RESUMO
In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.
Assuntos
Implantação do Embrião , Gastrulação , Células-Tronco Pluripotentes , Animais , Feminino , Humanos , Gravidez , Diferenciação Celular , Embrião de Mamíferos , Desenvolvimento Embrionário , Organogênese , Células-Tronco Pluripotentes/metabolismo , PrimatasRESUMO
Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at ~10 Å further reveal two conformations of the "double-head" myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases.
Assuntos
Músculo Esquelético/metabolismo , Sarcômeros/química , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinina/química , Actinina/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Animais , Microscopia Crioeletrônica , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Ligação Proteica , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Tropomiosina/química , Tropomiosina/metabolismoRESUMO
Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.
Assuntos
Carcinogênese , Proteínas de Drosophila , Proteólise , Ubiquitina-Proteína Ligases , Animais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Carcinogênese/genética , Humanos , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/embriologia , Genes Supressores de Tumor , Ubiquitinação , Proteínas do Grupo Polycomb/metabolismo , Proteínas do Grupo Polycomb/genética , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 1/genéticaRESUMO
The intercalated disc (ID) is a highly specialized structure that connects cardiomyocytes via mechanical and electrical junctions. Although described in some detail by light microscopy in the 19th century, it was in 1966 that electron microscopy images showed that the ID represented apposing cell borders and provided detailed insight into the complex ID nanostructure. Since then, much has been learned about the ID and its molecular composition, and it has become evident that a large number of proteins, not all of them involved in direct cell-to-cell coupling via mechanical or gap junctions, reside at the ID. Furthermore, an increasing number of functional interactions between ID components are emerging, leading to the concept that the ID is not the sum of isolated molecular silos but an interacting molecular complex, an "organelle" where components work in concert to bring about electrical and mechanical synchrony. The aim of the present review is to give a short historical account of the ID's discovery and an updated overview of its composition and organization, followed by a discussion of the physiological implications of the ID architecture and the local intermolecular interactions. The latter will focus on both the importance of normal conduction of cardiac action potentials as well as the impact on the pathophysiology of arrhythmias.
Assuntos
Miocárdio , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/fisiologia , Miocárdio/metabolismo , Junções Comunicantes/metabolismo , Arritmias CardíacasRESUMO
Eukaryotic transcription factors (TFs) form complexes with various partner proteins to recognize their genomic target sites. Yet, how the DNA sequence determines which TF complex forms at any given site is poorly understood. Here, we demonstrate that high-throughput in vitro DNA binding assays coupled with unbiased computational analysis provide unprecedented insight into how different DNA sequences select distinct compositions and configurations of homeodomain TF complexes. Using inferred knowledge about minor groove width readout, we design targeted protein mutations that destabilize homeodomain binding both in vitro and in vivo in a complex-specific manner. By performing parallel systematic evolution of ligands by exponential enrichment sequencing (SELEX-seq), chromatin immunoprecipitation sequencing (ChIP-seq), RNA sequencing (RNA-seq), and Hi-C assays, we not only classify the majority of in vivo binding events in terms of complex composition but also infer complex-specific functions by perturbing the gene regulatory network controlled by a single complex.
Assuntos
DNA/química , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Interactions between notochord and sclerotome are required for normal embryonic spine patterning, but whether the postnatal derivatives of these tissues also require interactions for postnatal intervertebral disc (IVD) growth and maintenance is less established. We report here the comparative analysis of four conditional knockout mice deficient for TonEBP, a transcription factor known to allow cells to adapt to changes in extracellular osmotic pressure, in specific compartments of the IVD. We show that TonEBP deletion in nucleus pulposus (NP) cells does not affect their survival or aggrecan expression, but promoted cell proliferation in the NP and in adjacent vertebral growth plates (GPs). In cartilage end plates/GPs, TonEBP deletion induced cell death, but also structural alterations in the adjacent NP cells and vertebral bodies. Embryonic or postnatal TonEBP loss generated similar IVD changes. In addition to demonstrating the requirement of TonEBP in the different compartments of the IVD, this comparative analysis uncovers the in vivo interdependency of the different IVD compartments during the growth of the postnatal IVD-vertebral units.
