RESUMO
While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of ß1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.
Assuntos
Trato Gastrointestinal/embriologia , Crista Neural/citologia , Animais , Adesão Celular , Diferenciação Celular , Matriz Extracelular/fisiologia , Trato Gastrointestinal/inervação , Integrina beta1/fisiologia , Camundongos , Músculo Liso/embriologiaRESUMO
SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions. Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues. Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1-/- mice retinas. Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.
Assuntos
Caveolina 1/fisiologia , Terapia Genética , Vetores Genéticos/uso terapêutico , Glaucoma/terapia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Retina/patologia , alfa-Globulinas/genética , Animais , Apoptose , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Caveolina 1/deficiência , Caveolina 1/genética , DNA Complementar/genética , Dependovirus/genética , Quinase 1 de Adesão Focal/fisiologia , Técnicas de Silenciamento de Genes , Genes Reporter , Genes Sintéticos , Glaucoma/metabolismo , Glaucoma/patologia , Integrina beta1/fisiologia , Pressão Intraocular , Injeções Intravítreas , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 11/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Tirosina Quinases/fisiologia , Regulação para CimaRESUMO
Integrin beta- like 1 (ITGBL1), an extracellular matrix protein, plays an oncogenic role in diverse forms of cancers. To this end, we examined the importance of ITGBL1 in gastric cancer (GC). The upregulated expression of ITGBL1 in GC was associated with a poor prognosis. Moreover, upregulation of ITGBL1 enhanced cell mobility while silencing it exerted an opposite effect. Up-regulation of ITGBL1 significantly promoted phosphorylation of Akt, decreased the ratio of phosphorylated Akt in AGS/ITGBL1-shRNA and N87/ITGBL1-shRNA cells, enhanced cell mobility and proliferation. Silencing ITGBL1 had an opposite effect on Akt phosphorylation, cell mobility, and proliferation. These findings show that ITGBL1 regulates mobility and proliferation of GC likely through activation of Akt signaling.
Assuntos
Proliferação de Células/fisiologia , Integrina beta1/fisiologia , Invasividade Neoplásica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Integrina beta1/genética , Fosforilação , Prognóstico , Regulação para CimaRESUMO
Collagen type 1 (COL1) is a ubiquitously existing extracellular matrix protein whose high density in breast tissue favors metastasis and chemoresistance. COL1-binding of MDA-MB-231 and MCF-7 breast cancer cells is mainly dependent on ß1-integrins (ITGB1). Here, we elucidate the signaling of chemoresistance in both cell lines and their ITGB1-knockdown mutants and elucidated MAPK pathway to be strongly upregulated upon COL1 binding. Notably, Discoidin Domain Receptor 1 (DDR1) was identified as another important COL1-sensor, which is permanently active but takes over the role of COL1-receptor maintaining MAPK activation in ITGB1-knockdown cells. Consequently, inhibition of DDR1 and ERK1/2 act synergistically, and sensitize the cells for cytostatic treatments using mitoxantrone, or doxorubicin, which was associated with an impaired ABCG2 drug efflux transporter activity. These data favor DDR1 as a promising target for cancer cell sensitization, most likely in combination with MAPK pathway inhibitors to circumvent COL1 induced transporter resistance axis. Since ITGB1-knockdown also induces upregulation of pEGFR in MDA-MB-231 cells, inhibitory approaches including EGFR inhibitors, such as gefitinib appear promising for pharmacological interference. These findings provide evidence for the highly dynamic adaptation of breast cancer cells in maintaining matrix binding to circumvent cytotoxicity and highlight DDR1 signaling as a target for sensitization approaches.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Colágeno Tipo I/metabolismo , Receptor com Domínio Discoidina 1/fisiologia , Integrina beta1/fisiologia , Proteínas de Neoplasias/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Transporte Biológico/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Receptor com Domínio Discoidina 1/antagonistas & inibidores , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/biossíntese , Receptores ErbB/genética , Quinase 1 de Adesão Focal/metabolismo , Gefitinibe/farmacologia , Gefitinibe/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Indazóis/farmacologia , Integrina beta1/genética , Integrina beta4/biossíntese , Integrina beta4/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Células MCF-7 , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Mitoxantrona/metabolismo , Mitoxantrona/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Piperazinas/farmacologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
