RESUMO
BACKGROUND: Hematuria is an established sign of glomerular disease and can be associated with kidney failure, but there has been limited scientific study of this trait. METHODS: Here, we combined genetic data from the UK Biobank with predicted gene expression and splicing from GTEx kidney cortex samples (n = 65) in a transcriptome-wide association study (TWAS) to identify additional potential biological mechanisms influencing hematuria. RESULTS: The TWAS using kidney cortex identified significant associations for 5 genes in terms of expression and 3 significant splicing events. Notably, we identified an association between the skipping of COL4A4 exon 27, which is genetically predicted by intronic rs11898094 (minor allele frequency 13%), and hematuria. Association between this variant was also found with urinary albumin excretion. We found independent evidence supporting the same variant predicting this skipping event in glomeruli-derived mRNA transcriptomics data (n = 245) from NEPTUNE. The functional significance of loss of exon 27 was demonstrated using the split NanoLuc-based α3α4α5(IV) heterotrimer assay, in which type IV collagen heterotrimer formation was quantified by luminescence. The causal splicing variant for this skipping event is yet to be identified. CONCLUSIONS: In summary, by integrating multiple data types, we identify a potential splicing event associated with hematuria and albuminuria.
RESUMO
COL4A3/A4/A5 mutations have been identified as critical causes of Alport syndrome and other genetic chronic kidney diseases. However, the underlying pathogenesis remains unclear, and specific treatments are lacking. Here, we constructed a transgenic Alport syndrome mouse model by generating a mutation (Col4a3 p.G799R) identified previously from one large Alport syndrome family into mice. We observed that the mutation caused a pathological decrease in intracellular and secreted collagen IV α3α4α5 heterotrimers. The mutant collagen IV α3 chains abnormally accumulated in the endoplasmic reticulum and exhibited defective secretion, leading to persistent endoplasmic reticulum stress in vivo and in vitro. RNA-seq analysis revealed that the MyD88/p38 MAPK pathway plays key roles in mediating subsequent inflammation and apoptosis signaling activation. Treatment with tauroursodeoxycholic acid, a chemical chaperone drug that functions as an endoplasmic reticulum stress inhibitor, effectively suppressed endoplasmic reticulum stress, promoted secretion of the α3 chains, and inhibited the activation of the MyD88/p38 MAPK pathway. Tauroursodeoxycholic acid treatment significantly improved kidney function in vivo. These results partly clarified the pathogenesis of kidney injuries associated with Alport syndrome, especially in glomeruli, and suggested that tauroursodeoxycholic acid might be useful for the early clinical treatment of Alport syndrome.
Assuntos
Apoptose , Autoantígenos , Colágeno Tipo IV , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Camundongos Transgênicos , Mutação , Nefrite Hereditária , Ácido Tauroquenodesoxicólico , Proteínas Quinases p38 Ativadas por Mitógeno , Animais , Ácido Tauroquenodesoxicólico/farmacologia , Ácido Tauroquenodesoxicólico/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Nefrite Hereditária/genética , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/patologia , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Apoptose/efeitos dos fármacos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Masculino , Rim/patologia , Rim/efeitos dos fármacos , Rim/metabolismoRESUMO
Aminopeptidase A is one of the most potent enzymes within the renin-angiotensin system in terms of angiotensin II degradation. Here, we examined whether there is a kidney phenotype and any compensatory changes in other renin angiotensin system enzymes involved in the metabolism of angiotensin II associated with aminopeptidase A deficiency. Kidneys harvested from aminopeptidase A knockout mice were examined by light and electron microscopy, immunohistochemistry and immunofluorescence. Kidney angiotensin II levels and the ability of renin angiotensin system enzymes in the glomerulus to degrade angiotensin II ex vivo, their activities, protein and mRNA levels in kidney lysates were evaluated. Knockout mice had increased blood pressure and mild glomerular mesangial expansion without significant albuminuria. By electron microscopy, knockout mice exhibited a mild increase of the mesangial matrix, moderate thickening of the glomerular basement membrane but a striking appearance of knob-like structures. These knobs were seen in both male and female mice and persisted after the treatment of hypertension. In isolated glomeruli from knockout mice, the level of angiotensin II was more than three-fold higher as compared to wild type control mice. In kidney lysates from knockout mice angiotensin converting enzyme activity, protein and mRNA levels were markedly decreased possibly as a compensatory mechanism to reduce angiotensin II formation. Thus, our findings support a role for aminopeptidase A in the maintenance of glomerular structure and intra-kidney homeostasis of angiotensin peptides.
