Download Development of characterization of a immortal and

Development and Characterization of an Immortal and
Differentiated Murine Trabecular Meshwork Cell Line
Ernst R. Tamm,1'2 Paul Russell? and Joram Piatigorsky1
PURPOSE. TO
study mouse trabecular meshwork (TM) and to develop a murine TM cell line.
Mouse TM in situ was studied by light and electron microscopy (EM). In addition, TM was
isolated from the H-2Kb-tsA58 transgenic mouse strain in which promoter sequences of the major
histocompatibility complex H-2K0 class 1 gene are fused to sequences of the SV40 mutant
temperature-sensitive (ts) strain tsA58. The promoter is inducible by interferon (IFN)-y, and the
tsA58 gene product is active at 33°C (permissive conditions), but not at 37°C (nonpermissive
conditions). The TM explant was cultured in permissive conditions. Outgrowing cells were
passaged through two rounds of single-cell cloning. One clonal cell line (MUTM-NEI/1) was
characterized in nonpermissive conditions by EM, immunohistochemistry, reverse transcriptionpolymerase chain reaction (RT-PCR), and northern blot hybridization. In addition, MUTM-NEI/1
cells were transfected with plasmid DNA.
METHODS.
The mouse eye has a circumferentially oriented outflow vessel and a TM that is subdivided
in an outer juxtacanalicular or cribriform part and an inner lamellated or trabecular part. From the
TM of the H-2Kb-tsA58 mouse, a clonal cell line (MUTM-NEI/1) was established. In permissive
conditions, MUTM-NEI/1 cells remained proliferative through at least 80 generations without
change in phenotype. In nonpermissive conditions, proliferation was slower, and MUTM-NEI/1
cells differentiated and synthesized collagen types I, III, IV, and VI; laminin; and fibronectin.
MUTM-NEI/1 cells were immunoreactive for vimentin, aB-crystallin, and neural cell adhesion
molecule (NCAM), but not for desmin or cytokeratin. Less than 10% of MUTM-NEI/1 cells stained
for osmooth muscle actin, whereas after 3 days of treatment with transforming growth factor-/3,
almost all cells were positive. MUTM-NEI/1 cells expressed mRNA for NCAM, aquaporin 1,
myocilin/trabecular meshwork glucocorticoid-inducible protein, and aB-crystallin, which was
increased after oxidative stress. MUTM-NEI/1 cells could be successfully transfected with plasmid DNA.
RESULTS.
CONCLUSIONS. The architecture of the murine outflow system is comparable to that in primates. The
MUTM-NEI/1 cell line is a clonal, immortal, and differentiated TM cell line that will be an important
tool for study of the expression of TM genes. (Invest Ophthalmol Vis Sci. 1999;4O:1392-l4O3)
P
rimary open-angle glaucoma (POAG) is commonly associated with intraocular pressure GOP) that is too high
for the health of the optic nerve head. The reason for
elevated IOP in POAG is an increase in outflow resistance in
the trabecular meshwork (TM), the major site of aqueous
humor outflow.1 A broad variety of structural changes have
been described in the TM of glaucomatous eyes. Still, the major
From the 'Laboratory of Molecular and Developmental Biology
and the 'Laboratory of Mechanisms of Ocular Diseases, National Eye
Institute, National Institutes of Health, Bethesda, Maryland.
Supported by Grants Ta 115/8-1 and Ta 115/11-1 from the Deutsche Forschungsgemeinschaft, Bonn, Germany; and the American
Health Assistance Foundation, Rockville, Maryland (all to ERT).
Presented in part at the annual meeting of the Association for
Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May
1997.
Submitted for publication August 20, 1998; revised January 22,
1999; accepted February 16, 1999.
Proprietary interest category: N.
2
Present address: Department of Anatomy II, University of Erlangen-Niirnberg, Universitatsstrasse 19, D-91054 Erlangen, Germany.
Reprint requests: Ernst R. Tamm, Department of Anatomy II,
University of Erlangen-Niirnberg, Universitiitsstrasse 19, D-91054 Erlangen, Germany.
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reason for the increase in trabecular resistance is unknown.2 A
possible candidate in some cases of POAG is myocilin/trabecular meshwork-induced glucocorticoid response protein
(MYOC/TIGR) that is produced in large amounts by cultured
human TM cells after long-term treatment with dexamethasone.3 Mutations in the coding sequences of MYOC/TIGR have
been described in patients with juvenile glaucoma, an autosomal dominant hereditary form of POAG, and in a small percentage of patients with nonhereditary late-onset POAG.4"6 Until
now, other TM genes that are causatively involved in the
pathogenesis of POAG have not been isolated.
A major obstacle for die identification and the characterization of such genes has been the lack of a suitable animal
model; species with a disease similar to POAG in humans have
not been identified.7 A possibly appropriate animal model is
one using the transgenic or gene knockout technique and the
development of animals that express modified forms of candidate genes or that are deficient in such genes. Because such
studies are usually performed in mice, we were interested in
whether the structure and the cell biology of the murine TM is
comparable to that in humans. Although some aspects of the
biology of the murine TM have been studied,89 a detailed
analysis of its morphology is lacking.
