GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2007) 12, 969-979. doi:10.1111/j.1365-2443.2007.01102.x
© 2007 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, B.-C.
Right arrow Articles by Yue, B. Y.J.T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Park, B.-C.
Right arrow Articles by Yue, B. Y.J.T.

Interaction between two glaucoma genes, optineurin and myocilin

 Bum-Chan Park, Martin Tibudan, Mishan Samaraweera, Xiang Shen and Beatrice Y.J.T. Yue*

Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, IL, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Myocilin (MYOC) and optineurin (OPTN) are two genes linked to glaucoma, a major blinding disease. To investigate the possible molecular interactions between MYOC and OPTN genes, we over-expressed MYOC and examined its effect on the level of endogenous OPTN in human trabecular meshwork (TM) cells and vice versa. We noted that over-expressing MYOC did not affect the OPTN level, whereas OPTN over-expression induced an up-regulation of the endogenous MYOC. This induction was also observed in other ocular and non-ocular cell types including PC12 cells. The endogenous levels of both OPTN and MYOC genes were in addition found increased when PC12 cells underwent differentiation upon treatment with nerve growth factor (NGF). Over-expression of OPTN resulted in prolonged turnover rate of MYOC mRNA but had little effect on the promoter activity of the MYOC gene. The over-expressed OPTN was localized in the cytoplasm, not translocated into the nucleus. These results indicate that interaction exists between OPTN and MYOC genes. Regulation of MYOC expression by OPTN is achieved primarily through control of the mRNA stability.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Glaucoma is a major blinding disease characterized by progressive loss of axons and retinal ganglion cells. The most common form of this disease, primary open angle glaucoma (POAG), is age related and is frequently associated with elevated intraocular pressure (IOP). The IOP is controlled by a balance between the production and outflow of the aqueous humor contained in the anterior chamber. The trabecular meshwork (TM), a specialized eye tissue neighboring the cornea, is the major site for regulation of the aqueous humor outflow (Bill 1975).

Genetic studies have established that POAG is genetically heterogeneous, caused by several susceptibility genes (Wiggs et al. 2000; Fan et al. 2006) and perhaps also environmental factors (Wang et al. 2001). To date, three genes, myocilin (MYOC), optineurin (OPTN) and WD40-repeat36 (WDR36) have been identified for POAG, respectively, as the GLC1A gene on 1q23-q24 (Sheffield et al. 1993; Stone et al. 1997), GLC1E gene on 10p14-p15 (Sarfarazi et al. 1998; Rezaie et al. 2002) and GLC1G gene on 5q22.1 (Monemi et al. 2005). Multiple mutations in MYOC have been observed in approximately 2%–4% of adult-onset POAG cases (Stone et al. 1997; Fingert et al. 1999; Alward et al. 2002) and in as high as 36% of juvenile-onset POAG patients (Shimizu et al. 2000). OPTN mutations have been associated mainly with normal pressure glaucoma, a subset of POAG (Rezaie et al. 2002). Mutations in OPTN have been found in 16.7% of families with hereditary POAG. The exact mechanisms why and how mutations of these glaucoma genes would lead to pathology remain obscure. It is nevertheless recognized that interactions between these and perhaps also other yet-to-be-identified glaucoma genes may be a major factor in the disease development. Genes such as cytochrome P450 1B1 (CYP1B1) that linked to primary congenital glaucoma (Stoilov et al. 1997), and apolipoprotein E (APOE) have been reported to be possible modifiers (Copin et al. 2002; Vincent et al. 2002) of MYOC phenotypes. Recent large scale studies of patients and control subjects indicated that POAG may have a polygenic etiology and that gene-to-gene interactions exist (Fan et al. 2005; Funayama et al. 2006). Common polymorphisms in MYOC, OPTN and APOE were identified that might interactively contribute to POAG (Fan et al. 2005).

In the present study, we examined the possible molecular interactions between MYOC and OPTN genes. We found that OPTN, upon over-expression, was capable of increasing the endogenous MYOC messenger RNA (mRNA) level in TM and several other cell types. To determine the possible mechanisms that mediate MYOC up-regulation, the MYOC mRNA stability was monitored and promoter assay for MYOC was carried out. The results indicated that OPTN has a regulatory role in MYOC expression at the post-transcriptional level.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
MYOC expression is increased by over-expression of OPTN

To determine if over-expression of MYOC affects the endogenous OPTN mRNA level or vice versa, we transiently transfected human TM cells with either pTargeT-MYOC or pTargeT-OPTN. Cells transfected in parallel with empty pTargeT vector served as a mock control. Total RNA was extracted from all three types of transfectants at various post-transfection time points. Reverse transcriptase-relative quantitative polymerase chain reaction (RT-RQPCR) for MYOC and OPTN was performed. The MYOC mRNA level was increased as expected in pTargeT-MYOC transfected cells. Over-expression of MYOC, however, did not affect the endogenous OPTN mRNA level (data not shown). By contrast, the endogenous MYOC mRNA level was dramatically induced in OPTN-over-expressing cells compared to mock controls (Fig. 1A). The induction was by 13.8-fold for the earliest 6 h post-transfection time point and was increased to 26.4-fold within 12 h. The high MYOC level was maintained for at least 72 h (Fig. 1A). Unlike MYOC, the endogenous expression level of two other glaucoma genes, WDR36 and CYP1B1, was not modified by the OPTN over-expression (Fig. 1B).


Figure 1
View larger version (57K):
[in this window]
[in a new window]

  		Figure 1  Expression of glaucoma genes after OPTN over-expression in human TM cells. RT-RQPCR analyses of OPTN and MYOC (A) or OPTN, WDR36 and CYP1B1 (B) mRNA levels were performed after transfection with pTargeT (mock control; lane C) or pTargeT-OPTN (lane O) for 6, 12, 24, 48 and 72 h. Marker VI (Roche) and GeneRulerTM 100 bp ladder are shown in lane M of (A) and (B), respectively. Densitometric analyses were performed and the band intensity of each glaucoma gene (upper band) was normalized to that of 18S (lower band). The expected sizes of human OPTN, MYOC, WDR36, CYP1B1 and 18S products are, respectively, 512, 465, 598, 510 and 315 bp. The data for pTargeT-OPTN-transfected samples at each time point are expressed as ratios relative to those of mock controls. Three independent experiments were performed, yielding similar results.

