Involvement of ZEB1 and Snail1 in excessive production of extracellular matrix in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) due to corneal endothelial cell degeneration is a major cause of corneal transplantation. It is characterized by abnormal deposition of extracellular matrix (ECM), such as corneal guttae, accompanied by a loss of endothelial cells. Although recent studies have revealed several genomic factors, the molecular pathophysiology of FECD has not yet been revealed. In this study, we establish a cellular in vitro model by using immortalized corneal endothelial cells obtained from late-onset FECD and control patients and examined the involvement of epithelial mesenchymal transition (EMT) on excessive ECM production. We demonstrate that the EMT-inducing genes ZEB1 and SNAI1 were highly expressed in corneal endothelial cells in FECD and were involved in excessive production of ECM proteins, such as type I collagen and fibronectin through the transforming growth factor (TGF)-ß signaling pathway. Furthermore, we found that SB431542, a specific inhibitor of TGF-ß type I ALK receptors, suppressed the expression of ZEB1 and Snail1 followed by reduced production of ECM. These findings suggest that increased expression levels of ZEB1 and Snail1 in FECD cells were responsible for an increased responsiveness to TGF-ß present in the aqueous humor and excessive production of ECM. In addition, these results suggest that the regulation of EMT-related genes by blocking the TGF-ß signaling pathway may be a feasible therapeutic strategy for FECD.

Corneal endothelial cell fate is maintained by LGR5 through the regulation of hedgehog and Wnt pathway.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a target of Wnt signaling, is reportedly a marker of intestine, stomach, and hair follicle stem cells in mice. To gain a novel insight into the role of LGR5 in human corneal tissue, we performed gain- and loss-of-function studies. The findings of this study show for the first time that LGR5 is uniquely expressed in the peripheral region of human corneal endothelial cells (CECs) and that LGR5((+)) cells have some stem/progenitor cell characteristics, and that in human corneal endothelium, LGR5 is the target molecule and negative feedback regulator of the Hedgehog (HH) signaling pathway. Interestingly, the findings of this study show that persistent LGR5 expression maintained endothelial cell phenotypes and inhibited mesenchymal transformation (MT) through the Wnt pathway. Moreover, R-spondin-1, an LGR5 ligand, dramatically accelerated CEC proliferation and also inhibited MT through the Wnt pathway. These findings provide new insights into the underlying homeostatic regulation of human corneal endothelial stem/progenitor cells by LGR5 through the HH and Wnt pathways.

Endothelial mesenchymal transformation mediated by IL-1ß-induced FGF-2 in corneal endothelial cells.

This review describes the molecular mechanism of endothelial mesenchymal transformation (EMT) mediated by fibroblast growth factor-2 (FGF-2) in corneal endothelial cells (CECs). Corneal fibrosis is not frequently observed in corneal endothelium/Descemet's membrane complex; but when this pathologic tissue is produced, it causes a loss of vision by physically blocking light transmittance. Herein, we will address the cellular activities of FGF-2 and its signaling pathways during the EMT process. Furthermore, we will discuss the role of inflammation on FGF-2-mediated EMT. Interleukin-1ß (IL-1ß) greatly upregulates FGF-2 production in CECs, thus leading to FGF-2-mediated EMT; the whole spectrum of the injury-mediated inflammation (IL-1ß pathway) and the subsequent EMT process (FGF-2 pathway) will be briefly discussed. Intervention in the two pathways will provide the means to block EMT before inflammation causes an irreversible change, such as the production of retrocorneal fibrous membrane observed in human eyes.

The role of nerve growth factor in hyperosmolar stress induced apoptosis.

Nerve growth factor (NGF) is a neurotrophic factor that plays an important role in the differentiation and growth of neuronal cells. It is also regarded as an inflammatory mediator in non-neuronal tissues under physiological stress conditions. The mechanisms of NGF production and its roles in hyperosmolar stress conditions have not been established. In this study, we show that NGF levels in cultured human corneal epithelial cells (HCECs) were up-regulated during hyperosmolar stress by IL-1beta, but not TNF-alpha. NF-kappaB activity, but not AP-1, increased significantly under hyperosmolar conditions, and NF-kappaB was involved in IL-1beta-induced NGF production. IL-1beta-induced NGF production reduced JNK phosphorylation and HCEC apoptosis. These changes were accompanied by down-regulated Bax and caspase-3, -8, -9 activities. NGF siRNA and the tyrosine kinase inhibitor K252a significantly enhanced Bax up-regulation. Thus, up-regulated NGF under hyperosmolar stress conditions may contribute, at least in part, to reduced HCEC apoptosis. This conclusion suggests that enhanced NGF expression may be beneficial in recovering corneal damage due to chronic hyperosmolar stress.

