|Molecular Vision 2002;
Received 12 December 2001 | Accepted 15 August 2002 | Published 28 August 2002
Bioinformatics and reanalysis of subtracted expressed sequence tags from the human ciliary body: Identification of novel biological functions
1Area de Genética, Facultad de Medicina, Universidad de Castilla-La Mancha, 02071-Albacete, Spain; 2Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT, USA
Correspondence to: Dr. Miguel Coca-Prados, Yale University School of Medicine, Department of Ophthalmology and Visual Science 330 Cedar Street, New Haven, CT, 06510; Phone: (203) 785-2742; FAX: (203) 785-6123; email: Miguel.Coca-Prados@yale.edu
Purpose: The ciliary body is largely known for its major roles in the regulation of aqueous humor secretion, intraocular pressure, and accommodation of the lens. In this review article we applied bioinformatics to re-examine hundreds of expressed sequence tags (ESTs) previously isolated by subtractive hybridization from a human ciliary body library . The DNA sequences of these clones have been recently added to the web site of NEIBank.
Methods: DNA sequence comparisons of subtracted ESTs were performed against all entries in the last available release of the non-redundant database containing GenBank, EMBL, DDBJ and PDB sequences using the BlastN program accessed through NCBI's BLAST services on the internet (NCBI). Sequences were also compared and mapped using the Blast search program provided through the Internet by the Human Genome Project (UCSC).
Results: A total number of 284 independent ESTs were classified in 17 functional groups. Analysis of their relationships allowed to define the expression of five major groups of known genes: (i) protein synthesis, folding, secretion and degradation (20%); (ii) energy supply and biosynthesis (12%); (iii) contractility and cytoskeleton structure (6%); (iv) cellular signaling and cell cycle regulation (7%); and (v) nerve cell related tasks (2%), including neuropeptide processing and putative non-visual phototransduction and circadian rhythm control. The largest group contain unidentified sequences, a total of 105 sequences, accounting for 37% of ESTs. The unidentified sequences show similarity to genomic non-coding regions, or genes of unknown function.
Conclusions: The most highly represented EST, correspond to myocilin, a gene involved in glaucoma. The data also confirms the secretory functions of the ciliary epithelium, and its high metabolism; the presence of a neuroendocrine peptidergic system presumably involved in the regulation of the intraocular pressure and/or aqueous humor secretion. Additional genes may be related to a non-visual phototransduction cascade and/or to circadian rhythms. Overall this initial group of subtracted ESTs can lead to uncover novel physiological functions of the ciliary body in normal and in disease, as well as novel candidate genes for ocular diseases.
The ciliary body (CB) is a multicellular tissue consistent of a bilayer of secretory neuroepithelial cells, an underlying stroma containing blood vessels, and smooth muscle cells postsynaptically innervated by adrenergic and parasympathetic nerve fibers. The two secretory ciliary epithelial cell layers originate from the neural crest during embryogenesis, then become apposed at their apices when the invagination of the optic vesicle occurs. The cells forming the bilayer, the pigmented (PE), and non-pigmented (NPE) ciliary epithelial cells, are polarized and coupled to each other by distinct gap junction proteins [2,3]. NPE and PE cells express different gene repertoires giving raise to phenotypic differences observable for example on the presence of distinct Na+, K+-ATPase α and β subunit isoforms , neurotransmitter receptors, and carrier proteins [5,6]. Phenotypic differences are also manifested among NPE cells along the distinct regions of ciliary epithelium, (that is, the pars plicata, pars plana, and ora serrata) which have been suggested to be functionally diverse. The NPE cell layer forms a true tight blood-aqueous barrier between the stroma and the posterior chamber of the anterior segment of the eye. This barrier is established by the presence of tight junctions at the apical plasma membranes of NPE cells, and it prevents the paracellular passage of proteins between NPE cells. In contrast, the PE cells, which lack tight junctions, are considered a "leaky" epithelium .
The aqueous humor fluid is secreted by the ciliary epithelium, and it is responsible for the nourishment of the avascular tissues of the anterior segment of the eye, including the lens, cornea, and trabecular meshwork. Electrophysiological approaches have been extensively applied to study the transport systems expressed by the ciliary epithelium underlying aqueous humor dynamics . The present model of aqueous humor secretion predicts a regulatory mechanism mediated by the coordinated effects of unidirectional secretion and reabsorption of solute and water at the basolateral plasma membrane of NPE cells [5,9,10].
The rate of secretion and drainage of aqueous humor out of the eye through distinct outflow pathways determine the proper level of intraocular pressure (IOP), which follows a circadian rhythm influenced by a light-dark cycle . Elevated IOP is a high risk factor in developing primary open-angle glaucoma, a chronic eye disease that can lead to optic nerve damage, retinal ganglion cell death and blindness.
The identification of factors that determine normal and abnormal aqueous humor formation, aqueous humor outflow, IOP and their relationships is essential for understanding and treating glaucoma. The genetic and molecular events underlying both physiological and pathological states of the ciliary body are currently poorly understood. In part, due to the anatomical complexity and the presence of multicellular components of this tissue in vivo. During the last few years a number of expression cDNA libraries have been generated from the intact ciliary body and from cultured ciliary epithelial cells. These libraries have served to isolate and identify a number of important genes encoding enzymes involved in detoxification and protection of the anterior segment of the eye from oxidative stress [6,12]. Moreover, through the analysis of cDNA clones isolated by different means from ciliary body libraries, we identified several genes encoding key enzymes involved in the synthesis and processing of regulatory neuropeptides, many of which are restricted to neuroendocrine cells or tissues [13,14]. Overall, these findings revealed that the ciliary body is a multifunctional tissue.
