title: NGF increases VEGF expression and promotes cell proliferation via ERK1/2 and AKT signaling in Müller cells.
abstract: OBJECTIVE
  Nerve growth factor (NGF) is a classic neuroprotective factor that contributes to angiogenesis under pathological conditions, which might be mediated by the upregulation of vascular endothelial growth factor (VEGF). Retinal Müller cells are a critical source of growth factors, including NGF and VEGF, and express the receptor for NGF, indicating the functional significance of NGF signaling in Müller cells. The aim of this study is to explore the effect of NGF on the production of other growth factors and cellular proliferation in Müller cells and to further detect the potential mechanism of these effects.
  
  
  METHODS
  Primary Müller cells from C57BL/6J mice were isolated and identified with glutamine synthetase (GS) immunofluorescence (IF), a specific marker for Müller cells. TrkA, a high affinity receptor for NGF, was detected with IF staining in the primary Müller cells. Then, the cultured cells were stimulated with recombinant mouse NGF, and the supernatants and the cellular lysate were collected at different time points. VEGF secretion in the supernatant was detected with an enzyme-linked immunosorbent assay (ELISA). The signaling activation in the Müller cells was accessed by western blot using specific phosphorylated antibodies. In addition, cell proliferation was analyzed with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Furthermore, K252a, U0126, and LY294002, the inhibitors for TrkA, extracellular signal-regulated kinases 1/2 (ERK1/2), and phosphatidylinositol 3-kinase (PI3K)/AKT, respectively, were used in combination with NGF in the assays analyzing VEGF expression and cell proliferation.
  
  
  RESULTS
  Primary mouse Müller cells were successfully cultured and confirmed with GS positive staining. The IF results showed that the TrkA receptor was abundantly expressed on Müller cells. The ELISA results revealed that NGF significantly promoted the production and secretion of VEGF in Müller cells after 12 or 24 h of stimulation, with more elevation after 24 h. Furthermore, NGF activated ERK1/2 and PI3K/AKT signaling, which was shown by the marked upregulation of phosphorylation in the western blot. As expected, K252a, the inhibitor of TrkA, a high-affinity NGF receptor, suppressed the activation, showing little phosphorylation of ERK1/2 and PI3K/AKT signaling. Importantly, the VEGF levels were decreased after the inhibitors for TrkA, ERK1/2, and PI3K/AKT were used compared with NGF alone. In addition, the MTT assay showed that NGF promoted the proliferation of the Müller cells, which was also blocked by the TrkA, ERK1/2, and PI3K/AKT inhibitors.
  
  
  CONCLUSIONS
  The results showed that NGF enhanced the secretion of VEGF and promoted cell proliferation via the ERK1/2 and PI3K/AKT pathways in Müller cells, indicating that NGF is involved in angiogenesis-related factor generation and gliosis in Müller cells.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P. R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P. R. China.
authors.forename: Chang
authors.initials: C
authors.lastname: He
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P. R. China.
authors.forename: Tian
authors.initials: T
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P. R. China.
authors.forename: Zijing
authors.initials: Z
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P. R. China.
authors.forename: Lingli
authors.initials: L
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, P. R. China.
authors.forename: Xialin
authors.initials: X
authors.lastname: Liu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D063928:Ependymoglial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D020932:Nerve Growth Factor / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D020917:Receptor, trkA / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D051057:Proto-Oncogene Proteins c-akt