Protein phosphorylation in cultured rat RPE. Effects of protein kinase C activation

Incubation of confluent cultures of rat retinal pigment epithelium (RPE) with 32P-orthophosphate resulted in the incorporation of 32P into proteins, RNA and the nucleoside phosphates ADP, GDP, ATP and GTP. RPE cultures incubated with phorbol-12-myristate-13-acetate (PMA), a known activator of protein kinase C, did not significantly change the incorporation of 32P into total protein, RNA or the nucleoside phosphates ADP, GDP, ATP and GTP. However, PMA exposure specifically increased phosphorylation of five proteins with molecular weights of 80 kilodaltons (K), 56K, 35K, 33K, and 29K having isoelectric points between 4.3 and 6.5. PMA treated cultures also showed dephosphorylation of two proteins having molecular weights of about 33K. The observed increase in 80K phosphorylation suggests that protein kinase C is present and activated by PMA in the RPE.     

Protein kinase C activation during corneal endothelial wound repair.

In previous studies, the authors have shown that the two forms of cell translocation that occur during corneal endothelial monolayer wound repair can be pharmacologically separated. Epidermal growth factor (EGF) enhanced the breaking of cell-cell contacts and movement of individual cells from the wound edge, while indomethacin, an inhibitor of PGE2 synthesis, promoted cell enlargement and spreading of the confluent monolayer sheet into the wound defect. From these findings, the authors hypothesized that the two forms of cell translocation were stimulated by different but coordinately regulated second messenger systems. The current studies used selected protein kinase C (PKC) stimulators and inhibitors, Rh-phalloidin staining of actin filaments, and immunofluorescent localization of PKC to show that: (1) PKC acts as a mediator of the EGF-induced enhancement of the migratory response; (2) the enhanced migratory response results, at least in part, from short-term EGF stimulation of PKC; (3) PKC is a mediator of the EGF-induced alterations in the actin cytoskeleton; and (4) PKC becomes activated in cells at the wound edge during normal, endogenously stimulated wound repair. The results of these studies provide suggestive evidence that wounding of the corneal endothelial monolayer must produce an endogenous, EGF-like stimulation of PKC activity in cells at the wound edge. One effect of PKC activation that must contribute to stimulation of individual cell migration is the induction of cytoplasmic changes that lead to alterations in actin filament organization.     

Calcium- and phospholipid-dependent protein kinase C and phosphatidylinositol kinase: two major phosphorylation systems in the cornea

The signaling messengers for the regulation of intracellular calcium-dependent functions involve a phospholipid-sensitive protein kinase (protein kinase C) modulated by diacylglycerol, which is a product of phosphoinositide degradation. We found that protein kinase C activity is two times higher in the epithelium than in the stroma-endothelial layers of the rabbit cornea. 12-0-tetradecanoylphorbol-13-acetate (TPA) and phosphatidylserine stimulate protein kinase C at low Ca2+ concentrations. The TPA-phosphatidylserine stimulated activity of corneal epithelium was recovered in the soluble fraction; in the membrane-bound fraction, a very active phosphatidylinositol kinase accounted for half the basal phosphorylation of the corneal epithelium.     

Vascular endothelial growth factor upregulates expression of ADAMTS1 in endothelial cells through protein kinase C signaling

PURPOSE: ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs) has been demonstrated to inhibit angiogenesis in vivo and to suppress endothelial cell proliferation in vitro. The purpose of this study was to investigate the expression of ADAMTS1 in endothelial cells and in a mouse model of ischemia-induced retinal neovascularization and to study the regulation of ADAMTS1 expression in endothelial cells by vascular endothelial growth factor (VEGF). In addition, the potential function of endothelial cell-derived ADAMTS1 on cell proliferation was investigated. METHODS: Expression of ADAMTS1 in human retinal endothelial cells (HRECs), human umbilical vein endothelial cells (HUVECs), and the mouse model of ischemia-induced retinal neovascularization was assayed by real-time PCR and Western blot analysis. The effect of ADAMTS1 on endothelial cell proliferation was evaluated using siRNA knockdown and [3H] thymidine incorporation. RESULTS: ADAMTS1 mRNA and protein levels were increased in a mouse model of ischemia-induced retinal neovascularization, and VEGF induced time- and dose-dependent increases in ADAMTS1 mRNA and protein expression in endothelial cells. This upregulation was inhibited by the VEGF receptor (VEGFR)2 inhibitor SU1498, anti-VEGFR2 neutralizing antibody, and the phospholipase C (PLC)-gamma inhibitor U73122. VEGF upregulation of ADAMTS1 expression was completely abolished by the inhibition of protein kinase C by calphostin C and largely blocked by the specific inhibition of PKCbeta. Knockdown of endogenous ADAMTS1 resulted in increased proliferation of endothelial cells. CONCLUSIONS: These results indicate that VEGF significantly induces ADAMTS1 expression in endothelial cells in a PKC-dependent fashion. ADAMTS1 expression is also increased, along with VEGF expression, in vivo in ischemia-induced retinal neovascularization. In addition, ADAMTS1 appears to be an endogenous regulator of endothelial cell proliferation. Therefore, VEGF upregulation of ADAMTS1, a potent angiogenesis inhibitor, may represent a mechanism for feedback inhibition of angiogenesis and retinal neovascularization.     

The role of protein tyrosine phosphorylation in the cell-cell interactions, junctional permeability and cell cycle control in post-confluent bovine corneal endothelial cells

Cell-cell interaction, junctional permeability and contact inhibition are important mechanisms that allow corneal endothelial cells to maintain stable corneal hydration status and also keep these cells in non-proliferative status. Protein tyrosine phosphatases (PTPs) are well known to play an important role in regulating cell-cell contacts, growth and differentiation. Inhibition of PTPs activity by a general PTP inhibitor has been proved to trigger post-confluent rat corneal endothelial cells to reenter cell cycles. In this study, we aimed to evaluate whether protein tyrosine phosphorylation is involved in cell-cell interactions, junctional permeability and cell cycle control in post-confluent, contact inhibited bovine corneal endothelial cells. Confluent cultures of bovine corneal endothelial cells were treated with different concentrations of general phosphatase inhibitor, sodium orthovanadate (vanadate) for several different durations. Protein tyrosine phosphorylation was confirmed by Western blot analysis. Immunocytochemistry was used to evaluate the effect of vanadate on adherens-type junctional proteins by staining of p120, N-cadherin and alpha-catenin. Paracelluar permeability was evaluated by transepithelial electric resistance. The effect of vanadate on cell cycle progression was confirmed by immunostaining of Ki67. Western blot analysis was used to evaluate the expression level of cell-cycle-associated proteins, including PCNA, cyclin D1, cyclin E and cyclin A. Time-dependent effects of vanadate on protein tyrosine phosphorylation were confirmed by Western blot analysis. ICC demonstrated the effect of vanadate on the disruption of p120, N-cadherin and alpha-catenin. Time- and dose-effects of vanadate on the severity of p120 disruption were also found. TER demonstrated the time- and dose-effect of vanadte on paracellular permeability. Although cell-cell junctions can be broken through by vanadate, no significant increase of Ki67 positive cells was found among the control group and all groups with different concentrations/durations of vanadate treatment. Western blot also showed no change of PCNA, cyclin D1, cyclin E and cyclin A after treatment with vanadate. In conclusion, protein tyrosine phosphatase inhibition can induce time-dependent release of cell-cell contacts and increase transepithelial permeability in post-confluent cultures of bovine corneal endothelial cells. However, such phenomenon is not enough to promoted cell cycle progression as seen in rat corneal endothelial cells.     

A reevaluation of corneal endothelial permeability to fluorescein

The permeability of the corneal endothelium and its aqueous-cornea distribution ratio were reevaluated in the rabbit eye. Both parameters were determined in an individual eye by applying the dye first by iontophoresis and then by intravitreal injection, which allows the influence of fluorescein glucuronide on the fluorophotometric measurements to be excluded. The corneal endothelial permeability coefficient was 5.13 +/- 1.64 X 10(-4) cm min-1, and the aqueous-cornea distribution ratio was 0.25 +/- 0.06 (mean +/- S.D., n = 11) on the average, and the former was considerably greater than the previous results, while the latter was considerably smaller.     

Human corneal endothelial permeability to fluorescein and fluorescein glucuronide

The corneal endothelial permeability coefficient (Pac) for fluorescein and fluorescein glucuronide was determined in ten normal young volunteers. After oral administration of fluorescein, the apparent concentrations of both dyes in the corneal stroma and the anterior chamber were measured by differential fluorometry. The apparent dye levels calculated directly from the in vivo fluorometric measurements were converted to the true ones, based on the result of a normalization experiment performed in rabbit eyes. The value of Pac averaged 5.44 +/- 1.77 X 10(-4) cm/min for fluorescein and 3.77 +/- 1.10 X 10(-4) cm/min for fluorescein glucuronide (mean +/- SD, N = 20); the former was significantly greater than the latter (paired t-test, P less than 0.001). The aqueous-cornea distribution ratio was 0.50 +/- 0.14 for fluorescein and 0.66 +/- 0.16 for fluorescein glucuronide; the latter was significantly greater than the former (paired t-test, P less than 0.001). It was suggested that the previously reported values of Pac for fluorescein in the human eye were underestimates.     

Influence of interleukin-1 beta induction and mitogen-activated protein kinase phosphorylation on optic nerve ligation-induced matrix metalloproteinase-9 activation in the retina

Ischemic damage to the retina is a multifaceted process that results in irreversible loss of ganglion cells and blinding disease. Although the mechanisms underlying ischemia-induced ganglion cell death in the retina are not clearly understood, we have recently reported that retinal damage induced by ligation of the optic nerve results in increased matrix metalloproteinase-9 (MMP-9) synthesis and promotes ganglion cell loss. In this study, we have investigated the roles of IL-1beta and mitogen activated protein kinases in MMP-9 induction in the retina. Optic nerve ligation led to a transient increase in IL-1beta and MMP-9 levels and phosphorylation of p42/p44 mitogen activated protein kinases (extracellular signal-regulated kinases, ERK1 and ERK2) in the retina. We found no significant increase in phosphorylation of p38 MAP kinase or c-jun N-terminal kinases indicating that ERK1/2 plays a major role in MMP-9 induction. Intravitreal injection of IL-1 receptor antagonist (IL-1Ra) or MAP kinase inhibitor U0126 significantly decreased both ERK1/2 phosphorylation and MMP-9 induction suggesting that interruption of this cascade might attenuate retinal damage. In support of this, intravitreal injection of IL-1Ra and U0126 offered significant protection against optic nerve-induced retinal damage. These results suggest that optic nerve ligation-induced IL-1beta promotes retinal damage by increasing MMP-9 synthesis in the retina.     

Protein kinase C regulation of corneal endothelial cell proliferation and cell cycle

PURPOSE: The purpose of this study was to determine the role of protein kinase C (PKC) in corneal endothelial cell proliferation. METHODS: Immunocytochemistry and Western blotting were used to define the PKC isoforms expressed in primary cultures of rat corneal endothelial cells. For proliferation studies, primary cultures of rat corneal endothelial cells were serum-starved for 48 hours and incubated for 2 hours with the PKC inhibitors staurosporine (10(-9) to 10(-7) M), chelerythrine (10(-9) to 5 x 10(-8) M), or calphostin C (10(-9) to 10(-7) M). Individual PKC isoforms were inhibited using PKCalpha antisense oligonucleotide transfection or exposure for 1 hour to myristoylated, pseudosubstrate-derived peptide inhibitors against PKCalpha, -alphassgamma, -epsilon, and -delta (10(-8) to 10(-6) M). Cells were then stimulated with 2.5% serum for 24 hours. Cell proliferation was measured with bromodeoxyuridine (BrDU) and Ki67 immunocytochemistry. Protein level of cyclin E was determined by Western blotting. RESULTS: PKCalpha, -ssII, -delta, -epsilon, -iota, -eta, -gamma, and -theta were detected in corneal endothelial cells. Maximum inhibition of PKC with staurosporine, calphostin C, and chelerythrine reduced cell proliferation to 7%, 31%, and 48% of control, respectively. Myristoylated peptide inhibition of PKCalpha and -epsilon reduced cell proliferation to 57% and 59% of control, respectively. PKCalpha antisense oligonucleotide reduced cell proliferation to 35% of control. Cyclin E protein level was decreased to 70%, 38%, 57%, and 43% of control in cells treated with calphostin C, staurosporine, chelerythrine, and PKCalpha antisense, respectively. CONCLUSIONS: PKC activity, in particular PKCalpha and -epsilon activity, is important in promoting corneal endothelial cell proliferation. Inhibition of PKC activity prohibits G1/S-phase progression and reduces cyclin E protein levels.     

cAMP-dependent phosphorylation of betaig-h3 protein in human corneal endothelial cells.

PURPOSE. To determine the identity of a major membrane/ extracellular matrix (ECM)-associated 66-kDa protein in human corneal endothelial cells and its phosphorylation in vivo. METHODS. A membrane/ECM-associated 66 kDa protein was purified from human corneal endothelial cells of 50-80 year-old donors by a three-step procedure that included preparative SDS-PAGE, preparative isoelectric focusing (IEF) and HPLC using a C-18 column. The phosphorylation of the 66-kDa protein was determined by both endogenous kinases and exogenous protein kinase A in the presence of endogenous or added cAMP, or added Walsh inhibitor (a specific inhibitor of protein kinase A). The phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. The phosphoamino acids were identified following hydrolysis of the purified phosphorylated 66-kDa protein and thin layer chromatography with standard phosphoamino acids. RESULTS. Following purification, the 66-kDa protein showed a single band on SDS-PAGE, a single species on two dimensional (2D)-gel electrophoresis and a single peak during C-18 HPLC. The partial N-terminal sequence of the 66-kDa protein matched with that of the 68-kDa beta ig-h3 protein (minus the signal peptide) of lung adenocarcinoma cells. Furthermore, on Western blot analysis, the 66-kDa protein immunoreacted with anti-beta ig-h3 past signal peptide (residue nos.24-32) antibody but not with the anti-beta ig-3 signal peptide (residue nos.1-9) antibody. During the phosphorylation of endothelial proteins by endogenous kinases or added protein kinase A in the presence of endogenous or added cAMP, the 66 kDa protein was the major substrate with its Ser residues phosphorylated in both cytosolic- and membrane/ECM-fractions. CONCLUSIONS. The human corneal endothelial 66-kDa protein is identical to the 68-kDa beta ig-h3 protein (minus signal peptide) from lung adenocarcinoma cells. The corneal protein exists in a phosphorylated state in vivo with its Ser residues phosphorylated by a cAMP-dependent protein kinase A.     

The changes of protein kinase C for human retinal

To study the effect of the subretinal fluid (SRF) on proliferation of retinal pigment epithelium (RPE) cells and retinal glial (RG) cells and associated activation and translocation of protein kinase C (PKC) as well as the application of PKC inhibitor. ·METHODS: RPE and RG cells were disintegrated to obtain PKC activity of cytoplasm and cellular membrane after being treated by the subretinal fluid (SRF) from the different stages of PVR patients (grade B and C) or being treated with PKC specific activator [phorbol-12-myris-tate-13-acetate (PMA)] or normal vitreous or DMEM culture medium. PKC activity in cytoplasm and cellular membrane was measured using radioactive isotope 32P labeling in a specific reaction of phosphorylation on PKC substrate. In addition, the PKC inhibitor, dequalinium chloride, was used to pretreat the RPE and RG cells before the cells exposed to SRF or PMA or normal vitreous. 3H-TdR (tritiated thymidine) was used to measure the levels of proliferation of RPE and RG cells with or without the activation and translocation. ·RESULTS: SRF and PMA promoted the proliferation of RPE and RG cells. SRF and PMA activated PKC in the cytoplasm of RPE and RG cells and the activated cytoplasm PKC translocated to the cellular membrane of RPE or RG cells. The cell proliferation or PKC activation or translocation was not equally active in RPE as in RG cells. However, PKC inhibitor which attenuated the cell proliferation did not show significant differenceoninhibitionof RPEandRGcell proliferation( >0.05). ·CONCLUSION: SRF can lead to the activation and translocation of PKC in RPE and RG cells, which promote the proliferation of RPE and RG cells. Dequalinium chloride can inhibit PKC activation and translocation hence slow down the cells proliferation     

Immunohistochemical assessment of the glial mitogen-activated protein kinase activation in glaucoma

PURPOSE: To determine whether retinal glial cells exhibit an activated phenotype in glaucomatous human eyes and whether the mitogen-activated protein kinases (MAPKs) are associated with glial activation in glaucoma. METHODS: Activated phenotypes of retinal macroglia (astrocytes and Müller cells) and microglia were identified by morphologic assessment and immunostaining for the cell markers glial fibrillary acidic protein (GFAP) and HLA-DR, respectively, in 30 eyes obtained from glaucomatous donor eyes in comparison with normal control eyes from 20 age-matched donors. Cellular localization of the activated forms of MAPKs, including extracellular signal-regulated kinases (ERK), c-Jun amino(N)-terminal kinase (JNK), and p38, were studied in the retina of these eyes by immunoperoxidase staining and double immunofluorescence labeling with phosphorylation site-specific antibodies. RESULTS: Retinal astrocytes and Müller cells exhibited a hypertrophic morphology and increased immunostaining for GFAP in the glaucomatous retina. Although an increase was detectable in the number and size of cells positive for HLA-DR immunostaining in the glaucomatous retina compared with the control retina, microglial activation was not as prominent or widespread as the macroglial activation detected in the same eyes. The intensity of immunostaining and the number of immunostained cells for the activated MAPKs were greater in retina sections from glaucomatous eyes than in control eyes, being most prominent for phospho-ERK. Double immunofluorescence labeling demonstrated that the increased retinal immunostaining for phospho-ERK was predominantly, but not exclusively, localized to glial cells, whereas, the immunostaining for phospho-JNK or phospho-p38 was mainly associated with nonglial cells. CONCLUSIONS: These findings provide evidence that retinal glial cells undergo activation in the glaucomatous human retina. A prominent and persistent activation of ERK in activated glial cells suggests that this signaling pathway is probably associated with the induction and/or maintenance of the activated glial phenotype in glaucoma. Because MAPKs are involved in determination of ultimate cell fate, their differential activity in neuronal and activated glial cells in the glaucomatous retina may be associated, in part, with the differential susceptibility of these cell types to glaucomatous injury.     

Plasma VEGF and soluble VEGF receptor FLT-1 in proliferative retinopathy: relationship to endothelial dysfunction and laser treatment

PURPOSE: To study plasma levels of vascular endothelial growth factor (VEGF, an index of angiogenesis), its soluble receptor (sFlt-1) and von Willebrand factor (vWf, an index of endothelial damage or dysfunction) in patients with proliferative retinopathy and corresponding changes in plasma levels after pan-retinal photocoagulation (PRP). METHODS: Eighteen patients (10 men; age, 57+/-16 years, mean +/- SD) with proliferative retinopathy secondary to diabetes (n = 13) and ischemic retinal vein occlusion (n = 5) with no previous PRP therapy were studied. Blood samples were obtained before and at 4 months after the last PRP session. Baseline (prelaser) plasma levels of VEGF, sFlt-1, and vWf (all by ELISA) were compared with levels in 16 diabetic patients with background retinopathy ("hospital controls"), and 18 healthy, age- and sex-matched "healthy controls." RESULTS: Patients with proliferative retinopathy had significantly raised plasma VEGF when compared with both control groups (P = 0.001). Patients with proliferative retinopathy and hospital controls had significantly raised plasma vWf levels when compared with healthy controls (P = 0.012). There was no difference in sFlt-1 levels between patients and controls (P = 0.162). After PRP, there was a significant reduction in plasma VEGF levels at 4 months' follow-up (P < 0.001), but no significant changes in plasma sFlt-1 or vWf levels. Patients with complete resolution of neovascularization had a trend toward lower median VEGF levels (80 versus 150 pg/ml, P = 0.062), but vWf levels (P = 0.50) and sFlt-1 (P = 0.479) were not statistically different. Baseline VEGF and sFlt-1 levels were significantly correlated (Spearman r = 0.505, P = 0.032) but after PRP at 4 months' follow-up, this was no longer significant (r = -0.269, P = 0.28). CONCLUSIONS: In this pilot study, patients with proliferative retinopathy demonstrate elevated peripheral markers of angiogenesis and endothelial dysfunction, suggesting a role for these processes in the pathogenesis of this condition. A fall in levels of VEGF after successful laser treatment may provide an opportunity for monitoring disease progression or relapse via a blood sample.