Investigative Ophthalmology & Visual Science, June 1999, Vol. 40, No. 7
Copyright © Association for Research in Vision and Ophthalmology
IOVS, June 1999, Vol. 40, No. 7
In the present study, we investigated the structure of the
murine outflow tissues by light microscopy and EM. In addition, to have an experimental tool for studies on gene expression in the TM, we developed and characterized a clonal,
immortal, and differentiated murine TM cell line (MUTM-NEI/
1). TM was isolated from the H-2Kb-tsA58 transgenic mouse
strain in which the 5'-flanking sequences of the mouse major
histocompatibility complex H-2Kb class 1 gene are fused to the
early-region coding sequences of the simian virus 40 (SV 40)
mutant temperature-sensitive (ts) strain tsA58, which encodes
a thermolabile large tumor (T) antigen.10 The promoter is
inducible by interferon (TFN)-y, and the tsA58 gene product is
active at 33°C (permissive conditions) but not at 37°C (nonpermissive conditions). Our results indicate that the murine
TM shared essential structural elements with the TM of primates and that MUTM-NEI/1 cells, when grown in nonpermissive conditions express typical characteristics of cultured human primary TM cells.
MATERIALS AND METHODS
EM of Mouse TM
Normal FVB/N and C57BL/6J mice (age, 2-4 months) were
killed by cervical dislocation. The eyes were enucleated and
fixed overnight in a mixture of 2% glutaraldehyde, 4% paraformaldehyde, and 20% sucrose in cacodylate buffer (pH 7.4).
After fixation, the eyes were dehydrated through graded alcohols and embedded in JB-4 glycol methacrylate (Piano, Wetzlar,
Germany) or in Epon (Roth, Karlsruhe, Germany). Meridional
semithin sections were cut through the pupillary- optic nerve
plane on a microtome and stained with hematoxylin and eosin
or with Richardson's stain.11 Ultrathin sections were treated
with lead citrate and uranyl acetate and viewed using an electron microscope (model 902; Zeiss, Oberkochen, Germany).
Cell Culture of Mouse TM
H-2Kb-tsA48 heterozygote mice (genetic background CBA/
ca X C57B1/10) were obtained from Charles River (Wilmington, MA) and mated to FVB/N mice. Tail DNA from the resultant hybrid mice was isolated and assayed for the presence of
the transgene using sense and antisense primers (5-CCATCTCCACAGTTTCACTTCTGCA-3' and 5'-GCAGTACATTGCATCAACACCAGGA-3') and the following reaction conditions: denaturation at 94°C for 90 seconds, followed by 35 cycles of 94°C
for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, and a
final extension step at 72°C for 5 minutes. Mice harboring the
transgene were killed at 2 months of age by cervical dislocation, and the eyes were enucleated. Methods for securing
animal tissue were humane, included proper consent and approval, and complied with the National Institutes of Health
Guidelines on the Care and Use of Animals in Research and the
ARVO Statement for the Use of Animals in Ophthalmic and
Vision Research. The eyes were bisected along the equator and
the posterior half removed. The anterior half was placed on a
sterile petri dish, corneal side down. Under a dissecting microscope (X40), the zonule was cut with fine scissors and the lens
removed. One branch of a fine forceps was inserted in the
suprachoroidal space of the specimen. The ciliary body was
grasped and gently pulled away from the sclera while its
attachments to the sclera were simultaneously cut with a fine
knife. After removing of the ciliary body and the attached iris,
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Murine Trabecular Meshwork Cell Line
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the outflow tissue could be easily distinguished against the
white background of the sclera as a pigmented circumferentially oriented band. This band was cut free, placed in a 35-mm
sterile, laminin-coated plastic petri dish, and covered with a
coverslip and 1 ml Dulbecco's modified Eagle's medium
(DMEM). The medium, which was changed every second day,
was supplemented with 20% fetal bovine serum (FBS), 20
/Ag/ml gentamicin (all Life Technologies, Gaithersburg, MD)
and recombinant murine IFN-y (Sigma, St. Louis, MO) at a final
concentration of 20 U/ml. Coating was performed by adding
laminin from basement membrane of Engelbreth-HolmSwarm sarcoma (Sigma) to the culture medium at a final concentration of 10 /ng/ml. Cultures were incubated at 33°C in
humidified air enriched with 10% CO2. Confluent cultures
were trypsinized with 0.25% trypsin for a few minutes and
transferred to uncoated culture flasks at split ratios of 1:4 to
1:10. For experimental assays, confluent cultures grown at
33°C (permissive conditions) were transferred to 37°C in the
absence of IFN-y (nonpermissive conditions).
