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This study aimed to evaluate photobiomodulation (PBM) adjunct effect on pain, swelling, and face temperature in individuals with oral cleft submitted to alveolar ridge bone grafting (ABG). The sample consisted of 46 patients with either incomplete or complete unilateral cleft lip and palate aged from 9 to 18 years, both sexes, who underwent ABG with chin donor area. Subjects were randomly divided into 3 groups: (1) Control (C): ABG (n=17); (2) PBM: ABG with PBM (n=14); (3) Simulated photobiomodulation (SPBM): ABG with SPBM (n=15). Aluminum Gallium Arsenide (GaAlAs) infrared laser was used at 808±10 nm and power of 100 mW±20%. GaAlAs infrared laser was applied on 14 points in the subject's face at 4J per point, at 2 times: Immediate postoperative period and 24 hours postoperative. The authors evaluated pain, swelling, and face temperature at 24 hours postoperative; at 7-day postoperative, the authors evaluated pain, medication use, and swelling decreasing perception. No statistically significant difference occurred between groups C, PBM, and SPBM. At 24 hours postoperative, pain values showed statistically significant difference (P=0.04) between group C and SPBM. PBM at the tested parameters did not reduce pain and swelling and did not significantly change face temperature of individuals submitted to ARBG.
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Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.
Assuntos
Proteínas de Membrana/metabolismo , Teratogênicos/toxicidade , Talidomida/toxicidade , Células HEK293 , Humanos , Especificidade por SubstratoRESUMO
BACKGROUND: TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway. METHODS: We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays. RESULTS: We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8. CONCLUSIONS: In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.
Assuntos
Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Proteínas do Tecido Nervoso/genética , Proteína Supressora de Tumor p53/genética , Regiões 3' não Traduzidas , Animais , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Proteínas de Transporte/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , MicroRNAs/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The p63 transcription factor, homolog to the p53 tumor suppressor gene, plays a crucial role in epidermal and limb development, as its mutations are associated to human congenital syndromes characterized by skin, craniofacial and limb defects. While limb and skin-specific p63 transcriptional targets are being discovered, little is known of the post-translation modifications controlling ΔNp63α functions. Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions. Furthermore we show that Fibroblast Growth Factor-8 (FGF8), a morphogenetic signaling molecule essential for embryonic limb development, increases the binding of ΔNp63α to the tyrosine kinase c-Abl as well as the levels of ΔNp63α acetylation. Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway. This mutant ΔNp63α protein displays promoter-specific loss of DNA binding activity and consequent altered expression of development-associated ΔNp63α target genes. Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity. Hence, limb malformation-causing p63 mutations, such as the K193E mutation, are likely to result in aberrant limb development via the combined action of altered protein stability and altered promoter occupancy.
Assuntos
Anormalidades Congênitas/genética , Fator 8 de Crescimento de Fibroblasto/genética , Proteínas Proto-Oncogênicas c-abl/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Fatores de Transcrição de p300-CBP/genética , Animais , Linhagem Celular , Anormalidades Congênitas/embriologia , Anormalidades Congênitas/patologia , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Fator 8 de Crescimento de Fibroblasto/biossíntese , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/patologia , Camundongos , Mutação , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/biossíntese , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/metabolismo , Fatores de Transcrição de p300-CBP/biossíntese , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1-ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations.
