RESUMEN
Mechanical and metabolic cues independently contribute to the regulation of cell and tissue homeostasis. However, how they cross-regulate each other during this process remains largely unknown. Here, we show that cellular metabolism can regulate integrin rigidity-sensing via the sphingolipid metabolic pathway controlled by the amino acid transporter and integrin coreceptor CD98hc (SLC3A2). Genetic invalidation of CD98hc in dermal cells and tissue impairs rigidity sensing and mechanical signaling downstream of integrins, including RhoA activation, resulting in aberrant tissue mechanical homeostasis. Unexpectedly, we found that this regulation does not occur directly through regulation of integrins by CD98hc but indirectly, via the regulation of sphingolipid synthesis and the delta-4-desaturase DES2. Loss of CD98hc decreases sphingolipid availability preventing proper membrane recruitment, shuttling and activation of upstream regulators of RhoA including Src kinases and GEF-H1. Altogether, our results unravel a novel cross-talk regulation between integrin mechanosensing and cellular metabolism which may constitute an important new regulatory framework contributing to mechanical homeostasis.
Asunto(s)
Fibroblastos/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Mecanotransducción Celular , Complejos Multienzimáticos/genética , Oxidorreductasas/genética , Esfingolípidos/biosíntesis , Animales , Dermis/citología , Dermis/metabolismo , Fibroblastos/citología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/deficiencia , Regulación de la Expresión Génica , Homeostasis , Lipogénesis , Ratones , Ratones Transgénicos , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/metabolismo , Cultivo Primario de Células , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Skin homeostasis relies on fine-tuning of epidermis-dermis interactions and is affected by aging. While extracellular matrix (ECM) proteins, such as integrins, are involved in aging, the molecular basis of the skin changes needs to be investigated further. Here, we showed that integrin co-receptor, SLC3A2, required for cell proliferation, is expressed at the surface of resting dermal fibroblasts in young patients and is reduced drastically with aging. In vivo SLC3A2 dermal fibroblast deletion induced major skin phenotypes resembling premature aging. Knockout mice (3 months old) presented strong defects in skin elasticity due to altered ECM assembly, which impairs epidermal homeostasis. SLC3A2 dermal fibroblast loss led to an age-associated secretome profile, with 77% of identified proteins belonging to ECM and ECM-associated proteins. ECM not only contributes to skin mechanical properties, but it is also a reservoir of growth factors and bioactive molecules. We demonstrate that dermal fibroblast SLC3A2 is required for ECM to fully exert its structural and reservoir role allowing proper and efficient TGF-ß localization and activation. We identified SLC3A2 as a protective controller of dermal ECM stiffness and quality required to maintain the epidermis to dermis interface as functional and dynamic.
Asunto(s)
Envejecimiento Prematuro/genética , Dermis/patología , Epitelio/fisiología , Fibroblastos/fisiología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Animales , Proliferación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Homeostasis , Humanos , Ratones , Ratones Noqueados , Transporte de Proteínas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Age-related bone loss is associated with an increased oxidative stress which is worsened by estrogen fall during menauposis. This observation has drawn attention to autophagy, a major cellular catabolic process, able to alleviate oxidative stress in osteoblasts (OB) and osteocytes (OST), two key bone cell types. Moreover, an autophagy decline can be associated with aging, suggesting that an age-related autophagy deficiency in OB and/or OST could contribute to skeletal aging and osteoporosis onset.In the present work, autophagy activity was analyzed in OST and OB in male and female mice according to their age and hormonal status. In OST, autophagy decreases with aging in both sexes. In OB, although a 95% decrease in autophagy is observed in OB derived from old females, this activity remains unchanged in males. In addition, while ovariectomy has no effect on OB autophagy levels, orchidectomy appears to stimulate this process. An inverse correlation between autophagy and the oxidative stress level was observed in OB derived from males or females. Finally, using OB-specific autophagy-deficient mice, we showed that autophagy deficiency aggravates the bone loss associated with aging and estrogen deprivation.Taken together, our data indicate that autophagic modulation in bone cells differs according to sex and cell type. The lowering of autophagy in female OB, which is associated with an increased oxidative stress, could play a role in osteoporosis pathophysiology and suggests that autophagy could be a new therapeutic target for osteoporosis in women.