For cell cloning, 96-well microtiter plates were used. Into
the wells, 0.2 ml of a cell suspension was added, so that only
approximately one in three wells received a viable cell. When
colonies were seen in phase-contrast optics, they were examined to ensure only single colonies were present. When these
single colonies grew to a sufficient size, they were trypsinized
and transferred to larger culture vessels. Certain of these cell
clones were selected because they showed a cellular phenotype comparable to that of human primary TM cell cultures.
These cell clones were again cloned using the same procedure.
For assessing growth rates, 2 X 104 cells were plated in
96-well microtiter plates (6 wells for each time point) and
incubated at either 33°C or 37°C. At each time point (1 hour,
23 hours, 62 hours, and 73 hours), the number of viable cells
in each well was determined using the protocol of Brasaemle
and Attie.12 The number of viable cells at each time point
(mean ± SD) was plotted against time.
For EM, cells were grown in uncoated plastic petri dishes,
fixed in 4% glutaraldehyde, postfixed in 1% osmium tetroxide,
and embedded in epon. Polymerization was performed at
60°C.
For treatment with transforming growth factor-/3, (TGFj3,), confluent cultures were treated for 3 days with 1 ng/ml
human recombinant TGF-/3, (Sigma) which was added to
DMEM supplemented with 20% FBS and 20 jixg/ml gentamicin
(37°C).
Immunohistochemistry
Cells were grown to confluence at 33°C in tissue culture
chambers mounted on microslides (Permanox; Laboratory Tek;
Nunc, Naperville, IL) and transferred to 37°C for 5 to 7 clays.
For the detection of collagen types I, III, IV, and VI; laminin;
fibronectin; and neural cell adhesion molecule (NCAM), the
cells were fixed with a mixture of ether-ethanol (1:1, 10 minutes at — 20°C), for vimentin, desmin, and cytokeratins; with
methanol (5 minutes at — 20°C); and with 4% phosphate-buffered saline (PBS)-paraformaldehyde for aB-crystallin (room
temperature for 20 minutes). After fixation, the slides were
washed three times (5 minutes each) in PBS and preincubated
with SuperBlock (Pierce, Rockford, IL, 30 minutes). After preincubation, the slides were incubated overnight at room temperature with the primary antibodies listed in Table 1. After
three washings in PBS, biotinylated secondary antibodies
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Tamm et al.
TABLE
IOVS, June 1999, Vol. 40, No. 7
1. Antisera Used for Immunocytochemistry
Source
Host Species
Dilution
Chemicon (Temecula, CA)
Research Diagnostics (Flanders, NJ)
Research Diagnostics
Dako (Carpinteria, CA)
Sigma (St. Louis, MO)
Sigma
Boehringer Mannheim (Mannheim, Germany)
Sigma
Sigma
Sigma
J. Horwitz (UCLA, CA)
Chemicon
Rabbit
Rabbit
Rabbit
Rabbit
Rabbit
Mouse
Mouse
Mouse
Mouse
Mouse
Rabbit
1:50
1:50
1:50
1:50
1:30
1:400
1:10
1:20
1:100
1:200
1:1000
1:100
Primary Antisera
Collagen type I
Collagen types III, IV, VI
Laminin
Fibronectin (cellular)
Vimentin
Desmin
Pan-cytokeratin
a-Smooth muscle actin
aB-Crystallin
Neural cell adhesion molecule
Rat
Rabbit antibodies were polyclonal; mouse or rat antibodies were monoclonal.
against rabbit, rat, or mouse (depending on the origin of the
primary antibody) immunoglobulins (Vector, Burlingame, CA)
were added for 1 hour. The slides were washed again, covered
with streptavidin-fluorescein isothiocyanate (Vector) for 1
hour, mounted in fluorescent mounting medium (Dako,
Carpinteria, CA), and viewed with a microscope (Zeiss). Control experiments were performed using either PBS or preimmune serum instead of the primary antibody.
RNA Analysis
Total RNA was isolated from cultured MUTM-NEI/1 cells
using RNAzol (Tel Test, Friendswood, TX). For reverse transcription-polymerase chain reaction (RT-PCR), a one-step
RT-PCR kit (PCR-Superscript, Life Technologies) was used
according to the manufacturer's protocol. The RT-PCR reaction was performed in a total volume of 50 /xl for 30 minutes
at 44°C (the RT step), followed by melting at 94°C for 2
minutes, then 30 cycles of 50 seconds' melting at 94°C, 75
seconds' annealing at 55°C, and 2 minutes' extension at
72°C. After the last cycle, the polymerization step was extended for 10 minutes so that all strands were completed.
The primers were designed according to the published
structures of the mouse genes encoding for aquaporin-1/
CHIP28,13 NCAM,14 myoc/tigr 1516 and glyceraldehyde-3phosphate dehydrogenase (G3PDH)17 and were added to a
final concentration of 0.2 micromole. The sequences of the
primer pairs were 5'-TGGAGGAGTGAAAGTGAG-3' and 5'CAAACACACACACACCAG-3' (position 1113-1793; product
size 680 bp) for aquaporin-l/CHIP28, 5'-ACTCCTCTACCCTCACCATC-3' and 5'-GCCTCGTCGTTTTTATCC-3' (position
391-1012, product size 621 bp) for NCAM, 5'-AGCCTATAACAATCTCCTTC-3' and 5'-TCTCATCCACACTCCATAC-3'
(position 472-869, product size 397 bp) for myoc/tigr, and
5'-CCCTTCATTGACCTCAAC-3' and 5'-TTCACACCCATCACAAAC-3' (position 146-447, product size 301 bp) for
G3PDH, respectively. Control experiments were performed
for each primer pair and RNA sample by omitting the RT
step (30 minutes at 44°C). The primer sequences for myoc/
tigr crossed exon-intron boundaries. The RT-PCR amplification products were gel purified and sequenced with fluorescent dideoxynucleotides on an automated sequencer
(Applied Biosystems model 310; Perkin Elmer, Hayward, CA).
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For northern blot experiments, RNA was separated on a
2.2-M formaldehyde-1.2% agarose gel and blotted onto a
membrane (Duralon; Stratagene, La Jolla, CA). After the
transfer, the blot was cross linked (UV Stratalinker; Stratagene). Blots were hybridized with a 244-bp BamHl-Hindlll
restriction fragment isolated from the mouse aB-crystallin
gene (+2849 to +3092) containing much of exon III.18 The
probe was labeled with 32P-dCTP using a random prime kit
(Life Technologies). Prehybridizations were performed at
55°C for 1 hour and hybridizations at 55°C overnight using
Hybrisol (Oncor, Gaithersburg, MD), according to the manufacturer's instructions (including 10% formamide in the
hybridization solution). Membranes were washed twice for
15 minutes each with 2X SSC-0.1% SDS at 55°C and twice
with 0.2X SSC-0.1% SDS at room temperature and autoradiographed (XAR5 film; Eastman Kodak; Rochester, NY) at
— 80°C with an intensifying screen (1-2 days). To monitor
the integrity of RNA, the relative amounts of RNA loaded on
the gel, and the efficiency of transfer, membranes were
hybridized to a cDNA probe for guinea pig 18S rRNA. mRNA
size was estimated by reference to the mobility of RNA size
markers (Life Technologies) stained with methylene blue.
For oxidative stress experiments, confluent cultures were
washed three times with serum-free medium and subsequently
incubated with serum-free medium containing 200 (xM H2O2
for 1 hour. After this time the medium was changed and regular
culture medium was added. Control cultures were incubated
for 1 hour in serum-free medium without H2O2.
Transfection Experiments
Cells grown at 33°C were transfected with 2 fxg to 6 /xg
pCMV/3 plasmid (Clontech) using lipofectAMINE (Life Technologies) according to the manufacturer's protocol. The cells
were transferred to 37°C, and after 48 hours, cellular extract
was prepared and a j3-galactosidase assay performed as described previously.19'20
RESULTS
Mouse TM In Situ
In all sections examined, a circumferential outflow vessel,
equivalent to Schlemm's canal in primates, was visible (Fig.
IOVS, June 1999, Vol. 40, No. 7
Murine Trabecular Meshwork Cell Line
1395
FIGURE 1. Morphology of mouse TM in situ. (A) Light microscopy shows that a circumferential outflow vessel (asterisk), equivalent to Schlemm's
canal in primates, is present in the chamber angle. The vessel is covered by a thin layer of TM (arrows; semithin section, hematoxylin-eosin stain).
(B). Electron microscopy of mouse TM consists of two to four trabecular lamellae that are covered by a single layer of flat endothelial-like cells
(arrows'). Between Schlemm's canal (asterisk) and the trabecular lamellae, there is a 2- to-5-jnm thick layer of loose connective tissue equivalent
to the juxtacanalicular or cribriform meshwork in primate eyes (arrowheads). (C, D) Electron microscopy of the cribriform meshwork. Flat
polygonal tnibecular cells (arrows) are embedded in afinefilamentousmatrix (arrowheads). The inner wall of the endothelium (E) of Schlemm's
canal (asterisk) expresses distinct giant vacuoles (V). Scale bar, (A) 28 /urn; <B) 3 3 /xm; (C, D) 830 nm. CB, ciliary body; S, sclera.
1A). This vessel was covered by a thin layer of TM. Electron
microscopy showed that the meshwork consisted of two to
four trabecular lamellae (Fig. IB). Each lamella had a core of
collagenous fibrils with the typical 64-nm periodicity and was
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covered by a single layer of flat endothelial-like cells that rested
on a basal lamina. The inner wall of the endothelium of
Schlemm's canal expressed distinct giant vacuoles (Fig. 1C).
Between the inner wall endothelium and the trabecular lamel-
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Tamm et al.
