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The expression of the extracellular sulfatase SULF2 has been associated with increased hepatocellular carcinoma (HCC) growth and poor patient survival. However, the molecular mechanisms underlying SULF2-associated tumor growth remain unclear. To address this gap, here we developed a transgenic mouse overexpressing Sulf2 in hepatocytes under the control of the transthyretin promoter. In this model, Sulf2 overexpression potentiated diethylnitrosamine-induced HCC. Further analysis indicated that the transcription factor GLI family zinc finger 1 (GLI1) mediates Sulf2 expression during HCC development. A cross of the Sulf2-overexpressing with Gli1-knockout mice revealed that Gli1 inactivation impairs SULF2-induced HCC. Transcriptomic analysis revealed that Sulf2 overexpression is associated with signal transducer and activator of transcription 3 (STAT3)-specific gene signatures. Interestingly, the Gli1 knockout abrogated SULF2-mediated induction of several STAT3 target genes, including suppressor of cytokine signaling 2/3 (Socs2/3); Pim-1 proto-oncogene, Ser/Thr kinase (Pim1); and Fms-related tyrosine kinase 4 (Flt4). Human orthologs were similarly regulated by SULF2, dependent on intact GLI1 and STAT3 functions in HCC cells. SULF2 overexpression promoted a GLI1-STAT3 interaction and increased GLI1 and STAT3 enrichment at the promoters of their target genes. Interestingly, the SULF2 overexpression resulted in GLI1 enrichment at select STAT3 consensus sites, and vice versa. siRNA-mediated STAT3 or GLI1 knockdown reduced promoter binding of GLI1 and STAT3, respectively. Finally, chromatin-capture PCR confirmed long-range co-regulation of SOCS2 and FLT3 through changes in promoter conformation. These findings define a mechanism whereby SULF2 drives HCC by stimulating formation of a GLI1-STAT3 transcriptional complex.
Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Fator de Transcrição STAT3/metabolismo , Sulfatases/fisiologia , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Carcinogênese , Humanos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Ligação Proteica , Proto-Oncogene Mas , Fatores de Transcrição STAT , Sulfatases/metabolismo , TransativadoresRESUMO
Sulfatase 2 (SULF2) is a heparan sulfate editing enzyme that regulates the milieu of growth factors and cytokines involved in a variety of cellular processes. We used a murine model of diet-induced steatohepatitis to assess the effect of SULF2 downregulation on the development of nonalcoholic steatohepatitis (NASH) and liver fibrosis. Wild-type B6;129 mice (WT) and Sulf2-knockout B6;129P2-SULF2Gt(PST111)Byg mice (Sulf2-KO) were fed a fast-food diet (FFD) rich in saturated fats, cholesterol, and fructose or a standard chow diet (SC) ad libitum for 9 mo. WT mice on FFD showed a threefold increase in hepatic Sulf2 mRNA expression, and a 2.2-fold increase in hepatic SULF2 protein expression compared with WT mice on SC. Knockout of Sulf2 led to a significant decrease in diet-mediated weight gain and dyslipidemia compared with WT mice on FFD. Knockout of Sulf2 also abrogated diet-induced steatohepatitis and hepatic fibrosis compared with WT mice on FFD. Furthermore, expression levels of the profibrogenic receptors TGFßR2 and PDGFRß were significantly decreased in Sulf2-KO mice compared with WT mice on FFD. Together, our data suggest that knockout of Sulf2 significantly downregulates dyslipidemia, steatohepatitis, and hepatic fibrosis in a diet-induced mouse model of NAFLD, suggesting that targeting of SULF2 signaling may be a potential therapeutic mechanism in NASH.NEW & NOTEWORTHY We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.
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Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Sulfatases/genética , Animais , Dieta Ocidental , Regulação para Baixo , Dislipidemias/genética , Fast Foods , Feminino , Resistência à Insulina , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , RNA Interferente Pequeno/genética , Aumento de Peso/genéticaRESUMO
A variety of approaches have been developed for the derivation of hepatocyte-like cells from pluripotent stem cells. Currently, most of these strategies employ step-wise differentiation approaches with recombinant growth-factors or small-molecule analogs to recapitulate developmental signaling pathways. Here, we tested the efficacy of a small-molecule based differentiation protocol for the generation of hepatocyte-like cells from human pluripotent stem cells. Quantitative gene-expression, immunohistochemical, and western blot analyses for SOX17, FOXA2, CXCR4, HNF4A, AFP, indicated the stage-specific differentiation into definitive endoderm, hepatoblast and hepatocyte-like derivatives. Furthermore, hepatocyte-like cells displayed morphological and functional features characteristic of primary hepatocytes, as indicated by the production of ALB (albumin) and α-1-antitrypsin (A1AT), as well as glycogen storage capacity by periodic acid-Schiff staining. Together, these data support that the small-molecule based hepatic differentiation protocol is a simple, reproducible, and inexpensive method to efficiently drive the differentiation of human pluripotent stem cells towards a hepatocyte-like phenotype, for downstream pharmacogenomic and regenerative medicine applications.
