RESUMEN
Osteosarcomas are immune-resistant and metastatic as a result of elevated nonsense-mediated RNA decay (NMD), reactive oxygen species (ROS), and epithelial-to-mesenchymal transition (EMT). Although vitamin D has anti-cancer effects, its effectiveness and mechanism of action against osteosarcomas are poorly understood. In this study, we assessed the impact of vitamin D and its receptor (VDR) on NMD-ROS-EMT signaling in in vitro and in vivo osteosarcoma animal models. Initiation of VDR signaling facilitated the enrichment of EMT pathway genes, after which 1,25(OH)2D, the active vitamin D derivative, inhibited the EMT pathway in osteosarcoma subtypes. The ligand-bound VDR directly downregulated the EMT inducer SNAI2, differentiating highly metastatic from low metastatic subtypes and 1,25(OH)2D sensitivity. Moreover, epigenome-wide motif and putative target gene analysis revealed the VDR's integration with NMD tumorigenic and immunogenic pathways. In an autoregulatory manner, 1,25(OH)2D inhibited NMD machinery genes and upregulated NMD target genes implicated in anti-oncogenic activity, immunorecognition, and cell-to-cell adhesion. Dicer substrate siRNA knockdown of SNAI2 revealed superoxide dismutase 2 (SOD2)-mediated antioxidative responses and 1,25(OH)2D sensitization via non-canonical SOD2 nuclear-to-mitochondrial translocalization leading to overall ROS suppression. In a mouse xenograft metastasis model, the therapeutically relevant vitamin D derivative calcipotriol inhibited osteosarcoma metastasis and tumor growth shown for the first time. Our results uncover novel osteosarcoma-inhibiting mechanisms for vitamin D and calcipotriol that may be translated to human patients.
RESUMEN
In this review, we summarize the most recent advances in vitamin D cancer research to provide molecular clarity, as well as its translational trajectory across the cancer landscape. Vitamin D is well known for its role in regulating mineral homeostasis; however, vitamin D deficiency has also been linked to the development and progression of a number of cancer types. Recent epigenomic, transcriptomic, and proteomic studies have revealed novel vitamin D-mediated biological mechanisms that regulate cancer cell self-renewal, differentiation, proliferation, transformation, and death. Tumor microenvironmental studies have also revealed dynamic relationships between the immune system and vitamin D's anti-neoplastic properties. These findings help to explain the large number of population-based studies that show clinicopathological correlations between circulating vitamin D levels and risk of cancer development and death. The majority of evidence suggests that low circulating vitamin D levels are associated with an increased risk of cancers, whereas supplementation alone or in combination with other chemo/immunotherapeutic drugs may improve clinical outcomes even further. These promising results still necessitate further research and development into novel approaches that target vitamin D signaling and metabolic systems to improve cancer outcomes.
Asunto(s)
Antineoplásicos , Neoplasias , Deficiencia de Vitamina D , Humanos , Vitamina D/metabolismo , Proteómica , Vitaminas/uso terapéutico , Neoplasias/tratamiento farmacológico , Deficiencia de Vitamina D/complicaciones , Deficiencia de Vitamina D/tratamiento farmacológico , Receptores de Calcitriol/metabolismoRESUMEN
The 24th Workshop on Vitamin D was held September 7-9, 2022 in Austin, Texas and covered a wide diversity of research in the vitamin D field from across the globe. Here, we summarize the meeting, individual sessions, awards and presentations given.