IOVS, June 1999, Vol. 40, No. 7
FIGURE 2. Mouse TM cell culture, phase-contrast optics. (A) Outgrowth of cells from a TM explant (arrow) derived from the H-2Kb-tsA58
transgenic mouse. In permissive conditions (IFN-y, 33°C) MUTM-NEI/1 cells have a characteristic endothelial cell-like phenotype (B) and form a
cobblestone-like cell layer at confluence (C). (D) In nonperniissive conditions (no IFN-y, 37°C), MUTM-NEI/1 cells change their phenotype. They
predominantly arrange themselves in parallel, slightly separated from each other, and become more spindle shaped with long, tapering fusiform
ends (arrows). Magnification, X100,
lae, there was a 2- to 5-jLtm-thick layer of loose connective
tissue containing flat polygonal cells with small cytoplasmic
processes, which were embedded in afinefibrillar matrix (Fig.
ID), [n C57BL/6J mice, the cells of the TM contained melanin
granules.
Mouse TM Cell Culture: MUTM-NEI/1 Cells
Initial outgrowth of cells was observed 5 days after placing
TM from the H-2Kb-tsA58 transgenic mouse as an explant in
culture. The cells grew from all sides of the explant (Fig.
2A). Confluence was reached 14 days after the initial outgrowth. After subculturing the initial culture at a split ratio
of 1:4, confluence was again reached after 48 hours. At this
stage, single cell cloning was performed as described in the
Material and Methods section and repeated two times. The
resultant clonal cell lines were screened in phase-contrast
optics for the typical growth pattern of cultured TM cells
and one (MUTM-NEI/1) was selected for further characterization. In permissive conditions (IFN-y, 33°Q MUTM-NEI/1
cells had a characteristic endothelial cell-like phenotype
(Fig. 2B) and formed a cobblestone-like cell layer at confluence (Fig. 2C). Shortly after reaching confluence, the cells
overgrew each other and formed an irregular multilayer.
When confluent cultures were transferred to nonpermissive
conditions (no IFN-y, 37°C), MUTM-NEI/1 cells changed
their phenotype after 24 hovirs. They predominantly ar-
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ranged themselves in parallel, slightly separated from each
other, and became more spindle shaped with long, tapering
fusiform ends (Fig. 2D). In addition, cell growth was markedly slower than in permissive conditions (Fig. 3). Whereas
the doubling time was at 23-3 hours when cells were grown
in permissive conditions, it was at 62.2 hours when cells
were transferred to nonpermissive conditions.
EM
In nonpermissive conditions, the perinuclear cytoplasm of
MUTM-NEI/1 cells contained numerous elongated mitochondria and aggregates of rough endoplasmic reticulum that were
often dilated and filled with amorphous material (Fig. 4A, 4B).
Many of the cells contained lysosomes filled with electrondense material (Fig. 4A). Near the periphery, accumulations of
glycogen, lipid droplets, and numerous membrane-associated
vesicles or caveolae were seen. MUTM-NEI/1 cells synthesized
a matrix of extracellular fine fibrils that were aggregated into
loose bundles. The fibrils measured 5 ani to 20 nm in cross
sections and showed no apparent evidence of periodicity. Most
of the extracellular matrix was present near the elongated ends
of the cells, where the fibrils came in close contact with the
cellular membrane. In areas of contact, the cell membrane
formed dense bands with intracellular, 5- to 6-nm-thick (actin)
filaments attached to it (Fig. 4C).
IOVS, June 1999, Vol. 40, No. 7
Murine Trabecular Meshwork Cell Line 1397
4
60
80
FIGURE 3- Growth of MUTM-NEI/1 cells. For assessing growth rates,
2 X 104 cells were plated in 96-well microtiter plates (6 wells for each
time point) and incubated in either permissive (IFN-y, 33°Q or nonpermissive conditions (no IFN--y, 37°C). At each time point (1 hour, 23
hours, 52 hours, and 73 hours), the number of viable cells per well was
determined (mean ± SD) and plotted against time.
amplified PCR-product appeared to be the same, regardless of
whether mRNA from cells grown in permissive or nonpermissive conditions was used as a template. For aquaporin-1/
CHIP28, more PCR product could be amplified using mRNA
from cells grown in nonpermissive conditions than from those
grown in permissive conditions. For myoc/tigr, a PCR-product
was only detected when mRNA from cells grown in nonpermissive conditions was tested. In control experiments that
were performed simultaneously for each primer pair and RNA
sample by omitting the RT step (30 minutes at 44°C), no PCR
product could be amplified.
Northern blot analysis of RNA from MUTM-NEI/1 cells
grown in nonpermissive conditions showed a distinct band,
approximately 0.8 kb in length, after hybridization with an
aB-crystallin DNA probe (Fig. 8) Oxidative stress caused an
accumulation of aB-crystallin mRNA after 8 to 24 hours. In
addition, there was a faint band approximately 2.4 kb in length,
which probably consisted of preprocessed aB-crystallin
mRNA. This high-molecular-weight band was not significantly
affected after oxidative stress.
Transfection Experiments
Immunohistochemistry
Confluent MUTM-NEI/1 cultures showed positive immunostaining for collagen types I, III, IV, and VI when grown in
nonpermissive conditions. Labeling for types I and III collagen
was confined to relatively thick strands of extracellular matrix
that spread irregularly between the individual cells (Fig. 5A,
5B). In contrast, type IV and VI collagen stained as a dense
network of fine extracellular fibrils that covered the whole
culture dish (Fig. 5C, 5D). In addition, confluent cultures
stained for laminin and fibronectin (Fig. 5E, 5F). Antibodies
against laminin labeled both intracellular vesicles and extracellular fibrils, whereas staining for fibronectin was present in
extracellular fibrils only. No extracellular material was positively stained in MUTM-NEI/1 cultures grown in permissive
conditions (not shown).
MUTM-NEI/1 cells showed immunoreactivity for vimentin
(Fig. 6A) and aB-crystallin (Fig. 6B). Staining for vimentin
appeared to be equally intense in all cells. This was not the case
for aB-crystallin, were some cells were more intensely labeled
than others. No staining was observed using antibodies against
desmin or cytokeratins. In confluent cultures grown in nonpermissive conditions, most cells were negative when stained
for a-smooth muscle (sm) actin. Only occasionally, elongated
cells that usually grew on top of the others were positively
stained (Fig. 6C). In contrast, after treatment with TGF-/3, for 3
days, almost all cells expressed stress fibers that were immunoreactive for a-sm-actin (Fig. 6D). In addition, treatment with
TGF-/3, resulted in the MUTM-NEI/1 cells becoming flatter and
more polygonal. After staining with antibodies against NCAM,
the surface of the MUTM-NEI/1 cells showed intense immunoreactivity (Fig. 6E). NO positive immunostaining was observed
in control experiments (Fig. 6F).
RT-PCR and Northern Blot Analysis
Primers specific for aquaporin-l/CHIP28, NCAM, myoc/tigr,
and G3PDH amplified distinct PCR products with the expected
size (Fig. 7). The identity of each product was confirmed by
sequence analysis. For NCAM and G3PDH, the amount of the
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Forty-eight hours after transfection with plasmid DNA (CMV/3),
activity for the reporter gene j3-galactosidase could be readily
detected in cell extracts from MUTM-NEI/1 cells. The activity
was positively correlated with the amount of DNA transfected
(Fig. 9).
DISCUSSION
It is generally agreed on that only in higher primates including
humans has a true TM developed that consists of interlacing
connective tissue beams or lamellae that are covered by a
single layer of endothelial-like cells and that lines the wall of an
organized circumferential outflow vessel (Schlemm's canal). In
lower monkeys (prosimiae) and most other mammalian species
including those commonly used in eye research (e.g.,rabbitor
cow) the meshwork is not trabecular or lamellated but rather
resembles a loose connective tissue that is reticular in structure.21"25 In addition, these species do not have an outflow
vessel like Schlemm's canal, but rather small vascular loops
(the aqueous plexus) that protrude into the meshwork. Our
study shows that the tissues responsible for aqueous humor
outflow in mouse eyes have marked similarities in morphology
to that found in higher primates. There is a continuous circumferential outflow vessel equivalent to Schlemm's canal, which
is covered by a well-developed TM that consists of connective
tissue beams lined by a single layer of endothelial-like cells.
Other morphologic similarities to higher primates are the giant
vacuoles that are formed by the inner wall endothelium of the
mouse Schlemm's canal and the fact that the outermost parts of
the meshwork differ in structure from the trabecular inner
parts. These parts of the mouse meshwork do not form lamellae but consist of a loose layer of elongated polygonal cells that
are embedded in afinefibrillarmatrix, similar to the cribriform
or juxtacanalicular part of the meshwork in higher primates.
Based on all these morphologic similarities, we believe that the
molecular principles that govern aqueous flow through the TM
may be similar between the mouse eye and those of higher
primates.
IOVS, June 1999, Vol. 40, No. 7
FIGURE 4. EM of MUTM-NET/1 cells
grown in nonpermissive conditions
(no IFN-y, 37°Q. (A) The cytoplasm of
MUTM-NEI/1 cells is filled with numerous elongated mitochondria (black arrowheads) and aggregates of rough
endoplasmic reticulum (asterisks). In
addition, the cells contain lysosomes
filled with electron-dense material
(black arrows), lipid droplets (white
arrow), and numerous membrane-associated vesicles or caveolae (white
arrowhead). (B) Gsternae of rough
endoplasmic reticulum (asterisks) are
often dilated and filled with amorphous material. (C) Near die elongated ends of MUTM-NEI/1 cells, extracellular fibrils (arrows) come in
close contact widi die cellular membrane. In areas of contact, the cell
membrane forms dense bands with intracellular, 5- to 6-nm-diick (actin) filaments (arrowheads) attached to it.