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Diferenciação Celular/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Endoderma/citologia , Endoderma/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Humanos , Fígado/efeitos dos fármacos , Células-Tronco Pluripotentes/citologiaRESUMO
Upamostat is an orally available small-molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2, trypsin 3 (PRSS1/2/3), and the urokinase-type plasminogen activator (uPA). These enzymes are expressed in many cancers, especially during tissue remodeling and subsequent tumor cell invasion. Opaganib (ABC294640), a novel, orally available small molecule is a selective inhibitor of the phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P) by sphingosine kinase 2 (SPHK2). Both sphingosine kinase 1 (SPHK1) and SPHK2 are known to regulate the proliferation-inducing compound S-1-P. However, SPHK2 is more critical in cancer pathogenesis. The goal of this project was to investigate the potential antitumor effects of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) xenografts in nude mice. PAX165, a patient-derived xenograft (PDX) from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2, and PRSS3. Four groups of 18 mice each were treated with upamostat, opaganib, both, or vehicle. Mouse weights and PAX165 tumor volumes were measured. Tumor volumes in the upamostat, opaganib, and upamostat plus opaganib groups were significantly decreased compared to the control group.
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Nuclear actin - which is immunologically distinct from cytoplasmic actin - has been documented in a number of differentiated cell types, and cardiac isoforms of troponin I (cTnI) and troponin T (cTnT) have been detected in association with nuclei of adult human cardiac myocytes. It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. We first tested the hypothesis that nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. We also tested the hypothesis that of these five proteins, only actin is present in nuclei of multipotent, bone marrow-derived mesenchymal stem cells (BM-MSCs) from adult rats in culture, but that cTnC, cTnI, cTnT and cTm appear early and uniquely following cardiomyogenic differentiation. Here we show that nuclear actin is present within nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs. We furthermore show that cTnC, cTnI, cTnT and cTm are not only present in myofilaments of ventricular cardiomyocytes in culture but are also within their nuclei; significantly, these four proteins appear between days 3 and 5 in both myofilaments and nuclei of BM-MSCs treated to differentiate into cardiomyogenic cells. These observations indicate that cardiac troponin and tropomyosin could have important cellular function(s) beyond Ca(2+)-regulation of contraction. While the roles of nuclear-associated actin, troponin subunits and tropomyosin in cardiomyocytes are not known, we anticipate that the BM-MSC culture system described here will be useful for elucidating their function(s), which likely involve cardiac-specific, Ca(2+)-dependent signaling in the nucleus.
Assuntos
Actinas/metabolismo , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/citologia , Tropomiosina/metabolismo , Troponina C/metabolismo , Troponina I/metabolismo , Troponina T/metabolismo , Actinas/genética , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Cálcio/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Tropomiosina/genética , Troponina C/genética , Troponina I/genética , Troponina T/genéticaRESUMO
The PAX1 gene plays an important role in the development of the parathyroid glands and the thymus. Mouse knockout models of PAX1, PAX3, and PAX9 have been found to have hypoplastic or absent parathyroid glands. To our knowledge, there are no reported cases of PAX1-associated hypoparathyroidism in humans. We present a case of hypoparathyroidism in a 23-month-old boy with a homozygous pathogenic variant in the PAX1 gene (PAX1 NM_006192.5 c.463_465del variant), predicted to cause an in-frame deletion of asparagine at position 155 (p.Asn155del) of the PAX1 protein. The hypoparathyroidism was unmasked after the patient developed significant hypocalcemia while receiving GoLYTELY (polyethylene glycol 3350, sodium sulfate anhydrous, sodium bicarbonate, sodium chloride, potassium chloride) for bowel cleanout. The patient had mild and asymptomatic hypocalcemia prior to hospitalization. The patient was noted to have inappropriately normal parathyroid hormone (PTH) level at the time of documented hypocalcemia thereby suggesting a diagnosis of hypoparathyroidism. Plain language summary: The first human case of hypoparathyroidism associated with a rare genetic disorder: a case report of PAX1 gene mutation The paired box (PAX) gene family is important for embryo development. One subfamily, PAX1, is necessary for development of the spinal column, thymus (important for the immune system development), and parathyroid (helps regulate the amount of calcium in the body). We present the case of a 23-month-old boy with known PAX1 gene mutation who came in with episodes of vomiting and poor growth. His presentation was thought to be most likely related to constipation. He was started on bowel cleanout medication and intravenous fluids. However, his calcium that had been mildly low subsequently dropped to very low levels. The level of parathyroid hormone (which helps regulate calcium levels) was inappropriately normal, meaning that his body was unable to make more, and was consistent with hypoparathyroidism. He was treated with calcium supplements and vitamin D and calcium levels normalized. He continues to be on calcium and vitamin D and calcium levels have remained stable. Doctors should keep this complication in mind when treating patients with PAX1 gene mutation.
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Purpose: To highlight the importance of the utility of clinical exome sequencing, and show how it led to the diagnosis of nonsyndromic autosomal dominant optic atrophy arising from an autosomal dominant variant in AFG3L2. Observations: A healthy father and daughter of East African heritage experienced the onset of vision loss in the first decade of life due to optic atrophy. No additional neurologic or neuroimaging abnormalities were detected. Clinical exome sequencing was initially performed and provided a negative result. Reanalysis of the sequencing data revealed an autosomal dominant pathogenic variant in AFG3L2, c.1064C>T (p.Thr355Met), a gene that was recently identified to be associated with non-syndromic optic atrophy. This variant has previously been reported in a patient with optic atrophy, motor disturbances, and an abnormal brain MRI. Conclusions: As the causes of dominant optic atrophy continue to expand, accurate genetic diagnosis is aided by an iterative reanalysis process for individuals and families when initial exome and genome testing does not provide an answer.
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Mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders caused by deficient levels and/or activity of glycosaminoglycan (GAG)-degradative enzymes. MPS are characterized by accumulation of the mucopolysaccharides heparan sulfate, dermatan sulfate, keratan sulfate, or chondroitin sulfate in tissues. We report the case of a 38-year-old woman with a history of joint restriction and retinitis pigmentosa who developed bivalvular heart failure requiring surgery. It was not until pathological examination of surgically excised valvular tissue that a diagnosis of MPS I was made. Her musculoskeletal and ophthalmologic symptoms, when placed in the context of MPS I, painted the diagnostic picture of a genetic syndrome that was overlooked until a diagnosis was made in late middle age.
⢠A 38-year-old woman with heart failure had heart valve surgery. Examining her cardiac valve tissue under the microscope suggested a metabolic disorder called mucopolysaccharidosis type I (MPS I). ⢠MPS I is due to defective breakdown of sugar molecules (called glycosaminoglycans or GAGs for short) in the body which then can accumulate, causing dysfunction. ⢠Our patient had short stature, a curved spine, stiff joints, and a degenerative eye disease called retinitis pigmentosa, all of which were due to her undiagnosed MPS I. ⢠Most patients with MPS I are discovered on newborn screening when they are babies, or at very young ages due to severe symptoms related to the disease. ⢠Our patient had a form of MPS I that was less severe, and the first symptom she received medical care for was her eye symptoms. ⢠A diagnosis of MPS I made in middle adulthood is unusual for MPS I, and so is an important learning case for providers as there were clues hidden in her medical history that suggested a genetic or inherited syndrome. ⢠Our genetics specialists were able to make a definitive diagnosis of MPS I and begin treatment with enzyme replacement therapy, as well as provide information for the patient about her risk of passing this disease on to children.
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Clinical genetic testing enables the detection of specific gene mutations and variants that predispose individuals and their family members to disease. In recent years, tremendous strides have been made in the variety of clinically useful tests. Targeted testing for specific mutations that cause well-known syndromes enables the efficient diagnosis of genetic diseases with cutaneous manifestations. Testing for specific genes, however, may not always reveal a diagnosis. Expanded options are available. This review outlines the major types of available technology with a focus on those tests most useful for pediatric dermatologists.
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Dermatologia , Criança , Testes Genéticos , Humanos , MutaçãoRESUMO
BACKGROUND: The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients. RESULTS: We demonstrated that the expression profile of stemness markers was altered in BM-MSCs derived from old rats. BM-MSCs from young rats (4 months) expressed Oct-4, Sox-2 and NANOG, but we failed to detect Sox-2 and NANOG in BM-MSCs from older animals (15 months). Chondrogenic, osteogenic and adipogenic potential is compromised in old BM-MSCs. Stimulation with a cocktail mixture of bone morphogenetic protein (BMP-2), fibroblast growth factor (FGF-2) and insulin-like growth factor (IGF-1) induced cardiomyogenesis in young BM-MSCs but not old BM-MSCs. Significant differences in the expression of gap junction protein connexin-43 were observed between young and old BM-MSCs. Young and old BM-MSCs fused with neonatal ventricular cardiomyocytes in co-culture and expressed key cardiac transcription factors and structural proteins. Cells from old animals expressed significantly lower levels of VEGF, IGF, EGF, and G-CSF. Significantly higher levels of DNA double strand break marker γ-H2AX and diminished levels of telomerase activity were observed in old BM-MSCs. CONCLUSION: The results suggest age related differences in the differentiation capacity of BM-MSCs. These changes may affect the efficacy of BM-MSCs for use in stem cell therapy.
Assuntos
Células-Tronco Adultas/metabolismo , Envelhecimento , Células-Tronco Mesenquimais/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Fatores Etários , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/citologia , Proteína Morfogenética Óssea 2/farmacologia , Células Cultivadas , Conexina 43/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/metabolismo , RatosRESUMO
Transforming growth factor-ß (TGF-ß) activates hepatic stellate cells (HSCs), which drive liver fibrosis via the production and deposition of extracellular matrix (ECM). We aimed to elucidate the mechanistic role of sulfatase-2 (SULF2) in liver fibrosis. To this end, we induced liver fibrosis in wild-type (WT) and SULF2 knockout (Sulf2-KO) mice (6-8 weeks-old) via bile duct ligation (BDL), intraperitoneal injection of carbon tetrachloride (CCl4) or thioacetamide (TAA). The levels of fibrosis in the liver sections were assessed via Sirius red and Masson's trichrome staining, immunohistochemistry and immunoblotting for α-smooth muscle actin (α-SMA) and hydroxyproline. To evaluate the interaction between TGF-ß and SULF2, we transfected human HSCs with scrambled control shRNA and shRNA constructs targeting SULF2 and measured α-SMA expression following treatment with TGF-ß1 ligand. We show here that knockout of SULF2 significantly decreases collagen content, as well as bands of bridging fibrosis, as demonstrated by Sirius red, Masson's trichrome and α-SMA staining after BDL, CCl4 and TAA injection in Sulf2-KO versus WT mice. In all three models of liver fibrosis, we observed significantly lower levels of hydroxyproline in the Sulf2-KO mice compared to the WT mice. HSCs with reduced levels of SULF2 failed to significantly express α-SMA and collagen type I following treatment with TGF-ß1. Furthermore, SULF2 co-localizes with TGFBR3 and the in vitro knockdown of SULF2 in HSCs decreases the release of TGF-ß1 from TGFBR3. Together, these data suggest that SULF2 regulates liver fibrosis via the TGF-ß signaling pathway. Pharmacologic inhibition of SULF2 may represent a novel therapeutic approach to improve liver fibrosis.
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The geographic and racial-ethnic inequities in health in the US are concerning, and their reduction is a priority for the national health initiative: Health People 2010. Though voluntary, life-style factors, such as smoking, are partially a cause of unevenness in health outcomes, targeting tobacco to minority youth raises questions about the extent to which behaviors are exacerbated by the local retail environment. In this case study, a GIS analysis of neighborhoods where businesses sold tobacco to minors was conducted with 2005 Florida Department of Health, and US census data, finding that in Miami, underage tobacco sales were significantly more concentrated in Hispanic majority neighborhoods.
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Comércio/legislação & jurisprudência , Grupos Minoritários , Nicotiana , Características de Residência , Adolescente , Comércio/estatística & dados numéricos , Florida , Sistemas de Informação Geográfica , HumanosRESUMO
Adult stem cells are critical for organ-specific regeneration and self-renewal with advancing age. The prospect of being able to reverse tissue-specific post-injury sequelae by harvesting, culturing and transplanting a patient's own stem and progenitor cells is exciting. Mesenchymal stem cells have emerged as a reliable stem cell source for this treatment modality and are currently being tested in numerous ongoing clinical trials. Unfortunately, the fervor over mesenchymal stem cells is mitigated by several lines of evidence suggesting that their efficacy is limited by natural aging. This article discusses the mechanisms and manifestations of age-associated deficiencies in mesenchymal stem cell efficacy. A consideration of recent experimental findings suggests that the ecological niche might be responsible for mesenchymal stem cell aging.