Assuntos
Membrana Basal Glomerular , Glutamil Aminopeptidase , Rim , Angiotensina II/metabolismo , Animais , Feminino , Membrana Basal Glomerular/metabolismo , Glutamil Aminopeptidase/genética , Glutamil Aminopeptidase/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Knockout , Sistema Renina-Angiotensina/genéticaRESUMO
BACKGROUND: Hemicentins (HMCNs) are a family of extracellular matrix proteins first identified in Caenorhabditis elegans, with two orthologs (HMCN1 and 2) in vertebrates. In worms, HMCN is deposited at specific sites where it forms long, fine tracks that link two tissues by connecting adjacent basement membranes (BMs). By generating CRISPR/Cas9-mediated Hmcn1 and Hmcn2 knockout mice, we tested the hypothesis that HMCNs perform similar functions in mammals. RESULTS: Hmcn1 -/- mice were viable and fertile. Using new, knockout mouse-validated HMCN1 antibodies, HMCN1 was detected in wild-type mice as fine tracks along the BM of hair and whisker follicles, in the sclera of the eyes, and in the lumen of some lymphoid conduits. It was also observed in the mesangial matrix of the kidney glomerulus. However, HMCN1 deficiency did not affect the functions of these tissues, including adherence of coat hairs and whiskers, the sieving function of lymphoid conduits, or the immune response to injected antigens. HMCN2 deficiency did not lead to any discernible phenotypes on its own or when combined with HMCN1 deficiency. CONCLUSION: That Hmcn1 -/- , Hmcn2 -/- , and Hmcn1/2 double knockout mice did not display any overt phenotypes implicates compensation by other members of the fibulin family.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Animais , Membrana Basal/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas da Matriz Extracelular/genética , Folículo Piloso/citologia , Folículo Piloso/metabolismo , Rim/metabolismo , Camundongos , Camundongos Knockout , Esclera/citologia , Esclera/metabolismoRESUMO
Background Laminin α5ß2γ1 (LM-521) is a major component of the GBM. Mutations in LAMB2 that prevent LM-521 synthesis and/or secretion cause Pierson syndrome, a rare congenital nephrotic syndrome with diffuse mesangial sclerosis and ocular and neurologic defects. Because the GBM is uniquely accessible to plasma, which permeates endothelial cell fenestrae, we hypothesized that intravenous delivery of LM-521 could replace the missing LM-521 in the GBM of Lamb2 mutant mice and restore glomerular permselectivity.Methods We injected human LM-521 (hLM-521), a macromolecule of approximately 800 kD, into the retro-orbital sinus of Lamb2-/- pups daily. Deposition of hLM-521 into the GBM was investigated by fluorescence microscopy. We assayed the effects of hLM-521 on glomerular permselectivity by urinalysis and the effects on podocytes by desmin immunostaining and ultrastructural analysis of podocyte architecture.Results Injected hLM-521 rapidly and stably accumulated in the GBM of all glomeruli. Super-resolution imaging showed that hLM-521 accumulated in the correct orientation in the GBM, primarily on the endothelial aspect. Treatment with hLM-521 greatly reduced the expression of the podocyte injury marker desmin and attenuated the foot process effacement observed in untreated pups. Moreover, treatment with hLM-521 delayed the onset of proteinuria but did not prevent nephrotic syndrome, perhaps due to its absence from the podocyte aspect of the GBM.Conclusions These studies show that GBM composition and function can be altered in vivovia vascular delivery of even very large proteins, which may advance therapeutic options for patients with abnormal GBM composition, whether genetic or acquired.
