Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling
et al. (2012) Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through
Activation of Rho Signaling. PLoS ONE 7(9): e44400. doi:10.1371/journal.pone.0044400
Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling
Shiri Diskin 0
Wei-Sheng Chen 0
Zhiyi Cao 0
Smita Gyawali 0
Haiyan Gong 0
Andrea Soza 0
Alfonso Gonza lez 0
Noorjahan Panjwani 0
Kin-Sang Cho, Schepens Eye Research Institute, Harvard Medical School, United States of America
0 1 Program in Cell, Molecular & Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University , Boston , Massachusetts, United States of America, 2 New England Eye Center and Department of Ophthalmology, Tufts University , Boston , Massachusetts, United States of America, 3 Department of Ophthalmology, Boston University School of Medicine, Boston, Massachusetts, United States of America, 4 Departamento de Inmunolog a Cl nica y Reumatolog a, Facultad de Medicina, Centro de Regulaci o n Celular y Patolog a and Centro de Envejecimiento y Regeneracio n, Facultad de Ciencias Biolo gicas, Pontificia Universidad Cato lica de Chile , Santiago , Chile , 5 Millennium Institute for Fundamental and Applied Biology , Santiago , Chile , 6 Department of Biochemistry, Tufts University , Boston, Massachusetts , United States of America
Purpose: The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling. Methods: Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-b1 integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined. Principal Findings: We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-b1 integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632. Conclusions/Significance: The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow.
Funding: This work was supported by National Institutes of Health Grants R03EY015168 (N.P.), a core grant for vision research P30EY13078, Mass Lions Eye
Research fund and a grant from Research to Prevent Blindness. A.G. was funded by Grants FONDECYT (National Fund for Scientific and Technological
Development) #1050715 and FONDAP (Fund for Advanced Research in Priority Areas (Centers of Excellence FONDAP) #19800001. New England Eye Center
(NEEC) is not a company. It is a nonprofit physician group dedicated to providing patients with an unprecedented level of care and responsive service. NEEC is the
Ophthalmology Department of Tufts University School of Medcine. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Primary Open Angle Glaucoma (POAG) is a major cause for
irreversible blindness. Factors that lead to the development of
POAG are not yet fully known. It is clear, however, that elevated
intraocular pressure is a major causal risk factor . Elevation in
intraocular pressure is due to the dysfunction of outflow pathway
tissues resulting in inadequate clearance of aqueous humor.
Trabecular meshwork (TM) cell-matrix adhesion is crucial for the
maintenance of the outflow pathway. In the short term,
experimental procedures that cause loss of TM cell contact with
the beams lead to a sharp increase in aqueous outflow [2,3,4]. In
the long term, beams denuded of cells (typical of POAG eyes) tend
to collapse on one another, blocking the outflow channels .
Also, off-target effects of glucocorticoid decrease outflow facility by
increasing cell rigidity in TM cells [6,7,8]. In recent years, a large
body of research in the field of POAG has focused on the role of
Rho signaling in the regulation of outflow facility through
regulation of the TM actin cytoskeleton. The emerging paradigm
is that the inhibition of Rho signaling leads to elevation in outflow
facility, while induction of Rho signaling leads to increased
resistance to outflow [2,9,10,11]. In cultured TM cells, inhibitors
of the Rho signaling cascade cause TM cell rounding, loss of stress
fibers and focal adhesions and retraction of cell processes [9,12]. In
perfused human and animal eyes, inhibitors of Rho signaling cause
TM cell rounding and detachment from the beams concomitant
with a marked elevation in outflow facility [2,4]. A recent phase 2b
clinical trial that utilizes a novel and potent Rho kinase inhibitor,
AR-12286, further strengthens the therapeutic importance of
relaxing the TM by targeting Rho signaling .
In non-ocular studies, a carbohydrate-binding protein,
galectin8 (Gal8), has been shown to form high-affinity interactions with
integrins, modulate cell-matrix interactions, and promote cell
spreading by activating PI3K and the small GTPases, Ras and
Rac [14,15,16]. Little is known about the role of the
carbohydratemediated recognition systems in TM cell adhesion and signaling.
In a recent study, we have observed that TM cells adhere to Gal8
substratum and that b1 integrins derived from TM cells bind to
Gal8 in a carbohydrate-dependent fashion . The role of Gal8
in the regulation of Rho signaling that modulates stress fiber
formation and focal adhesion assembly has, thus far, not been
investigated in any cell type. In the current study, we underscore
for the first time the function of Gal8 in modulating the Rho
signaling pathway in TM cells. We demonstrate here that: b1
integrin function-blocking antibody inhibits the adhesion and
spreading of TM cells on Gal8-coated wells; cells adhered to Gal8
accumulate phosphorylated myosin light chain 2 (MLC2), and
accumulation of phosphorylated MLC2 is associated with stress
fiber formation that is abolished by the presence of either the Rho
inhibitor, C3 transferase, or the Rho-kinase (ROCK) inhibitor,
Y27632. These data lead us to propose that Gal8 promotes
cytoskeletal rearrangement in TM cells through interaction with
b1 integrins leading to activation of the Rho/ROCK/MLC2
Galectin-8 is Expressed in Human TM Tissue and in
Cultured TM Cells
Multiple techniques including RT-PCR, Western blot and
immunohistochemical staining were used to detect Gal8
expression in TM. In immunohistochemical staining, anti-Gal8 reacted
strongly with the TM cells on beams (Figure 1Ai, arrows) and with
TM cells in the juxtacanalicular region (Figure 1Ai, arrowheads).
Some staining was observed in the ECM in all parts of the tissue
and in the wall of Schlemms canal (Figure 1Ai, SC). No staining
was observed when sections were not exposed to a primary
antibody (Figure 1Aii) or exposed to nonimmune goat IgG (data
not shown). To assess the expression of Gal8 in human cultured
TM cells, RT-PCR experiments were performed on total RNA
preparations from two different donors. All RNA preparations
produced the expected size Gal8 (191 bp) product (Figure 1Bi). In
all cases, when reaction mixtures lacked RT, no components were
amplified (Figure 1Bi). Bands were isolated from the gel and
sequenced. The product was found by BLAST analysis to be 98%
identical to the published Gal8 cDNA sequence . To assess the
abundance of Gal8 expression in TM, the expression level of Gal8
was compared with that of GAPDH by quantitative RT-PCR.
The qPCR experiments were performed in triplicates on whole
RNA extracts from cultured TM cells derived from two different
donors. In each case, a robust Gal8 signal was detected (Ct: 37.37
and 33.38 for Gal8 and GAPDH, respectively) (Figure 1Bii). To
verify Gal8 protein expression, TM cell lysates were incubated
with b-lactose conjugated beads, proteins bound to the beads were
eluted first with sucrose, a non-competing sugar, and then with
blactose, a competing sugar. Both the lactose eluate (Figure 1Biv,
lane L) and the unfractionated total extract (Figure 1Biv, lane T)
contained a major anti-Gal8-reactive 36-kDa band, which is the
published molecular weight for human Gal8 . This band was
absent from the sucrose eluate as well as from the unbound
fraction (Figure 1Biv, lanes S and UB). Many other protein bands
in both the total extract and unbound fractions that were detected
by Ponceau S staining (Figure 1Biii, lanes UB and T) were not
stained with anti-Gal8, thereby attesting to the high specificity of
the antibody. No bands were visible in control immunoblots that
were not exposed to primary antibody (data not shown).
TM Cell Adhesion and Spreading on Gal8 is Mediated by
In a recent study, we observed that TM cells adhere to and
spread on Gal8 substratum and that b1 integrins derived from TM
cells bind to Gal8 in a carbohydrate-dependent fashion .
Because b1 integrins are known to play a central role in cell
adhesion and spreading [18,19,20], we sought to determine
whether the adhesion and spreading of TM cells to Gal8 is
mediated by b1 integrins. For this, we conducted the cell adhesion
assay in the presence and the absence of a function-blocking
antib1 integrin antibody (JB1A). Anti-b1 integrin antibody inhibited
cell adhesion to both Gal8 and fibronectin (positive control) by
about 20% (Figure 2A). The extent of inhibition of cell adhesion to
fibronectin by anti-b1 integrin observed in our study is
substantially lower than the published values for a number of
nonocular cell types [21,22,23], but is consistent with a published
study reporting that anti-b1 integrin antibodies attenuate TM cell
adhesion to fibronectin by 1020% . F-actin staining revealed
that anti-b1 integrin antibody abolished cell spreading on
Gal8coated wells, whereas control IgG had no such effect (Figures 2B
and 2C). Taken together, these data suggest that TM cell adhesion
and, in particular spreading on Gal8, is mediated by one or more
Galectin-8 Promotes Rearrangement of Cytoskeleton and
Modulates Rho Signaling in TM Cells
Having established that Gal8 modulates TM cell spreading, it
was of interest to determine whether the lectin has the capacity
to influence the structure of cytoskeleton and Rho signaling. To
determine whether Gal8 promotes rearrangement of the actin
cytoskeleton, TM cells incubated on slides coated with Gal8 for
varying periods (30 min to 2 hr) were stained with
rhodaminelabeled phalloidin. Slides coated with human fibronectin and
poly-L-lysine served as positive and negative controls,
respectively. Forces generated by actin polymerization drive cells to
form membrane protrusion. Actin punctate staining indicates the
initiation of actin polymerization. Cells incubated on Gal8 for
30 min showed membrane protrusions and the actin cytoskeleton
showed punctate staining (Figure 3Ai). Following 1 hr
incubation, initial stress fibers were seen (Figure 3Aii), and at 2 hr, the
cells were fully spread and contained stress fibers aligned along
the longitudinal axes of the cells (Figure 3Aiii). A very similar
transition from rounded cells with punctate actin staining to
spread cells with stress fibers was observed in cells adhered to
fibronectin (Figure 3Aivvi). Cells with robust stress fibers were
quantified at different time points. After 2 hr incubation, more
than 95% of TM cells formed stress fibers on Gal-8 as well as
fibronectin substrate (Figure 3B). In contrast, most cells adhered
to poly-L-lysine retained their rounded shape and punctate actin
staining up to 2 hr incubation in serum-free medium
(Figure 3Aviiix). These results suggest that the interaction
between integrins and Gal8 which leads to cell spreading is
indeed mediated through rearrangement of the actin cytoskeleton
and formation of stress fibers. To determine whether stress fiber
formation in TM cells adhered to Gal8 is mediated by Rho
GTPase; we conducted the cytoskeletal rearrangement assay in
the presence of a Rho specific inhibitor, C3 transferase. Cells
incubated on Gal8 for 2 hr, showed fully developed stress fibers
(Figure 3Aiii). When cells were incubated on Gal8-coated glass
slides in the presence of C3 transferase, the formation of actin
stress fibers was significantly reduced in a dose-dependent
manner (Figure 4A). Cells were spread, but only a few isolated
stress fibers formed, and actin staining became predominantly
punctate. In addition, some cells were characterized by collapse
of the cell body and protrusion of dendrite-like extensions
A key regulator of actin cytoskeleton is the small GTPase Rho
[24,25,26]. One of the effector proteins of Rho is the Rho-kinase,
also known as Rho associated coiled coil kinase (ROCK). To assess
the involvement of ROCK in the formation of stress fibers in TM
cells adhered to Gal8, we conducted the cytoskeletal
rearrangement assay in the presence of a well characterized ROCK
inhibitor, Y27632. Y28632 significantly inhibited stress-fiber
formation in a dose-dependent manner (Figure 4C). At 20 mM
of Y27632, very few stress fibers formed in TM cells adhered to
Gal8. Instead, almost every cell had protrusion of dendrite-like
Figure 2. TM cell adhesion and spreading on Gal8 is mediated by b1 integrins. A: Normal human TM cells were incubated on microtiter
wells coated with BSA, fibronectin, or Gal8 in DPBS, in the presence and absence of a function-blocking anti-b1 integrin antibody (JB1A), or control
mouse IgG. Following incubation at 37uC for 30 min, cells were fixed and stained with crystal violet. Attached cells in fibronectin-coated wells are set
as 100% (positive control); attached cells in other wells are presented as percent of positive control. Data are expressed as mean6SEM and analyzed
with one-way ANOVA. *P,0.05 vs IgG; **P,0.01 vs media or IgG; ***P,0.001 vs media. B and C: Cell spreading assay. TM cells were fixed with 4%
paraformaldehyde after adhesion for 30 min. F-actin was stained with rhodamine-labeled phalloidin and cell nuclei were labeled with DAPI. Random
fields of each experimental condition were photographed, and spread areas of individual cells were quantified with ImageJ. Representative
micrographs of TM cells incubated in the presence and the absence of anti-b1 integrin antibody are shown in C. Data are presented as Boxwhisker
plot (after Tukey) and analyzed with one-way ANOVA. ***P,0.001 vs media or IgG. This experiment was performed three times with reproducible
results. Bar: 100 mm.
Figure 3. Galectin-8 promotes cytoskeletal rearrangement in TM cells. A: Normal human TM cells were plated on eight-chamber glass slides
coated with 20 mg/ml of recombinant human Gal8 (iiii), 20 mg/ml of fibronectin (ivvi), or 100 mg/ml of poly-L-lysine (viiix) in serum-free DMEM at
37uC for 30 min (i, iv, vii), 1 hr (ii, v, viii), and 2 hr (iii, vi, ix). Following the incubation period, cells were fixed with 4% paraformaldehyde and stained
with rhodamine-labeled phalloidin. Bar: 50 mm. B: Quantification of stress fiber formation. Random fields were photographed, and cells with robust
stress fibers were counted. N = 225 to 362. Data are expressed as mean6SEM and analyzed with one-way ANOVA. ***P,0.001 vs poly-L-lysine at
different time points. This experiment was performed three times with reproducible results.
extensions (Figure 4D). At the highest concentration of the
inhibitors used, C3 transferase and Y28632 inhibited the stress
fiber formation by 50% and 95% respectively. The different effects
of the two inhibitors may be due to the characteristics of the
inhibitors. The ROCK inhibitor, Y27632, changes TM cell
morphology within 2 hr. However, for the Rho inhibitor, C3
transferase, it takes at least 4 hr to changes TM cell morphology.
We did not extend the incubation time beyond 2 hr because TM
cells also produce other matrix proteins which may complicate
interpretation of data. Despite these profound effects, neither
Y27632 nor C3 transferase were cytotoxic to TM cells (Figure S1).
Neither inhibitor had any effect on the actin cytoskeleton of cells
adhered to poly-L-lysine (data not shown). Together, these results
suggest that Gal8 promotes stress fiber formation in TM cells
through activation of Rho signaling and that the pathway for this
effect runs through ROCK. Activated ROCK promotes the
Figure 4. Rho and ROCK inhibitors inhibit Galectin-8 induced stress fiber formation. A and C: Serum-starved human TM cells were
incubated on chamber glass slides coated with recombinant human Gal8 in the presence of the Rho inhibitor, C3 transferase or ROCK inhibitor,
Y27632, at different concentrations. After 2 hr, cells were stained with rhodamine-labeled phalloidin, and cells with robust stress fibers were
enumerated. Data are expressed as mean6SEM. B and D: Cells were treated C3 transferase at 2 mg/ml (B) or Y27632 at 20 mM (D), stained with
rhodamine-labeled phalloidin, and random fields were photographed. Note that cells treated with Y27632 or C3 transferase are not spread and
exhibit dendrite-like structures. Bar: 50 mm.
accumulation of phosphorylated MLC2 which, in turn, promotes
the formation of stress fibers . To find whether MLC2 is
phosphorylated in TM cells adhered to Gal8, we analyzed the
phospho-MLC2 content of the cells with a MLC2 (Thr18/Ser19)
antibody. TM cells adhered to Gal8 showed a time-dependent
accumulation of phosphorylated MLC2 (Figure 5A). The extent of
accumulating phosphorylated MLC2 on Gal8-coated wells was
similar to that seen in cells adhered to fibronectin (Figure S2).
Elevated level of phospho-MLC2 was detected at 1 hour and
robust stress fibers were detected at 2 hours on Gal8 substrate.
This is in line with the current understanding that stress fiber
formation is downstream of, and, is modulated by phosphorylated
MLC2. Treatment with Y27632 and C3 transferase abolished
phosphorylation of MLC2 of TM cells on Gal8 substrate
(Figure 5B). Taken together, these data suggest a novel activity
for Gal8 involving induction of the Rho/ROCK/MLC2 pathway
leading to cell spreading and stress fiber formation.
Perturbation of the outflow of the aqueous humor leads to
elevation in intraocular pressure, which is one of the major causal
risk factors for vision loss in POAG . The dynamic structure of
the TM cells actin cytoskeleton with its focal adhesions is an
important determinant of outflow facility. Reagents that disrupt
the actin cytoskeleton such as cytochalasins, lantrunculin and H-7,
cause elevation in outflow facility as well as loss of TM cell contact
with the beams and neighboring cells [28,29,30,31]. Our data
support the hypothesis that Gal8 can regulate TM actin
cytoskeleton. TM cell adhesion to Gal8 was followed by cell
spreading and actin cytoskeleton rearrangement, showing gradual
actin polymerization into stress fibers over time in adhered cells. A
key regulator of actin cytoskeleton is the small GTPase Rho
[24,25,26]. Activated (GTP-bound) Rho interacts with ROCK
and activates its kinase activity. This in turn, phosphorylates and
inactivates MLC phosphatase, and also phosphorylates MLC
directly. The phosphorylation of MLC leads to rearrangement of
cytoskeleton and stress fiber formation . Recent studies have
shown that phosphorylation of MLC in TM cells is dependent on
activation of the Rho/ROCK signaling pathway [10,12]. In the
current study, we demonstrate for the first time that Gal8 can
modulate Rho GTPase signaling in TM cells. Our findings that
spreading of TM cells on Gal8 is associated with the accumulation
of phosphorylated MLC2 and that inhibitors of Rho as well as of
ROCK attenuate TM cell spreading and stress fiber formation,
suggest that Gal8 has the ability to participate in the regulation of
the Rho/ROCK/MLC2 signaling pathway in TM cells. Rho
signaling has been intimately linked to the regulation of outflow
facility. Specific inhibitors, like ROCK, Y27632 [9,11] and H1152
 have been shown to increase outflow facility in whole monkey
and porcine eyes in a time- and dose-dependent manner. Likewise,
in a perfused human eye, dominant negative RhoA  and
dominant negative ROCK  caused a marked increase in
outflow facility, TM cell rounding and detachment from the
beams. In cultured TM cells, delivery of a dominant negative
RhoA  and dominant negative ROCK  caused disruption of
the actin cytoskeleton, TM cell rounding, loss of stress fibers and
The current study has focused on characterization of Gal8
effects on normal TM cells. The relevance of these effects to the
pathogenesis of POAG needs to be investigated further. The
LGALS8 gene encodes six different isoforms of Gal8, resulting from
alternative splicing and the use of multiple polyadenylation signals
. Different isoforms differ in the length of their hinge peptide
. It has been shown that isoforms with a short hinge peptide
are severely impaired in their biological activity, they are
profoundly less capable of promoting cell adhesion and of
inducing outside-in signaling . In a recent study conducted
in our lab , glycogene expression patterns were compared
between normal and glaucomatous TM tissues, using a specialized
glycogene microarray, GLYCOv2 (Consortium for Functional
Glycomics). While this study revealed no differences in Gal8
expression levels between normal and glaucomatous TM tissues,
the Gal8 probes used in the GLYCOv2 microarray were not
designed to detect the alternatively spliced isoforms of Gal8 with
the different hinge peptide lengths. Thus, one cannot eliminate the
possibility that normal and glaucomatous TM may express distinct
isoforms of Gal8 resulting in different effects on the tissues;
longerhinged Gal8 possess increased glycan-binding capacity, which may
decrease TM cell relaxation and decrease aqueous outflow in
POAG patients. In addition, a recent study has reported that
nonsynonymous single nucleotide polymorphism of Gal8 (F19Y) is
strongly associated with autoimmune diseases, such as rheumatoid
arthritis and myasthenia gravis . In these autoimmune
diseases, Gal8 expression level is not affected. Thus, a possibility
that Gal8 polymorphism may be involved in the pathogenesis of
glaucoma cannot be ignored. On the other hand, a different
regulatory mechanism may involve not Gal8 itself but its
countereceptors. TM cells treated with dexamethasone show
elevated expression levels of a5 integrin  which we previously
found to bind to Gal8 . It is possible that glaucomatous TM
cells bearing more countereceptors for Gal8 are more susceptible
to its effects.
In conclusion, data presented in the current study suggest
a novel function for Gal8 in activation of Rho signaling and the
regulation of TM cell cytoskeleton. It lays a foundation for
exploration of a possible role for Gal8 in regulation of aqueous
outflow and the pathogenesis of POAG.
Materials and Methods
Human eyes with no known history of eye disease were obtained
from the National Disease Research Interchange (Philadelphia,
PA) within 24 hr postmortem. The eyes were bisected by an
equatorial incision and the anterior segment of the eye was cut into
four quadrants. Tissues were fixed in formaldehyde (4%, 3 hr,
25uC) and were then processed for paraffin embedding. For
immunostaining, longitudinal tissue sections (5 mm) were
deparaffinized and sequentially treated with a basic pH antigen retrieval
reagent (R&D systems, Minneapolis, MN), normal horse serum
(R&D systems), goat anti-human Gal8 (2 hr, 37uC, R&D Systems;
the goat antibody is specific for Gal8 and it does not recognize
other galectins including Gal3 or Gal1), biotinylated anti-goat IgG
(1 hr, 25uC, R&D Systems), a freshly prepared solution of
avidinbiotin-complex (R&D Systems Cell and Tissue Staining Kit,
30 min, 25uC) and a diaminobenzidine/H2O2 reagent (R&D
systems). Control sections were processed the same way except
that either the step involving incubation with the primary antibody
was omitted or primary antibody was substituted with nonimmune
goat serum (R&D systems).
Human TM Cell Cultures
Normal human cadaver eyes (donor age 7189 years) were
obtained from the Central Florida Lions Eye Bank (Tampa, FL).
All eyes were enucleated within 6 hr postmortem and tissues were
explanted for culture within 24 hr postmortem. TM cells were
grown in tissue culture as described previously . Cultures were
propagated in DMEM supplemented with 10% FBS and cells
were used at third to fourth passage. Consistent with published
studies, these cells expressed aquaporin1 and CD44, and, when
treated with Dexamethasone, markedly upregulated the
expression of myocilin. (Data not shown, expression levels of CD44 and
myocilin were detected by Western blot and RT-PCR analyses,
respectively, and the expression of aquaporin1 was detected in
a microarray study using 48.5 Illumina HEEBO oligo microarrays
[Microarrays, Inc. Nashville, TN]). For some in vitro experiments,
primary human TM cells purchased from ScienCell (Carlsbad,
CA) were used.
RT-PCR Amplification of Galectin-8
Confluent cultures of TM cells from two different donors were
subjected to RNA extraction using the RNEasy RNA extraction
kit (Qiagen, Valencia, CA). On-column DNase (Qiagen) digestion
was performed during RNA purification to avoid any DNA
contamination. Reverse transcription was performed on total
RNA (1 mg) with random primers (Invitrogen) in the presence or
the absence of reverse transcriptase (RT) (SuperScript II,
Invitrogen). Primer sequences: 59 ccc/tgt/tct/ctt/gag/ctt/cg 39
and 59 cac/tgg/gga/agg/agt/tgt/gt 39, were chosen for an
expected product size of 191 bp. PCR products were
electrophoresed on 1% agarose gel, and sequenced at the Tufts
University Core Facility.
To assess the abundance of Gal8 expression in TM, the
expression level of Gal8 was compared with that of GAPDH by
quantitative RT-PCR. Quantitative RT-PCR was performed
using the Mx4000 real-time PCR machine (Stratagene, La Jolla,
CA). Briefly, cDNA was synthesized from 25 ng total RNA using
the High Capacity cDNA Archive Kit (Applied Biosystems, Foster
City, CA) according to the manufacturers instructions. PCR was
carried out in triplicates using inventory gene-specific primers
(Gal8:HS00180706; GAPDH: HS99999905, Applied Biosystems)
and the TaqMan Universal PCR Master Mix containing ROX as
a passive reference. Reactions performed in the absence of
template served as negative controls. Fluorescent signals were
recorded once per cycle with a detector corresponding to FAM.
To normalize the non-PCR related fluctuations between wells,
each fluorescent reporter signal was measured against the ROX
(internal reference dye) signal. Amplification plots showing the
increase in the FAM fluorescence with each cycle of PCR (DRn)
were generated and threshold cycle values (Ct) were calculated for
all samples. The Ct value represented the PCR cycle number at
which the fluorescence was detectable above a threshold based on
the variability of the baseline data during the first 15 cycles. All Ct
values were obtained in the exponential phase.
Western Blot Analysis of Galectin-8
To detect the expression of Gal8 in TM cells, 0.6 ml of cold
radioimmunoprecipitation (RIPA) buffer containing a protease
inhibitor cocktail (Roche Applied Science, Mannheim, Germany)
was added to washed confluent cell cultures. Since Gal8 is
a carbohydrate binding protein with high affinity towards
blactose, a b-lactose affinity chromatography assay was used. After
30 min on ice, cell extracts were clarified by centrifugation and
were incubated with Sepharose beads conjugated with b-lactose
(1 hr, 4uC; EY labs, San Mateo, CA). Following the incubation
period, the beads were washed with PBS containing 0.1% Triton
X-100 (PBS-T), eluted first with a non-competing sugar, sucrose
(100 mM in PBS-T) towards which Gal8 has no affinity, and then
with b-lactose (100 mM in PBS-T). Eluates were dialyzed and
then analysed by SDS-PAGE. Protein blots were stained with
Ponceau S (Sigma) and were then processed for immunostaining
using goat anti-human Gal8 as a primary antibody (1 hr, 25uC;
R&D Systems), peroxidase-labeled anti-goat IgG (Vector Labs) as
a secondary antibody, and a chemiluminescence detection system
(Perkin Elmer, Boston, MA).
Preparation of Recombinant Human
Glutathione-STransferase Tagged Galectin-8
Recombinant human glutathione-S-transferase (GST) tagged
Gal8, was produced and purified as previously described  with
a few modifications. Lysates of bacteria expressing GST-Gal8 (2L
culture) were chromatographed on a b-lactose-conjugated
Sepharose column (EY Labs; 1 ml bed volume). After allowing Gal8 to
bind to the affinity matrix, the gel bed was washed first with wash
buffer I (20 ml of 20 mM Tris-HCl pH 7.4, 150 mM NaCl,
2 mM EDTA, 0.2 mM PMSF, 4 mM b-mercaptoethanol) and
then with 10 ml of wash buffer II (PBS containing 0.2 mM PMSF,
4 mM b-mercaptoethanol). GST-Gal8 was eluted from the
column with 10 ml wash buffer II containing 100 mM b-lactose.
Fractions containing the lectin were dialyzed against PBS
containing 2% glycerol and 4 mM b-mercaptoethanol and stored at
Quantification of Cell Adhesion
To assess the adhesion and spreading of TM cells onto different
substrates, 96-well microtiter plates were coated with Gal8 (20 mg/
ml), human fibronectin from placenta (Sigma) (20 mg/ml), and
poly-L-lysine (Sigma) (100 mg/ml). After 2 hr, plates were blocked
with 1% BSA/Dulbeccos PBS (DPBS) (1 hr, 25uC). Confluent
TM cultures were dissociated with Accutase (Invitrogen),
resuspended in 0.2% BSA/DPBS, and plated on microtiter plates
coated with Gal8 and other substrates described above (30,000
cells/well, 3 wells/group). Following incubation at 37uC for
30 min, cells were washed with PBS, stained/fixed with 0.2%
crystal violet (Sigma) in PBS containing 20% methanol (15 min,
25uC), and washed thoroughly with distilled water to remove
unbound stain. Bound stain was solubilized with 1% SDS in
distilled water for 1 hr, and the absorbance was measured at
595 nm by the FilterMax F5 Multi-Mode Microplate Reader
(Molecular Devices, Sunnyvale, CA). To assess the involvement of
b1-integrins in the adhesion of TM cells on Gal8, cells were
incubated in the presence of an anti-integrin b1
functionalblocking antibody, JB1A (1:50 dilution; 10 min, 25uC; EMD
Millipore, Billerica, MA). The JB1A antibody binds to a specific
regulatory epitope (amino acids: 82 to 87) on b1 integrin that is
distinct from the RGD binding site (amino acids: 140164) .
The antibody binding to this epitope locks the integrin in an
inactive conformation and does not allow it to be activated
regardless of the nature of the ligand presented . This antibody
has been shown to interfere with the adhesion of various cell types
to a number of ECM substrates including fibronectin and collagen
Quantification of Cell Spreading and F-actin Staining
Human TM cells plated on Gal8 substrates were incubated in
the presence of JB1A antibody for 30 min as described in the
previous section. At the end of incubation period, cells were fixed
with 4% paraformaldehyde in PBS (15 min, 25uC), washed with
PBS three times, and permeabilized with 0.5% Triton X-100 in
PBS (5 min, 25uC). After washing again with PBS three times,
non-specific binding sites were blocked with 1% BSA in PBS
(20 min, 25uC), the cells were stained with rhodamine-labeled
phalloidin (100 nM in PBS; Cytoskeleton, Denvor, CO), mounted
in Vectashield mounting medium with DAPI (Vector
Laboratories, Burlingame, CA). Six to eight different fields of each
experimental condition were photographed, and the spread areas
of individual cells were quantified by ImageJ software (National
Institute of Health, Bethesda, MD).
Detection of Cytoskeletal Changes in Human TM Cells
Adhered to Galectin-8
Eight-chamber glass slides were coated with Gal8 (20 mg/ml),
human fibronectin from placenta (Sigma) (20 mg/ml), and
poly-Llysine (Sigma) (100 mg/ml). TM cultures were dissociated with
Accutase and plated on substrate-coated slides (5000 cells/
chamber in serum-free DMEM). Following incubation at 37uC
for 0.5, 1, or 2 hr, cells were washed, fixed and stained with
rhodamine-labeled phalloidin as previously described. To assess
the involvement of Rho signaling in stress fiber formation in cells
adhered to Gal8, TM cells were serum starved overnight and
treated with different concentrations of ROCK inhibitor, Y27632
(Abcam, Cambridge, MA) or the Rho-specific inhibitor, C3
transferase from clostridium botulinum (Cytoskeleton) for 4 hr.
This enzyme is a highly specific inhibitor of Rho that does not
affect other Rho family members such as Rac or cdc42. It is
a ribosyltransferase that by modifying the Asn 41 residue of Rho,
renders it biologically inactive .
Detection of Myosin Light Chain Phosphorylation in TM
Cells Adhered to Galectin-8
In an effort to further confirm the involvement of Rho signaling
in stress fiber formation in TM cells adhered to Gal8, confluent
TM cell cultures were dissociated with Accutase and plated on
Gal8-coated dishes (100 mm). After incubation at 37uC for 0.5, 1
and 2 hr, cells were lysed with cell lysis buffer (Cell Signaling
Technology, Danvers, MA) supplemented with the complete
protease inhibitor cocktail (Roche Applied Science) and
PhosSTOP phosphatase inhibitor cocktail (Roche), and subjected to
electrophoresis in 12% SDS-PAGE gels. Protein blots of the gels
were blocked with OdysseyH blocking buffer (OBB) (Li-COR,
Biosceiences, Lincoln, NE) and were then probed with rabbit
antiphospho-myosin light chain 2 (Thr18/Ser19) primary antibody
(1:500 dilution in OBB, 4uC, overnight; Cell Signaling
Technology), followed by anti-rabbit IRDye 800CW secondary antibody
(Li-COR) (1:10,000 dilution in OBB, 45 min, room temperature).
Membranes were scanned by an OdysseyH Infrared Imaging
System using Image Studio v2.0 software (Li-COR). After image
acquisition, membranes were stripped with the NewBlot
nitrocellulose stripping buffer (Li-Cor) and re-probed with rabbit
antiMRCL3/MRLC2/MYL9 (FL-172) primary antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) (1:400 dilution) followed by
antirabbit IRDye 800CW secondary antibody. Relative band intensity
was quantified by ImageJ.
Figure S1 Rho and ROCK inhibitors are not cytotoxic to
TM cells. A: Cells were incubated on glass slides coated with
recombinant human Gal8 in the absence (a) or the presence of:
Y27632 at 20 mM (b), or C3 transferase at 2 mg/ml (c) or tert-butyl
hydroperoxide (tBH) at 3.5 mM (d). Following the incubation
period, cells were washed and stained with ethidium homodimer
III (red) which stains dead cells and Hoechst 33342 (blue) which
stains nuclei of both living and dead cells. In the left panel are
representative micrographs from each group showing no
significant cell death in the presence of Y27632 (b) or (c) C3 transferase
and significant cell death in the presence of tBH (d). Random fields
of each experimental condition were photographed, and dead cells
were counted manually. Percent dead cells for cells adhered to
Gal8 in the presence of the different inhibitors are shown in panel
B. Data are shown as mean 6 SEM and analyzed by one-way
ANOVA. N = 3.
Figure S2 Galectin-8 promotes phosphorylation of
myosin light chain. Normal human TM cells were incubated on
Gal8-coated 100-mm dishes for 0.5, 1, 2 and 4 hr. Following
incubation, cells were lysed, and protein extracts were subjected to
affinity chromatography using a phosphoprotein affinity column.
Bound fraction was electrophoresed on SDS-polyacrylamide gel
and gel blots were stained with Ponceau S (A) and were then
processed for immunostaining with anti-myosin light chain
antibody (B). Phosphoproteins isolated from cells incubated on
fibronectin and poly-L-lysine for 4 hr served as positive and
negative controls respectively. The expected MLC band of 20-kDa
appeared in the phosphorylated fraction of all cell lysates.
Approximate band intensity was quantified by image analysis
software and normalized to Ponceau S staining. The accumulation
of phosphorylated MLC over time in TM cells adhered to Gal8 is
plotted in panel C. Comparison of phosphorylated MLC content
in cells adhered to different substrates following 4 hr incubation is
shown in panel D. Note that by 4 hr, MLC phosphorylation is
similar in cells adhered to Gal8 and to fibronectin. Lys,
poly-Llysine; FN, human fibronectin.
Conceived and designed the experiments: SD WSC NP. Performed the
experiments: SD WSC. Analyzed the data: SD ZC NP. Contributed
reagents/materials/analysis tools: WSC SG HG AS AG. Wrote the paper:
SD WSC NP.
1. Weinreb RN , Khaw PT ( 2004 ) Primary open-angle glaucoma . Lancet 363 : 1711 - 1720 .
2. Vittitow JL , Garg R , Rowlette LL , Epstein DL , O'Brien ET , et al. ( 2002 ) Gene transfer of dominant-negative RhoA increases outflow facility in perfused human anterior segment cultures . Mol Vis 8 : 32 - 44 .
3. Johnson M ( 2006 ) ' What controls aqueous humour outflow resistance?' . Exp Eye Res 82 : 545 - 557 .
4. Rao PV , Deng P , Maddala R , Epstein DL , Li CY , et al. ( 2005 ) Expression of dominant negative Rho-binding domain of Rho-kinase in organ cultured human eye anterior segments increases aqueous humor outflow . Mol Vis 11 : 288 - 297 .
5. Polansky J , Alvarado J ( 1994 ) Cellular mechanisms influencing the aqueous humor outflow pathway . In: DM A , FA J, editors. Principles and practice of ophthalmology: basic science . Philadelphia: W. B. Saunders Co . 226 - 251 .
6. Clark AF , Wilson K , McCartney MD , Miggans ST , Kunkle M , et al. ( 1994 ) Glucocorticoid-induced formation of cross-linked actin networks in cultured human trabecular meshwork cells . Invest Ophthalmol Vis Sci 35 : 281 - 294 .
7. Clark AF , Brotchie D , Read AT , Hellberg P , English-Wright S , et al. ( 2005 ) Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue . Cell Motil Cytoskeleton 60 : 83 - 95 .
8. Filla MS , Schwinn MK , Nosie AK , Clark RW , Peters DM ( 2011 ) Dexamethasone-associated cross-linked actin network formation in human trabecular meshwork cells involves beta3 integrin signaling . Invest Ophthalmol Vis Sci 52 : 2952 - 2959 .
9. Koga T , Koga T , Awai M , Tsutsui J , Yue BY , et al. ( 2006 ) Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells . Exp Eye Res 82 : 362 - 370 .
10. Rao PV , Deng P , Sasaki Y , Epstein DL ( 2005 ) Regulation of myosin light chain phosphorylation in the trabecular meshwork: role in aqueous humour outflow facility . Exp Eye Res 80 : 197 - 206 .
11. Tian B , Kaufman PL ( 2005 ) Effects of the Rho kinase inhibitor Y-27632 and the phosphatase inhibitor calyculin A on outflow facility in monkeys . Exp Eye Res 80 : 215 - 225 .
12. Rao PV , Deng P-F , Kumar J , Epstein DL ( 2001 ) Modulation of Aqueous Humor Outflow Facility by the Rho Kinase- Specific Inhibitor Y-27632. Invest Ophthalmol Vis Sci 42 : 1029 - 1037 .
13. Williams RD , Novack GD , van Haarlem T , Kopczynski C ( 2011 ) Ocular hypotensive effect of the Rho kinase inhibitor AR-12286 in patients with glaucoma and ocular hypertension . Am J Ophthalmol 152 : 834 - 841 e831.
14. Carcamo C , Pardo E , Oyanadel C , Bravo-Zehnder M , Bull P , et al. ( 2006 ) Galectin-8 binds specific beta1 integrins and induces polarized spreading highlighted by asymmetric lamellipodia in Jurkat T cells . Exp Cell Res 312 : 374 - 386 .
15. Levy Y , Arbel-Goren R , Hadari YR , Eshhar S , Ronen D , et al. ( 2001 ) Galectin8 functions as a matricellular modulator of cell adhesion . J Biol Chem 276 : 31285 - 31295 .
16. Levy Y , Ronen D , Bershadsky AD , Zick Y ( 2003 ) Sustained induction of ERK, protein kinase B, and p70 S6 kinase regulates cell spreading and formation of Factin microspikes upon ligation of integrins by galectin-8, a mammalian lectin . J Biol Chem 278 : 14533 - 14542 .
17. Diskin S , Cao Z , Leffler H , Panjwani N ( 2009 ) The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading . Glycobiology 19 : 29 - 37 .
18. Chen N , Chen C-C , Lau LF ( 2000 ) Adhesion of Human Skin Fibroblasts to Cyr61 Is Mediated through Integrin alpha 6beta 1 and Cell Surface Heparan Sulfate Proteoglycans . J Biol Chem 275 : 24953 - 24961 .
19. Wu Y , Chen L , Zheng PS , Yang BB ( 2002 ) beta 1-Integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a Cterminal domain of PG-M/versican . J Biol Chem 277 : 12294 - 12301 .
20. Zhou L , Zhang SR , Yue BY ( 1996 ) Adhesion of human trabecular meshwork cells to extracellular matrix proteins. Roles and distribution of integrin receptors . Invest Ophthalmol Vis Sci 37 : 104 - 113 .
21. Akimov SS , Krylov D , Fleischman LF , Belkin AM ( 2000 ) Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin . J Cell Biol 148 : 825 - 838 .
22. Veevers-Lowe J , Ball SG , Shuttleworth A , Kielty CM ( 2011 ) Mesenchymal stem cell migration is regulated by fibronectin through alpha5beta1-integrin-mediated activation of PDGFR-beta and potentiation of growth factor signals . J Cell Sci 124 : 1288 - 1300 .
23. Summers L , Kielty C , Pinteaux E ( 2009 ) Adhesion to fibronectin regulates interleukin-1 beta expression in microglial cells . Mol Cell Neurosci 41 : 148 - 155 .
24. Barry ST , Flinn HM , Humphries MJ , Critchley DR , Ridley AJ ( 1997 ) Requirement for Rho in integrin signalling . Cell Adhes Commun 4 : 387 - 398 .
25. Ridley AJ ( 1999 ) Rho family proteins and regulation of the actin cytoskeleton . Prog Mol Subcell Biol 22 : 1 - 22 .
26. Uehata M , Ishizaki T , Satoh H , Ono T , Kawahara T , et al. ( 1997 ) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension . Nature 389 : 990 - 994 .
27. Kaibuchi K ( 1999 ) Regulation of cytoskeleton and cell adhesion by rho targets . In: Jeanteur P, editor. Progress in molecular and subcellular biology . New York ,: Springer-Verlag. 23 - 34 .
28. Kaufman PL , Erickson KA ( 1982 ) Cytochalasin B and D dose-outflow facility response relationships in the cynomolgus monkey . Invest Ophthalmol Vis Sci 23 : 646 - 650 .
29. Peterson JA , Tian B , McLaren JW , Hubbard WC , Geiger B , et al. ( 2000 ) Latrunculins' Effects on Intraocular Pressure , Aqueous Humor Flow, and Corneal Endothelium . Invest Ophthalmol Vis Sci 41 : 1749 - 1758 .
30. Tian B , Gabelt BT , Geiger B , Kaufman PL ( 1999 ) Combined effects of H-7 and cytochalasin B on outflow facility in monkeys . Exp Eye Res 68 : 649 - 655 .
31. Tian B , Kaufman PL , Volberg T , Gabelt BT , Geiger B ( 1998 ) H-7 disrupts the actin cytoskeleton and increases outflow facility . Arch Ophthalmol 116 : 633 - 643 .
32. Bidon N , Brichory F , Bourguet P , Le Pennec JP , Dazord L ( 2001 ) Galectin-8: a complex sub-family of galectins (Review) . Int J Mol Med 8 : 245 - 250 .
33. Levy Y , Auslender S , Eisenstein M , Vidavski RR , Ronen D , et al. ( 2006 ) It depends on the hinge: a structure-functional analysis of galectin-8, a tandemrepeat type lectin . Glycobiology 16 : 463 - 476 .
34. Diskin S , Kumar J , Cao Z , Schuman JS , Gilmartin T , et al. ( 2006 ) Detection of differentially expressed glycogenes in trabecular meshwork of eyes with primary open-angle glaucoma . Invest Ophthalmol Vis Sci 47 : 1491 - 1499 .
35. Pal Z , Antal P , Srivastava SK , Hullam G , Semsei AF , et al. ( 2012 ) Nonsynonymous single nucleotide polymorphisms in genes for immunoregulatory galectins: Association of galectin-8 (F19Y) occurrence with autoimmune diseases in a Caucasian population . Biochim Biophys Acta.
36. Dickerson JE , Jr., Steely HT , Jr., English-Wright SL , Clark AF ( 1998 ) The effect of dexamethasone on integrin and laminin expression in cultured human trabecular meshwork cells . Exp Eye Res 66 : 731 - 738 .
37. Stamer WD , Seftor RE , Williams SK , Samaha HA , Snyder RW ( 1995 ) Isolation and culture of human trabecular meshwork cells by extracellular matrix digestion . Curr Eye Res 14 : 611 - 617 .
38. Shiokawa S , Yoshimura Y , Sawa H , Nagamatsu S , Hanashi H , et al. ( 1999 ) Functional Role of Arg-Gly-Asp (RGD)-Binding Sites on 1 Integrin in Embryo Implantation Using Mouse Blastocysts and Human Decidua . Biol Reprod 60 : 1468 - 1474 .
39. Ni H , Wilkins JA ( 1998 ) Localisation of a novel adhesion blocking epitope on the human beta 1 integrin chain . Cell Adhes Commun 5 : 257 - 271 .
40. Dallabrida SM , Falls LA , Farrell DH ( 2000 ) Factor XIIIa supports microvascular endothelial cell adhesion and inhibits capillary tube formation in fibrin . Blood 95 : 2586 - 2592 .
41. Shen CX , Stewart S , Wayner E , Carter W , Wilkins J ( 1991 ) Antibodies to different members of the beta 1 (CD29) integrins induce homotypic and heterotypic cellular aggregation . Cell Immunol 138 : 216 - 228 .
42. Stupack DG , Stewart S , Carter WG , Wayner EA , Wilkins JA ( 1991 ) B lymphocyte fibronectin receptors: expression and utilization . Scand J Immunol 34 : 761 - 769 .
43. Wilde C , Aktories K ( 2001 ) The Rho-ADP-ribosylating C3 exoenzyme from Clostridium botulinum and related C3-like transferases . Toxicon 39 : 1647 - 1660 .