Scale bar (A) 2.12 /am; (insert) 700
run; (B, C) 430 nm.
Primary cell cultures from mouse TM have been established in the past 26 and were reported to have similarities with
primary cultures established from the TM of other species,
(e.g., human, monkey, cow, and pig). 27 " 31 Regardless of the
species, TM cells are growth-arrested in situ, and the growth of
primary TM cell cultures is usually very limited. In addition,
primary TM cultures can contain different cell populations 32
and, if initiated from the eyes of different donors, may be
subject to interindividual differences. To have a reliable tool to
study TM cell and molecular biology, we were therefore interested in developing an immortal and clonal cell line from
mouse TM. Immortalized human TM cell lines have been ob-
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tained in the past by transformation with the large SV40 T
antigen. 33 Similar to other SV40-transformed cell lines, transformed human TM cell lines are not well differentiated and do
not express all characteristics of TM cells. 34 To avoid the
problem of dedifferentiation, we developed the mouse TM cell
line (MUTM-NEI/1) from the H-2Kb-tsA58 transgenic mouse
strain in which the 5'-flanking sequences of the mouse major
histocompatibility complex H-2Kb class 1 gene are fused to the
early-region coding sequences of the simian SV 40 mutant ts
strain tsA58, which encodes a thermolabile large T antigen.1"
The promoter is inducible by IFN-y, and the tsA58 gene product is active at 33°C (permissive conditions) but not at 37°C
IOVS, June 1999, Vol. 40, No. 7
Murine Trabecular Meshwork Cell Line
1399
FicURii 5. Immunohistochemistry of MUTM-NH/l cultures grown in nonpermissive conditions (no IFN--y, 37°C). The cultures show positive
labeling of fibrillar extracellular matrix when stained with antibodies against collagen type I (A), collagen type HI (B), collagen type IV (C), and
collagen type VI (D). In addition, the cultures stain for laminin (E) and fibroneclin (F). Antibodies against laminin label both intnicellular vesicles
and extracellular fibrils, whereas staining for fibronectin is present in extracellular fibrils only. Magnification, X275.
(nonpermissive conditions). It has been reported that cell lines
initiated from this mouse strain grow immortally and dedifferentiated in permissive conditions but differentiate back to the
original phenotype in nonpermissive conditions.""*0 Our results clearly indicate that the same is true for the MUTM-NEI/1
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cell line. Electron microscopy shows that MUTM-NEl/l cells
stay healthy when transferred to nonpermissive conditions,
expressing all morphologic signs of active protein biosynthesis
and synthesizing extracellular matrix. The synthesis of extracellular matrix compounds is regarded as a characteristic fea-
1400
Tamm et al.
IOVS, June 1999, Vol. 40, No. 7
FIGURK 6. Immunohistochemistry of MUTM-NET/1 cultures grown in nonpermissive conditions (no lFN-y, 37°C). MUTM-NEI/1 cells show
immunoreactivity for vimtntin (A) and aB-crystallin (B). Most cells were negative when stained for a-sm-actin. Only occasionally, elongated cells
that usually grow on top of the others were positively stained (C). In contrast, after treatment with TGF-fi, for 3 days, almost all cells expressed
stress fibers that were immunoreactive for a-sm-actin (D). After staining with antibodies against NCAM, the surface of the MUTM-NEI/1 cells
showed intense immunoreactivity (E). No positive immunostaining was observed in control experiments (F). Magnification X275.
ture of human TM cells that deposit, both in situ and in vitro,
collagen types I, III, and VT and fibronectin and the basement
membrane components, collagen type IV and laminin.41"48
Our findings show that all these extracellular matrix compo-
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nents are synthesized by MUTM-NEI/1 cells when grown in
nonpermissive conditions. This is in marked contrast to other
matrix-producing cell types (e.g., fibroblasts) that stop matrix
synthesis after SV40 transformation.49"52
Murine Trabecular Meshwork Cell Line
IOVS, June 1999, Vol. 40, No. 7
CHIP 28
33°
37°
NCAM
33°
TIGR
37°
33°
RT 0 RT 0 RT 0 RT 0
37°
G3PDH
33°
37C
RT 0 RT 0 RT 0 RT 0
bp
FIGURE 7. Agurose gel electrophoresis of RT-PCR-ampIified aquaporin-1/
CHJP28, NCAM, MYOC/TIGR, and
G3PDH mRNA in MUTM-NEI/1 cells
grown in both permissive (lFN-y,
33°C) and nonpermissive conditions
(no lFN-y, 37°C). Five hundred nanograms total RNA were amplified in
each reaction; 20 \x\ (from 50 /u,l total) of each product were examined
on the gel and compared with
100-bp standard size markers. Control experiments were performed by
omitting the RT step (0).
bp
700 —
600 —
500 —
400 —
In addition to components of the extracellular matrix,
MUTM-NEI/1 cells express a considerable number of other
typical TM proteins. One of those proteins is the small heat
shock protein, aB-crystallin, In human TM in situ, aB-crystallin
is concentrated in the cribriform area, 3 4 '" which is the part of
the meshwork that provides most of the resistance against
aqueous humor outflow.5'' aB-crystallin is also expressed in
primary human TM cell cultures, especially if they derive from
the cribriform region. By contrast aB-crystallin is not expressed
in SV40-transformed human TM cell lines. 3 "' 53 Similar to primary human TM cultures, mRNA for aB-crystallin can be induced in MUTM-NEI/1 cells under oxidative stress. 34 Other
typical TM genes that are expressed in human TM in situ and
by MUTM-NEI/1 cells include NCAM;55'56 aquaporin-1/
CH1P28,57>5H a water channel; and myoc/tigr, 3 a protein of
unknown function that is mutated in hereditary juvenile glaucoma and in some cases of late-onset POAG. In contrast to
kb
1401
NCAM, aquaporin-1 and myoc/tigr mRNAs are only expressed
in nonpermissive conditions, a fact that may indicate that these
genes are markers for differentiated TM cells.
It has been reported that TM cells have some contractile
properties in situ and in vitro. 3 2 ' 5 9 ' 6 1 In accordance with this,
some cells of the human and bovine TM express a-sm-actin. 2 5 3 2 * 5 2 "^ In primary human TM cultures, the expression of
ct-sm-actin can be induced on treatment with TGF-/3,.f"' Our
results show that this is also the case for MUTM-NET/1 cells.
0.6
•
0.5
•-
0.4
-
Co 8h 12h 24h
2.37 —
1.35 —
0.3 •
0.2
0.1 •
18S —
1 0 fig
FIGURE 8. Northern blot for cdJ-crystallin mRNA in MUTM-NEI/1 cells
grown at 37°C without INI7-7 after oxidative stress (1 hour serum-free
medium with 200 ptM hydrogen peroxide) and under control conditions. Twenty micrograms total RNA were loaded on each lane. The
size of molecular markers is expressed in kilobases. Integrity and
relative amounts of RNA were controlled by reprobing the membrane
with a cDNA probe specific for 18S ribosomal RNA.
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2.5 ug
6.0
pCMV[3
FIGURE 9. Transient transfection of MUTM-NEI/1 cells with plasmid
pCMVfJ. /3-Galactosidase activities in transfected cells were determined
using 50 (i\ cell extract and measuring optical density ( 0 0 ) at a
wavelength of 460 A. Transfections were carried out three times, and
the mean ± SD is shown.
1402
Tamm et al.
MUTM-NEI/1 cells express vimentin, the typical intermediate
filament of TM cells,65 but not desmin the characteristic intermediate filament of ciliary muscle cells or cytokeratins, which
are found in ciliary epithelial cells.66"69
Although all the MUTM-NEI/1 proteins that were investigated in this study are typical for TM cells, we are aware of the
fact that none of these proteins is specific for TM per se.
Nevertheless, we believe that the combined expression of all
these TM markers necessarily supports the idea that the
MUTM-NEI/1 cell line, when grown in nonpermissive conditions, consists of well differentiated TM cells. Furthermore, our
findings indicate that the H-2kb-tsA58 transgenic mouse strain
may be very useful in establishing immortal and differentiated
cell lines from other ocular cell populations that are difficult to
cultivate, such as corneal epithelium or endothelium, lens
epithelium or retinal pigmented epithelium.
The fact that MUTM-NEI/1 cells can be transfected by
plasmid DNA indicates that this cell line may be valuable in
studying the transcriptional regulation of TM genes. Such studies might involve the identification of DNA regulatory sequences important for the control of these genes. Possible
candidates might be genes that show an increase in expression
in POAG, such as type VI collagen,45 aB-crystallin, and myoc/
tigr.70 Because MUTM-NEI/1 cells are of murine origin, the
regulation of TM genes and their products can be directly
studied both in MUTM-NEI/1 cells and in transgenic mice using
a single homologous system.
The MUTM-NEI/1 cell line will be an invaluable tool for
elucidating the complex pattern of TM gene expression including that of potential candidate genes for POAG.
Acknowledgments
The authors thank Elke Kretzschmar (Department of Anatomy, University of Erlangen-Niirnberg, Germany) for excellent help with electron
microscopy; Rick Dreyfuss (Visual Arts and Photography, National
Institutes of Health, Bethesda, Maryland) and Marco Gosswein (Department of Anatomy, University of Erlangen-Niirnberg, Germany) for
expert assistance with photography; Joseph Horwitz (University of
California, Los Angeles) for providing the antibodies for aB-crystallin,
and Terete Borras (Duke University Eye Center, Durham, North Carolina) for providing the guinea pig 18S ribosomal RNA cDNA probe; and
Ales Cvekl, Marc Kantorow, and Todd Kays for helpful discussions.
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