IOVS Advertisement
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1167/iovs.08-2590 on December 5, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:1801-1807.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
doi:10.1167/iovs.08-2590
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
iovs.08-2590v1
50/4/1801    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koh, S.-W. M.
Right arrow Articles by Ku, C. Y. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koh, S.-W. M.
Right arrow Articles by Ku, C. Y. P.

Functional CNTF Receptor {alpha} Subunit Restored by Its Recombinant in Corneal Endothelial Cells in Stored Human Donor Corneas: Connexin-43 Upregulation

Shay-Whey M. Koh,1,2 Jordan Celeste,1 and C. Y. Paul Ku1

1From the Departments of Ophthalmology and Visual Sciences and 2Physiology, University of Maryland, Baltimore, Maryland.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Ciliary neurotrophic factor (CNTF) is undergoing testing in human clinical trials to rescue degenerating retina, whereas studies show that the CNTF-binding {alpha}-subunit of the CNTF receptor (CNTFR{alpha}) is released from injured tissues. Here, the recombinant human (rh) CNTFR{alpha} was shown to restore functional CNTFR{alpha} in human corneal endothelial (CE) cells that lost endogenous CNTFR{alpha} during corneal storage

METHODS. In CE cells of stored human donor corneas, endogenous CNTFR{alpha} levels were quantified (by Western blot analysis), CNTF stimulation leading to the upregulation of connexin-43 was demonstrated, and the effectiveness of rhCNTFR{alpha} (8.3 nM) in augmenting the CNTF (0.83 nM) effect was tested. Paired human donor corneas were used as vehicle versus CNTF-treated or CNTF- versus (rhCNTFR{alpha}+CNTF)-treated (24 hours, 37°C), followed by analysis of CE cell connexin-43 mRNA and protein by semiquantitative RT-PCR and Western blot analysis, respectively. After 90-minute incubation with stored human corneas, rhCNTFR{alpha} incorporation into the CE membrane fraction was demonstrated by Western blot analysis

RESULTS. CE cell CNTFR{alpha} levels decreased as corneal storage time increased. CE cell connexin-43 mRNA levels in CNTF-treated and (rhCNTFR{alpha}+CNTF)-treated paired corneas averaged (mean ± SEM) 0.26 ± 0.08 and 0.58 ± 0.21, respectively (P = 0.029; eight pairs; storage time ≥25 days). rhCNTFR{alpha} augmentation was confirmed at the protein level. In corneas with short storage times (≤9 days) that retained abundant endogenous CNTFR{alpha}, rhCNTFR{alpha} decreased the effectiveness of CNTF. rhCNTFR{alpha} was incorporated into CE membranes

CONCLUSIONS. rhCNTFR{alpha} acted as a surrogate to the lost endogenous membrane-bound CNTFR{alpha} in CNTF signaling, suggesting the potential of an adjuvant rhCNTFR{alpha} therapy in CNTF-therapy.

Ciliary neurotrophic factor (CNTF) was discovered in an extract of eye tissues consisting of ciliary body, iris, and choroid and was characterized as a survival factor for chick ciliary ganglion neurons.1 2 It has since been shown to exert neurotrophic activities in various neuronal injury models, including axotomy-induced motor neuron degeneration and retinal ganglion cell apoptosis in vivo.3 4 Recently, because the beneficial effects of CNTF treatment have been observed in a variety of animal models of photoreceptor cell degeneration,5 a phase 1 (safety) human clinical trial was conducted in which encapsulated cells engineered to secrete CNTF were implanted into the vitreous cavity of the eyes of patients with retinitis pigmentosa.6 Although the precise CNTF targets have not been identified, this therapy demonstrated a trend of beneficial effects on the visual outcome of the participants. Because CNTFR{alpha}, the CNTF-binding subunit of the CNTF receptor complex, has been localized to retinal pigment epithelial cells, rods and cones, inner nuclear cells, and retinal ganglion cells,7 these cells are potential targets of CNTF therapy. However, to the best of our knowledge, the status of CNTFR{alpha} in the CNTF therapy-targeted tissues has never been addressed. It is a distinct possibility that CNTFR{alpha} is lost during the progression of degenerative eye diseases, which CNTF therapy is designed to treat, and the inclusion of CNTFR{alpha} in CNTF therapy may further improve the outcome. This possibility must be addressed given that phase 2 and 3 clinical trials are under way or have been conducted to test the efficacy of CNTF in the treatment of macular degeneration, retinitis pigmentosa, Usher type 2 and 3, and choroideremia.8 9 10 11