Assuntos
Disco Intervertebral , Fatores de Transcrição NFATC , Animais , Camundongos , Regulação da Expressão Gênica , Disco Intervertebral/metabolismo , Camundongos Knockout , Pressão Osmótica , Fatores de Transcrição/metabolismo , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismoRESUMO
During development, unique combinations of transcription factors and signaling pathways carve the nascent eye-antennal disc of the fruit fly Drosophila melanogaster into several territories that will eventually develop into the compound eye, ocelli, head epidermis, bristles, antenna and maxillary palpus of the adult head. Juxtaposed patterns of Hedgehog (Hh) and Decapentaplegic (Dpp) initiate compound eye development, while reciprocal domains of Dpp and Wingless (Wg) induce formation of the antennal and maxillary palp fields. Hh and Wg signaling, but not Dpp, contribute to the patterning of the dorsal head vertex. Here, we show that combinatorial reductions of the Pax6 transcription factor Twin of Eyeless and either the Wg pathway or the Mirror (Mirr) transcription factor trigger a transformation of the ocelli into a compound eye and the neighboring head epidermis into an antenna. These changes in fate are accompanied by the ectopic expression of Dpp, which might be expected to trigger these changes in fate. However, the transformation of the field cannot be replicated by increasing Dpp levels alone despite the recreation of adjacent Hh-Dpp and Wg-Dpp domains. As such, the emergence of these ectopic organs occurs through a unique regulatory path.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento , Cabeça , Proteínas Hedgehog , Animais , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cabeça/embriologia , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Proteína Wnt1/metabolismo , Proteína Wnt1/genética , Padronização Corporal/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
A fundamental goal of developmental biology is to understand how cell and tissue fates are specified. The imaginal discs of Drosophila are excellent model systems for addressing this paradigm as their fate can be redirected when discs regenerate after injury or when key selector genes are misregulated. Here, we show that when Polycomb expression is reduced, the wing selector gene vestigial is ectopically activated. This leads to the inappropriate formation of the Vestigial-Scalloped complex, which forces the eye to transform into a wing. We further demonstrate that disrupting this complex does not simply block wing formation or restore eye development. Instead, immunohistochemistry and high-throughput genomic analysis show that the eye-antennal disc unexpectedly undergoes hyperplastic growth with multiple domains being organized into other imaginal discs and tissues. These findings provide insight into the complex developmental landscape that tissues must navigate before adopting their final fate.
Assuntos
Proteínas de Drosophila , Discos Imaginais , Animais , Proteínas de Drosophila/genética , Drosophila , Genômica , Hiperplasia , Proteínas do Grupo Polycomb/genéticaRESUMO
Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Comunicação Celular , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Proteínas Hedgehog/metabolismo , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The prognosis and treatment outcomes of heart failure (HF) patients rely heavily on disease etiology, yet the majority of underlying signaling mechanisms are complex and not fully elucidated. Phosphorylation is a major point of protein regulation with rapid and profound effects on the function and activity of protein networks. Currently, there is a lack of comprehensive proteomic and phosphoproteomic studies examining cardiac tissue from HF patients with either dilated dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM). Here, we used a combined proteomic and phosphoproteomic approach to identify and quantify more than 5,000 total proteins with greater than 13,000 corresponding phosphorylation sites across explanted left ventricle (LV) tissue samples, including HF patients with DCM vs. nonfailing controls (NFC), and left ventricular infarct vs. noninfarct, and periinfarct vs. noninfarct regions of HF patients with ICM. Each pair-wise comparison revealed unique global proteomic and phosphoproteomic profiles with both shared and etiology-specific perturbations. With this approach, we identified a DCM-associated hyperphosphorylation cluster in the cardiomyocyte intercalated disc (ICD) protein, αT-catenin (CTNNA3). We demonstrate using both ex vivo isolated cardiomyocytes and in vivo using an AAV9-mediated overexpression mouse model, that CTNNA3 phosphorylation at these residues plays a key role in maintaining protein localization at the cardiomyocyte ICD to regulate conductance and cell-cell adhesion. Collectively, this integrative proteomic/phosphoproteomic approach identifies region- and etiology-associated signaling pathways in human HF and describes a role for CTNNA3 phosphorylation in the pathophysiology of DCM.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Animais , Camundongos , Humanos , Cardiomiopatia Dilatada/metabolismo , Ventrículos do Coração/metabolismo , Fosforilação , Proteômica , Miocárdio/metabolismo , Insuficiência Cardíaca/metabolismo , alfa Catenina/metabolismoRESUMO
The development of modern birds provides a window into the biology of their dinosaur ancestors. We investigated avian postnatal development and found that sterile inflammation drives formation of the pygostyle, a compound structure resulting from bone fusion in the tail. Inflammation is generally induced by compromised tissue integrity, but here is involved in normal bone development. Transcriptome profiling and immuno/histochemistry reveal a robust inflammatory response that resembles bone fracture healing. The data suggest the involvement of necroptosis and multiple immune cell types, notably heterophils (the avian equivalent of neutrophils). Additionally, nucleus pulposus structures, heretofore unknown in birds, are involved in disc remodeling. Anti-inflammatory corticosteroid treatment inhibited vertebral fusion, substantiating the crucial role of inflammation in the ankylosis process. This study shows that inflammation can drive developmental skeletogenesis, in this case leading to the formation of a flight-adapted tail structure on the evolutionary path to modern avians.
Assuntos
Aves , Inflamação , Animais , Evolução Biológica , Coluna Vertebral , NeutrófilosRESUMO
The early specification and rapid growth of extraembryonic membranes are distinctive hallmarks of primate embryogenesis. These complex tasks are resolved through an intricate combination of signals controlling the induction of extraembryonic lineages and, at the same time, safeguarding the pluripotent epiblast. Here, we delineate the signals orchestrating primate epiblast and amnion identity. We encapsulated marmoset pluripotent stem cells into agarose microgels and identified culture conditions for the development of epiblast- and amnion-spheroids. Spatial identity mapping authenticated spheroids generated in vitro by comparison with marmoset embryos in vivo. We leveraged the microgel system to functionally interrogate the signalling environment of the post-implantation primate embryo. Single-cell profiling of the resulting spheroids demonstrated that activin/nodal signalling is required for embryonic lineage identity. BMP4 promoted amnion formation and maturation, which was counteracted by FGF signalling. Our combination of microgel culture, single-cell profiling and spatial identity mapping provides a powerful approach to decipher the essential cues for embryonic and extraembryonic lineage formation in primate embryogenesis.
Assuntos
Microgéis , Ativinas , Âmnio , Animais , Callithrix , Diferenciação Celular , Camadas Germinativas , SefaroseRESUMO
The eye-antennal disc of Drosophila is composed of three cell layers: a columnar epithelium called the disc proper (DP); an overlying sheet of squamous cells called the peripodial epithelium (PE); and a strip of cuboidal cells that joins the other two cellular sheets to each other and comprises the outer margin (M) of the disc. The M cells play an important role in patterning the eye because it is here that the Hedgehog (Hh), Decapentaplegic (Dpp) and JAK/STAT pathways function to initiate pattern formation. Dpp signaling is lost from the margin of eyes absent (eya) mutant discs and, as a result, the initiation of retinal patterning is blocked. Based on these observations, Eya has been proposed to control the initiation of the morphogenetic furrow via regulation of Dpp signaling within the M. We show that the failure in pattern formation surprisingly results from M cells prematurely adopting a head epidermis fate. This switch in fate normally takes place during pupal development after the eye has been patterned. Our results suggest that the timing of cell fate decisions is essential for correct eye development.