Asunto(s)
Autofagia/fisiología , Osteoblastos/patología , Osteoporosis/patología , Caracteres Sexuales , Animales , Femenino , Masculino , Ratones , Osteocitos/patologíaRESUMEN
CD98hc functions as an amino acid (AA) transporter (together with another subunit) and integrin signaling enhancer. It is overexpressed in highly proliferative cells in both physiological and pathological conditions. CD98hc deletion induces strong impairment of cell proliferation in vivo and in vitro Here, we investigate CD98hc-associated AA transport in cell survival and proliferation. By using chimeric versions of CD98hc, the two functions of the protein can be uncoupled. Although recovering the CD98hc AA transport capacity restores the in vivo and in vitro proliferation of CD98hc-null cells, reconstitution of the integrin signaling function of CD98hc is unable to restore in vitro proliferation of those cells. CD98hc-associated transporters (i.e. xCT, LAT1, and y(+)LAT2 in wild-type cells) are crucial to control reactive oxygen species and intracellular AA levels, thus sustaining cell survival and proliferation. Moreover, in CD98hc-null cells the deficiency of CD98hc/xCT cannot be compensated, leading to cell death by ferroptosis. Supplementation of culture media with ß-mercaptoethanol rescues CD98hc-deficient cell survival. Under such conditions null cells show oxidative stress and intracellular AA imbalance and, consequently, limited proliferation. CD98hc-null cells also present reduced intracellular levels of branched-chain and aromatic amino acids (BCAAs and ARO AAs, respectively) and induced expression of peptide transporter 1 (PEPT1). Interestingly, external supply of dipeptides containing BCAAs and ARO AAs rescues cell proliferation and compensates for impaired uptake of CD98hc/LAT1 and CD98hc/y(+)LAT2. Our data establish CD98hc as a master protective gene at the cross-road of redox control and AA availability, making it a relevant therapeutic target in cancer.
Asunto(s)
Aminoácidos/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Estrés Oxidativo , Sistema de Transporte de Aminoácidos y+/genética , Sistema de Transporte de Aminoácidos y+/metabolismo , Sistema de Transporte de Aminoácidos y+L , Aminoácidos/genética , Animales , Transporte Biológico Activo/fisiología , Línea Celular , Supervivencia Celular/fisiología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/genética , Cadenas Ligeras de la Proteína-1 Reguladora de Fusión/metabolismo , Eliminación de Gen , Ratones , Células Madre Embrionarias de Ratones/citología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Bone remodeling is a tightly controlled mechanism in which osteoblasts (OB), the cells responsible for bone formation, osteoclasts (OC), the cells specialized for bone resorption, and osteocytes, the multifunctional mechanosensing cells embedded in the bone matrix, are the main actors. Increased oxidative stress in OB, the cells producing and mineralizing bone matrix, has been associated with osteoporosis development but the role of autophagy in OB has not yet been addressed. This is the goal of the present study. We first show that the autophagic process is induced in OB during mineralization. Then, using knockdown of autophagy-essential genes and OB-specific autophagy-deficient mice, we demonstrate that autophagy deficiency reduces mineralization capacity. Moreover, our data suggest that autophagic vacuoles could be used as vehicles in OB to secrete apatite crystals. In addition, autophagy-deficient OB exhibit increased oxidative stress and secretion of the receptor activator of NFKB1 (TNFSF11/RANKL), favoring generation of OC, the cells specialized in bone resorption. In vivo, we observed a 50% reduction in trabecular bone mass in OB-specific autophagy-deficient mice. Taken together, our results show for the first time that autophagy in OB is involved both in the mineralization process and in bone homeostasis. These findings are of importance for mineralized tissues which extend from corals to vertebrates and uncover new therapeutic targets for calcified tissue-related metabolic pathologies.
Asunto(s)
Autofagia , Huesos/metabolismo , Osteoblastos/citología , Animales , Remodelación Ósea , Resorción Ósea , Línea Celular Tumoral , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis , Ratones , Ratones Transgénicos , Microscopía Confocal , Subunidad p50 de NF-kappa B/metabolismo , Osteoclastos/metabolismo , Estrés Oxidativo , Ligando RANK/metabolismo , Ratas , Microtomografía por Rayos XRESUMEN
CD98hc (SLC3A2) is the heavy chain component of the dimeric transmembrane glycoprotein CD98, which comprises the large neutral amino acid transporter LAT1 (SLC7A5) in cells. Overexpression of CD98hc occurs widely in cancer cells and is associated with poor prognosis clinically, but its exact contributions to tumorigenesis are uncertain. In this study, we showed that genetic deficiency of CD98hc protects against Ras-driven skin carcinogenesis. Deleting CD98hc after tumor induction was also sufficient to cause regression of existing tumors. Investigations into the basis for these effects defined two new functions of CD98hc that contribute to epithelial cancer beyond an intrinsic effect of CD98hc on tumor cell proliferation. First, CD98hc increased the stiffness of the tumor microenvironment. Second, CD98hc amplified the capacity of cells to respond to matrix rigidity, an essential factor in tumor development. Mechanistically, CD98hc mediated this stiffness sensing by increasing Rho kinase (ROCK) activity, resulting in increased transcription mediated by YAP/TAZ, a nuclear relay for mechanical signals. Our results suggest that CD98hc contributes to carcinogenesis by amplifying a positive feedback loop, which increases both extracellular matrix stiffness and resulting cellular responses. This work supports a rationale to explore the use of CD98hc inhibitors as cancer therapeutics.
Asunto(s)
Carcinogénesis/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Integrinas/metabolismo , Proteínas ras/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinogénesis/patología , Proteínas de Ciclo Celular , Proliferación Celular/fisiología , Células Cultivadas , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Mecanotransducción Celular , Ratones , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Piel/metabolismo , Piel/patología , Factores de Transcripción/metabolismo , Microambiente Tumoral/fisiología , Proteínas Señalizadoras YAP , Quinasas Asociadas a rho/metabolismoRESUMEN
Degradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth, its survival, and its motility. Emerging evidence suggests that autophagy limits several signaling pathways by degrading kinases, downstream components, and transcription factors; however, this often occurs under stressful conditions. Our recent studies revealed that constitutive autophagy temporally and spatially controls the RHOA pathway. Specifically, inhibition of autophagosome degradation induces the accumulation of the GTP-bound form of RHOA. The active RHOA is sequestered via SQSTM1/p62 within autolysosomes, and accordingly fails to localize to the spindle midbody or to the cell surface, as we demonstrate herein. As a result, all RHOA-downstream responses are deregulated, thus driving cytokinesis failure, aneuploidy and motility, three processes that directly have an impact upon cancer progression. We therefore propose that autophagy acts as a degradative brake for RHOA signaling and thereby controls cell proliferation, migration, and genome stability.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/fisiología , Proliferación Celular , Proteínas de Choque Térmico/metabolismo , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Animales , Autofagia/genética , Movimiento Celular/fisiología , Células Cultivadas , Citocinesis/genética , Citocinesis/fisiología , Ratones , Fagosomas/metabolismo , Proteína Sequestosoma-1RESUMEN
Skin aging is linked to reduced epidermal proliferation and general extracellular matrix atrophy. This involves factors such as the cell adhesion receptors integrins and amino acid transporters. CD98hc (SLC3A2), a heterodimeric amino acid transporter, modulates integrin signaling in vitro. We unravel CD98hc functions in vivo in skin. We report that CD98hc invalidation has no appreciable effect on cell adhesion, clearly showing that CD98hc disruption phenocopies neither CD98hc knockdown in cultured keratinocytes nor epidermal ß1 integrin loss in vivo. Instead, we show that CD98hc deletion in murine epidermis results in improper skin homeostasis and epidermal wound healing. These defects resemble aged skin alterations and correlate with reduction of CD98hc expression observed in elderly mice. We also demonstrate that CD98hc absence in vivo induces defects as early as integrin-dependent Src activation. We decipher the molecular mechanisms involved in vivo by revealing a crucial role of the CD98hc/integrins/Rho guanine nucleotide exchange factor (GEF) leukemia-associated RhoGEF (LARG)/RhoA pathway in skin homeostasis. Finally, we demonstrate that the deregulation of RhoA activation in the absence of CD98hc is also a result of impaired CD98hc-dependent amino acid transports.
Asunto(s)
Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Queratinocitos/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Factores de Edad , Animales , Proteína Tirosina Quinasa CSK , Adhesión Celular/genética , Movimiento Celular/genética , Proliferación Celular , Proteínas de Unión al ADN/metabolismo , Epidermis/metabolismo , Epidermis/patología , Cadena Pesada de la Proteína-1 Reguladora de Fusión/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Folículo Piloso/metabolismo , Homeostasis , Integrinas/metabolismo , Queratinocitos/patología , Ratones , Ratones Transgénicos , Especies Reactivas de Oxígeno/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal , Fenómenos Fisiológicos de la Piel , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Factores de Transcripción/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteína de Unión al GTP rhoA , Familia-src Quinasas/metabolismoRESUMEN
RATIONALE: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. OBJECTIVE: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. METHODS AND RESULTS: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of α2ß1 and α6ß1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by α2ß1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin γ1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins α2 or α6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. CONCLUSIONS: We show that, in endothelial cells, ligation of α2ß1 and α6ß1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.
Asunto(s)
Células Endoteliales/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa6beta1/metabolismo , Integrinas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Receptores Notch/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Membrana Basal , Proteínas de Unión al Calcio , Adhesión Celular , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Laminina/metabolismo , Proteínas de la Membrana/fisiología , Neovascularización Fisiológica , Receptor Cross-TalkRESUMEN
Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that alpha2beta1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, alpha2beta1 engagement alters assembly of mature focal adhesions by alpha5beta1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of alpha5beta1 to signal for GTP loading of Rac is preserved, the joint engagement of alpha2beta1 interferes with membrane anchorage of Rac. Adapting the 'split-ubiquitin' sensor to screen for membrane-proximal alpha2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by alpha2beta1 integrin. Altogether, our data establish that alpha2beta1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.
Asunto(s)
Antígenos CD/metabolismo , Células Endoteliales/metabolismo , Integrina alfa2beta1/metabolismo , Laminina/farmacología , Glicoproteínas de Membrana/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Animales , Antígenos CD/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Femenino , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibronectinas/farmacología , Adhesiones Focales/genética , Adhesiones Focales/metabolismo , Humanos , Integrina alfa2beta1/agonistas , Integrina alfa5beta1/agonistas , Integrina alfa5beta1/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Tetraspanina 29RESUMEN
Expression of the lama3 gene, encoding the laminin alpha3A chain, is restricted to specialized epithelia. We previously showed that lama3 gene expression is controlled by an epithelial enhancer through the cooperative effect of AP-1 binding sites. In fibroblasts, there is no lama3 expression because of the recruitment of a repressor complex absent or inactive in epithelial cells. In this paper, we show evidence that this repression of the lama3 gene is relieved by exogenous and UV-induced USF-1 through its interaction with a non-canonical E-box site. Using a chromatin immunoprecipitation assay, we find that UV stress induces USF to bind to the lama3 promoter in vivo. We further demonstrate that this loss of cell specificity is directly related to the accessibility of the E-box, resulting in a strong induction in fibroblasts, while expression remains constitutively high in keratinocytes. This accessibility appears to be dependent upon the recruitment of a fibroblastic repressor complex. Therefore, we speculate that anchorage of this repressor complex in fibroblasts modifies the enhancer geometry, allowing USF to interact under stress-inducing conditions with its heptameric binding site.