Although the mechanism has not been established, studies describe the circumstances in which CNTFR{alpha} is lost from the tissues under stress. CNTFR{alpha} is lost from the fibers of the optic nerve after optic nerve injury, which may explain why CNTF is not effective in promoting regeneration of the injured optic nerve.12 Although CNTF does not have a signal peptide sequence for secretion and has been postulated as an injury factor released only after injury13 and CNTFR{alpha} lacks a transmembrane domain and is anchored to the cell membrane through a glycosylphosphatidylinositol anchor,14 we have previously demonstrated that CNTF is released in a complex with CNTFR{alpha} by sublethal oxidative stress-injured corneal endothelial (CE) cells in corneal organ cultures.15 In addition, CNTFR{alpha} is released by the skeletal muscle in response to peripheral nerve injury.16 Furthermore, CNTFR{alpha} has been detected in cerebrospinal fluid samples of patients with degenerative diseases of the central nervous system,16 and the level detected in the urine of patients with amyotrophic lateral sclerosis is 4.4-fold that of healthy persons.17

Recombinant human (rh) CNTFR{alpha} has been shown to bind with CNTF at a 1:1 stoichiometry and transduces signal as the membrane-anchored CNTFR{alpha}.18 CNTF in combination with CNTFR{alpha} (but not alone) transduces signals upregulating connexin-43 expression in glioma and astrocytes, cells that do not express CNTFR{alpha}.19 20 The present study was designed to demonstrate that rhCNTFR{alpha} can restore in cells with diminished endogenous CNTFR{alpha} functional levels of CNTFR{alpha}, augmenting the effect of CNTF on these cells.

The corneal endothelium, a neural crest-derived tissue,21 22 consists of a single cell layer of CE cells. CE cells in fresh human donor corneas express CNTFR{alpha}.23 As a common clinical practice, human donor corneas for transplantation are stored in the preservation medium in the eye bank (for only a few days) before they are transplanted into recipient eyes. We have found in CE cells in stored human donor corneas a functional CNTF/CNTFR{alpha} signaling pathway that induces the expression of vasoactive intestinal peptide,23 which is a trophic24 and differentiation-maintaining factor of CE cells.25 We have also found that the level of CE cell CNTFR{alpha} gradually diminishes during human donor cornea storage. The diminishing level of CNTFR{alpha} in stored human donor corneas in which the CE cells remain viable provides a unique opportunity to examine whether functional CNTFR{alpha} can be restored by rhCNTFR{alpha}.

Whereas CNTF/CNTFR{alpha} signaling leads to the upregulation of connexin-43 expression in astrocytes and in C6 glioma cells,18 19 connexin-43 is expressed in the corneal endothelium of rat,26 27 rabbit,28 and human.29 Recently, connexin-43 has been shown to play an important role in wound repair of the corneal endothelium.30 In the present study we demonstrated that stimulation of the CNTF/CNTFR{alpha} signaling pathway in the corneal endothelium also upregulated connexin-43 expression and that, in the presence of rhCNTFR{alpha}, CE cells that lost endogenous CNTFR{alpha} during human donor cornea storage were more responsive to stimulation by CNTF.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Media
Media used were as follows: medium A—Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 20 mM HEPES; medium B—DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 0.292 mg/mL L-glutamine; complete medium B—medium B plus 5% fetal calf serum and fungizone (250 ng/mL amphotericin; Invitrogen, Grand Island, NY).

Human Donor Corneas
Viable human corneoscleral explants (human donor corneas) stored in medium (Optisol-GS; Bausch & Lomb Surgical, Irvine, CA) at 4°C were obtained from the Lions Eye Institute for Transplant and Research (Tampa, FL). These corneas were deemed not suitable for transplantation because of their less than optimal CE cell densities and the advanced ages of the donors. In addition, using the same procedure as that used by the eye bank, fresh human donor corneoscleral explants were retrieved from cadavers (within 30 hours of death) in the Anatomy Board of the State of Maryland (Baltimore, MD). Cadavers were deidentified and were not considered human subjects by the Human Research Protection Office of the University of Maryland School of Medicine.

CNTF and CNTF/CNTFR{alpha} Treatments of Human Donor Corneas
Paired human donor corneas were removed from the storage medium (Optisol-GS; Bausch & Lomb Surgical)-containing vials and incubated in 3.5 mL complete medium B in 35-mm Petri dishes for 24 hours at 37°C in 5% CO2-95% air. For testing the effect of CNTF on upregulating connexin-43 expression, complete medium B containing either no (right eye) or 0.83 nM rhCNTF (catalog no. 257-NT; R&D Systems, Minneapolis, MN; left eye) was used to treat the paired human donor corneas. For showing CNTFR{alpha} augmentation of the CNTF effect, complete medium B containing 0.83 nM CNTF (right eye) or 0.83 nM CNTF plus 8.3 nM rhCNTFR{alpha} (catalog no. 303-CR; R&D Systems; left eye) was used to treat paired human donor corneas.

