RESUMEN
Dysregulated macrophage responses and changes in tissue metabolism are hallmarks of chronic inflammation in the skin. However, the metabolic cues that direct and support macrophage functions in the skin are poorly understood. Here, we show that during sterile skin inflammation, the epidermis and macrophages uniquely depend on glycolysis and the TCA cycle, respectively. This compartmentalisation is initiated by ROS-induced HIF-1α stabilization leading to enhanced glycolysis in the epidermis. The end-product of glycolysis, lactate, is then exported by epithelial cells and utilized by the dermal macrophages to induce their M2-like fates through NF-κB pathway activation. In addition, we show that psoriatic skin disorder is also driven by such lactate metabolite-mediated crosstalk between the epidermis and macrophages. Notably, small-molecule inhibitors of lactate transport in this setting attenuate sterile inflammation and psoriasis disease burden, and suppress M2-like fate acquisition in dermal macrophages. Our study identifies an essential role for the metabolite lactate in regulating macrophage responses to inflammation, which may be effectively targeted to treat inflammatory skin disorders such as psoriasis.
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Ácido Láctico , Psoriasis , Ratones , Animales , Ácido Láctico/metabolismo , Ácido Láctico/farmacología , Piel/metabolismo , Macrófagos/metabolismo , Inflamación/metabolismo , Psoriasis/metabolismoRESUMEN
Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics.
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Folículo Piloso , MicroARNs , Animales , Ratones , Actomiosina/metabolismo , Cabello , Folículo Piloso/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células Madre/metabolismoRESUMEN
Transfer RNA (tRNA)-derived fragments (tRFs) are an emerging class of conserved small non-coding RNAs that play important roles in post-transcriptional gene regulation. High-throughput sequencing of multiple biological samples have identified heterogeneous species of tRFs with distinct functionalities. These small RNAs have garnered a lot of scientific attention due to their ubiquitous expression and versatility in regulating various biological processes. In this review, we highlight our current understanding of tRF biogenesis and their regulatory functions. We summarize the diverse modes of biogenesis through which tRFs are generated and discuss the mechanism through which different tRF species regulate gene expression and the biological implications. Finally, we conceptualize research areas that require focus to strengthen our understanding of the biogenesis and function of tRFs.
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ARN Pequeño no Traducido/metabolismo , ARN de Transferencia/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/patología , Plantas/genética , Plantas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Pequeño no Traducido/genética , ARN de Transferencia/genética , Ribonucleoproteínas/metabolismoRESUMEN
Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.
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Diferenciación Celular , MicroARNs/metabolismo , ARN de Transferencia/metabolismo , Animales , Células Cultivadas , Células HCT116 , Humanos , Ratones , MicroARNs/genética , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Estabilidad del ARN , ARN de Transferencia/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.
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Citoplasma/metabolismo , Epidermis/metabolismo , Planarias/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Epitelio/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Homeostasis , Modelos Biológicos , Planarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regeneración , Cicatrización de HeridasRESUMEN
Kindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue α5ß1 integrin activation defects in kindlin-2-knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)-PINCH-parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity.
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Adhesiones Focales/metabolismo , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Isoformas de Proteínas , Transducción de SeñalRESUMEN
The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and ΔNp63. To investigate the role of ΔNp63 in epidermal morphogenesis we generated ΔNp63 knock-in mice in which the ΔNp63-specific exon is replaced by GFP. Homozygous ΔNp63(gfp/gfp) animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. ΔNp63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of ΔNp63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to ΔNp63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of ΔNp63 as demonstrated by ChIP experiments. The analysis of ΔNp63(gfp/gfp) knockout mice reaffirms the indispensable role of the ΔN isoform of p63 in epithelial biology and confirms that ΔNp63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate.