The ß1 integrin subunit contributes to pancreatic beta cell growth and function through communication with the extracellular matrix (ECM). The effects of in vitro and in vivo ß1 integrin knockout have been extensively studied in mature islets, yet no study to date has examined how the loss of ß1 integrin during specific stages of pancreatic development impacts beta cell maturation. Beta-cell-specific tamoxifen-inducible Cre recombinase (MIP-CreERT) mice were crossed with mice containing floxed Itgb1 (ß1 integrin) to create an inducible mouse model (MIPß1KO) at the second transition stage (e13.5) of pancreas development. By e19.5-20.5, the expression of beta-cell ß1 integrin in fetal MIPß1KO mice was significantly reduced and these mice displayed decreased beta cell mass, density and proliferation. Morphologically, fetal MIPß1KO pancreata exhibited reduced islet vascularization and nascent endocrine cells in the ductal region. In addition, decreased ERK phosphorylation was observed in fetal MIPß1KO pancreata. The expression of transcription factors needed for beta-cell development was unchanged in fetal MIPß1KO pancreata. The findings from this study demonstrate that ß1 integrin signaling is required during a transition-specific window in the developing beta-cell to maintain islet mass and vascularization.
Assuntos
Células Secretoras de Insulina/citologia , Integrina beta1/fisiologia , Pâncreas/embriologia , Animais , Diferenciação Celular , Feminino , Desenvolvimento Fetal , Integrinas/deficiência , Masculino , Camundongos , Camundongos Knockout , Pâncreas/irrigação sanguíneaRESUMO
Epithelial fusion is a key process of morphogenesis by which tissue connectivity is established between adjacent epithelial sheets. A striking and poorly understood feature of this process is "zippering," whereby a fusion point moves directionally along an organ rudiment. Here, we uncover the molecular mechanism underlying zippering during mouse spinal neural tube closure. Fusion is initiated via local activation of integrin ß1 and focal anchorage of surface ectoderm cells to a shared point of fibronectin-rich basement membrane, where the neural folds first contact each other. Surface ectoderm cells undergo proximal junction shortening, establishing a transitory semi-rosette-like structure at the zippering point that promotes juxtaposition of cells across the midline enabling fusion propagation. Tissue-specific ablation of integrin ß1 abolishes the semi-rosette formation, preventing zippering and causing spina bifida. We propose integrin-mediated anchorage as an evolutionarily conserved mechanism of general relevance for zippering closure of epithelial gaps whose disturbance can produce clinically important birth defects.
Assuntos
Embrião de Mamíferos/fisiologia , Células Epiteliais/fisiologia , Adesões Focais , Integrina beta1/fisiologia , Crista Neural/embriologia , Tubo Neural/embriologia , Neurulação , Actomiosina/metabolismo , Animais , Fusão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Crista Neural/metabolismo , Crista Neural/fisiologia , Tubo Neural/metabolismo , Tubo Neural/fisiologiaRESUMO
Integrin dimers α3/ß1, α6/ß1 and α6/ß4 are the mammary epithelial cell receptors for laminins, which are major components of the mammary basement membrane. The roles of specific basement membrane components and their integrin receptors in the regulation of functional gland development have not been analyzed in detail. To investigate the functions of laminin-binding integrins, we obtained mutant mice with mammary luminal cell-specific deficiencies of the α3 and α6 integrin chains generated using the Cre-Lox approach. During pregnancy, mutant mice displayed decreased luminal progenitor activity and retarded lobulo-alveolar development. Mammary glands appeared functional at the onset of lactation in mutant mice; however, myoepithelial cell morphology was markedly altered, suggesting cellular compensation mechanisms involving cytoskeleton reorganization. Notably, lactation was not sustained in mutant females, and the glands underwent precocious involution. Inactivation of the p53 gene rescued the growth defects but did not restore lactogenesis in mutant mice. These results suggest that the p53 pathway is involved in the control of mammary cell proliferation and survival downstream of laminin-binding integrins, and underline an essential role of cell interactions with laminin for lactogenic differentiation.