RT-PCR and Semiquantitative RT-PCR
Total RNA was isolated from CE cells scraped from human corneas (RNA-Bee; Tel-Test, Friendswood, TX) and subjected to reverse transcription (RT) using a kit (RETROscript; catalog no. 1710; Ambion, Austin, TX). RT products were subjected to PCR using the primer set corresponding to the gene sequences of human connexin-43 (5'-CCTTCTTGCTGATCCAGTGGTAC-3' [forward] and 5'-ACCAAGGACACCACCAGCAT-3' [reverse]).31 Two negative controls were conducted: a reagent control in which the PCR reaction was conducted in the absence of the RT product and a PCR control in which RNA was not subjected to RT. PCR used RT products derived from 10% of isolated human RNA. Between an initial 94°C (2-minute) and a final 72°C (5-minute) treatment, 25 to 26 thermocycles (20 seconds at 94°C, 25 seconds at 54°C, and 40 seconds at 72°C) were conducted. The identity of the specific band of appropriate size (154 bp) revealed after agarose gel electrophoresis of the PCR product was proven by sequencing (Biopolymer Laboratory, University of Maryland) and was demonstrated to be identical with the sequence for human connexin-43. For quantifying mRNA levels, semiquantitative PCR was conducted in the presence of the18S primers and the 18S competomers (Universal 18S internal standards; catalog no. 1718; Ambion) in the ratio of either 2:8 or 3:7. RT-PCR products were electrophoresed (2% agarose gel), and the optical densities of bands were measured using a densitometer (NucleoVision; NucleoTech, San Carlos, CA).

CE Cell Extract
Corneal endothelium was scraped from the corneas using a razor blade and homogenized in a glass homogenizer in the solubilization buffer containing 50 mM HEPES, pH 7.2, 10% glycerol, 1.25% triton X-100, 300 mM NaCl, 0.2 mM EGTA, 0.2 mM MgSO4, 100 µM sodium orthovanadate, and protease inhibitor cocktail (one tablet of Complete Mini [Roche Diagnostics, Mannheim, Germany] per 10 mL buffer).

CE Membrane Preparation
CE cells scraped from the corneal endothelium with a razor blade were homogenized in a glass homogenizer in a buffer (10 mM Tris-HCl, pH 7.3 containing 5 mM MgCl2) at 4°C. The homogenate was centrifuged at 4°C for 10 minutes at 12,000g to remove unbroken cells and nuclei. Crude membrane fractions were prepared by centrifugation of the supernatants at 100,000g for 60 minutes. Final pellets were dispersed in 2x sample buffer for Western blot analysis.

Western Blot Analysis
Samples of CE cell extracts and membrane preparation were electrophoresed under reducing conditions using preformed Tris/glycine polyacrylamide gradient gels (NuPage; Novex, San Diego, CA) and electrophoretically transferred to nitrocellulose membranes. For the detection of CNTFR{alpha}, nitrocellulose membranes were immunostained with an affinity-purified goat anti-human CNTFR{alpha} primary antibody (catalog no. AF-303-NA; R&D Systems) and an anti-goat IgG-alkaline phosphatase conjugate secondary antibody (catalog no. 401512; Calbiochem, La Jolla, CA). CNTFR{alpha} on nitrocellulose membranes was detected by a chromogenic method using an alkaline phosphatase substrate solution made from tablets (Fast Red TR/Naphthol AS-MX; Sigma, St Louis, MO). For the detection of connexin-43 and actin (internal standard), chemiluminescence was used with affinity-purified rabbit anti-connexin-43 (catalog no. AB19012; Chemicon, Temecula CA) and mouse monoclonal anti-actin (catalog no. CP01; Ab-1; Calbiochem, La Jolla, CA) primary antibodies, horseradish peroxidase-linked anti-mouse and anti-rabbit IgG secondary antibody, and horseradish peroxidase substrate (ECL kit; Amersham Pharmacia, Piscataway, NJ).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Loss of CNTFR{alpha} in CE Cells in Human Donor Corneas Stored in Storage Medium
We previously demonstrated the presence of CNTFR{alpha} (53 kDa) in CE cells from each of the 12 fresh human corneas from 12 donors with postmortem times of less than 24 hours.23 In the present study, the status of CE cell CNTFR{alpha} in human donor corneas that had been in storage medium (Optisol-GS; Bausch & Lomb Surgical) for varying lengths of time was examined, with Western blot analysis and actin as the internal standard. Although persistently expressed in CE cells of stored corneas, CNTFR{alpha} levels in CE cells decreased as the time of storage increased (Fig. 1A) . Figure 1B shows the normalized CNTFR{alpha} (against the actin internal standard) levels of nine corneas of nine human donors. There was a general correlation between the level of CNTFR{alpha} and the storage time of the corneas (Fig. 1B) . However, there was no correlation between CNTFR{alpha} level and the age of the donor (data not shown). Furthermore, there was disintegration of the cell-cell adhesion in the corneal endothelial cell layer in corneas under long-term storage, as revealed by light microscopic examination of Alizarin red S (a dye that binds to the calcium-enriched cell-cell adhesion)-stained, flat-mounted corneas (data not shown).


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 1. Diminishing CNTFR{alpha} level in CE cells in human donor corneas during storage in medium. (A) CNTFR{alpha}- and actin-immunoreactive molecules in CE cells of corneas in storage for 6 and 13 days demonstrated in a Western blot. Each lane contained cell extract from a corneal endothelium of one cornea. (B) The level of normalized CNTFR{alpha} (against the actin internal standard) decreased in a days-of-preservation-dependent manner.

 
CNTF-Upregulated Connexin-43 mRNA in CE Cells in Human Donor Corneas
The presence of CE cell connexin-43 mRNA was established and was followed by the demonstration of CNTF upregulation of connexin-43 mRNA expression in CE cells with endogenous CNTFR{alpha}. Total RNA isolated from pooled CE cells from six corneas of three donors was subjected to RT-PCR using the connexin-43 primer set. Gel electrophoresis of RT-PCR products demonstrated a specific band of the appropriate size (154 bp) in the positive and not in the two negative controls (Fig. 2A) . Sequencing of the 154-bp band revealed it to be identical with the sequence for human connexin-43 (nucleotides 668–821; accession no. M65188). CNTF upregulation of connexin-43 mRNA expression was demonstrated by semiquantitative RT-PCR. As shown in Figure 2B , one pair of human corneas that had been stored in storage medium (Optisol-GS; Bausch & Lomb Surgical) for 11 days and used as a control (right eye) compared with CNTF (0.83 nM)-treated (left eye) demonstrated the effect of CNTF in increasing the connexin-43 mRNA level.


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 2. Connexin-43 mRNA and CNTF-upregulated connexin-43 mRNA in CE cells with endogenous CNTFR{alpha}. Agarose gel (2%) electrophoresis of RT-PCR product of connexin-43 mRNA isolated from pooled CE cells of three pairs of fresh human donor corneas (A) and that of semiquantitative RT-PCR products of connexin-43 and 18S (as the internal standard) mRNA isolated from paired human donor corneas in storage for 11 days in storage medium. (B) Paired human corneas treated as control versus CNTF (0.83 nM)-treated. +, RT products; NC1, reagent negative control (no RT products); NC2, PCR control (RNA not subjected to RT); M, DNA size marker; bp, base pair.

 
rhCNTFR{alpha} Augmentation of CNTF-Upregulated Connexin-43 mRNA Expression in CE Cells of Human Donor Corneas Stored in Storage Medium
We have hypothesized that the loss of CNTFR{alpha} from CE cells during extended storage of the human donor corneas in storage medium (Optisol-GS; Bausch & Lomb Surgical; Fig. 1 ) can be compensated by rhCNTFR{alpha}. Thus, rhCNTFR{alpha} was expected to augment the effect of CNTF in upregulating connexin-43 expression in CE cells in human donor corneas in long-term storage. As shown in Figure 3A , semiquantitative RT-PCR of connexin-43 mRNA demonstrated that CE cells in (CNTFR{alpha}+CNTF)-treated human donor corneas (storage time, 33 days) expressed higher connexin-43 mRNA levels than those in CNTF-treated paired corneas. In eight pairs of human donor corneas (storage time, 25 days and longer), the normalized connexin-43 (against the 18S internal standard) mRNA levels in the CE cells in CNTF-treated corneas and those in the (CNTFR{alpha}+CNTF)-treated paired corneas averaged (mean ± SEM) 0.26 ± 0.08 and 0.58 ± 0.21, respectively (P = 0.029; Fig. 3B ).


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3. rhCNTFR{alpha} (8.3 nM)-augmented effect of CNTF (0.83 nM) in upregulating the connexin-43 mRNA level in CE cells in human donor corneas in storage for 25 days and longer. Paired human donor corneas in storage for 25, 27, 28, 30, 33 (two pairs), 36, and 43 days were treated with CNTF (right eye) and (CNTFR{alpha}+CNTF) (left eye), followed by analysis of connexin-43 mRNA. (A) Agarose gel (2%) electrophoresis of RT-PCR products of CE cell connexin-43 and 18S (as the internal standard) mRNA. (B) Normalized connexin-43 cDNA (against the 18S internal standard) levels in CE cells of paired corneas treated with CNTF versus (CNTFR{alpha}+CNTF) of all eight pairs averaged (mean ± SEM) 0.26 ± 0.08 and 0.58 ± 0.21, respectively (P = 0.029).

 
CNTFR{alpha} augmentation of CNTF upregulated connexin-43 expression in CE cells, as confirmed at the protein level by Western blot analysis with the use of actin as the internal standard. Figures 4A and 4B demonstrated that in the CNTF (0.8 nM) treatment of one pair of human donor corneas in storage for 23 days, the presence of CNTFR{alpha} (8.3 nM) increased CNTF effectiveness to 168% that observed in the absence of CNTFR{alpha}.


