production by 2.5 x 10(4) cells after exposure to IL-1beta with or without NS-398 in serum-free medium. RESULTS: Consistent with previous results, NS-398 reduced IL-1beta-stimulated PGE(2) by approximately 98% (P <0.001) and IL-6 by approximately 65% (P <0.001). IL-1beta increased nuclear and cytoplasmic p65 ( approximately 8-fold [P <0.001] and approximately 2.5-fold [P <0.03], respectively) over control levels. NS-398 reduced IL-1beta-stimulated nuclear and cytoplasmic p65 to control levels. IL-1beta increased phospho-IkappaBalpha in whole cell extracts by a maximum of approximately 9.5 times (P = 0.0001), and this was inhibited significantly by NS-398 (P Assuntos
Inibidores de Ciclo-Oxigenase 2/farmacologia
, Gengiva/efeitos dos fármacos
, NF-kappa B/antagonistas & inibidores
, Nitrobenzenos/farmacologia
, Periodontite/metabolismo
, Sulfonamidas/farmacologia
, Adolescente
, Linhagem Celular
, Núcleo Celular/metabolismo
, Dinoprostona/biossíntese
, Feminino
, Fibroblastos/efeitos dos fármacos
, Fibroblastos/metabolismo
, Gengiva/citologia
, Gengiva/metabolismo
, Humanos
, Proteínas I-kappa B/metabolismo
, Interleucina-1beta/farmacologia
, Interleucina-6/biossíntese
, Fosforilação/efeitos dos fármacos
, Fator de Transcrição RelA/antagonistas & inibidores
, Ativação Transcricional/efeitos dos fármacos
RESUMO
tGolgin-1 (golgin-245, trans golgi p230) and golgin-97 are members of a family of peripheral membrane proteins of unknown function that localize to the trans Golgi network (TGN) through a conserved C-terminal GRIP domain. We have probed for GRIP protein function by assessing the consequences of overexpressing isolated GRIP domains. By semi-quantitative immunofluorescence microscopy we found that high level expression of epitope-tagged, GRIP domain-containing fragments of tGolgin-1 or golgin-97 specifically altered the characteristic pericentriolar distribution of TGN integral membrane and coat components. Concomitantly, vesicular transport from the TGN to the plasma membrane and furin-dependent cleavage of substrate proteins in the TGN were inhibited. Mutagenesis of a conserved tyrosine in the tGolgin-1 GRIP domain abolished these effects. GRIP domain overexpression had little effect on the distribution of most Golgi stack resident proteins and no effect on markers of other organelles. Electron microscopy analyses of GRIP domain-overexpressing cells revealed distended perinuclear vacuoles and a proliferation of multivesicular late endosomes to which the TGN resident protein TGN46 was largely mislocalized. These studies, the first to address the function of GRIP domain-containing proteins in higher eukaryotes, suggest that some or all of these proteins and/or their ligands function in maintaining the integrity of the TGN by regulating resident protein localization.