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1.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198528

RESUMO

Intracellular free zinc ([Zn2+]i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn2+]i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn2+ have been identified, proteins that are involved in Zn2+ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn2+ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn2+]i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn2+]i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca2+ oscillations. Assays with fluorescent Zn2+ indicators revealed that the basal [Zn2+]i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn2+]i level in the presence of 1 µM Zn2+ in inflammatory FLSs. Among the 17 out of 24 known Zn2+ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn2+]i level. Taken together, Zn2+ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn2+]i in inflammatory FLSs.


Assuntos
Proteínas de Transporte de Cátions/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/genética , Sinoviócitos/metabolismo , Canal de Cátion TRPA1/genética , Regulação para Cima/genética , Zinco/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/patologia , Espaço Intracelular/metabolismo , Sinoviócitos/patologia , Canal de Cátion TRPA1/metabolismo
2.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199748

RESUMO

BACKGROUND: Psoriasis, a chronic inflammatory disease affecting 2-3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. METHODS: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. RESULTS: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a "psoriatic phenotype" in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. CONCLUSION: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.


Assuntos
Conexinas/genética , Regulação da Expressão Gênica , Psoríase/genética , Adulto , Idoso , Biópsia , Conexinas/metabolismo , Conexinas/farmacologia , Epiderme/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células HaCaT , Humanos , Mediadores da Inflamação , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Queratinócitos/patologia , Pessoa de Meia-Idade , Modelos Biológicos , Oligopeptídeos/farmacologia , Peptidoglicano/isolamento & purificação , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Psoríase/patologia , Staphylococcus aureus/fisiologia , Adulto Jovem
3.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070360

RESUMO

Adenosine is a cellular metabolite with diverse derivatives that possesses a wide range of physiological roles. We investigated the molecular mechanisms of adenosine and cordycepin for their promoting effects in wound-healing process. The mitochondrial energy metabolism and cell proliferation markers, cAMP responsive element binding protein 1 (CREB1) and Ki67, were enhanced by adenosine and cordycepin in cultured dermal fibroblasts. Adenosine and cordycepin stimulated adenosine receptor signaling via elevated cAMP. The phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, mammalian target of rapamycin (mTOR) and glycogen synthase kinase 3 beta (Gsk3b) and Wnt target genes such as bone morphogenetic protein (BMP) 2/4 and lymphoid enhancer binding factor (Lef) 1 were activated. The enhanced gene expression by adenosine and cordycepin was abrogated by adenosine A2A and A2B receptor inhibitors, ZM241385 and PSH603, and protein kinase A (PKA) inhibitor H89, indicating the involvement of adenosine receptor A2A, A2B and PKA. As a result of Wnt/ß-catenin pathway activation, the secretion of growth factors such as insulin-like growth factor (IGF)-1 and transforming growth factor beta (TGFß) 3 was increased, previously reported to facilitate the wound healing process. In addition, in vitro fibroblast migration was also increased, demonstrating their possible roles in facilitating the wound healing process. In conclusion, our data strongly demonstrate that adenosine and cordycepin stimulate the Wnt/ß-catenin signaling through the activation of adenosine receptor, possibly promoting the tissue remodeling process and suggest their therapeutic potential for treating skin wounds.


Assuntos
Adenosina/farmacologia , Desoxiadenosinas/farmacologia , Fibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Linhagem Celular , Fibroblastos/patologia , Humanos , Pele/lesões , Pele/metabolismo , Pele/patologia , Cicatrização/efeitos dos fármacos , beta Catenina/metabolismo
4.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073402

RESUMO

The development of scaffolds mimicking the extracellular matrix containing bioactive substances has great potential in tissue engineering and wound healing applications. This study investigates melatonin-a methoxyindole present in almost all biological systems. Melatonin is a bioregulator in terms of its potential clinical importance for future therapies of cutaneous diseases. Mammalian skin is not only a prominent melatonin target, but also produces and rapidly metabolizes the multifunctional methoxyindole to biologically active metabolites. In our methodology, chitosan/collagen (CTS/Coll)-contained biomaterials are blended with melatonin at different doses to fabricate biomimetic hybrid scaffolds. We use rat tail tendon- and Salmo salar fish skin-derived collagens to assess biophysical and cellular properties by (i) Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR), (ii) thermogravimetric analysis (TG), (iii) scanning electron microscope (SEM), and (iv) proliferation ratio of cutaneous cells in vitro. Our results indicate that melatonin itself does not negatively affect biophysical properties of melatonin-immobilized hybrid scaffolds, but it induces a pronounced elevation of cell viability within human epidermal keratinocytes (NHEK), dermal fibroblasts (NHDF), and reference melanoma cells. These results demonstrate that this indoleamine accelerates re-epithelialization. This delivery is a promising technique for additional explorations in future dermatotherapy and protective skin medicine.


Assuntos
Bandagens , Quitosana/química , Colágeno/química , Derme/metabolismo , Epiderme/metabolismo , Fibroblastos/metabolismo , Queratinócitos/metabolismo , Melatonina , Linhagem Celular , Derme/patologia , Avaliação Pré-Clínica de Medicamentos , Epiderme/patologia , Fibroblastos/patologia , Humanos , Queratinócitos/patologia , Melatonina/química , Melatonina/farmacocinética , Melatonina/farmacologia
5.
Nat Commun ; 12(1): 3709, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140509

RESUMO

Fibrotic skin disease represents a major global healthcare burden, characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix. Fibroblasts are found to be heterogeneous in multiple fibrotic diseases, but fibroblast heterogeneity in fibrotic skin diseases is not well characterized. In this study, we explore fibroblast heterogeneity in keloid, a paradigm of fibrotic skin diseases, by using single-cell RNA-seq. Our results indicate that keloid fibroblasts can be divided into 4 subpopulations: secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory. Interestingly, the percentage of mesenchymal fibroblast subpopulation is significantly increased in keloid compared to normal scar. Functional studies indicate that mesenchymal fibroblasts are crucial for collagen overexpression in keloid. Increased mesenchymal fibroblast subpopulation is also found in another fibrotic skin disease, scleroderma, suggesting this is a broad mechanism for skin fibrosis. These findings will help us better understand skin fibrotic pathogenesis, and provide potential targets for fibrotic disease therapies.


Assuntos
Colágeno/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Queloide/metabolismo , Mesoderma/citologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Colágeno/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica/genética , Ontologia Genética , Humanos , Queloide/genética , Queloide/patologia , Ligantes , Mesoderma/metabolismo , Mesoderma/patologia , RNA-Seq , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Análise de Célula Única , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia
6.
Curr Cardiol Rep ; 23(7): 82, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34081224

RESUMO

PURPOSE OF REVIEW: Cardiac fibroblast activation contributes to fibrosis, maladaptive remodeling and heart failure progression. This review summarizes the latest findings on cardiac fibroblast activation dynamics derived from single-cell transcriptomic analyses and discusses how this information may aid the development of new multispecific medicines. RECENT FINDINGS: Advances in single-cell gene expression technologies have led to the discovery of distinct fibroblast subsets, some of which are more prevalent in diseased tissue and exhibit temporal changes in response to injury. In parallel to the rapid development of single-cell platforms, the advent of multispecific therapeutics is beginning to transform the biopharmaceutical landscape, paving the way for the selective targeting of diseased fibroblast subpopulations. Insights gained from single-cell technologies reveal critical cardiac fibroblast subsets that play a pathogenic role in the progression of heart failure. Combined with the development of multispecific therapeutic agents that have enabled access to previously "undruggable" targets, we are entering a new era of precision medicine.


Assuntos
Miocárdio , Medicina de Precisão , Fibroblastos/patologia , Fibrose , Coração , Humanos , Miocárdio/patologia
7.
Methods Mol Biol ; 2277: 187-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080153

