Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

CCS, conserved core structures; SRS, substrate recognition sequence. aReported as DNA polymorphism.

bThis mutation is also reported in subjects with Peters’ anomaly.

cThis mutation is also reported in subjects with juvenile-onset open angle glaucoma (JOAG). dThis mutation is associated with reduced penetrance in primary congenital glaucoma.

eThis mutation is reported only in subjects with adult-onset open angle glaucoma (POAG). f This mutation is reported in subjects with Rieger’s anomaly.

g This mutation is reported only in subjects with Peters’ anomaly.

hThis mutation is reported only in subjects with juvenile-onset open angle glaucoma (JOAG).

Table 3

Common CYP1B1 Mutations in PCG, JOAG, and POAG Subjects

these ocular conditions can be established. Further confirmation of CYP1B1 mutations in JOAG or POAG subjects from other population with low degree of consanguinity may further clarify this issue.

The above studies indicate that CYP1B1 may contribute to a broader range of glaucoma phenotypes than the PCG phenotype alone. This could in turn signify that either direct interaction of the CYP1B1 protein with other POAG causative proteins of myocilin (21), optineurin (22), WDR36 (23), and/or indirect ability of CYP1B1 to metabolize a wide range of endogenous substrates are crucial for maintaining the physiology of eye at different stages of life and/or for normal eye development. Any interference in this delicate balance could lead to various ocular disorders including PCG, Peter’s and Rieger’s anomalies, JOAG, and POAG. Many more detailed studies are still needed before the exact role of CYP1B1 in eye disorders other than PCG is fully understood.

Biochemical and Functional Properties of CYP1B1

The CYP1B1 gene belongs to family 1 and subfamily B1 of the cytochrome P450 (CYP) superfamily of enzymes that are involved in oxidative metabolism of a wide variety of endogenous or xenobiotic compounds. Although it was originally anticipated that CYP1B1 would be another drug-metabolizing enzyme, there is now compelling evidence that this enzyme is also involved in very early stages of eye development. This in turn perhaps supports the hypothesis that members of the cytochrome P450 superfamily may control the processes of growth and differentiation. The metabolic activity of CYP1B1 could either generate or degrade an active-signaling molecule, for example, a morphogen, which affects the downstream transcriptional expression of a set of genes associated with the proper development of anterior segment of the eye (24). Localization studies of CYP1B1 in humans and mice show similar expression mainly in the ciliary epithelium, cornea, and retina (25). As the expression of CYP1B1 in the anterior chamber structures is restricted to non-pigmented ciliary epithelium (NPCE), the bioactive-signaling molecule may be secreted from NPCE into the aqueous humor for modulating the proper development of TM (24).

The Cyp1b1 in mouse is constitutively expressed in extrahepatic tissues as well as inducible by, and active in metabolism of, polycyclic aromatic hydrocarbons. Ever since the discovery of Cyp1b1 in mouse (26) it has proven to be most interesting to investigators in the field of xenobiotic metabolism, chemical carcinogenesis and cancer. An ortholog was quickly identified in the human genome and was also shown to be present in many different types of cancers (27–33) and responsible for xenobiotic metabolism and procarcinogen activation (34,35). The mouse Cyp1b1 enzyme, and its human ortholog (CYP1B1) are both constitutively expressed in extrahepatic tissues (36).

The cytochromes of the P450 superfamily are all monooxygenase enzymes that oxidize lipophilic molecules and are found in almost every phylum in which they have been sought. In mammals, they oxidize lipophilic molecules of endogenous (37) as well as exogenous (xenobiotics) origin, serving as a terminal oxidase (38). Considerable attention has been paid to a number of the 57 different forms of human cytochrome P450 and their associated metabolism of xenobiotics, because of the ability of many such compounds to serve as carcinogen precursors (39). Perhaps, the greatest number

of studies on CYP1B1 deals with its role in carcinogen activation and presence in neoplastic cells and tissues.

In 1997, we found three truncating mutations of CYP1B1 in the DNA of PCG individuals (3). Further analysis of the impact of these mutations on the CYP1B1 structure and function indicated that they resulted in loss of hemoproteins nature and ability of the protein to function as a normal monooxygenase enzyme (6). The recognition that the human ortholog might be involved in eye disease prompted our consideration of this enzyme as having a role in normal eye development (40). These observations served as impetus for an in-depth examination for the role of CYP1B1 hemoprotein in PCG. CYP1B1 transcripts were detected in embryonic and fetal tissue cDNA libraries by a number of investigators (27,41,42) supporting the concept of involvement of this hemoprotein in normal development and in agreement with gene defects in PCG subjects. However, in a number of instances, siblings homozygous for the same mutations frequently failed to develop the disease phenotype. The condition, known as incomplete penetrance, exceeded 50% in some multigenerational families (19). In those instances, the deduced sequences of mutations in the CYP1B1 predicted full-length proteins with single amino acid changes.

We prepared expression systems for four of these mutant forms exclusively found in individuals with PCG. The four mutations were cloned into the plasmid, pCWori+, for transformation of an Escherichia coli expression system (43). Two of the expressed CYP1B1 mutant proteins, G61E and R469W, have been studied and compared with the properties of the wild-type enzyme (42). These missense mutations of CYP1B1 are expressed as intact hemoproteins as compared to truncating mutations (deletion or insertion) that result in an expression of non-functional proteins. In fact, intact hemoproteins were observed when plasmids coding for the G61E or R469W mutated CYP1B1 cDNAs were expressed in the transformed bacteria. Both mutated proteins had spectral profiles characteristic of the cytochrome P450 holoenzyme. However, a higher proportion of a denatured form (cytochrome P420) was found in each expression system. Stability measurements at 4 °C revealed that G61E was much less stable than the wild-type protein, losing 50% of its hemoprotein characteristics in 24 h at 4 °C. In contrast, R469W was as stable as the wild-type enzyme (42).

Comparison of enzymatic activities of the mutant forms with the wild-type demonstrated other differences. Both of the mutant enzymes had lower specific activities than the wild-type; G61E had about 50% of the specific activities toward steroid hormones as substrates (testosterone, progesterone, and estradiol) while R469W had only about 30% of the activities (42). Further, isomeric specificities were not preserved in metabolites of some substrates, such as ß-estradiol, in which several metabolites were produced. With the wild-type protein, the ratio of 4-hydroxy-estradiol to 2-hydroxy-estradiol formed was 2.5, while that of G61E and R469W were 1.1 and 1.4, respectively. This would suggest that if the function of CYP1B1 in normal eye development is to synthesize a specific developmentally critical metabolite, then the normal development might be compromised in the mutants. How then might some siblings escape the disease phenotype? CYP1B1 and other family 1 cytochrome P450 forms are all inducible proteins. Induction of these proteins involve activation of the aryl hydrocarbon (AH) receptor by binding of ligands such as any one of a large number of polycyclic aromatic

hydrocarbons (44–47), as well as by compounds found in certain vegetables (48). We suggested that perhaps exposure to compounds capable of elevating levels of the mutant CYP1B1 provided sufficient threshold activity of this enzyme to permit normal eye development (42).

The mouse was chosen as a model for studies on eye development and the role of CYP1B1 in PCG. In humans, the limited availability of eye tissues and the difficulty of controlling conditions of exposure to inducing agents make such studies difficult. The validity of such a model was indicated by the observation that creation of a knockout (Cyp1b1−/−) mouse (29) resulted in a strain in which the eyes contained similar abnormalities as found in individuals with PCG (49). The question we asked was are there developmental changes (spatial and temporal) in cytochrome P450 expression during ontogeny? To answer this question, we obtained and examined four panels of mouse cDNA, at E7 (7-day-old embryo), E11, E15.5, and E17, covering the range from embryo to late fetal development. The mouse has 93 different cytochrome P450 genes as compared with 57 in humans. Using primers selective for 40 of the mouse genes, we were able to show the constitutive expression of 27 of the genes during ontogeny, a considerable number in the absence of inducing agents. This would suggest involvement of these genes in processes associated with the development of the organism. Many of those genes are known to be involved in homeostasis and in vitamin and hormone metabolism. However, members of cytochromes P450s (Cyps) family 1, family 2, and family 3 are generally considered as xenobiotic metabolizing enzymes, and 11 Cyps out of 19 of these examined differentially appeared at the different ontogenic stages (50).

Many of the cytochrome P450 forms have broad, overlapping substrate specificities, and the question was raised as to why, in the absence of Cyp1b1 in mouse, the other two forms of the Cyp1 family, namely Cyp1a1 or Cyp1a2, did not serve to provide the necessary function as compensatory activity? These three forms have highly conserved orthologs throughout vertebrate species and metabolize many of the same substrates. The answer was soon apparent. Cyp1a1 was only present at E7 and was absent from the other later developmental stages. In contrast, Cyp1a2 never appeared at all during in utero development while Cyp1b1 did not appear until E11, after Cyp1a1 disappeared, and remained through late fetal development (50).

If Cyp1b1 has a developmental function, what sort of function would that be? A logical supposition would be that its function probably utilizes its monooxygenase properties. In that case, it would either oxidatively convert some endogenous substrate to a developmentally active metabolite or oxidatively eliminate the biological activity of a developmentally functional or detrimental molecule. Attempts to determine the nature of such a molecule were based on the recognition that orthologous forms of CYP1B1 exist over a range of vertebrate species from the bony fish (zebra fish or fugu) to human, all with a common ancestral gene and a high degree of sequence identity. Between mouse and human, the sequences of the whole proteins are 81% identical. If one considers the six substrate recognitions sites (SRSs), the sequence identities are as much as 90% in several and are 100% in two of the sites (51,52).

We attempted to determine the nature of the endogenous substrate utilized by CYP1B1 in eye development. We reasoned that retention of orthologs in vertebrates and the demonstrated role in mouse and human eye development indicate a conser-

vation of function, and this may also indicate use of a common endogenous substrate. Thus, we began to screen lipophilic potential endogenous substrates for production of metabolites by mouse Cyp1b1 and human CYP1B1; a potential substrate would have to be metabolized by both, and a compound metabolized by only one of the two orthologs would be eliminated from the consideration of being a key biomolecule responsible for proper eye development. In this manner, we were able to eliminate the steroids, testosterone, progesterone, and ß-estradiol, all of which were metabolized by human CYP1B1 (42) but only poorly by Cyp1b1 in the mouse (52).

We have examined the possibility of arachidonic acid metabolism being the conserved substrate of the CYP1B1 orthologs. This would be an ideal choice, as metabolites of arachidonic acid have been amply demonstrated to have a wide range of biological activities (53–61). However, although human CYP1B1 metabolized this substrate readily and had a unique pattern of metabolites that differed from other family 1 cytochromes P450, Cyp1b1 handled this endogenous compound very poorly (51).

Another biologically active compound, retinoic acid (RA), is a known active morphogen and is readily demonstrated to influence embryonic and fetal development (62–67). Indeed, RA is found in eye tissues, in which the proportions that can be released from the various structures change with age, declining in the retina from E14 to the adult eye by a factor of four (68). It is synthesized from vitamin A (all-trans- retinol or ROL) by two oxidative steps, the first forming all-trans-retinal (RAL) and the second all-trans-RA. Synthesis of RA has generally been considered to be by two families of dehydrogenases, retinol dehydrogenase and retinal dehydrogenase (69,70). RAL is a product released by light from the 11-cis-retinaldehyde chromophore of rhodopsin and is rapidly converted to RA, an irreversible reaction (70). To function properly as a morphogen, it is necessary for RA to be efficiently inactivated so that only the target cells and tissues respond to it. A cytochrome P450 enzyme was found to serve in this capacity, CYP26 (71), oxidizing RA to the 4-oxo RA. In the past decade, CYP26 has been shown to be a family of related RA oxidizing enzymes (72–76), all functioning to inactivate RA. These differ in time of appearance and location within the retina. Cyp26a1 is found in the eye as early as E9 and is seen as a band in the retina at P1 that is inducible by RA; in contrast, Cyp26b1 is not seen until induction by RA (77).

A number of other studies have examined the role of human family 1–3 forms of cytochrome P450 in retinoid metabolism (see Table 4). Cytochromes P450 of other species also have been found to metabolize the retinoids, including rabbit, mouse, and rat (78–80). We compared the metabolism of these compounds by the human ortholog with that of mouse Cyp1b1. Both human and mouse orthologs could oxidize ROL to RAL and RAL to RA. However, we found neither could oxidize RA. The specific activity of the mouse enzyme was higher than the human ortholog in ROL oxidizing efficiency, but the human ortholog was more efficient in oxidation of RAL (51). From this, we concluded that if the role of CYP1B1 in eye development involves the retinoids, it would be in synthesis of the active RA and not in its removal. At present, the extent to which the retinoid metabolism by CYP1B1 is involved in eye development and function is unclear. Further studies are needed in which the contribution to retinoid

244 Sarfarazi et al.

Table 4

Human Cytochromes P450 Families 1–3 Implicated in Retinoid Metabolism

Activity is ROH to RAL, RAL to RA, and RA to other metabolites. NA, no activity.

synthesis by CYP1B1 orthologs in the eye is determined and its roles in the different eye structures established to link this enzyme to eye development.

Based on the EST Profile Viewer function in the Unigene database, of the 23 forms of cytochrome P450 in families 1–3, nine are expressed in the adult eye tissues. These include CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2R1, CYP2U1, CYP3A4, and CYP3A5. Interestingly, all of these forms, except for CYP2R1 and CYP2U1, which have only recently been identified, have been shown to be capable of metabolism of retinoids (see Table 4). The relative expression (transcripts per million of ESTs) in the human eye for these forms, taken from the database, indicates CYP1B1 to be fivefold higher in eye than the next highest form. Of those forms, only CYP1B1 has orthologs in vertebrates.

CYP1B1 Summary

PCG is a disease in which there appears to be developmental defects in the anterior chamber, specifically in the region of the TM. The observation that the Cyp1b1−/− mouse possesses abnormalities in eye structure similar to the PCG human eye defects indicates the mouse could be a good model in which to study this disease. Interestingly, it has been indicated that the elevations in IOP observed in PCG individuals were not seen in the Cyp1b1−/− mouse (49). In a recent study, CYP1B1 protein distribution in human eye structures has been demonstrated (25). Our recent examination of the distribution of the mouse ortholog, Cyp1b1, in the adult mouse eye indicated

a similar distribution of the hemoproteins (24). In both species, the hemoprotein is normally absent from the TM. This suggests that the action of CYP1B1 on development and function of the TM must be by some metabolite produced by the hemoprotein that is expressed in nearby structures and distributes to the TM. A detailed distribution of the hemoprotein with ontogeny is currently in progress in our laboratories, which will hopefully contribute to understanding of its role in the anterior chamber of the eye.

As CYP1B1 is expressed in anterior and posterior parts of the eye, it may play significant functional roles in both regions. Its role in the anterior segment is suggested by the association of CYP1B1 mutations with PCG (81), as mentioned above, and its recent association with other anomalies of the anterior segment dysgeneses of tissues derived from the neural crest, such as Rieger’s anomaly and Peter’s anomaly (12–14). As CYP1B1 is not detected in the TM region, a direct interaction between CYP1B1 and other potential glaucoma gene products in this region seems improbable. We believe that CYP1B1 synthesizes a key-signaling molecule, which is then secreted out from the NPCE into the aqueous humor and further modulates transcriptional activation of downstream genes. In the posterior part of eye, CYP1B1 is expressed in the retinal tissues. Therefore, the presence of CYP1B1 mutations in POAG subjects (juvenile and adult onset) may indicate a possibility for its physiological role in homeostasis of the retina. This delayed clinical manifestation of CYP1B1 mutation in adult POAG patients, as compared to the PCG cases, could be due to compensatory activity of some other gene products or temporary induction of the lower activity of mutant CYP1B1 forms by the environmental agents and/or diets during the fetal and early childhood period. The subsequent lowering of CYP1B1 activity in the retinal region could cause accumulation of toxic endogenous or exogenous molecules, thus leading to toxicity and damage to retinal ganglion cells (RGCs) during the adult stages of life. Hence, in the adult eye, CYP1B1 may be playing a homeostatic role that is not only maintaining the aqueous humor outflow but also retinal physiology. The retinal region shows a positive expression for CYP1B1 as well as other POAG genes identified so far (MYOC, OPTN, and WDR36) and hence could also indicate the possibility of direct interaction of these proteins with CYP1B1. The co-occurrence of mutation in CYP1B1 and other glaucoma-associated genes in an individual may amplify the glaucomatous effect during adulthood either acting independently or synergistically.

OPTINEURIN (OPTN)

One of the earliest genetic loci to be identified for adult-onset POAG was the GLC1E locus on 10p14 region. We originally identified this locus in a very large kindred with many family members affected with normal-tension glaucoma (NTG) (82). Our subsequent mutation screening of several candidate genes from the GLC1E region (83,84) identified mutations in the affected members of the original family linked to this locus. The gene was named optineurin (for “optic neuropathy-inducing” protein or OPTN), and mutations in this gene were initially observed in 16.7% of our hereditary NTG families (22). Since this initial report, many different groups have screened for OPTN in their glaucoma population and confirmed the presence of additional sequenceassociated alterations with the glaucoma phenotype (85–89). So far, a total of 15 OPTN