RESUMO
Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.
The biggest problem confronting oligonucleotide therapeutics is a lack of compounds capable of targeting compounds to diseased tissues. This paper reports a major advance targeting the transferrin receptor to increase the delivery of morpholine oligomers to muscle cells in vivo. This work suggests the possibility for improved treatments of muscular dystrophy and other diseases.
Assuntos
Distrofina , Éxons , Morfolinos , Distrofia Muscular de Duchenne , Oligonucleotídeos Antissenso , Animais , Camundongos , Distrofina/genética , Éxons/genética , Camundongos Endogâmicos mdx , Morfolinos/farmacologia , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/farmacologia , Receptores da Transferrina/genéticaRESUMO
Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/ß pairs in a 5'-3' to 3'-5' orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.
Assuntos
Globinas/genética , Região de Controle de Locus Gênico/genética , Peixe-Zebra/genética , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/fisiologia , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes de Troca , Subunidade p45 do Fator de Transcrição NF-E2/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
Erythropoietin (Epo) and its cognate receptor (EpoR) are required for maintaining adequate levels of circulating erythrocytes during embryogenesis and adulthood. Here, we report the functional characterization of the zebrafish epo and epor genes. The expression of epo and epor was evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and whole-mount in situ hybridization, revealing marked parallels between zebrafish and mammalian gene expression patterns. Examination of the hypochromic mutant, weissherbst, and adult hypoxia-treated hearts indicate that zebrafish epo expression is induced by anemia and hypoxia. Overexpression of epo mRNA resulted in severe polycythemia, characterized by a striking increase in the number of cells expressing scl, c-myb, gata1, ikaros, epor, and betae1-globin, suggesting that both the erythroid progenitor and mature erythrocyte compartments respond to epo. Morpholino-mediated knockdown of the epor caused a slight decrease in primitive and complete block of definitive erythropoiesis. Abrogation of STAT5 blocked the erythropoietic expansion by epo mRNA, consistent with a requirement for STAT5 in epo signaling. Together, the characterization of zebrafish epo and epor demonstrates the conservation of an ancient program that ensures proper red blood cell numbers during normal homeostasis and under hypoxic conditions.
Assuntos
Eritropoetina/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Anemia/metabolismo , Anemia/patologia , Animais , Sequência Conservada , DNA Complementar/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Células Eritroides/citologia , Eritropoese , Eritropoetina/química , Eritropoetina/genética , Eritropoetina/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Dados de Sequência Molecular , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Fator de Transcrição STAT5/metabolismo , Alinhamento de Sequência , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The zebrafish (Danio rerio) is a well-established vertebrate model for studying hematopoiesis. The major advantages of this system include robust experimental techniques in both genetics and embryology, which have been utilized to model many aspects of human development and disease. Although much is known about the transcription factors involved in the terminal differentiation of peripheral blood lineages, little is known about the development and maintenance of the hematopoietic stem cell (HSC). This review will focus on the current knowledge of the transcriptional regulation of the HSC in the context of the zebrafish. Future studies using new technologies in the zebrafish model will enhance our understanding of the molecular networks regulating HSC pluripotency and differentiation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Fatores de Transcrição/fisiologia , Animais , Desenvolvimento Embrionário/genética , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Peixe-ZebraRESUMO
The Cres (cystatin-related epididymal spermatogenic) gene encodes the defining member of a new subgroup within the family 2 cystatins of cysteine protease inhibitors. Cres expression is highly tissue- and cell-specific, with messenger RNA (mRNA) present in the testicular round/elongating spermatids, proximal caput epididymal epithelium, gonadotroph cells in the anterior pituitary gland, and corpus luteum of the ovary. To begin to elucidate the molecular mechanisms controlling the tissue- and cell-specific expression of the Cres gene, transgenic mice were generated containing 1.6 kilobases (kb) of the mouse Cres promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. A CAT enzyme-linked immunosorbent assay detected CAT protein in the testis, epididymis, isolated cauda epididymal spermatozoa, and anterior pituitary gland from mice heterozygous and homozygous for the transgene. However, reverse transcription (RT)-PCR did not detect CAT mRNA in any regions of the epididymis, suggesting that the CAT protein detected in the epididymis was from spermatozoa. RT-PCR also did not detect CAT mRNA in the ovary. RT-PCR analysis of the testes from mice of different postnatal ages showed CAT mRNA first detected at day 22, which correlated with the first appearance of Cres mRNA and with the presence of round spermatids. These studies demonstrate that 1.6 kb of Cres promoter contains the DNA elements necessary for germ cell and pituitary gland-specific expression but lacks critical sequences necessary for expression in the epididymis and ovary.
Assuntos
Cistatinas/genética , Adeno-Hipófise/fisiologia , Regiões Promotoras Genéticas , Espermatozoides/fisiologia , Animais , Sequência de Bases , Cloranfenicol O-Acetiltransferase/genética , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Genes Reporter , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , Ovário/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , EspermatogêneseRESUMO
The mammalian epididymis plays a critical role in sperm maturation, a function dependent on testicular androgens. However, the function of the initial segment, the most proximal part of the epididymis, is also dependent on luminal factors of testicular origin. Efferent duct ligation (EDL), which prevents luminal testicular fluid from reaching the epididymis, results in changes in gene expression within this region. Cystatin-related epididymal spermatogenic (cres) gene and gamma-glutamyl transpeptidase (GGT) mRNA IV are highly expressed in the initial segment and are regulated by luminal testicular factors. EDL results in decreased expression of both genes. To evaluate these promoters in the context of their native physiological state, an in vivo electroporation procedure was used. Significant differences were observed in vivo compared to previous in vitro results. Whereas two C/EBP sites were necessary for transcriptional activity from a 135-base-pair (bp) cres promoter in vitro, only the 5' site displayed functional activity in the in vivo system. A 135-bp GGT promoter IV construct was sufficient for reporter gene expression in vitro. However, in vivo, substantial expression was not observed until the construct was extended to 530 bp. Three polyoma enhancer activator 3 (PEA3) sites were found to be necessary for in vivo reporter gene expression from this construct. A cis-acting negative regulatory element between -530 and -681 bp was also identified that was not previously recognized in the in vitro studies. These studies demonstrate the utility of in vivo electroporation for elucidating promoter elements that may not be identified when traditional in vitro methods are used.
Assuntos
Eletroporação/métodos , Epididimo/fisiologia , Regiões Promotoras Genéticas/genética , Epitélio Seminífero/fisiologia , Animais , Clonagem Molecular/métodos , Epididimo/citologia , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Masculino , Mutagênese , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/citologiaRESUMO
Microarray analysis was carried out to identify genes with enriched expression in the initial segment region of the mouse epididymis. A set of approximately 15 000 clones developed at the National Institutes for Aging and consisting of expressed sequence tags (ESTs) derived from pre- and peri-implantation embryos, Embryonic Day 12.5 female gonad/mesonephros, and newborn ovary were hybridized with probes generated against the initial segment (epididymal region 1) and the remainder of the epididymis (epididymal regions 2-5). The median values for the normalized ratios of region 1 to regions 2-5 from three independent experiments were averaged for each gene/EST using Genespring 5.0 software. The majority of clones showed a ratio of 1.0, suggesting they were expressed at similar levels in all epididymal regions. In addition, 123 clones exhibited 2-fold or higher expression in the initial segment, including Cres3, prostein, lipocalin 2, ALEX3, synaptotagmin-like 4, erm, and milk fat globule factor, whereas 216 clones, including elafin-like 1, lactotransferrin, Sin3B, zinc-finger protein 91, and membrane-type frizzled-related protein, showed 2-fold or higher expression in epididymal regions 2-5. Northern blot analyses of 12 clones predicted by microarray analysis to be either enriched in the initial segment (n = 8), enriched in epididymal regions 2-5 (n = 2), or similar in all regions (n = 2) were carried out. All clones exhibited the expected region-specific expression, thus confirming the microarray results. The studies presented here show a global survey of region-specific gene expression in the epididymis, identifying 15287 sequences, the majority of which have not previously been shown to be expressed in this organ.
Assuntos
Epididimo/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reprodutibilidade dos TestesRESUMO
Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.
Assuntos
Epididimo/metabolismo , Infertilidade/genética , Infertilidade/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Northern Blotting , Western Blotting , DNA Complementar/biossíntese , DNA Complementar/genética , Expressão Gênica , Genótipo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Biossíntese de ProteínasRESUMO
The cystatins are a superfamily of cysteine protease inhibitors. Several genes including Cres (cystatin-related epididymal spermatogenic), testatin, and cystatin T, have been identified that are related to the family 2 cystatins but lack critical consensus sites important for cysteine protease inhibition. In addition, these genes are primarily expressed in the reproductive tract suggesting they may have evolved to perform tissue-specific functions distinct from that of the typical cystatins. This review describes the CRES subgroup within the family 2 cystatins including potential new members and their putative functions in the reproductive tract.
Assuntos
Cistatinas/classificação , Cistatinas/fisiologia , Sequência de Aminoácidos , Animais , Cistatinas/genética , Humanos , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a K(i) of 25 nM. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.
Assuntos
Cistatinas/farmacologia , Inibidores de Serina Proteinase/farmacologia , Subtilisinas/antagonistas & inibidores , Animais , Ligação Competitiva , Catepsina B/antagonistas & inibidores , Linhagem Celular , Cistatina C , Cistatinas/química , Cistatinas/genética , Epididimo/química , Feminino , Cinética , Masculino , Camundongos , Ovário/química , Papaína/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Adeno-Hipófise/química , Pró-Proteína Convertase 2 , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Relação Estrutura-Atividade , Subtilisinas/genética , Testículo/químicaRESUMO
The cystatin-related epididymal spermatogenic (CRES) and recently identified testatin and cystatin T proteins define a new subgroup within the family 2 cystatins of cysteine protease inhibitors. Members of the CRES subgroup are predominantly expressed in reproductive tissues and lack critical cystatin active-site sequences implying divergent functions. To determine whether there are additional members of the subgroup, we searched nucleotide databases and identified two novel genes that we designated Cres2 and Cres3. These genes, like other subgroup members, encode proteins with four conserved cysteine residues and predicted molecular weights characteristic of family 2 cystatins but have divergent cystatin inhibitory sequences. Furthermore, the genes exhibited reproductive-specific expression with Cres2 exclusively expressed in the epithelial cells of the proximal and midcaput epididymal regions and Cres3 expressed in the proximal caput epididymal epithelium, Sertoli cells of the testis, and early follicles and corpora lutea in the ovary. Additional studies showed that, like Cres, both Cres2 and Cres3 genes are dependent on testicular factors for epididymal expression. Taken together, CRES2 and CRES3 represent new members of a subgroup of cystatin family 2 proteins that likely carry out tissue-specific functions distinct from that of typical cystatins.
Assuntos
Cistatinas/genética , Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Corpo Lúteo/química , Cistatinas/análise , Cistatinas/química , DNA Complementar/análise , Epididimo/química , Epitélio/química , Feminino , Regulação da Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Dados de Sequência Molecular , Orquiectomia , Folículo Ovariano/química , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Células de Sertoli/química , Testículo/química , Distribuição TecidualRESUMO
The CRES (cystatin-related epididymal spermatogenic) protein defines a new subgroup in the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, unlike the ubiquitous expression of cystatin C, the Cres gene is preferentialy expressed in postmeiotic germ cells, the proximal caput epididymidis, and anterior pituitary gonadotrophs. Furthermore, CRES protein lacks two of the three consensus sites necessary for the cystatin inhibition of C1 cysteine proteases. Therefore, CRES may perform unique and tissue-specific functions in the reproductive and neuroendocrine systems. In the present review, we describe our studies on: 1. the Cres gene promoter and the transcriptional regulatory protein and their associated DNA binding sites that may be important for tissue-specific expression; and 2. the biochemical function of CRES protein. In brief, Northern blot, gel shift analyses, and transient transfection assays demonstrated that the C/EBP beta (CCAAT/enhancer binding protein) transcription factor is the predominant C/EBP family member expressed in the epididymis and gonadotroph cells and is necessary for high levels of Cres expression in these two tissues. In other studies, analyses of transgenic mice expressing a CAT reporter gene driven by 1.6 kb of Cres promoter revealed CAT mRNA and protein only in the germ cells. These studies suggest that the 1.6 kb of Cres 5' flanking sequence contains the required DNA elements for expression in the testis, but lacks the elements to correctly target expression of the reporter gene in the epididymis. Alternatively, repressor elements may be present. Finally, in vitro protease assays were performed to determine if CRES functions as a protease inhibitor. In contrast to cystain C, CRES did not inhibit the C1 cysteine protease papain but rather inhibited at nanomolar concentrations the serine protease PC2, a prohormone processing enzyme. Therefore, CRES is a new cross-class inhibitor that may regulate PC2 of PC2-like proteases and suggests a role for CRES in the regulation of prohormone and proprotein processing.
Assuntos
Cistatinas/genética , Regulação da Expressão Gênica , Espermatogênese/fisiologia , Sequência de Aminoácidos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/biossíntese , Cistatinas/classificação , Cistatinas/fisiologia , Epididimo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Homologia de Sequência de AminoácidosRESUMO
Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the human CRES gene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouse CRES: Northern blot and in situ hybridization experiments showed that CRES is highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.