Assuntos
Anormalidades Múltiplas/tratamento farmacológico , Anormalidades Múltiplas/metabolismo , Anormalidades do Olho/tratamento farmacológico , Anormalidades do Olho/metabolismo , Membrana Basal Glomerular/metabolismo , Laminina/genética , Laminina/uso terapêutico , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/metabolismo , Distúrbios Pupilares/tratamento farmacológico , Distúrbios Pupilares/metabolismo , Anormalidades Múltiplas/genética , Animais , Desmina/metabolismo , Modelos Animais de Doenças , Anormalidades do Olho/complicações , Anormalidades do Olho/genética , Injeções Intravenosas , Laminina/administração & dosagem , Camundongos , Síndromes Miastênicas Congênitas , Síndrome Nefrótica/complicações , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/genética , Permeabilidade/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/ultraestrutura , Proteinúria/etiologia , Proteinúria/prevenção & controle , Distúrbios Pupilares/complicações , Distúrbios Pupilares/genética , Proteínas Recombinantes/uso terapêuticoRESUMO
1-O-acylceramide is a new class of epidermal cer-amide (Cer) found in humans and mice. Here, we report an ESI linear ion-trap (LIT) multiple-stage MS (MSn) approach with high resolution toward structural characterization of this lipid family isolated from mice. Molecular species desorbed as the [M + H]+ ions were subjected to LIT MS2 to yield predominately the [M + H - H2O]+ ions, followed by MS3 to cleave the 1-O-acyl residue to yield the [M + H - H2O - (1-O-FA)]+ ions. The structures of the N-acyl chain and long-chain base (LCB) of the molecule were determined by MS4 on [M + H - H2O - (1-O-FA)]+ ions that yielded multiple sets of specific ions. Using this approach, isomers varied in the 1-O-acyl (from 14:0- to 30:0-O-acyl) and N-acyl chains (from 14:0- to 34:1-N-acyl) with 18:1-sphingosine as the major LCB were found for the entire family. Minor isomers consisting of 16:1-, 17:1-, 18:2-, and 19:1-sphingosine LCBs with odd fatty acyl chain or with monounsaturated N- or O-fatty acyl substituents were also identified. An estimation of more than 700 1-O-acylceramide species, largely isobaric isomers, are present, underscoring the complexity of this Cer family.
Assuntos
Ceramidas/química , Ceramidas/isolamento & purificação , Epiderme/química , Lipídeos/isolamento & purificação , Animais , Isomerismo , Lipídeos/química , Camundongos , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Esfingosina/química , Esfingosina/isolamento & purificaçãoRESUMO
The pathophysiology of many proteinuric kidney diseases is poorly understood, and microRNAs (miRs) regulation of these diseases has been largely unexplored. Here, we tested whether miR-378a-3p is a novel regulator of glomerular diseases. MiR-378a-3p has two predicted targets relevant to glomerular function, the glomerular basement membrane matrix component, nephronectin (NPNT), and vascular endothelial growth factor VEGF-A. In zebrafish (Danio rerio), miR-378a-3p mimic injection or npnt knockdown by a morpholino oligomer caused an identical phenotype consisting of edema, proteinuria, podocyte effacement, and widening of the glomerular basement membrane in the lamina rara interna. Zebrafish vegf-A protein could not rescue this phenotype. However, mouse Npnt constructs containing a mutated 3'UTR region prevented the phenotype caused by miR-378a-3p mimic injection. Overexpression of miR-378a-3p in mice confirmed glomerular dysfunction in a mammalian model. Biopsies from patients with focal segmental glomerulosclerosis and membranous nephropathy had increased miR-378a-3p expression and reduced glomerular levels of NPNT. Thus, miR-378a-3p-mediated suppression of the glomerular matrix protein NPNT is a novel mechanism for proteinuria development in active glomerular diseases.
Assuntos
Proteínas da Matriz Extracelular/genética , Membrana Basal Glomerular/metabolismo , Glomerulonefrite Membranosa/genética , Glomerulosclerose Segmentar e Focal/genética , MicroRNAs/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Biópsia , Modelos Animais de Doenças , Regulação para Baixo , Proteínas da Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes/métodos , Membrana Basal Glomerular/patologia , Glomerulonefrite Membranosa/patologia , Glomerulonefrite Membranosa/urina , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/urina , Humanos , Masculino , Camundongos , MicroRNAs/genética , Morfolinos/metabolismo , Podócitos/metabolismo , Podócitos/patologia , Proteinúria/genética , Proteinúria/patologia , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Fatty acids in the epidermis can be incorporated into complex lipids or exist in a free form, and they are crucial to proper functions of the epidermis and its appendages, such as sebaceous glands. Epidermal fatty acids can be synthesized de novo by keratinocytes or taken up from extracutaneous sources in a process that likely involves protein transporters. Several proteins that are expressed in the epidermis have been proposed to facilitate the uptake of long-chain fatty acids (LCFA) in mammalian cells, including fatty acid translocase/CD36, fatty acid binding protein, and fatty acid transport protein (FATP)/very long-chain acyl-CoA synthetase. In this review, we will discuss the mechanisms by which these candidate transporters facilitate the uptake of fatty acids. We will then discuss the clinical implications of defects in these transporters and relevant animal models, including the FATP4 animal models and ichthyosis prematurity syndrome, a congenital ichthyosis caused by FATP4 deficiency. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.
Assuntos
Aniridia , Proteínas de Transporte de Ácido Graxo , Ácidos Graxos , Ictiose , Doenças do Prematuro , Rim/anormalidades , Transtornos Psicomotores , Pele , Animais , Aniridia/genética , Aniridia/metabolismo , Aniridia/patologia , Transporte Biológico Ativo/genética , Modelos Animais de Doenças , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Feminino , Humanos , Ictiose/genética , Ictiose/metabolismo , Ictiose/patologia , Doenças do Prematuro/genética , Doenças do Prematuro/metabolismo , Doenças do Prematuro/patologia , Rim/metabolismo , Rim/patologia , Masculino , Transtornos Psicomotores/genética , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/patologia , Pele/metabolismo , Pele/patologiaRESUMO
Fatty acid transport protein 4 (FATP4) is one of a family of six transmembrane proteins that facilitate long- and very long-chain fatty acid uptake. FATP4 is expressed in several tissues, including skin. Mutations in human SLC27A4, which encodes FATP4, cause ichthyosis prematurity syndrome, characterized by a thick desquamating epidermis and premature birth. Mice lacking FATP4, which genetically model the human disease, are born with tight, thick skin and a defective skin barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene expression of FATP4 in suprabasal keratinocytes. Sebaceous glands in Fatp4 null skin grafted onto nude mice were found to be dystrophic and enwrapped by thick layers of epithelial cells. Consistent with these results, transgene-rescued Fatp4 null mice showed a subnormal level of FATP4 expression in sebocytes and exhibited abnormal development of both sebaceous glands and meibomian glands, specialized sebaceous glands of the eyelids. Sebum from these mice contained a reduced level of type II diester wax, a major mouse sebum lipid species, and showed perturbations in mass spectrometric profiles of diester wax and cholesteryl ester species. In addition, these mice showed an impaired ability to repel water and regulate body temperature after water immersion. Taken together, our results suggest that FATP4 plays crucial roles in the development and maturation of both sebaceous and meibomian glands, as well as in the formation and composition of sebum, likely by regulating the trafficking of fatty acids necessary for proper synthesis of sebum lipids.
Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Ictiose/metabolismo , Doenças do Prematuro/metabolismo , Glândulas Tarsais/metabolismo , Sebo/metabolismo , Animais , Aniridia , Modelos Animais de Doenças , Proteínas de Transporte de Ácido Graxo/genética , Humanos , Ictiose/genética , Ictiose/patologia , Doenças do Prematuro/genética , Doenças do Prematuro/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Rim/anormalidades , Glândulas Tarsais/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transtornos Psicomotores , Transplante de Pele , Transplante HomólogoRESUMO
Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective epidermal barrier; they die neonatally due to dehydration and restricted movements. Both the skin phenotype and the lethality are rescued by transgene-driven expression of FATP4 solely in suprabasal keratinocytes. Here we show that Fatp4 mutants exhibit epidermal hyperplasia resulting from an increased number of proliferating suprabasal cells. In addition, barrier formation initiates precociously but never progresses to completion. To investigate possible mechanisms whereby Fatp4 influences skin development, we identified misregulated genes in Fatp4 mutants. Remarkably, three members of the epidermal growth factor (EGF) family (Ereg, Areg, and Epgn) showed increased expression that was associated with elevated epidermal activation of the EGF receptor (EGFR) and STAT3, a downstream effector of EGFR signaling. Both Tyrphostin AG1478, an EGFR tyrosine kinase inhibitor, and curcumin, an inhibitor of both STAT3 and EGFR, attenuated STAT3 activation/nuclear translocation, reduced skin thickening, and partially suppressed the barrier abnormalities. These data identify FATP4 activity as negatively influencing EGFR activation and the resulting STAT3 signaling during normal skin development. These findings have important implications for understanding the pathogenesis of ichthyosis prematurity syndrome, a disease recently shown to be caused by FATP4 mutations.
Assuntos
Receptores ErbB , Proteínas de Transporte de Ácido Graxo/genética , Proteínas de Transporte de Ácido Graxo/metabolismo , Fator de Transcrição STAT3/metabolismo , Animais , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Epirregulina , Receptores ErbB/genética , Receptores ErbB/metabolismo , Receptores ErbB/fisiologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Mutação/efeitos dos fármacos , Fenótipo , Quinazolinas , Fator de Transcrição STAT3/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Pele/metabolismo , Dermatopatias/genética , Dermatopatias/metabolismo , Dermatopatias/patologia , TirfostinasRESUMO
Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j-dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.
Assuntos
Linfócitos B , Citocinas/metabolismo , Epiderme/patologia , Transtornos Linfoproliferativos/fisiopatologia , Receptores Notch/deficiência , Secretases da Proteína Precursora do Amiloide/deficiência , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Proliferação de Células , Epiderme/enzimologia , Epiderme/metabolismo , Feminino , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Queratinócitos/metabolismo , Contagem de Leucócitos , Longevidade , Transtornos Linfoproliferativos/genética , Camundongos , Gravidez , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Análise de Sobrevida , Fatores de Tempo , Linfopoietina do Estroma do TimoRESUMO
Lipidomic analyses by mass spectrometry (MS) of epidermal ceramides, a large family of lipids crucial to the permeability barrier of the skin, have been reported previously. To ensure the accuracy of lipid identification, we describe here the isolation of mouse newborn epidermal lipids followed by fractionation with solid-phase extraction columns, and lipidomic analyses by high-resolution MS for structural identification. We also describe here the employment of thin layer chromatography, an old but useful tool, in facilitating the structural characterization of the epidermal lipid species by MS.
Assuntos
Ceramidas/análise , Lipidômica/métodos , Pele/química , Animais , Animais Recém-Nascidos , Cromatografia em Camada Fina , Epiderme/química , Espectrometria de Massas , Camundongos , Extração em Fase SólidaRESUMO
CONTEXT: Mutations in LAMB2, encoding the basement membrane protein, laminin ß2, are associated with an autosomal recessive disorder characterized by congenital nephrotic syndrome, ocular abnormalities, and neurodevelopmental delay (Pierson syndrome). CASE DESCRIPTION: This report describes a 12-year-old boy with short stature, visual impairment, and developmental delay who presented with macroscopic hematuria and albuminuria. He had isolated growth hormone deficiency, optic nerve hypoplasia, and a small anterior pituitary with corpus callosum dysgenesis on his cranial magnetic resonance imaging, thereby supporting a diagnosis of optic nerve hypoplasia syndrome. Renal histopathology revealed focal segmental glomerulosclerosis. Using next-generation sequencing on a targeted gene panel for steroid-resistant nephrotic syndrome, compound heterozygous missense mutations were identified in LAMB2 (c.737G>A p.Arg246Gln, c.3982G>C p.Gly1328Arg). Immunohistochemical analysis revealed reduced glomerular laminin ß2 expression compared to control kidney and a thin basement membrane on electron microscopy. Laminin ß2 is expressed during pituitary development and Lamb2-/- mice exhibit stunted growth, abnormal neural retinae, and here we show, abnormal parenchyma of the anterior pituitary gland. CONCLUSION: We propose that patients with genetically undefined optic nerve hypoplasia syndrome should be screened for albuminuria and, if present, screened for mutations in LAMB2.
Assuntos
Albuminúria/genética , Hipopituitarismo/genética , Laminina/genética , Mutação , Hipoplasia do Nervo Óptico/genética , Criança , Humanos , Masculino , FenótipoRESUMO
The role of Notch signaling during skin development was analyzed using Msx2-Cre to create mosaic loss-of-function alleles with precise temporal and spatial resolution. We find that gamma-secretase is not involved in skin patterning or cell fate acquisition within the hair follicle. In its absence, however, inner root sheath cells fail to maintain their fates and by the end of the first growth phase, the epidermal differentiation program is activated in outer root sheath cells. This results in complete conversion of hair follicles to epidermal cysts that bears a striking resemblance to Nevus Comedonicus. Sebaceous glands also fail to form in gamma-secretase-deficient mice. Importantly, mice with compound loss of Notch genes in their skin phenocopy loss of gamma-secretase in all three lineages, demonstrating that Notch proteolysis accounts for the major signaling function of this enzyme in this organ and that both autonomous and nonautonomous Notch-dependent signals are involved.
Assuntos
Padronização Corporal/genética , Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese , Pele/crescimento & desenvolvimento , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Diferenciação Celular , Linhagem da Célula , Cisto Epidérmico/patologia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Folículo Piloso/anatomia & histologia , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/ultraestrutura , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Modelos Biológicos , Receptores Notch , Glândulas Sebáceas/anormalidades , Pele/anatomia & histologiaRESUMO
Fatty acid transport protein 4 (FATP4) is an acyl-CoA synthetase that is required for normal permeability barrier in mammalian skin. FATP4 (SLC27A4) mutations cause ichthyosis prematurity syndrome, a nonlethal disorder. In contrast, Fatp4-/- mice die neonatally from a defective barrier. Here we used electron microscopy and lipidomics to characterize defects in Fatp4-/- mice. Mutants showed lamellar body, corneocyte lipid envelope, and cornified envelope abnormalities. Lipidomics identified two lipids previously speculated to be present in mouse epidermis, sphingosine ß-hydroxyceramide and monoacylglycerol; mutants displayed decreased proportions of these and the two ceramide classes that carry ultralong-chain, amide-linked fatty acids (FAs) thought to be critical for barrier function, unbound ω-O-acylceramide and bound ω-hydroxyceramide, the latter constituting the major component of the corneocyte lipid envelope. Other abnormalities included elevated amounts of sphingosine α-hydroxyceramide, phytosphingosine non-hydroxyceramide, and 1-O-acylceramide. Acyl chain length alterations in ceramides also suggested roles for FATP4 in esterifying saturated non-hydroxy and ß-hydroxy FAs with at least 25 carbons and saturated or unsaturated ω-hydroxy FAs with at least 30 carbons to CoA. Our lipidomic analysis is the most thorough such study of the Fatp4-/- mouse skin barrier to date, providing information about how FATP4 can contribute to barrier function by regulating fatty acyl moieties in various barrier lipids.
Assuntos
Ceramidas/metabolismo , Proteínas de Transporte de Ácido Graxo/fisiologia , Ácidos Graxos/metabolismo , Queratinócitos/metabolismo , Monoglicerídeos/metabolismo , Pele/metabolismo , Animais , Lipídeos/análise , Camundongos , Camundongos TransgênicosRESUMO
The corneocyte lipid envelope (CLE), a monolayer of ω-hydroxyceramides whose function(s) remain(s) uncertain, is absent in patients with autosomal recessive congenital ichthyoses with mutations in enzymes that regulate epidermal lipid synthesis. Secreted lipids fail to transform into lamellar membranes in certain autosomal recessive congenital ichthyosis epidermis, suggesting the CLE provides a scaffold for the extracellular lamellae. However, because cornified envelopes are attenuated in these autosomal recessive congenital ichthyoses, the CLE may also provide a scaffold for subjacent cornified envelope formation, evidenced by restoration of cornified envelopes after CLE rescue. We provide multiple lines of evidence that the CLE originates as lamellar body-limiting membranes fuse with the plasma membrane: (i) ABCA12 patients and Abca12-/- mice display normal CLEs; (ii) CLEs are normal in Netherton syndrome, despite destruction of secreted LB contents; (iii) CLEs are absent in VSP33B-negative patients; (iv) limiting membranes of lamellar bodies are defective in lipid-synthetic autosomal recessive congenital ichthyoses; and (v) lipoxygenases, lipase activity, and LIPN co-localize within putative lamellar bodies.
Assuntos
DNA/genética , Eritrodermia Ictiosiforme Congênita/genética , Metabolismo dos Lipídeos/genética , Lipídeos/genética , Mutação , Pele/metabolismo , Animais , Análise Mutacional de DNA , Humanos , Eritrodermia Ictiosiforme Congênita/metabolismo , Eritrodermia Ictiosiforme Congênita/patologia , Pele/patologiaRESUMO
The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5ß2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Membrana Basal Glomerular/patologia , Nefrite Hereditária/genética , Síndrome Nefrótica/genética , Distúrbios Pupilares/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Colágeno Tipo IV/química , Colágeno Tipo IV/genética , Anormalidades do Olho/metabolismo , Anormalidades do Olho/patologia , Membrana Basal Glomerular/metabolismo , Humanos , Laminina/química , Laminina/genética , Mutação , Síndromes Miastênicas Congênitas , Nefrite Hereditária/metabolismo , Nefrite Hereditária/patologia , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Distúrbios Pupilares/metabolismo , Distúrbios Pupilares/patologiaRESUMO
Fatty acid transport protein (FATP) 4 is one of a family of six FATPs that facilitate long- and very-long-chain fatty acid uptake. Mice lacking FATP4 are born with tight, thick skin and a defective barrier; they die neonatally because of dehydration and restricted movements. Mutations in SLC27A4, the gene encoding FATP4, cause ichthyosis prematurity syndrome (IPS), characterized by premature birth, respiratory distress, and edematous skin with severe ichthyotic scaling. Symptoms of surviving patients become mild, although atopic manifestations are common. We previously showed that suprabasal keratinocyte expression of a Fatp4 transgene in Fatp4 mutant skin rescues the lethality and ameliorates the skin phenotype. Here we tested the hypothesis that FATP1, the closest FATP4 homolog, can compensate for the lack of FATP4 in our mouse model of IPS, as it might do postnatally in IPS patients. Transgenic expression of FATP1 in suprabasal keratinocytes rescued the phenotype of Fatp4 mutants, and FATP1 sorted to the same intracellular organelles as endogenous FATP4. Thus, FATP1 and FATP4 likely have overlapping substrate specificities, enzymatic activities, and biological functions. These results suggest that increasing expression of FATP1 in suprabasal keratinocytes could normalize the skin of IPS patients and perhaps prevent the atopic manifestations.
