RESUMO
Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.
Assuntos
Proteínas da Matriz do Complexo de Golgi/genética , Proteoma/genética , Proteômica , Estresse Fisiológico/genética , Matriz Extracelular/genética , Complexo de Golgi/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Transdução de Sinais/genéticaRESUMO
OBJECTIVES: Activation of fibroblasts is a hallmark of fibrotic processes. Besides cytokines and growth factors, fibroblasts are regulated by the extracellular matrix environment through receptors such as integrins, which transduce biochemical and mechanical signals enabling cells to mount appropriate responses according to biological demands. The aim of this work was to investigate the in vivo role of collagen-fibroblast interactions for regulating fibroblast functions and fibrosis. METHODS: Triple knockout (tKO) mice with a combined ablation of integrins α1ß1, α2ß1 and α11ß1 were created to address the significance of integrin-mediated cell-collagen communication. Properties of primary dermal fibroblasts lacking collagen-binding integrins were delineated in vitro. Response of the tKO mice skin to bleomycin induced fibrotic challenge was assessed. RESULTS: Triple integrin-deficient mice develop normally, are transiently smaller and reveal mild alterations in mechanoresilience of the skin. Fibroblasts from these mice in culture show defects in cytoskeletal architecture, traction stress generation, matrix production and organisation. Ablation of the three integrins leads to increased levels of discoidin domain receptor 2, an alternative receptor recognising collagens in vivo and in vitro. However, this overexpression fails to compensate adhesion and spreading defects on collagen substrates in vitro. Mice lacking collagen-binding integrins show a severely attenuated fibrotic response with impaired mechanotransduction, reduced collagen production and matrix organisation. CONCLUSIONS: The data provide evidence for a crucial role of collagen-binding integrins in fibroblast force generation and differentiation in vitro and for matrix deposition and tissue remodelling in vivo. Targeting fibroblast-collagen interactions might represent a promising therapeutic approach to regulate connective tissue deposition in fibrotic diseases.
RESUMO
Our skin is continuously exposed to mechanical challenge, including shear, stretch, and compression. The extracellular matrix of the dermis is perfectly suited to resist these challenges and maintain integrity of normal skin even upon large strains. Fibroblasts are the key cells that interpret mechanical and chemical cues in their environment to turnover matrix and maintain homeostasis in the skin of healthy adults. Upon tissue injury, fibroblasts and an exclusive selection of other cells become activated into myofibroblasts with the task to restore skin integrity by forming structurally imperfect but mechanically stable scar tissue. Failure of myofibroblasts to terminate their actions after successful repair or upon chronic inflammation results in dysregulated myofibroblast activities which can lead to hypertrophic scarring and/or skin fibrosis. After providing an overview on the major fibrillar matrix components in normal skin, we will interrogate the various origins of fibroblasts and myofibroblasts in the skin. We then examine the role of the matrix as signaling hub and how fibroblasts respond to mechanical matrix cues to restore order in the confusing environment of a healing wound.
Assuntos
Cicatriz Hipertrófica , Cicatrização , Diferenciação Celular , Cicatriz Hipertrófica/patologia , Matriz Extracelular/patologia , Fibroblastos/patologia , Humanos , Miofibroblastos/patologia , Pele/patologia , Fator de Crescimento Transformador beta1RESUMO
Myeloid cells can be beneficial as well as harmful in tissue regenerative responses. The molecular mechanisms by which myeloid cells control this critical decision of the immune system are not well understood. Using two different models of physiological acute or pathological chronic skin damage, in this study we identified myeloid cell-restricted STAT3 signaling as important and an injury context-dependent regulator of skin fibrosis. Targeted disruption of STAT3 signaling in myeloid cells significantly accelerated development of pathological skin fibrosis in a model of chronic bleomycin-induced tissue injury, whereas the impact on wound closure dynamics and quality of healing after acute excision skin injury was minor. Chronic bleomycin-mediated tissue damage in control mice provoked an antifibrotic gene signature in macrophages that was characterized by upregulated expression of IL-10, SOCS3, and decorin. In contrast, in STAT3-deficient macrophages this antifibrotic repair program was abolished whereas TGF-ß1 expression was increased. Notably, TGF-ß1 synthesis in cultured control bone marrow-derived macrophages (BMDMs) was suppressed after IL-10 exposure, and this suppressive effect was alleviated by STAT3 deficiency. Accordingly, coculture of IL-10-stimulated control BMDMs with fibroblasts suppressed expression of the TGF-ß1 downstream target connective tissue growth factor in fibroblasts, whereas this suppressive effect was lost by STAT3 deficiency in BMDMs. Our findings highlight a previously unrecognized protective role of myeloid cell-specific STAT3 signaling in immune cell-mediated skin fibrosis, and its regulatory pathway could be a potential target for therapy.
Assuntos
Macrófagos/imunologia , Células Mieloides/fisiologia , Fator de Transcrição STAT3/metabolismo , Dermatopatias/imunologia , Pele/patologia , Doença Aguda , Animais , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Fibrose , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regeneração , Fator de Transcrição STAT3/genética , Transdução de Sinais , Dermatopatias/induzido quimicamente , Transcriptoma , Fator de Crescimento Transformador beta/metabolismo , CicatrizaçãoRESUMO
Integrins are a family of transmembrane proteins, involved in substrate recognition and cell adhesion in cross-talk with the extra cellular matrix. In this study, we investigated the influence of integrin α2ß1 on tendons, another collagen type I-rich tissue of the musculoskeletal system. Morphological, as well as functional, parameters were analyzed in vivo and in vitro, comparing wild-type against integrin α2ß1 deficiency. Tenocytes lacking integrin α2ß1 produced more collagen in vitro, which is similar to the situation in osseous tissue. Fibril morphology and biomechanical strength proved to be altered, as integrin α2ß1 deficiency led to significantly smaller fibrils as well as changes in dynamic E-modulus in vivo. This discrepancy can be explained by a higher collagen turnover: integrin α2ß1-deficient cells produced more matrix, and tendons contained more residual C-terminal fragments of type I collagen, as well as an increased matrix metalloproteinase-2 activity. A greatly decreased percentage of non-collagenous proteins may be the cause of changes in fibril diameter regulation and increased the proteolytic degradation of collagen in the integrin-deficient tendons. The results reveal a significant impact of integrin α2ß1 on collagen modifications in tendons. Its role in tendon pathologies, like chronic degradation, will be the subject of future investigations.
Assuntos
Colágeno/metabolismo , Integrina alfa2beta1/deficiência , Metaloproteinase 2 da Matriz/metabolismo , Tendões/metabolismo , Tenócitos/metabolismo , Animais , Fenômenos Biomecânicos , Células Cultivadas , Colágeno/ultraestrutura , Feminino , Fibroblastos/metabolismo , Gelatinases/metabolismo , Integrina alfa2beta1/genética , Integrina alfa2beta1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteína-Lisina 6-Oxidase/metabolismo , Tendões/citologia , Tendões/enzimologia , Tendões/ultraestruturaRESUMO
Collagen receptors GPVI (also known as GP6) and integrin α2ß1 are highly expressed on blood platelets and megakaryocytes, their immediate precursors. After vessel injury, subendothelial collagen becomes exposed and induces platelet activation to prevent blood loss. Collagen types I and IV are thought to have opposite effects on platelet biogenesis, directing proplatelet formation (PPF) towards the blood vessels to prevent premature release within the marrow cavity. We used megakaryocytes lacking collagen receptors or treated megakaryocytes with blocking antibodies, and could demonstrate that collagen-I-mediated inhibition of PPF is specifically controlled by GPVI. Other collagen types competed for binding and diminished the inhibitory signal, which was entirely dependent on receptor-proximal Src family kinases, whereas Syk and LAT were dispensable. Adhesion assays indicate that megakaryocyte binding to collagens is mediated by α2ß1, and that collagen IV at the vascular niche might displace collagen I from megakaryocytes and thus contribute to prevention of premature platelet release into the marrow cavity and thereby directionally promote PPF at the vasculature.
Assuntos
Plaquetas/metabolismo , Colágeno Tipo I/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Animais , Medula Óssea/metabolismo , Adesão Celular , Diferenciação Celular , Matriz Extracelular/metabolismo , Feminino , Fêmur/metabolismo , Imuno-Histoquímica , Masculino , Megacariócitos/citologia , Camundongos Endogâmicos C57BL , Fenótipo , Receptores de Colágeno/metabolismoRESUMO
Cartilage oligomeric matrix protein (COMP) is an abundant component in the extracellular matrix (ECM) of load-bearing tissues such as tendons and cartilage. It provides adaptor functions by bridging different ECM structures. We have previously shown that COMP is also a constitutive component of healthy human skin and is strongly induced in fibrosis. It binds directly and with high affinity to collagen I and to collagen XII that decorates the surface of collagen I fibrils. We demonstrate here that lack of COMP-collagen interaction in the extracellular space leads to changes in collagen fibril morphology and density, resulting in altered skin biomechanical properties. Surprisingly, COMP also fulfills an important intracellular function in assisting efficient secretion of collagens, which were retained in the endoplasmic reticulum of COMP-null fibroblasts. Accordingly, COMP-null mice showed severely attenuated fibrotic responses in skin. Collagen secretion was fully restored by introducing wild-type COMP. Hence, our work unravels a new, non-structural and intracellular function of the ECM protein COMP in controlling collagen secretion.
Assuntos
Proteína de Matriz Oligomérica de Cartilagem/genética , Colágenos Fibrilares/metabolismo , Pele/metabolismo , Animais , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático , Feminino , Fibroblastos/metabolismo , Fibrose , Camundongos Endogâmicos C57BL , Pele/patologiaRESUMO
RATIONALE: Analyzing the molecular heterogeneity of different forms of organ fibrosis may reveal common and specific factors and thus identify potential future therapeutic targets. OBJECTIVES: We sought to use proteome-wide profiling of human tissue fibrosis to (1) identify common and specific signatures across end-stage interstitial lung disease (ILD) cases, (2) characterize ILD subgroups in an unbiased fashion, and (3) identify common and specific features of lung and skin fibrosis. METHODS: We collected samples of ILD tissue (n = 45) and healthy donor control samples (n = 10), as well as fibrotic skin lesions from localized scleroderma and uninvolved skin (n = 6). Samples were profiled by quantitative label-free mass spectrometry, Western blotting, or confocal imaging. MEASUREMENTS AND MAIN RESULTS: We determined the abundance of more than 7,900 proteins and stratified these proteins according to their detergent solubility profiles. Common protein regulations across all ILD cases, as well as distinct ILD subsets, were observed. Proteomic comparison of lung and skin fibrosis identified a common upregulation of marginal zone B- and B1-cell-specific protein (MZB1), the expression of which identified MZB1+/CD38+/CD138+/CD27+/CD45-/CD20- plasma B cells in fibrotic lung and skin tissue. MZB1 levels correlated positively with tissue IgG and negatively with diffusing capacity of the lung for carbon monoxide. CONCLUSIONS: Despite the presumably high molecular and cellular heterogeneity of ILD, common protein regulations are observed, even across organ boundaries. The surprisingly high prevalence of MZB1-positive plasma B cells in tissue fibrosis warrants future investigations regarding the causative role of antibody-mediated autoimmunity in idiopathic cases of organ fibrosis, such as idiopathic pulmonary fibrosis.
Assuntos
Citocinas/análise , Fibrose/patologia , Doenças Pulmonares Intersticiais/patologia , Plasma/química , Proteoma/análise , Dermatopatias/patologia , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , ProteômicaRESUMO
Integrin-linked kinase (ILK) is an adaptor protein required to establish and maintain the connection between integrins and the actin cytoskeleton. This linkage is essential for generating force between the extracellular matrix (ECM) and the cell during migration and matrix remodelling. The mechanisms by which ILK stability and turnover are regulated are unknown. Here we report that the E3 ligase CHIP-heat shock protein 90 (Hsp90) axis regulates ILK turnover in fibroblasts. The chaperone Hsp90 stabilizes ILK and facilitates the interaction of ILK with α-parvin. When Hsp90 activity is blocked, ILK is ubiquitinated by CHIP and degraded by the proteasome, resulting in impaired fibroblast migration and a dramatic reduction in the fibrotic response to bleomycin in mice. Together, our results uncover how Hsp90 regulates ILK stability and identify a potential therapeutic strategy to alleviate fibrotic diseases.
Assuntos
Movimento Celular/fisiologia , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Actinas/metabolismo , Animais , Bleomicina/toxicidade , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/induzido quimicamente , Fibrose/metabolismo , Adesões Focais/fisiologia , Proteínas de Choque Térmico HSP90/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/genética , Pele/efeitos dos fármacos , Pele/patologia , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Integrins are transmembrane receptors composed of one α subunit and one ß subunit and are involved in cellular growth, differentiation, and apoptosis. The collagen-binding integrins α1ß1 and α2ß1 have been shown to regulate wound and tumor vascularization by different mechanisms. In this study, we assessed wound and tumor vascularization in mice with genetic ablation of both integrin subunits α1 and α2, which resulted in loss of integrins α1ß1 and α2ß1. Wound angiogenesis was investigated in excisional wounds that were inflicted on the back skin of control and mice lacking integrin α1ß1 and α2ß1. Mutant mice displayed reduced wound angiogenesis, which correlated with decreased macrophage numbers at 3 and 7 days after injury, and showed significantly attenuated vascularization of sponge implants. Angiogenesis induced by tumors arising from intradermal injection of B16 F1 melanoma cells was also reduced in comparison to controls 7 days after injection. This reduction in angiogenesis correlated with increased levels and activity of circulating matrix metalloproteinase 9 and elevated angiostatin levels in plasma of mutant mice, which reduced endothelial cell proliferation. Ex vivo mutant aortic ring explants developed significantly fewer and thinner aortic sprouts with fewer branch points than controls because of impaired endothelial cell proliferation. In conclusion, the loss of integrins α1ß1 and α2ß1 in mice results in reduced wound and tumor angiogenesis by cell-autonomous and extrinsic mechanisms.
Assuntos
Integrina alfa1beta1/metabolismo , Integrina alfa2beta1/metabolismo , Neoplasias/irrigação sanguínea , Cicatrização/fisiologia , Ferimentos e Lesões/patologia , Animais , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Integrina alfa1beta1/genética , Integrina alfa2beta1/genética , Melanoma/irrigação sanguínea , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/etiologia , Neoplasias/patologia , Neovascularização Patológica , Pele/irrigação sanguínea , Pele/lesões , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/irrigação sanguínea , Ferimentos e Lesões/etiologiaRESUMO
Cell-matrix adhesions provide structural stability to the tissue and regulate intracellular signalling pathways that are important for cell fate decisions of the different cell populations within the skin. As a consequence of these central functions, genetic or functional impairment of various key protein components of matrix adhesions plays a causative role in the aetiology or pathophysiology in a large variety of skin disorders. Research towards understanding the molecular composition of these adhesions as well as the mechanisms by which they transmit signals is therefore of obvious importance. In this essay, we discuss the roles of integrin-linked kinase, a key component of cell-matrix adhesions, in the (patho)physiology of skin and in particular highlight its role in regulating mechanical tension and matrix remodelling both in the epidermis and in the dermis.
Assuntos
Regulação da Expressão Gênica , Integrinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fenômenos Fisiológicos da Pele , Actinas/metabolismo , Animais , Diferenciação Celular , Citoesqueleto/metabolismo , Derme/metabolismo , Epiderme/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Proteínas de Choque Térmico HSP90/metabolismo , Cabelo/fisiologia , Humanos , Camundongos , Transdução de Sinais , Pele/metabolismo , Células-Tronco/citologia , Estresse MecânicoRESUMO
OBJECTIVE: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS: Tropisetron suppressed collagen synthesis induced by transforming growth factor ß1 (TGFß1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFß1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.
Assuntos
Colágeno/biossíntese , Indóis/farmacologia , Receptores Nicotínicos/metabolismo , Escleroderma Sistêmico/tratamento farmacológico , Células 3T3 , Adulto , Idoso , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Derme/efeitos dos fármacos , Derme/metabolismo , Derme/patologia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Fibrose/patologia , Humanos , Camundongos , Pessoa de Meia-Idade , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Tropizetrona , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Transforming growth factor ß (TGF-ß) signaling is a core pathway of fibrosis, but the molecular regulation of the activation of latent TGF-ß remains incompletely understood. Here, we demonstrate a crucial role of WNT5A/JNK/ROCK signaling that rapidly coordinates the activation of latent TGF-ß in fibrotic diseases. WNT5A was identified as a predominant noncanonical WNT ligand in fibrotic diseases such as systemic sclerosis, sclerodermatous chronic graft-versus-host disease, and idiopathic pulmonary fibrosis, stimulating fibroblast-to-myofibroblast transition and tissue fibrosis by activation of latent TGF-ß. The activation of latent TGF-ß requires rapid JNK- and ROCK-dependent cytoskeletal rearrangements and integrin αV (ITGAV). Conditional ablation of WNT5A or its downstream targets prevented activation of latent TGF-ß, rebalanced TGF-ß signaling, and ameliorated experimental fibrosis. We thus uncovered what we believe to be a novel mechanism for the aberrant activation of latent TGF-ß in fibrotic diseases and provided evidence for targeting WNT5A/JNK/ROCK signaling in fibrotic diseases as a new therapeutic approach.
Assuntos
Fibroblastos , Fibrose , Fator de Crescimento Transformador beta , Proteína Wnt-5a , Quinases Associadas a rho , Proteína Wnt-5a/metabolismo , Proteína Wnt-5a/genética , Animais , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/genética , Camundongos , Humanos , Fibroblastos/metabolismo , Fibroblastos/patologia , Quinases Associadas a rho/metabolismo , Quinases Associadas a rho/genética , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/genética , Camundongos Knockout , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Sistema de Sinalização das MAP Quinases , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Transdução de Sinais , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/genéticaRESUMO
Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.
Assuntos
Cicatriz , Colágeno , Fibroblastos , Cicatrização , Peixe-Zebra , Humanos , Animais , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Colágeno/metabolismo , Cicatrização/efeitos dos fármacos , Cicatriz/metabolismo , Cicatriz/patologia , Cicatriz/tratamento farmacológico , Pele/metabolismo , Pele/patologia , Pele/efeitos dos fármacos , Fibrose , Peptídeos/farmacologia , Peptídeos/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/efeitos dos fármacosRESUMO
The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.
Assuntos
Colágeno Tipo XII/metabolismo , Colágeno/metabolismo , Derme/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Animais , Membrana Basal/metabolismo , Proteína de Matriz Oligomérica de Cartilagem , Pré-Escolar , Colágeno/química , Colágeno/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo XII/química , Colágeno Tipo XII/genética , Derme/ultraestrutura , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Fibroblastos/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Células HEK293 , Humanos , Lactente , Queratinócitos/metabolismo , Proteínas Matrilinas , Camundongos , Microscopia Imunoeletrônica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Mice with a combined deficiency in the α2ß1 and α11ß1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2ß1 or α11ß1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.
Assuntos
Nanismo/genética , Nanismo/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Integrina alfa2beta1/genética , Integrinas/genética , Receptores de Colágeno/genética , Animais , Densidade Óssea/genética , Osso e Ossos/citologia , Osso e Ossos/fisiologia , Colágeno/metabolismo , Matriz Extracelular/fisiologia , Feminino , Hormônio do Crescimento/metabolismo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Homeostase/fisiologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Osteoblastos/fisiologia , Transdução de Sinais/fisiologiaRESUMO
CD4 T cells play a key role in immunological memory. We have demonstrated that professional memory CD4 T cells reside and rest in the bone marrow (BM). However, the molecular mechanisms of their establishment in the BM and their maintenance remain unclear. We here show that memory CD4 T cells express high levels of CD49b and that CD49b-deficient or -blocked memory CD4 T-cell precursors fail to migrate from blood into the marrow of the bone, and they especially fail to transmigrate through sinusoidal endothelial cells of the BM. In the marrow, memory CD4 T cells and the precursors contact stromal cells expressing collagen II that are specific ligands for CD49b. Interestingly, memory CD4 T cells on day 117 of an immune response also dock on IL-7(+)/collagen XI(+) stromal cells, whereas memory precursors on day 12 do not. These results indicate that the collagen receptor CD49b is required for the migration of memory CD4 T-cell precursors into their survival niches of the bone marrow.
Assuntos
Medula Óssea/metabolismo , Integrina alfa2/metabolismo , Células Precursoras de Linfócitos T/imunologia , Células Estromais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Medula Óssea/patologia , Antígenos CD4/metabolismo , Comunicação Celular , Diferenciação Celular/genética , Movimento Celular/genética , Células Cultivadas , Colágeno Tipo II/metabolismo , Colágeno Tipo XI/metabolismo , Memória Imunológica/genética , Integrina alfa2/genética , Interleucina-7/genética , Interleucina-7/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Migração Transendotelial e Transepitelial/genéticaRESUMO
OBJECTIVE: Integrin α2ß1 functions as a major receptor for type I collagen on different cell types, including fibroblasts and inflammatory cells. Although in vitro data suggest a role for α2ß1 integrin in regulating both cell attachment and expression of matrix-degrading enzymes such as matrix metalloproteinases (MMPs), mice that lack the α2 integrin subunit (Itga2(-/-) mice) develop normally and are fertile. We undertook this study to investigate the effect of Itga2 deficiency in 2 different mouse models of destructive arthritis: the antigen-induced arthritis (AIA) mouse model and the human tumor necrosis factor α (TNFα)-transgenic mouse model. METHODS: AIA was induced in the knee joints of Itga2(-/-) mice and wild-type controls. Human TNF-transgenic mice were crossed with Itga2(-/-) mice and were assessed clinically and histopathologically for signs of arthritis, inflammation, bone erosion, and cartilage damage. MMP expression, proliferation, fibroblast attachment, and ERK activation were determined. RESULTS: Under arthritic conditions, Itga2 deficiency led to decreased severity of joint pathology. Specifically, Itga2(-/-) mice showed less severe clinical symptoms and dramatically reduced pannus formation and cartilage erosion. Mice lacking α2ß1 integrin exhibited reduced MMP-3 expression, both in their sera and in fibroblast-like synoviocytes (FLS), due to impaired ERK activation. Further, both the proliferation and attachment of FLS to cartilage were partially dependent on α2ß1 integrin in vitro and in vivo. CONCLUSION: Our findings suggest that α2ß1 integrin contributes significantly to inflammatory cartilage destruction by promoting fibroblast proliferation and attachment and MMP expression.
Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Cartilagem Articular/patologia , Integrina alfa2beta1/deficiência , Sinovite/metabolismo , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Cartilagem Articular/metabolismo , Adesão Celular , Proliferação de Células , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Endogamia , Integrina alfa2beta1/genética , Masculino , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Joelho de Quadrúpedes/patologia , Sinovite/genética , Sinovite/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Mesangial cells and podocytes express integrins α1ß1 and α2ß1, which are the two major collagen receptors that regulate multiple cellular functions, including extracellular matrix homeostasis. Integrin α1ß1 protects from glomerular injury by negatively regulating collagen production, but the role of integrin α2ß1 in renal injury is unclear. Here, we subjected wild-type and integrin α2-null mice to injury with adriamycin or partial renal ablation. In both of these models, integrin α2-null mice developed significantly less proteinuria and glomerulosclerosis. In addition, selective pharmacological inhibition of integrin α2ß1 significantly reduced adriamycin-induced proteinuria, glomerular injury, and collagen deposition in wild-type mice. This inhibitor significantly reduced collagen synthesis in wild-type, but not integrin α2-null, mesangial cells in vitro, demonstrating that its effects are integrin α2ß1-dependent. Taken together, these results indicate that integrin α2ß1 contributes to glomerular injury by positively regulating collagen synthesis and suggest that its inhibition may be a promising strategy to reduce glomerular injury and proteinuria.
Assuntos
Injúria Renal Aguda/patologia , Doxorrubicina/farmacologia , Integrina alfa2beta1/metabolismo , Glomérulos Renais/lesões , Injúria Renal Aguda/metabolismo , Albuminúria/fisiopatologia , Análise de Variância , Animais , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Integrina alfa2beta1/efeitos dos fármacos , Testes de Função Renal , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Distribuição Aleatória , Receptores de Colágeno/metabolismoRESUMO
Cellular receptors for collagens belong to the family of ß(1) integrins. In the epidermis, integrin α(2)ß(1) is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α(2)ß(1) receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α(2)ß(1) in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α(2)ß(1).