Asunto(s)
Deficiencia de Vitamina D , Vitamina D , Humanos , VitaminasRESUMEN
Osteosarcomas are immune-resistant and metastatic as a result of elevated nonsense-mediated RNA decay (NMD), reactive oxygen species (ROS), and epithelial-to-mesenchymal transition (EMT). Although vitamin D has anti-cancer effects, its effectiveness and mechanism of action against osteosarcomas are poorly understood. In this study, we assessed the impact of vitamin D and its receptor (VDR) on the NMD-ROS-EMT signaling axis in in vitro and in vivo osteosarcoma animal models. Initiation of VDR signaling facilitated the enrichment of EMT pathway genes, after which 1,25(OH) 2 D, the active vitamin D derivative, inhibited the EMT pathway in osteosarcoma subtypes. The ligand-bound VDR directly downregulated the EMT inducer SNAI2 , differentiating highly metastatic from low metastatic subtypes and 1,25(OH) 2 D sensitivity. Moreover, epigenome-wide motif and putative target gene analysis revealed the VDRâ™s integration with NMD tumorigenic and immunogenic pathways. In an autoregulatory manner, 1,25(OH) 2 D inhibited NMD machinery genes and upregulated NMD target genes implicated in anti-oncogenic activity, immunorecognition, and cell-to-cell adhesion. Dicer substrate siRNA knockdown of SNAI2 revealed superoxide dismutase 2 (SOD2)-mediated antioxidative responses and 1,25(OH) 2 D sensitization via non-canonical SOD2 nuclear-to-mitochondrial translocalization leading to overall ROS suppression. In a mouse xenograft metastasis model, the therapeutically relevant vitamin D derivative calcipotriol inhibited osteosarcoma metastasis and tumor growth shown for the first time. Our results uncover novel osteosarcoma-inhibiting mechanisms for vitamin D and calcipotriol that may be translated to human patients.
RESUMEN
Tissue wounding induces cutaneous sensory axon regeneration via hydrogen peroxide (H2O2) that is produced by the epithelial NADPH oxidase, Duox1. Sciatic nerve injury instead induces axon regeneration through neuronal uptake of the NADPH oxidase, Nox2, from macrophages. We therefore reasoned that the tissue environment in which axons are damaged stimulates distinct regenerative mechanisms. Here, we show that cutaneous axon regeneration induced by tissue wounding depends on both neuronal and keratinocyte-specific mechanisms involving H2O2 signaling. Genetic depletion of H2O2 in sensory neurons abolishes axon regeneration, whereas keratinocyte-specific H2O2 depletion promotes axonal repulsion, a phenotype mirrored in duox1 mutants. Intriguingly, cyba mutants, deficient in the essential Nox subunit, p22Phox, retain limited axon regenerative capacity but display delayed Wallerian degeneration and axonal fusion, observed so far only in invertebrates. We further show that keratinocyte-specific oxidation of the epidermal growth factor receptor (EGFR) at a conserved cysteine thiol (C797) serves as an attractive cue for regenerating axons, leading to EGFR-dependent localized epidermal matrix remodeling via the matrix-metalloproteinase, MMP-13. Therefore, wound-induced cutaneous axon de- and regeneration depend on the coordinated functions of NADPH oxidases mediating distinct processes following injury.
Asunto(s)
Axones , Peróxido de Hidrógeno , NADPH Oxidasas , Regeneración Nerviosa , Cicatrización de Heridas , Proteínas de Pez Cebra , Animales , Axones/fisiología , Peróxido de Hidrógeno/metabolismo , Queratinocitos/fisiología , NADPH Oxidasas/genética , NADPH Oxidasas/fisiología , Regeneración Nerviosa/genética , Células Receptoras Sensoriales/fisiología , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiología , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
The ability of an animal to regenerate lost tissue and body parts has obviously life-saving implications. Understanding how this ability became restricted or active in specific animal lineages will help us understand our own regeneration. According to phylogenic analysis, the glial cell line-derived neurotrophic factor (GDNF) signaling pathway, but not other family members, is conserved in axolotls, a salamander with remarkable regenerative capacity. Furthermore, comparing the pro-regenerative Spiny mouse to its less regenerative descendant, the House mouse, revealed that the GDNF signaling pathway, but not other family members, was induced in regenerating Spiny mice. According to GDNF receptor expression analysis, GDNF may promote hair follicle neogenesis - an important feature of skin regeneration - by determining the fate of dermal fibroblasts as part of new hair follicles. These findings support the idea that GDNF treatment will promote skin regeneration in humans by demonstrating the GDNF signaling pathway's ancestral and cellular nature.
RESUMEN
We propose that GDNF, a glial cell line-derived neurotrophic factor, can promote hair follicle neogenesis and skin regeneration after wounding by directing the fate of dermal fibroblasts. Our hypothesis is largely based on detailed GDNF and receptor analysis during skin regenerative stages, as well as the induction of GDNF receptors after wounding between the pro-regenerative spiny mouse (genus Acomys) and its less-regenerative descendant, the house mouse (Mus musculus). To characterize the GDNF-target cells, we will conduct a series of lineage-tracing experiments in conjunction with single-cell RNA and assay for transposase-accessible chromatin sequencing experiments. The heterogenetic dynamics of skin regeneration have yet to be fully defined, and this research will help to advance the fields of regenerative medicine and biology. Finally, we believe that stimulating the GDNF signalling pathway in fibroblasts from less-regenerative animals, such as humans, will promote skin regeneration, morphogenesis and scarless wound healing.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial , Piel , Animales , Fibroblastos , Cabello , Regeneración , Factor de Crecimiento Transformador beta , Cicatrización de Heridas/genéticaRESUMEN
The relationship between the active form of vitamin D3 (1,25-dihydroxyvitamin D, 1,25(OH)2D) and reactive oxygen species (ROS), two integral signaling molecules of the cell, is poorly understood. This is striking, given that both factors are involved in cancer cell regulation and metabolism. Mitochondria (mt) dysfunction is one of the main drivers of cancer, producing more mitochondria, higher cellular energy, and ROS that can enhance oxidative stress and stress tolerance responses. To study the effects of 1,25(OH)2D on metabolic and mt dysfunction, we used the vitamin D receptor (VDR)-sensitive MG-63 osteosarcoma cell model. Using biochemical approaches, 1,25(OH)2D decreased mt ROS levels, membrane potential (ΔΨmt), biogenesis, and translation, while enforcing endoplasmic reticulum/mitohormetic stress adaptive responses. Using a mitochondria-focused transcriptomic approach, gene set enrichment and pathway analyses show that 1,25(OH)2D lowered mt fusion/fission and oxidative phosphorylation (OXPHOS). By contrast, mitophagy, ROS defense, and epigenetic gene regulation were enhanced after 1,25(OH)2D treatment, as well as key metabolic enzymes that regulate fluxes of substrates for cellular architecture and a shift toward non-oxidative energy metabolism. ATACseq revealed putative oxi-sensitive and tumor-suppressing transcription factors that may regulate important mt functional genes such as the mTORC1 inhibitor, DDIT4/REDD1. DDIT4/REDD1 was predominantly localized to the outer mt membrane in untreated MG-63 cells yet sequestered in the cytoplasm after 1,25(OH)2D and rotenone treatments, suggesting a level of control by membrane depolarization to facilitate its cytoplasmic mTORC1 inhibitory function. The results show that 1,25(OH)2D activates distinct adaptive metabolic responses involving mitochondria to regain redox balance and control the growth of osteosarcoma cells. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMEN
MicroRNAs (miRNAs) are a major class of conserved non-coding RNAs that have a wide range of functions during development and disease. Biogenesis of canonical miRNAs depend on the cytoplasmic processing of pre-miRNAs to mature miRNAs by the Dicer endoribonuclease. Once mature miRNAs are generated, the miRNA-induced silencing complex (miRISC), or miRISC, incorporates one strand of miRNAs as a template for recognizing complementary target messenger RNAs (mRNAs) to dictate post-transcriptional gene expression. Besides regulating miRNA biogenesis, Dicer is also part of miRISC to assist in activation of the complex. Dicer associates with other regulatory miRISC co-factors such as trans-activation responsive RNA-binding protein 2 (Tarbp2) to regulate miRNA-based RNA interference. Although the functional role of miRNAs within epidermal keratinocytes has been extensively studied within embryonic mouse skin, its contribution to the normal function of hair follicle bulge stem cells (BSCs) during post-natal hair follicle development is unclear. With this question in mind, we sought to ascertain whether Dicer-Tarpb2 plays a functional role within BSCs during induced anagen development by utilizing conditional knockout mouse models. Our findings suggest that Dicer, but not Tarbp2, functions within BSCs to regulate induced anagen (growth phase) development of post-natal hair follicles. These findings strengthen our understanding of miRNA-dependency within hair follicle cells during induced anagen development.
RESUMEN
Glial-cell-derived neurotrophic factor (GDNF) is a well-studied neuroregenerative factor; however, the degree to which it supports hair formation and skin wound repair is not known. By using a Gfra1 (GDNF family receptor alpha 1) knock-in reporter mouse line, GDNF signaling was found to occur within hair bulge stem cells (BSCs) during the initiation of the hair cycle and early stages of hair formation after depilation. Both recombinant and transgene overexpression of GDNF promoted BSC colony growth, hair formation, and skin repair after wounding through enhanced self-renewal of BSCs and commitment of BSC-derived progenitors into becoming epidermal cells at the injury site. Conditional ablation of Gfra1 among BSCs impaired the onset of the hair cycle, while conditional ablation of the GDNF family member signal transducer, Ret, within BSCs prevented the onset of the hair cycle and depilation-induced anagen development of hair follicles. Our findings reveal that GDNF promotes hair formation and wound repair and that bulge stem cells are critical mediators of both.
RESUMEN
Sunlight exposure of the skin is associated with both risks and benefits. On one hand, sunlight ultraviolet (UV) radiation can cause skin cancer through signature DNA mutations. On the other hand, it can be absorbed in the skin by 7-dehydrocholesterol to instigate endogenous synthesis of vitamin D to regulate anticancer effects. Thus, protecting one's skin from sunlight to avoid skin cancer may lead to impaired vitamin D levels arguing for sensible sun exposure practices. To limit cancer, vitamin D metabolites can promote uncharacterized and diverse sets of events such as repair responses to DNA damage, apoptosis of malignant cells, and suppression of immune surveillance, proliferation and angiogenesis. Recent findings also suggest that part of the anticancer effects of vitamin D within squamous cell carcinoma-a type of skin cancer most directly linked to sun exposure-involves the DDIT4-mTOR catabolic signalling pathway to enhance cell autophagy. As mTOR activity and cellular metabolism are modulated as part of the DNA damage response, insights into the means by which mTOR can be controlled by vitamin D to suppress cancer is of molecular and clinical importance. Overall, the research so far suggests that presence of vitamin D through sunlight exposure and supplementation are beneficial for human health in the face of cancer.
Asunto(s)
Neoplasias Cutáneas , Vitamina D , Humanos , Piel , Luz Solar , Rayos Ultravioleta , VitaminasRESUMEN
Cutaneous squamous cell carcinoma (SCC) is one of the most common non-melanoma skin cancers worldwide. While its exact tumorigenesis mechanisms is far from well-established and less satisfied therapeutic strategy can be clinically used nowadays. In this study, we intended to investigate the role of DNA damage-inducible transcript 4 (DDIT4) in human SCC. Firstly, we identified DDIT4 is significantly suppressed in human SCC tissue and cultured A431 cell line, and reduced DDIT4 accelerates keratinocytes proliferation but impedes the autophagy flux through mTORC1 pathway by affecting the downstream S6 Kinase1, 4E-BP1, Beclin1 and LC3 II/I. While 1,25(OH)2 D3 enhanced DDIT4 expression and activated autophagy and inhibit mTORC1 to take the effect of anti-proliferation and activating autophagy. Further, formation of direct vitamin D receptor (VDR)-DDIT4 transcription complex was verified by ChIP-qPCR, which showed the molecular mechanism of how 1,25(OH)2 D3 promotes DDIT4 transcription. Thirdly, xenograft tumor-bearing mice model treated by gradient concentrations of 1,25(OH)2 D3 revealed the obvious anti-carcinoma effect of 1,25(OH)2 D3 in vivo and DDIT4 acted the molecular vector of 1,25(OH)2 D3 through mTORC1. Lastly, elevated DDIT4 expression was verified in human actinic keratoses tissue, and chronic long-term ultraviolet (UV) irradiation on mouse disclosed UV could promote DDIT4 expression inside epidermis. Conclusively, our research suggested a novel molecular mechanism about the human SCC tumorigenesis and the pharmacological mechanism about how 1,25(OH)2 D3 take its anti-carcinoma role on human SCC, as well as a striking paradoxes that how UV irradiation plays the tumorigenesis effect but synchronously take a protective role in the early stage of SCC carcinogenesis.
Asunto(s)
Anticarcinógenos/farmacología , Calcitriol/farmacología , Carcinoma de Células Escamosas/metabolismo , Factores de Transcripción/metabolismo , Animales , Autofagia , Línea Celular Tumoral , Proliferación Celular , Colecalciferol/metabolismo , Daño del ADN , Femenino , Humanos , Queratinocitos/citología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Receptores de Calcitriol/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Rayos UltravioletaRESUMEN
Although the functions of H2O2 in epidermal wound repair are conserved throughout evolution, the underlying signaling mechanisms are largely unknown. In this study we used human keratinocytes (HEK001) to investigate H2O2-dependent wound repair mechanisms. Scratch wounding led to H2O2 production in two or three cell layers at the wound margin within â¼30â min and subsequent cysteine modification of proteins via sulfenylation. Intriguingly, exogenous H2O2 treatment resulted in preferential sulfenylation of keratinocytes that adopted a migratory phenotype and detached from neighboring cells, suggesting that one of the primary functions of H2O2 is to stimulate signaling factors involved in cell migration. Based on previous findings that revealed epidermal growth factor receptor (EGFR) involvement in H2O2-dependent cell migration, we analyzed oxidation of a candidate upstream target, the inhibitor of κB kinase α (IKKα; encoded by CHUK), as a mechanism of action. We show that IKKα is sulfenylated at a conserved cysteine residue in the kinase domain, which correlates with de-repression of EGF promoter activity and increased EGF expression. Thus, this indicates that IKKα promotes migration through dynamic interactions with the EGF promoter depending on the redox state within cells.
Asunto(s)
Movimiento Celular , Quinasa I-kappa B/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cisteína/metabolismo , Factor de Crecimiento Epidérmico/genética , Humanos , Peróxido de Hidrógeno , Quinasa I-kappa B/química , Oxidación-Reducción , Regiones Promotoras Genéticas/genética , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Pez CebraRESUMEN
Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Osteocitos/metabolismo , Empalme del ARN , Transcripción Genética , Vitamina D/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Osteocitos/efectos de los fármacos , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Precursores del ARN , Vitamina D3 24-Hidroxilasa/genética , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.
Asunto(s)
Estudios de Asociación Genética , Glicoproteínas/genética , Mutación , Fenotipo , Animales , Huesos/metabolismo , Proteínas de Unión al Calcio , Mapeo Cromosómico , Modelos Animales de Enfermedad , Metabolismo Energético/genética , Exoma , Femenino , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Riñón/metabolismo , Riñón/fisiopatología , Pruebas de Función Renal , Masculino , Ratones , Ratones Noqueados , Osteítis Deformante/genética , Osteítis Deformante/metabolismo , Osteítis Deformante/patología , Esqueleto/anomalíasRESUMEN
Mice and human patients with impaired vitamin D receptor (VDR) signaling have normal developmental hair growth but display aberrant post-morphogenic hair cycle progression associated with alopecia. In addition, VDR-/- mice exhibit impaired cutaneous wound healing. We undertook experiments to determine whether the stress-inducible regulator of energy homeostasis, DNA damage-inducible transcript 4 (Ddit4), is involved in these processes. By analyzing hair cycle activation in vivo, we show that VDR-/- mice at day 14 exhibit increased Ddit4 expression within follicular stress compartments. At day 29, degenerating VDR-/- follicular keratinocytes, but not bulge stem cells, continue to exhibit an increase in Ddit4 expression. At day 47, when normal follicles and epidermis are quiescent and enriched for Ddit4, VDR-/- skin lacks Ddit4 expression. In a skin wound healing assay, the re-epithelialized epidermis in wildtype (WT) but not VDR-/- animals harbor a population of Ddit4- and Krt10-positive cells. Our study suggests that VDR regulates Ddit4 expression during epidermal homeostasis and the wound healing process, while elevated Ddit4 represents an early growth-arresting stress response within VDR-/- follicles.
Asunto(s)
Folículo Piloso/metabolismo , Repitelización/fisiología , Receptores de Calcitriol/metabolismo , Factores de Transcripción/metabolismo , Animales , Epidermis/metabolismo , Masculino , Ratones , Ratones Noqueados , Unión Proteica , Repitelización/genética , Receptores de Calcitriol/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Estrés Fisiológico , Factores de Transcripción/genética , Cicatrización de Heridas/fisiologíaRESUMEN
Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxel-induced peripheral neuropathy remain elusive, and therapies that prevent or alleviate this condition are not available. We established a zebrafish in vivo model to study the underlying mechanisms and to identify pharmacological agents that may be developed into therapeutics. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin before induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and up-regulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Thus, our studies provide evidence that the epidermis plays a critical role in this condition, and we provide a previously unidentified candidate for therapeutic interventions.
Asunto(s)
Antineoplásicos/efectos adversos , Epitelio/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Paclitaxel/efectos adversos , Nervios Periféricos/efectos de los fármacos , Aletas de Animales/citología , Aletas de Animales/inervación , Animales , Axones/efectos de los fármacos , Embrión no Mamífero/efectos de los fármacos , Expresión Génica , Humanos , Queratinocitos/efectos de los fármacos , Metaloproteinasa 13 de la Matriz/genética , Piel/citología , Piel/efectos de los fármacos , Piel/inervación , Percepción del Tacto/efectos de los fármacos , Pruebas de Toxicidad , Pez CebraRESUMEN
Skin wounds need to be repaired rapidly after injury to restore proper skin barrier function. Hydrogen peroxide (H2O2) is a conserved signaling factor that has been shown to promote a variety of skin wound repair processes, including immune cell migration, angiogenesis and sensory axon repair. Despite growing research on H2O2 functions in wound repair, the downstream signaling pathways activated by this reactive oxygen species in the context of injury remain largely unknown. The goal of this study was to provide a comprehensive analysis of gene expression changes in the epidermis upon exposure to H2O2 concentrations known to promote wound repair. Comparative transcriptome analysis using RNA-seq data from larval zebrafish and previously reported microarray data from a human epidermal keratinocyte line shows that H2O2 activates conserved cell migration, adhesion, cytoprotective and anti-apoptotic programs in both zebrafish and human keratinocytes. Further assessment of expression characteristics and signaling pathways revealed the activation of three major H2O2-dependent pathways, EGF, FOXO1, and IKKα. This study expands on our current understanding of the clinical potential of low-level H2O2 for the promotion of epidermal wound repair and provides potential candidates in the treatment of wound healing deficits.
Asunto(s)
Peróxido de Hidrógeno/toxicidad , Transducción de Señal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Pez Cebra/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Perfilación de la Expresión Génica , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/metabolismo , Inhibidor NF-kappaB alfa/genética , Inhibidor NF-kappaB alfa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Transcriptoma/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Pez Cebra/crecimiento & desarrolloRESUMEN
Congenital vertebral malformations caused by embryonic segmentation defects are relatively common in humans and domestic animals. Although reverse genetics approaches in mice have provided information on the molecular mechanisms of embryonic somite segmentation, hypothesis-driven approaches cannot adequately reflect human dysmorphology within the population. In a N-ethyl-N-nitrosourea (ENU) mutagenesis project in Kyoto, the Oune mutant rat strain was isolated due to a short and kinked caudal vertebra phenotype. Skeletal staining of heterozygous rats showed partial loss of the cervical vertebrae as well as hemivertebrae and fused vertebral blocks in lumbar and sacral vertebrae. In homozygous embryos, severe displacement of the whole vertebrae was observed. The Oune locus was genetically mapped to rat chromosome 1 using 202 backcross animals and 50 genome-wide microsatellite markers. Subsequently, a miss-sense mutation in the Tbx6 gene was identified in the critical region. Although the mutation is located within the T-box domain near a predicted dimmer-interface, in vitro experiments revealed that the Tbx6 variant retains normal DNA binding ability and translational efficiency. However, the variant has decreased transcriptional activation potential in response to Notch-mediated signaling. Recently, it was reported that a dominant type of familial spondylocostal dysostosis is caused by a stoploss mutation in TBX6. Thus, we propose that partial dysfunction of Tbx6 leads to similar congenital vertebral malformations in both humans and rats. The Oune strain could be a unique animal model for dominant spondylocostal dysostosis and is useful for molecular dissection of the pathology of congenital vertebral malformations in humans.