RESUMO

Mitochondria, similar to living cells and organelles, have a negative membrane potential, which ranges between (-108) and (150) mV as compared to (-70) and (-90) mV of the plasma membrane. Therefore, permeable lipophilic cations tend to accumulate in the mitochondria. Those cations which exhibit fluorescence activity after accumulation into energized systems are widely used to decipher changes in membrane potential by imaging techniques. Here we describe the use of two different dyes for labeling mitochondrial membrane potential (Δψm) in live cells. One is the lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide (JC-1), which alters reversibly its color from green (J-monomer, at its low concentration in the cytosol) to red (J-aggregates, at its high concentration in active mitochondria) with increasing mitochondrial membrane potential (Δψm). The other is MitoTracker® Orange, a mitochondrion-selective probe which passively diffuses across the plasma membrane and accumulates in active mitochondria depending on their Δψm. We show that in addition to changes in Δψm, these specific dyes can be used to follow alterations in mitochondrial distribution and mitochondrial network connectivity. We suggest that JC-1 is a preferable probe to compare between different cell types and cell state, as a red to green ratio of fluorescence intensities is used for analysis. This ratio depends only on the mitochondrial membrane potential and not on other cellular and/or mitochondrial-dependent or independent factors that may alter, for example, due to treatment or disease state. However, in cells labeled either with green or red fluorescence protein, JC-1 cannot be used. Therefore, other dyes are preferable. We demonstrate various applications of JC-1 and MitoTracker Orange staining to study mitochondrial abnormalities in different cell types derived from schizophrenia patients and healthy subjects.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Esquizofrenia/metabolismo , Benzimidazóis/química , Carbocianinas/química , Técnicas de Cultura de Células , Fibroblastos/metabolismo , Fibroblastos/patologia , Corantes Fluorescentes/química , Humanos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Mitocôndrias/patologia , Estudo de Prova de Conceito , Esquizofrenia/patologia , Xantenos/química
8.
FASEB J ; 35(7): e21705, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34105826

RESUMO

Keloids are fibrotic lesions that grow unceasingly and invasively and are driven by local mechanical stimuli. Unlike other fibrotic diseases and normal wound healing, keloids exhibit little transformation of dermal fibroblasts into α-SMA+ myofibroblasts. This study showed that asporin is the most strongly expressed gene in keloids and its gene-ontology terms relate strongly to ECM metabolism/organization. Experiments with human dermal cells (HDFs) showed that asporin overexpression/treatment abrogated the HDF ability to adopt a perpendicular orientation when subjected to stretching tension. It also induced calcification of the surrounding 3D collagen matrix. Asporin overexpression/treatment also prevented the HDFs from remodeling the surrounding 3D collagen matrix, leading to a disorganized network of thick, wavy collagen fibers that resembled keloid collagen architecture. This in turn impaired the ability of the HDFs to contract the collagen matrix. Asporin treatment also made the fibroblasts impervious to the fibrous collagen contraction of α-SMA+ myofibroblasts, which normally activates fibroblasts. Thus, by calcifying collagen, asporin prevents fibroblasts from linearly rearranging the surrounding collagen; this reduces both their mechanosensitivity and mechanosignaling to each other through the collagen network. This blocks fibroblast activation and differentiation into the mature myofibroblasts that efficiently remodel the extracellular matrix. Consequently, the fibroblasts remain immature, highly proliferative, and continue laying down abundant extracellular matrix, causing keloid growth and invasion. Notably, dermal injection of asporin-overexpressing HDFs into murine wounds recapitulated keloid collagen histopathological characteristics. Thus, disrupted interfibroblast mechanocommunication may promote keloid progression. Asporin may be a new diagnostic biomarker and therapeutic target for keloids.


Assuntos
Colágeno/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Queloide/prevenção & controle , Mecanotransdução Celular , Animais , Células Cultivadas , Progressão da Doença , Proteínas da Matriz Extracelular/genética , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Queloide/genética , Queloide/metabolismo , Queloide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pele/metabolismo
9.
FASEB J ; 35(7): e21695, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34160101

RESUMO

Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1ß/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies.