Recent work supports the view that the ciliary body is a "neuroendocrine gland" displaying neuroendocrine characteristics linked to metabolic and physiological processes including secretion of aqueous humor, the regulation of intraocular pressure, and metabolism of steroid hormones. Among the neuroendocrine markers identified in the ciliary body and in cultured ciliary epithelial cells figured the proprotein convertases (PCs) 1 and 2, and the acidic protein 7B2 . The PCs and 7B2 mediate the processing of prohormone and proneuropeptides in the secretory pathway of neuroendocrine cells. Many of the biologically active peptides so far identified, specifically, neurotensin, natriuretic peptides, angiotensin, and endothelins, are known to influence blood pressure in the cardiovascular system, or to display hypotensive and hypertensive biological activities in experimental animals. Thus, these same peptides may regulate either the IOP or the secretion of aqueous humor through signaling pathways linked to their cognate receptors. These receptors are expressed either in the same peptide-producing cells (that is, PE and NPE cells) at the site of inflow and in the cells of the TM in the outflow system. Additional cell targets for these peptides are the vascular endothelium in the stroma, and smooth muscle cells in the ciliary muscle.
More recently, it has been reported the intriguing expression of mRNA and proteins in the human ciliary epithelium of components of a non-visual phototransduction cascade including rhodopsin, rhodopsin kinase, and arrestin , which may be linked to circadian entrainment tasks such as aqueous humor secretion and IOP. One should note, in this respect, that the PE and NPE ciliary epithelial cell layers share same embryological origin that the retinal pigment epithelium (RPE) and the retina respectively. Whether the ciliary epithelium of the human eye may be photoreceptive remains to be determined, but in recent years opsin-based pigments have been shown not to be exclusively restricted to the rod and cone photoreceptors of the retina .
Altogether these findings offer a new insight into the physiology of the ciliary body and of the eye. Regarding the molecular basis of eye diseases, our previous transcriptional analysis of the ciliary body contributed to the identification of the first gene known to be involved in glaucoma, myocilin [1,17,18], and the gene responsible for different types of inherited corneal dystrophies, β-ig-h3 .
The advent of the postgenomic era has opened a new way to explore the function of many tissues, through the analysis of a broad sample of transcripts or by focusing on transcripts differentially expressed in certain organs. Expressed Sequence Tags (ESTs) constitute a valuable tool to identify transcribed genes in different tissues, allowing a deeper understanding of the biological functions in which such tissues are involved. This strategy has been used to investigate the biology of several organs such as brain, muscle, liver and most recently to isolate and identify ESTs from the human ciliary body. In this review we have taken advantage of the vast amount of information derived from the Human Genome Project to advance the identification and mapping of 284 ESTs obtained by subtractive hybridization. Hence, it is expected that most of these ESTs are enriched in the ciliary body, although not exclusively restricted to this tissue. Those genes that are differentially or preferentially expressed in the ciliary body are potentially of great interest since they may be candidates, in some inherited pathologies of the eye, including glaucoma.
Human ciliary body subtracted (CBS) cDNA clones
Here, 284 cDNA clones isolated previously by subtractive hybridization between two directional cDNA libraries constructed in λUni-ZAP XR phagemid vectors (Stratagene, La Jolla, CA) , are re-examined. The target library is a human cDNA library constructed from the ciliary bodies of a pair of eyes of a 34-year-old female donor and the driver library is a cDNA library constructed from a human ciliary epithelial cell line, ODM-2 [1,20].
Sequence comparisons of all the subtracted ESTs from the ciliary body are performed against all entries in the last available release of the non-redundant database containing GenBank, EMBL, DDBJ and PDB sequences using the BlastN program  accessed through NCBI's BLAST services on the internet (NCBI). Sequences are also compared and mapped using the Blat search program provided through the Internet by the Human Genome Project (UCSC). We used the freeze of August 6, 2001. The average length of the cDNA sequences used for comparison is about 160 nucleotides. In general, putative identification is assigned to sequences with more than 90% identical bases through their length. Unknown sequences are assigned to Unigene clusters defined by the dbEST division of GenBank. Each cluster is automatically generated on the basis of overlapping ESTs obtained from different cDNA libraries, and ideally corresponds to one gene. Currently, the dbEST Human Ciliary Body section of NEIBank has incorporated the 268 subtracted ESTs sequences deposited in GenBank database in 1995.
Enrichment of genes expressed in the ciliary body
A number of techniques have been developed over the past few years to isolate and identify differentially expressed genes in tissues or cells. These included subtraction of cDNA libraries , differential display , RNA fingerprinting by arbitrarily primed PCR , representational difference cDNA analysis [25,26], subtractive hybridization for differential screening of arrayed cDNA clones or libraries [27,28], large scale generation of expressed sequence tags (ESTs) from cDNA libraries , serial analysis of gene expression (SAGE) , and suppression subtractive hybridization . However, none of these techniques are exempt of pitfalls.