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Diferenciación Celular/fisiología , Epitelio/embriología , Ratones Noqueados , Morfogénesis/fisiología , Fosfoproteínas/metabolismo , Isoformas de Proteínas/metabolismo , Transactivadores/metabolismo , Animales , Biomarcadores/metabolismo , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Epidermis/anatomía & histología , Epidermis/embriología , Epidermis/metabolismo , Células Epiteliales/citología , Células Epiteliales/fisiología , Epitelio/anatomía & histología , Epitelio/metabolismo , Matriz Extracelular/metabolismo , Humanos , Uniones Intercelulares/metabolismo , Queratinocitos/citología , Queratinocitos/fisiología , Ratones , Fosfoproteínas/genética , Isoformas de Proteínas/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Transactivadores/genéticaRESUMEN
Focal adhesions (FAs), sites of tight adhesion to the extracellular matrix, are composed of clusters of transmembrane integrin adhesion receptors and intracellular proteins that link integrins to the actin cytoskeleton and signaling pathways. Two integrin-binding proteins present in FAs, kindlin-1 and kindlin-2, are important for integrin activation, FA formation, and signaling. Migfilin, originally identified in a yeast two-hybrid screen for kindlin-2-interacting proteins, is a LIM domain-containing adaptor protein found in FAs and implicated in control of cell adhesion, spreading, and migration. By binding filamin, migfilin provides a link between kindlin and the actin cytoskeleton. Here, using a combination of kindlin knockdown, biochemical pulldown assays, fluorescence microscopy, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP), we have established that the C-terminal LIM domains of migfilin dictate its FA localization, shown that these domains mediate an interaction with kindlin in vitro and in cells, and demonstrated that kindlin is important for normal migfilin dynamics in cells. We also show that when the C-terminal LIM domain region is deleted, then the N-terminal filamin-binding region of the protein, which is capable of targeting migfilin to actin-rich stress fibers, is the predominant driver of migfilin localization. Our work details a correlation between migfilin domains that drive kindlin binding and those that drive FA localization as well as a kindlin dependence on migfilin FA recruitment and mobility. We therefore suggest that the kindlin interaction with migfilin LIM domains drives migfilin FA recruitment, localization, and mobility.
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Moléculas de Adhesión Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Adhesiones Focales/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Células Cultivadas , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Inactivación de Genes , Humanos , Queratinocitos/metabolismo , Ratones , Proteínas Musculares/deficiencia , Proteínas Musculares/genética , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
Integrin-ß1-null keratinocytes can adhere to fibronectin through integrin αvß6, but form large peripheral focal adhesions and exhibit defective cell spreading. Here we report that, in addition to the reduced avidity of αvß6 integrin binding to fibronectin, the inability of integrin ß6 to efficiently bind and recruit kindlin-2 to focal adhesions directly contributes to these phenotypes. Kindlins regulate integrins through direct interactions with the integrin-ß cytoplasmic tail and keratinocytes express kindlin-1 and kindlin-2. Notably, although both kindlins localize to focal adhesions in wild-type cells, only kindlin-1 localizes to the integrin-ß6-rich adhesions of integrin-ß1-null cells. Rescue of these cells with wild-type and chimeric integrin constructs revealed a correlation between kindlin-2 recruitment and cell spreading. Furthermore, despite the presence of kindlin-1, knockdown of kindlin-2 in wild-type keratinocytes impaired cell spreading. Our data reveal unexpected functional consequences of differences in the association of two homologous kindlin isoforms with two closely related integrins, and suggest that despite their similarities, different kindlins are likely to have unique functions.
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Antígenos de Neoplasias/metabolismo , Integrina beta1/metabolismo , Integrinas/metabolismo , Queratinocitos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Adhesión Celular/fisiología , Fibronectinas/metabolismo , Citometría de Flujo , Adhesiones Focales , Técnicas de Inactivación de Genes , Humanos , Integrina beta1/genética , Queratinocitos/citología , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Alineación de SecuenciaRESUMEN
In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.
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Citoesqueleto de Actina/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Adhesiones Focales/enzimología , Citoesqueleto de Actina/ultraestructura , Animales , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula , Movimiento Celular/fisiología , Forma de la Célula , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Quinasa 2 de Adhesión Focal/análisis , Quinasa 2 de Adhesión Focal/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Integrina beta1/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Ratones , Microtúbulos/metabolismo , Fosforilación , Proteínas Represoras/metabolismo , Transducción de SeñalRESUMEN
Stem cells have been shown to play an important role in regenerative medicine due to their proliferative and differentiation potential. The challenge, however, lies in regulating and controlling their potential for this purpose. Stem cells are regulated by growth factors as well as an array of biochemical and mechanical signals. While the role of biochemical signals and growth factors in regulating stem cell homeostasis is well explored, the role of mechanical signals has only just started to be investigated. Stem cells interact with their niche or to other stem cells via adhesion molecules that eventually transduce mechanical cues to maintain their homeostatic function. Here, we present a comprehensive review on our current understanding of the influence of the forces perceived by cell adhesion molecules on the regulation of stem cells. Additionally, we provide insights on how this deeper understanding of mechanobiology of stem cells has translated toward therapeutics.