Assuntos
Integrinas/fisiologia , Lactação , Glândulas Mamárias Animais/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Sobrevivência Celular , Citoesqueleto/fisiologia , Progressão da Doença , Feminino , Deleção de Genes , Hormônios/fisiologia , Integrina alfa3/fisiologia , Integrina alfa6/fisiologia , Integrina beta1/fisiologia , Integrina beta4/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Mutantes , Mutação , Células-Tronco Neoplásicas/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Ovário/fisiologia , Fenótipo , Gravidez , Prenhez , Prognóstico , Ligação Proteica , Multimerização ProteicaRESUMO
Navigating a changing environment requires associating stimuli and actions with their likely outcomes and modifying these associations when they change. These processes involve the orbitofrontal cortex (OFC). Although some molecular mediators have been identified, developmental factors are virtually unknown. We hypothesized that the cell adhesion factor ß1-integrin is essential to OFC function, anticipating developmental windows during which ß1-integrins might be more influential than others. We discovered that OFC-selective ß1-integrin silencing before adolescence, but not later, impaired the ability of mice to extinguish conditioned fear and select actions based on their likely outcomes. Early-life knock-down also reduced the densities of dendritic spines, the primary sites of excitatory plasticity in the brain, and weakened sensitivity to cortical inputs. Notwithstanding these defects in male mice, females were resilient to OFC (but not hippocampal) ß1-integrin loss. Existing literature suggests that resilience may be explained by estradiol-mediated transactivation of ß1-integrins and tropomyosin receptor kinase B (trkB). Accordingly, we discovered that a trkB agonist administered during adolescence corrected reward-related decision making in ß1-integrin-deficient males. In sum, developmental ß1-integrins are indispensable for OFC function later in life.SIGNIFICANCE STATEMENT The orbitofrontal cortex (OFC) is a subregion of the frontal cortex that allows organisms to link behaviors and stimuli with anticipated outcomes, and to make predictions about the consequences of one's behavior. Aspects of OFC development are particularly prolonged, extending well into adolescence, likely optimizing organisms' abilities to prospectively calculate the consequences of their actions and select behaviors appropriately; these decision making strategies improve as young individuals mature into adulthood. Molecular factors are not, however, well understood. Our experiments reveal that a cell adhesion protein termed "ß1-integrin" is necessary for OFC neuronal maturation and function. Importantly, ß1-integrins operate during a critical period equivalent to early adolescence in humans to optimize the ability of organisms to update expectancies later in life.
Assuntos
Antecipação Psicológica/fisiologia , Integrina beta1/fisiologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Córtex Pré-Frontal/fisiologia , Animais , Antecipação Psicológica/efeitos dos fármacos , Antipsicóticos/farmacologia , Clozapina/farmacologia , Condicionamento Clássico , Espinhas Dendríticas/fisiologia , Meio Ambiente , Estradiol/fisiologia , Extinção Psicológica , Medo/psicologia , Feminino , Masculino , Camundongos , Plasticidade Neuronal/fisiologia , Receptor trkB/metabolismo , Reforço Psicológico , Resiliência PsicológicaRESUMO
RATIONALE: Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the ß-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown. OBJECTIVE: To test the requirement of talin-dependent activation of ß1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function. METHODS AND RESULTS: EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring ß1-integrin activation in talin-deficient cells with a ß1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins. CONCLUSIONS: Talin-dependent activation of EC ß1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.
Assuntos
Antígenos CD/fisiologia , Caderinas/fisiologia , Células Endoteliais/fisiologia , Integrina beta1/fisiologia , Talina/fisiologia , Animais , Antígenos CD/análise , Caderinas/análise , Feminino , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Junções Intercelulares/metabolismo , Masculino , CamundongosRESUMO
OBJECTIVES: To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin ß1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). METHODS: Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion. RESULTS: Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively). CONCLUSIONS: Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.
Assuntos
Compostos Alílicos/farmacologia , Dissulfetos/farmacologia , Integrina beta1/fisiologia , Pós-Condicionamento Isquêmico , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Molécula-1 de Adesão Celular Endotelial a Plaquetas/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Integrina beta1/análise , Integrina beta1/genética , Masculino , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , RNA Mensageiro/análise , SuínosRESUMO
Colonic epithelial cells are highly polarised with a lumen-facing apical membrane, termed the brush border, and a basal membrane in contact with the underlying extracellular matrix (ECM). This polarity is often maintained in cancer tissue in the form of neoplastic glands and has prognostic value. We compared the cellular polarity of several ex vivo spheroid colonic cancer cultures with their parental tumours and found that those grown as non-attached colonies exhibited apical brush border proteins on their outer cellular membranes. Transfer of these cultures to an ECM, such as collagen, re-established the centralised apical polarity observed in vivo. The multidrug resistance protein ABCB1 also became aberrantly polarised to outer colony membranes in suspension cultures, unlike cultures grown in collagen, where it was polarised to central lumens. This polarity switch was dependent on the presence of serum or selected serum components, including epidermal growth factor (EGF), transforming growth factor-ß1 (TGF-ß1) and insulin-like growth factor-1 (IGF-1). The apical/basal orientation of primary cancer colon cultures cultured in collagen/serum was modulated by α2ß1 integrin signalling. The polarisation of ABCB1 in colonies significantly altered drug uptake and sensitivity, as the outward polarisation of ABCB1 in suspension colonies effluxed substrates more effectively than ECM-grown colonies with ABCB1 polarised to central lumens. Thus, serum-free suspension colonies were more resistant to a variety of anti-cancer drugs than ECM-grown colonies. In conclusion, the local stroma, or absence thereof, can have profound effects on the sensitivity of colorectal cultures to drugs that are ABCB1 substrates. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Polaridade Celular/fisiologia , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/fisiologia , Actinas/metabolismo , Colágeno/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Combinação de Medicamentos , Matriz Extracelular/metabolismo , Humanos , Integrina beta1/fisiologia , Laminina , Proteoglicanas , Transdução de Sinais/fisiologia , Esferoides Celulares/patologia , Células Tumorais CultivadasRESUMO
The molecular mechanisms underlying resistance to radiotherapy in breast cancer cells remain elusive. Previously, we reported that elevated ß1-integrin is associated with enhanced breast cancer cell survival postirradiation, but how ß1-integrin conferred radioresistance was unclear. Ionizing radiation (IR) induced cell killing correlates with the efficiency of DNA double-strand break (DSB) repair, and we found that nonmalignant breast epithelial (S1) cells with low ß1-integrin expression have a higher frequency of S-phase-specific IR-induced chromosomal aberrations than the derivative malignant breast (T4-2) cells with high ß1-integrin expression. In addition, there was an increased frequency of IR-induced homologous recombination (HR) repairosome focus formation in T4-2 cells compared with that of S1 cells. Cellular levels of Rad51 in T4-2 cells, a critical factor in HR-mediated DSB repair, were significantly higher. Blocking or depleting ß1-integrin activity in T4-2 cells reduced Rad51 levels, while ectopic expression of ß1-integrin in S1 cells correspondingly increased Rad51 levels, suggesting that Rad51 is regulated by ß1-integrin. The low level of Rad51 protein in S1 cells was found to be due to rapid degradation by the ubiquitin proteasome pathway (UPP). Furthermore, the E3 ubiquitin ligase RING1 was highly upregulated in S1 cells compared to T4-2 cells. Ectopic ß1-integrin expression in S1 cells reduced RING1 levels and increased Rad51 accumulation. In contrast, ß1-integrin depletion in T4-2 cells significantly increased RING1 protein levels and potentiated Rad51 ubiquitination. These data suggest for the first time that elevated levels of the extracellular matrix receptor ß1-integrin can increase tumor cell radioresistance by decreasing Rad51 degradation through a RING1-mediated proteasomal pathway.
Assuntos
Integrina beta1/fisiologia , Integrina beta1/efeitos da radiação , Rad51 Recombinase/metabolismo , Neoplasias da Mama , Linhagem Celular Tumoral , Sobrevivência Celular , DNA , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA/fisiologia , Feminino , Recombinação Homóloga/fisiologia , Humanos , Integrina beta1/metabolismo , Rad51 Recombinase/fisiologia , Radiação Ionizante , Reparo de DNA por Recombinação/fisiologiaRESUMO
Treatment of neuropathic pain (NP) continues to be a clinical challenge and the underlying mechanisms of NP remain elusive. More evidence suggests that glial cell line-derived neurotrophic factor (GDNF) has potent anti-nociceptive effects on NP, but the underlying mechanisms are still largely unknown. Recent data have shown that integrin ß1 plays an important part in NP induction, and that the activity of integrin ß1 signaling is associated with the phosphorylation of the conserved threonines in the cytoplasmic domain and recruitment of focal adhesion kinase (FAK) to the integrin ß1 tail and phosphorylation. We assessed the effect of GDNF on integrinß1/FAK signaling in NP states. Immunostaining results showed that integrin ß1 was mainly observed in the superficial dorsal horn in the spinal cord of rats, and was mostly expressed in intrinsic neurons. Expression of p-integrin ß1 and the phosphorylation of integrin ß1-associated FAK, but not integrin ß1 itself, was up-regulated after chronic constriction injury (CCI), which could be reversed by GDNF, and the effect of GDNF on integrin ß1/FAK signaling was inhibited by pre-treatment with RET function-blocking antibody (RET Ab). Moreover, pre-treatment with RET Ab could antagonize the effect of GDNF on inhibiting the NP induced by CCI. These data suggest that GDNF can regulate integrin ß1 activity via a RET-related mechanism.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Integrina beta1/fisiologia , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Analgésicos/farmacologia , Animais , Quinase 1 de Adesão Focal/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Integrina beta1/metabolismo , Integrinas/metabolismo , Integrinas/fisiologia , Masculino , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/fisiologia , Neuralgia/fisiopatologia , Neuroglia/metabolismo , Neuroglia/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-ret/metabolismo , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/lesões , Transdução de Sinais/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Cell adhesion to the extracellular matrix or to surrounding cells plays a key role in cell proliferation and differentiation and is critical for proper tissue homeostasis. An important pathway in adhesion-dependent cell proliferation is the Hippo signaling cascade, which is coregulated by the transcription factors Yes-associated protein 1 (YAP1) and transcriptional coactivator with PDZ-binding motif (TAZ). However, how cells integrate extracellular information at the molecular level to regulate YAP1's nuclear localization is still puzzling. Herein, we investigated the role of ß1 integrins in regulating this process. We found that ß1 integrin-dependent cell adhesion is critical for supporting cell proliferation in mesenchymal cells both in vivo and in vitro ß1 integrin-dependent cell adhesion relied on the relocation of YAP1 to the nucleus after the down-regulation of its phosphorylated state mediated by large tumor suppressor gene 1 and 2 (LATS1/2). We also found that this phenotype relies on ß1 integrin-dependent local activation of the small GTPase RAC1 at the plasma membrane to control the activity of P21 (RAC1)-activated kinase (PAK) of group 1. We further report that the regulatory protein merlin (neurofibromin 2, NF2) interacts with both YAP1 and LATS1/2 via its C-terminal moiety and FERM domain, respectively. PAK1-mediated merlin phosphorylation on Ser-518 reduced merlin's interactions with both LATS1/2 and YAP1, resulting in YAP1 dephosphorylation and nuclear shuttling. Our results highlight RAC/PAK1 as major players in YAP1 regulation triggered by cell adhesion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Genes da Neurofibromatose 2/fisiologia , Integrina beta1/fisiologia , Neurofibromina 2/metabolismo , Fosfoproteínas/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Adesão Celular , Proteínas de Ciclo Celular , Proliferação de Células , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Neurofibromina 2/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Sinalização YAP , Quinases Ativadas por p21/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Purpose: Previous research showed that the absence of ß1-integrin from the mouse lens after embryonic day (E) 13.5 (ß1MLR10) leads to the perinatal apoptosis of lens epithelial cells (LECs) resulting in severe microphthalmia. This study focuses on elucidating the molecular connections between ß1-integrin deletion and this phenotype. Methods: RNA sequencing was performed to identify differentially regulated genes (DRGs) in ß1MLR10 lenses at E15.5. By using bioinformatics analysis and literature searching, Egr1 (early growth response 1) was selected for further study. The activation status of certain signaling pathways (focal adhesion kinase [FAK]/Erk, TGF-ß, and Akt signaling) was studied via Western blot and immunohistochemistry. Mice lacking both ß1-integrin and Egr1 genes from the lenses were created (ß1MLR10/Egr1-/-) to study their relationship. Results: RNA sequencing identified 120 DRGs that include candidates involved in the cellular stress response, fibrosis, and/or apoptosis. Egr1 was investigated in detail, as it mediates cellular stress responses in various cell types, and is recognized as an upstream regulator of numerous other ß1MLR10 lens DRGs. In ß1MLR10 mice, Egr1 levels are elevated shortly after ß1-integrin loss from the lens. Further, pErk1/2 and pAkt are elevated in ß1MLR10 LECs, thus providing the potential signaling mechanism that causes Egr1 upregulation in the mutant. Indeed, deletion of Egr1 from ß1MLR10 lenses partially rescues the microphthalmia phenotype. Conclusions: ß1-integrin regulates the appropriate levels of Erk1/2 and Akt phosphorylation in LECs, whereas its deficiency results in the overexpression of Egr1, culminating in reduced cell survival. These findings provide insight into the molecular mechanism underlying the microphthalmia observed in ß1MLR10 mice.
Assuntos
Apoptose/fisiologia , Integrina beta1/fisiologia , Cristalino/metabolismo , Cristalino/patologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Fibrose , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Análise de Sequência de RNA , Estresse Fisiológico/fisiologiaRESUMO
Some bona fide adult adipocytes arise de novo from a bone marrow-derived myeloid lineage. These studies further demonstrate that adipose tissue stroma contains a resident population of myeloid cells capable of adipocyte and multilineage mesenchymal differentiation. These resident myeloid cells lack hematopoietic markers and express mesenchymal and progenitor cell markers. Because bone marrow mesenchymal progenitor cells have not been shown to enter the circulation, we hypothesized that myeloid cells acquire mesenchymal differentiation capacity in adipose tissue. We fabricated a 3-dimensional fibrin matrix culture system to define the adipose differentiation potential of adipose tissue-resident myeloid subpopulations, including macrophages, granulocytes and dendritic cells. Our data show that multilineage mesenchymal potential was limited to adipose tissue macrophages, characterized by the acquisition of adipocyte, osteoblast, chondrocyte and skeletal muscle myocyte phenotypes. Fibrin hydrogel matrices stimulated macrophage loss of hematopoietic cell lineage determinants and the expression of mesenchymal and progenitor cell markers, including integrin ß1. Ablation of integrin ß1 in macrophages inhibited adipocyte specification. Therefore, some bona fide adipocytes are specifically derived from adipose tissue-resident macrophages via an integrin ß1-dependent hematopoietic-to-mesenchymal transition, whereby they become capable of multipotent mesenchymal differentiation. The requirement for integrin ß1 highlights this molecule as a potential target for controlling the production of marrow-derived adipocytes and their contribution to adipose tissue development and function.
Assuntos
Integrina beta1/metabolismo , Integrina beta1/fisiologia , Células-Tronco Mesenquimais/fisiologia , Adipócitos/citologia , Adipogenia , Tecido Adiposo/citologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Células Cultivadas , Fibrina/metabolismo , Fibrina/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , Células Mieloides , Células-Tronco/citologiaRESUMO
Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating ß1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/fisiologia , Neurogênese/fisiologia , Planárias/fisiologia , Regeneração/fisiologia , Animais , Padronização Corporal , Adesão Celular , Diferenciação Celular , Proliferação de Células , Hibridização In Situ , Neurônios/citologia , Filogenia , Interferência de RNA , Transdução de Sinais , Células-Tronco/citologiaAssuntos
Adesão Celular/fisiologia , Granulócitos/patologia , Integrina beta1/fisiologia , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/patologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Doença Crônica , Humanos , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/metabolismoRESUMO
Animals capable of adult regeneration require specific signaling to control injury-induced cell proliferation, specification and patterning, but comparatively little is known about how the regeneration blastema assembles differentiating cells into well-structured functional tissues. Using the planarian Schmidtea mediterranea as a model, we identify ß1-integrin as a crucial regulator of blastema architecture. ß1-integrin(RNAi) animals formed small head blastemas with severe tissue disorganization, including ectopic neural spheroids containing differentiated neurons normally found in distinct organs. By mimicking aspects of normal brain architecture but without normal cell-type regionalization, these spheroids bore a resemblance to mammalian tissue organoids synthesized in vitro We identified one of four planarian integrin-alpha subunits inhibition of which phenocopied these effects, suggesting that a specific receptor controls brain organization through regeneration. Neoblast stem cells and progenitor cells were mislocalized in ß1-integrin(RNAi) animals without significantly altered body-wide patterning. Furthermore, tissue disorganization phenotypes were most pronounced in animals undergoing brain regeneration and not homeostatic maintenance or regeneration-induced remodeling of the brain. These results suggest that integrin signaling ensures proper progenitor recruitment after injury, enabling the generation of large-scale tissue organization within the regeneration blastema.
Assuntos
Encéfalo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/fisiologia , Planárias/fisiologia , Regeneração , Células-Tronco/citologia , Animais , Padronização Corporal/genética , Diferenciação Celular , Proliferação de Células , Neurogênese , Neurônios/citologia , Interferência de RNA , Transdução de SinaisRESUMO
Maspin is a non-inhibitory member of the serpin family that affects cell behaviours related to migration and survival. We have previously shown that peptides of the isolated G α-helix (G-helix) domain of maspin show bioactivity. Migration, invasion, adhesion and proliferation of vascular smooth muscle cells (VSMC) are important processes that contribute to the build-up of atherosclerotic plaques. Here we report the use of functional assays of these behaviours to investigate whether other maspin-derived peptides impact directly on VSMC; focusing on potential anti-atherogenic properties. We designed 18 new peptides from the structural moieties of maspin above ten amino acid residues in length and considered them beside the existing G-helix peptides. Of the novel peptides screened those with the sequences of maspin strand 4 and 5 of beta sheet B (S4B and S5B) reduced VSMC migration, invasion and proliferation, as well as increasing cell adhesion. A longer peptide combining these consecutive sequences showed a potentiation of responses, and a 7-mer contained all essential elements for functionality. This is the first time that these parts of maspin have been highlighted as having key roles affecting cell function. We present evidence for a mechanism whereby S4B and S5B act through ERK1/2 and AMP-activated protein kinase (AMPK) to influence VSMC responses.