In an earlier report the identification of approximately 90 independent ESTs isolated by subtractive hybridization, from a human ciliary body library constructed from the eyes of a single human donor , is documented. One of the goals in subtractive hybridization technology is to identify genes that are enriched in the target tissue but not in the tissue or cell used as carrier. The hypothesis is that clones isolated by subtractive hybridization are unique and restricted to the target tissue, and therefore biologically important, because they may reveal a key physiological function or property specific of the tissue. This review article extends the search up to 195 additional subtracted ESTs clones from the human ciliary body. Using northern blotting it was previously shown that subtracted ESTs are preferentially enriched in the target tissue (for example, ciliary body) but not in the cell line used as carrier [1,18,32,33].
Figure 1 shows the distribution of a total of 284 ESTs into 17 functional groups. 179 ESTs (63%) are homologous to genes of known function, and the rest comprised of 105 sequences (37%) of unknown function. Among the latter, 20 (7%) did not show any significant similarity with other sequences deposited in the data bases searched, while 48 (17%) are similar to cDNAs or RNAs of known sequence but undetermined function, and 37(13%) corresponded to sequences located in large clones of genomic human DNA. Sequences related to protein synthesis, degradation and secretion accounted for 20% of the total. Twelve percent of the ESTs identified transcripts involved in metabolic pathways, 7% are related to cytoskeletal or contractile proteins and 2% are nerve cell-related sequences, including members of a non-visual phototransduction system and circadian rhythm regulators. Antioxidant-related molecules represented 1% of the clones. It is included a glaucoma-related group of ESTs composed by four clones encoding myocilin (1%).
Table 1 and Table 2 summarize the putative identification and chromosome mapping of the known and unknown subtracted ESTs respectively, classified in the above mentioned functional groups. Identity is determined using BlastN and Blat programs. A few messengers are represented in the subtracted library by more than one clone, indicating that they corresponded to genes highly enriched in the ciliary body. In addition to four ESTs encoding myocilin (CBS-424, CBS-582, CBS-591 and CBS-670) we found three ESTs corresponding to the myosin light polypeptide kinase (CBS-152, CBS-427 and CBS-549). Eight ESTs are represented twice.
Analysis of a group of ESTs similar to transcripts involved in neuronal, neuroendocrine and phototransduction functions
One of the most interesting groups of ESTs identified corresponded to transcripts of genes expressed almost exclusively in nerve cells. It is composed of six members: phospholipase C, beta 4 (PLC; CBS-140 and CBS-369); GABA(A) receptor associated protein (GAGAARAP; CBS-291); carboxypeptidase E (CPE; CBS-294); Odd Oz/ten-m homolog 2 (CBS-659); synaptogyrin I (CBS-577); and short-chain dehydrogenase/reductase 1-like (CBS-390). PLC beta takes part in the photoreceptor signaling cascade . EST CBS-390 is 130 nucleotide long and contains a segment of 49 nucleotides 100% identical to the short-chain dehydrogenase/reductase 1, an enzyme expressed in photoreceptor cells that play a role in the reduction of all-trans-retinal during bleached visual pigment regeneration. On the basis of this finding we can speculate that the ciliary epithelium expresses a gene that can play a role in a non-visual phototransduction pathway, as has recently been proposed . We can not rule out a role of this putative gene in the general retinal metabolism as has been suggested for the short-chain dehydrogenase/reductase 1 in other non-ocular tissues . CPE is a neuropeptide-processing enzyme and is restricted to neuroendocrine tissues and cells . Its gene regulation and expression has been studied in the human cultured ciliary epithelium . The identification of GABAARAP, which is believed to link GABAA receptors to cytoskeleton, suggests the presence of this type of inhibitory receptor in the ciliary body. Gene Odz2 is involved in the development of the nervous system, and interestingly, is expressed in the photoreceptor cells of Drosophila . Synaptogyrin I is a component of synaptic vesicles, although it can also be present at low levels in other cells. Considering the fact that the ciliary epithelium originates from the embryonary neural crest and the neuroendocrine features of the adult ciliary epithelium, the presence of such transcripts in this tissue is not surprising, although other possibilities can not be ruled out at this time. For instance, the transcripts of these genes can be present in axons innervating the ciliary body.
Circadian rhythm proteins
Several clones matched to genes involved in the regulation of circadian rhythms. ESTs CBS-15 and CBS-18 have a length of about 175 nucleotides and show two segments of 30 and 41 nucleotides, which share more than 90% identity with the suprachiasmatic nucleus circadian oscillatory protein (SCOP) of Rattus norvegicus. SCOP is expressed in a circadian manner in the suprachiasmatic nucleus (SCN) and can play a role in the intracellular signaling in this nucleus . The presence of the SCOP-like EST suggest that the ciliary body cancontribute to the regulation of cyclical physiologic processes such as IOP. EST CBS-203 is 159 nucleotides long and has a segment of 72 nucleotides that is identical to acetylserotonin O-methytransferase-like protein (ASMTL). This molecule shares homology with hydroxyindole-O-methyltransferase (HIOMT) an enzyme responsible of catalyzing the last step in the synthesis of melatonin, and a neurohormone involved in circadian rhythms. It is believed that HIOMT is exclusively expressed in brain, retina, and pineal gland. Our data indicate that a related gene is expressed in the ciliary body.
Synthesis, folding, secretion and degradation of proteins
Considered together these ESTs accounted for 21% of the clones. Twenty-seven sequences (9.5%) correspond to genes devoted to transcription, translation and nuclear transport of polypeptides. An intense synthesis of proteins requires the activity of a team of molecules responsible for its correct folding. Hence, is not surprising that 7 ESTs (2%) are identified as heat shock or heat shock-related proteins. ESTs CBS-381 and CBS-634 are identified as heat shock protein 27; CBS-147, CBS-519 and CBS-527 are members of the 70 kD heat shock protein, and CBS-293 is a component of the 90 kD heat shock protein. Clone CBS-458 corresponds to crystallin alpha B; a member of the small heat sock protein family. Eleven ESTs (3.8%) are derived from genes involved in secretion and vesicular trafficking and 17(5.9%) are related to molecules that participate in protein degradation, mainly proteinases and proteinase-inhibitors. The abundance of ESTs corresponding to genes taking part in secretion and vesicular trafficking can be explained by the intense secretory activity of the ciliary epithelium, directly related to aqueous humor production and secretion of different polypeptides.
We establish three groups of genes dedicated to protein degradation: (i) genes encoding intracellular proteases of the ubiquitin/proteasome pathway; (ii) genes encoding extracellular proteases and (iii) genes encoding extracellular proteinase inhibitors. Members of the first group participate in the breakdown of various proteins as a component of the balance between the synthesis and degradation pathways necessary to maintain certain cellular activities. ESTs CBS-23, CBS-249, CBS-385 and CBS-494 correspond to components of the ubiquitin system, while CBS-418 CBS-425 and CBS-641 encode proteasome subunits z, HC8 and 26S, respectively. In addition, CBS-560 is identified as the large subunit of calpain 1, a calcium-dependent intracellular protease. Cathepsins D and O (CBS-265 and CBS-620, respectively), serine protease 15 (CBS-322) or insulin-degrading enzyme (CBS-334) are components of the second group. The third group is composed of alpha-2 macroglobulin (CBS-16), matrix-associated serine protease inhibitor (CBS-550), also known as placental protein-5, and tissue inhibitor of metalloproteinase 2 (CBS-442). Previous work have demonstrated that the human ciliary epithelium synthesizes and secretes cathepsin D and alpha-2 macroglobulin as well as alpha-1 antitrypsin to the aqueous humor . A balance between proteinases and their inhibitors in the aqueous humor can play a role in the processing, targeting and turnover of protein messengers and/or membrane receptors involved in the modulation of the aqueous humor outflow or in other biological functions, coordinated by the ciliary epithelium.
Matrix metalloproteinases and their inhibitors are responsible for the control of extracellular matrix degradation. Aberrations in this enzyme system might be involved in the extracellular matrix alterations associated with glaucoma . Hence, the ciliary body can control the status of the extracellular matrix of the trabecular meshwork through the secretion of metalloproteinases inhibitors. The presence of matrix metalloproteinase inhibitor 2 in the aqueous humor has been demonstrated .
Cytoskeleton and contractility
The group of cDNAs related to cytoskeleton components and molecules involved in contractility is composed of 17 (5.9%) different clones. Some of the identified molecules are structural components of the cytoskeleton or contribute to its organization. They are represented, among others, by the beta polypeptide of tubulin (CBS-51), gelsolin (CBS-37), echinoderm microtubule-associated protein/like (CBS-570), Wiskott-Aldrich syndrome protein (CBS-253), and profiling 1 (CBS-315). It is likely that most of the ESTs identifying contractile molecules originate from transcripts present in the ciliary muscle. For instance clones CBS-513, CBS-139, CBS-82, and CBS-152 encode tropomyosin 1 (alpha), heavy polypeptide 11 of smooth muscle myosin, smooth muscle actin alpha-2, and myosin light polypeptide kinase, respectively. The abundance of these transcripts in the ciliary body is indicated by the fact that the last mentioned protein is represented with three ESTs (CBS-152, CBS-427, and CBS549), while two ESTs (CBS-82 and CBS-498) are found to encode smooth muscle actin alpha-2.
Cell signaling/cell differentiation and cell cycle/apoptosis
ESTs included in these two groups are abundantly represented among the subtracted clones. Eleven sequences are found to be similar to genes involved in cell signaling, while six are identified as cell cycle regulators. The relative abundance of ESTs taking part in cell signaling (6%) indicates the relevance of such events in the ciliary body, which supports the idea of this tissue as a key coordinator of the physiology of the eye. Among these molecules some are related to GTP-binding regulatory proteins, such as CBS-269, CBS-327 and CBS-457. EST CBS-623 is similar to a protein possesing a pleckstrin-homology-domain containing a phosophatidylinositol 3,4,5-triphosphate-binding motif (Plekha3). Members of the receptor tyrosine kinase signaling pathways are also present. cDNAs CBS-197 and CBS-319 are similar to receptor-like tyrosine kinase (RYK) and receptor type protein tyrosine phosphatase G, respectively. On the other hand, the expression of genes that play a role in the regulation of the cell cycle, indicate that they form part of the molecular network that controls cell differentiation and proliferation in the ciliary body. Representative members of this group are ESTs CBS-213 and CBS-221, which are homologous to cyclin-dependent kinase inhibitor 1B and apoptosis related protein APR-3, respectively.
Eight ESTs (3%) are related to molecules of the immune system. Some of them, such as CBS-143, similar to MHC class I, are not restricted to immune cells. Others, such as CBS-606, homologous to peroxiredoxin or natural killer-enhancing factor, are expressed in immune cells. Whether these ESTs are derived from transcripts of immune cells present in the ciliary body, and enriched during the subtraction process, or reflect the presence of transcripts produced by ciliary body cells is unknown. We have demonstrated that the ciliary processes express the gene encoding complement component C4 and that the protein is secreted into the aqueous humor . This peculiarity may be related to the immuno-isolation of the anterior segment of the eye due to the blood-aqueous barrier.
Metabolic pathways, iron metabolism and melanin synthesis
Overall, this is the third largest group of sequences. About 9% of the clones are related to genes involved in energy production or biosynthesis. The presence of ESTs identifying housekeeping genes among the subtracted molecules may be due to its high relative abundance in the target cDNA population and/or to the possibility that they escaped hybridization because of the formation of highly stable secondary structures in their ssDNAs and RNAs. Transcripts of genes responsible for energy production can likely arise from two main places in the ciliary body: the ciliary muscle and the ciliary epithelium. In fact, three ESTs corresponded to muscular isoforms of different enzymes: pyruvate kinase (CBS-635), phosphorylase kinase alpha-1 (CBS-77) and ATP synthase (CBS-492). The intense expression of these genes provides the energy required to maintain essential functions of the ciliary body, such as contraction of the ciliary muscle, essential for visual accommodation, and synthesis and secretion carried out by the ciliary epithelium.
Some of the ESTs identified encoded genes involved in specialized biosynthetic metabolic activities such as prostaglandin production, iron transport and storage (1%) or melanin synthesis in the NPE (1%). CBS-671 is similar to transferring receptor, CBS-358 and CBS-646 are homologous to the two components of ferritin: the heavy polypeptide chain 1 and the L-chain, respectively. Ferritin is the major iron storage protein in the cytoplasm and regulates the levels of intracellular iron . Iron is an obligate requirement for essential metabolic processes, and too little can lead to cell death. The expression in the ciliary body of these genes may provide the ciliary epithelium with the iron needed to maintain its high metabolic activity. An excess of iron results in cellular toxicity, probably through the generation of free radicals . Ferritin is also a critical regulator of oxidative stress due to its capacity to sequester iron in a redox inactive form. In fact, mutations in the L-ferritin gene are responsible of the hereditary hyperferritinemia cataract syndrome .
Three clones showing similarity to proteins involved in melanogenesis are also identified. Clones CBS-353, CBS-421 and CBS-516, are homologous to the heavy polypeptide 12 of myosin VA, tyrosinase related protein 1 and tyrosinase-related protein-2/DOPAchrome tautomerase, respectively. The transport of melanosomes in mammals is a process dependent on myosin VA, an unconventional myosin. The identification of these ESTs in the subtracted library raise the possibility that melanin is synthesized in the adult ciliary body. It is thought that the melanocytes in the mammalian eye produce melanin only during fetal development and in the very young individual. However, there is some evidence indicating that iridial melanocytes can produce melanin in adult individuals . Thus, our data supports the existence of an active melanin synthesis in the adult ciliary body.
Three cDNAs are included in this group. They are identified as selenoprotein P (CBS-71), glutathione peroxidase 3, plasma (CBS-566) and 15 kD selenoprotein (CBS-416 and CBS-432), and accounted for 1% of the ESTs. Eye tissues are subjected to a high oxidative stress following exposure to UV radiation and to the intense aerobic metabolism occurring specially in the secretory epithelium of the ciliary body. Glutathione peroxidases catalyze the reduction of different peroxides, and contain a selenocysteine as the active site. The ciliary epithelium expresses high levels of this enzyme, exhibiting differences in its abundance along the different regions .
Twenty nine ESTs (10.2%) show similarity with genes that carry out diverse biological functions. They are classified as members of the miscellaneous group, including glucose transport-like protein III (CBS-220), brain and reproductive organ-expressed protein (CBS-460), protein similar to retinal degeneration B beta (CBS-472), basigin (CBS-217), and pigment epithelium-derived factor (PEDF; CBS-211). We have demonstrated that PEDF is abundantly expressed in the human ciliary epithelium, secreted into the aqueous humor and highly accumulated in the vitreous humor . It shows neurotrophic activity both in the retina and in the central nervous system and in addition is the most potent inhibitor of angiogenesis in the mammalian eye .
ESTs unidentified or matching genes of unknown function
This is the largest group of ESTs (105), accounting for 37% of the sequences (see Table 2). Twenty clones showed similarity to mRNAs or clones of unknown function, many of them encoding hypothetical proteins. Ten show identity to human KIAA clones. These clones encode large unidentified proteins with sequences stored in the HUGE database [47,48]. Finally, some sequences showed similarity with human clones of genomic DNA. We found a few ESTs located in non-coding regions such as introns of known genes and discovered that they overlapped or are in close proximity to other described ESTs. For instance, CBS-323 and CBS-330 are located in introns of diacylglycerol kinase gamma and serotonin receptor 2A, respectively. The location of these sequences may be explained in different ways. They can arise by differential splicing, or may correspond to unidentified genes located in introns, such as it has been described for the blood clotting factor VIII gene (F8C), which contains in its intron 22 two internal genes, F8A and F8B . It is unlikely that they reflect the presence of contaminant genomic DNA in the target cDNA library used in the subtractive hybridization because attempts to amplify intronic sequences using PCR did not produce any positive signal (data not shown). In addition, most of the subtracted clones located within genomic DNA regions overlapped with ESTs obtained independently from other different cDNA libraries, indicating that they likely identify transcripts generated from novel genes. In fact, 6 of these clones (CBS-631, 658, 28, 264, 292 and 365) are located inside exon sequences predicted by the assembly program of the USCF human genome browser (data not shown). The future characterization of this group of sequences will allow the identification of unique functions of the ciliary body, particularly of its epithelium.
Conclusions and future directions
During the last decade a substantial amount of effort has been directed in analyzing differentially expressed genes in the ciliary body as a strategy to better understand the physiology of this tissue and to identify candidate genes for ocular diseases. To accomplish such goals the construction of subtractive cDNA libraries from the ciliary body using a procedure originally described by Swaroop et al 1991 , it has been instrumental . This attempt is useful in obtaining putative differentially expressed genes, although it presented some limitations including: (i) the inability to easily isolate full-length clones, (ii) the difficulty of specifically isolate differentially represented clones and (iii) the whole process, from the cDNA library construction to the analysis of isolated clones, was very laborious and time consuming.
More recently, new technologies have emerged such as DNA arrays and quantitative PCR, and they can be used globally to identify and measure gene expression in the ciliary body. For instance the non-redundant ciliary body cDNA subtracted sequences can be included in DNA arrays, that would be useful to study variation in the expression of selected genes under different physiological, and particularly pathological conditions such as glaucoma. DNA microarrays, although very attractive today, are however very expensive, their accuracy is limited by the purity and quantity of the experimental RNA in each hybridization, and the experiments need to be performed repeatedly to ensure accuracy and reproducibility of the data . To date, DNA microarray technology in combination with quantitative PCR, may be a powerful and a rapid approach to investigate changes in expression of thousand of genes in the ciliary body, for example during development and in glaucoma. This type of analysis will permit the identification of sets of genes that may be part of signaling pathways helping in designing new therapies to treat glaucoma.
Among other approaches to study glaucoma, the potential and powerful benefit of the identification of inbred mouse strains (including DBA/2Nnia, DBA/2J, AKXD-28/Ty), that spontaneously develop secondary forms of glaucoma , has become apparent. Furthermore, the generation of mouse strains with mutations in glaucoma genes is helping to understand the multifactorial components (genetic, enviromental and developmental) associated with the different subset of glaucomas [53,54]. These and other mouse strains will help to understand the function of glaucoma genes in the susceptible eye tissues where physiological alterations may be induced (that is, the anterior segment, retinal ganglion cells, and optic nerve). The development of an electrophysiologic approach (the servo-null micropipette system) adapted to measure continuously monitoring IOP in the mouse  proved also useful to investigate the effects of inhibitors of transport systems of the ciliary epithelium on IOP . Most recently, a goniolens for studying the genetic and clinical etiology of anterior segment abnormalities and glaucoma in mice has been developed . Future studies may be extended, using the mouse model, to investigate the endocrine factors that may be involved in the diurnal changes of IOP and aqueous humor secretion.
As has been shown, despite a vast effort carried out during the last decade to analyze the molecular biology of the ciliary body, the knowledge of the mechanisms that underlies both physiological and pathological processes occurring in this tissue is still incomplete. The advent of new sequence information and analytical tools, derived from the Human Genome Project, has opened new opportunities to explore gene expression in different tissues, including the ciliary body.
The identification of genes either differentially expressed in the ciliary body or preferentially restricted in this tissue, may reveal important clues regarding the mechanisms involved in the regulation of IOP and in the development of prevalent ocular pathologies such as glaucoma. In this regard, all sequences contained in the dbEST human ciliary body section of NEIBank, are ESTs reported in this work. The NEIBank is an initiative to create a repository database of the genes expressed in the distinct tissues of the human eye [58,59], including lens , iris , retina , and RPE/choroid .
This information can serve to identify genes that are also represented in other cDNA libraries from distinct ocular tissues. Although we are not aware yet of a ciliary body-specific gene, we have identified cDNAs that are preferentially restricted to the ciliary body when compared to other ocular tissues [6,12,33]. The myocilin gene is an example. This gene although ubiquitously expressed, its mRNA is abundantly found in iris, ciliary body and trabecular meshwork [18,64]. Thus, the identification of genes that may be restricted to a tissue or cell, may provide the specific target site to repair its potential abnormal function in disease by augmenting or adding a missing function, for example, through viral gene therapy .
The analysis carried out shows that the ciliary body expresses a repertoire of genes responsible of five major biological functions: (i) protein synthesis, secretion and degradation; (ii) energy production and biosynthesis; (iii) contractility and maintenance of cytoskeleton structure; (iv) cell signaling and cell cycle regulation; and (v) putative nerve cell-related functions including non-visual photransduction and circadian rhythm tasks. The first three groups of functions correlate very well with known histological and physiological properties of the ciliary body. For instance, it is well known that NPE cells contain numerous mitochondria and rough and smooth endoplasmic reticulum, characteristic of high metabolically active and secretory cells. The high metabolic rate provides the energy necessary to maintain the intense biosynthetic and secretory activities of the ciliary epithelium and to contract the ciliary muscle. A substantial energy supply is also required to afford the active solute transport carried out by Na+/K+-ATPase, HCO3-/Cl- exchanger, Cl- and K+ channels, and Na+-K+-Cl- cotransporter . There is a greater development of intracellular organelles and a higher metabolic rate in the NPE compared with the PE [67,68], probably reflecting the dominant role of NPE in aqueous humor formation. On the other hand, the relative abundance of transcripts related to contractility probably reflects the function of the ciliary muscle, which is directly responsible for visual accommodation. Figure 2 summarizes these major activities as well as the functional relationships between them in the context of the anatomy of the anterior segment of the eye.
As mentioned above, the high relative abundance of ESTs related to protein synthesis, secretion and degradation, indicates that theses activities occupy a prominent site among the functions of the ciliary body, probably consuming a substantial amount of metabolic energy. The answer to why the ciliary epithelium affords such intense protein synthesis and secretion may be explained by aqueous humor dynamics. The rate flow of aqueous humor calculated for the rabbit is 2.48 μl/min, corresponding to a renewal rate of some 1.5% , which is fairly uniform among mammalian species, including in humans . Hence, the aqueous humor is completely replaced approximately every 66 min. To maintain constant the protein concentration in the aqueous humor, the persistent drainage of molecules must be balanced by continuous synthesis and secretion. The synthesis of proteins, by the ciliary epithelium, such as complement component C4, transferring, proteinases and proteinases inhibitors, and their secretion into the aqueous humor has been demonstrated [1,39,70]. In addition, it has been shown that some proteins in the vitreous may be secreted by the ciliary epithelium . However, not all the proteins present in the aqueous humor are secreted by the ciliary epithelium. Albumin, the major protein of this fluid, enters the posterior chamber of the eye directly via the iris root [72,73].
The ciliary body also produces a repertoire of intracellular polypeptides. The relative abundance of ESTs related to intracellular and extracellular proteases is not surprising because they perform many tasks vital to the cell. Several components of the ubiquitin/proteasome system are detected. This system accomplishes the degradation of misfolded and malfunctioning proteins. In addition, ubiquitination regulates a host of critical cellular functions, usually by mediating the selective degradation of master regulatory proteins by proteasomes, such as the progression of the cell cycle, the induction of the inflammatory response, and antigen presentation . The apparent intense production of both intracellular and secreted proteins would require a repertoire of molecules dedicated to guarantee its correct folding. This may explain the profusion of ESTs similar to heat shock proteins.
Other groups of identified ESTs highlighted specialized metabolic functions such as iron transport and storage and melanin synthesis. An abundant iron supply is required to maintain the strong metabolic rate. Two main sources of oxidative stress challenge the eye: the intense aerobic metabolism and the exposure to UV light. As a defense the ciliary body expresses a set of antioxidative genes and genes encoding enzymes involved in detoxification. The detoxification enzyme systems in the ciliary body are of great interest, because its role is to remove a wide array of xenobiotics and environmental toxins to which the eye is exposed. An important group of detoxification enzymes are the cytochrome P450 (CYP450) involved in xenobiotic metabolism, and one of them, CYP1B1, is associated to primary congenital glaucoma .
The ciliary epithelium produces and secretes a number of neuropeptide-processing enzymes and neuropeptides to the aqueous humor, behaving as a neuroendocrine tissue. It can regulate the IOP and other biological processes by targeting factors to the outflow facility and other structures bathed by the aqueous humor [13,14].
Recently, molecular evidence of the expression in the human NPE epithelium of components of a non-visual phototransduction pathway, putatively linked to circadian entrainment tasks, has been obtained . They include components of the phototransduction cascade, such as rhodopsin, and components linked to its deactivation, such as rhodopsin kinase, recoverin, and visual arrestin. The detection of these genes in the ciliary epithelium is not due to cross contamination from the retina, since their cellular distribution along the nonpigmented ciliary epithelium have been confirmed by indirect immunofluorescence at the pars plicata region of the ciliary epithelium, an area distant from the retina . We have obtained additional data indicating both the presence of this non-visual phototransduction system and the involvement of the ciliary body in the control of circadian rhythms. Transcripts of PLC β4 are present in the ciliary body as revealed by two ESTs (CBS-140 and CBS-369). In this tissue it may play a role in the phototransduction system, since in photoreceptors it is involved in the rod and rod-bipolar signaling cascades , hydrolyzing phosphatidylinositol 4,5-biphosphate to inositol triphosphate and diacylglycerol. PLC β4 is expressed in the brain and in neurons of the retina (bipolar cells, horizontal cells, and ganglion cells), modulating the visual response in mice. On the other hand, the identification of CBS-390 as a short-chain dehydrogenase/reductase 1-like molecule, suggests the presence in the ciliary body of a putative gene involved in the regeneration of the visual pigment. The expression of a related gene in the ciliary epithelium, the cellular retinaldehyde-binding protein (CRALBP) , a component of the visual cycle involved in the transport of 11-cis-retinaldehyde or 11-cis-retinol, retinoid metabolism and visual pigment regeneration has been also demonstrated.
Evidence of the existence of a circadian clock in the ciliary body is provided by the identification of SCOP-like transcripts (CBS-15 and CBS-18) and by the identification of an EST (CBS-203) showing a segment identical to ASMTL protein, an enzyme involved in the synthesis of melatonin. Melatonin is an endogenous mediator of photoperiodic information and a molecular component of circadian time keeping systems [76,77]. Additional findings support this idea including: (i) Functional receptors of the neurohormone melatonin have been detected in rabbit iris-ciliary processes ; (ii) Melatonin is present in blood, but also in the retina, iris, ciliary body and aqueous humor [79,80]; (iii) The human ciliary body synthesizes and secretes melatonin to the aqueous humor . Hence, the possibility exists that IOP, which follows a circadian rhythm, is regulated by circadian mechanisms lying in the ciliary body.
Evidence of the expression of genes in the ciliary body that are associated to nerve cell physiology are available. They include synaptogirin I, GABARAP and Odz2. Synaptogyrin I, is a protein abundant in synaptic vesicles, that belongs to a family of non neuronal components. The human gene encoding synaptogirin I (SYNGR1) can be alternatively transcribed into three mRNAs of 4.5, 1.3, and 0.9 kb. The most abundant transcript (4.5 kb) is highly expressed in neurons of the central nervous system and at much lower levels in other cells. In vitro studies show that synaptogyrin I can be a strong regulator of neurotransmitter release  but a more general role of this gene in vesicular traffic in the ciliary epithelium can not be discounted. GABARAP is a putative linker protein between cytoskeleton and GABAA receptors, located at postsynaptic sites [83,84]. This finding suggests the presence of GABA receptors in the ciliary body. The gene Odz2, shows an expression restricted to the nervous system and belongs to a family of genes involved in development . Interestingly, the Drosophila odz gene is expressed in the morphogenetic furrow and in photoreceptor R7 cells in eye imaginal discs , which implicates it in the development of the eye. Our data indicate that this gene is expressed in the adult human ciliary body, perhaps playing a role in the maintenance of the adult differentiated phenotype. The presence of these transcripts is not surprising if we consider the embryological origin of the ciliary epithelium from the neural crest. However, further investigation is required to determine the actual function of these transcripts in the ciliary body. The activity of some of these molecules is dependent on a group of interacting proteins involved in cell signaling. Our data shows the presence of a repertoire of ESTs of this type. For instance, PLC β4 is activated by G proteins and several ESTs similar to that class of polypeptides have been identified.
The identified subtracted ESTs represent a valuable tool to study eye diseases because they can identify candidate genes that can be used in either functional approaches or positional cloning strategies. In fact four of these ESTS (CBS-424, CBS-582, CBS-591 and CBS-670) played a fundamental role in the identification of the first human gene involved in primary open-angle glaucoma, myocilin .
The largest group of ESTs is comprised of unidentified sequences or sequences of unknown function (Table 2). It is interesting to note that more than 3 million ESTs have been deposited in the EST database division of GenBank, isolated from a variety of different tissues. However, 20 sequences reported here are not previously found (Table 2), indicating that they may correspond to genes whose expression can be restricted to the ciliary body, and therefore can be responsible of the most unique functions of this tissue. Thirty-seven sequences are located in regions of non-coding genomic DNA. They do not represent cloning artifacts, since many of them have been reported as ESTs by other groups in libraries prepared from other tissues. On the contrary they may contribute to the identification of novel genes or to characterize alternative splicing in certain genes [86,87]. Alignments of ESTs to the draft sequence of the human genome, suggest that about 60% of the human genes have multiple splicing variants .
Figure 2 summarizes the major functions that we propose for the ciliary body on the basis of the data presented here, offering in some cases a new framework to further investigate the molecular physiology and pathology of this tissue. From this data it is clear that in addition to the known role of the ciliary epithelium in the secretion of the aqueous humor, it shows an intense protein synthesis and secretion into the aqueous humor. Some of the secreted molecules can be targeted to tissues directly bathed by the fluid such as the lens, iris, cornea and trabecular meshwork or even to the retina by diffusion through the vitreous humor. We propose that some of the secreted proteins and peptides, such as neuropeptides, proteinases, and proteinase inhibitors, can play important roles in the regulation and coordination of biological functions including control of the IOP through the regulation of the aqueous humor outflow. It also can be responsible for maintaining the balance of synthesis and degradation or drainage of secreted proteins. As a part of this coordinating role, we have shown additional evidence of the expression of genes potentially involved in a non-visual phototransduction system and in regulation of circadian rhythm tasks.
Finally, three promising and exciting areas of research of the normal and disease ciliary body can be considered and they are related to the fields of Neuroendocrinology and Neurobiology. One emerging area is related to the role of the ciliary epithelium in the metabolism of corticosteroids , sex steroid hormones (estrogen, androgen, and progesterone) (unpublished data), and involvement in the development of glaucoma.
The second area of research relates to the properties of the ciliary body as an endocrine gland within the eye. The function of the endocrine hormones released by the ciliary epithelium, may provide key information on their intracrine, autocrine and paracrine signaling mechanisms in aqueous secretion, intraocular pressure and other eye functions. A third area of research of great interest is related to the identification of putative photosensory receptors and clock genes in the ciliary epithelium. If the ciliary body contains cells that are photosensory, it will provide the physiological bases to link the circadian rhythms of IOP and aqueous secretion, and the light-dark cycle.
This work is supported in part by research grants from the National Institutes of Health/National Eye Institute [EY04873 and EY00785 (for core facilities)], and Research to Prevent Blindness (to M.C.-P.), by research funds from the University of Castilla-La Mancha (011.1252) and by grants from the "Consejeria de Sanidad de la Junta de Comunidades de Castilla-La Mancha" [PREG00107] and "Consejerí a de Ciencia y Tecnologí a de la Junta de Comunidades de Castilla-La Mancha" [PAI-02-049] (to J. E.). We thank Mr. José-Daniel Aroca-Aguilar for assistance with the preparation of tables.
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