Assuntos
Ectoderma/embriologia , Fator 8 de Crescimento de Fibroblasto/metabolismo , Proteínas de Homeodomínio/metabolismo , Deformidades Congênitas dos Membros/metabolismo , Peptidilprolil Isomerase/metabolismo , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Animais , Padronização Corporal , Linhagem Celular , Modelos Animais de Doenças , Ectoderma/metabolismo , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/genética , Humanos , Botões de Extremidades/embriologia , Deformidades Congênitas dos Membros/patologia , Camundongos , Peptidilprolil Isomerase de Interação com NIMA , Fosfoproteínas/genética , Estabilidade Proteica , Transativadores/genéticaRESUMO
The transcription factor interferon regulatory factor 6 (IRF6) regulates craniofacial development and epidermal proliferation. We recently showed that IRF6 is a component of a regulatory feedback loop that controls the proliferative potential of epidermal cells. IRF6 is transcriptionally activated by p63 and induces its proteasome-mediated down-regulation, thereby limiting keratinocyte proliferative potential. We hypothesized that IRF6 may also be involved in skin carcinogenesis. Hence, we analyzed IRF6 expression in a large series of squamous cell carcinomas (SCCs) and found a strong down-regulation of IRF6 that correlated with tumor invasive and differentiation status. IRF6 down-regulation in SCC cell lines and primary tumors correlates with methylation on a CpG dinucleotide island located in its promoter region. To identify the molecular mechanisms regulating IRF6 potential tumor suppressive activity, we performed a genome-wide analysis by combining ChIP sequencing for IRF6 binding sites and gene expression profiling in primary human keratinocytes after siRNA-mediated IRF6 depletion. We observed dysregulation of cell cycle-related genes and genes involved in differentiation, cell adhesion, and cell-cell contact. Many of these genes were direct IRF6 targets. We also performed in vitro invasion assays showing that IRF6 down-regulation promotes invasive behavior and that reintroduction of IRF6 into SCC cells strongly inhibits cell growth. These results indicate a function for IRF6 in suppression of tumorigenesis in stratified epithelia.
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Carcinoma de Células Escamosas/patologia , Regulação Neoplásica da Expressão Gênica , Fatores Reguladores de Interferon/fisiologia , Proteínas Supressoras de Tumor , Fenômenos Fisiológicos Celulares/genética , Proliferação de Células , Metilação de DNA , Humanos , Fatores Reguladores de Interferon/genética , Queratinócitos/patologia , Invasividade Neoplásica/genética , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.
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The objective of this study was to evaluate the efficacy of photobiomodulation in the bone regeneration of critical-sized defects (CSD) filled with inorganic bovine bone associated or not with collagen membranes. The study has been conducted on 40 critical defects in the calvaria of male rats, divided into four experimental groups (n = 10): (1) DBBM (deproteinized bovine bone mineral); (2) GBR (DBBM+collagen membrane); (3) DBBM+P (DBBM+photobiomodulation); and (4) GBR+P (GBR+photobiomodulation). At 30 days postoperative, the animals were euthanized, and after the tissue had been processed, histological, histometric, and statistical analyses were performed. The analyses have taken into account newly formed bone area (NBA), linear bone extension (LBE), and residual particle area (RPA) as variables. The Kruskal-Wallis test has been performed, followed by the Dwass-Steel-Critchlow-Fligner test for comparison between groups (p < 0.05). When the DBBM+P group was compared to the DBBM group, it was possible to observe significant statistical differences in all the variables analyzed (p < 0.05). The application of photobiomodulation in guided bone regeneration (GBR+P) has shown a decrease in the median value for the RPA variable (26.8) when compared to the GBR group (32.4), with a significant statistical difference; however, for NBA and LBE, the therapy has not provided significant results.
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Tight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the DeltaNp63alpha protein. We found that MDM2 binds DeltaNp63alpha in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for DeltaNp63alpha nuclear export and subsequent degradation, whereas the MDM2 ring-finger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of DeltaNp63alpha by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the alpha and beta tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous DeltaNp63alpha in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative DeltaNp63alpha protein.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas F-Box/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/efeitos da radiação , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA/genética , Doxorrubicina/farmacologia , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Humanos , Camundongos , Mutação/genética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Interferente Pequeno/genética , Transativadores/genética , Fatores de Transcrição , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Raios Ultravioleta/efeitos adversosRESUMO
PURPOSE: This study evaluated the effect of two photobiomodulation therapy protocols on bone regeneration in criticalsize bone defects grafted with inorganic bovine bone. MATERIALS AND METHODS: A critical-size defect was created in 30 adult male rat calvaria, which were divided equally and randomly into three experimental groups (n = 10): (1) DBBM (deproteinized bovine bone mineral); (2) DBBM + PBMT 4 J (4 J; photobiomodulation therapy; GaAlAs, 730 nm, 100 mW, 140 J/cm2); and (3) DBBM + PBMT 6 J (6 J; GaAlAs, 730 nm, 100 mW, 210 J/cm2). Animals were euthanized after 30 days. The neoformed bone area (NBA), linear bone extension (LBE), and area of the remaining particles (ARP) were evaluated. The data were subjected to nonparametric Kolmogorov-Smirnov test and ANOVA, followed by Tukey post hoc test to identify differences between the groups (P < .05). RESULTS: The 6 J group showed the highest average NBA (48.57% ± 28.22%) and demonstrated a statistically significant difference in NBA and LBE. A higher mean ARP was found in the DBBM group (38.73 ± 6.95) than in the groups irradiated by photobiomodulation therapy, with statistically significant differences (P < .05). CONCLUSION: The 6 J protocol showed the best results, promoting greater bone formation with greater resorption of residual particles.
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Produtos Biológicos , Terapia com Luz de Baixa Intensidade , Masculino , Animais , Bovinos , Ratos , Cicatrização , MineraisRESUMO
The TP53 tumor suppressor gene is known as the guardian of the genome, playing a pivotal role in controlling genome integrity, and its functions are lost in more than 50% of human tumors due to somatic mutations. This percentage rises to 90% if mutations and alterations in the genes that code for regulators of p53 stability and activity are taken into account. Renal cell carcinoma (RCC) is a clear example of cancer that despite having a wild-type p53 shows poor prognosis because of the high rate of resistance to radiotherapy or chemotherapy, which leads to recurrence, metastasis and death. Remarkably, the fact that p53 is poorly mutated does not mean that it is functionally active, and increasing experimental evidences have demonstrated this. Therefore, RCC represents an extraordinary example of the importance of p53 pathway alterations in therapy resistance. The search for novel molecular biomarkers involved in the pathways that regulate altered p53 in RCC is mandatory for improving early diagnosis, evaluating the prognosis and developing novel potential therapeutic targets for better RCC treatment.
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The p53-related transcription factor p63 is critically important for basic cellular functions during development of the ectoderm and derived structure and tissues, including skin, limb, palate, and hair. On the one side, p63 is required to sustain the proliferation of keratinocyte progenitors, while on the other side it is required for cell stratification, commitment to differentiate, cell adhesion, and epithelial-mesenchymal signaling. Molecules that are components or regulators of the p63 pathway(s) are rapidly being identified, and it comes with no surprise that alterations in the p63 pathway lead to congenital conditions in which the skin and other ectoderm-derived structures are affected. In this paper, we summarize the current knowledge of the molecular and cellular regulations centered on p63, derived from the comprehension of p63-linked human diseases and the corresponding animal models, as well as from cellular models and high-throughput molecular approaches. We point out common themes and features, that allow to speculate on the possible role of p63 downstream events and their potential exploitation in future attempts to correct the congenital defect in preclinical studies.
Assuntos
Anormalidades Congênitas/genética , Ectoderma/crescimento & desenvolvimento , Ectoderma/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Cabelo/crescimento & desenvolvimento , Humanos , Redes e Vias Metabólicas , Camundongos , Palato/crescimento & desenvolvimento , Pele/crescimento & desenvolvimento , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
The superfamily of TRIM (TRIpartite Motif-containing) proteins is one of the largest groups of E3 ubiquitin ligases. Among them, interest in TRIM8 has greatly increased in recent years. In this review, we analyze the regulation of TRIM8 gene expression and how it is involved in many cell reactions in response to different stimuli such as genotoxic stress and attacks by viruses or bacteria, playing a central role in the immune response and orchestrating various fundamental biological processes such as cell survival, carcinogenesis, autophagy, apoptosis, differentiation and inflammation. Moreover, we show how TRIM8 functions are not limited to ubiquitination, and contrasting data highlight its role either as an oncogene or as a tumor suppressor gene, acting as a "double-edged weapon". This is linked to its involvement in the selective regulation of three pivotal cellular signaling pathways: the p53 tumor suppressor, NF-κB and JAK-STAT pathways. Lastly, we describe how TRIM8 dysfunctions are linked to inflammatory processes, autoimmune disorders, rare developmental and cardiovascular diseases, ischemia, intellectual disability and cancer.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitinação/genética , HumanosRESUMO
p63, a transcription factor related to the p53 tumor suppressor, plays a key role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (DeltaN) a transactivation domain. DeltaNp63alpha is the most widely expressed isoform, at all stages of development and in adult tissues. It supports the regenerative capacity of basal keratinocytes and its upregulation is a hallmark of human squamous carcinomas. To get insight into the complex biology of DeltaNp63alpha, we set out to identify DeltaNp63alpha interacting proteins by co-immunoprecipitation in mammalian cells and mass spectrometry analysis. A total of 49 potential DeltaNp63alpha binding proteins, including several heterogeneous ribonucleoproteins (hnRNPs), were identified. Integration of the proteomic data with a Human Coexpression Network highlighted 5 putative p63 protein interactors whose expression is significantly comodulated with p63: hnRNPA/B, hnRNPK, hnRNPQ, FUS/TLS and Keratin 5. hnRNPA/B was already described as a p63 partner, but the others were novel. Interaction of DeltaNp63alpha with hnRNPQ, hnRNPK and FUS/TLS was confirmed by reciprocal co-immunoprecipitations in human keratinocytes. The finding that DeltaNp63alpha exists in complexes with several RNA-binding proteins lays the premises for the analysis of the role of DeltaNp63alpha in mRNA metabolism and transport.
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Mapeamento de Interação de Proteínas/métodos , Espectrometria de Massas em Tandem/métodos , Transativadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Bases de Dados de Proteínas , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Isoformas de Proteínas , Proteínas/química , Proteínas/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/metabolismo , Transativadores/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/químicaRESUMO
The p53 gene family network plays a pivotal role in the control of many biological processes and therefore the right balance between the pro-apoptotic and pro-survival isoforms is key to maintain cellular homeostasis. The stability of the p53 tumor suppressor protein and that of oncogenic ΔNp63α, is crucial to control cell proliferation. The aberrant expression of p53 tumor suppressor protein and oncogenic ΔNp63α contributes to tumorigenesis and significantly affects anticancer drug response. Recently, we demonstrated that TRIM8 increases p53 stability, potentiating its tumor suppressor activity. In this paper, we show that TRIM8 simultaneously reduces the level of the pro-proliferative ΔNp63α protein, in both a proteasomal and caspase-1 dependent way, thereby playing a critical role in the cellular response to DNA damaging agents. Moreover, we provided evidence that ΔNp63α in turn, suppresses TRIM8 gene expression by preventing p53-mediated transactivation of TRIM8, therefore suggesting the existence of a negative feedback loop. These findings indicate that TRIM8 exerts its anticancer power through a joint action that provides on one hand, the activation of the p53 tumor suppressor role, and on the other the quenching of the oncogenic ΔNp63α protein activity. The enhancement of TRIM8 activity may offer therapeutic benefits and improve the management of chemoresistant tumors.
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ΔNp63α is finely and strictly regulated during embryogenesis and differentiation. ΔNp63α is the only p63 isoform degraded by the proteasome after Ubiquitin and SUMO (Small Ubiquitin-like MOdifier) conjugation. Here, we show that p63 ubiquitylation per se is not the signal triggering p63 proteasomal degradation. Taking advantage of natural ΔNp63α mutants isolated by patients with Split Hand and Foot Malformation IV syndrome, we found that SUMO and Ub modifications are not redundant and both are required to guarantee efficient ΔNp63α degradation. Here, we present evidence that sumoylation and ubiquitylation of ΔNp63α are strongly intertwined, and none of the two can efficiently occur if the other is impaired.
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Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Deformidades Congênitas dos Membros/genética , Peso Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
The repair of bone defects raises the interest of investigators in several health specialties. Grafting techniques with bone substitutes and laser therapies have been investigated to replace autogenous bone and accelerate the bone healing process. Objective To evaluate the effect of photobiomodulation therapy (PBMT) associated with guided bone regeneration (GBR) in critical size defects. Material and Methods The study was conducted on 80 male rats (Rattus norvegicus albinus, Wistar) submitted to surgical creation of a critical size defect on the calvaria, divided into eight study groups: group C (control - only blood clot); group M (collagen membrane); group PBMT (photobiomodulation therapy); group AB (autogenous bone); group AB+PBMT; group AB+M; group PBMT+M; group AB+PBMT+M. The animals were killed 30 days postoperatively. After tissue processing, bone regeneration was evaluated by histomorphometric analysis and statistical analyses were performed (Tukey test, p<0.05). Results All groups had greater area of newly formed bone compared to group C (9.96±4.49%). The group PBMT+M (achieved the greater quantity of new bone (64.09±7.62%), followed by groups PBMT (47.67±8.66%), M (47.43±15.73%), AB+PBMT (39.15±16.72%) and AB+PBMT+M (35.82±7.68%). After group C, the groups AB (25.10±16.59%) and AB+M (22.72±13.83%) had the smallest quantities of newly formed bone. The area of remaining particles did not have statistically significant difference between groups AB+M (14.93±8.92%) and AB+PBMT+M (14.76±6.58%). Conclusion The PBMT utilization may be effective for bone repair, when associated with bone regeneration techniques.
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Regeneração Óssea/efeitos da radiação , Regeneração Tecidual Guiada/métodos , Terapia com Luz de Baixa Intensidade/métodos , Animais , Autoenxertos , Regeneração Óssea/fisiologia , Colágeno/análise , Masculino , Osteogênese/fisiologia , Osteogênese/efeitos da radiação , Distribuição Aleatória , Ratos Wistar , Valores de Referência , Reprodutibilidade dos Testes , Crânio/fisiologia , Crânio/efeitos da radiação , Crânio/cirurgia , Resultado do Tratamento , Cicatrização/fisiologia , Cicatrização/efeitos da radiaçãoRESUMO
The ARF/MDM2/p53 pathway is a principal defense mechanism to protect the organism from uncontrolled effects of deregulated oncogenes. Oncogenes activate ARF, which interacts with and inhibits the ubiquitin ligase MDM2, resulting in p53 stabilization and activation. Once stabilized and activated, p53 can either induce or repress a wide array of different gene targets, which in turn can regulate cell cycle, DNA repair, and a number of apoptosis-related genes. Here we show that, unlike p53, p63, a member of the p53 family, directly interacts with p14(ARF). Through this interaction ARF inhibits p63-mediated transactivation and transrepression. In p63-transfected cells, ARF, which normally localizes into nucleoli, accumulates in the nucleoplasm. Based on these observations, we suggest that stimuli inducing p14(ARF) expression can, at the same time, activate p53 and impair p63 transcriptional activity, altering the pattern of p53 target gene expression. Here we show, for the first time, a physical and functional link between the p14(ARF) tumor suppressor protein and p63, a member of the p53 family.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Células COS , Chlorocebus aethiops , Camundongos , Mutação , Células NIH 3T3 , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Transativadores/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
p63 is a transcription factor structurally related to the p53 tumor suppressor. The C-terminal region differs from p53's in that it contains a sterile alpha motif (SAM) domain and is subject to multiple alternative splicings. The N-terminal region is present in the transactivation (TA) and DeltaN configurations, with the latter lacking the transcriptional activation domain 1. Single amino acid substitutions and frameshift mutations of p63 cause the human ankyloblepharon ectodermal dysplasia clefting (AEC) or ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndromes. We have systematically compared the activities of the wild-type p63 isoforms and of the natural mutants in activation and repression assays on three promoters modulated by p53. We found that p63 proteins with an altered SAM domain or no SAM domain-the beta isoforms, the EEC frameshift mutant, and the missense AEC mutations-all showed a distinctly higher level of activation of the MDM2 promoter and decreased repression on the HSP70 promoter. Fusion of SAM to the GAL4 DNA-binding domain repressed a heterologous promoter. A second activation domain, TA2, corresponding to exons 11 to 12, was uncovered by comparing the activation of DeltaN isoforms on natural promoters and in GAL4 fusion systems. In colony formation assays, the AEC mutants, but not the EEC frameshift, were consistently less efficient in suppressing growth, in both the TA version and the DeltaN version, with respect to their p63alpha counterparts. These data highlight the modularity of p63, identifying the SAM domain as a dominant transcriptional repression module and indicating that the AEC and EEC frameshift mutants are characterized by a subversion of the p63 transcriptional potential.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana , Mutação , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Transcrição Gênica , Processamento Alternativo , Animais , Linhagem Celular , Proteínas de Ligação a DNA , Éxons/genética , Genes Supressores de Tumor , Humanos , Fosfoproteínas/genética , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transativadores/genética , Fatores de Transcrição , Proteínas Supressoras de TumorRESUMO
A fotobiomodulação sistêmica (FBM-S) consiste em uma técnica que utiliza o laser de baixa intensidade no espectro vermelho da luz para irradiação sistêmica. Seus benefícios incluem efeito analgésico, antioxidante sistêmico e anti-inflamatório, ativação de células imunológicas, melhora na cicatrização, vasodilatação e aumento da microcirculação. A técnica original, que utiliza cateter e fibra óptica para irradiação sistêmica, é uma técnica invasiva, por isso a fotobiomodulação sistêmica transdérmica foi desenvolvida como uma alternativa. Assim, o objetivo dessa revisão de literatura é discutir os efeitos, aplicações, protocolos e efeitos colaterais desta terapia modificada. Para tanto, foi realizada uma busca na literatura nas bases de dados Pubmed, Bireme, Embase, Scopus, Science Direct, Web of Science e CENTRAL, sem restrição de idioma no período entre 2010 e 2021. Encontraram-se seis estudos sendo um na área da Odontologia. Os resultados desses estudos sugerem que a FBS-S pode ser utilizada para o tratamento de condições sistêmicas. Em Odontologia, no entanto, a literatura ainda é escassa e mais estudos clínicos randomizados controlados são necessários para comprovar seus efeitos e estabelecer um protocolo clínico para sua utilização.
Systemic photobiomodulation (PBM-S) is a technique that uses low-level laser in the red spectrum of light for systemic irradiation. Its benefits include analgesic, systemic antioxi-dant, and anti-inflammatory effect, activation of immune cells, improved healing, vasodilation, and increased microcirculation. The original technique, which uses catheter and optical fibers for systemic irradiation is an invasive technique. Thus, the transdermal systemic photobiomodulation was developed as an alternative. The purpose of this literature review is to discuss the effects, applications, protocols, and side effects of this modified therapy. A literature search was carried out on Pubmed, Bireme, Embase, Scopus, Science Direct, Web of Science, and CENTRAL databases, with no language restriction in the period be-tween 2010 and 2021. Six studies were found, one in the area of Dentistry. The results of these studies suggest that PBM-S can be used for the treatment of systemic conditions. In Dentistry, however, the literature is still scarce and more randomized controlled clinical trials are needed to prove its effects and establish a protocol for its use.