RESUMO
Oxidative stress and neuroinflammation play important roles in Alzheimer's disease (AD). ABCG2 is a transporter protein expressed in the brain and involved in GSH transport. To study the roles of Abcg2 in oxidative stress and AD, we cross-bred Tg-SwDI and Abcg2-KO mice and generated Tg-SwDI/Abcg2-KO (double-tg) mice. Brain tissues from double-tg, Tg-SwDI, wild-type, and Abcg2-KO mice at various ages were analyzed. Aß40 and Aß42 were detected in Tg-SwDI and double-tg mice. Total brain GSH was decreased and levels of lipid/DNA oxidation were increased in 3-month double-tg compared to Tg-SwDI mice. Low brain GSH was still detected in 9-month double-tg mice. Increased HMOX-1 and MCP-5 expression was observed in 9-month double-tg mice but not in Tg-SwDI mice compared to WT and Abcg2-KO mice. Increased HMOX-1 and decreased ICAM-1 expression were observed in 12-month double-tg mice compared to Tg-SwDI mice. The levels of Nrf-2 expression and activity were decreased in 6-month double-tg mice. Behavioral tests show impaired cognitive/memory performance of 9-month double-tg compared to Tg-SwDI mice as well as WT and Abcg2-KO mice. These results suggest that Abcg2 deficiency increases oxidative stress and alters inflammatory response in the brain and exacerbates cognitive/memory deficit in double-tg mice at different developmental stages.
Assuntos
Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Transtornos Cognitivos/genética , Transtornos Cognitivos/psicologia , Estresse Oxidativo/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Envelhecimento/genética , Envelhecimento/psicologia , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/deficiência , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Citocinas/metabolismo , DNA/genética , DNA/isolamento & purificação , Progressão da Doença , Encefalite/genética , Encefalite/patologia , Ensaio de Imunoadsorção Enzimática , Glutationa/metabolismo , Heme Oxigenase-1/metabolismo , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/metabolismo , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fator 2 Relacionado a NF-E2/fisiologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Alzheimer's disease (AD) is characterized by accumulation and deposition of Abeta peptides in the brain. Abeta deposition in cerebrovessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Since Abeta can be transported across blood-brain barrier (BBB), aberrant Abeta trafficking across BBB may contribute to Abeta accumulation in the brain and CAA development. Expression analyses of 273 BBB-related genes performed in this study showed that the drug transporter, ABCG2, was significantly upregulated in the brains of AD/CAA compared with age-matched controls. Increased ABCG2 expression was confirmed by Q-PCR, Western blot, and immunohistochemistry. Abcg2 was also increased in mouse AD models, Tg-SwDI and 3XTg. Abeta alone or in combination with hypoxia/ischemia failed to stimulate ABCG2 expression in BBB endothelial cells; however, conditioned media from Abeta-activated microglia strongly induced ABCG2 expression. ABCG2 protein in AD/CAA brains interacted and coimmunoprecipitated with Abeta. Overexpression of hABCG2 reduced drug uptake in cells; however, interaction of Abeta(1-40) with ABCG2 impaired ABCG2-mediated drug efflux. The role of Abcg2 in Abeta transport at the BBB was investigated in Abcg2-null and wild-type mice after intravenous injection of Cy5.5-labeled Abeta(1-40) or scrambled Abeta(40-1). Optical imaging analyses of live animals and their brains showed that Abcg2-null mice accumulated significantly more Abeta in their brains than wild-type mice. The finding was confirmed by immunohistochemistry. These results suggest that ABCG2 may act as a gatekeeper at the BBB to prevent blood Abeta from entering into brain. ABCG2 upregulation may serve as a biomarker of CAA vascular pathology in AD patients.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Angiopatia Amiloide Cerebral/metabolismo , Proteínas de Neoplasias/biossíntese , Fragmentos de Peptídeos/metabolismo , Regulação para Cima/fisiologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Barreira Hematoencefálica/patologia , Linhagem Celular , Células Cultivadas , Angiopatia Amiloide Cerebral/genética , Angiopatia Amiloide Cerebral/patologia , Resistência a Múltiplos Medicamentos , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genéticaRESUMO