Assuntos
Epiderme/metabolismo , Proteínas de Transporte de Ácido Graxo/fisiologia , Animais , Aniridia/etiologia , Aniridia/genética , Ictiose/etiologia , Ictiose/genética , Doenças do Prematuro/etiologia , Doenças do Prematuro/genética , Rim/anormalidades , Camundongos , Transtornos Psicomotores/etiologia , Transtornos Psicomotores/genética , Fator de Transcrição STAT3/fisiologiaRESUMO
FA transport protein 4 (FATP4), one member of a multigene family of FA transporters, was proposed as a major FA transporter in intestinal lipid absorption. Due to the fact that Fatp4(-/-) mice die because of a perinatal skin defect, we rescued the skin phenotype using an FATP4 transgene driven by a keratinocyte-specific promoter (Fatp4(-/-);Ivl-Fatp4(tg/+) mice) to elucidate the role of intestinal FATP4 in dietary lipid absorption. Fatp4(-/-);Ivl-Fatp4(tg/+) mice and wild-type littermates displayed indistinguishable food consumption, growth, and weight gain on either low or high fat (Western) diets, with no differences in intestinal triglyceride (TG) absorption or fecal fat losses. Cholesterol absorption and intestinal TG absorption kinetics were indistinguishable between the genotypes, although Western diet fed Fatp4(-/-);Ivl-Fatp4(tg/+) mice showed a significant increase in enterocyte TG and FA content. There was no compensatory upregulation of other FATP family members or any other FA or cholesterol transporters in Fatp4(-/-);Ivl-Fatp4(tg/+) mice. Furthermore, although serum cholesterol levels were lower in Fatp4(-/-);Ivl-Fatp4(tg/+) mice, there was no difference in hepatic VLDL secretion in-vivo or in hepatic lipid content on either a chow or Western diet. Taken together, our studies find no evidence for a physiological role of intestinal FATP4 in dietary lipid absorption in mice.
Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Absorção Intestinal/fisiologia , Metabolismo dos Lipídeos , Animais , Colesterol/metabolismo , Gorduras na Dieta/administração & dosagem , Proteínas de Transporte de Ácido Graxo/deficiência , Proteínas de Transporte de Ácido Graxo/genética , Feminino , Absorção Intestinal/genética , Mucosa Intestinal/metabolismo , Cinética , Metabolismo dos Lipídeos/genética , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Triglicerídeos/metabolismoRESUMO
Activating Ras mutations occur in a large portion of human tumors. Yet, the signaling pathways involved in Ras-induced tumor formation remain incompletely understood. The mitogen-activated protein kinase pathways are among the best studied Ras effector pathways. The p38 mitogen-activated protein kinase isoforms are important regulators of key biological processes including cell proliferation, differentiation, survival, inflammation, senescence, and tumorigenesis. However, the specific in vivo contribution of individual p38 isoforms to skin tumor development has not been elucidated. Recent studies have shown that p38delta, a p38 family member, functions as an important regulator of epidermal keratinocyte differentiation and survival. In the present study, we have assessed the effect of p38delta deficiency on skin tumor development in vivo by subjecting p38delta knockout mice to a two-stage 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate chemical skin carcinogenesis protocol. We report that mice lacking p38delta gene exhibited a marked resistance to development of 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin papillomas, with increased latency and greatly reduced incidence, multiplicity, and size of tumors compared with wild-type mice. Our data suggest that the underlying mechanism for reduced susceptibility to skin carcinogenesis in p38delta-null mice involves a defect in proliferative response associated with aberrant signaling through the two major transformation-promoting pathways: extracellular signal-regulated kinase 1/2-activator protein 1 and signal transducer and activator of transcription 3. These findings strongly suggest an in vivo role for p38delta in promoting cell proliferation and tumor development in epidermis and may have therapeutic implication for skin cancer.