 
The increase in endogenous MYOC message level by OPTN over-expression was likewise observed in two other human ocular [retinal pigment epithelium (RPE) and corneal stromal fibroblast (CSF)] and two human non-ocular [HeLa and human embryonic kidney (HEK)] cell lines (Fig. 2A) at both 6- and 24-h post-transfection time points (Fig. 2A). The fold of induction of MYOC was variable, ranging from 7.1 to 38.9 depending on cell types.


Figure 2
View larger version (59K):
[in this window]
[in a new window]

  		Figure 2  Expression level of OPTN and MYOC after OPTN transfection in various cell types. (A) RT-RQPCR analyses of OPTN and MYOC expression in RPE, CSF, HeLa and HEK cells transfected with pTargeT (mock control; lane C) or pTargeT-OPTN (lane O) for 6 or 24 h. GeneRulerTM 100 bp ladders are shown in lane M. Densitometric analyses were performed and the band intensity of OPTN (upper band, 512 bp) or MYOC (upper band, 465 bp) was normalized to that of 18S (lower band, 315 bp). The data for pTargeT-OPTN-transfected samples at each time point are expressed as ratios relative to those of mock controls. (B) RT-PCR analyses of forced expressed human OPTN, endogenous rat MYOC and 18S expression in PC12 and RGC5 cells after transfection with pTargeT (mock control; lane C) or pTargeT-OPTN (lane O) for 6 or 24 h. GeneRulerTM 100 bp ladders are shown in lane M. The expected sizes of human OPTN, rat MYOC and 18S products are, respectively, 512, 586 and 315 bp.

 
Experiments were also carried out with two rat neuronal cell lines, rat pheochromocytoma (PC12) and rat retinal ganglion cell line 5 (RGC5). Again, the cells were transfected with pTargeT-OPTN for 6 and 24 h and the message level of the forced expressed human OPTN, the endogenous rat MYOC and 18S was analyzed by RT-PCR using appropriate primer sets (Fig. 2B). The endogenous rat MYOC level was relatively low in PC12 and RGC5 cells. More thermal cycling (43 cycles for PC12 and 50 cycles for RGC5) was necessary to detect the MYOC PCR product in both compared to other cell types (26 cycles) (Table 1). It was nevertheless evident that the endogenous rat MYOC level was also increased by over-expression of OPTN (Fig. 2B).


View this table:
[in this window]
[in a new window]

  		Table 1  Primer sequences, anticipated sizes of PCR products and PCR or RQPCR conditions
 
PC12 cells have been shown, upon treatment with nerve growth factor (NGF), to differentiate and display a neuronal phenotype (Greene & Tischler 1976). In agreement with previously reported findings, 2 days after NGF treatment, PC12 cells developed neurites that were on average 3–4 times longer than the size of cell body (data not shown). Coincide with the differentiation, the expression of endogenous OPTN and MYOC was increased by 8.8 and 5.0-fold, respectively (Fig. 3).


Figure 3
View larger version (65K):
[in this window]
[in a new window]

  		Figure 3  RT-RQPCR analyses of endogenous rat OPTN and rat MYOC expression in PC12 cells without (control; –NGF) or with NGF (+NGF) treatment for 2 days. Densitometric data, determined as in Fig. 2A, are given under the images. GeneRulerTM 100 bp ladders are shown in lane M. The expected sizes of rat OPTN, rat MYOC and 18S products are, respectively, 524, 488 and 315 bp.

 
Turnover of MYOC mRNA

Up-regulation of endogenous MYOC mRNA after OPTN over-expression could result from decreased turnover, increased transcription, or both. To investigate whether OPTN over-expression influences the stability of MYOC mRNA, human RPE cells transfected with either pTargeT or pTargeT-OPTN for 24 h were subjected to 5 µg/mL actinomycin D (ActD) treatment to block de novo transcription. Total RNA was extracted at various time points and RT-RQPCR for MYOC was performed. ActD reduced the MYOC expression with time in cells transfected with empty vector (Fig. 4). The half life of the endogenous MYOC mRNA in mock transfected cells was calculated to be 28.4 ± 4.9 h (n = 3, Fig. 4). In OPTN-transfected cells, the high expression level of MYOC mRNA was not diminished by ActD throughout the 48-h time course examined. An attempt was made to extend the time course to 72 h but the cells started to round up and die at that point (data not shown). We thus concluded that the half life of the MYOC transcript in OPTN-over-expressing cells was at least 48 h and that the OPTN over-expression enhanced the MYOC mRNA stability.


Figure 4
View larger version (57K):
[in this window]
[in a new window]

  		Figure 4  Effects of OPTN over-expression on MYOC mRNA turnover. Human RPE cells transfected with either pTargeT or pTargeT-OPTN for 24 h were treated with ActD (5 µg/mL) for 0, 8, 24, 32 or 48 h. GeneRulerTM 100 bp ladders are shown in lane M. Upon RT-RQPCR, the band intensity of MYOC (upper band, 465 bp) was normalized to that of 18S (lower band, 315 bp) and values were obtained for each sample. The data is presented as ratio of each value relative to that of the 0 h (no ActD treatment) sample. Three independent experiments were performed. Data compiled from these experiments were used to calculate the half-life of the MYOC transcript.

 
Promoter activity of the MYOC gene

To evaluate whether OPTN has an effect on promoter activity of the MYOC gene, we generated secreted alkaline phosphatase (SEAP) reporter plasmids that encompass different 5'-flanking promoter fragments of the human MYOC gene. The 5'-flanking fragments started from –5194, –3519, –2646 or –1046 relative to the putative transcription start site (Nguyen et al. 1998) and all ended at position +67 (Fig. 5A). All promoter constructs were co-transfected into human TM cells along with pmaxGFP, a green fluorescent protein (GFP) expression vector, as well as either pTargeT empty vector or pTargeT-OPTN. After 48 h, culture media collected were measured for SEAP activity (Fig. 5B). The promoter activity, after normalization to the GFP fluorescence intensity, was compared to negative controls using the promoterless pSEAP2-Basic vector. Constructs pSEAP2-PMYOC53, pSEAP2-PMYOC36, pSEAP2-PMYOC27 and pSEAP2-PMYOC11 had similar promoter activities, each displaying 23.8-, 24.5-, 18.6- and 20.4-fold, respectively, higher activity than that of pSEAP2-Basic (Fig. 5B). The shortest 1.1 kb fragment, –1046 and +67, was already sufficient to exhibit the full promoter activity as demonstrated previously (Kirstein et al. 2000). Additional experiments further indicated that the MYOC promoter activity was not significantly varied regardless of whether the cells were co-transfected with pTargeT or pTargeT-OPTN (Fig. 5B).


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Figure 5  MYOC promoter studies. (A) A schematic representation of four MYOC promoter-SEAP constructs and a promoterless SEAP-Basic construct is shown. The position of the transcription start site is indicated with an arrow. Numbers are given relative to the putative transcription start site (+1). (B) Each of the five reporter constructs and pmaxGFP were co-transfected into human TM cells along with either pTargeT (mock control; –) or pTargeT-OPTN (+). The SEAP reporter activity was normalized to the GFP fluorescence intensity. Transfection was done in triplicate and the experiments were repeated at least 3 times. The results are shown as mean ± SEM (n = 3) values relative to those of the promoterless reporter construct pSEAP2-Basic. Data from pTargeT-OPTN (+) co-transfected samples were not significantly different from those of mock controls. Parallel experiments using positive control pSEAP2-Control yielded SEAP activity more than 100-fold higher (not shown) than that of pSEAP2-Basic.

 
The OPTN, MYOC and SEAP expression levels in these cells were verified by RT-RQPCR. As anticipated, OPTN was up-regulated in cells co-transfected with pTargeT-OPTN and all promoter constructs (Fig. 6). The MYOC transcript level also was elevated accordingly. However, the SEAP activity from the promoter constructs was not induced by OPTN co-transfection. This confirmed that OPTN elicited little effect on the MYOC promoter activity. The up-regulation of MYOC mRNA by OPTN in transfection experiments was therefore not likely via the transcriptional activation. The possibility that the OPTN response element in the MYOC promoter may be beyond the –5194 to +67 regions however, could be not excluded.


Figure 6
View larger version (61K):
[in this window]
[in a new window]

  		Figure 6  RT-RQPCR analyses of OPTN, MYOC and SEAP expression in human TM cells transfected with each MYOC promoter-reporter construct, pmaxGFP and pTargeT (lane C) or pTargeT-OPTN (lane O). The OPTN (upper band, 512 bp), MYOC (upper band, 465 bp) and SEAP (upper band, 607 bp) intensity was normalized to that of 18S (lower band, 315 bp) and the data of pTargeT-OPTN-transfected samples were expressed relative to those of pTargeT-transfected controls. GeneRulerTM 100 bp ladder markers are shown in lane M.

 
OPTN is not localized in the nucleus

The localization of OPTN in a cell after over-expression might provide a clue as to whether the endogenous MYOC mRNA is up-regulated transcriptionally or post-transcriptionally. We first performed immunofluorescence staining in pTargeT-OPTN-transfected cells. In both TM and RPE cells, a strong perinuclear staining was observed but the over-expressed OPTN in general had a diffuse cytoplasmic distribution pattern (Fig. 7A), similar to that described previously (Park et al. 2006b). No staining was found in the nucleus. To investigate further, nuclear extracts of RPE cells transfected with either pTargeT or pTargeT-OPTN were fractionated on sucrose density gradient. Two marker proteins, nucleolin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were detected by Western blotting at the correct molecular sizes (110 and 36 kDa, respectively), respectively, in each expected nuclear or cytosolic fraction (Fig. 7B). OPTN was also detected at the anticipated size (74 kDa) with a much higher expression level in the over-expressing sample. It was found only in the cytosolic fraction, not in the nuclear fraction of either mock or OPTN-transfected cells (Fig. 7B) even after overloading or long exposure of the blots.


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		Figure 7  Localization of over-expressed OPTN. (A) Immunofluorescence staining of TM and RPE cells. Cells transfected with pTargeT-OPTN were fixed and immunostained with anti-OPTN. Images were taken using a 63 x oil objective with AXIOSCOPE and METAMORPH software. Bar, 20 µm. (B) Western blot analyses of isolated nuclear and cytosol fractions. RPE cells transfected with pTargeT (lane C) or pTargeT-OPTN (lane O) for 16 h were subjected to nuclear extraction. Fifteen micrograms of protein extracts from cytosol and nuclear fractions were immunoblotted with anti-OPTN, anti-nucleolin or anti-GAPDH.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The present study demonstrates that molecular interaction between the two glaucoma genes, MYOC and OPTN, does exist. Recent genetic studies suggest that glaucoma genes may interactively contribute to POAG (Fan et al. 2006; Funayama et al. 2006). For example, primary congenital glaucoma CYP1B1 gene has been shown to be a modifier of MYOC expression in juvenile-onset POAG cases (Vincent et al. 2002). Individuals carrying both MYOC and CYP1B1 mutations had early disease onset compared with carriers of MYOC mutation alone (Vincent et al. 2002). A promoter polymorphism of MYOC, when interacted with an APOE polymorphism, was associated with increased IOP in patients with POAG (Copin et al. 2002). Among POAG genes, a potential interaction between OPTN and MYOC was also reported by a polymorphism study (Fan et al. 2005). In the current study, we provide evidence of interaction between OPTN and MYOC, showing that OPTN, when over-expressed, promoted the MYOC mRNA level in many cell types including the TM (Figs 1 and 2A,B). Furthermore, in PC12 cells both OPTN and MYOC genes are up-regulated upon differentiation (Fig. 3). To our knowledge, this is the first report to investigate the interaction between two major POAG genes at the molecular level.

Our results showed that co-transfection of pTargeT-OPTN along with MYOC promoter constructs in SEAP reporter assays neither altered the MYOC promoter activity (Fig. 5B) nor the SEAP message level (Fig. 6). In addition, the over-expressed OPTN was not translocated into the nucleus (Fig. 7) to exert influence on gene transcription. We therefore concluded that the increase in endogenous MYOC mRNA subsequent to OPTN over-expression may not be related to transcriptional activation. Instead, the data suggest that the up-regulation of MYOC transcript by OPTN may be due to an increase in the MYOC mRNA stability. ActD decay experiments revealed that the half-life of the endogenous MYOC transcript is much prolonged by OPTN over-expression (Fig. 4).

It is well established that the mRNA turnover in mammalian cells is a tightly regulated process. The rate of mRNA degradation is determined by the orchestrated interaction between the sequences of the nucleic acid (cis-element on the mRNA sequences) and the proteins that bind them (trans-acting RNA binding proteins). The cis-elements include the 5'-cap structure, 5'-untranslated region (UTR), the protein coding region, 3'-UTR and the 3'-polyadenylate [poly(A)] tail (Guhaniyogi & Brewer 2001). In mammalian cells, the deadenylation-dependent pathway of mRNA decay is initiated with removal of the poly(A) tail. The shortened tails bind fewer poly(A) binding proteins and other proteins in the ribonucleoprotein complexes that followed by decapping and degradation of the body of mRNA by exonucleases. The deadenylation-independent pathway starts with endoribonucleolytic cleavage. The cleaved mRNA products are then subjected to exonucleolytic activities (Guhaniyogi & Brewer 2001).

The cis–trans interactions can be altered in response to factors including cytokines and hormones as well as physiological stress such as hypoxia and aging (Guhaniyogi & Brewer 2001). Differentiation, proliferation and apoptosis are known to be associated with modifications of the rate of mRNA degradation. Deregulation of mRNA stability has also been associated with cancer, inflammatory disease and Alzheimer's disease (Hollams et al. 2002). In addition, aberrant expression of RNA-binding proteins has been shown to be involved in regulation of mRNA stability in human diseases (Hollams et al. 2002). For instance, over-expression of human coding region determinant binding protein, known to bind and stabilize c-Myc mRNA (Doyle et al. 1998), has been reported in 30% and 50% of human breast cancers and colon cancers, respectively (Doyle et al. 2000; Ross et al. 2001).

In the case of MYOC mRNA, OPTN may act as an RNA-binding protein to inhibit the mRNA decay or protect endonucleolytic cleavage sites. It may also stabilize MYOC mRNA by coordinating with other RNA-binding proteins to protect it from degradation. The OPTN gene does have a putative zinc finger sequence (Li et al. 1998a; Schwamborn et al. 2000) that has been shown to be one of the RNA-binding domains (Hollams et al. 2002). Further research, however, is needed to determine whether OPTN protein binds to MYOC mRNA and to map the interaction domains.

More than 70 mutations in the MYOC gene have been identified in POAG patients (Gong et al. 2004). Most of the mutations are mis-sense substitutions in the C-terminal olfactomedin-like domain. These mutations or absence of the olfactomedin domain in MYOC have been found to trigger mis-folding of the MYOC protein and suppression of its secretion (Caballero & Borras 2001; Jacobson et al. 2001). The mutants accumulate in the endoplasmic reticulum (ER) (Joe et al. 2003; Liu & Vollrath 2004), causing cell death through ER stress (Joe et al. 2003; Liu & Vollrath 2004; Yam et al. 2007). Such a gain of function, not haploinsufficiency, is considered to be the basis for MYOC mutation-related pathology.

Whether and how up-regulation of wild type MYOC would directly lead to glaucoma is less clear. MYOC expression is known to be up-regulated in TM cells by glucocorticoids such as dexamethasone (Polansky et al. 1997) and steroid induced glaucoma cases have been reported in patients after glucocorticoid treatments (Fingert et al. 2002). Dexamethasone administration has also been shown to lead to IOP elevation in animals including rabbit and primate (Knepper et al. 1985; Pang et al. 2001; Fingert et al. 2001). Results from our laboratory indicate that MYOC protein is associated with mitochondria (Wentz-Hunter et al. 2002; Sakai et al. 2007) and that the mitochondrial membrane potential is decreased in TM cells upon MYOC over-expression or dexamethasone treatment (Sakai et al. 2007). Furthermore, transfection of MYOC into TM cells has been shown to cause a loss of actin stress fibers, compromise cell adhesion and sensitize cells to apoptotic process (Wentz-Hunter et al. 2004). Based on these observations, it is postulated that the up-regulation of MYOC may result in TM cell vulnerability, which, with additional stress or challenge, may lead to pathology (Wentz-Hunter et al. 2002; Sakai et al. 2007).

Herein we uncover another MYOC overabundance scenario that occurs in consequence of OPTN over-expression or up-regulation such as after NGF-induced differentiation in PC12 cells. OPTN expression has also been noted previously to increase by cytokines including tumor necrosis factor-{alpha}, and interferon {alpha}/ß and {gamma} (Li et al. 1998a; Schwamborn et al. 2000). Irrespective of the OPTN-inducing cues, the increase in the endogenous MYOC level ensued may, as discussed above, have pathologic consequences.

In summary, the current study documents molecular interactions between two POAG genes, OPTN and MYOC. The expression of endogenous MYOC is up-regulated after forced expression of OPTN or upon induced differentiation in PC12 cells. The up-regulation is mediated primarily by an increase in the MYOC mRNA stability. OPTN thus appears to have a regulatory role in MYOC expression at the post-transcriptional level.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Plasmid construction

Plasmid pTargeT-OPTN was constructed as previously described (Park et al. 2006b). Human MYOC open reading frame (ORF) was PCR-amplified against pRSET-MYOC (Park et al. 2006a) and cloned into pTargeT (Promega, Madison, WI, USA) to generate pTargeT-MYOC. Human genomic DNA was used as the template to construct by PCR plasmids for 5'-flanking region of the human MYOC gene. The promoter fragments included a 5261 bp-piece containing sequences from –5194 to +67 (–5194/+67), a 3586 bp- (–3519/+67), a 2713 bp- (–2646/+67) and 1113 bp- (–1046/+67) pieces. Human genomic DNA was extracted from cultured CSF by DNA extraction kit (Stratagene, La Jolla, CA, USA). The resulting PCR fragments were digested with BglII and HindIII and cloned into promoterless pSEAP2-Basic (BD Biosciences, San Jose, CA, USA) at the same restriction sites, yielding pSEAP2-PMYOC53, pSEAP2-PMYOC36, pSEAP2-PMYOC27 and pSEAP2-PMYOC11. Sequencing was followed to verify all the constructs. All the primers used are listed in Table 1.

Cell culture and transfection

Normal human eyes from donors 17, 19, 20, 23, 26, 29, 39, 44, 49 and 54 years of age were obtained from the Illinois Eye Bank (Chicago, IL, USA). TM tissues excised from these eyes were cultured on Falcon Primaria flasks with complete medium containing Dulbecco's modified Eagle's minimum essential medium (DMEM; Invitrogen, Carlsbad, CA, USA), 10% fetal bovine serum (FBS), 5% calf serum, essential and non-essential amino acids and antibiotics. When the cells reached confluence, they were trypsinized and subcultured. Second- or third-passaged cells were used for experiments. The TM cells displayed a polygonal morphology with a growth pattern distinct from that of fibroblastic and corneal endothelial cultures. They stained positively with DiI-acetylated low density lipoprotein (DiI-AcLDL, Invitrogen), consistent with previous reports that TM cells possess receptors for modified LDL (Chang et al. 1991; Stamer et al. 1998). In parallel experiments, human umbilical vein endothelial cells (positive control) stained strongly, while CSF (negative control) did not react, with DiI-AcLDL as expected (data not shown).

Human CSF cultures were established as previously described (Wentz-Hunter et al. 2003). RPE (ARPE-19), HeLa, HEK and PC12 cells were purchased from American Type Cell Culture (Manassas, VA, USA). RGC5 cells were obtained from the Ophthalmology departmental core facility, deposited originally by Dr Paul Knepper (Choi et al. 2005). These cells were grown and maintained in complete medium. For differentiation, PC12 cells were transferred to RPMI-1640 medium (Invitrogen) supplemented with 1% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin 1 day prior to the treatment with 100 ng/mL of NGF (Sigma, St. Louis, MO, USA) for 2 days.

Transient transfection was carried out using FuGENE6 reagent (Roche, Indianapolis, IN, USA) as per the manufacturer's instruction with minor modifications. Briefly, cells were plated at 70%–80% confluency overnight. Cells were washed with phenol red-free DMEM (Invitrogen) and incubated for 2 h in DMEM containing 2.5% FBS. FuGENE6 reagent mixed with plasmid DNA at a 3 : 1 ratio was added to the cells for the indicated time periods (Park et al. 2006b). Two micrograms of plasmid DNA per mL of the final transfection mixture was used unless otherwise noted.

RT-RQPCR

Total RNA was isolated from cells transfected with pTargeT or pTargeT-OPTN using RNeasy mini kit (Qiagen, Valencia, CA, USA). cDNA was prepared employing random hexamers according to the manufacturer's instruction. Aliquots of cDNA samples were PCR-amplified using 50 µM of target gene-specific primer pair and optimal ratio of 18S primers to competimers (QuantumRNA universal 18S internal standard kit; Ambion, Austin, TX, USA). The number of thermal cycle and the optimal ratio of 18S primers to competimers for each target gene were determined prior to RQPCR experiments according to manufacturer's instructions. The sequence of primer pair for target genes, the anticipated sizes of PCR products and RQPCR or PCR conditions such as cycle numbers and optimal 18S ratios are listed in Table 1. PCR products, along with Marker VI (Roche) or GeneRulerTM 100 bp ladder markers (Fermentas, Hanover, MD, USA) were resolved on 1.2% agarose gels. The gel image captured with Gel Doc 2000 image analyzer (Bio-Rad, Hercules, CA, USA) was analyzed by densitometry using Image station 440 (Eastman Kodak Company, Rochester, NY, USA).

mRNA turnover assay

RPE cells in 6-well plates were transfected with pTargeT or pTargeT-OPTN for 24 h and treated with 5 µg/mL of ActD for 0, 8, 24, 32 and 48 h. The cells were subjected to total RNA extraction and RT-RQPCR. After agarose gel electrophoresis, densitometry was performed to determine the half-life of MYOC mRNA. The experiment was carried out 3 times.

SEAP assay

Human TM cells in 24 well plates were co-transfected for 48 h with a reporter plasmid (pSEAP2-PMYOC53, pSEAP2-PMYOC36, pSEAP2-PMYOC27, pSEAP2-PMYOC11, pSEAP2-Control or pSEAP2-Basic), pmaxGFP (Amaxa, Gaithersburg, MD, USA) and either pTargeT or pTargeT-OPTN at a 38 : 1 : 1 ratio. Plasmids pSEAP2-Control, driven by the SV40 early promoter and enhancer, was used as a positive control. The use of the SV40 promoter and enhancer in primary cultures has been reported in other primary cell cultures including human embryonic lung fibroblasts (Ducrest et al. 2002), human aortic fibroblasts (Gonzalez-Nicolini et al. 2006) and human CSF (Li et al. 1998b). The promoterless pSEAP2-Basic was used as a negative control for the SEAP assay. The plasmid pmaxGFP was used to normalize for the transfection efficiency. After transfection, aliquots of medium samples were analyzed for the SEAP activity in a luminometer (Optocomp II; MGM instruments, Hamden, CA, USA) according to the manufacturer's instruction. Cells were lysed in 200 µL of cell lysis reagent (CelLyticTM-M; Sigma) containing protease inhibitor cocktail. To quantify GFP fluorescence, 50 µL of the cell lysate was added to the MICROTEST 96-well assay plate (BD Biosciences) and the fluorescence intensity was measured on GENios Pro microplate reader (Tecan, Durham, NC, USA). The resulting GFP fluorescence was used to normalize the SEAP activity. Assays were performed in triplicate, and each experiment was repeated at least 3 times.

Immunofluorescence staining

TM and RPE cells (8000 cells/well) plated onto Lab-Tek 8-well CC2 glass chamber slides (Nalge Nunc, Rochester, NY, USA) were transfected with pTargeT-OPTN. The cells were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton X-100 for 4 min. Upon blocking with 3% bovine serum albumin for 30 min, the cells were incubated for 1 h at room temperature with anti-OPTN (1 : 200, Cayman Chemical, Ann Arbor, MI, USA). They were further incubated for 45 min with FITC-conjugated secondary antibody (1 : 200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA). Photographs were taken using a 63 x oil objective on an Axioscope (Carl Zeiss MicroImaging, Thornwood, NY, USA) with the aid of METAMORPH software (Molecular Devices, Downingtown, PA, USA).

Nuclear extraction

RPE (8 x 106) cells transfected with pTargeT or pTargeT-OPTN for 16 h were washed and harvested. The nuclei were purified using Nuclei PURE Prep kit (Sigma) according to manufacturer's instructions. The isolated nuclei were lysed in CelLyticTM-M reagent containing protease inhibitor cocktail. Cytosol fractions were also collected. Fifteen micrograms of protein extracts from each fraction was resolved on 10% SDS-polyacrylamide gels and immunoblotted with anti-OPTN (1 : 2000), anti-nucleolin (1 : 2000; Santa Cruz Biotech, Santa Cruz, CA, USA) or anti-GAPDH (1 : 5000; Trevigen, Gaithersburg, MD, USA). The blot was then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (1 : 10 000; Jackson ImmunoResearch Laboratories). The protein bands were detected using SuperSignal Substrate (Pierce, Rockford, IL, USA). For repeated probing, the blot was stripped for 1 h at room temperature with ImmunoPure IgG Elution buffer (Pierce).


   Acknowledgements
 
This work was supported in part by grants EY05628 (BYJTY), EY03890 (BYJTY) and core grant EY01792 from the National Institutes of Health, Bethesda, Maryland, and in part by McGraw Foundation, Northbrook, Illinois.


   Footnotes
 
Communicated by: Noriko Osumi

* Correspondence: E-mail: beatyue{at}uic.edu


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Alward, W.L., Kwon, Y.H., Khanna, C.L., Johnson, A.T., Hayreh, S.S., Zimmerman, M.B., Narkiewicz, J., Andorf, J.L., Moore, P.A., Fingert, J.H., Sheffield, V.C. & Stone, E.M. (2002) Variations in the myocilin gene in patients with open-angle glaucoma. Arch. Ophthalmol. 120, 1189–1197.[Abstract/Free Full Text]

Bill, A. (1975) The drainage of aqueous humor. Invest. Ophthalmol. Vis. Sci. 14, 1–3.[Free Full Text]

Caballero, M. & Borras, T. (2001) Inefficient processing of an olfactomedin-deficient myocilin mutant: potential physiological relevance to glaucoma. Biochem. Biophys. Res. Commun. 282, 662–670.[CrossRef][Medline]

Chang, I.L., Elner, S.G., Yue, B.Y.J.T., Cornicelli, J.A., Kawa, J.E. & Elner, V.M. (1991) Expression of modified low-density lipoprotein receptors by trabecular meshwork cells. Curr. Eye Res. 10, 1101–1112.[Medline]

Choi, J., Miller, A.M., Nolan, M.J., Yue, B.Y.J.T., Thotz, S.T., Clark, A.F., Agarwal, N. & Knepper, P.A. (2005) Soluble CD44 is cytotoxic to trabecular meshwork and retinal ganglion cells in vitro. Invest. Ophthalmol. Vis. Sci. 46, 214–222.[Abstract/Free Full Text]

Copin, B., Brezin, A.P., Valtot, F., Dascotte, J.C., Bechetoille, A. & Garchon, H.J. (2002) Apolipoprotein E-promoter single-nucleotide polymorphisms affect the phenotype of primary open-angle glaucoma and demonstrate interaction with the myocilin gene. Am. J. Hum. Genet. 70, 1575–1581.[CrossRef][Medline]

Doyle, G.A., Betz, N.A., Leeds, P.F., Fleisig, A.J., Prokipcak, R.D. & Ross, J. (1998) The c-Myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins. Nucleic Acids Res. 26, 5036–5044.[Abstract/Free Full Text]

Doyle, G.A., Bourdeau-Heller, J.M., Coulthard, S., Meisner, L.F. & Ross, J. (2000) Amplification in human breast cancer of a gene encoding a c-Myc mRNA-binding protein. Cancer Res. 60, 2756–2759.[Abstract/Free Full Text]

Ducrest, A.L., Amacker, M., Lingner, J. & Nabholz, M. (2002) Detection of promoter activity by flow cytometric analysis of GFP reporter expression. Nucleic Acids Res. 30, e65.[Abstract/Free Full Text]

Fan, B.J., Wang, D.Y., Fan, D.S., Tam, P.O., Lam, D.S., Tham, C.C., Lam, C.Y., Lau, T.C. & Pang, C.P. (2005) SNPs and interaction analyses of myocilin, optineurin, and apolipoprotein E in primary open angle glaucoma patients. Mol. Vis. 11, 625–631.[Medline]

Fan, B.J., Wang, D.Y., Lam, D.S.C. & Pang, C.P. (2006) Gene mapping for primary open angle glaucoma. Clin. Biochem. 39, 249–258.[CrossRef][Medline]

Fingert, J.H., Clark, A.F., Craig, J.E., Alward, W.L., Snibson, G.R., McLaughlin, M., Tuttle, L., Mackey, D.A., Sheffield, V.C. & Stone, E.M. (2001) Evaluation of the myocilin (MYOC) glaucoma gene in monkey and human steroid-induced ocular hypertension. Invest. Ophthalmol. Vis. Sci. 42, 145–152.[Abstract/Free Full Text]

Fingert, J.H., Heon, E., Liebmann, J.M. et al. (1999) Analysis of myocilin mutations in 1703 glaucoma patients from five different populations. Hum. Mol. Genet. 8, 899–905.[Abstract/Free Full Text]

Fingert, J.H., Stone, E.M., Sheffield, V.C. & Alward, W.L. (2002) Myocilin glaucoma. Surv. Ophthalmol. 47, 547–561.[CrossRef][Medline]

Funayama, T., Mashima, Y., Ohtake, Y., Ishikawa, K., Fuse, N., Yasuda, N., Fukuchi, T., Murakami, A., Hotta, Y. & Shimada, N. (2006) SNPs and interaction analyses of noelin 2, myocilin, and optineurin genes in Japanese patients with open-angle glaucoma. Invest. Ophthalmol. Vis. Sci. 47, 5368–5375.[Abstract/Free Full Text]

Gong, G., Kosoko-Lasaki, O., Haynatzki, G.R. & Wilson, M.R. (2004) Genetic dissection of myocilin glaucoma. Hum. Mol. Genet. 13, R91–R102.[Abstract/Free Full Text]

Gonzalez-Nicolini, V., Sanchez-Bustamante, C.D., Hartenbach, S. & Fussenegger, M. (2006) Adenoviral vector platform for transduction of constitutive and regulated tricistronic or triple-transcript transgene expression in mammalian cells and microtissues. J. Gene Med. 8, 1208–1222.[CrossRef][Medline]

Greene, L.A. & Tischler, A.S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424–2428.[Abstract/Free Full Text]

Guhaniyogi, J. & Brewer, G. (2001) Regulation of mRNA stability in mammalian cells. Gene 265, 11–23.[CrossRef][Medline]

Hollams, E.M., Giles, K.M., Thomson, A.M. & Leedman, P.J. (2002) mRNA stability and the control of gene expression: implications for human disease. Neurochem. Res. 27, 957–980.[CrossRef][Medline]

Jacobson, N., Andrews, M., Shepard, A.R., Nishimura, D., Searby, C., Fingert, J.H., Hageman, G., Mullins, R., Davidson, B.L., Kwon, Y.H., Alward, W.L., Stone, E.M., Clark, A.F. & Sheffield, V.C. (2001) Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor. Hum. Mol. Genet. 10, 117–125.[Abstract/Free Full Text]

Joe, M.K., Sohn, S., Hur, W., Moon, Y., Choi, Y.R. & Kee, C. (2003) Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem. Biophys. Res. Commun. 312, 592–600.[CrossRef][Medline]

Kirstein, L., Cvekl, A., Chauhan, B.K. & Tamm, E.R. (2000) Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor. Genes Cells 5, 661–676.[Abstract]

Knepper, P.A., Collins, J.A. & Frederick, R. (1985) Effects of dexamethasone, progesterone, and testosterone on IOP and GAGs in the rabbit eye. Invest. Ophthalmol. Vis. Sci. 26, 1093–1100.[Abstract/Free Full Text]

Li, Y., Kang, J. & Horwitz, M.S. (1998a) Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor {alpha}-inducible cellular protein containing leucine zipper domains. Mol. Cell Biol. 18, 1601–1610.[Abstract/Free Full Text]

Li, Y., Zhou, L., Twining, S.S., Sugar, J. & Yue, B.Y.J.T. (1998b) Involvement of Sp1 elements in the promoter activity of the {alpha}1-proteinase inhibitor gene. J. Biol. Chem. 273, 9959–9965.[Abstract/Free Full Text]

Liu, Y. & Vollrath, D. (2004) Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum. Mol. Genet. 13, 1193–1204.[Abstract/Free Full Text]

Monemi, S., Spaeth, G., DaSilva, A., Popinchalk, S., Ilitchev, E., Liebmann, J., Ritch, R., Heon, E., Crick, R.P., Child, A. & Sarfarazi, M. (2005) Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1. Hum. Mol. Genet. 14, 725–733.[Abstract/Free Full Text]

Nguyen, T.D., Chen, P., Huang, W.D., Chen, H., Johnson, D. & Polansky, J.R. (1998) Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J. Biol. Chem. 273, 6341–6350.[Abstract/Free Full Text]

Pang, I.H., Moll, H., McLaughlin, M.A., Knepper, P.A., De Santis, L., Epstein, D.L. & Clark, A.F. (2001) Ocular hypotensive and aqueous outflow-enhancing effects of AL-3037A (sodium ferri ethylenediaminetetraacetate). Exp. Eye Res. 73, 815–825.[CrossRef][Medline]

Park, B.C., Shen, X., Fautsch, M.P., Tibudan, M., Johnson, D.H. & Yue, B.Y.J.T. (2006a) Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins. Mol. Vis. 12, 832–840.[Medline]

Park, B.C., Shen, X., Samaraweera, M. & Yue, B.Y.J.T. (2006b) Studies of optineurin, a glaucoma gene: Golgi fragmentation and cell death from overexpression of wild type and mutant optineurin in two ocular cell types. Am. J. Pathol. 169, 1976–1989.[Abstract/Free Full Text]

Polansky, J.R., Fauss, D.J., Chen, P., Chen, H., Lutjen-Drecoll, E., Johnson, D., Kurtz, R.M., Ma, Z.D., Bloom, E. & Nguyen, T.D. (1997) Cellular pharmacology and molecular biology of the trabecular meshwork inducible glucocorticoid response gene product. Ophthalmologica 211, 126–139.[Medline]

Rezaie, T., Child, A., Hitchings, R., Brice, G., Miller, L., Coca-Prados, M., Heon, E., Krupin, T., Ritch, R., Kreutzer, D., Crick, P.P. & Sarfarazi, M. (2002) Adult-onset primary open-angle glaucoma caused by mutations in optineurin. Science 295, 1077–1079.[Abstract/Free Full Text]

Ross, J., Lemm, I. & Berberet, B. (2001) Overexpression of an mRNA-binding protein in human colorectal cancer. Oncogene 20, 6544–6550.[CrossRef][Medline]

Sakai, H., Shen, X., Koga, T., Park, B.C., Noskina, Y., Tibudan, M. & Yue, B.Y.J.T. (2007) Mitochondrial association of myocilin, product of a glaucoma gene, in human trabecular meshwork cells. J. Cell. Physiol. In press.

Sarfarazi, M., Child, A., Stoilova, D., Brice, G., Desai, T., Trifan, O.C., Poinoosawmy, D. & Crick, R.P. (1998) Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15-p14 region. Am. J. Hum. Genet. 62, 641–652.[CrossRef][Medline]

Schwamborn, K., Weil, R., Courtois, G., Whiteside, S.T. & Israel, A. (2000) Phorbol esters and cytokines regulate the expression of the NEMO-related-protein, a molecule involved in a NF-{kappa}B-independent pathway. J. Biol. Chem. 275, 22780–22789.[Abstract/Free Full Text]

Sheffield, V.C., Stone, E.M., Alward, W.L., Drack, A.V., Johnson, A.T., Streb, L.M. & Nichols, B.E. (1993) Genetic linkage of familial open angle glaucoma to chromosome 1q21-q31. Nat. Genet. 4, 47–50.[Medline]

Shimizu, S., Lichter, P.R., Johnson, A.T., Zhou, Z., Higashi, M., Gottfredsdottir, M., Othman, M., Moroi, S.E., Rozsa, F.W., Schertzer, R.M., Clarke, M.S., Schwartz, A.L., Downs, C.A., Vollrath, D. & Richards, J.E. (2000) Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma. Am. J. Ophthalmol. 130, 165–177.[CrossRef][Medline]

Stamer, W.D., Roberts, B.C., Howell, D.N. & Epstein, D.L. (1998) Isolation, culture, and characterization of endothelial cells from Schlemm's canal. Invest. Ophthalmol. Vis. Sci. 39, 1804–1812.[Abstract/Free Full Text]

Stoilov, I., Akarsu, A.N. & Sarfarazi, M. (1997) Identification of three different truncating mutations in cytochrome P450 1B1 (CYP1B1) as the principal cause of primary congenital glaucoma (Buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum. Mol. Genet. 6, 641–647.[Abstract/Free Full Text]

Stone, E.M., Fingert, J.H., Alward, L.M., Nguyen, T.D., Polansky, J.R., Sunden, L.F., Nishimura, D., Clark, A.F., Nystuen, A., Nichols, B.E., Mackey, D.A., Ritch, R., Kalenak, J.W., Craven, E.R. & Sheffield, V.C. (1997) Identification of a gene that causes primary open angle glaucoma. Science 275, 668–670.[Abstract/Free Full Text]

Vincent, A.L., Billingsley, G., Buys, Y., Levin, A.V., Priston, M., Trope, G., Williams-Lyn, D. & Heon, E. (2002) Digenic inheritance of early-onset glaucoma: CYP1B1, a potential modifier gene. Am. J. Hum. Genet. 70, 448–460.[CrossRef][Medline]

Wang, N., Chintala, S.K., Fini, M.E. & Schuman, J.S. (2001) Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype. Nat. Med. 7, 304–309.[CrossRef][Medline]

Wentz-Hunter, K., Shen, X., Okazaki, K., Tanihara, H. & Yue, B.Y.J.T. (2004) Overexpression of myocilin in cultured human trabecular meshwork cells. Exp. Cell Res. 297, 39–48.[CrossRef][Medline]

Wentz-Hunter, K., Shen, X. & Yue, B.Y.J.T. (2003) Distribution of myocilin, a glaucoma gene product, in human corneal fibroblasts. Mol. Vis. 9, 308–314.[Medline]

Wentz-Hunter, K., Ueda, J., Shimizu, N. & Yue, B.Y.J.T. (2002) Myocilin is associated with mitochondria in human trabecular meshwork cells. J. Cell. Physiol. 190, 46–53.[CrossRef][Medline]

Wiggs, J.L., Allingham, R.R., Hossain, A., Kern, J., Auguste, J., DelBono, E.A., Broomer, B., Graham, F.L., Hauser, M., Pericak-Vance, M. & Haines, J.L. (2000) Genome-wide scan for adult onset primary open angle glaucoma. Hum. Mol. Genet. 9, 1109–1117.[Abstract/Free Full Text]

Yam, G.H., Gaplovska-Kysela, K., Zuber, C. & Roth, J. (2007) Aggregated myocilin induces Russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma. Am. J. Pathol. 170, 100–109.[Abstract/Free Full Text]

Received: 4 January 2007
Accepted: 22 May 2007




This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
X. Shen, T. Koga, B.-C. Park, N. SundarRaj, and B. Y. J. T. Yue
Rho GTPase and cAMP/Protein Kinase A Signaling Mediates Myocilin-induced Alterations in Cultured Human Trabecular Meshwork Cells
J. Biol. Chem., January 4, 2008; 283(1): 603 - 612.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, B.-C.
Right arrow Articles by Yue, B. Y.J.T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Park, B.-C.
Right arrow Articles by Yue, B. Y.J.T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS