Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

Background

The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new therapeutics. This requires further in-depth knowledge of the similarities and differences between mouse and human RPE.

Methods

We performed a microarray study to identify and functionally annotate RPE specific gene expression in mouse and human RPE. We used a meticulous method to determine C57BL/6J mouse RPE signature genes, correcting for possible RNA contamination from its adjacent layers: the choroid and the photoreceptors. We compared the signature genes, gene expression profiles and functional annotations of the mouse and human RPE.

Results

We defined sets of mouse (64), human (171) and mouse–human interspecies (22) RPE signature genes. Not unexpectedly, our gene expression analysis and comparative functional annotation suggested that, in general, the mouse and human RPE are very similar. For example, we found similarities for general features, like “organ development” and “disorders related to neurological tissue”. However, detailed analysis of the molecular pathways and networks associated with RPE functions, suggested also multiple species-specific differences, some of which may be relevant for the development of AMD. For example, CFHR1, most likely the main complement regulator in AMD pathogenesis was highly expressed in human RPE, but almost absent in mouse RPE. Furthermore, functions assigned to mouse and human RPE expression profiles indicate (patho-) biological differences related to AMD, such as oxidative stress, Bruch’s membrane, immune-regulation and outer blood retina barrier.

Conclusion

These differences may be important for the development of new therapeutic strategies and translational studies in age-related macular degeneration.     

The Gene for Human Glutaredoxin (GLRX) Is Localized to Human Chromosome 5q14

Glutaredoxin is a small protein (12 kDa) catalyzing glutathione-dependent disulfide oxidoreduction reactions in a coupled system with NADPH, GSH, and glutathione reductase. A cDNA encoding the human glutaredoxin gene (HGMW-approved symbol GLRX) has recently been isolated and cloned from a human fetal spleen cDNA library. The screening of a human genomic library in Charon 4A led to the identification of three genomic clones. Using fluorescencein situhybridization to metaphase chromosomes with one genomic clone as a probe, the human glutaredoxin gene was localized to chromosomal region 5q14. This localization at chromosome 5 was in agreement with the somatic cell hybrid analysis, using DNA from a human–hamster and a human–mouse hybrid panel and using a human glutaredoxin cDNA as a probe.     

人类Grx3蛋白与转录因子p65相互作用的研究

巯基氧化还原酶在生物体的系统内担当着非常重要的角色,其主要介导的是巯基和二硫键之间的相互转化的反应。人类Grx3蛋白作为一种巯基氧化还原酶在谷氧还蛋白系统中有着非常重要的作用。已有的研究表明,Grx3蛋白对NF-κB信号通路的活性有十分显著的影响。p65(RELA)是人类机体内十分重要的一种蛋白质,其在NF-κB信号通路有着重要的调控作用,几乎调控着NF-κB信号通路中的所有下游反应。通过大肠杆菌提纯表达的p65与Grx3在体外条件下进行偶联实验,为p65与Grx3存在相互作用提供了强有力的证据,同时也为Grx3蛋白对NF-κB信号通路的影响机制提供了一个可能的解释。     

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.     

The Gene Encoding Protein Kinase SEK1 Maps to Mouse Chromosome 11 and Human Chromosome 17

We report the mapping of the human and mouse genes encoding SEK1 (SAPK/ERK kinase-1), a newly identified protein kinase that is a potent physiological activator of the stress-activated protein kinases. The human SERK1 gene was assigned to human chromosome 17 using genomic DNAs from human–rodent somatic cell hybrid lines. A specific human PCR product was observed solely in the somatic cell line containing human chromosome 17. The mouseSerk1gene was mapped to chromosome 11, closely linked toD11Mit4,using genomic DNAs from a (C57BL/6J ×Mus spretus)F₁×M. spretusbackcross.     

Safety and Proof-of-Concept Study of Oral QLT091001 in Retinitis Pigmentosa Due to Inherited Deficiencies of Retinal Pigment Epithelial 65 Protein (RPE65) or Lecithin:Retinol Acyltransferase (LRAT)

Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001). Safety and visual outcomes of a once-daily oral dose of 40 mg/m²/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44%) showed a ≥20% increase and 4 of 18 (22%) showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67%) and 5 (28%) of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI) than non-responders (3.5 ± 1.2 μm). This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01014052","term_id":"NCT01014052"}}NCT01014052     

The Gene for PAX7, a Member of the Paired-Box-Containing Genes, Is Localized on Human Chromosome Arm 1p36

The murine Pax-7 gene and the cognate human gene, formerly designated HuP1, are members of the multi-gene paired-box-containing class of developmental regulatory genes first identified in Drosophila. By analysis of somatic cell hybrids segregating human chromosomes, the gene encoding PAX7 was localized to human chromosome 1. Fluorescence in situ hybridization confirmed this assignment and allowed mapping of the gene to the terminal region of the short arm (1p36) of the chromosome. Additionally, these results confirm the extensive homology between human chromosome 1p and the distal segment of mouse chromosome 4, extending from bands C5 through E2.     

The Zinc Finger Protein Zfr1p Is Localized Specifically to Conjugation Junction and Required for Sexual Development in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.     

The Human Intercellular Adhesion Molecule 3 (ICAM3) Gene Is Located in the 19p13.2-p13.3 Region, Close to the ICAM1 Gene

The chromosomal location of the intercellular adhesion molecule 3 (ICAM3) gene, coding for a lymphocyte function-associated antigen (LFA)-1 counterreceptor and selectively expressed by human leukocytes, was analyzed by in situ hybridization with the cDNA coding sequence as a probe. This sequence mapped to the p13.2-p13.3 region of chromosome 19, close to the ICAM1 gene chromosomal location.     

CDADC1在人视网膜色素上皮细胞中功能研究

目的:研究细胞增殖相关基因CDADC1在人视网膜色素上皮细胞ARPE19中的表达及对ARPE19细胞增殖的影响。方法:采用基因重组技术构建荧光表达载体pEGFP-C1-CDADC1和真核表达载体pcDNA3.1-myc-CDADC1,用脂质体转染法转染ARPE19细胞,观察GFP-CDADC1融合蛋白在ARPE19细胞的表达定位,流式细胞仪测定CDADC1转染后对ARPE19细胞生长周期、凋亡的影响。结果:FP-CDADC1融合蛋白亚细胞定位显示,CDADC1低表达于ARPE19细胞胞浆,高表达于细胞核;pcD-NA3.1-myc-CDADC1瞬时转染ARPE19细胞显示24小时细胞无明显改变,48小时后重组质粒转染组S期细胞占细胞总数的19.37%,pcDNA3.1-myc空载质粒转染组S期细胞占10.87%,而空白对照组S期细胞占3.33%,重组质粒转染组与两对照组之间的差异有统计学意义(P<0.01)。结论:CDADC1在增生性玻璃体视网膜病变(PVR)发生和发展过程中可促进DNA的合成,引起细胞增生。     

CDADC1在人视网膜色素上皮细胞中功能研究

目的：研究细胞增殖相关基因CDADC1在人视网膜色素上皮细胞ARPE19中的表达及对ARPE19细胞增殖的影响。方法：采用基因重组技术构建荧光表达载体pEGFP－C1－CDADC1和真核表达载体pcDNA3．1-myc-CDADC1,用脂质体转染法转染ARPE19细胞,观察GFP—CDADC1融合蛋白在ARPE19细胞的表达定位,流式细胞仪测定CDADC1转染后对ARPE19细胞生长周期、凋亡的影响。结果：FP—CDADC1融合蛋白亚细胞定位显示,CDADC1低表达于ARPE19细胞胞浆,高表达于细胞核;pcD—NA3．1-myc-CDADC1瞬时转染ARPE19细胞显示24小时细胞无明显改变,48小时后重组质粒转染组S期细胞占细胞总数的19．37％,pcDNA3．1-myc空载质粒转染组S期细胞占10．87％,而空白对照组S期细胞占3．33％,重组质粒转染组与两对照组之间的差异有统计学意义（P〈0．01）。结论：CDADC1在增生性玻璃体视网膜病变（PVR）发生和发展过程中可促进DNA的合成,引起细胞增生。     

小鼠受精抗原1基因与人类同源基因的关系

参照已报道小鼠受精抗原1（FA1）基因序列设计引物,运用PCR和PCR产物克隆测序等方法,对人受精抗原1（FA1）基因进行了克隆,并对两者进行了比较,结果显示:（1）已报道的小鼠FA1基因序列在其可读框内可能存在两处错误。（2）人OTK27基因可能与小鼠FA1基因同源,即OTK27基因就是人hFA1基因,或FA1基因就是小鼠otk27基因。 Abstract:The cloning of human fertilization antigen 1 gene（FA1） ,the supposed counterpart gene of mouse fertilization antigen 1 gene (FA1),was performed using the PCR and PCR products cloned sequencing methods.The result shows that there might be two mistakes in the mouse FA1 gene open reading frame (ORF),and human OTK27 gene and mouse FA1 gene might be homogeneous genes in the two species.     

小鼠受精抗原1基因与人类同源基因的关系

参照已报道小鼠受精抗原1（FA1）基因序列设计引物,运用PCR和PCR产物克隆测序等方法,对人受精抗原1（hFAI)基因进行了克隆,并对两进行了比较,结果显示：（1）已报道的小鼠FA1基因序列在其可读框内可能存在两处错误。（2）人OTK27基因可能与小鼠FA1基因同源,即OTK27基因就是人hFA1基因,或FA1基因就是小鼠otk27基因。     

视紫红质及RPE65蛋白在光致视网膜变性疾病中的作用机制

视紫红质是感光细胞中的一种视色素,在光线的接收和视觉电位的产生方面具有重要的生理作用,由视紫红质介导的过度光信号传导是光性视网膜变性的主要原因。近年的研究表明,视网膜色素上皮细胞中的RPE65蛋白作为影响视紫红质再生的关键因素,与视网膜光损伤的易感性密切相关。就视紫红质和RPE65蛋白在光致视网膜变性中的作用机理作一探讨。     

The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho-32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.