Assuntos
Olho Composto de Artrópodes/citologia , Proteínas de Drosophila/metabolismo , Proteínas do Olho/metabolismo , Animais , Diferenciação Celular , Olho Composto de Artrópodes/crescimento & desenvolvimento , Olho Composto de Artrópodes/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Janus Quinases/metabolismo , Morfogênese , Mutação , Fatores de Transcrição STAT/metabolismoRESUMO
Degeneration of the intervertebral disc (IVD) results in a range of symptomatic (i.e., painful) and asymptomatic experiences. Components of the degenerative environment, including structural disruption and inflammatory cytokine production, often correlate with pain severity. However, the role of inflammation in the activation of pain and degenerative changes has been complex to delineate. The most common IVD injury model is puncture; however, it initiates structural damage that is not representative of the natural degenerative cascade. In this study, we utilized in vivo injection of lipopolysaccharide (LPS), a pro-inflammatory stimulus, into rat caudal IVDs using 33G needles to induce inflammatory activation without the physical tissue disruption caused by puncture using larger needles. LPS injection increased gene expression of pro-inflammatory cytokines (Tnfa, Il1b) and macrophage markers (Inos, Arg1), supported by immunostaining of macrophages (CD68, CCR7, Arg1) and systemic changes in blood cytokine and chemokine levels. Disruption of the IVD structural integrity after LPS injection was also evident through changes in histological grading, disc height, and ECM biochemistry. Ultimately, intradiscal inflammatory stimulation led to local mechanical hyperalgesia, demonstrating that pain can be initiated by inflammatory stimulation of the IVD. Gene expression of nociceptive markers (Ngf, Bdnf, Cgrp) and immunostaining for neuron ingrowth (PGP9.5) and sensitization (CGRP) in the IVD were also shown, suggesting a mechanism for the pain exhibited. To our knowledge, this rat IVD injury model is the first to demonstrate local pain behavior resulting from inflammatory stimulation of caudal IVDs. Future studies will examine the mechanistic contributions of inflammation in mediating pain.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Ratos , Animais , Degeneração do Disco Intervertebral/metabolismo , Lipopolissacarídeos/toxicidade , Lipopolissacarídeos/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Punção Espinal , Disco Intervertebral/metabolismo , Dor/etiologia , Dor/metabolismo , Citocinas/metabolismo , Inflamação/metabolismoRESUMO
Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Dor Lombar , Núcleo Pulposo , Camundongos , Animais , Núcleo Pulposo/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Notocorda/metabolismo , Dor Lombar/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Análise de Sequência de RNARESUMO
Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1ß, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD.
Assuntos
Apoptose , Inflamação , Degeneração do Disco Intervertebral , Fatores Inibidores da Migração de Macrófagos , Núcleo Pulposo , Ratos Sprague-Dawley , Animais , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/metabolismo , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Apoptose/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/patologia , Ratos , Masculino , Humanos , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Feminino , Isoxazóis/farmacologia , Adulto , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.
Assuntos
Desdiferenciação Celular , Reprogramação Celular , Degeneração do Disco Intervertebral , Notocorda , Núcleo Pulposo , Núcleo Pulposo/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/patologia , Animais , Reprogramação Celular/genética , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/metabolismo , Ratos , Notocorda/metabolismo , Notocorda/citologia , Humanos , Modelos Animais de Doenças , Fator 3 de Transcrição de Octâmero/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Análise de Célula Única , Proteínas com Domínio T/metabolismo , Proteínas com Domínio T/genética , Células CultivadasRESUMO
The accumulation of metabolites in the intervertebral disc is considered an important cause of intervertebral disc degeneration (IVDD). Lactic acid, which is a metabolite that is produced by cellular anaerobic glycolysis, has been proven to be closely associated with IVDD. However, little is known about the role of lactic acid in nucleus pulposus cells (NPCs) senescence and oxidative stress. The aim of this study was to investigate the effect of lactic acid on NPCs senescence and oxidative stress as well as the underlying mechanism. A puncture-induced disc degeneration (PIDD) model was established in rats. Metabolomics analysis revealed that lactic acid levels were significantly increased in degenerated intervertebral discs. Elimination of excessive lactic acid using a lactate oxidase (LOx)-overexpressing lentivirus alleviated the progression of IVDD. In vitro experiments showed that high concentrations of lactic acid could induce senescence and oxidative stress in NPCs. High-throughput RNA sequencing results and bioinformatic analysis demonstrated that the induction of NPCs senescence and oxidative stress by lactic acid may be related to the PI3K/Akt signaling pathway. Further study verified that high concentrations of lactic acid could induce NPCs senescence and oxidative stress by interacting with Akt and regulating its downstream Akt/p21/p27/cyclin D1 and Akt/Nrf2/HO-1 pathways. Utilizing molecular docking, site-directed mutation and microscale thermophoresis assays, we found that lactic acid could regulate Akt kinase activity by binding to the Lys39 and Leu52 residues in the PH domain of Akt. These results highlight the involvement of lactic acid in NPCs senescence and oxidative stress, and lactic acid may become a novel potential therapeutic target for the treatment of IVDD.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Ratos , Animais , Degeneração do Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Disco Intervertebral/metabolismo , Senescência CelularRESUMO
Intervertebral disc degeneration is closely related to abnormal phenotypic changes in disc cells. However, the mechanism by which disc cell phenotypes are maintained remains poorly understood. Here, Hedgehog-responsive cells were found to be specifically localized in the inner annulus fibrosus and cartilaginous endplate of postnatal discs, likely activated by Indian Hedgehog. Global inhibition of Hedgehog signaling using a pharmacological inhibitor or Agc1-CreERT2-mediated deletion of Smo in disc cells of juvenile mice led to spontaneous degenerative changes in annulus fibrosus and cartilaginous endplate accompanied by aberrant disc cell differentiation in adult mice. In contrast, Krt19-CreER-mediated deletion of Smo specifically in nucleus pulposus cells led to healthy discs and normal disc cell phenotypes. Similarly, age-related degeneration of nucleus pulposus was accelerated by genetic inactivation of Hedgehog signaling in all disc cells, but not in nucleus pulposus cells. Furthermore, inactivation of Gli2 in disc cells resulted in partial loss of the vertebral growth plate but otherwise healthy discs, whereas deletion of Gli3 in disc cells largely corrected disc defects caused by Smo ablation in mice. Taken together, our findings not only revealed for the first time a direct role of Hedgehog-Gli3 signaling in maintaining homeostasis and cell phenotypes of annuls fibrosus and cartilaginous endplate, but also identified disc-intrinsic Hedgehog signaling as a novel non-cell-autonomous mechanism to regulate nucleus pulposus cell phenotype and protect mice from age-dependent nucleus pulposus degeneration. Thus, targeting Hedgehog signaling may represent a potential therapeutic strategy for the prevention and treatment of intervertebral disc degeneration.
Assuntos
Anel Fibroso , Degeneração do Disco Intervertebral , Disco Intervertebral , Camundongos , Animais , Degeneração do Disco Intervertebral/genética , Proteínas Hedgehog/genética , FenótipoRESUMO
Secreted signaling molecules act as morphogens to control patterning and growth in many developing tissues. Since locally produced morphogens spread to form a concentration gradient in the surrounding tissue, spreading is generally thought to be the key step in the non-autonomous actions. Here, we review recent advances in tool development to investigate morphogen function using the role of decapentaplegic (Dpp)/bone morphogenetic protein (BMP)-type ligand in the Drosophila wing disc as an example. By applying protein binder tools to distinguish between the roles of Dpp spreading and local Dpp signaling, we found that Dpp signaling in the source cells is important for wing patterning and growth but Dpp spreading from this source cells is not as strictly required as previously thought. Given recent studies showing unexpected requirements of long-range action of different morphogens, manipulating endogenous morphogen gradients by synthetic protein binder tools could shed more light on how morphogens act in developing tissues.