Alzheimer's disease is characterized by accumulation and deposition of Aß peptides in the brain. Aß deposition generates reactive-oxygen species (ROS), which are involved in Alzheimer's inflammatory and neurodegenerative pathology. We have previously observed that, in Alzheimer's disease brain, ABCG2 is up-regulated and AP-1 is activated, but NF-κB is not activated. In the present study, we examine the roles and mechanism of ABCG2 on ROS generation, inflammatory gene expression and signaling, heme homeostasis and Aß production in cell models and on inflammatory signaling and Aß deposition in Abcg2-knockout and wild-type mice. Our results show that ABCG2 plays a protective role against oxidative stress by decreasing ROS generation, enhancing antioxidant capacity, regulating heme level, and inhibiting inflammatory response in cell models. ABCG2 inhibits NF-κB activation but has less effect on AP-1 activation induced by ROS. This results in inhibition of interleukin-8 and growth-related oncogene (GRO) expression induced by ROS via NF-κB pathway. Abcg2 deficiency increased Aß deposition and NF-κB activation in the brains of Abcg2-knockout mice compared with controls. These findings suggest that ABCG2 may relieve oxidative stress and inflammatory response via inhibiting NF-κB signaling pathway in cell models and brain tissues and thus may play a potential protective role in Alzheimer's neuroinflammatory response.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Doença de Alzheimer/metabolismo , Proteínas de Neoplasias/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Quimiocina CXCL1/biossíntese , Quimiocina CXCL2/biossíntese , Hemina/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Inflamação/metabolismo , Interleucina-8/biossíntese , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/metabolismo , terc-Butil Hidroperóxido/toxicidadeRESUMO
Alzheimer's disease (AD) is characterized by overproduction of A beta derived from APP cleavage via beta- and gamma-secretase pathway. Recent evidence has linked altered cholesterol metabolism to AD pathogenesis. In this study, we show that AD brain had significant cholesterol retention and high beta- and gamma-secretase activities as compared to age-matched non-demented controls (ND). Over one-half of AD patients had an apoE4 allele but none of the ND. beta- and gamma-secretase activities were significantly stimulated in vitro by 40 and 80 microM cholesterol in AD and ND brains, respectively. Both secretase activities in AD brain were more sensitive to cholesterol (40 microM) than those of ND (80 microM). Filipin-stained cholesterol overlapped with BACE and A beta in AD brain sections. Cholesterol (10-80 microM) added to N2a cultures significantly increased cellular cholesterol, beta- and gamma-secretase activities and A beta secretion. Similarly, addition of cholesterol (20-80 microM) to cell lysates stimulated both in vitro secretase activities. Ergosterol slightly decreased beta-secretase activity at 20-80 microM, but strongly inhibited gamma-secretase activity at 40 microM. Cholesterol depletion reduced cellular cholesterol, beta-secretase activity and A beta secretion. Transcription factor profiling shows that several key nuclear receptors involving cholesterol metabolism were significantly altered in AD brain, including decreased LXR-beta, PPAR and TR, and increased RXR. Treatment of N2a cells with LXR, RXR or PPAR agonists strongly stimulated cellular cholesterol efflux to HDL and reduced cellular cholesterol and beta-/gamma-secretase activities. This study provides direct evidence that cholesterol homeostasis is impaired in AD brain and suggests that altered levels or activities of nuclear receptors may contribute to cholesterol retention which likely enhances beta- and gamma-secretase activities and A beta production in human brain.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Encéfalo/metabolismo , Colesterol/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Linhagem Celular Tumoral , Células Cultivadas , Ativação Enzimática/fisiologia , Feminino , Humanos , Masculino , CamundongosRESUMO
Bradykinin is an important autacoid produced in the kidney, regulating both renal function and blood pressure. In vivo studies in anesthetized rabbits, revealed that BK induced diuresis (UV), natriuresis (U(Na)V) and was not associated with renal hemodynamic changes. These diuretic and natriuretic effects were blocked by the BK-B2 antagonist HOE-140. BK also inhibits vasopressin (AVP)-stimulated water flow (L(p)) in microperfused rabbit cortical collecting ducts (rCCD), in a concentration-dependent fashion, consistent with its in vivo diuretic effects. BK-B1 antagonist Leu8-des-Arg9-BK did not alter the effect of BK on Lp, but HOE-140 completely blocked the inhibitory effects of BK on Lp. While BK did not increase [Ca2+]i in fura-2 loaded freshly microdissected rCCD, BK increased [Ca2+]i in immortalized cultured rCCD cells demonstrating different signaling mechanisms are activated by BK in microdissected versus cultured rCCD. In microperfused rCCD, neither the protein kinase C inhibitor staurosporine nor the phospholipase C (PLC) inhibitor U-73,122 attenuated the BK response arguing against activation of PLC/PKC by BK in rCCD. We conclude: (1) BK induces UV and U(Na)V by a BK-B(2) receptor; (2) BK inhibits AVP-stimulated Lp by a BK-B2 receptor suggesting that its effects on Lp are not via a PLC/PKC; (3) finally, BK raises [Ca2+]i in rCCD cells by a BK-B2 receptor mechanism.