Assuntos
Fibroblastos/patologia , Dermatopatias Infecciosas/patologia , Pele/patologia , Infecções Estafilocócicas/complicações , Staphylococcus aureus/patogenicidade , Cicatrização , Adulto , Idoso , Estudos de Casos e Controles , Movimento Celular , Proliferação de Células , Células Cultivadas , Feminino , Fibroblastos/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Pele/microbiologia , Dermatopatias Infecciosas/microbiologia , Infecções Estafilocócicas/microbiologia , Adulto Jovem
10.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064331

RESUMO

Metastasis is the process whereby cancer cells migrate from the primary tumour site to colonise the surrounding or distant tissue or organ. Metastasis is the primary cause of cancer-related mortality and approximately half of all cancer patients present at diagnosis with some form of metastasis. Consequently, there is a clear need to better understand metastasis in order to develop new tools to combat this process. MicroRNAs (miRNAs) regulate gene expression and play an important role in cancer development and progression including in the metastatic process. Particularly important are the roles that miRNAs play in the interaction between tumour cells and non-tumoral cells of the tumour microenvironment (TME), a process mediated largely by circulating miRNAs contained primarily in extracellular vesicles (EVs). In this review, we outline the accumulating evidence for the importance of miRNAs in the communication between tumour cells and the cells of the TME in the context of the pre-metastatic and metastatic niche.


Assuntos
MicroRNAs/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , RNA Neoplásico/genética , Microambiente Tumoral/genética , Animais , Comunicação Celular , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , MicroRNAs/classificação , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Neoplásico/metabolismo , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia
11.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070950

RESUMO

Fifty-five to two hundred CGG repeats (called a premutation, or PM) in the 5'-UTR of the FMR1 gene are generally unstable, often expanding to a full mutation (>200) in one generation through maternal inheritance, leading to fragile X syndrome, a condition associated with autism and other intellectual disabilities. To uncover the early mechanisms of pathogenesis, we performed metabolomics and proteomics on amniotic fluids from PM carriers, pregnant with male fetuses, who had undergone amniocentesis for fragile X prenatal diagnosis. The prenatal metabolic footprint identified mitochondrial deficits, which were further validated by using internal and external cohorts. Deficits in the anaplerosis of the Krebs cycle were noted at the level of serine biosynthesis, which was confirmed by rescuing the mitochondrial dysfunction in the carriers' umbilical cord fibroblasts using alpha-ketoglutarate precursors. Maternal administration of serine and its precursors has the potential to decrease the risk of developing energy shortages associated with mitochondrial dysfunction and linked comorbidities.


Assuntos
Transtorno Autístico/genética , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Mitocôndrias/genética , Mutação , Serina/deficiência , Regiões 5' não Traduzidas , Adulto , Amniocentese , Líquido Amniótico/química , Transtorno Autístico/diagnóstico , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Ciclo do Ácido Cítrico/genética , Feminino , Feto , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Expressão Gênica , Teste de Complementação Genética , Heterozigoto , Humanos , Masculino , Metabolômica/métodos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Gravidez , Cultura Primária de Células , Proteômica/métodos , Serina/biossíntese , Repetições de Trinucleotídeos
12.
Methods Mol Biol ; 2274: 353-363, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050485

RESUMO

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). Necroptotic cells release a variety of cellular and nuclear factors, referred to as danger-associated molecular patterns (DAMPs). We recently developed a förster resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation based on FRET). SMART comprises a fragment of MLKL, and it monitors necroptosis, but not apoptosis or necrosis. We performed live-cell imaging for secretion activity (LCI-S) to observe the release of high-mobility group box 1 (HMGB1) from necroptotic cells at single-cell resolution. Moreover, we combined SMART and LCI-S imaging techniques and found two different modes of HMGB1 release from necroptotic cells. Thus, SMART and LCI-S are valuable tools for investigating intimate cross talk between necroptosis and DAMP release at single-cell resolution.


Assuntos
Alarminas/metabolismo , Fibroblastos/patologia , Transferência Ressonante de Energia de Fluorescência/métodos , Proteína HMGB1/metabolismo , Necroptose , Proteínas Quinases/metabolismo , Imagem com Lapso de Tempo/métodos , Fibroblastos/metabolismo , Humanos
13.
Am J Hum Genet ; 108(6): 1095-1114, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33991472