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PURPOSE: Osteonecrosis of the jaws (ONJ) is a clinical condition that is characterized by a nonhealing breach in the oral mucosa resulting in exposure of bone and has been increasingly reported in patients receiving bisphosphonate (BP) therapy. Although the pathogenesis and natural history of ONJ remain ill-defined, it appears that the oral soft tissues play a critical role in the development of this condition. We examined the effects of the nitrogen-containing BPs pamidronate and zoledronate on primary human gingival fibroblasts. MATERIALS AND METHODS: Primary gingival fibroblasts were exposed to clinically relevant doses of pamidronate and zoledronate. Cellular proliferation was measured with an MTS/PMS reagent-based kit (Promega, Madison, WI), scratch wound assays were performed to measure cellular migration, and apoptosis was measured by use of terminal deoxynucleotidyl transferase-mediated dUTP-FITC end labeling and caspase assays. The BP-exposed cells were treated with 10-ng/mL recombinant human platelet-derived growth factor BB (rhPDGF-BB) and 50-µmol/L geranylgeraniol (GGOH). RESULTS: Gingival fibroblasts are significantly more sensitive to inhibition of proliferation by zoledronate compared with pamidronate. Exposure of these cells to pamidronate but not zoledronate resulted in an increase in cellular apoptosis. Furthermore, exposure of gingival fibroblasts to pamidronate or zoledronate resulted in a decrease in cellular migration. We show that these defects are due to a loss of cell-substratum adhesion and a reduction of F-actin bundles. Finally, we show that the addition of rhPDGF-BB and GGOH in vitro is able to partially rescue the cell proliferation, migration, and adhesion defects. CONCLUSION: The cytotoxic effects of BPs on oral fibroblasts and their significant reversal by the addition of GGOH and rhPDGF-BB provide both the potential mechanism and treatment options for ONJ.
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Conservadores de la Densidad Ósea/efectos adversos , Difosfonatos/efectos adversos , Diterpenos/farmacología , Encía/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Cicatrización de Heridas/efectos de los fármacos , Actinas/metabolismo , Apoptosis , Becaplermina , Caspasa 3/metabolismo , Adhesión Celular/efectos de los fármacos , Técnicas de Cultivo de Célula , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Encía/citología , Humanos , Imidazoles/efectos adversos , Etiquetado Corte-Fin in Situ , Pamidronato , Proteínas Proto-Oncogénicas c-sis , Proteínas Recombinantes/farmacología , Ácido ZoledrónicoRESUMEN
Generating high-quality electron microscopy images of the skin and keratinocytes can be challenging. Here we describe a simple protocol for scanning electron microscopy (SEM) of murine skin. The protocol enables characterization of the ultrastructure of the epidermis, dermis, hair follicles, basement membrane, and cell-cell junctions. We detail the specific steps for sample preparation and highlight the critical need for proper orientation of the sample for ultrathin sectioning. We also describe the isolation and preparation of primary keratinocyte monolayers for SEM. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2021).
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Queratinocitos/ultraestructura , Microscopía Electrónica de Rastreo/métodos , Piel/diagnóstico por imagen , Animales , Membrana Basal/ultraestructura , Dermis/diagnóstico por imagen , Células Epidérmicas/ultraestructura , Epidermis/diagnóstico por imagen , Folículo Piloso/diagnóstico por imagen , Queratinocitos/metabolismo , RatonesRESUMEN
Macrophages are highly responsive to the environmental cues and are the primary responders to tissue stress and damage. While much is known about the role of macrophages during inflammatory disease progression; the initial series of events that set up the inflammation remains less understood. In this study, we use next generation sequencing (NGS) of embryonic skin macrophages and the niche cells - skin epithelia and stroma in the epidermis specific knockout of integrin beta 1 (Itgß1) model to uncover specific roles of each cell type and identify how these cell types communicate to initiate the sterile inflammatory response. We demonstrate that while the embryonic skin fibroblasts in the Itgß1 knockout skin are relatively inactive, the keratinocytes and macrophages are the critical responders to the sterile inflammatory cues. The epidermis expresses damage associated molecular patterns (DAMPs), stress response genes, pro-inflammatory cytokines, and chemokines that aid in eliciting the inflammatory response. The macrophages, in-turn, respond by acquiring enhanced M2-like characteristics expressing ECM remodeling and matrisome signatures that exacerbate the basement membrane disruption. Depletion of macrophages by blocking the CSF1 receptor (CSF1R) results in improved basement membrane integrity and reduced ECM remodeling activity in the KO skin. Further, blocking the skin inflammation with celecoxib reveals that the acquired fate of macrophages in the KO skin is dependent on its interaction with the epidermal compartment through COX2 dependent cytokine production. Taken together, our study highlights a critical crosstalk between the epithelia and the dermal macrophages that shapes macrophage fate and initiates sterile inflammation in the skin. The insights gained from our study can be extrapolated to other inflammatory disorders to understand the early events that set up the disease.
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Dermatitis/etiología , Dermatitis/metabolismo , Susceptibilidad a Enfermedades , Epitelio/metabolismo , Macrófagos/metabolismo , Animales , Biomarcadores , Epidermis/metabolismo , Epidermis/patología , Matriz Extracelular/metabolismo , Femenino , Feto , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Modelos BiológicosRESUMEN
Vinculin, a mechanotransducer associated with both adherens junctions (AJs) and focal adhesions (FAs), plays a central role in force transmission through cell-cell and cell-substratum contacts. We generated the conditional knockout (cKO) of vinculin in murine skin that results in the loss of bulge stem cell (BuSC) quiescence and promotes continual cycling of the hair follicles. Surprisingly, we find that the AJs in vinculin cKO cells are mechanically weak and impaired in force generation despite increased junctional expression of E-cadherin and α-catenin. Mechanistically, we demonstrate that vinculin functions by keeping α-catenin in a stretched/open conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, at the AJs. Altogether, our data provide mechanistic insights into the hitherto-unexplored regulatory link between the mechanical stability of cell junctions and contact-inhibition-mediated maintenance of BuSC quiescence.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Uniones Adherentes/fisiología , Folículo Piloso/fisiología , Mecanotransducción Celular , Células Madre/fisiología , Vinculina/fisiología , alfa Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Adhesión Celular , Femenino , Folículo Piloso/citología , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre/citología , Proteínas Señalizadoras YAP , alfa Catenina/genéticaRESUMEN
alphabeta1 integrins have been implicated in the survival, spreading, and migration of cells and tissues. To explore the underlying biology, we identified conditions where primary beta1 null keratinocytes adhere, proliferate, and display robust alphavbeta6 integrin-induced, peripheral focal contacts associated with elaborate stress fibers. Mechanistically, this appears to be due to reduced FAK and Src and elevated RhoA and Rock activities. Visualization on a genetic background of GFPactin shows that beta1 null keratinocytes spread, but do so aberrantly, and when induced to migrate from skin explants in vitro, the cells are not able to rapidly reorient their actin cytoskeleton toward the polarized movement. As judged by RFPzyxin/GFPactin videomicroscopy, the alphavbeta6-actin network does not undergo efficient turnover. Without the ability to remodel their integrin-actin network efficiently, alphabeta1-deficient keratinocytes cannot respond dynamically to their environment and polarize movements.
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Adhesión Celular/fisiología , Citoesqueleto/fisiología , Integrinas/fisiología , Queratinocitos/fisiología , Actinas/metabolismo , Animales , Western Blotting , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Proteínas del Citoesqueleto/fisiología , Citoesqueleto/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente/instrumentación , Técnica del Anticuerpo Fluorescente/métodos , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Adhesiones Focales/metabolismo , Proteínas de Homeodominio/metabolismo , Indoles/metabolismo , Integrinas/genética , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Proteínas con Homeodominio LIM , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Faloidina/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Factores de Tiempo , Factores de Transcripción , Trasplantes , Tirosina/metabolismoRESUMEN
Skin is the primary barrier against the external environment and develops a robust immune network for its surveillance. The origin of the resident immune cells of the skin has become a focus of interest over past a decade. Fate mapping studies have revealed that the macrophages home into the skin as early as E12.5 and are derived from the yolk sac and fetal liver. The resident γδT cells are born in the thymus and home to the skin by E16.5. Recent work from our lab has shown that the embryonic macrophages can actively remodel the extracellular matrix in skin suggesting that the skin immune system can be activated long before exposure to foreign antigens. In this chapter, we present a detailed protocol for isolating monocytes, macrophages, and epidermal dendritic T cell populations from embryonic skin.
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Sistema Inmunológico/citología , Piel/citología , Animales , Matriz Extracelular/fisiología , Femenino , Hígado/citología , Macrófagos/citología , Ratones , Monocitos/citología , Linfocitos T/citología , Saco Vitelino/citologíaRESUMEN
The extracellular matrix (ECM) is a complex network of proteins and proteoglycans secreted by keratinocytes, fibroblasts and immune cells. The function of the skin ECM has expanded from being a scaffold that provides structural integrity, to a more dynamic entity that is constantly remodeled to maintain tissue homeostasis. The ECM functions as ligands for cell surface receptors such as integrins, dystroglycans, and toll-like receptors (TLRs) and regulate cellular signaling and immune cell dynamics. The ECM also acts as a sink for growth factors and cytokines, providing critical cues during epithelial morphogenesis. Dysregulation in the organization and deposition of ECMs lead to a plethora of pathophysiological conditions that are exacerbated by aberrant ECM-immune cell interactions. In this review, we focus on the interplay between ECM and immune cells in the context of skin diseases and also discuss state of the art therapies that target the key molecular players involved.
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PURPOSE: Bisphosphonates (BPs) are a widely used class of drugs that are effective in the treatment and prevention of osteoporosis, hypercalcemia of malignancy, and bone metastases associated with multiple myeloma, breast cancer, and other solid tumors. In the past several years there have been numerous reports describing the occurrence of osteonecrosis of the jaws (ONJ) associated with these drugs. Whether the ONJ lesion initiates in the oral mucosa or derives from the underlying bone is not well understood. In this report we describe the effect of pamidronate, a second-generation BP, on oral mucosal cells. MATERIALS AND METHODS: Murine oral keratinocytes were isolated and exposed to pamidronate at a range of clinically relevant doses. Cellular proliferation was measured using a MTS/PMS reagent-based kit and wound healing was examined with a scratch assay. To determine whether oral keratinocytes undergo apoptosis following exposure to pamidronate, TUNEL, caspase-3, and DAPI apoptosis assays were performed. RESULTS: We show that BP pretreatment of oral mucosal cells inhibits proliferation and wound healing at clinically relevant doses, and that this inhibition is not due to cellular apoptosis. CONCLUSIONS: To our knowledge this is the first report investigating the effect of nitrogen-containing BPs on oral mucosal cells. This study suggests that BPs inhibit oral keratinocyte wound healing which may play a significant role in the initiation of ONJ.
Asunto(s)
Conservadores de la Densidad Ósea/toxicidad , Difosfonatos/toxicidad , Mucosa Bucal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Apoptosis , Conservadores de la Densidad Ósea/química , Caspasa 3/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Difosfonatos/química , Colorantes Fluorescentes/metabolismo , Etiquetado Corte-Fin in Situ , Indoles/metabolismo , Enfermedades Maxilomandibulares/inducido químicamente , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Ratones , Mucosa Bucal/enzimología , Nitrógeno , Osteonecrosis/inducido químicamente , PamidronatoRESUMEN
Epidermal knockout of integrin ß1 results in complete disorganization of the basement membrane (BM), resulting in neonatal lethality. Here, we report that this disorganization is exacerbated by an early embryonic inflammatory response involving the recruitment of tissue-resident and monocyte-derived macrophages to the dermal-epidermal junction, associated with increased matrix metalloproteinase activity. Remarkably, the skin barrier in the integrin ß1 knockout animals is intact, suggesting that this inflammatory response is initiated in a sterile environment. We demonstrate that the molecular mechanism involves de novo expression of integrin αvß6 in the basal epidermal cells, which activates a TGF-ß1 driven inflammatory cascade resulting in upregulation of dermal NF-κB in a Tenascin C-dependent manner. Importantly, treatment of ß1 KO embryos in utero with small molecule inhibitors of TGF-ßR1 and NF-κB results in marked rescue of the BM defects and amelioration of immune response, revealing an unconventional immuno-protective role for integrin ß1 during BM remodeling.