Assuntos
Líquidos Corporais/metabolismo , Eletrólitos/metabolismo , Rim/metabolismo , Receptor B2 da Bradicinina/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Líquidos Corporais/efeitos dos fármacos , Bradicinina/análogos & derivados , Bradicinina/antagonistas & inibidores , Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina , Células Cultivadas , Rim/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Coelhos , Fatores de TempoRESUMO
Neutrophil gelatinase-associated lipocalin (NGAL) is a novel member of the lipocalin family and may be a new human oncogene product, but function of NGAL is not clear in the cancer. It was recently found that NGAL was overexpressed in the progression of malignant transformation from human immortalized esophageal epithelial cell line SHEE to esophageal carcinoma cell line SHEEC. This indicated that cell line SHEEC was a good model for exploring functions of NGAL in the carcinogenesis. The effects of blocking transcription of NGAL gene on invasion, division and proliferation of SHEEC cells were studied by antisense blocking RNA technique and tumor formation in nude mice. The results showed that the antisense blocking of transcription of NGAL gene not only decreased effectively the activity of MMP-9 and MMP-2 secreted by SHEEC cells, but suppressed significantly also the invasion of these cells in nude mice. However, the telomere length, the content of the cellular topoisomerase II-alpha and cellular proliferation index (PI) of the SHEEC cells have not been changed markedly. These results indicate that NGAL is possibly involved in invasion of tumor cells by regulating activity of MMP-9 and MMP-2, but is not apparently related with division and proliferation of tumor cells in SHEEC.
Assuntos
Proteínas de Fase Aguda , Proteínas de Transporte/genética , Neoplasias Esofágicas/patologia , Proteínas Oncogênicas , Animais , Western Blotting , Proteínas de Transporte/metabolismo , Divisão Celular/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Antissenso/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Humanos , Lipocalina-2 , Lipocalinas , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Transplante de Neoplasias , Plasmídeos/genética , Proteínas Proto-Oncogênicas , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
BACKGROUND: Aß transport (flux) across the blood-brain barrier (BBB) is thought to contribute to the pathogenesis of Alzheimer's disease as well as to elimination of toxic amyloid from the brain by immunotherapy. Several BBB transporters have been implicated in Aß exchange between brain parenchyma and the circulation, including efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2. Here we describe an application of in vivo optical imaging methods to study Aß transport across the BBB in wild-type or animals deficient in specific efflux transporters. METHODS/DESIGN: Synthetic human Aß1-40 or scrambled Aß40-1 peptides were labeled with the near-infrared fluorescent tracer, Cy5.5. The free tracer or Cy5.5-labeled peptides were injected intravenously into Abcb1-KO or Abcg2-KO mice or their corresponding wild-type controls. The animals were imaged prospectively at different time points over a period of 8 hours using eXplore Optix small animal imager. At the end of the observation, animals were sacrificed by perfusion, their brains were imaged ex-vivo and sectioned for immunofluorescence analyses. DISCUSSION: After appropriate circulation time, the fluorescence concentration in the head ROI measured in vivo was close to background values in both wild-type and Abcb1-KO or Abcg2-KO mice injected with either free dye or scrambled Aß40-1-Cy5.5. In animals injected with Aß1-40-Cy5.5, the deficiency in either Abcb1 or Abcg2 resulted in significant increases in fluorescence concentration in the head ROIs 2 hours after injection compared to wild-type animals. Fluorescence decay (elimination rate) over 2-8 hours after injection was similar between wild-type (t1/2 = 1.97 h) and Abcg2-KO (t1/2 = 2.34 h) and was slightly faster (t1/2 = 1.38 h) in Abcb1-KO mice. In vivo time-domain imaging method allows prospective, dynamic analyses of brain uptake/elimination of fluorescently-labeled compounds, including Aß. Deficiency of either of the two major efflux pumps, Abcb1 and Abcg2, implicated in Aß trafficking across the BBB, resulted in increased accumulation of peripherally-injected Aß1-40 in the brain.
RESUMO
OBJECTIVE The double transgenic mouse model (APPswe/PS1dE9) of Alzheimer's disease (AD) has been widely used in experimental studies. ß-Amyloid (Aß) peptide is excessively produced in AD mouse brain, which affects synaptic function and the development of central nervous system. However, little has been reported on characterization of this model. The present study aimed to characterize this mouse AD model and its wild-type counterparts by biochemical and functional approaches. METHODS Blood samples were collected from the transgenic and the wild-type mice, and radial arm water maze behavioral test was conducted at the ages of 6 and 12 months. The mice were sacrificed at 12-month age. One hemisphere of the brain was frozen-sectioned for immunohistochemistry and the other hemisphere was dissected into 7 regions. The levels of Aß1-40, Aß1-42 and 8-hydroxydeoxyguanosine (8-OHdG) in blood or/and brain samples were analyzed by ELISA. Secretase activities in brain regions were analyzed by in vitro assays. RESULTS The pre-mature death rate of transgenic mice was approximately 35% before 6-month age, and high levels of Aß(1-40) and Aß(1-42) were detected in these dead mice brains with a ratio of 1:10. The level of blood-borne Aß at 6-month age was similar with that at 12-month age. Besides, Aß(1-40) level in the blood was significantly higher than Aß(1-42) level at the ages of 6 and 12 months (ratio 2.37:1). In contrast, the level of Aß(1-42) in the brain (160.6 ng/mg protein) was higher than that of Aß(1-40) (74 ng/mg protein) (ratio 2.17:1). In addition, the levels of Aß(1-40) and Aß(1-42) varied markedly among different brain regions. Aß(1-42) level was significantly higher than Aß(1-40) level in cerebellum, frontal and posterior cortex, and hippocampus. Secretase activity assays did not reveal major differences among different brain regions or between wild-type and transgenic mice, suggesting that the transgene PS1 did not lead to higher γ-secretase activity but was more efficient in producing Aß(1-42) peptides. 8-OHdG, the biomarker of DNA oxidative damage, showed a trend of increase in the blood of transgenic mice, but with no significant difference, as compared with the wild-type mice. Behavioral tests showed that transgenic mice had significant memory deficits at 6-month age compared to wild-type controls, and the deficits were exacerbated at 12-month age with more errors. CONCLUSION These results suggest that this mouse model mimics the early-onset human AD and may represent full-blown disease at as early as 6-month age for experimental studies.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Precursor de Proteína beta-Amiloide/genética , Modelos Animais de Doenças , Transtornos da Memória/genética , 8-Hidroxi-2'-Desoxiguanosina , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/mortalidade , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangue , Humanos , Aprendizagem em Labirinto , Transtornos da Memória/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismoRESUMO
The homeostatic function of prostaglandin E(2) (PGE(2)) is dependent on a balance of EP receptor-mediated events. A disruption in this balance may contribute to the progression of renal injury. Although PGE(2) excretion is elevated in diabetes, the expression of specific EP receptor subtypes has not been studied in the diabetic kidney. Therefore, the purpose of this study was to characterize the expression profile of four EP receptor subtypes (EP(1-4)) in 16-wk streptozotocin (STZ) and B6-Ins2(Akita) type I diabetic mice. In diabetic mice, the ratio of kidney weight to body weight was increased twofold compared with controls, blood glucose was elevated, but urine albumin was only increased in B6-Ins2(Akita) mice. The excretion of PGE(2) and its metabolite was augmented two- to fourfold as determined by enzyme immunoassay. Accordingly, renal cyclooxygenases were also increased in diabetic mice, with isoform-specific and regional differences in each model. Finally, there was altered EP(1-4) receptor expression in diabetic kidneys, with significant differences between STZ and B6-Ins2(Akita) mice (fold-control). In STZ mice, cortical EP(1) increased by 1.6, EP(3) increased by 2.3, and EP(4) decreased by 0.63; yet in B6-Ins2(Akita) mice, cortical EP(1) increased by 2.4, but there was a general decrease in the remaining subtypes. Similarly, in the STZ medulla EP(3) increased by 3.6, but both EP(1) and EP(3) increased by 5.5 and 1.95, respectively, in B6-Ins2(Akita) mice. Therefore, knowing the pattern of change in relative EP receptor expression in the kidney could be useful in identifying specific EP targets for the prevention of various components of diabetic kidney disease.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Rim/metabolismo , Receptores de Prostaglandina E/biossíntese , Animais , Western Blotting , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Diabetes Mellitus Tipo 1/genética , Nefropatias Diabéticas/patologia , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , RNA/análise , RNA/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We investigated the role of the prostaglandin E(2) (PGE(2)) EP(1) receptor in modulating urine concentration as it is expressed along the renal collecting duct where arginine-vasopressin (AVP) exerts its anti-diuretic activity, and in the paraventricular and supraoptic nuclei of the hypothalamus where AVP is synthesized. The urine osmolality of EP(1)-null mice (EP(1)(-/-)) failed to match levels achieved by wild-type (WT) counterparts upon water deprivation (WD) for 24 h. This difference was reflected by higher plasma osmolality in WD EP(1)(-/-) mice. Along the collecting duct, the induction and subapical to plasma membrane translocation of the aquaporin-2 water channel in WD EP(1)(-/-) mice appeared equivalent to that of WD WT mice as determined by quantitative RT-PCR and immunohistochemistry. However, medullary interstitial osmolalities dropped significantly in EP(1)(-/-) mice following WD. Furthermore, urinary AVP levels of WD EP(1)(-/-) mice were significantly lower than those of WD WT mice. This deficit could be traced back to a blunted induction of hypothalamic AVP mRNA expression in WD EP(1)(-/-) mice as determined by quantitative RT-PCR. Administration of the AVP mimetic [deamino-Cys(1),D-Arg(8)]-vasopressin restored a significant proportion of the urine concentrating ability of WD EP(1)(-/-) mice. When mice were water loaded to suppress endogenous AVP production, urine osmolalities increased equally for WT and EP(1)(-/-) mice. These data suggest that PGE(2) modulates urine concentration by acting at EP(1) receptors, not in the collecting duct, but within the hypothalamus to promote AVP synthesis in response to acute WD.
Assuntos
Capacidade de Concentração Renal/genética , Receptores de Prostaglandina E/deficiência , Privação de Água/fisiologia , Animais , Aquaporina 2/biossíntese , Arginina Vasopressina/biossíntese , Desamino Arginina Vasopressina/farmacologia , Camundongos , Receptores de Prostaglandina E Subtipo EP1RESUMO
BACKGROUND & OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) was a novel member of the lipocalin family. The authors previously found that NGAL was overexpressed in the progress of malignant transformation from human immortalized esophageal epithelial cell line SHEE to esophageal carcinoma cell line SHEEC. However, the regulation mechanism of NGAL overexpression was not known. The objective of this study was to clone 5'-untranslated region(5'-UTR) and 3'untranslated region (3'-UTR) of NGAL in SHEEC and to analyze their structural characters. METHODS: 5'-UTR and 3'-UTR of NGAL were cloned from SHEEC using rapid amplification of cDNA ends(RACE). After sequencing the alignment of their nucleotides was analyzed by BLAST database of NCBI and the potential cis-acting elements in the 3'-UTR were identified by computer analysis. RESULTS: The authors cloned and sequenced 69 bp 5'-UTR and 147 bp 3'-UTR of NGAL gene on the basis of the previous works and did not find any base pair mutation. CONCLUSION: NGAL gene from SHEEC had the entire 5'-UTR and 3'-UTR.
Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Proteínas de Fase Aguda , Proteínas de Transporte/genética , Neoplasias Esofágicas/genética , Proteínas Oncogênicas , Regiões 3' não Traduzidas/isolamento & purificação , Regiões 5' não Traduzidas/isolamento & purificação , Clonagem Molecular , Humanos , Lipocalina-2 , Lipocalinas , Proteínas Proto-Oncogênicas , RNA Neoplásico/análise , Células Tumorais CultivadasRESUMO
Autosomal recessive polycystic kidney disease (ARPKD) is a common hereditary renal cystic disease in infants and children. By genetic linkage analyses, the gene responsible for this disease, termed polycystic kidney and hepatic disease 1 (PKHD1), was mapped on human chromosome 6p21.1-p12, and has been further localized to a 1-cM genetic interval flanked by the D6S1714/D6S243 (telomeric) and D6S1024 (centromeric) markers. We recently identified a novel gene in this genetic interval from kidney cDNA, using cloning strategies. The gene PKHD1 (PKHD1-tentative) encodes a novel 3396-amino-acid protein with no apparent homology with any known proteins. We named its gene product "tigmin" because it contains multiple TIG domains, which usually are seen in proteins containing immunoglobulin-like folds. PKHD1 encodes an 11.6-kb transcript and is composed of 61 exons spanning an approximately 365-kb genomic region on chromosome 6p12-p11.2 adjacent to the marker D6S1714. Northern blot analyses demonstrated that the gene has discrete bands with one peak signal at approximately 11 kb, indicating that PKHD1 is likely to have multiple alternative transcripts. PKHD1 is highly expressed in adult and infant kidneys and weakly expressed in liver in northern blot analysis. This expression pattern parallels the tissue involvement observed in ARPKD. In situ hybridization analysis further revealed that the expression of PKHD1 in the kidney is mainly localized to the epithelial cells of the collecting duct, the specific tubular segment involved in cyst formation in ARPKD. These features of PKHD1 make it a strong positional candidate gene for ARPKD.
Assuntos
Rim Policístico Autossômico Recessivo/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Estrutura Terciária de Proteína , Receptores de Superfície Celular/química , Receptores de Superfície Celular/fisiologiaRESUMO
Mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene have been shown to cause autosomal recessive polycystic kidney disease (ARPKD), but the cellular functions of the gene product (PKHD1) remain uncharacterized. To illuminate its properties, the spatial and temporal expression patterns of PKHD1 were determined in mouse, rat, and human tissues by using polyclonal Abs and mAbs recognizing various specific regions of the gene product. During embryogenesis, PKHD1 is widely expressed in epithelial derivatives, including neural tubules, gut, pulmonary bronchi, and hepatic cells. In the kidneys of the pck rats, the rat model of which is genetically homologous to human ARPKD, the level of PKHD1 was significantly reduced but not completely absent. In cultured renal cells, the PKHD1 gene product colocalized with polycystin-2, the gene product of autosomal dominant polycystic disease type 2, at the basal bodies of primary cilia. Immunoreactive PKHD1 localized predominantly at the apical domain of polarized epithelial cells, suggesting it may be involved in the tubulogenesis and/or maintenance of duct-lumen architecture. Reduced PKHD1 levels in pck rat kidneys and its colocalization with polycystins may underlie the pathogenic basis for cystogenesis in polycystic kidney diseases.