Open Access
Biochemistry and Molecular Biology  |   July 2016
PKC-ζ Regulates Thrombin-Induced Proliferation of Human Müller Glial Cells
Author Affiliations & Notes
  • Irene Lee-Rivera
    Instituto de Fisiología Celular Universidad Nacional Autónoma de México, México City, México
  • Edith López
    Instituto de Fisiología Celular Universidad Nacional Autónoma de México, México City, México
  • Miriam Gabriela Carranza-Pérez
    Instituto de Fisiología Celular Universidad Nacional Autónoma de México, México City, México
  • Ana María López-Colomé
    Instituto de Fisiología Celular Universidad Nacional Autónoma de México, México City, México
  • Correspondence: Ana María López-Colomé, Instituto de Fisiología Celular, UNAM, Apartado Postal 70-253, Ciudad Universitaria, México City 04510, Mexico; acolome@ifc.unam.mx
Investigative Ophthalmology & Visual Science July 2016, Vol.57, 3769-3779. doi:10.1167/iovs.16-19574
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      Irene Lee-Rivera, Edith López, Miriam Gabriela Carranza-Pérez, Ana María López-Colomé; PKC-ζ Regulates Thrombin-Induced Proliferation of Human Müller Glial Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(8):3769-3779. doi: 10.1167/iovs.16-19574.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: To investigate the effect of thrombin on the proliferation of human Müller glial cells (MCs) and define the possible signaling mechanisms involved in this process.

Methods: Protease-activated receptor (PARs 1–4) expression was analyzed using RT-PCR and Western blot in the MIO–M1 Müller cell line (MC). Müller cell proliferation was assessed by the MTS reduction method. Wound healing and immunoreactivity to Ki67 antigen were used to dissociate proliferation and migration. Cell migration was examined using transwell migration assays. The involvement of extracellular signal–regulated kinase (ERK1/2) phosphorylation/activation in thrombin-induced human MC proliferation was determined by Western blot. Intracellular pathways involved in ERK1/2 activation were analyzed by pharmacologic inhibition.

Results: We first demonstrated that human MCs express PARs 1 to 4. Our results show that thrombin dose-dependently stimulates MC proliferation by 44%, with a calculated Ec50 of 0.86 nM. Müller cell maximal proliferation required sustained thrombin treatment for 72 hours, in contrast to our previous findings in RPE cells showing maximal thrombin-induced proliferation at 24-hour stimulation. We demonstrate that thrombin induces MC cell proliferation through the Ras-independent activation of the Raf/MEK/ERK cascade, under the control of protein kinase C (PKC)-ζ.

Conclusions: The breakdown of blood–retina barrier (BRB) exposes MCs to thrombin contained in serum. Our findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.

Müller glial cells (MCs) are the main type of glia in the retina, and play a key role in the maintenance and survival of all neuronal cell types, as well as a central role in blood–retina barrier (BRB) maintenance. In pathological conditions, MCs modulate the inflammatory response and monitor the alteration of retinal physiology, from light-induced damage to pathologies such as glaucoma, diabetic retinopathy, macular degeneration, and retinal detachment.1,2 Despite improvement of surgical techniques, procedures aimed to control these diseases3 fail due to subretinal fibrosis, characterized by proliferation and/or infiltration of transdifferentiated RPE and glial cells.4 Particularly, the hypertrophy and growth of MCs onto retinal surfaces inhibit the regeneration of photoreceptors,5 and MC growth into the vitreous generates contractile cellular membranes that cause redetachment. In spite of this evidence, little is known regarding MC participation in retinal scarring or the mechanisms involved in MC proliferation. 
A prominent consequence of BRB breakdown due to retinal detachment or retinal surgery is the exposure of retinal cells to serine/threonine protease thrombin contained in serum. Under these conditions, Müller glia and retinal astrocytes release factors such as vascular endothelial growth factor (VEGF) and cytokines that contribute to further disrupt the BRB6 and to promote MC proliferation.7 Recent studies have shown that thrombin activity is increased in the vitreous from proliferative vitreoretinopathy (PVR) patients,8 suggesting thrombin involvement in the pathogenesis of PVR, characterized by the proliferation, dedifferentiation, and migration of MCs and transdifferentiation of RPE cells into the vitreous and the assembly of contractile cellular membranes on retinal surfaces, thus promoting retinal detachment.9 It has been previously shown that thrombin stimulates glial cell proliferation in a dose-dependent manner10,11 and that it has an inhibitory effect on inwardly rectifying K+ current (IK(IR)).12 
Intracellular thrombin signaling is triggered by the activation of protease-activated receptors (PARs), a family of G-protein–coupled receptors (GPCRs) activated by the proteolytic cleavage of the extracellular N-terminal domain, which unmasks a new N-sequence that functions as intramolecular ligand. Four members of this family have been identified: PAR 1, PAR 3, and PAR 4, activated by thrombin, and PAR 2, activated by trypsin and other serine proteases. Protease-activated receptor 1 is the prototype of this receptor family, and its cleavage at the Arg41–Ser42 bond by thrombin exposes a new N-terminus (S42FLLRN47) that acts as a tethered ligand.13 
Protease-activated receptors have been linked to the activation of a number of physiological responses by interacting with GPCR Gα subunits Gq11α, G12/13α, and Gαi. Protease-activated receptor 1 coupling to Gqα activates phospholipase C-β (PLC-β), with the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), and the activation of conventional/novel (c/n) protein kinase C (PKC). Giα inhibits adenylyl cyclase, while the Gβγ subunits activate phosphoinositide 3-kinase (PI3K) and other lipid-modifying enzymes, protein kinases, and ion channels. Finally, the α-subunit of G12/13 activates Rho guanosine triposphatase (GTPase) known to regulate the assembly and organization of the actin cytoskeleton.14 The activation of PAR 1 promotes signaling through mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase (ERK1/2), PKC, and phosphatidylinositol-3 kinase (PI3K) cascades, required for cyclin D1 expression and proliferation.15 Our previous work has shown that α-thrombin induces the proliferation of RPE cells through the joint activation of the MAPK and PKC-ζ pathways.16 
Müller human cell line MIO-M1 (Moorfields/Institute of Ophthalmology-Müller 1) has been shown to express Müller glial markers, such as cellular retinaldehyde-binding protein (CRALBP), epidermal growth factor receptor (EGF-R), vimentin, glutamine synthetase, and α-smooth muscle actin (α-SMA), and to exhibit the morphology of human MCs in primary culture, including polarization, the orientation of microtubules, and the presence of glycogen particles. MIO–M1 cells also exhibit the electrophysiological properties described for primary mammalian MCs.17 These morphologic and physiological features support MIO–M1 cells as a suitable model for studying MC function in vitro. 
The purpose of this study was to investigate the effect of thrombin on human MC proliferation using MIO–M1 cell line as a model. We first demonstrated that human MCs are sensitive to thrombin since they express PARs 1 to 4 at the mRNA and the protein level. Our results show that thrombin promotes in vitro wound healing by stimulating MC proliferation, but not MC migration. This effect was mediated by thrombin-induced ERK1/2 phosphorylation and required the promotion of PKC-ζ signaling. 
Materials and Methods
Reagents
All reagents used were cell culture grade. Thrombin, hirudin, manumycin, Ro-32-042, PKC-ζ pseudosubstrate, and Y27632 were purchased from Calbiochem/EMD Millipore (Billerica, MA, USA). D-Phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) was from Enzo Life Sciences (New York, NY, USA). Wortmannin and all other reagents were from Sigma-Aldrich Corp. (St. Louis, MO, USA). 
Cell Culture
MIO–M1 cells were a kind donation from G.A. Limb (Institute of Ophthalmology and Moorfields Eye Hospital, London, UK). Cells were grown as previously described.17 
Fetal bovine serum (FBS), 10%, was included only for cell propagation. For all assays, cultures were serum deprived for 24 hours prior to the experiment. 
The viability of MIO–M1 cells was assessed by trypan blue exclusion in cultures maintained in the presence (10%) or absence of serum for 24 hours and up to 8 days as previously described.15 After dissociating the cells with 0.1% tripsin–EDTA (Invitrogen, Carlsbad, CA, USA), trypan blue (0.08%) was added to cells in serum-free Dulbeco minimum essential medium (DMEM). Trypan-stained and live cells were counted by optical microscopy in a Neubauer chamber and viability was expressed as percentage of live cells with reference to the total number of cells. 
MIO–M1 Cell Proliferation Measurement
MIO–M1 cells were cultured as described above and stimulated with thrombin (0–4 U/mL) in serum-free minimum essential medium (MEM) for 24 hours and up to 7 days. Proliferation was quantified in nonconfluent cultures using the colorimetric MTS [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction method according to the manufacturer's instructions (Cell Titer 96 Aqueous One Solution Reagent; Promega, Madison, WI, USA); absorbance at 490/630 nm was corrected for background measurement. Cultures maintained in serum-free DMEM were used as negative control (Ctl), and their values were arbitrarily set as 100% (basal) proliferation. 
Cell Stimulation
MIO–M1 cells from semiconfluent six-well plates were serum deprived for 24 hours. The synchronized cultures were then stimulated with thrombin (2 U/mL) for the indicated time period. When tested, inhibitors were added to the medium 30 minutes prior to stimulation with 2 U/mL thrombin. Inhibitors were used at the following concentrations: hirudin, 4 U/mL; PPACK, 500 nM; pertussis toxin, 100 ng/mL; wortmannin, 100 nM; manumycin A, 20 μM; Ro32-4032, 10 μM; PKC-ζ pseudosubstrate, 25 μM; Rho kinase (ROCK) inhibitor Y27632, 10 μM. These concentrations have been previously shown not to be toxic.15,16,18 
Wound Healing Assays
MIO–M1 cells were grown to confluence in six-well plates (Costar; Corning, Inc., Washington, DC, USA) in DMEM containing 10% FBS. After serum deprivation for 24 hours, cell monolayers were scratched mechanically with a pipette tip drawing a 1.2-mm line. Scratched monolayers were washed with serum-free DMEM and incubated with thrombin (2 U/mL) for the indicated time period; DMEM containing 10% FBS and serum-free DMEM were used as positive or negative control wells, respectively. After incubation at 37°C (24 hours–5 days), cells were visually examined by phase-contrast microscopy (×100). For Ki-67 labeling experiments, cells were fixed in acetone. Slides were blocked with FBS 5% in PBS, washed, and incubated with anti-Ki67 antibody (Abcam, 16667; Cambridge, UK) 1:200 in PBS 0.3% Triton X-100 overnight. Secondary antibody was FITC anti-rabbit (Jackson Labs, 111-095-003; Sacramento, CA, USA). When indicated, MIO–M1 cells were preincubated with hirudin 4 U/mL or PPACK (500 nM) for 30 minutes at 37°C prior to stimulation with thrombin (2 U/mL in serum-free DMEM) for the indicated time period. Cytosine β-arabino-furanoside (10 μM) or RPE–conditioned medium (CM; conditioned medium from human-derived RPE cell line ARPE-19 stimulated with 2 U/mL thrombin for 24 hours) was added 30 minutes prior to thrombin stimulation, and was present throughout the duration of stimulation (24 hours–5 days). The ARPE-19 (American Type Culture Collection, CRL-2302; Bethesda, MD, USA) cell line was propagated in DMEM/F12 medium containing 4% FBS, as described previously.19 Thrombin-conditioned medium was prepared by incubating cells in serum-free DMEM for 24 hours and stimulating with thrombin (2 U/mL) for another 24 hours. 
Transwell Migration Assays
Assays were performed as previously described.20 The inferior surface of Boyden transwell migration inserts (Falcon Multiwell Cell Culture Plates; BD Biosciences, Franklin Lakes, NJ, USA) was coated with either collagen I (50 μg/mL) or collagen IV (10 μg/mL) for 1 hour at room temperature. Inserts without substrate were also used for migration assays as a control. Cell chemotaxis was induced by the inclusion of thrombin (2 U/mL), 10% FBS DMEM, or serum-free DMEM in the inferior chamber of the migration plate. After incubation for 48 hours at 37°C, nonmigrating cells were removed from the upper side of the membrane insert with a sterile cotton swab, and the cells that migrated to the inferior face of the membrane were stained with crystal violet 0.05%, photographed, and manually counted. Data were graphed as a percentage of control (basal levels). 
Quantitation of PAR (1–4) mRNA
Semiquantitative RT-PCR was used for measuring PAR (1–4) mRNA expression. Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Messenger RNA retrotranscription was performed using 1 μg (PAR 1–3) or 3 μg (PAR 4) total RNA, 20 U murine Moloney leukemia virus retrotranscriptase (MMLV-RT, Invitrogen), 1 U RNase inhibitor (RNAseout, Invitrogen), and 0.25 μg oligo dT primer (Invitrogen), following the manufacturer's recommendations. Complementary DNA (5 μL) was used as substrate for PCR. This reaction was performed using manufacturer's recommended buffer, 2.5 U recombinant Taq polymerase (Altaenzymes, Alberta, Canada), 0.4 μM each oligonucleotide and deoxynucleotide triphospate (dNTPs). Primers, MgCl2 concentrations, and annealing temperatures are shown in Table 1. Protease-activated receptors were amplified for 28 cycles and β-actin for 21 cycles. Densitometric analysis was performed using ImageJ software (http://imagej.nih.gov/ij/ [in the public domain]). Actin amplification was used as loading control; PAR expression was normalized to actin expression in the same sample. 
Table 1
 
Primers and PCR Conditions
Table 1
 
Primers and PCR Conditions
Western Blot Analysis
Following stimulation, samples were processed as described previously.20 Protein in the lysates was quantified using Bradford reagent, and 20 μg total protein was used for PAR expression analysis and 5 μg for ERK phosphorylation blots. Samples were resolved by 10% (PAR expression) or 12.5% (ERK phosphorylation) SDS/PAGE and electrotransferred onto polyvinyldiene difluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). After blocking for 1 hour at room temperature with 5% nonfat milk, 3% bovine serum albumin in Tween-Tris-buffered saline (TBS), the PVDF membranes were incubated overnight at 4°C as described in Table 2. Secondary HRP-conjugated antibodies (Invitrogen) were incubated for 1 hour and membranes were developed using the Immobilon Western AP Chemiluminescent Substrate (EMD Millipore, Billerica, MA, USA). Kodak film images were digitized using an Alpha Digi-Doc system (Alpha-Innotech, San Leandro, CA, USA), and densitometric analysis was performed using ImageJ software (http://imagej.nih.gov/ij/). Anti-β-actin was used as loading control, and expression was normalized in relation to these values. Data were graphed as a percentage of control (basal levels). 
Table 2
 
Western Blot Antibodies
Table 2
 
Western Blot Antibodies
Statistical Analysis
Results shown represent mean ± SEM. Raw data for analysis were obtained from at least three independent experiments as specified in the figure legends. Student's t-test was applied to results in which only two groups (negative control versus thrombin stimulation, or thrombin stimulation versus inhibitor) were compared. One-way ANOVA with Bonferroni's multiple comparison test was used to compare results within different groups. Prism V5.0 for MacOSX program from GraphPad (La Jolla, CA, USA) was used. 
Results
MIO–M1 Cells Express Protease-Activated Receptors
In order to ascribe thrombin effects to specific receptor activation, we analyzed the expression of PARs in MIO–M1 cells using RT-PCR (Figs. 1A, 1B) and Western blot (Fig. 1C). Our results showed that MIO–M1 cells express all types of PAR (1–4) receptors. 
Figure 1
 
PAR expression in Müller glial cells. mRNA expression in MIO–M1 cells was analyzed by RT-PCR using 1 μg RNA (A) PAR 1, PAR 2, and PAR 3, or 3 μg RNA (B) PAR 4. Representative gels are shown. Results are expressed as the mean ± SEM of three independent experiments. (C) PAR protein expression was assessed by Western blot. Results are expressed as the mean ± SEM of 11 independent experiments.
Figure 1
 
PAR expression in Müller glial cells. mRNA expression in MIO–M1 cells was analyzed by RT-PCR using 1 μg RNA (A) PAR 1, PAR 2, and PAR 3, or 3 μg RNA (B) PAR 4. Representative gels are shown. Results are expressed as the mean ± SEM of three independent experiments. (C) PAR protein expression was assessed by Western blot. Results are expressed as the mean ± SEM of 11 independent experiments.
MIO–M1 Cell Viability Is Not Affected by Serum Starvation
The viability of MIO–M1 cells was assessed by trypan blue exclusion in cultures maintained in the presence (10%) or absence of serum for 24 hours and up to 8 days as previously described.15 Comparison of cell viability in cultures containing serum and those deprived of serum for 24 hours or up to 8 days showed that starvation does not affect cell viability compared to control cultures in the presence of serum. Control cultures had 95 ± 1.7% viable cells, whereas cultures deprived of serum for 24 hours and up to 6 days had 90 ± 0.2% and 76 ± 8.65% viable cells following 8 days of serum deprivation. 
Thrombin Induces MIO–M1 Cell Proliferation
To investigate the possible role of thrombin in gliosis and PVR pathogenesis, we analyzed the effect of thrombin on MIO–M1 cell proliferation. Following serum deprivation for 24 hours, cells were stimulated with 0.1, 0.3, 0.5, 1, and 2 U/mL thrombin (0.88, 2.64, 4.4, 8.8, and 17.6 nM) in serum-free DMEM, and proliferation was assessed by MTS assay at 24, 48, and 72 hours and 5 days in the presence of thrombin. The slope of the curves in Figure 2A indicates that the rate of thrombin-induced proliferation is independent from the concentration of thrombin applied, although it depends on the duration of the stimulus. For all doses of thrombin, maximal stimulation was attained at 72 hours of stimulation and was sustained up to 5 days. An exception to the former was observed for stimulation with the lowest dose of thrombin (0.88 nM), possibly due to the fact that this dose corresponds to the EC50 value calculated for thrombin effect, as shown in Figure 2B. Saturation curve for thrombin-induced MC proliferation at 72 hours revealed a maximum increase in proliferation of 44% over the negative control, with a calculated Ec50 = 0.863 nM (Fig. 2B). The thrombin effect on proliferation was shown to be specific, since it was prevented by the thrombin inhibitors hirudin and PPACK (Fig. 2C). 
Figure 2
 
Thrombin induces specific dose-dependent proliferation in Müller glial cells. Cell proliferation was measured using an MTS reduction method from 24 hours to 5 days. Results are expressed as the mean ± SEM of 11 independent experiments. (A) Thrombin-induced proliferation rate does not change as a function of time, but the maximum level is attained 72 hours after stimulation (B). The maximum level of proliferation is 44% over control, and it is dose dependent with an Ec50 = 0.863 nM. (C). Thrombin-induced proliferation is specific, since it can be inhibited by thrombin inhibitors hirudin and PPACK. Student's t-test, with reference to control. *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test, with reference to thrombin-stimulated cells. #P < 0.001; Control, unstimulated cells; T, thrombin (2 U/mL); H, hirudin (4 U/mL); P, PPACK (100 nM). Results are expressed as the mean ± SEM of four independent experiments.
Figure 2
 
Thrombin induces specific dose-dependent proliferation in Müller glial cells. Cell proliferation was measured using an MTS reduction method from 24 hours to 5 days. Results are expressed as the mean ± SEM of 11 independent experiments. (A) Thrombin-induced proliferation rate does not change as a function of time, but the maximum level is attained 72 hours after stimulation (B). The maximum level of proliferation is 44% over control, and it is dose dependent with an Ec50 = 0.863 nM. (C). Thrombin-induced proliferation is specific, since it can be inhibited by thrombin inhibitors hirudin and PPACK. Student's t-test, with reference to control. *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test, with reference to thrombin-stimulated cells. #P < 0.001; Control, unstimulated cells; T, thrombin (2 U/mL); H, hirudin (4 U/mL); P, PPACK (100 nM). Results are expressed as the mean ± SEM of four independent experiments.
Thrombin Induces Wound Healing Through Proliferation, Not Migration
We next analyzed the possible effect of thrombin on Müller cell contribution to wound healing using MIO–M1 cell monolayers deprived of serum for 24 hours in an in vitro scratch assay. Visual inspection by light phase-contrast microscopy (×100) following the addition of 2 U/mL thrombin or 10% FBS (positive control) for 24 hours at 37°C revealed that thrombin-treated cells filled the gap in scratched monolayers by either proliferation or migration, to a lower extent than FBS control, whereas the scratched area remained cell free up to 5 days of incubation in negative control wells incubated in serum-free DMEM (Fig. 3A). Furthermore, we demonstrated that wound healing does not depend on soluble factors released by RPE cells, since the addition of thrombin-treated RPE-conditioned media had no effect. 
Figure 3
 
In vitro wound healing of MC is driven by proliferation but not by migration. (A) Thrombin-induced wound healing is driven by proliferation since it is prevented by cytosine β-arabino-furanoside and does not depend on soluble factors released by RPE cells. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); T+H, thrombin (2 U/mL) + hirudin (4 U/mL); T+P, thrombin (2 U/mL) + PPACK (100 nM); T+A, thrombin (2 U/mL) + cytosine β-arabino-furanoside (10 uM); CM, thrombin-treated RPE-conditioned medium. (B) MIO–M1 cells do not migrate in response to thrombin. Cells were allowed to migrate into the lower chamber, which was coated with either collagen I (Col I; 50 μg/mL) or collagen IV (Col IV; 10 μg/mL) for 48 hours. Noncoated inserts were used as a no-substrate control (w/o substrate). Representative fields are shown. (C) Migrating cells on the lower chamber were stained and counted in three independent experiments. ***P < 0.001; 1-way ANOVA with Bonferroni's multiple comparison test was used. Comparison was referenced to unstimulated control. #P < 0.001; comparison was referenced to collagen I or collagen IV. Results are expressed as the mean ± SEM of three independent experiments.
Figure 3
 
In vitro wound healing of MC is driven by proliferation but not by migration. (A) Thrombin-induced wound healing is driven by proliferation since it is prevented by cytosine β-arabino-furanoside and does not depend on soluble factors released by RPE cells. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); T+H, thrombin (2 U/mL) + hirudin (4 U/mL); T+P, thrombin (2 U/mL) + PPACK (100 nM); T+A, thrombin (2 U/mL) + cytosine β-arabino-furanoside (10 uM); CM, thrombin-treated RPE-conditioned medium. (B) MIO–M1 cells do not migrate in response to thrombin. Cells were allowed to migrate into the lower chamber, which was coated with either collagen I (Col I; 50 μg/mL) or collagen IV (Col IV; 10 μg/mL) for 48 hours. Noncoated inserts were used as a no-substrate control (w/o substrate). Representative fields are shown. (C) Migrating cells on the lower chamber were stained and counted in three independent experiments. ***P < 0.001; 1-way ANOVA with Bonferroni's multiple comparison test was used. Comparison was referenced to unstimulated control. #P < 0.001; comparison was referenced to collagen I or collagen IV. Results are expressed as the mean ± SEM of three independent experiments.
In order to ascribe thrombin-induced wound healing to either cell proliferation or cell migration, the proliferation inhibitor cytosine β-arabino-furanoside was included in the wound healing assay. As shown in Figure 3, addition of this nucleoside prevents both thrombin- and FBS-induced gap closure, thus suggesting that proliferation is indeed responsible for the observed wound healing. To further confirm this assumption, thrombin effect on MC migration was analyzed using Boyden transwell chambers (Figs. 3B, 3C). Results showed that thrombin was unable to induce MC cell migration, supporting the notion that thrombin induces MIO–M1 wound healing primarily by promoting cell proliferation but not cell migration. In this regard, we determined if either collagen I or collagen IV could influence MC migration. As can be observed in Figure 3C, neither substrate had an effect on thrombin-induced migration. Additionally, wound healing assays performed in culture plates coated with fibronectin (1 μg/mL) or laminin (2 μg/mL) (not shown) showed that, as found for collagen-coated or naked plates, no difference in migration of thrombin-stimulated and negative control cells was observed. 
We next investigated if MC proliferation was restricted to the border of the wound by labeling the cells with Ki-67, a nuclear protein widely used as a marker for cell proliferation.21 Figure 4 shows that 48 hours after scratching, Ki-67–positive cells were not restricted to the wound region, but randomly distributed within the field. These results indicate that thrombin-induced wound healing is not driven by juxtacrine signals exclusively present in the cells that limit the wound but is a rather general phenomenon, at least in vitro. 
Figure 4
 
Thrombin-induced proliferation is not limited to the edge of the wound. Ki67 labeling of cell cultures, wounded and treated with thrombin (2 U/mL) for 48 hours, shows that proliferation is not limited to those cells in the edge of the wound but is a rather general phenomenon. Representative fields are shown of three independent experiments. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; Thr, thrombin (2 U/mL).
Figure 4
 
Thrombin-induced proliferation is not limited to the edge of the wound. Ki67 labeling of cell cultures, wounded and treated with thrombin (2 U/mL) for 48 hours, shows that proliferation is not limited to those cells in the edge of the wound but is a rather general phenomenon. Representative fields are shown of three independent experiments. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; Thr, thrombin (2 U/mL).
Thrombin Induces Müller Cell Proliferation by Activating the MAPK Cascade
The extracellular signal–regulated kinase (ERK1/2) cascade is the prototype mammalian MAPK signaling cascade that regulates a number of processes, including proliferation and migration. In order to define the molecular mechanisms underlying thrombin stimulation of MC proliferation, we assessed the involvement of the MAPK signaling pathway by testing the effect of thrombin on ERK1/2 phosphorylation/activation. As shown in Figure 5A, thrombin stimulates the phosphorylation of ERK Thr202/Tyr204, thus activating this signaling pathway. Thrombin-induced ERK phosphorylation was fast, starting at 5 minutes and sustained up to 120 minutes in the presence of thrombin. As shown in Figure 5B, the MEK inhibitor U0126 induced the dose-dependent inhibition of thrombin-induced proliferation. Consistently, the inhibition of MEK by PD98059 (50 μM) had the same effect (results not shown). Thrombin induction of ERK phosphorylation was shown to be specific, since it was prevented by the thrombin inhibitors hirudin and PPACK (Fig. 5C). 
Figure 5
 
MEK/ERK signaling pathway is activated by thrombin in human MCs. (A) Time course of ERK1/2 activation (pThr202/Tyr204) by thrombin (2 U/mL). Thormbin-induced ERK1/2 activation was sustained up to 4 hours. (B) MEK was inhibited by U0126 (12.5–50 uM), demonstrating specificity of ERK1/2 phosphorylation induced by thrombin. (C) Specificity (thrombin inhibitors). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); Hir, thrombin 2 U/mL + hirudin 4 U/mL; PP, thrombin 2 U/mL + PPACK (100 nM). Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of three independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Figure 5
 
MEK/ERK signaling pathway is activated by thrombin in human MCs. (A) Time course of ERK1/2 activation (pThr202/Tyr204) by thrombin (2 U/mL). Thormbin-induced ERK1/2 activation was sustained up to 4 hours. (B) MEK was inhibited by U0126 (12.5–50 uM), demonstrating specificity of ERK1/2 phosphorylation induced by thrombin. (C) Specificity (thrombin inhibitors). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); Hir, thrombin 2 U/mL + hirudin 4 U/mL; PP, thrombin 2 U/mL + PPACK (100 nM). Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of three independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Signaling Pathways Involved in Thrombin-Induced ERK1/2 Activation
Extensive evidence has shown PAR 1 coupling to Giα, Gqα, and G12/13.13 Additionally, Gβγ subunit–induced activation of PI3K has also been demonstrated. Along this line, our previous work in RPE cells has characterized a PKC-ζ–dependent pathway, which promotes cyclin D1 expression,16,22 essential for cell proliferation. In order to identify the mechanisms involved in thrombin induction of MC proliferation, we analyzed the participation of these signaling pathways on ERK1/2 phosphorylation. We assessed signaling from adenylyl cyclase using pertussis toxin (PTX; Fig. 6A). Pertussis toxin catalyzes the ADP-ribosylation of the αi subunits of the heterotrimeric G-protein, preventing its interaction with GPCRs,23 in this case, with thrombin receptor. Rho kinase was inhibited using Rho kinase inhibitor Y27632, a selective inhibitor that interacts with ATP binding site in the catalytic site (Fig. 6B).24 Phosphoinositide 3-kinase was studied using wortmannin, a nonspecific, covalent inhibitor, and LY294002, a morpholine-containing chemical compound that, even though it is somewhat less potent than wortmannin, is a reversible inhibitor of PI3K whereas wortmannin acts irreversibly25 (Fig. 6C). Ras pathway was addressed by using manumycin, a farnesyltransferase inhibitor, which inhibits Ras incorporation to the cell membrane.26 The role of c/n PKC isoforms was investigated by using Ro32-4032, a selective cell-permeable PKC inhibitor that displays selectivity for conventional PKC isoforms over novel and atypical PKC isoforms27 (Fig. 6D). Results in Figure 6 show that thrombin-induced ERK1/2 phosphorylation was prevented exclusively by the inhibition of atypical PKC-ζ using a pseudosubstrate28 (Fig. 6D). 
Figure 6
 
Signaling pathways induced by thrombin involved in ERK1/2 activation. (A) Inhibition of adenylyl cyclase (AC). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); PTX, pertussis toxin (100 ng/mL). (B) Inhibition of Rho kinase. Y5, ROCK inhibitor Y27632, 5 mM; Y10, Y27632, 10 mM. (C) Inhibition of PI3K. LY, LY294002, 10 mM; W, wortmannin, 100 mM. (D) Inhibition of Ras, c/n PKC, and PKC-ζ. MA, manumycin, 20 mM; Ro, Ro32-4032, 10 mM; PKC-ζ pseudosubstrate, 25 mM. Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of five independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Figure 6
 
Signaling pathways induced by thrombin involved in ERK1/2 activation. (A) Inhibition of adenylyl cyclase (AC). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); PTX, pertussis toxin (100 ng/mL). (B) Inhibition of Rho kinase. Y5, ROCK inhibitor Y27632, 5 mM; Y10, Y27632, 10 mM. (C) Inhibition of PI3K. LY, LY294002, 10 mM; W, wortmannin, 100 mM. (D) Inhibition of Ras, c/n PKC, and PKC-ζ. MA, manumycin, 20 mM; Ro, Ro32-4032, 10 mM; PKC-ζ pseudosubstrate, 25 mM. Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of five independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Discussion
Proliferative eye diseases that eventually lead to blindness are an important cause of failure in surgery aimed at correcting retinal detachment.29 The pathogenesis of these diseases, such as PVR, involves distinct cell types, including RPE cells and MCs. Particularly, the transdifferentiation of RPE cells together with MC activation and hypertrophy onto the retinal surface is centrally involved in the formation of contractile epiretinal membranes.30 In fact, subretinal glial scars have been estimated to occur in 15.7% of all human retinal detachments. Despite numerous attempts to reduce the incidence of these diseases, no effective pharmacologic treatment has been found to date. 
Thrombin promotes the proliferation of a variety of cell types through the activation of PARs.31 The present study demonstrates, for the first time, that human Müller cells express the protease-activated receptors PAR 1, 2, 3, and 4 (Fig. 1) required for thrombin promotion of intracellular signaling. 
Our results show that thrombin induces the proliferation of human MIO–M1 cells by 44% over control values. However, in contrast with the early onset of thrombin-induced proliferation of RPE cells (which peaks at 24 hours),20 the induction of MIO–M1 cell proliferation reached maximum levels 4 days post stimulation (Fig. 2A), in keeping with previous findings.10 These in vitro data are consistent with in vivo studies in a model of retinal detachment showing that MC proliferation peaks at 3 to 4 days after detachment, and continues at a low rate for as long as the retina remains detached.32 Importantly, we demonstrated that human MCs are highly sensitive to thrombin, since the calculated EC50 for proliferation was 0.863 nM, 10 times lower than the normal prothrombin concentration in adult human blood (Fig. 2B),33 which would allow thrombin induction of proliferation upon the brief exposure to blood during retinal detachment surgery. 
The in vitro analysis of MC contribution to wound repair, measured as the capacity to fill the gap in scratch assays, showed that thrombin promotes wound closure through the stimulation of MC proliferation (Fig. 3A), since experiments in Boyden chambers clearly demonstrated that MCs do not migrate in response to thrombin (Figs. 3B, 3C). It is noticeable that, in contrast to our findings in RPE cells,20 MC proliferation was not induced by cytokines or growth factors known to be released by RPE upon wounding but is directly promoted by PAR activation, since RPE-conditioned media had no effect on in vitro wound healing. 
Evidence has been provided showing that upon retinal detachment, MC nuclei migrate within cell processes at the outer retina, incorporate bromodeoxyuridine, divide, and form the glial scars characteristic of retinal detachment.34 Our present results are consistent with these findings, since results from both studies show that thrombin induces MC proliferation but not migration. It is noticeable, therefore, that the antiproliferative agent 5-fluorouracil (5-FU) did not significantly reduce the occurrence of PVR in human patients.35 However, this result could be attributed to the short duration of drug application, restricted to the surgical removal of scar tissue or, alternatively, to the very short half-life of antiproliferative drugs used in clinical eye procedures. Furthermore, studies in a rabbit model of retinal detachment showed that the intravitreal administration of long–half-life antiproliferative compounds into the eye at the time of detachment surgery effectively reduced both intraretinal proliferation and the formation of subretinal glial scars.30,36 These studies, together with our present results, support the assumption that thrombin stimulation of MC proliferation, in conditions that alter the BRB, could contribute significantly to the formation of subretinal glial scars. 
Müller cells are known to release a number of growth factors and cytokines in order to restore homeostasis upon injury.3739 Results from in vivo studies indicate that MCs become Ki-67 positive and proliferate in response to insults such as retinal detachment, laser injury, or the formation of macular holes36,40,41; however, the juxtacrine or paracrine origin of signals that trigger proliferation has not been determined. In the present study we assessed the location of Ki-67 antibody–labeled cells following thrombin stimulation (Fig. 4). We found that proliferating cells are not restricted to the wound boundary but are randomly distributed among the cell population, suggesting that proliferation is likely induced by the release of diffusible factors by MCs into the culture media. 
Mitogen-activated protein kinase signaling is known to play a central role in cell proliferation. Our results show that thrombin induces a fast increase in ERK1/2 phosphorylation (Fig. 5A). Analysis of the G-protein–linked intracellular pathways involved in ERK1/2 activation showed that neither adenylyl cyclase (Fig. 6A) nor the Rho/ROCK pathways (Fig. 6B) are responsible for thrombin-induced ERK1/2 phosphorylation. 
The canonical signaling pathway leading to MEK/ERK signaling requires the activation of the monomeric GTPase Ras and the downstream activation of the serine/threonine kinase Raf-1, which subsequently activates MEK/ERK1/2.42 In keeping with our previous findings in RPE cells, we demonstrated that thrombin promotion of ERK phosphorylation in human MCs bypasses Ras since it is not prevented by manumycin (Fig. 6D), which suppresses the membrane anchoring and activation of Ras.16,43 In this regard, in contrast with the noncanonical Ras-independent phosphorylation of ERK1/2 in RPE cells and intestinal epithelial cells, shown to depend on cPKC or nPKC isoenzyme activity,16,44 we found that thrombin-induced activation of ERK1/2 and proliferation in MCs does not require c/n PKC activity but depends on atypical PKC-ζ activation (Fig. 6D). Since G-βγ/ PI3K is a well-known activator of PKC-ζ,45,46 we examined the effect of PI3K inhibition on thrombin-induced ERK phosphorylation (Fig. 6C). Results demonstrate that, unlike our observations in RPE cells showing that ERK phosphorylation is driven by PI3K/ PKC-ζ activity,16 thrombin-induced ERK1/2 phosphorylation in MCs does not require PI3K activity. As an alternative mechanism, the activation of MEK/ERK could be elicited by the direct activation of Raf-1 by PKC-ζ,44 which in turn could be mediated by the phosphorylation of 14-3-3 scaffold protein by PKC-ζ, since 14-3-3 binds both PKC-ζ and Raf-1 in a ternary complex.47 
Collectively, these findings suggest a cell-specific control of molecular pathways leading to proliferation within the eye and further support an important role for thrombin in the pathogenesis of PVR induced by injury or retinal surgery. 
Acknowledgments
The authors thank G.A. Limb (University College London, UK) for the donation of MIO–M1 cells and Sergio Jaime-Rodríguez for technical assistance. 
Supported in part by Grants 254333 from Consejo Nacional de Ciencia y Tecnologia (CONACyT) and IN20015 from Programa de Apoyo a Proyectos de Investigación e Inovación Tecnoloógica (PAPIIT) to AML-C. 
Disclosure: I. Lee-Rivera, None; E. López, None; M.G. Carranza-Pérez, None; A.M. López-Colomé, None 
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Figure 1
 
PAR expression in Müller glial cells. mRNA expression in MIO–M1 cells was analyzed by RT-PCR using 1 μg RNA (A) PAR 1, PAR 2, and PAR 3, or 3 μg RNA (B) PAR 4. Representative gels are shown. Results are expressed as the mean ± SEM of three independent experiments. (C) PAR protein expression was assessed by Western blot. Results are expressed as the mean ± SEM of 11 independent experiments.
Figure 1
 
PAR expression in Müller glial cells. mRNA expression in MIO–M1 cells was analyzed by RT-PCR using 1 μg RNA (A) PAR 1, PAR 2, and PAR 3, or 3 μg RNA (B) PAR 4. Representative gels are shown. Results are expressed as the mean ± SEM of three independent experiments. (C) PAR protein expression was assessed by Western blot. Results are expressed as the mean ± SEM of 11 independent experiments.
Figure 2
 
Thrombin induces specific dose-dependent proliferation in Müller glial cells. Cell proliferation was measured using an MTS reduction method from 24 hours to 5 days. Results are expressed as the mean ± SEM of 11 independent experiments. (A) Thrombin-induced proliferation rate does not change as a function of time, but the maximum level is attained 72 hours after stimulation (B). The maximum level of proliferation is 44% over control, and it is dose dependent with an Ec50 = 0.863 nM. (C). Thrombin-induced proliferation is specific, since it can be inhibited by thrombin inhibitors hirudin and PPACK. Student's t-test, with reference to control. *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test, with reference to thrombin-stimulated cells. #P < 0.001; Control, unstimulated cells; T, thrombin (2 U/mL); H, hirudin (4 U/mL); P, PPACK (100 nM). Results are expressed as the mean ± SEM of four independent experiments.
Figure 2
 
Thrombin induces specific dose-dependent proliferation in Müller glial cells. Cell proliferation was measured using an MTS reduction method from 24 hours to 5 days. Results are expressed as the mean ± SEM of 11 independent experiments. (A) Thrombin-induced proliferation rate does not change as a function of time, but the maximum level is attained 72 hours after stimulation (B). The maximum level of proliferation is 44% over control, and it is dose dependent with an Ec50 = 0.863 nM. (C). Thrombin-induced proliferation is specific, since it can be inhibited by thrombin inhibitors hirudin and PPACK. Student's t-test, with reference to control. *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test, with reference to thrombin-stimulated cells. #P < 0.001; Control, unstimulated cells; T, thrombin (2 U/mL); H, hirudin (4 U/mL); P, PPACK (100 nM). Results are expressed as the mean ± SEM of four independent experiments.
Figure 3
 
In vitro wound healing of MC is driven by proliferation but not by migration. (A) Thrombin-induced wound healing is driven by proliferation since it is prevented by cytosine β-arabino-furanoside and does not depend on soluble factors released by RPE cells. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); T+H, thrombin (2 U/mL) + hirudin (4 U/mL); T+P, thrombin (2 U/mL) + PPACK (100 nM); T+A, thrombin (2 U/mL) + cytosine β-arabino-furanoside (10 uM); CM, thrombin-treated RPE-conditioned medium. (B) MIO–M1 cells do not migrate in response to thrombin. Cells were allowed to migrate into the lower chamber, which was coated with either collagen I (Col I; 50 μg/mL) or collagen IV (Col IV; 10 μg/mL) for 48 hours. Noncoated inserts were used as a no-substrate control (w/o substrate). Representative fields are shown. (C) Migrating cells on the lower chamber were stained and counted in three independent experiments. ***P < 0.001; 1-way ANOVA with Bonferroni's multiple comparison test was used. Comparison was referenced to unstimulated control. #P < 0.001; comparison was referenced to collagen I or collagen IV. Results are expressed as the mean ± SEM of three independent experiments.
Figure 3
 
In vitro wound healing of MC is driven by proliferation but not by migration. (A) Thrombin-induced wound healing is driven by proliferation since it is prevented by cytosine β-arabino-furanoside and does not depend on soluble factors released by RPE cells. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); T+H, thrombin (2 U/mL) + hirudin (4 U/mL); T+P, thrombin (2 U/mL) + PPACK (100 nM); T+A, thrombin (2 U/mL) + cytosine β-arabino-furanoside (10 uM); CM, thrombin-treated RPE-conditioned medium. (B) MIO–M1 cells do not migrate in response to thrombin. Cells were allowed to migrate into the lower chamber, which was coated with either collagen I (Col I; 50 μg/mL) or collagen IV (Col IV; 10 μg/mL) for 48 hours. Noncoated inserts were used as a no-substrate control (w/o substrate). Representative fields are shown. (C) Migrating cells on the lower chamber were stained and counted in three independent experiments. ***P < 0.001; 1-way ANOVA with Bonferroni's multiple comparison test was used. Comparison was referenced to unstimulated control. #P < 0.001; comparison was referenced to collagen I or collagen IV. Results are expressed as the mean ± SEM of three independent experiments.
Figure 4
 
Thrombin-induced proliferation is not limited to the edge of the wound. Ki67 labeling of cell cultures, wounded and treated with thrombin (2 U/mL) for 48 hours, shows that proliferation is not limited to those cells in the edge of the wound but is a rather general phenomenon. Representative fields are shown of three independent experiments. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; Thr, thrombin (2 U/mL).
Figure 4
 
Thrombin-induced proliferation is not limited to the edge of the wound. Ki67 labeling of cell cultures, wounded and treated with thrombin (2 U/mL) for 48 hours, shows that proliferation is not limited to those cells in the edge of the wound but is a rather general phenomenon. Representative fields are shown of three independent experiments. FBS, fetal bovine serum 10%; Ctl, unstimulated cells; Thr, thrombin (2 U/mL).
Figure 5
 
MEK/ERK signaling pathway is activated by thrombin in human MCs. (A) Time course of ERK1/2 activation (pThr202/Tyr204) by thrombin (2 U/mL). Thormbin-induced ERK1/2 activation was sustained up to 4 hours. (B) MEK was inhibited by U0126 (12.5–50 uM), demonstrating specificity of ERK1/2 phosphorylation induced by thrombin. (C) Specificity (thrombin inhibitors). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); Hir, thrombin 2 U/mL + hirudin 4 U/mL; PP, thrombin 2 U/mL + PPACK (100 nM). Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of three independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Figure 5
 
MEK/ERK signaling pathway is activated by thrombin in human MCs. (A) Time course of ERK1/2 activation (pThr202/Tyr204) by thrombin (2 U/mL). Thormbin-induced ERK1/2 activation was sustained up to 4 hours. (B) MEK was inhibited by U0126 (12.5–50 uM), demonstrating specificity of ERK1/2 phosphorylation induced by thrombin. (C) Specificity (thrombin inhibitors). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); Hir, thrombin 2 U/mL + hirudin 4 U/mL; PP, thrombin 2 U/mL + PPACK (100 nM). Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of three independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Figure 6
 
Signaling pathways induced by thrombin involved in ERK1/2 activation. (A) Inhibition of adenylyl cyclase (AC). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); PTX, pertussis toxin (100 ng/mL). (B) Inhibition of Rho kinase. Y5, ROCK inhibitor Y27632, 5 mM; Y10, Y27632, 10 mM. (C) Inhibition of PI3K. LY, LY294002, 10 mM; W, wortmannin, 100 mM. (D) Inhibition of Ras, c/n PKC, and PKC-ζ. MA, manumycin, 20 mM; Ro, Ro32-4032, 10 mM; PKC-ζ pseudosubstrate, 25 mM. Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of five independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Figure 6
 
Signaling pathways induced by thrombin involved in ERK1/2 activation. (A) Inhibition of adenylyl cyclase (AC). FBS, fetal bovine serum 10%; Ctl, unstimulated cells; T2, thrombin (2 U/mL); PTX, pertussis toxin (100 ng/mL). (B) Inhibition of Rho kinase. Y5, ROCK inhibitor Y27632, 5 mM; Y10, Y27632, 10 mM. (C) Inhibition of PI3K. LY, LY294002, 10 mM; W, wortmannin, 100 mM. (D) Inhibition of Ras, c/n PKC, and PKC-ζ. MA, manumycin, 20 mM; Ro, Ro32-4032, 10 mM; PKC-ζ pseudosubstrate, 25 mM. Representative Western blots are shown. Values from nonstimulated cells were arbitrarily set as 100% (Ctl). Results are expressed as the mean ± SEM of five independent experiments. Student's t-test with reference to unstimulated control; *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test with reference to thrombin stimulation, @@@P < 0.0005.
Table 1
 
Primers and PCR Conditions
Table 1
 
Primers and PCR Conditions
Table 2
 
Western Blot Antibodies
Table 2
 
Western Blot Antibodies
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