RESUMEN
How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.
Asunto(s)
Elementos de Facilitación Genéticos , Regiones Promotoras Genéticas , Factores de Transcripción SOXB1/genética , Animales , Cromatina , Regulación del Desarrollo de la Expresión Génica , Ratones , Factores de Transcripción/metabolismoRESUMEN
Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1ß binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1ß showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.
Asunto(s)
Señalización del Calcio , Fibroblastos , Encía , Canales Catiónicos TRPV , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genética , Animales , Humanos , Ratones , Fibroblastos/metabolismo , Encía/metabolismo , Encía/citología , Calcio/metabolismo , Sistema de Señalización de MAP Quinasas , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Interleucina-1/metabolismo , Interleucina-1/farmacología , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologíaRESUMEN
Transient Receptor Potential Vanilloid-type 4 (TRPV4) is a mechanosensitive, Ca2+ -permeable plasma membrane channel that associates with focal adhesions, influences collagen remodeling, and is associated with fibrotic processes through undefined mechanisms. While TRPV4 is known to be activated by mechanical forces transmitted through collagen adhesion receptors containing the ß1 integrin, it is not understood whether TRPV4 affects matrix remodeling by altering ß1 integrin expression and function. We tested the hypothesis that TRPV4 regulates collagen remodeling through its impact on the ß1 integrin in cell-matrix adhesions. In cultured fibroblasts derived from mouse gingival connective tissues, which exhibit very rapid collagen turnover, we found that higher TRPV4 expression is associated with reduced ß1 integrin abundance and adhesion to collagen, reduced focal adhesion size and total adhesion area, and reduced alignment and compaction of extracellular fibrillar collagen. The reduction of ß1 integrin expression mediated by TRPV4 is associated with the upregulation of miRNAs that target ß1 integrin mRNA. Our data suggest a novel mechanism by which TRPV4 modulates collagen remodeling through post-transcriptional downregulation of ß1 integrin expression and function.
Asunto(s)
Integrina beta1 , Canales Catiónicos TRPV , Animales , Ratones , Uniones Célula-Matriz , Colágeno , Adhesiones FocalesRESUMEN
Disturbed remodeling of the extracellular matrix (ECM) is frequently observed in several high-prevalence pathologies that include fibrotic diseases of organs such as the heart, lung, periodontium, liver, and the stiffening of the ECM surrounding invasive cancers. In many of these lesions, matrix remodeling mediated by fibroblasts is dysregulated, in part by alterations to the regulatory and effector systems that synthesize and degrade collagen, and by alterations to the functions of the integrin-based adhesions that normally mediate mechanical remodeling of collagen fibrils. Cell-matrix adhesions containing collagen-binding integrins are enriched with regulatory and effector systems that initiate localized remodeling of pericellular collagen fibrils to maintain ECM homeostasis. A large cadre of regulatory molecules is enriched in cell-matrix adhesions that affect ECM remodeling through synthesis, degradation, and contraction of collagen fibrils. One of these regulatory molecules is Transient Receptor Potential Vanilloid-type 4 (TRPV4), a mechanically sensitive, Ca2+-permeable plasma membrane channel that regulates collagen remodeling. The gating of Ca2+ across the plasma membrane by TRPV4 and the consequent generation of intracellular Ca2+ signals affect several processes that determine the structural and mechanical properties of collagen-rich ECM. These processes include the synthesis of new collagen fibrils, tractional remodeling by contractile forces, and collagenolysis. While the specific mechanisms by which TRPV4 contributes to matrix remodeling are not well-defined, it is known that TRPV4 is activated by mechanical forces transmitted through collagen adhesion receptors. Here, we consider how TRPV4 expression and function contribute to physiological and pathological collagen remodeling and are associated with collagen adhesions. Over the long-term, an improved understanding of how TRPV4 regulates collagen remodeling could pave the way for new approaches to manage fibrotic lesions.
Asunto(s)
Matriz Extracelular , Canales Catiónicos TRPV , Membrana Celular , Uniones Célula-Matriz , Colágeno , Integrinas , Canales Catiónicos TRPV/genética , HumanosRESUMEN
Tissue fibrosis manifests as excessive deposition of compacted, highly aligned collagen fibrils, which interfere with organ structure and function. Cells in collagen-rich lesions often exhibit marked overexpression of discoidin domain receptor 1 (DDR1), which is linked to increased collagen compaction through the association of DDR1 with the Ca2+ -dependent nonmuscle myosin IIA (NMIIA). We examined the functional relationship between DDR1 and the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+ -permeable ion channel that is implicated in collagen compaction. Fibroblasts expressing high levels of DDR1 were used to model cells in lesions with collagen compaction. In these cells, the expression of the ß1 integrin was deleted to simplify studies of DDR1 function. Compared with DDR1 wild-type cells, high DDR1 expression was associated with increased Ca2+ influx through TRPV4, enrichment of TRPV4 in collagen adhesions, and enhanced contractile activity mediated by NMIIA. At cell adhesion sites to collagen, DDR1 associated with TRPV4, which enhanced DDR1-mediated collagen alignment and compaction. We conclude that DDR1 regulates Ca2+ influx through the TRPV4 channel to promote critical, DDR1-mediated processes that are important in lesions with collagen compaction and alignment.
Asunto(s)
Calcio , Receptor con Dominio Discoidina 1 , Calcio/metabolismo , Calcio de la Dieta , Uniones Célula-Matriz/metabolismo , Colágeno/metabolismo , Receptor con Dominio Discoidina 1/genética , Miosinas/metabolismo , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
In healthy connective tissues, mechanosensors trigger the generation of Ca2+ signals, which enable cells to maintain the structure of the fibrillar collagen matrix through actomyosin contractile forces. Transient receptor potential vanilloid type 4 (TRPV4) is a mechanosensitive Ca2+ -permeable channel that, when expressed in cell-matrix adhesions of the plasma membrane, regulates extracellular matrix (ECM) remodeling. In high prevalence disorders such as fibrosis and tumor metastasis, dysregulated matrix remodeling is associated with disruptions of Ca2+ homeostasis and TRPV4 function. Here, we consider that ECM polymers transmit cell-activating mechanical signals to TRPV4 in cell adhesions. When activated, TRPV4 regulates fibrillar collagen remodeling, thereby altering the mechanical properties of the ECM. In this review, we integrate functionally connected processes of matrix remodeling to highlight how TRPV4 in cell adhesions and matrix mechanics are reciprocally regulated through Ca2+ signaling.