Figure 4
View larger version (39K):
[in this window]
[in a new window]

  		FIGURE 4. rhCNTFR{alpha} augmentation and inhibition of CNTF-induced connexin-43 expression in CE cells of human donor corneas in long- and short-term storage, respectively. CE cells from paired human donor corneas in long-term storage for 23 days (A, B) and short-term storage for 9 (C, D) and 3 (E, F) days used as CNTF versus (CNTFR{alpha}+CNTF)-treated were analyzed by Western blot analysis for connexin-43 and actin (as internal standard). Normalized (against actin) connexin-43 levels in CE cells of the (CNTFR{alpha}+CNTF)-treated donor corneas in storage for 23, 9, and 3 days were 168%, 65%, and 68% of those in their respective paired corneas that were treated with CNTF alone. Lanes contained CE cell extract obtained from the whole cornea. Concentrations of CNTF and CNTFR{alpha} were 0.83 and 8.3 nM, respectively.

 
The possibility that rhCNTFR{alpha} competed with endogenous CNTFR{alpha} for binding with CNTF in CE cells that retained abundant endogenous CNTFR{alpha} was examined. In two pairs of human donor corneas in short-term storage for 9 and 3 days (therefore retaining endogenous CNTFR{alpha}), rhCNTFR{alpha} (8.3 nM) decreased the effectiveness of CNTF (0.83 nM) in upregulating connexin-43 protein levels to 65% and 68% those observed in paired corneas treated with CNTF alone, respectively (Figs. 4C 4D and 4E 4F) .

Increased CE Cell Membrane-Bound CNTFR{alpha} Found in Long-term Stored Human Donor Corneas after Short-term Incubation with rhCNTFR{alpha}
Increased CE cell membrane CNTFR{alpha} levels resulting from short-term (90-minute) incubation with rhNTFR{alpha} was demonstrated in human donor corneas in storage medium (Optisol-GS; Bausch & Lomb Surgical) for 40 days and longer. As shown in Figure 5 , the membrane fractions isolated from paired human donor corneas in storage medium (Optisol-GS; Bausch & Lomb Surgical) for 40 (two pairs), 41 (three pairs), and 42 (two pairs) days before treatment with CNTF (right eye) versus (CNTFR{alpha}+CNTF) (left eye) were subjected to Western blot analysis for CNTFR{alpha}. Results demonstrated a higher CNTFR{alpha} level in CE cell membrane fractions from (CNTFR{alpha}+CNTF)-treated corneas than that from CNTF-treated paired corneas (Figs. 5A 5B) . The normalized CNTFR{alpha} (against a 71-kDa molecule revealed by Ponceau stain as the most abundant protein molecule in the membrane fraction) levels in the CE cells of all 14 corneas demonstrated that the presence of CNTFR{alpha} in the CNTF-incubation increased the membrane-bound CNTFR{alpha} levels from (mean ± SEM) 1.80 ± 0.12 to 5.75 ± 1.00 (P = 0.001; Fig. 5B ).


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 5. Short-term (90-minute) incubation with rhCNTFR{alpha} increased the level of membrane-bound CNTFR{alpha} in CE cells in human donor corneas in storage for 40 days and longer. (A) Western blot analysis and Ponceau staining of electrophoresed CE cell membrane proteins pooled from two pairs of human donor corneas (40 days in storage), each used as CNTF (right eye) versus (CNTFR{alpha}+CNTF) (left eye). (B) Normalized CNTFR{alpha} (against a 71-kDa molecule that was the most abundant protein revealed by Ponceau staining of the blots) levels in CE cells of 14 paired-human-donor corneas stored in medium for 40 (two pairs), 41 (three pairs), and 42 (two pairs) days were (mean ± SEM) 1.80 ± 0.12 and 5.75 ± 1.00 in CNTF- and (CNTFR{alpha}+CNTF)-treated corneas, respectively (P = 0.001).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
On binding of CNTF to the endogenous membrane-bound CNTFR{alpha}, the two β subunits (gp130 and LIFRβ) of the receptor dimerize, forming a trimeric receptor complex that activated the Janus family of tyrosine kinases Jak1/Jak2, leading to tyrosine phosphorylation, dimerization, and nuclear translocation of signal transducer and activator of transcription STAT3, which then bound to specific responsive elements in the promoter regions of the CNTF-responsive genes.32 33 CNTF-responsive genes included genes of vasoactive intestinal peptide34 35 and connexin-43.18 19 We have recently demonstrated CNTF induction of vasoactive intestinal peptide in CE cells of human donor corneas preserved in storage medium (Optisol-GS; Bausch & Lomb Surgical).23

In mature astrocytes, which do not express CNTFR{alpha}, CNTF and the recombinant CNTFR{alpha} in combination induce connexin-43 expression in a JAK/STAT pathway-dependent manner.20 Nevertheless, the mechanism of CNTF in combination with the recombinant CNTFR{alpha}-transducing signals through the JAK/STAT pathway has not been established. In the present study, CNTF and the rhCNTFR{alpha} might have formed a complex before modulating the two β subunits (gp130 and LIFRβ), similar to that of IL-6 and its soluble receptor (sIL-6R), forming a complex to stimulate cells that lack the endogenous membrane IL-6 receptor.36 In contrast, rhCNTFR{alpha} might have functioned as a surrogate to the membrane-bound CNTFR{alpha} and transduced the CNTF signal because (in the absence of CNTF) rhCNTFR{alpha} can bind to LIFRβ and to gp130 in vitro.37 38 Regardless of the mechanism, the insertion of rhCNTFR{alpha} in the membrane was involved because rhNTFR{alpha} became membrane bound after 90-minute incubation with the endogenous CNTFR{alpha}-depleted CE cells (Fig. 5) .