RESUMO

Latent transforming growth factor ß (TGFß)-binding proteins (LTBPs) are microfibril-associated proteins essential for anchoring TGFß in the extracellular matrix (ECM) as well as for correct assembly of ECM components. Variants in LTBP2, LTBP3, and LTBP4 have been identified in several autosomal recessive Mendelian disorders with skeletal abnormalities with or without impaired development of elastin-rich tissues. Thus far, the human phenotype associated with LTBP1 deficiency has remained enigmatic. In this study, we report homozygous premature truncating LTBP1 variants in eight affected individuals from four unrelated consanguineous families. Affected individuals present with connective tissue features (cutis laxa and inguinal hernia), craniofacial dysmorphology, variable heart defects, and prominent skeletal features (craniosynostosis, short stature, brachydactyly, and syndactyly). In vitro studies on proband-derived dermal fibroblasts indicate distinct molecular mechanisms depending on the position of the variant in LTBP1. C-terminal variants lead to an altered LTBP1 loosely anchored in the microfibrillar network and cause increased ECM deposition in cultured fibroblasts associated with excessive TGFß growth factor activation and signaling. In contrast, N-terminal truncation results in a loss of LTBP1 that does not alter TGFß levels or ECM assembly. In vivo validation with two independent zebrafish lines carrying mutations in ltbp1 induce abnormal collagen fibrillogenesis in skin and intervertebral ligaments and ectopic bone formation on the vertebrae. In addition, one of the mutant zebrafish lines shows voluminous and hypo-mineralized vertebrae. Overall, our findings in humans and zebrafish show that LTBP1 function is crucial for skin and bone ECM assembly and homeostasis.


Assuntos
Colágeno/metabolismo , Cútis Laxa/etiologia , Variação Genética , Proteínas de Ligação a TGF-beta Latente/genética , Adolescente , Alelos , Animais , Células Cultivadas , Criança , Pré-Escolar , Cútis Laxa/patologia , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Masculino , Linhagem , Pele/metabolismo , Pele/patologia , Peixe-Zebra
14.
Am J Hum Genet ; 108(6): 1069-1082, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34022130

RESUMO

BCAS3 microtubule-associated cell migration factor (BCAS3) is a large, highly conserved cytoskeletal protein previously proposed to be critical in angiogenesis and implicated in human embryogenesis and tumorigenesis. Here, we established BCAS3 loss-of-function variants as causative for a neurodevelopmental disorder. We report 15 individuals from eight unrelated families with germline bi-allelic loss-of-function variants in BCAS3. All probands share a global developmental delay accompanied by pyramidal tract involvement, microcephaly, short stature, strabismus, dysmorphic facial features, and seizures. The human phenotype is less severe compared with the Bcas3 knockout mouse model and cannot be explained by angiogenic defects alone. Consistent with being loss-of-function alleles, we observed absence of BCAS3 in probands' primary fibroblasts. By comparing the transcriptomic and proteomic data based on probands' fibroblasts with those of the knockout mouse model, we identified similar dysregulated pathways resulting from over-representation analysis, while the dysregulation of some proposed key interactors could not be confirmed. Together with the results from a tissue-specific Drosophila loss-of-function model, we demonstrate a vital role for BCAS3 in neural tissue development.


Assuntos
Mutação com Perda de Função , Perda de Heterozigosidade , Proteínas de Neoplasias/genética , Transtornos do Neurodesenvolvimento/etiologia , Adolescente , Adulto , Animais , Movimento Celular , Criança , Pré-Escolar , Drosophila , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Lactente , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/metabolismo , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Proteoma/análise , Adulto Jovem
15.
FASEB J ; 35(6): e21586, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33960016

RESUMO

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Only 10% of all cases are familial form, the remaining 90% are sporadic form with unknown genetic background. The etiology of sporadic AD is still not fully understood. Pathogenesis and pathobiology of this disease are limited due to the limited number of experimental models. We used primary culture of fibroblasts derived from patients diagnosed with sporadic form of AD for investigation of dynamic properties of mitochondria, including fission-fusion process and localization of mitochondria within the cell. We observed differences in mitochondrial network organization with decreased mitochondrial transport velocity, and a drop in the frequency of fusion-fission events. These studies show how mitochondrial dynamics adapt to the conditions of long-term mitochondrial stress that prevails in cells of sporadic form of AD.


Assuntos
Doença de Alzheimer/patologia , Fibroblastos/patologia , Mitocôndrias/patologia , Doenças Mitocondriais/complicações , Dinâmica Mitocondrial , Estresse Fisiológico , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/etiologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
16.
FASEB J ; 35(6): e21640, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33991130

RESUMO

Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.


Assuntos
Colesterol/metabolismo , Citotoxinas/efeitos adversos , Difosfonatos/farmacologia , Inibidores Enzimáticos/farmacologia , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Fibroblastos/citologia , Substâncias Protetoras/farmacologia , Células A549 , Proteínas de Bactérias/efeitos adversos , Toxinas Bacterianas/efeitos adversos , Proliferação de Células , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Células HeLa , Proteínas Hemolisinas/efeitos adversos , Humanos , Estreptolisinas/efeitos adversos
17.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946884

RESUMO

Triple-negative breast cancer (TNBC) is an aggressive breast tumor subtype characterized by poor clinical outcome. In recent years, numerous advancements have been made to better understand the biological landscape of TNBC, though appropriate targets still remain to be determined. In the present study, we have determined that the expression levels of FGF2 and S100A4 are higher in TNBC with respect to non-TNBC patients when analyzing "The Invasive Breast Cancer Cohort of The Cancer Genome Atlas" (TCGA) dataset. In addition, we have found that the gene expression of FGF2 is positively correlated with S100A4 in TNBC samples. Performing quantitative PCR, Western blot, CRISPR/Cas9 genome editing, promoter studies, immunofluorescence analysis, subcellular fractionation studies, and ChIP assays, we have also demonstrated that FGF2 induces in TNBC cells the upregulation and secretion of S100A4 via FGFR1, along with the ERK1/2-AKT-c-Rel transduction signaling. Using conditioned medium from TNBC cells stimulated with FGF2, we have also ascertained that the paracrine activation of the S100A4/RAGE pathway triggers angiogenic effects in vascular endothelial cells (HUVECs) and promotes the migration of cancer-associated fibroblasts (CAFs). Collectively, our data provide novel insights into the action of the FGF2/FGFR1 axis through S100A4 toward stimulatory effects elicited in TNBC cells.


Assuntos
Fator 2 de Crescimento de Fibroblastos/fisiologia , Proteínas de Neoplasias/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Proteína A4 de Ligação a Cálcio da Família S100/fisiologia , Transdução de Sinais/fisiologia , Neoplasias de Mama Triplo Negativas/fisiopatologia , Antígenos de Neoplasias/fisiologia , Movimento Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Feminino , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neovascularização Patológica/fisiopatologia , Comunicação Parácrina , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-rel/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/irrigação sanguínea , Células Tumorais Cultivadas
18.
Toxicol Lett ; 347: 58-66, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961985

RESUMO

For smoking-induced pulmonary fibrosis (PF), a serious disease endangering human health, there is no effective clinical treatment. Aberrant epithelium-fibroblast cross-talk is involved in formation of the excessive extracellular matrix (ECM) that contributes to PF. Circular RNAs have been associated with various pulmonary diseases. However, the mechanisms of circRNAs in PF are not clear. Herein, our goals were to investigate the involvement of circRNA_0026344 in the aberrant epithelium-fibroblast cross-talk induced by cigarette smoke (CS) and to define its mechanism. Chronic exposure (16 weeks) of BALB/c mice to 500 mg/m3 CS induced lung injury and fibrosis in lung tissues. From HBE cells, circRNA_0026344 was selected by microarray analysis and verified as that with the most severe down-regulation caused by cigarette smoke extract (CSE). The regulatory relationship between circRNA_0026344 and miR-21 was assessed by use of bioinformatics, RNA pull-down assays, and qRT-PCR. We found that miR-21 binding sites were present in circRNA_0026344 and, in HBE cells, it could act as a sponge for miR-21. When pcDNA3.0-circRNA_0026344, a high expression plasmid of circRNA_0026344, was transfected into HBE cells, the CSE-induced up-regulation of miR-21 levels was reversed. In MRC-5 cells, HBE-secreted exosomal miR-21 decreased levels of Smad7 and activated the TGF-ß1/Smad3 pathway. By using the Targetscan database, the presence of species-conserved miR-21 binding sites in the Smad7 3'UTR region were predicted. We verified, by use of a luciferase reporter gene, that miR-21 bound to the 3'UTR region of Smad7 mRNA to inhibit its transcription. In conclusion, the results reveal that, in CS-induced pulmonary fibrosis, circRNA_0026344, via exosomal miR-21 regulation of Smad7, is involved in aberrant cross-talk of epithelium-fibroblasts. These results will be useful for the discovery of early biomarkers and for providing therapeutic targets for smoking-induced pulmonary fibrosis.


Assuntos
Comunicação Celular , Células Epiteliais/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , RNA Circular/metabolismo , Proteína Smad7/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/patologia , Exossomos/genética , Exossomos/patologia , Fibroblastos/patologia , Regulação da Expressão Gênica , Humanos , Pulmão/patologia , Masculino , Camundongos Endogâmicos BALB C , MicroRNAs/genética , MicroRNAs/metabolismo , Fibrose Pulmonar/etiologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , RNA Circular/genética , Transdução de Sinais , Proteína Smad7/genética , Fumaça , Produtos do Tabaco , Fator de Crescimento Transformador beta1/metabolismo
19.
Toxicol Appl Pharmacol ; 422: 115559, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961903

RESUMO

The occurrence and development of silicosis is related to the interaction of multiple cells through signal transmission caused by silica dust. Including inflammatory changes reduced by macrophages and phenotypic transdifferentiation reduced by lung fibroblasts. As a communication medium between cells, exosomes have become a hot research topic. To explore the role of exosomal proteins in the occurrence and development of silicosis and the possible intervention targets, this study conducted proteomic analysis of macrophage-derived exosomes induced by silica, to identify specific proteins for intervention. In this study, we used proteomic analysis to screen exosomal protein profiles from the RAW264.7 macrophages exposed to silica. A total of 291 proteins were differentially expressed, of which 178 were upregulated and 113 were downregulated. By performing functional annotation and analysis of the differentially expressed proteins, we identified proteins SPP1, HMGB3, and HNRNPAB, which were consistent with the proteomics analysis. The involvement of SPP1 protein in fibrosis was studied further. Knocking down the expression of SPP1 in exosomes resulted in a decrease in fibrosis-related indicators. These results help to understand that exosomal protein can mediate cell communication and play a key role in the transition from fibroblasts to myofibroblasts. Further, this study also provided strategies and scientific basis for future studies on the intervention of silicosis.


Assuntos
Comunicação Celular , Transdiferenciação Celular , Exossomos/efeitos dos fármacos , Fibroblastos/metabolismo , Macrófagos/efeitos dos fármacos , Osteopontina/metabolismo , Dióxido de Silício/toxicidade , Silicose/metabolismo , Animais , Proliferação de Células , Exossomos/genética , Exossomos/metabolismo , Fibroblastos/patologia , Macrófagos/metabolismo , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Células NIH 3T3 , Osteopontina/genética , Proteoma , Proteômica , Células RAW 264.7 , Silicose/genética , Silicose/patologia
20.
Methods Mol Biol ; 2262: 397-409, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977491

RESUMO

Costello syndrome (CS), characterized by a developmental delay and a failure to thrive, is also associated with an impaired lipid and energy metabolism. White adipose tissue is a central sensor of whole-body energy homeostasis, and HRAS hyperactivation may affect adipocyte differentiation and mature adipocyte homeostasis. An extremely useful tool for delineating in vitro intrinsic cellular signaling leading to metabolic alterations during adipogenesis is mouse embryonic fibroblasts, known to differentiate into adipocytes in response to adipogenesis-stimulating factors. Here, we describe in detail the isolation and maintenance of CS HRAS G12V mouse embryonic fibroblasts, their differentiation into adipocytes, and an assessment of adipocyte differentiation.


Assuntos
Adipócitos/patologia , Diferenciação Celular , Síndrome de Costello/patologia , Modelos Animais de Doenças , Fibroblastos/patologia , Mutação , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Adipócitos/metabolismo , Adipogenia , Animais , Síndrome de Costello/genética , Síndrome de Costello/metabolismo , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fibroblastos/metabolismo , Homeostase , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout
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