ERK/p44p42 Mitogen-Activated Protein Kinase Mediates EGF-Stimulated Proliferation of Conjunctival Goblet Cells in Culture

Harvard University, Cambridge, Massachusetts, United States
Investigative ophthalmology & visual science (Impact Factor: 3.4). 05/2008; 49(8):3351-9. DOI: 10.1167/iovs.08-1677
Source: PubMed


To determine whether activation of the ERK pathway by EGF leads to rat and human goblet cell proliferation.
The conjunctiva was removed from male Sprague-Dawley rats. Human conjunctiva was removed during ocular surgery. The tissue was minced and goblet cells were grown. The cells were stimulated with EGF (10(-7) M) for 1 and 5 minutes and Western blot analysis was performed with an antibody against phosphorylated EGFR, to measure the activation of the EGF receptor (EGFR). The cells were incubated with EGF (10(-7) M) for 24 hours, and cell proliferation was measured by WST-8. Inhibitors were added either 20 minutes before EGF or 2 hours after. The cells were stimulated with EGF (10(-7) M) for 1 minute to 24 hours. The number of cells expressing phosphorylated ERK (pERK) in the nucleus and Ki-67 was determined by immunofluorescence.
EGF increased the activation of EGFR in rat conjunctival goblet cells. EGF-stimulated proliferation was inhibited by the EGFR inhibitor AG1478 and the MEK inhibitor U0126 in rat and human cultured goblet cells. EGF caused the translocation of pERK to the nucleus in a biphasic manner. Inhibition of the second peak with U0126 prevented proliferation. EGF-stimulated goblet cells progressed through the cell cycle expressing pERK in the nucleus.
EGF stimulated human and rat conjunctival goblet cell proliferation by activating the EGFR. EGFR stimulated ERK causing its biphasic translocation to the nucleus. The second peak response is responsible for cell proliferation, but the role of the first peak is not known.

Download full-text


Available from: Robin R Hodges, Oct 03, 2015
63 Reads
  • Source
    • "Absence of EGFR activation in CL 1-5 cells under dcEF was validated by Western blotting against phosphorylated EGFR (Tyr1068), which is shown in Figure 10A. Tyr1068 is a common residue of EGFR that is phosphorylated when EGFR is activated by EGF [66–68]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells' response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.
    PLoS ONE 08/2013; 8(8):e73418. DOI:10.1371/journal.pone.0073418 · 3.23 Impact Factor
  • Source
    • "Proliferation of EPC cultured for 7 days were measured with a Cell Counting Kit-8 (Takeuchi et al., 2003) (Dojindo Molecular Technologies, Gaithersburg, MD, USA) and bromodeoxyuridine (BrdU) assay separately. The tetrazolium compound WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2, 4-disulphophenyl)-2H-tetrazolium, monosodium salt] and an electron coupling reagent were added to the medium to determine the number of viable EPCs (Moriya et al., 2007; Shatos et al., 2008). The tetrazolium compound is bioreduced by cells into a coloured formazan product that is soluble in the tissue culture medium. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs. EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg L(-1)) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 microg mL(-1)), phosphatidylinositol-3-kinase (PI3K) inhibitor (LY294002, 5 microM), nitric oxide (NO) synthase inhibitor (L-N(G)-nitro-arginine methyl ester (L-NAME), 100 microM) or sodium nitroprusside (SNP, 25 microM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined. Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and LY294002. The PPARgamma agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs.
    British Journal of Pharmacology 11/2009; 158(8):1865-73. DOI:10.1111/j.1476-5381.2009.00450.x · 4.84 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The authors determined the role of the protein kinase C (PKC) isoforms cPKCalpha and nPKCepsilon in EGF-stimulated proliferation of cultured rat and human conjunctival goblet cells. Rat and human conjunctivas were minced, and goblet cells were allowed to grow. Passage 1 cells were serum starved for 24 to 48 hours and were incubated with the PKC inhibitors calphostin C and Gö 6983 (10(-10)-10(-7) M) for 20 minutes before stimulation with EGF (10(-7) M) for 24 hours. The presence and localization of PKC isoforms in cultured rat goblet cells were determined by Western blot analysis and immunofluorescence microscopy, respectively. Cultured rat goblet cells were serum starved and incubated with adenoviruses containing genes for dominant-negative cPKCalpha (Ad DNPKCalpha, 10(4) pfu), dominant-negative nPKCepsilon (Ad DNPKCepsilon, 10(4) pfu), and wild-type cPKCalpha (Ad WTPKCalpha, 10(7) pfu), and proliferation was measured. In rat goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C, whereas Gö 6983 inhibited proliferation by 53%+/-15%. In human goblet cells, EGF-stimulated proliferation was completely inhibited by calphostin C. PKCalpha, -betaI, -betaII, -delta, -epsilon, -iota/lambda, -theta, -gamma, and -zeta were found in cultured rat goblet cells. Ad DNPKCalpha and Ad DNPKCepsilon inhibited EGF-stimulated proliferation in rat goblet cells by 78%+/-6% and 92%+/-8%, respectively. Incubation with Ad WTPKCalpha alone significantly increased proliferation. cPKCalpha and nPKCepsilon play key roles in conjunctival goblet cell proliferation.
    Investigative ophthalmology & visual science 10/2008; 50(2):614-20. DOI:10.1167/iovs.08-2467 · 3.40 Impact Factor
Show more