Involvement of two distinct ubiquitin E3 ligase systems for p27 degradation in corneal endothelial cells.

PURPOSE: p27(Kip1) (p27) is an important regulator of G(1) progression. For cells to proliferate, p27 must undergo proteolysis. FGF-2 enables phosphorylation of p27 at both the Thr-187 and Ser-10 sites, an event that is prerequisite for polyubiquitination. This study was undertaken to determine whether degradation of the two phosphorylated p27s is mediated by a distinct ubiquitin E3 ligase complex at different subcellular locations. METHODS: Expression of p27, KPC1, KPC2, Skp1, Skp2, and Cul1 was analyzed by immunoblot analysis. Association of p27 with ubiquitin E3 ligase was determined with coimmunoprecipitation followed by immunoblot analysis. Inhibitors were used to inhibit proteasomal degradation and nuclear export of the phosphorylated p27. DNA synthesis was measured by BrdU incorporation into DNA. RESULTS: Among ubiquitin ligase complex proteins, Cul1, KPC1, and KPC2 were constitutively expressed, whereas expression of Skp1 and Skp2 was temporally induced by FGF-2. Skp1, Skp2, and Cul1 were involved in polyubiquitination of phosphorylated p27 at Thr-187 (pp27Thr187) in nuclei. Maximum association of pp27Thr187 with the ubiquitin E3 ligase occurred 24 hours after FGF-2 stimulation. pp27Ser10 used the cytoplasmic ubiquitin E3 ligases KPC1 and KPC2, with maximum protein interaction observed at 8 hours. MG132 effectively blocked degradation of both pp27Thr187 and pp27Ser10, whereas leptomycin B blocked the nuclear export of pp27Ser10. Both inhibitors blocked BrdU incorporation into DNA. CONCLUSIONS: The findings demonstrate distinct polyubiquitination pathways for pp27Thr187 and pp27Ser10; the former is ubiquitinated through the nuclear ubiquitin E3 ligase system during late G(1) phase; the latter by cytosolic ubiquitin E3 ligase during early G(1) phase.

The Effect of a p38 Mitogen-Activated Protein Kinase Inhibitor on Cellular Senescence of Cultivated Human Corneal Endothelial Cells.

Purpose: We have begun a clinical trial of a cell-based therapy for corneal endothelial dysfunction in Japan. The purpose of this study was to investigate the usefulness of a p38 MAPK inhibitor for prevention cellular senescence in cultivated human corneal endothelial cells (HCECs). Methods: HCECs of 10 donor corneas were divided and cultured with or without SB203580 (a p38 MAPK inhibitor). Cell density and morphology were evaluated by phase-contrast microscopy. Expression of function-related proteins was examined by immunofluorescent staining. Cellular senescence was evaluated by SA-ß-gal staining and Western blotting for p16 and p21. Senescence-associated factors were evaluated by membrane blotting array, quantitative PCR, and ELISA. Results: Phase-contrast microscopy showed a significantly higher cell density for HCECs cultured with SB203580 than without SB203580 (2623 ± 657 cells/mm2 and 1752 ± 628 cells/mm2, respectively). The HCECs cultured with SB203580 maintained a hexagonal morphology and expressed ZO-1, N-cadherin, and Na+/K+-ATPase in the plasma membrane, whereas the control HCECs showed an altered staining pattern for these marker proteins. HCECs cultured without SB203580 showed high positive SA-ß-gal staining, a low nuclear/cytoplasm ratio, and expression of p16 and p21. IL-6, IL-8, CCL2, and CXCL1 were observed at high levels in low cell density HCECs cultured without SB203580. Conclusions: Activation of p38 MAPK signaling due to culture stress might be a causative factor that induces cellular senescence; therefore, the use of p38 MAPK inhibitor to counteract senescence may achieve sufficient numbers of HCECs for tissue engineering therapy for corneal endothelial dysfunction.