According to the specification and use manual provided by the manufacturer, the rhCNTFR{alpha} (324 amino acids) used in the present study was expressed in Sf21 insect cells and was glycosylated, resulting in a protein with an apparent molecular size of 52 kDa, which was similar to that of the endogenous CNTFR{alpha} (53 kDa) found in CE cells.23 The glycosylphosphatidyl inositol anchor of the endogenous CNTFR{alpha}14 is also the result of posttranslational modification adding the anchor to the protein.39 It is, therefore, likely that glycosylphosphatidyl inositol posttranslation modification also took place in the insect cell-produced rhCNTFR{alpha}, allowing it to be inserted in the cell membrane. This notion was in agreement with our finding that rhCNTFR{alpha} became enriched in the membrane fraction after only 90 minutes of incubation with endogenous CNTFR{alpha}-depleted CE cells (Fig. 5) . Although future studies will be necessary to determine whether insertion of the rhCNTFR{alpha} in the membrane requires the presence of CNTF, studies of other glycosylphosphatidyl inositol-anchored proteins indicate that these proteins by themselves can be exogenously incorporated onto the cell membrane.39 Transfer of glycosylphosphatidylinositol-anchored proteins (CD55 and CD59) contained in vesicles shed by normal erythrocytes in blood stored for transfusion to erythrocytes deficient in these proteins in patients have been demonstrated in vivo.40 It has also been demonstrated that CD59 in proteasomes, when transferred to CD59-deficient erythrocytes, retains its function and protects erythrocytes against complement-mediated hemolysis.41

In human donor corneas that had been stored for a short period (and that retained endogenous CNTFR{alpha}), the rhCNTFR{alpha}, while present in large excess over CNTF (10:1), actually decreased the effectiveness of CNTF (Figs. 4C 4D 4E 4F) , suggesting that rhCNTFR{alpha} competed with the endogenous CNTFR{alpha} for CNTF and that the complex of rhCNTFR{alpha}/CNTF was not as effective as the endogenous CNTFR{alpha}/CNTF complex in signal transduction. As a result, the endogenous CNTFR{alpha} level was inversely related to the effectiveness of rhCNTFR{alpha} in further increasing the CNTF-upregulated connexin-43 level.

Finally, the mechanism of CNTFR{alpha} loss during human donor corneal storage is unknown. In blood stored for transfusion, it has been demonstrated that levels of CD59 and another glycosylphosphatidylinositol-anchored, membrane-associated complement regulatory protein, the decay-accelerating factor (DAF), gradually decline during storage.42 Loss of glycosylphosphatidylinositol-anchored proteins is mediated by enrichment of these proteins in vesicles and the release of these vesicles from erythrocytes in stored blood.43 44

In conclusion, the present study demonstrated that rhCNTFR{alpha} can act as a surrogate to the lost endogenous membrane-bound CNTFR{alpha} in transducing the CNTF signal, suggesting an adjuvant role for the rhCNTFR{alpha} in CNTF therapy.


   Acknowledgements
 
The authors thank Dante Gloria for excellent technical support.


   Footnotes
 
Supported by National Institutes of Health Grant RO1EY-11607 and Research to Prevent Blindness, Inc.

Submitted for publication July 19, 2008; revised September 20, 2008; accepted February 17, 2009.

Disclosure: S.-W.M. Koh, None; J. Celeste, None; C.Y.P. Ku, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Shay-Whey M. Koh, Room 500C MSTF, 10 S. Pine Street, Baltimore, MD 21201; skoh{at}umaryland.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Adler R, Landa K, Manthorpe M, Varon S. Cholinergic neuronotrophic factors: intraocular distribution of trophic activity for ciliary neurons. Science. 1979;204:1434–1436.[Abstract/Free Full Text]
  2. Adler R, Varon S. Neuronal survival in intact ciliary ganglia in vivo and in vitro: CNTF as a target surrogate. Dev Biol. 1982;92:470–475.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  3. Sendtner A, Kreutzberg GW, Thoenen H. Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy. Nature. 1990;345:440–441.[CrossRef][Medline][Order article via Infotrieve]
  4. van Adel BA, Arnold JM, Phipps J, Doering LC, Ball AK. Ciliary neurotrophic factor protects retinal ganglion cells from axotomy-induced apoptosis via modulation of retinal glia in vivo. J Neurobiol. 2005;63:215–234.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  5. Wen R, Song Y, Kjellstrom S, et al. Regulation of rod phototransduction machinery by ciliary neurotrophic factor. J Neurosci. 2006;26:13523–13530.[Abstract/Free Full Text]
  6. Sieving PA, Caruso RC, Tao W, et al. Ciliary neurotrophic factor (CNTF) for human retinal degeneration: phase I trial of CNTF delivered by encapsulated cell intraocular implants. Proc Natl Acad Sci U S A. 2006;103:3896–3901.[Abstract/Free Full Text]
  7. Beltran WA, Zhang Q, Kijas JW, et al. Cloning, mapping, and retinal expression of the canine ciliary neurotrophic factor receptor alpha (CNTFR{alpha}). Invest Ophthalmol Vis Sci. 2003;44:3642–3649.[Abstract/Free Full Text]
  8. A phase II study of implants of encapsulated human NTC-201 cells releasing ciliary neurotrophic factor (CNTF) in participants with visual acuity impairment associated with atrophic macular degeneration. ;ClinicalTrials.gov Identifier: NCT00277134
  9. A study of an encapsulated cell technology (ECT) implant for patients with atrophic macular degeneration. ;ClinicalTrials.gov Identifier: NCT00447954
  10. A study of encapsulated cell technology (ECT) implant for participants with early stage retinitis pigmentosa. ;ClinicalTrials.gov Identifier: NCT00447980
  11. A study of encapsulated cell technology (ECT) implant for patients with late stage retinitis pigmentosa. ;ClinicalTrials.gov Identifier: NCT00447993
  12. Miotke JA, MacLennan AJ, Meyer RL. Immunohistochemical localization of CNTFR{alpha} in adult mouse retina and optic nerve following intraorbital nerve crush: evidence for the axonal loss of a trophic factor receptor after injury. J Comp Neurol. 2007;500:384–400.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  13. Sendtner M, Stockli KA, Thoenen H. Synthesis and localization of ciliary neurotrophic factor in sciatic nerve of the adult rat after lesion and during regeneration. J Cell Biol. 1992;118:1436–1453.
  14. Davis S, Aldrich TH, Valenzuela DM, et al. The receptor for ciliary neurotrophic factor. Science. 1991;253:59–63.[Abstract/Free Full Text]
  15. Koh SW. Ciliary neurotrophic factor released by corneal endothelium surviving oxidative stress ex vivo. Invest Ophthalmol Vis Sci. 2002;43:2887–2896.[Abstract/Free Full Text]
  16. Davis S, Aldrich TH, Ip NY, et al. Released form of CNTF receptor alpha component as a soluble mediator of CNTF responses. Science. 1993;259:1736–1739.[Abstract/Free Full Text]
  17. Weis J, Lie DC, Ragoss U, et al. Increased expression of CNTF receptor alpha in denervated human skeletal muscle. J Neuropathol Exp Neurol. 1998;57:850–857.[Web of Science][Medline][Order article via Infotrieve]
  18. Panayotatos N, Everdeen D, Liten A, Somogyi R, Acheson A. Recombinant human CNTF receptor alpha: production, binding stoichiometry, and characterization of its activity as a diffusible factor. Biochemistry. 1994;33:5813–5818.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  19. Ozog MA, Bechberger JF, Naus CC. Ciliary neurotrophic factor (CNTF) in combination with its soluble receptor (CNTFR{alpha}) increases connexin-43 expression and suppresses growth of C6 glioma cells. Cancer Res. 2002;62:3544–3548.[Abstract/Free Full Text]
  20. Ozog MA, Bernier SM, Bates DC, Chatterjee B, Lo CW, Naus CC. The complex of ciliary neurotrophic factor-ciliary neurotrophic factor receptor {alpha} upregulates connexin-43 and intercellular coupling in astrocytes via the Janus tyrosine kinase/signal transducer and activator of transcription pathway. Mol Biol Cell. 2004;15:4761–4774.[Abstract/Free Full Text]
  21. Johnston MC, Noden DM, Hazelton RD, Coulombre JL, Coulombre AJ. Origins of avian ocular and periocular tissues. Exp Eye Res. 1979;29:27–43.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  22. Hayashi K, Sueishi K, Tanaka K, et al. Immunohistochemical evidence of the origin of human corneal endothelial cells and keratocytes. Graefes Arch Clin Exp Ophthalmol. 1986;224:452–456.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  23. Koh SWM, Guo Y, Bernstein SL, Waschek JA, Liu X, Symes AJ. Vasoactive intestinal peptide induction by ciliary neurotrophic factor in donor human corneal endothelium in situ. Neurosci Lett. 2007;423:89–94.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  24. Koh SW, Waschek JA. Corneal endothelial cell survival in organ cultures under acute oxidative stress: effect of VIP. Invest Ophthalmol Vis Sci. 2000;41:4085–4092.[Abstract/Free Full Text]
  25. Koh SWM, Chandrasekara K, Abbondandolo CJ, Coll TJ, Rutzen AR. VIP and VIP gene silencing modulate differentiation marker N-cadherin and cell shape of corneal endothelium in human corneas ex vivo. Invest Ophthalmol Vis Sci. 2008;49:3491–3498.[Abstract/Free Full Text]
  26. Joyce NC, Harris DL, Zieske JD. Mitotic inhibition of corneal endothelium in neonatal rats. Invest Ophthalmol Vis Sci. 1998;39:2572–2583.[Abstract/Free Full Text]
  27. Laux-Fenton WT, Donaldson PJ, Kistler J, Green CR. Connexin expression patterns in the rat cornea: molecular evidence for communication compartments. Cornea. 2003;22:457–464.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  28. Williams KK, Watsky MA. Bicarbonate promotes dye coupling in the epithelium and endothelium of the rabbit cornea. Curr Eye Res. 2004;28:109–120.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  29. Williams K, Watsky M. Gap junctional communication in the human corneal endothelium and epithelium. Curr Eye Res. 2002;25:29–36.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  30. Nakano Y, Oyamada M, Dai P, Nakagami T, Kinoshita S, Takamatsu T. Connexin-43 knockdown accelerates wound healing but inhibits mesenchymal transition after corneal endothelial injury in vivo. Invest Ophthalmol Vis Sci. 2008;49:93–104.[Abstract/Free Full Text]
  31. Chen Z, Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li DQ. Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cells. 2004;22:355–366.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  32. Ip NY, Yancopoulos GD. The neurotrophins and CNTF: two families of collaborative neurotrophic factors. Annu Rev Neurosci. 1996;19:491–515.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  33. Segal RA, Greenberg ME. Intracellular signaling pathways activated by neurotrophic factors. Annu Rev Neurosci. 1996;19:463–489.[Web of Science][Medline][Order article via Infotrieve]
  34. Symes A, Rao MS, Lewis SE, Landis SC, Hyman SE, Fink JS. Ciliary neurotrophic factor coordinately activates transcription of neuropeptide genes in a neuroblastoma cell line. Proc Natl Acad Sci U S A. 1993;90:572–576.[Abstract/Free Full Text]
  35. Symes A, Lewis S, Corpus L, Rajan P, Hyman SE, Fink JS. STAT proteins participate in the regulation of the vasoactive intestinal peptide gene by the ciliary neurotrophic factor family of cytokines. Mol Endocrinol. 1994;8:1750–1763.[Abstract/Free Full Text]
  36. Rose-John S, Waetzig GH, Scheller J, Grötzinger J, Seegert D. The IL-6/sIL-6R complex as a novel target for therapeutic approaches. Exp Opin Ther Targets. 2007;11:613–624.[CrossRef]
  37. He W, Gong K, Zhu G, Smith DK, Ip NY. Membrane distal cytokine binding domain of LIFR interacts with soluble CNTFR in vitro. FEBS Lett. 2002;514:214–218.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  38. Man D, He W, Sze KH, et al. Solution structure of the C-terminal domain of the ciliary neurotrophic factor (CNTF) receptor and ligand free associations among components of the CNTF receptor complex. J Biol Chem. 2003;278:23285–23294.[Abstract/Free Full Text]
  39. Paulick MG, Bertozzi DR. The glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins. Biochemistry. 2008;47:6991–7000.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  40. Sloand EM, Mainwaring L, Keyvanfar K, et al. Transfer of glycosylphosphatidylinositol-anchored proteins to deficient cells after erythrocyte transfusion in paroxysmal nocturnal hemoglobinuria. Blood. 2004;104:3782–3788.[Abstract/Free Full Text]
  41. Babiker AA, Ronquist G, Nilsson UR, Nilsson B. Transfer of prostasomal CD59 to CD59-deficient red blood cells results in protection against complement-mediated hemolysis. Am J Reprod Immunol. 2002;47:183–192.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  42. Long KE, Yomtovian R, Kida M, Knez JJ, Medof ME. Time-dependent loss of surface complement regulatory activity during storage of donor blood. Transfusion. 1993;33:294–300.[CrossRef][Web of Science][Medline][Order article via Infotrieve]
  43. Bütikofer P, Kuypers FA, Xu CM, Chiu DT, Lubin B. Enrichment of two glycosyl-phosphatidylinositol-anchored proteins, acetylcholinesterase and decay accelerating factor, in vesicles released from human red blood cells. Blood. 1989;74:1481–1485.[Abstract/Free Full Text]
  44. Kriebardis AG, Antonelou MH, Stamoulis KE, Economou-Petersen E, Margaritis LH, Papassideri IS. RBC-derived vesicles during storage: ultrastructure, protein composition, oxidation, and signaling components. Transfusion. 2008;48:1943–1953.[CrossRef][Web of Science][Medline][Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
iovs.08-2590v1
50/4/1801    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koh, S.-W. M.
Right arrow Articles by Ku, C. Y. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koh, S.-W. M.
Right arrow Articles by Ku, C. Y. P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS