Pigment Epithelium-Derived Factor Promotes the Growth and Migration of Human Esophageal Squamous Cell Carcinoma.

Pigment epithelium-derived factor (PEDF) is an oncogene found in various types of cancers. However, how PEDF affects the development of human esophageal squamous cell carcinoma (ESCC) is unknown. This study investigates the role of PEDF in ESCC cell proliferation, migration, and cell cycle both in vitro and in vivo. The PEDF expression was examined in patient tumor samples and ESCC cell lines. Short hairpin RNA technology was used to inhibit the PEDF expression in ESCC EC9706 and KYSE150 cells. In vitro cell proliferation and migration assays were performed. The effects of PEDF on tumor growth and progression were examined in vivo in murine subcutaneous xenograft tumor models. It was found that PEDF was overexpressed in esophageal cancer cells and patient tumor tissues compared to normal control samples. PEDF enhanced cell cycle progression and inhibited cell apoptosis. Knock down of PEDF inhibited esophageal cell proliferation and migration in vitro. Moreover, Inhibition of PEDF significantly reduced tumor growth and tumor size in vivo. These results indicate that PEDF induce tumorigenesis in ESCC and can be a potential therapeutic target for cancer treatment.

Relationship of Fas, FasL, p53 and bcl-2 expression in human non-small cell lung carcinomas.

OBJECTIVE: Lack of surface Fas expression is a main route for apoptotic resistance which is considered an important mechanism of tumorigenesis and tumor progression. Fas and FasL expression in 110 non-small cell lung carcinomas (NSCLCs) were investigated to evaluate their roles in pulmonary carcinogenesis and to examine the clinicopathologic significance of Fas expression with its relationship with p53 and bcl-2 over- expression. METHODS: Immunohistochemical analysis using tissue microarray demonstrated that a large proportion of NSCLC patients (60%) showed lack of membranous Fas expression. The Fas-negative cases revealed the significantly lower survival rate than Fas-positive ones. Also, the loss of Fas receptor expression was found more frequently in advanced stage and higher nodal status. FasL protein was increased in most NSCLCs (89%) compared to normal lungs. RESULTS: p53 and bcl-2 overexpression showed no association with Fas expression. Conclusively, reduced membranous Fas expression as a mechanism of apoptotic resistance is considered to play an important part of the pulmonary carcinogenesis, which may predict poor survival and have a negative prognostic influence. CONCLUSION: Increased FasL expression is thought to be a basis for the immune evasion in NSCLCs. The rare bcl-2 overexpression suggests that this anti-apoptotic protein is unlikely to play a role in the apoptotic resistance of NSCLCs.

UBIAD1 mutation alters a mitochondrial prenyltransferase to cause Schnyder corneal dystrophy.

BACKGROUND: Mutations in a novel gene, UBIAD1, were recently found to cause the autosomal dominant eye disease Schnyder corneal dystrophy (SCD). SCD is characterized by an abnormal deposition of cholesterol and phospholipids in the cornea resulting in progressive corneal opacification and visual loss. We characterized lesions in the UBIAD1 gene in new SCD families and examined protein homology, localization, and structure. METHODOLOGY/PRINCIPAL FINDINGS: We characterized five novel mutations in the UBIAD1 gene in ten SCD families, including a first SCD family of Native American ethnicity. Examination of protein homology revealed that SCD altered amino acids which were highly conserved across species. Cell lines were established from patients including keratocytes obtained after corneal transplant surgery and lymphoblastoid cell lines from Epstein-Barr virus immortalized peripheral blood mononuclear cells. These were used to determine the subcellular localization of mutant and wild type protein, and to examine cholesterol metabolite ratios. Immunohistochemistry using antibodies specific for UBIAD1 protein in keratocytes revealed that both wild type and N102S protein were localized sub-cellularly to mitochondria. Analysis of cholesterol metabolites in patient cell line extracts showed no significant alteration in the presence of mutant protein indicating a potentially novel function of the UBIAD1 protein in cholesterol biochemistry. Molecular modeling was used to develop a model of human UBIAD1 protein in a membrane and revealed potentially critical roles for amino acids mutated in SCD. Potential primary and secondary substrate binding sites were identified and docking simulations indicated likely substrates including prenyl and phenolic molecules. CONCLUSIONS/SIGNIFICANCE: Accumulating evidence from the SCD familial mutation spectrum, protein homology across species, and molecular modeling suggest that protein function is likely down-regulated by SCD mutations. Mitochondrial UBIAD1 protein appears to have a highly conserved function that, at least in humans, is involved in cholesterol metabolism in a novel manner.

High glucose suppresses epidermal growth factor receptor/phosphatidylinositol 3-kinase/Akt signaling pathway and attenuates corneal epithelial wound healing.

OBJECTIVE: Patients with diabetes are at an increased risk for developing corneal complications and delayed wound healing. This study investigated the effects of high glucose on epidermal growth factor receptor (EGFR) signaling and on epithelial wound healing in the cornea. RESEARCH DESIGN AND METHODS: Effects of high glucose on wound healing and on EGFR signaling were investigated in cultured porcine corneas, human corneal epithelial cells, and human corneas using Western blotting and immunofluorescence. Effects of high glucose on reactive oxygen species (ROS) and glutathione levels and on EGFR pathways were assessed in porcine and primary human corneal epithelial cells, respectively. The effects of EGFR ligands and antioxidants on high glucose-delayed epithelial wound healing were assessed in cultured porcine corneas. RESULTS: High glucose impaired ex vivo epithelial wound healing and disturbed cell responses and EGFR signaling to wounding. High glucose suppressed Akt phosphorylation in an ROS-sensitive manner and decreased intracellular glutathione in cultured porcine corneas. Exposure to high glucose for 24 h resulted in an increase in ROS-positive cells in primary human corneal epithelial cells. Whereas heparin-binding EGF-like growth factor and antioxidant N-acetylcysteine had beneficial effects on epithelial wound closure, their combination significantly accelerated high glucose-delayed wound healing to a level similar to that seen in control subjects. Finally, Akt signaling pathway was perturbed in the epithelia of human diabetic corneas, but not in the corneas of nondiabetic, age-matched donors. CONCLUSIONS: High glucose, likely through ROS, impairs the EGFR-phosphatidylinositol 3-kinase/Akt pathway, resulting in delayed corneal epithelial wound healing. Antioxidants in combination with EGFR ligands may be promising potential therapeutics for diabetic keratopathy.

Cross talk between c-Met and epidermal growth factor receptor during retinal pigment epithelial wound healing.

PURPOSE: The authors sought to determine how hepatocyte growth factor (HGF) receptor c-Met and epidermal growth factor receptor (EGFR) cross talk in response to injury in human ARPE-19 cells. METHODS: A scratch wound was made on a cell monolayer of ARPE-19 cells using a sequence-comb or a pipet tip, and it was allowed to heal in the presence or absence of HGF and heparin-binding EGF-like growth factor (HB-EGF). The activation of EGFR was analyzed by immunoprecipitation with EGFR antibody, followed by Western blotting with phosphotyrosine-specific antibody. Phosphorylation of extracellular signal-regulated kinase (ERK) and AKT (a major substrate of phosphatidylinositol 3'-kinase (PI3K) was assessed by Western blotting. The release of c-Met ectodomain into the culture media was determined by Western blotting using an antibody against the extracellular region. Cell migration was assessed by Boyden chamber migration assay. RESULTS: ARPE-19 cells underwent spontaneous wound healing in basal medium, and exogenously added HB-EGF and HGF significantly enhanced wound closure. Basal and growth factor-enhanced wound closures were attenuated but not slowed by hydroxyurea, a cell proliferation inhibitor. RPE cells expressed all four erbBs, and wounding induced EGFR transactivation and downstream ERK and PI3K phosphorylation in ARPE-19 cells. HGF also induced EGFR tyrosine phosphorylation. The EGFR kinase inhibitor AG1478 blocked wound- and HGF-stimulated EGFR transactivation and attenuated spontaneous and growth factor-induced wound closure. Wounding and EGFR ligands induced the release of c-Met into the culture media. Moreover, pretreatment of cells with HB-EGF impaired ARPE-19 migration toward HGF in a matrix metalloproteinase inhibitor-sensitive manner. CONCLUSIONS: EGFR modulates HGF/c-Met activity by inducing c-Met ectodomain shedding, and HGF/c-Met transactivates EGFR, leading to an enhanced activation of downstream signaling pathways. Cross talk between EGFR and c-Met may play a key role in regulating RPE cell migration, proliferation, and wound healing.

Lysophosphatidic acid promoting corneal epithelial wound healing by transactivation of epidermal growth factor receptor.

PURPOSE: To identify the underlying mechanisms by which lipid mediator lysophosphatidic acid (LPA) acts as a growth factor in stimulating extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) during corneal epithelial wound healing. METHODS: Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence or absence of an epidermal growth factor receptor (EGFR) inhibitor (tyrphostin AG1478), a matrix metalloproteinase inhibitor (GM6001), or a heparin-binding EGF-like growth factor (HB-EGF) antagonist (CRM197) with or without LPA. EGFR activation was analyzed by immunoprecipitation using EGFR antibodies and Western blotting with phosphotyrosine antibodies. Phosphorylation of ERK and AKT (a major substrate of PI3K) was analyzed by Western blotting with antibodies specific to the phosphorylated proteins. Wound- and LPA-induced shedding of HB-EGF was assessed by measuring the release of alkaline phosphatase (AP) in a stable THCE cell line that expressed HB-EGF with AP inserted in the heparin-binding site. RESULTS: In organ and cell culture models, LPA enhanced corneal epithelial wound healing. LPA-stimulated and spontaneous wound closure was attenuated by AG1478, GM6001, or CRM197. Consistent with the effects on epithelial migration, these inhibitors, as well as the Src kinase inhibitor (PP2), retarded LPA-induced activation of EGFR and its downstream effectors ERK and AKT in THCE cells. Unlike exogenously added HB-EGF, LPA stimulated moderate EGFR phosphorylation; the level of phosphorylated EGFR was similar to that induced by wounding. However, LPA appeared to prolong wound-induced EGFR signaling. The release of HB-EGF assessed by AP activity increased significantly in response to wounding, LPA, or both, and the release of HB-EGF-AP induced by LPA was inhibited by PP2 and GM6001. CONCLUSIONS: LPA accelerates corneal epithelial wound healing through its ability to induce autocrine HB-EGF signaling. Transactivation of EGFR by LPA represents a convergent signaling pathway accessible to stimuli such as growth factors and ligands of G-protein-coupled receptors in response to pathophysiological challenge in human corneal epithelial cells.

SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells.