FGF-2-induced wound healing in corneal endothelial cells requires Cdc42 activation and Rho inactivation through the phosphatidylinositol 3-kinase pathway.

PURPOSE: Acquisition of elongated cells with pseudopodia is observed when corneal endothelial cells (CECs) are simultaneously treated with basic fibroblast growth factor (FGF)-2 and RhoA inhibitors. This study was designed to determine whether these phenotypes are migratory and whether Cdc42 activation and RhoA inactivation are involved in cell migration. METHODS: A scratch-induced directional migration assay was used to measure migratory rates. Activation of Cdc42 was determined by GTP pull-down assay. Transfection was performed using constitutively active (ca) or dominant negative (dn) Rho guanosine triphosphatase (GTPase) vectors. RESULTS: Stimulation with basic FGF-2 alone resulted in a 43% recovery of the wound area, whereas CECs treated with FGF-2 and Y27632 (inhibitor of Rho-associated kinase) achieved an 84% recovery of the wound area with a fast migratory speed (0.72 microm/min). The synergistic effects of FGF-2 and Y27632 were completely blocked by LY294002 (PI 3-kinase inhibitor). Under these conditions, activation of PI 3-kinase and Cdc42 were observed in the migratory cells. The involvement of activated Cdc42 and inactivated Rho in endothelial migration was determined by transfecting CECs with ca- or dnRho GTPase vectors. A high migratory rate (0.52 microm/min) was seen in CECs expressing caCdc42, whereas endothelial migration was completely inhibited in CECs expressing caRho. When cells expressing caCdc42 were treated with FGF-2, migration reached the maximum rate (0.69 microm/min), similar to that observed in cells treated with FGF-2 and Y27632. CONCLUSIONS: These findings suggest that endothelial migration is induced by activated Cdc42 and inactivated Rho via PI 3-kinase after FGF-2 stimulation and that Cdc42 activation is crucial for CECs to acquire the characteristic migratory phenotypes.

Cross-talk among Rho GTPases acting downstream of PI 3-kinase induces mesenchymal transformation of corneal endothelial cells mediated by FGF-2.

PURPOSE: Endothelial-mesenchymal transformation (EMT), in which the contact-inhibited corneal endothelial cells (CECs) become multilayers of spindle-shaped cells containing protrusive processes, is mediated by fibroblast growth factor (FGF)-2. The involvement in EMT of cross-talk among Rho GTPases mediated by FGF-2 was also investigated. METHODS: GTP pull-down assays were performed to identify the activated Rho GTPases. Transfection of CECs with either constitutively active (ca) or dominant negative (dn) Rho GTPase vectors was performed. Protein-protein interaction was investigated by coimmunoprecipitation and a yeast two-hybrid assay. RESULTS: The alteration of morphology and actin cytoskeleton caused by FGF-2 was mediated by active Rac and inactive Rho. Prolonged treatment of CECs with FGF-2 induced formation of protrusive processes through activated Cdc42. All FGF-2 actions were blocked by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002. Cells transfected with caRacG12V acquired elongated morphology; the actin cytoskeleton was reorganized to the cortex. Formation of protrusive processes was observed in the elongated cells expressing caCdc42G12V or dominant negative (dn)RhoT19N, whereas polygonal cells expressing dnRacT17N, caRhoG14V, or dnCdc42T17N had stress fibers. Further analysis demonstrated that Rac was associated with Cdc42 or Rho through a 32- or 30-kDa Dbl homology/pleckstrin homology-containing protein. CONCLUSIONS: These findings suggest that alteration of cell shape and actin cytoskeleton are closely linked to the sequential activation of Rho GTPases through PI 3-kinase in response to FGF-2 stimulation. Cortical actin is formed via active Rac and inactive Rho followed by formation of protrusive processes mediated by active Cdc42 and inactive Rho.

Regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation.

PURPOSE: To investigate the regulatory role of FGF-2 on type I collagen expression during endothelial mesenchymal transformation (EMT). METHODS: Corneal endothelial cells (CECs) treated with FGF-2 from the primary culture to the third passage were transformed and designated as fibroblastic CECs (fCECs). Steady state levels of both type I collagen RNAs were measured using reverse transcription-real-time PCR, and their half lives were determined in the presence of inhibitor of RNA synthesis. Limited proteolysis with pepsin was used to determine secretion of type I collagen. Protein-protein interaction was determined by coimmunoprecipitation, and subcellular localization was studied by immunofluorescence. RESULTS: fCECs were characterized by greatly stimulated proliferative potential, loss of contact inhibition, and multilayer fibroblastic cells. The steady state level of alpha1(I) collagen RNA was greatly upregulated through stabilization of the message in fCECs, whereas steady state level and half-life of the alpha2(I) collagen RNA were slightly increased compared with the corresponding levels in normal CECs. Of interest, fCECs predominantly secreted homotrimeric type I collagen, [alpha1(I)](3), with heterotrimeric type I collagen as a minor species. Type I collagen in fCECs was preferentially associated and colocalized with Hsp47 at Golgi apparatus as opposed to its association with protein disulfide isomerase in CECs. LY294002 (a specific PI 3-kinase inhibitor) greatly reduced the steady state levels and stability of alpha1(I) and alpha2(I) collagen RNAs and the secretion of type I collagen. CONCLUSIONS: FGF-2 directly mediates corneal EMT through the action of PI 3-kinase, which acts on posttranscriptional regulation by affecting the stability of type I collagen RNA.

Regulatory role of PI 3-kinase on expression of Cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells.

PURPOSE: FGF-2 is a potent mitogen of rabbit corneal endothelial cells (CECs). This study was undertaken to investigate whether PI 3-kinase participates in cell cycle regulation in response to stimulation with FGF-2 in CECs. METHODS: Cell proliferation was assayed by counting the cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of cyclin-dependent kinase 4 (Cdk4), p27(Kip1) (p27), phosphatidylinositol 3 (PI 3)-kinase, protein kinase B/Akt (Akt), and beta-actin was analyzed by immunoblot. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. LY294002 was used to inhibit PI 3-kinase. RESULTS: CEC required prolonged and continuous exposure to FGF-2. FGF-2 at 10 ng/mL markedly stimulated PI 3-kinase enzyme activity, and stimulation with FGF-2 also caused activation of Akt. LY294002 inhibited both cell proliferation and PI 3-kinase activity in a concentration-dependent manner. The role of PI 3-kinase in cell cycle stimulation was determined: FGF-2 markedly upregulated expression of Cdk4 and stimulated translocation of Cdk4 into nuclei, whereas LY294002 markedly blocked upregulation of Cdk4 expression, and the inhibitor facilitated nuclear export of Cdk4. In contrast, FGF-2 significantly downregulated expression of p27 and facilitated phosphorylation of p27. LY294002 completely blocked the action of FGF-2 on the expression and phosphorylation of p27. CONCLUSIONS: These data indicate that PI 3-kinase ultimately leads to activation of the cell cycle machinery in response to FGF-2. It does so by upregulating expression of Cdk4, facilitating the nuclear import of Cdk4, and sequestering Cdk4 in the nuclei as it simultaneously downregulates expression of p27 and facilitates the proteolysis of the molecule by phosphorylation.

Regulatory role of cAMP on expression of Cdk4 and p27(Kip1) by inhibiting phosphatidylinositol 3-kinase in corneal endothelial cells.

PURPOSE: Fibroblast growth factor (FGF)-2 is a potent mitogen of corneal endothelial cells (CECs). Results in an earlier study showed that FGF-2 activates phosphatidylinositol (PI) 3-kinase to stimulate the cell cycle machinery. The current study was designed to determine whether adenosine 3',5'-monophosphate (cAMP) antagonizes FGF-2 by inhibiting PI 3-kinase/Akt pathways, thus leading to regulation of cyclin-dependent kinase 4 (Cdk4) and p27(Kip1) (p27) expression. METHODS: Cell proliferation was assayed by counting cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of Cdk4, p27, PI 3-kinase, Akt, and beta-actin was analyzed by immunoblot analysis. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. RESULTS: 8-Bromoadenosine cAMP (8-Br-cAMP), a diffusible cAMP analogue, inhibited the PI 3-kinase/Akt signaling pathways. The 8-Br-cAMP and PI 3-kinase inhibitor (LY294002) produced equivalent stimulation and inhibition, respectively, of p27 and Cdk4 protein levels. They also equally inhibited cell proliferation, nuclear translocation of Cdk4, and phosphorylation of p27. Negative regulation of PI 3-kinase by 8-Br-cAMP was mediated by a direct inhibition of PI 3-kinase activity, which subsequently blocked phosphorylation of Akt at both the Ser473 and Thr308 sites. In addition, 8-Br-cAMP promoted a rapid turnover of Akt protein, and 8-Br-cAMP markedly reduced the half-life of Cdk4 protein. This inhibitory activity of cAMP was not mediated by PKA, but 8-Br-cAMP inhibited membrane localization of the p85 regulatory subunit of PI 3-kinase. CONCLUSIONS: These data support the hypothesis that cAMP inhibits the proliferation of CECs, preventing them from entering the S phase by negatively regulating PI 3-kinase.

FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells.

PURPOSE: Corneal endothelial cells (CECs) undergo endothelial to mesenchymal transformation (EMT) in response to FGF-2 stimulation. One phenotypic change that occurs during EMT is a change in cell shape from polygonal to elongated fibroblast-like cells. We investigated whether FGF-2 plays a role in this morphogenetic pathway by reorganizing actin cytoskeleton through the actions of phosphatidylinositol (PI) 3-kinase and the Ras related Rho family of small guanosine triphosphatases (GTPases). METHODS: Cell morphology was analyzed using phase contrast microscopy, and the organization of actin cytoskeleton and focal adhesions were analyzed by immunofluorescent staining. Expression of vinculin and beta-actin was determined by immunoblot analysis. Pharmacologic inhibitors (LY294002, C3 exoenzyme, Y27632, or PD98059) or neutralizing antibody to FGF-2, respectively, were used to block PI 3-kinase, Rho, Rho associated kinase, extracellular signal regulated kinase, or FGF-2 pathways. RESULTS: CECs treated with FGF-2 became smaller and lost their characteristic polygonal cell morphology. Such cell shape change was completely blocked by treatment with LY294002. CECs in culture have abundant stress fibers that are oriented radially across the cell. However, FGF-2 caused a loss of these stress fibers and focal adhesions. The modulated cells contained a cortical actin ring while LY294002 completely abolished this action of FGF-2 on actin cytoskeleton. Treatment of cells with C3 exoenzyme or Y27632 in the presence of FGF-2 induced spindle shaped cells with prominent pseudopodia which were rapidly formed upon exposure to the inhibitor. The expression level of vinculin was found to be similar in all experimental conditions but vinculin was mostly translocated to the cytoplasm in response to FGF-2 stimulation. CECs plated on Matrigel matrix demonstrated findings similar to those from cells plated on the conventional culture dishes, except that Matrigel facilitated the formation of pseudopodia. We further investigated in vivo actin organization using organ cultures of corneal endothelium (CE) on Descemet's membrane. The contact inhibited endothelial monolayer demonstrated a circumferential actin ring, and no stress fibers were observed. When CE was treated with FGF-2, a half population of CE lost its characteristic contact inhibited cobblestone morphology. Actin cortex was greatly disrupted in these modulated cells. Both neutralizing antibody to FGF-2 and LY294002 completely impeded the modulating activity of FGF-2 on the endothelial monolayer. When CE was simultaneously treated with FGF-2 and Y27632, the circumferential actin cortex was greatly disrupted and the endothelial monolayer was transformed into multi-layers of fibroblastic cells containing pseudopodia. Both LY294002 and neutralizing antibody to FGF-2 antagonized the actions of FGF-2 and Y27632. CONCLUSIONS: These data indicate that CECs in culture have constitutively active Rho activity as evidenced by stress fiber formation and that PI 3-kinase negatively regulates the formation of stress fibers and focal adhesions, perhaps antagonizing the Rho pathways. Formation of pseudopodia in response to FGF-2 and Y27632 may suggest that the Rho/ROCK pathway negatively regulates Cdc42.

Differential interaction of molecular chaperones with procollagen I and type IV collagen in corneal endothelial cells.

PURPOSE: Procollagen I is synthesized and intracellularly degraded in corneal endothelial cells (CEC), whereas type IV and VIII collagens are secreted into Descemet's membrane. In our previous study, we demonstrated that procollagen I synthesized by CEC is improperly folded and that the molecule was largely colocalized with protein disulfide isomerase (PDI) within the endoplasmic reticulum (ER). In the present study, we further investigated whether the alpha-subunit of prolyl 4-hydroxylase (P4Halpha) and glucose regulatory protein/immunoglobulin heavy chain binding protein (Grp78/BiP) were also involved in ER retention of procollagen I in CEC. METHODS: Immunocytochemical analysis was performed to determine the colocalization of procollagen I with molecular chaprones. Protein synthesis was measured by immunoblot analysis and the association between proteins was determined by coimmunoprecipitation followed by immunoblot analysis. mRNA was quantitated using RT-PCR. RESULTS: To study the interaction of procollagen I with certain molecular chaperones involved in the collagen biosynthetic pathway, we determined whether procollagen I colocalized with P4Halpha and Grp78/Bip, and then compared this molecular chaperone colocalization with their association with type IV collagen. Procollagen I was colocalized with either P4Halpha or Grp78/Bip to a much lesser degree than type IV collagen was colocalized with these same ER proteins. Colocalization between the molecular chaperones demonstrated that P4Halpha and Grp78/Bip were largely colocalized in the peripheral region of the ER, whereas colocalization of P4Halpha and PDI was mostly limited to a small region of the ER. When cells were treated with alpha,alpha-dipyridyl, the inhibitor did not affect the colocalization profiles of collagens with the molecular chaperones. However, the inhibitor markedly increased colocalization of P4Halpha and PDI, but it significantly decreased colocalization between P4Halpha and Grp78/Bip. When synthesis of the molecular chaperones was compared between CEC and corneal stromal fibroblasts (CSF), more Grp78/Bip and PDI were produced by CEC than by CSF. On the other hand, expression of Hsp47 was lower in CEC than it was in CSF. Coimmunoprecipitation was used to compare the association of P4Halpha or Grp78/Bip with collagens in CEC and CSF. The association of collagens (regardless of type) with P4Halpha or Grp78/Bip was much higher in CEC than in CSF. When the association of collagen molecules with respective molecular chaperones was compared in CEC, the degree of association between Grp78/Bip and procollagen I was similar to that between the molecular chaperone and type IV collagen. On the other hand, the degree of association between P4Halpha and type IV collagen was much higher than that between P4Halpha and procollagen I. CONCLUSIONS: These data suggest that procollagen I and type IV collagen may use different molecular chaperones in the ER, thus targeting their distinctive destinations.

Cdk4 and p27Kip1 play a role in PLC-gamma1-mediated mitogenic signaling pathway of 18 kDa FGF-2 in corneal endothelial cells.

PURPOSE: To determine whether PLC-gamma1 enzyme activity is essential for cell proliferation in response to FGF-2 stimulation and to investigate the effect of PLC-gamma1 activation on cell division and on processes that regulate cell cycle progression. METHODS: Cell proliferation was assayed using a colorimetric method to determine the number of viable cells. Subcellular localization of proteins was determined by immunocytochemical analysis, and expression of the proteins was analyzed by immunoblotting. PLC activity was determined by measuring the total inositol phosphates. RESULTS: When CEC were treated with FGF-2, a prolonged and continuous FGF-2 exposure was required for both PLC enzyme activation and cell proliferation. However, there was a long lag period between the PLC enzyme activation and cell proliferation: the maximum enzyme activity was reached 8 h after FGF-2 stimulation, but no cell proliferation was observed in the cells exposed to FGF-2 for 8 h. Using neutralizing anti-PLC-gamma1, PLC-beta1, or PLC-delta1 antibodies, we further demonstrated that PLC-gamma1 accounts for the hydrolysis of total phosphoinositides (PI) and cell proliferation mediated by FGF-2. The role of PLC-gamma1 linking to the cell cycle was then determined by the blockades of FGF-2 action on Cdk4 and p27-Kip1. Interestingly, FGF-2 both upregulates Cdk4 synthesis and facilitates the nuclear import of the molecule from the cytoplasm, whereas it facilitates the nuclear export of p27-Kip1 to the cytoplasm without affecting synthesis of the molecule. The neutralizing anti-PLC-gamma1 antibody completely abolishes the FGF-2 activity on Cdk4, both at the synthetic level and at the level of translocation, and the PLC-gamma1 antibody blocks the nuclear export of p27-Kip1. CONCLUSIONS: These data indicate that PLC-gamma1 ultimately leads to activation of the cell cycle machinery to induce cell proliferation mediated by FGF-2. It does so by upregulating Cdk4 expression and by facilitating the nuclear import of the molecule and nuclear export of the Cdk inhibitor (p27-Kip1) to the proteolysis site, the cytoplasm.

R-spondin1 regulates cell proliferation of corneal endothelial cells via the Wnt3a/ß-catenin pathway.

PURPOSE: To evaluate the effect of Roof plate-specific spondin 1 (R-spondin1) on the proliferation of corneal endothelial cells (CECs) and to determine whether the Wnt/ß-catenin pathway is involved in the activities of R-spondin1. METHODS: The proliferation of rabbit CECs (RCECs) and human CECs (HCECs) was measured by 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA. The effect of R-spondin1 on CEC density was evaluated in ex vivo organ-cultured rabbit and human corneal tissues. The cell density of HCECs cultured with R-spondin1 was also evaluated in vitro. The subcellular localization of function-associated markers of CECs (zona occludens 1 [ZO-1] and Na+/K+-ATPase) was determined by immunohistocytochemistry. The expression of cell cycle proteins and localization of ß-catenin were determined by immunoblotting. RESULTS: The in vitro proliferation of RCECs and HCECs increased by 1.2- to 1.3-fold in response to R-spondin1. The CEC densities of rabbit and human corneal tissues were increased significantly by R-spondin1 treatment. Na+/K+-ATPase and ZO-1 were well preserved on the plasma membranes. When HCECs were maintained in the presence of R-spondin1 for up to 90 days, the maximum cell density was observed at approximately 50 days, and the cell density was maintained for up to 90 days. R-spondin1 facilitated the nuclear import of ß-catenin in RCECs within 30 minutes, which subsequently upregulated cyclin D and downregulated p27, leading to G1/S progression by hyperphosphorylation of the retinoblastoma protein. CONCLUSIONS: The unique effects of R-spondin1 on the proliferation of CECs, regardless of species, indicate that R-spondin1 may play a key role in maintaining corneal endothelium homeostasis through the Wnt/ß-catenin pathway.

Involvement of cyclin D and p27 in cell proliferation mediated by ROCK inhibitors Y-27632 and Y-39983 during corneal endothelium wound healing.

PURPOSE: To investigate the molecular mechanism of Rho-associated kinase (ROCK) inhibitors Y-27632 and Y-39983 on corneal endothelial cell (CEC) proliferation and their wound-healing effect. METHODS: The expression of G1 proteins of the cell cycle and expression of phosphorylated Akt in monkey CECs (MCECs) treated with Y-27632 were determined by Western blotting. The effect of Y-39983 on the proliferation of MCECs and human CECs (HCECs) was evaluated by both Ki67 staining and incorporation of BrdU. As an in vivo study, Y-39983 was topically instilled in a corneal-endothelial partially injured rabbit model, and CEC proliferation was then evaluated. RESULTS: Investigation of the molecular mechanism of Y-27632 on CEC proliferation revealed that Y-27632 facilitated degradation of p27Kip1 (p27), and promoted the expression of cyclin D. When CECs were stimulated with Y-27632, a 1.7-fold increase in the activation of Akt was seen in comparison to the control after 1 hour. The presence of LY294002, the PI 3-kinase inhibitor, sustained the level of p27. When the efficacy of Y-39983 on cell proliferation was measured in a rabbit model, Y-39983 eye-drop instillation demonstrated rapid wound healing in a concentration range of 0.095 to 0.95 mM, whereas Y-27632 demonstrated rapid wound healing in a concentration range of 3 to 10 mM. CONCLUSIONS: These findings show that ROCK inhibitors employ both cyclin D and p27 via PI 3-kinase signaling to promote CEC proliferation, and that Y-39983 may be a more potent agent than Y-27632 for facilitating corneal endothelium wound healing.

Inhibition of TGF-ß signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine.

Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs) has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs) is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-ß) receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+)/K(+)-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-ß receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

The ROCK inhibitor eye drop accelerates corneal endothelium wound healing.

PURPOSE: To evaluate the effect of Rho kinase (ROCK)-inhibitor eye drops on a corneal endothelial dysfunction primate model and human clinical case series of corneal endothelial dysfunction. METHODS: As a corneal-endothelial partially injured model, the corneal endothelium of seven cynomolgus monkeys was damaged by transcorneal freezing; 10 mm of rock inhibitor Y-27632 was then applied topically 6 times daily. The phenotype of the reconstructed corneal endothelium was evaluated by immunohistochemical analysis and noncontact specular microscopy. For clinical study, the effect of Y-27632 eye drops after transcorneal freezing was evaluated in eight corneal endothelial dysfunction patients: four central corneal edema patients and four diffuse corneal edema patients. RESULTS: Slit-lamp microscopy revealed that both Y-27632-treated and -nontreated corneas became hazy after transcorneal freezing, and then recovered their transparency within 4 weeks. ROCK inhibitor Y-27632 promoted recovery of corneal endothelial cell density and wound healing in terms of both morphology and function. The percentage of ZO-1 and Na(+)/K(+)-ATPase positive cells in the regenerated area in the Y-27632 group was significantly higher than in the controls. Noncontact specular microscopy revealed that corneal endothelial cell density was significantly higher in the Y-27632 group compared with the controls at 4 weeks; cell density reached approximately 3000 cells/mm(2), as opposed to 1500 cells/mm(2) in the control group. In addition to the animal study findings, the clinical study findings showed that Y-27632 eye drops effectively improved corneal edema of corneal endothelial dysfunction patients with central edema. CONCLUSIONS: These findings show that rock inhibitor Y-27632 eye drops promote corneal endothelial wound healing in a primate animal model and suggest the possibility of Y-27632 as a novel therapeutic modality for certain forms of corneal endothelial dysfunction. (http://www.umin.ac.jp/ctr/ number, UMIN000003625.).

Corneal endothelial expansion promoted by human bone marrow mesenchymal stem cell-derived conditioned medium.

Healthy corneal endothelium is essential for maintaining corneal clarity, as the damage of corneal endothelial cells and loss of cell count causes severe visual impairment. Corneal transplantation is currently the only therapy for severe corneal disorders. The greatly limited proliferative ability of human corneal endothelial cells (HCECs), even in vitro, has challenged researchers to establish efficient techniques for the cultivating HCECs, a pivotal issue for clinical applications. The aim of this study was to evaluate conditioned medium (CM) obtained from human bone marrow-derived mesenchymal stem cells (MSCs) (MSC-CM) for use as a consistent expansion protocol of HCECs. When HCECs were maintained in the presence of MSC-CM, cell morphology assumed a hexagonal shape similar to corneal endothelial cells in vivo, as opposed to the irregular cell shape observed in control cultures in the absence of MSC-CM. They also maintained the functional protein phenotypes; ZO-1 and Na(+)/K(+)-ATPase were localized at the intercellular adherent junctions and pump proteins of corneal endothelium were accordingly expressed. In comparison to the proliferative potential observed in the control cultures, HCECs maintained in MSC-CM were found to have more than twice as many Ki67-positive cells and a greatly increased incorporation of BrdU into DNA. MSC-CM further facilitated the cell migration of HCECs. Lastly, the mechanism of cell proliferation mediated by MSC-CM was investigated, and phosphorylation of Akt and ERK1/2 was observed in HCECs after exposure to MSC-CM. The inhibitor to PI 3-kinase maintained the level of p27(Kip1) for up to 24 hours and greatly blocked the expression of cyclin D1 and D3 during the early G1 phase, leading to the reduction of cell density. These findings indicate that MSC-CM not only stimulates the proliferation of HCECs by regulating the G1 proteins of the cell cycle but also maintains the characteristic differentiated phenotypes necessary for the endothelial functions.

NF-κB is the transcription factor for FGF-2 that causes endothelial mesenchymal transformation in cornea.

PURPOSE: To determine the role of nuclear factor-κB (NF-κB) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1ß stimulation in corneal endothelial cells (CECs). METHODS: Expression and/or activation of IL-1 receptor-associated protein kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA kit, while binding of NF-κB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. RESULTS: Brief stimulation of CECs with IL-1ß upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after IL-1ß stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/ß and degrades IκB, subsequently leading to activation of NF-κB. The induction of FGF-2 by IL-1ß was completely blocked by inhibitors to NF-κB activation (sulfasalazine) or PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-κB is the transcription factor of FGF-2 as NF-κB binds the putative NF-κB binding site of the FGF-2 promoter. CONCLUSIONS: These data suggest that IL-1ß signaling combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene.