July 2008
Volume 49, Issue 7
Free
Cornea  |   July 2008
Association of Protein Tyrosine Phosphatases (PTPs)-1B with c-Met Receptor and Modulation of Corneal Epithelial Wound Healing
Author Affiliations
  • Azucena Kakazu
    From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana.
  • Guru Sharma
    From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana.
  • Haydee E. P. Bazan
    From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana.
Investigative Ophthalmology & Visual Science July 2008, Vol.49, 2927-2935. doi:10.1167/iovs.07-0709
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      Azucena Kakazu, Guru Sharma, Haydee E. P. Bazan; Association of Protein Tyrosine Phosphatases (PTPs)-1B with c-Met Receptor and Modulation of Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2008;49(7):2927-2935. doi: 10.1167/iovs.07-0709.

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

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Abstract

purpose. The purpose of this study was to investigate the expression and activity of protein tyrosine phosphatases (PTPs) in epithelium during corneal wound healing and to investigate how PTPs regulate activation of the c-Met receptor and the receptor’s proximal signaling.

methods. Rabbit corneas were injured by gentle scraping of the surface, leaving the limbal epithelium intact, and epithelium was collected at 1, 2, 3, and 7 days after injury. In organ culture models, epithelium was removed and corneas were incubated with hepatocyte growth factor (HGF), with or without the PTP inhibitor bpV(phen), and the PI-3K inhibitors wortmannin and LY294002. Human corneal epithelial (HCE) cells were stimulated with HGF with or without bpV(phen). Total cell lysates and cytosolic and membrane fractions were analyzed by Western blot. PTP activities were measured with specific substrates. PTP1B and SHP-2 genes were knocked down by interference RNA (siRNA).

results. PTP activity and expression increased during wound healing. The most abundant were SHP-2, PTP1B, and PTEN. HGF activated the c-Met receptor in HCE cells up to 30 minutes and was downregulated by 2 hours. Inhibition of PTPs increased HGF-promoted wound healing, HGF-activated phosphorylation of c-Met, and its downstream signal PI-3K/Akt, but not ERK1/2 or p70S6K. PTP1B and SHP-2 were bound to the c-Met. Part of the c-Met was colocalized in the endoplasmic reticulum with PTP1B. PTP1B phosphorylation increased when the c-Met receptor was deactivated, and gene knockdown of PTP1B increased c-Met activation. SHP-2 phosphorylation and binding to c-Met was higher during receptor activation, and SHP-2 gene silencing decreased receptor phosphorylation.

conclusions. Inhibition of PTP activity mimics the effect of HGF by activating the PI-3K/Akt signal involved in wound healing. PTP1B and SHP-2 are bound to the c-Met receptor to control its activity. Although the binding of PTP1B increases when there is a decrease in c-Met activation and acts as a negative regulator of the receptor, the increased binding and phosphorylation of SHP-2 coincide with maximal stimulation of c-Met, acting as a positive regulator.

Acentral theme in corneal epithelial repair is how growth factors modulate the complex, highly interactive wound healing process. 1 2 3 4 5 Regulation of cell proliferation, migration, adhesion, and apoptosis is fundamental to obtaining an adequate repair of the epithelium and to maintaining corneal transparency. Growth factors exert their action through binding to receptor tyrosine kinases (RTKs) that signal through lipid and protein kinases by specific phosphorylation–dephosphorylation reactions that will modulate overall wound healing. RTKs contain an N-terminal extracellular binding protein, a transmembrane domain, and a cytosolic C-terminal region with tyrosine kinase activity. In addition, many RTKs are coupled to a variety of adaptor proteins that enhance their responses. 6 One of these RTKs is the c-Met receptor, whose ligand is hepatocyte growth factor (HGF). HGF is a paracrine growth factor that is released by corneal stroma cells and the lacrimal gland after cornea injury and that acts on the c-Met in epithelial cells. 7 8  
Our previous studies showed that HGF activates a phosphatidylinositol-3 kinase (PI-3K)/Akt pathway involved in wound healing and survival 9 10 and the specific mitogen-activated kinases ERK1/2 and p38, which are important in epithelial cell proliferation and migration, respectively. 11 Very recently, we found that PKCα and PKCε are also activated by HGF and are involved in the wound healing response of epithelial cells. 12 Therefore, the activity of c-Met must be tightly regulated to maintain normal cellular responses. Aberrant dysfunction of the receptor could be responsible for disorders in epithelial repair. In fact, during corneal wound healing, the activation of PI-3K signaling is maintained for some time and then switched off, probably to avoid overactivation. 13 One set of mechanisms that regulate cell signaling is protein tyrosine phosphatases (PTPs), which are enzymes that catalyze the dephosphorylation of tyrosine-phosphorylated proteins. 14 15 16 17 PTPs can function as negative or positive regulators of signaling triggered by RTK. The PTPs constitute a very large family of phosphatases that are broadly classified into transmembrane, or receptor-like, and nontransmembrane, or non-receptor, PTPs. They are differentiated by their noncatalytic segments important for their cellular targeting. Nonreceptor PTPs are also structurally diverse, which allows them to target specific subcellular locations, including the cytosol, the plasma membrane, and the endoplasmic reticulum. They are also further divided according to their substrate specificity: tyrosine-specific PTPs (such as PTP1B, PTP1C [also known as SHP-1], and PTP1D [also known as SHP-2]); dual-specific phosphatases, which have catalytic action in both phospho-Tyr and phospho-Ser or Thr (including enzymes such as mitogen-activated protein kinase [MAPK] phosphatases [MKPs], and Cdc25 15 ); and the multiple-specific phosphatases (MSPs), which have additional substrate specificities (such as PTEN). The specificity of the PTPs relies also in the recognition of certain phosphopeptides for their action and in the selective expression of individual PTPs in cells and organelles. 
There is not much information on PTPs in the cornea, except for a report on the expression of the transmembrane PTP LAR in stroma cells of patients with keratoconus, 18 a few more recent studies on the expression of some PTPs in endothelial cells that may be involved in maintaining the cells in a nonproliferative state, 19 20 and the association between changes in MKP-1 expression and migratory responses to epithelial growth factor (EGF). 21 Information on how PTPs are involved in the regulation of the c-Met receptor in the corneal epithelium is not available. In this study, we found that the corneal epithelium expresses a selective pattern of PTPs during wound healing. Inhibition of PTPs increases c-Met activation and cell signaling stimulated by HGF and the rate of epithelial wound healing. Two nonreceptor PTPs, PTP1B and SHP-2, increase their expression after epithelial injury and are bound to the c-Met receptor. Although PTP1B is a negative regulator of c-Met activation, SHP-2 acts as an activator of the HGF receptor. 
Materials and Methods
Materials
Human recombinant double-chain HGF was a gift from Genentech (San Francisco, CA). The monoclonal antibodies PTP1B, SHP-1, SHP-2, LAR, and KAP and the secondary antibodies against mouse and rabbit conjugated horseradish peroxidase (HRP) were purchased from BD Biosciences PharMingen (San Diego, CA). The mouse monoclonal antibodies c-Met, phosphotyrosine (PY) clone 4G10, the phosphorylated form of Akt (Ser 437), the rabbit polyclonal antibodies against the p85α subunit of PI-3K, and the phosphorylated form of Met (Tyr 1234, Tyr 1235) were purchased from Upstate (Charlottesvile, VA). The rabbit polyclonal antibodies PTP1B, SHP-2, ERK1, the mouse monoclonal antibody of the phosphorylated form of p70S6K at Ser411, the mouse monoclonal PTEN and GAPDH antibodies, the PTP1B substrate, and the protein A or G agarose were from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal antibody SHP-2 in its phosphorylated form at Tyr542 and the biotinylated protein marker detection kit were from Cell Signaling Technology (Beverly, MA). The mouse monoclonal active form of ERK1/2, sodium orthovanadate (SOV), and phenyl arsine oxide were purchased from Sigma-Aldrich (St. Louis, MO). Inhibitors bpV(phen), LY294002, and wortmannin were from Calbiochem (La Jolla, CA). Another peptide inhibitor was purchased from Biomol (CinnGEL 2Me; Plymouth Meeting, PA). Validated PTP1B siRNA, validated SHP-2 siRNA, negative siRNA 1, and siPORT Neo FX transfection reagent were purchased from Ambion (Austin, TX). Mouse monoclonal (RL90) to protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) marker, was from Abcam (Cambridge, MA). Secondary antibodies used for immunostaining, goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 546, were from Invitrogen (Carlsbad, CA). 
Corneal Epithelial Wound Healing
In Vivo Model.
New Zealand albino rabbits of both sexes, weighing between 2 and 3 kg each, were used. The animals were treated in compliance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the experimental protocol was approved by the Institutional Animal Care and Use Committee, Louisiana State University Health Sciences Center. Rabbits were anesthetized with a mixture of ketamine/xylazine injected intramuscularly. The corneal epithelium was gently scraped with a sterile blade, leaving the limbal epithelium intact and enabling the stem cells to differentiate, migrate, and proliferate. This epithelium was used as control. Newly generated epithelial cells were removed at 1, 2, 3, and 7 days after injury by gentle scraping of the surface of the cornea and were then homogenized. At the end of each time point, the rabbits were killed with an overdose of pentobarbital. 
Corneal Organ Culture Wound Healing Model.
Rabbit eyes were obtained from Pel-Freez Biologicals (Roger, AR). The central part of the cornea was marked with a 7-mm surgical trephine, and the epithelium was removed with a battery-operated device (Algerbrush II; Alger Co., Lago Vista, TX), as previously described. 11 Wounded corneas were excised with a 1-mm rim and were positioned in a six-well plate on sterile nonstick balls (12-mm diameter). 9 The wells were filled to the cornea rim with Dulbecco modified Eagle medium/F12 medium with HGF (40 ng/mL), with or without the PTP inhibitor bpV(phen) (6 μM). In some wells, the PI-3K inhibitors 200 nM wortmannin and 20 μM LY294002 were added. Corneas incubated without HGF and inhibitors were used as 24-hour controls. To evaluate the wound healing, the corneas were stained with Alizarin red. 9 Photographs were taken on a dissecting microscope with an attached camera and were recorded by Adobe Photoshop software. Uncovered areas were analyzed by a computer digitizer and an image analysis program. 
PTP1B and SHP-2 siRNA Transfections
Human corneal epithelial (HCE) cells were transfected with PTP1B siRNA, SHP-2 siRNA, and negative control according to the manufacturer’s protocol. Briefly, for each well in a six-well plate, 5 μL siPORT NeoFX transfection reagent was diluted with 100:1 medium (Opti-MEM; Invitrogen) and incubated for 10 minutes at room temperature. Fifty picomoles each siRNA (PTP1B, SHP-2, and negative control) was diluted in 100:1 of the same medium. Both solutions were mixed together and incubated for an additional 10 minutes at room temperature to ensure the formation of siRNA-transfection reagent complex, and then the mixture was dispensed into the well. HCE cells suspended in KGM (2.3 × 105 cells/well) were overlaid on the transfection complexes. The plate was gently rocked back and forth to distribute the cells evenly into the complexes and was incubated at 37°C for 24 hours. The medium was replaced with fresh KGM and further incubated for 48 hours. For the experiments, cells were starved overnight and either treated or not treated with 4 or 6 μM bpV(phen) for 30 minutes before stimulation with 40 ng/mL HGF for 15 minutes or 2 hours. 
Cell Culture
HCE cells were used in this study. The HCE cell line was kindly provided by Roger Beuerman (LSU Eye Center, New Orleans, LA). The cell line was established using an HPV16-E6E7 vector, 22 maintained as previously described, 11 and used between passages 25 and 45. These cells responded to growth factors in a manner similar to the response of the primary cultures. 10 11 12 23 When the cells reached 80% to 90% confluence, they were switched to keratinocyte basal medium (KBM, Cambrex, Walkersville, MD) for 20 hours. Cells were then stimulated with 40 ng/mL HGF. In some experiments, the PTP inhibitor bpV(phen) was added before HGF. 
Cell Fractionation
Cells collected from culture or from rabbit corneas wounded in vivo were homogenized using a glass–glass homogenizer in 20 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol (DTT), 10 μg/mL leupeptin, 25 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.25 M sucrose and then centrifuged at 100,000g for 1 hour at 4°C. The supernatant, referred to as the cytosolic fraction, was collected, and the pellet was resuspended in the same buffer containing 1% Triton X-100, stirred for 1 hour at 4°C, and centrifuged as described. This supernatant is referred to as the membrane fraction. 
Immunoprecipitation and Western Blot
HCE cells were lysed in 50 mM Tris-HCl, pH 7.6, 5 mM EGTA, 1 mM sodium orthovanadate, 1% Triton X-100, 5 mM NaF, 1 mM sodium pyrophosphate, 5 mM β-glycerol-phosphate, 150 mM NaCl, 2 mM DTT, 10 μg/mL leupeptin, 25 μg/mL aprotinin, 1 μM microcystin LR, and 1 mM PMSF (lysis buffer) and were homogenized as described. The homogenate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and the pellet was discarded. 
HCE cell extracts containing 1 mg protein were immunoprecipitated by incubation with antibodies for c-Met or phosphotyrosine (PY) for 2 hours at 4°C. Immunocomplexes were captured with protein A or G agarose by overnight incubation. Agarose beads were collected by centrifugation and washed four times with lysis buffer. Immunoprecipitates and cell extracts or cell fractions were resolved by SDS-PAGE using precast gels from Invitrogen (4%–12%) and were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham, Piscataway, NJ). Biotinylated protein molecular weight standards were applied in one line of each gel. The membranes were blocked with 5% milk in Tris-buffered saline (TBS; 20 mM Tris-Cl, 150 mM NaCl, pH 7.6) plus 0.1% Tween-20 for 1 hour and then probed with specific primary antibodies, as described in Results, by incubation for 2 hours at room temperature or overnight at 4°C. Membranes were washed with the same buffer and were further incubated with appropriate HRP-conjugated secondary antibodies. To confirm similar loading, in appropriate experiments, the membranes were reprobed with control antibodies. Protein bands were visualized using chemiluminescence detection reagents (ECL-Plus; Amersham) and were quantified by densitometry. 
Immunofluorescence Staining
HCE cells were seeded in four-well glass slides and, on reaching 50% to 60% confluence, were starved for 24 hours in KBM. Cells were stimulated with HGF (40 ng/mL) for 15 minutes. In some experiments, the cells were pretreated for 30 minutes with 10 μM bpV(phen) before the growth factor was added. The cells were fixed with 2% paraformaldehyde for 30 minutes, then blocked with 10% normal goat serum and 1% BSA in phosphate-buffered saline (PBS) for 30 minutes and incubated overnight at 4°C with the primary antibodies. Next, the cells were incubated with the appropriate secondary antibodies. After each step, the slides were washed three times with PBS. No staining was observed when the primary antibody was omitted. Hoechst staining was performed to localize the nuclei. Cells were examined with a Nikon (Tokyo, Japan) fluorescence microscope (Eclipse TE 200), and the images were captured with a Photometrics camera (Cool Snap HQ; Tucson, AZ). 
Protein Tyrosine Phosphatase Assay
In a 96-well microtiter plate (half-area plate), 1 to 5 μg protein from HCE cell fractions were incubated for 30 minutes at 30°C with the PTP substrate (RRLIEDAEpYAARG) or with the specific substrate for PTP1B (DADEpYIPQQG) in a final volume of 25 μL assay buffer (25 mM HEPES, pH 7.2, 50 mM NaCl, 5 mM DTT, and 2.5 mM EDTA). For PTP1B assays, inhibitors were added at the concentrations specified. The reaction was stopped by the addition of 100 μL Malachite green solution. After a 15-minute interval for color development, activity was determined by reading the absorbency at 630 nm in a microtiter plate reader. Blanks were prepared without substrate. Reaction times and concentrations of substrate and protein were tested in advance to ensure the linearity of phosphate detection. 
Results
Changes in PTP Expression and Activity during Corneal Epithelial Wound Healing In Vivo
We first investigated whether changes occurred in PTP activity during corneal epithelial wound healing. Rabbit corneas were injured, as described in Materials and Methods, and tissue was collected 48 hours after wounding; cytosolic and membrane fractions were isolated as described in Materials and Methods. The activity of the total PTPs was determined using a tyrosine phosphopeptide as substrate. There was a significant increase in the activity of PTPs after injury, with higher increases in the membrane fraction (Fig. 1A) . Next, we determined the expression of several PTPs by Western blot in cytosolic and membrane fractions of epithelium from rabbit corneas 1, 2, 3, and 7 days after injury (Fig. 1B) . In the cytosolic fraction, rabbit corneal epithelial cells expressed the nonreceptor SHP-2 (also known as PTP1D or SH-PTP2) as well as PTEN, an MSP. Both PTPs increased their expression after injury. On the other hand, another nonreceptor PTP, SHP-1 (also known as PTP1C or SH-PTP1), and a dual-specific phosphatase called KAP had very low rates of expression in the cytosolic fraction but increased their expression 3 and 7 days after injury. Both were also expressed at different times after injury in the membrane fraction. The nonreceptor PTP1B was the major PTP expressed in the membrane fraction, and its expression increased 1 day after injury and continued to increase (3.5-fold over control) by day 3. At 7 days, PTP1B expression was decreased to almost the same levels as in controls. The epithelial membrane fraction also expressed LAR, a receptor-type PTP, at 3 days and 7 days after injury. We found no expression for other PTPs tested (VHR, S1RPα1, RPTPβ, RPTPα). 
PTP Inhibition Increases c-Met Phosphorylation in HCE Cells
Activation of signal transduction pathways in response to HGF stimulation is mediated by autophosphorylation of two tyrosine residues, Tyr1234 and Tyr1235, localized in the intracellular region of c-Met. 6 Using an antibody that recognizes these two phosphorylated residues, higher concentrations of HGF increased the tyrosine phosphorylation of c-Met, which appears as a specific band at 140 kDa in HCE cells (Fig. 2A) . In nonstimulated conditions, there was no visible tyrosine phosphorylation of c-Met. HGF at 40 ng/mL stimulated c-Met phosphorylation as early as 5 minutes, continued to increase its activation up to 30 minutes, and decreased it by 60 minutes (Fig. 2B) . By 2 hours there was low activity, and by 24 hours there was none. Results showed that in HCE cells, c-Met tyrosine phosphorylation stimulated by HGF was time and concentration dependent. With the use of bpV(phen), a potent PTP inhibitor, 24 we investigated the possibility of increasing the activation of c-Met by PTP inhibition. Cells were incubated in the presence of 1 and 10 μM bpV(phen), with or without HGF. Significantly increased levels of p-c-Met were found after incubation with 10 μM bpV(phen) (Fig. 2C) . Stimulation with 40 ng/mL HGF for 15 minutes further enhanced the activation of c-Met. The blots were reprobed with GAPDH, which showed no changes. Immunofluorescence also confirmed the presence of tyrosine-phosphorylated c-Met after treatment with the PTP inhibitor, which was further increased in the presence of HGF (Fig. 2D) . The results demonstrated that PTP inhibition enhances HGF-induced activation of c-Met and suggested that bpV(phen) acts by preventing the dephosphorylation and inactivation of c-Met through the inhibition of associated PTPs. 
Inhibition of PTP Increases Activation of the PI-3K/Akt-1 Pathway
We had shown previously that HGF stimulates PI-3K/Akt-1 signaling as well as that of p70S6K and ERK1/2. 9 With the use of HCE cells, we first studied the time-course activation of these kinases by HGF to determine whether they followed patterns similar to those of c-Met phosphorylation. Phosphorylation of Akt-1 at Ser437 was rapidly stimulated, and elevation was maintained up to 1 hour (Fig. 3A) . This stimulation is similar to that reported in rabbit corneal epithelial cells. 10 By 2 hours, the activation was lower, and at 24 hours it was similar to that of the control. The phosphorylation of p706SK and ERK1/2 followed the same profile as Akt-1, and there were no changes in any of the total proteins with HGF, demonstrating that tyrosine phosphorylation of c-Met is followed by activation of these kinases and that when c-Met is downregulated, the kinases are also deactivated. 
To investigate what role PTPs have in the signaling pathways activated by HGF, HCE cells were stimulated with 40 ng/mL HGF for 15 minutes, with or without bpV(phen) (Fig. 3B) . As previously shown, 1 μM inhibitor did not cause an increase in c-Met phosphorylation (Fig. 2C) ; therefore, for these experiments, 6 and 10 μM bpV(phen) were used. To determine the activation of PI-3K, the cell extract was immunoprecipitated with phosphotyrosine (PY) and was immunoblotted with the PI-3K regulatory subunit p85, which is bound to the c-Met complex and is tyrosine phosphorylated during stimulation. 6 HGF increased the amount of p85 present in anti-phosphotyrosine immunoprecipitates. The PTP inhibitor bpV(phen) also increased the phosphorylation of p85, which was further increased in cells stimulated with HGF. Similar patterns of activation were followed by Akt-1, a downstream target of PI-3K. However, the phosphorylation of p706SK and ERK1/2 was increased only by the higher concentration of the inhibitor, 10 μM, with no further increase in the presence of HGF. The results suggest that although the inhibition of PTPs affects various signaling pathways, the stimulation of PI-3K/Akt-1 linked to c-Met activation is more sensitive to regulation by PTPs. 
PTP1B and SHP-2 Complex with c-Met in HCE Cells and Are Tyrosine Phosphorylated
HCE cells were stimulated at different times with HGF, and the cell extracts were immunoprecipitated with c-Met or PY antibodies, as described in Materials and Methods. Immunoblots with PTP1B showed a positive band at 50 kDa corresponding to the phosphatase (Fig. 4A) . PTP1B bound to c-Met did not change with HGF stimulation up to 60 minutes, but continuous stimulation for 120 minutes and 24 hours produced an increase that ranged from 35% to 60% of PTP1B bound to c-Met. The results correlated well with the data in Figure 2B , in which there was no tyrosine phosphorylation of c-Met at those times. The blots were reprobed with c-Met to demonstrate that the changes were not caused by an increase in c-Met recovery from the immunoprecipitates. This demonstrates, for the first time, that PTP1B forms complexes with the c-Met receptor and that the bind increases after long-term stimulation with HGF. 
PTP1B is a phosphoprotein whose activity can be regulated by phosphorylation at Ser/Thr or Tyr residues. 25 26 To investigate the tyrosine phosphorylation state of PTP1B on HGF stimulation, cell extracts from parallel experiments were immunoprecipitated with PY antibody and immunoblotted with PTP1B antibody (Fig. 4A , lower panel). Nonstimulated HCE cells contain tyrosine-phosphorylated PTP1B that decreases at 15 minutes and 30 minutes after HGF stimulation but is elevated again at 60 minutes and remains higher up to 24 hours. The increase in phosphotyrosine content in PTP1B correlates with the decrease in c-Met phosphorylation after HGF stimulation (Fig. 2B) , suggesting that tyrosine phosphorylation activates the phosphatase. 
It had been previously shown in other systems that the tyrosine phosphatase SHP-2 can be recruited to the c-Met and, on stimulation, becomes tyrosine phosphorylated. 6 This particular PTP, contrary to PTP1B, serves as a positive activator of the receptor for sustained activation of ERK1/2. 27 We investigated whether SHP-2 is bound and tyrosine phosphorylated after HGF stimulation in HCE cells. Immunoblots with SHP-2 antibody of c-Met immunoprecipitates from HCE cell extracts showed a strong band of SHP-2 (Fig. 4B)that increased at 15 minutes of stimulation. Bound SHP-2 was also phosphorylated at Tyr542 after 15 and 30 minutes of stimulation. Immunoblotted controls with c-Met showed that the immunoprecipitates contained similar amounts of the receptor. 
PTP1B and SHP-2 Gene Knockdown Affect the Phosphorylation of c-Met
To obtain more clear evidence of the roles of PTP1B and SHP-2 on the phosphorylation of c-Met, the genes of these two PTPs were knocked down using validated siRNAs, as described in Materials and Methods. Conditions were optimized for the maximum transfection efficiency. Ninety-six hours after transfection, HCE cells were treated with HGF, 4 and 6 μM bpV(phen), or bpV(phen) plus HGF. These concentrations of inhibitor were chosen for the experiments to observe the changes in c-Met phosphorylation when the specific phosphatases were knocked down. The samples were analyzed by Western blot for protein expression using specific antibodies. HCE cells transfected with SHP-2 siRNA resulted in a 90% decrease in SHP-2 expression compared with negative control siRNA (Fig. 5A , upper bands). Cells transfected with PTP1B siRNA showed 70% inhibition (Fig. 5A , middle bands). GAPDH was used as a loading control. There was selectivity in the transfection, and HCE cells transfected with SHP-2 siRNA showed no changes in PTP1B expression compared with controls. Transfections with PTP1B siRNA showed only minimal changes in the expression of SHP-2. Figures 5B and 5Cshow how PTP1B and SHP-2-gene knockdown affected the phosphorylation of c-Met in the presence of bpV(phen). As already shown in Figure 2B , HGF at 15 minutes stimulated c-Met phosphorylation. Although 4 μM bpV(phen) alone had no effect, incubations in the presence of the inhibitor with 40 ng/mL HGF for 15 minutes increased the phosphorylation of c-Met, which was further increased (>80%) when PTP1B was knocked down. Decreased phosphorylation (20%) was observed when the cells were transfected with SHP-2 siRNA under the same conditions (Fig. 5C) . To further determine the role of PTP1B and SHP-2 in the activation of c-Met, HCE cells were incubated with 6 μM bpV(phen) for 2 hours, a time that showed the decrease in c-Met phosphorylation stimulated by HGF (Fig. 2B) . This concentration of the inhibitor stimulated the phosphorylation of c-Met at 2 hours, which was further increased (>60%) in the presence of HGF. When PTP1B was knocked down (Fig. 5B) , another threefold increase was observed, demonstrating that the activation of PTP1B is important to switch off c-Met activation after HGF stimulation. However, under the same conditions, the phosphorylation of c-Met decreased (35%) when the cells were transfected with SHP-2 siRNA (Fig. 5C) , implying that this phosphatase is a positive regulator of c-Met receptor in HCE cells. 
Cell Localization of PTP1B and c-Met
The presence of PTP1B bound to c-Met in HCE cells prompted us to investigate the localization of these proteins in the cells. Immunofluorescence of HCE cells showed a strong PTP1B-positive stain in the ER that coincided with the PDI antibody (a marker of the ER) when the figures were merged (Fig. 6A) . No changes in PTP1B distribution were noted when the cells were stimulated with HGF (not shown). Interestingly, c-Met was also found around the nuclei, with a considerable proportion of the growth factor receptor colocalized with PTP1B (Fig. 6B)
Inhibition of PTP1B and Increase in HGF-Stimulated Epithelial Wound Healing
Several vanadium derivative compounds had been shown to inhibit PTP activity. 28 In addition, derivatives of cinnamic acid had shown moderate selectivity for PTP1B inhibition. 29 The action of four inhibitors at different concentrations on PTP1B activity was tested on the membrane fraction of HCE cells (Table 1) . The most potent was bpV(phen), which at 6 μM produced 98% inhibition of the PTP1B activity; phenyl arsine oxide at 10 μM inhibited 56% of the activity, whereas a more specific PTP1B inhibitor (CinnGEL 2Me; Biomol) 29 and SOV were less potent. These results demonstrate that bpV(phen) at 6 μM is an excellent inhibitor of PTP1B in corneal epithelial cells. They also suggest that the increases in c-Met phosphorylation and PI-3K/Akt-1 signaling, after HGF stimulation in the presence of the inhibitor (Figs. 2C 3B) , could be attributed, in part, to the inhibition of this PTP. 
We then investigated the action of bpV(phen) on epithelial wound healing. Rabbit corneas in organ culture were wounded, as described in Materials and Methods. As previously reported, 9 HGF stimulated epithelial wound healing in this model 24 hours after the wound (Fig. 7) . In the presence of 6 μM bpV(phen), there was a further decrease in the wounded area. The PI-3K inhibitors wortmannin and LY294002, on the other hand, blocked the epithelial wound healing stimulated by the PTP inhibitor. These experiments demonstrated that the inhibition of PTP action increased HGF stimulation of corneal epithelial wound healing requiring the activation of PI-3K signaling. 
Discussion
Corneal epithelial injury in vivo is a complex process involving an array of growth factors and cytokines that activate a variety of cell signaling mechanisms to induce the repair of tissue. In this study, we found that after an epithelial wound, there was a significant increase in PTP activity in the cytosol and the membrane fraction of epithelial cells. Although the increase corresponded to total PTP activity, PTP1B expression increased significantly 2 days after the injury and made for a proportion of the increase in activity. After in vivo corneal epithelial injury, PTPs were expressed in different compartments and at different rates. Some of them, such as SHP-2 and PTEN, continued to increase their expression up to 7 days after injury, whereas others, such as PTP1B and KAP, had a peak at 3 days, suggesting that they regulate different cell signaling after injury. Therefore, selectivity at the level of PTPs is an important mechanism in regulating cell adhesion, migration, survival, and proliferation of corneal epithelial cells. For example, KAP had been reported to be involved in the phosphorylation of cyclin-dependent kinases, 30 and its increase in expression after injury could be linked to the inactivation of kinases and the inhibition of cell proliferation. This could explain the appearance of KAP in the cytosol at longer times, when the epithelium had been regenerated, and it may be important in controlling mitosis at the level of CDKs. 31 Another PTP that changed after injury was PTEN. This enzyme belongs to the MSP and is able to dephosphorylate protein tyrosine residues and the D-3 phosphate of inositol phospholipids. Although there is an increase of PTEN expression during the wound healing process, it is present in the cytosolic compartment and does not bind directly to RTK receptors, 13 suggesting that its regulation is at another level. LAR was expressed in epithelium at 3 and 7 days after injury. This enzyme corresponds to the receptor-like PTPs; it is known to bind to laminin and has been postulated to be involved in cell adhesion. 32 Very recently, it has been shown that LAR dephosphorylates c-Met in confluent hepatocytes. 33 This could suggest an important role in controlling the proliferation of the cells. It had also been reported to be a negative regulator of insulin signaling. 34 Further studies are warranted to explore the functions of PTEN and LAR in corneal epithelial repair. 
The nonreceptors PTP1B and SHP-2 were highly expressed during wound healing. We focused our attention on these two PTPs as candidates to modulate HGF signaling. 
PTP1B has been shown to regulate insulin and insulinlike growth factor-1 signaling in vivo and in vitro and has been implicated as a modulator of diabetes and obesity. 26 35 36 It also regulates the action of EGF, platelet-derived growth factor, and fibroblast growth factor, 37 38 and there is some recent evidence that it can regulate EGF receptors in corneal epithelial cells. 20 However, only one recent study links this PTP with c-Met, in which it was reported that PTP1B-deficient hepatocytes show an increase in c-Met phosphorylation. 39 To our knowledge, this is the first report to demonstrate an association between the c-Met receptor and PTP1B. Evidence of the involvement of PTP1B in HGF action is provided by showing that binding to c-Met increased at longer times of HGF stimulation and correlated well with the dephosphorylation of c-Met and the downregulation of signaling pathways. In addition, knocking down the expression of PTP1B increased c-Met phosphorylation. 
It is important to note that PTP1B bound to c-Met when there was no HGF stimulation. In experiments in kidney cells, interaction between insulin receptor and PTP1B occurs under basal conditions, and it is suggested that this interaction is important during insulin receptor biosynthesis in the ER to maintain the receptor in a nonactivated form. 40 In HCE cells, most of the PTP1B was localized in the ER. Therefore, this localization might be important for the regulation of c-Met receptors during their biosynthesis. In addition, to avoid overactivation of the receptor, c-Met could be internalized but must be also dephosphorylated to prevent the stimulation of intracellular signaling pathways in an anomalous way. In fact, immunostaining shows that a significant proportion of c-Met colocalized with PTP1B. Interaction of this phosphatase with c-Met at the plasma membrane is a less possible scenario because we could not detect immunostaining in the cell membrane. 
One way to regulate PTP1B is by phosphorylation. Several phosphorylation sites have been identified. 25 26 Although changes in PTP1B activity had been associated with changes in its phosphorylation, the precise mechanisms by which the phosphorylation of PTP1B can be important in activating and inhibiting RTKs are unclear. Our results show that PTP1B is tyrosine phosphorylated. This phosphorylation decrease, at 15 and 30 minutes after HGF stimulation, coincides with maximal activation of c-Met. This suggests a positive action, by which phosphorylated PTP1B might increase its phosphatase activity. This, in turn, allows c-Met to be dephosphorylated and inactivated at longer times. In fact, in knocking down PTP1B, there was a further increase in c-Met phosphorylation stimulated by HGF in the presence of bpV(phen). The results are in agreement with previous studies showing that, in fibroblasts, insulin stimulates tyrosine phosphorylation of PTP1B and activates the enzyme. 26  
Another important PTP in the regulation of the HGF receptor is SHP-2, which acts as a positive modulator of c-Met. 27 This is also demonstrated in HCE cells, in which SHP-2 gene silencing decreased the activation of c-Met induced by HGF in the presence of the PTP inhibitor. The HGF receptor binds to several adaptor proteins (such as Grb1, ShC, Crk, and Gab1) that, in turn, recruit other proteins involved in cell signaling. For example, the binding protein Gab1 recruits SHP-2. 6 This particular PTP is mainly cytosolic and increases its expression during wound healing. We had found a portion of the enzyme bound to the c-Met receptor in unstimulated cells that increased after 15 minutes of stimulation. That probably accounts for a very small proportion of the enzyme, which is difficult to detect in the membrane fraction without c-Met immunoprecipitation. In fact, our Western blot analysis of epithelium collected during wound healing showed a tenuous band in the membrane fraction. 
In epithelial kidney cells, SHP-2 activation is required for tubulogenesis of the cells and for sustained activation of ERK1/2. 27 Our data on HCE cells show that ERK1/2 was downregulated by 2 hours after HGF stimulation, whereas phosphorylation (activation) of SHP-2 lasted for 30 minutes on HGF stimulation. This suggests that SHP-2 controls HGF responses during the early phase of c-Met activation. The transient activation of SHP-2 may be insufficient for modulating cell proliferation stimulated by ERK1/2 in corneal epithelial cells. 11 It has also been suggested that SHP-2 could play a role in regulating cell spreading and migration. 41 In that context, we had previously shown that during the epithelial migration phase, there is an inhibition of ERK1/2 activation. 11 It is tempting to suggest that SHP-2 could be a regulator in that particular phase of epithelial wound healing. Future experiments will be needed to investigate this interesting point. 
Finally, we also demonstrated that inhibiting PTP activity with bpV(phen) increased HGF-stimulated epithelial wound healing, which was reverted when the PI-3K pathway was blocked. Time-response experiments with the PTP inhibitor in the presence of HGF demonstrated that c-Met phosphorylation correlated well with activation of the PI-3K/Akt signal pathway. Although these studies were conducted with a broad PTP inhibitor, we showed that bpV(phen) completely blocked PTP1B activity. This, along with our experiments with PTP1B siRNA, suggests that PTP1B is a key modulator of corneal epithelial wound healing stimulated by HGF. 
In summary, our data show that there is increased activity and expression of selective PTPs after corneal epithelial injury. Inactivation of PTPs selectively increases the PI-3K/Akt-1 pathway stimulated by HGF and the wound healing response of epithelium. SHP-2 binds to c-Met in HCE cells, its phosphorylation (activation) state coincides with the maximal c-Met activation, and gene knockdown of SHP-2 decreases the stimulated c-Met phosphorylation, all of which demonstrate a positive role of SHP-2 in the HGF action on HCE cells. We also demonstrate, for the first time, that PTP1B is bound to the c-Met receptor and that its tyrosine phosphorylation is downregulated when HGF increases the activity of c-Met and its downstream signals. PTP1B gene silencing increases the activation of c-Met, demonstrating an inhibitory role of this phosphatase. This new action of PTP1B on c-Met opens novel possibilities to explore the role of this particular phosphatase in regulating the actions of HGF, not only in corneal epithelium but in other tissues in which c-Met has important functions. 
 
Figure 1.
 
PTP activity and expression during in vivo epithelial wound healing. New Zealand rabbits were injured by gentle scraping of the cornea, leaving the limbal epithelium intact. Cornea epithelium from control and injured rabbits was collected, and cytosol and membrane fractions were prepared. (A) Total PTP activity was measured with 5 μg protein before and after 48-hour injury. Values represent the mean ± SD of three samples. *Significant differences with respect to control. (B) Samples were collected 1, 2, 3, and 7 days after injury, and the same amount of proteins was separated by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting with PTP antibodies. As loading controls for the cytosol fraction, a 35-kDa protein that appeared in the gels after blotting with some of the antibodies, and that did not change its expression during would healing, was used. In the membrane fraction, the phosphatase KAP also exhibited no change between 1 and 7 days after injury. Data represent results of one of two experiments.
Figure 1.
 
PTP activity and expression during in vivo epithelial wound healing. New Zealand rabbits were injured by gentle scraping of the cornea, leaving the limbal epithelium intact. Cornea epithelium from control and injured rabbits was collected, and cytosol and membrane fractions were prepared. (A) Total PTP activity was measured with 5 μg protein before and after 48-hour injury. Values represent the mean ± SD of three samples. *Significant differences with respect to control. (B) Samples were collected 1, 2, 3, and 7 days after injury, and the same amount of proteins was separated by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting with PTP antibodies. As loading controls for the cytosol fraction, a 35-kDa protein that appeared in the gels after blotting with some of the antibodies, and that did not change its expression during would healing, was used. In the membrane fraction, the phosphatase KAP also exhibited no change between 1 and 7 days after injury. Data represent results of one of two experiments.
Figure 2.
 
Tyrosine phosphorylation of c-Met after HGF stimulation. HCE cells were treated with HGF at concentrations and times indicated and then were lysed. Whole cell lysates were immunoblotted with Tyr1234- and Tyr1235-phosphorylated c-Met antibody (p-c-Met). The membranes were reblotted with GAPDH as gel loading control. (A) HCE cells were incubated with different concentrations of HGF for 15 minutes. (B, C) Stimulation was with 40 ng/mL HGF for different times. (D) HCE cells were treated with bpV(phen) and stimulated or not stimulated with HGF for 15 minutes and immunostained with p-c-Met. Hoechst was used for nuclear counterstain. The experiments were repeated three times with similar results.
Figure 2.
 
Tyrosine phosphorylation of c-Met after HGF stimulation. HCE cells were treated with HGF at concentrations and times indicated and then were lysed. Whole cell lysates were immunoblotted with Tyr1234- and Tyr1235-phosphorylated c-Met antibody (p-c-Met). The membranes were reblotted with GAPDH as gel loading control. (A) HCE cells were incubated with different concentrations of HGF for 15 minutes. (B, C) Stimulation was with 40 ng/mL HGF for different times. (D) HCE cells were treated with bpV(phen) and stimulated or not stimulated with HGF for 15 minutes and immunostained with p-c-Met. Hoechst was used for nuclear counterstain. The experiments were repeated three times with similar results.
Figure 3.
 
Effect of PTP inhibition on HGF-stimulated downstream kinases. (A) HCE cells were stimulated with 40 ng/mL HGF for different times, and the cell lysate was subjected to Western blot with p-Akt-1 followed by reblotting with Akt-1 antibody, p-p706SK followed by p706SK, and p-ERK1/2 followed by ERK1/2 antibody. (B) HCE cells were stimulated with HGF with or without 6 or 10 μM bpV (phen). To determine the phosphorylation of the p85 subunit of PI-3K, the samples were first immunoprecipitated with PY antibody and then immunoblotted with p85. Data from one of three typical experiments are shown.
Figure 3.
 
Effect of PTP inhibition on HGF-stimulated downstream kinases. (A) HCE cells were stimulated with 40 ng/mL HGF for different times, and the cell lysate was subjected to Western blot with p-Akt-1 followed by reblotting with Akt-1 antibody, p-p706SK followed by p706SK, and p-ERK1/2 followed by ERK1/2 antibody. (B) HCE cells were stimulated with HGF with or without 6 or 10 μM bpV (phen). To determine the phosphorylation of the p85 subunit of PI-3K, the samples were first immunoprecipitated with PY antibody and then immunoblotted with p85. Data from one of three typical experiments are shown.
Figure 4.
 
Bound to c-Met and phosphotyrosine content of PTP1B and SHP2. HCE cells were incubated in the absence or presence of 40 ng/mL HGF for different times and then collected in lysis buffer. Cell lysate (1 mg) was immunoprecipitated with monoclonal c-Met antibody (A, B) or with PY monoclonal antibody (A). Proteins were separated by 4% to 12% gradient gels and were transferred to PVDF membranes. Membranes were immunoblotted with the polyclonal PTP1B antibody (A) or with the polyclonal SHP-2 and the phosphorylated polyclonal p-SHP-2 antibody (B). Membranes were stripped and reprobed with c-Met antibody. Data represent one of three similar experiments.
Figure 4.
 
Bound to c-Met and phosphotyrosine content of PTP1B and SHP2. HCE cells were incubated in the absence or presence of 40 ng/mL HGF for different times and then collected in lysis buffer. Cell lysate (1 mg) was immunoprecipitated with monoclonal c-Met antibody (A, B) or with PY monoclonal antibody (A). Proteins were separated by 4% to 12% gradient gels and were transferred to PVDF membranes. Membranes were immunoblotted with the polyclonal PTP1B antibody (A) or with the polyclonal SHP-2 and the phosphorylated polyclonal p-SHP-2 antibody (B). Membranes were stripped and reprobed with c-Met antibody. Data represent one of three similar experiments.
Figure 5.
 
Effect of PTP1B and SHP-2 knockdown on c-Met phosphorylation. HCE cells were transfected with PTP1B, SHP-2, or negative control siRNA. Cells were treated with HGF, 4 or 6 μM bpV(phen), or bpV(phen) plus HGF. (A) Selectivity of transfection and knockdown expression was assayed by immunoblotting with PTP1B and SHP-2 antibodies. Cells transfected with PTP1B (B) or SHP-2 (C) and their controls were immunoblotted with p-c-Met antibody to show how the phosphorylation of c-Met is affected. GAPDH was used as loading control. The experiment was repeated once with similar results.
Figure 5.
 
Effect of PTP1B and SHP-2 knockdown on c-Met phosphorylation. HCE cells were transfected with PTP1B, SHP-2, or negative control siRNA. Cells were treated with HGF, 4 or 6 μM bpV(phen), or bpV(phen) plus HGF. (A) Selectivity of transfection and knockdown expression was assayed by immunoblotting with PTP1B and SHP-2 antibodies. Cells transfected with PTP1B (B) or SHP-2 (C) and their controls were immunoblotted with p-c-Met antibody to show how the phosphorylation of c-Met is affected. GAPDH was used as loading control. The experiment was repeated once with similar results.
Figure 6.
 
Localization of PTP1B and c-Met in HCE cells. The cells were processed for immunofluorescence, using PTP1B and c-Met antibodies. PDI (protein disulfide isomerase) antibody was used as an ER marker. Hoechst was used to stain the nuclei. (A) Colocalization of PTP1B with the ER marker is shown when the pictures are overlaid. (B) Colocalization of PTP1B with c-Met.
Figure 6.
 
Localization of PTP1B and c-Met in HCE cells. The cells were processed for immunofluorescence, using PTP1B and c-Met antibodies. PDI (protein disulfide isomerase) antibody was used as an ER marker. Hoechst was used to stain the nuclei. (A) Colocalization of PTP1B with the ER marker is shown when the pictures are overlaid. (B) Colocalization of PTP1B with c-Met.
Table 1.
 
Effect of Tyrosine Phosphatase Inhibitors on PTP1B Activity from Membrane Fractions of HCE Cells
Table 1.
 
Effect of Tyrosine Phosphatase Inhibitors on PTP1B Activity from Membrane Fractions of HCE Cells
Inhibitor Concentration (μM) Inhibition (%)
bpV(phen) 1 12.3 ± 2.6
6 98.3 ± 0.4
10 100.1 ± 0.2
PAO 1 25.6 ± 1.1
10 55.8 ± 1.8
CinnGel 2ME 1 12.6 ± 1.8
10 17.2 ± 2.7
20 30.2 ± 3.8
SOV 200 16.4 ± 3.0
1000 17.5 ± 3.5
Figure 7.
 
bpV(phen) increased HGF-stimulated epithelial wound healing that was reversed by PI-3K inhibitors. Corneas were injured and cultured in a serum-free medium for 24 hours in the presence of HGF (40 ng/mL) with or without 6 μM bpV(phen) or with or without 200 nM wortmannin or 20 μM LY294002. Corneas were stained with Alizarin red, and the remaining uncovered area was calculated. Images represent a cornea in each condition. Bars correspond to five to six corneas in each treatment. *P < 0.05 compared with control at 24 hours. **P < 0.05 compared with HGF. ***P < 0.05 compared with bpV(phen) + HGF (Student’s t-test analysis).
Figure 7.
 
bpV(phen) increased HGF-stimulated epithelial wound healing that was reversed by PI-3K inhibitors. Corneas were injured and cultured in a serum-free medium for 24 hours in the presence of HGF (40 ng/mL) with or without 6 μM bpV(phen) or with or without 200 nM wortmannin or 20 μM LY294002. Corneas were stained with Alizarin red, and the remaining uncovered area was calculated. Images represent a cornea in each condition. Bars correspond to five to six corneas in each treatment. *P < 0.05 compared with control at 24 hours. **P < 0.05 compared with HGF. ***P < 0.05 compared with bpV(phen) + HGF (Student’s t-test analysis).
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Figure 1.
 
PTP activity and expression during in vivo epithelial wound healing. New Zealand rabbits were injured by gentle scraping of the cornea, leaving the limbal epithelium intact. Cornea epithelium from control and injured rabbits was collected, and cytosol and membrane fractions were prepared. (A) Total PTP activity was measured with 5 μg protein before and after 48-hour injury. Values represent the mean ± SD of three samples. *Significant differences with respect to control. (B) Samples were collected 1, 2, 3, and 7 days after injury, and the same amount of proteins was separated by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting with PTP antibodies. As loading controls for the cytosol fraction, a 35-kDa protein that appeared in the gels after blotting with some of the antibodies, and that did not change its expression during would healing, was used. In the membrane fraction, the phosphatase KAP also exhibited no change between 1 and 7 days after injury. Data represent results of one of two experiments.
Figure 1.
 
PTP activity and expression during in vivo epithelial wound healing. New Zealand rabbits were injured by gentle scraping of the cornea, leaving the limbal epithelium intact. Cornea epithelium from control and injured rabbits was collected, and cytosol and membrane fractions were prepared. (A) Total PTP activity was measured with 5 μg protein before and after 48-hour injury. Values represent the mean ± SD of three samples. *Significant differences with respect to control. (B) Samples were collected 1, 2, 3, and 7 days after injury, and the same amount of proteins was separated by SDS polyacrylamide gel electrophoresis and analyzed by immunoblotting with PTP antibodies. As loading controls for the cytosol fraction, a 35-kDa protein that appeared in the gels after blotting with some of the antibodies, and that did not change its expression during would healing, was used. In the membrane fraction, the phosphatase KAP also exhibited no change between 1 and 7 days after injury. Data represent results of one of two experiments.
Figure 2.
 
Tyrosine phosphorylation of c-Met after HGF stimulation. HCE cells were treated with HGF at concentrations and times indicated and then were lysed. Whole cell lysates were immunoblotted with Tyr1234- and Tyr1235-phosphorylated c-Met antibody (p-c-Met). The membranes were reblotted with GAPDH as gel loading control. (A) HCE cells were incubated with different concentrations of HGF for 15 minutes. (B, C) Stimulation was with 40 ng/mL HGF for different times. (D) HCE cells were treated with bpV(phen) and stimulated or not stimulated with HGF for 15 minutes and immunostained with p-c-Met. Hoechst was used for nuclear counterstain. The experiments were repeated three times with similar results.
Figure 2.
 
Tyrosine phosphorylation of c-Met after HGF stimulation. HCE cells were treated with HGF at concentrations and times indicated and then were lysed. Whole cell lysates were immunoblotted with Tyr1234- and Tyr1235-phosphorylated c-Met antibody (p-c-Met). The membranes were reblotted with GAPDH as gel loading control. (A) HCE cells were incubated with different concentrations of HGF for 15 minutes. (B, C) Stimulation was with 40 ng/mL HGF for different times. (D) HCE cells were treated with bpV(phen) and stimulated or not stimulated with HGF for 15 minutes and immunostained with p-c-Met. Hoechst was used for nuclear counterstain. The experiments were repeated three times with similar results.
Figure 3.
 
Effect of PTP inhibition on HGF-stimulated downstream kinases. (A) HCE cells were stimulated with 40 ng/mL HGF for different times, and the cell lysate was subjected to Western blot with p-Akt-1 followed by reblotting with Akt-1 antibody, p-p706SK followed by p706SK, and p-ERK1/2 followed by ERK1/2 antibody. (B) HCE cells were stimulated with HGF with or without 6 or 10 μM bpV (phen). To determine the phosphorylation of the p85 subunit of PI-3K, the samples were first immunoprecipitated with PY antibody and then immunoblotted with p85. Data from one of three typical experiments are shown.
Figure 3.
 
Effect of PTP inhibition on HGF-stimulated downstream kinases. (A) HCE cells were stimulated with 40 ng/mL HGF for different times, and the cell lysate was subjected to Western blot with p-Akt-1 followed by reblotting with Akt-1 antibody, p-p706SK followed by p706SK, and p-ERK1/2 followed by ERK1/2 antibody. (B) HCE cells were stimulated with HGF with or without 6 or 10 μM bpV (phen). To determine the phosphorylation of the p85 subunit of PI-3K, the samples were first immunoprecipitated with PY antibody and then immunoblotted with p85. Data from one of three typical experiments are shown.
Figure 4.
 
Bound to c-Met and phosphotyrosine content of PTP1B and SHP2. HCE cells were incubated in the absence or presence of 40 ng/mL HGF for different times and then collected in lysis buffer. Cell lysate (1 mg) was immunoprecipitated with monoclonal c-Met antibody (A, B) or with PY monoclonal antibody (A). Proteins were separated by 4% to 12% gradient gels and were transferred to PVDF membranes. Membranes were immunoblotted with the polyclonal PTP1B antibody (A) or with the polyclonal SHP-2 and the phosphorylated polyclonal p-SHP-2 antibody (B). Membranes were stripped and reprobed with c-Met antibody. Data represent one of three similar experiments.
Figure 4.
 
Bound to c-Met and phosphotyrosine content of PTP1B and SHP2. HCE cells were incubated in the absence or presence of 40 ng/mL HGF for different times and then collected in lysis buffer. Cell lysate (1 mg) was immunoprecipitated with monoclonal c-Met antibody (A, B) or with PY monoclonal antibody (A). Proteins were separated by 4% to 12% gradient gels and were transferred to PVDF membranes. Membranes were immunoblotted with the polyclonal PTP1B antibody (A) or with the polyclonal SHP-2 and the phosphorylated polyclonal p-SHP-2 antibody (B). Membranes were stripped and reprobed with c-Met antibody. Data represent one of three similar experiments.
Figure 5.
 
Effect of PTP1B and SHP-2 knockdown on c-Met phosphorylation. HCE cells were transfected with PTP1B, SHP-2, or negative control siRNA. Cells were treated with HGF, 4 or 6 μM bpV(phen), or bpV(phen) plus HGF. (A) Selectivity of transfection and knockdown expression was assayed by immunoblotting with PTP1B and SHP-2 antibodies. Cells transfected with PTP1B (B) or SHP-2 (C) and their controls were immunoblotted with p-c-Met antibody to show how the phosphorylation of c-Met is affected. GAPDH was used as loading control. The experiment was repeated once with similar results.
Figure 5.
 
Effect of PTP1B and SHP-2 knockdown on c-Met phosphorylation. HCE cells were transfected with PTP1B, SHP-2, or negative control siRNA. Cells were treated with HGF, 4 or 6 μM bpV(phen), or bpV(phen) plus HGF. (A) Selectivity of transfection and knockdown expression was assayed by immunoblotting with PTP1B and SHP-2 antibodies. Cells transfected with PTP1B (B) or SHP-2 (C) and their controls were immunoblotted with p-c-Met antibody to show how the phosphorylation of c-Met is affected. GAPDH was used as loading control. The experiment was repeated once with similar results.
Figure 6.
 
Localization of PTP1B and c-Met in HCE cells. The cells were processed for immunofluorescence, using PTP1B and c-Met antibodies. PDI (protein disulfide isomerase) antibody was used as an ER marker. Hoechst was used to stain the nuclei. (A) Colocalization of PTP1B with the ER marker is shown when the pictures are overlaid. (B) Colocalization of PTP1B with c-Met.
Figure 6.
 
Localization of PTP1B and c-Met in HCE cells. The cells were processed for immunofluorescence, using PTP1B and c-Met antibodies. PDI (protein disulfide isomerase) antibody was used as an ER marker. Hoechst was used to stain the nuclei. (A) Colocalization of PTP1B with the ER marker is shown when the pictures are overlaid. (B) Colocalization of PTP1B with c-Met.
Figure 7.
 
bpV(phen) increased HGF-stimulated epithelial wound healing that was reversed by PI-3K inhibitors. Corneas were injured and cultured in a serum-free medium for 24 hours in the presence of HGF (40 ng/mL) with or without 6 μM bpV(phen) or with or without 200 nM wortmannin or 20 μM LY294002. Corneas were stained with Alizarin red, and the remaining uncovered area was calculated. Images represent a cornea in each condition. Bars correspond to five to six corneas in each treatment. *P < 0.05 compared with control at 24 hours. **P < 0.05 compared with HGF. ***P < 0.05 compared with bpV(phen) + HGF (Student’s t-test analysis).
Figure 7.
 
bpV(phen) increased HGF-stimulated epithelial wound healing that was reversed by PI-3K inhibitors. Corneas were injured and cultured in a serum-free medium for 24 hours in the presence of HGF (40 ng/mL) with or without 6 μM bpV(phen) or with or without 200 nM wortmannin or 20 μM LY294002. Corneas were stained with Alizarin red, and the remaining uncovered area was calculated. Images represent a cornea in each condition. Bars correspond to five to six corneas in each treatment. *P < 0.05 compared with control at 24 hours. **P < 0.05 compared with HGF. ***P < 0.05 compared with bpV(phen) + HGF (Student’s t-test analysis).
Table 1.
 
Effect of Tyrosine Phosphatase Inhibitors on PTP1B Activity from Membrane Fractions of HCE Cells
Table 1.
 
Effect of Tyrosine Phosphatase Inhibitors on PTP1B Activity from Membrane Fractions of HCE Cells
Inhibitor Concentration (μM) Inhibition (%)
bpV(phen) 1 12.3 ± 2.6
6 98.3 ± 0.4
10 100.1 ± 0.2
PAO 1 25.6 ± 1.1
10 55.8 ± 1.8
CinnGel 2ME 1 12.6 ± 1.8
10 17.2 ± 2.7
20 30.2 ± 3.8
SOV 200 16.4 ± 3.0
1000 17.5 ± 3.5
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