PURPOSE: The authors have previously demonstrated that wounding of human corneal epithelial cells (HCECs) transactivates epidermal growth factor (EGF) receptor (EGFR) and its downstream signaling pathways and that this EGFR signaling is required for epithelial wound healing. In this study, the authors sought to identify the underlying mechanisms for EGFR transactivation in response to wounding in HCECs. METHODS: SV40-immortalized HCEC (THCE) monolayer was wounded and allowed to heal in the presence or absence of a selective inhibitor of the Src family kinases PP2 and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Wound closure was monitored by photographing of the injury immediately or 24 hours after wounding. Activation of EGFR in THCE cells and in primary HCECs was analyzed by immunoprecipitation of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylation of extracellular signal-regulated kinase (ERK), AKT (a major substrate of phosphatidylinositol 3'-kinase [PI3K]), Src at tyrosine Y416, and EGFR at Y845 was analyzed by Western blotting with antibodies specific to phosphorylated proteins. Effects of PP2 on THCE cell migration were determined by Boyden chamber migration assay. RESULTS: Among several inhibitors tested, PP2 blocked wound-induced EGFR phosphorylation in THCE cells. PP2 at 12.5 microM effectively inhibited EGFR transactivation in response to wounding and to the phosphorylation of ERK and AKT in THCE cells and primary HCECs. Consistent with the inhibition of EGFR transactivation, PP2 also attenuated epithelial migration and wound closure with or without exogenously added HB-EGF. PP2 at a concentration as high as 50 microM exhibited no effects on HB-EGF induced ERK phosphorylation. On the other hand, AKT phosphorylation was much more sensitive to PP2 than ERK or EGFR phosphorylation because 3.13 microM PP2 effectively inhibited wound- or HB-EGF-induced AKT phosphorylation. CONCLUSIONS: These results suggest that Src kinase mediates wound-induced EGFR transactivation and participates in a pathway to activate the PI3K-AKT pathway downstream of EGFR in HCECs.

Role of ErbB2 in Corneal Epithelial Wound Healing.

PURPOSE: Human corneal epithelial cells (HCECs) were functionally depleted of erbB2 to elucidate its role in epidermal growth factor (EGF) receptor (EGFR) activation-dependent cell migration. METHODS: The retrovirus pBabe-5R, which encodes an erbB2 single-chain antibody with an endoplasmic reticulum (ER)-targeting sequence, and control pBabe-puro were used to infect THCE cells (an SV40-immortalized HCEC line). Several cell lines expressing 5R were selected along with a pBabe-puro control line. The depletion of erbB2 was verified by cell surface biotinylation of proteins, followed by streptavidin precipitation and subsequent detection of erbB2 by immunoblot analysis. Activation of erbBs was analyzed by immunoprecipitation using the phosphotyrosine antibody pY20, followed by Western blot analysis with erbB1 or erbB2 antibodies. Phosphorylation of extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K) was analyzed by Western blot with antibodies specific to phosphorylated proteins. Effects of erbB2 depletion on heparin-binding EGF-like growth factor (HB-EGF)-induced cell migration were determined by Boyden chamber migration assay and by scratch wound assay. RESULTS: Wounding induced erbB2 tyrosine phosphorylation. Expression of 5R encoding an erbB2 single-chain antibody with an endoplasmic reticulum-targeting sequence depleted the cell surface expression of erbB2 in HCECs. Wounding resulted in a rapid increase in the phosphorylation of erbB1 in both 5R-expressing and control cells, whereas wound-induced erbB2 phosphorylation in 5R-expressing cells was not detectable. Depletion of functional erbB2 attenuated the healing of scratch wounds in the presence of HB-EGF and impaired both chemotactic migration stimulated by HB-EGF and haptotactic migration toward a fibronectin-collagen I (3:1; FNC) coating mix. Expression of 5R affected both the intensity and the duration of wound-induced, EGFR-elicited ERK and PI3K activation. Inhibition of ERK and PI3K pathways in cultured porcine corneas impaired ex vivo epithelial wound healing. CONCLUSIONS: ErbB2 serves as a critical component that couples erbB receptor tyrosine kinase to the migration machinery of corneal epithelial cells.

Wound-induced HB-EGF ectodomain shedding and EGFR activation in corneal epithelial cells.

PURPOSE: Epithelial wound healing is, at least in part, mediated in an autocrine fashion by epidermal growth factor (EGF) receptor (EGFR)-ligand interactions. This study sought to identify the endogenous EGFR ligand and the mechanism by which it is generated in response to wounding in cultured porcine corneas and human corneal epithelial cells. METHODS: Epithelial debridement wounds in cultured porcine corneas and scratch wounds in an epithelial monolayer of SV40-immortalized human corneal epithelial (THCE) cells were allowed to heal in the presence of tyrphostin AG1478 (an EGFR inhibitor), GM6001 (a matrix metalloproteinase [MMP] inhibitor), or CRM197 (a diphtheria toxin mutant), with or without HB-EGF. The activation of EGFR and extracellular signal-regulated kinase (ERK) was analyzed by immunoprecipitation using EGFR antibodies and Western blot analysis with phosphotyrosine antibody. Wound induced HB-EGF shedding was assessed by isolation of secreted HB-EGF from wounded THCE cells and by measuring the release of alkaline phosphatase (AP) in THCE stable cell lines expressing HB-EGF-AP. RESULTS: In THCE cells, wound-induced EGFR phosphorylation and ERK activation. In both organ and cell culture models, epithelial wounds were healed in basal media and inhibition of EGFR activation by AG1478 blocked wound closure with or without exogenously added HB-EGF. GM6001 delayed wound closure. Its effects diminished in the presence of exogenous EGF or HB-EGF, suggesting that the MMP inhibitor primarily blocks the release of EGFR ligands. CRM197, a highly specific antagonist of HB-EGF, impaired epithelial wound closure, suggesting that HB-EGF is an endogenous ligand released on epithelial wounding. Consistent with the effects on epithelial migration, all inhibitors as well as HB-EGF function-blocking antibodies retarded wound-induced EGFR phosphorylation in cultured THCE cells. The release of HB-EGF in response to wounding was demonstrated by the fact that heparin-binding proteins isolated from wounded, but not control, THCE-conditioned medium stimulated EGFR and ERK phosphorylation and by the expression of HB-EGF-AP in THCE cells, in which wounding induced the release of AP activity in an MMP-inhibitor-sensitive manner. CONCLUSIONS: HB-EGF released on wounding acts as an autocrine-paracrine EGFR ligand. HB-EGF shedding and EGFR activation represent a critical event during corneal epithelial wound healing, suggesting a possible manipulation of wound healing during the early phases.

EGF-induced ERK phosphorylation independent of PKC isozymes in human corneal epithelial cells.

PURPOSE: To investigate the role of protein kinase C (PKC) isozymes in epithelial growth factor (EGF)-induced activation of extracellular signal-regulated kinase (ERK) and cell proliferation in cultured human corneal epithelial cells. METHODS: Simian virus (SV)40 stably transfected human corneal epithelial (THCE) cells were cultured in keratinocyte growth medium. PKC isozymes and phosphorylation of ERK in THCE cells were assessed by Western blot analysis. Translocation of the PKC isozyme was determined by subcellular fractionation followed by Western blot analysis. Cell proliferation was measured by incorporation of [(3)H]-thymidine into DNA. RESULTS: Six PKC isozymes-PKC-alpha, -betaI, -betaII, -delta, - epsilon, and - micro -were found in THCE cells. Phorbol 12-myristate 13-acetate (PMA) caused PKC-alpha, -betaI, and - epsilon, initially present in the cytoplasm, to be translocated to the membrane and nuclear subcellular fractions and PKC-delta to be depleted from the cytoskeleton. The PKC inhibitor GF109203X inhibited PMA-induced, but not basal or EGF-induced, phosphorylation of ERK, whereas the EGF receptor inhibitor tyrphostin AG1478 blocked basal and EGF-, but not PMA-, induced phosphorylation of ERK. Depletion of PMA-sensitive PKC isozymes including PKC-alpha, -betaI, -betaII, -delta, and - epsilon, inhibited PMA-, but not EGF-, induced phosphorylation of ERK. Depletion of these PKC isozymes blocked PMA-, but not EGF-, induced cell proliferation. CONCLUSIONS: Although activation of PKC by PMA results in activation of ERK, EGF-induced phosphorylation of ERK and/or cell proliferation is independent of the conventional and novel isozymes PKC-alpha, -betaI, -betaII, -delta, and - epsilon in human corneal epithelial cells.