title: TGF-β signaling is required for maintenance of retinal ganglion cell differentiation and survival.
abstract: OBJECTIVE
  To determine the role of TGF-β1 in the maintenance of retinal ganglion cell line (RGC-5) differentiation and integrity.
  
  
  METHODS
  RGC-5 cells were differentiated in media conditioned by human non-pigmented ciliary epithelial cells (HNPE) for 4 days before treatment with TGF-β1 for 24 h. Cells were examined for morphological changes and harvested for western blot and real-time PCR analysis. For study of apoptosis, differentiated RGC-5 cells were grown in serum-free medium for 24 h in the presence or absence of TGF-β1 and collected for Annexin V/Propidium iodide FACs analysis. The role of MAPK pathways in TGF-β1-dependent signaling was determined by treatment with specific inhibitors of ERK, JNK and p38.
  
  
  RESULTS
  Differentiation of RGC-5 cells in HNPE-conditioned media (CM) increased the neural cell markers, Brn-3c, NF-160, Thy1.2, Tau and PGP9.5. Treatment with TGF-β1 significantly increased the length of neurites extended by differentiated RGC-5s, concomitant with increased expression of NF-160 and PGP9.5, but not Brn-3c, Thy1.2 or Tau. TGF-β1 also decreased RGC-5 cell apoptosis in serum-free medium. p38 phosphorylation, but not smad2/3, JNK or ERK phosphorylation, was increased in TGF-β1 treated cells. Specific inhibition of p38 signaling reversed TGF-β1 induced neurite growth.
  
  
  CONCLUSIONS
  These findings demonstrate the induction of RGC-5 cell differentiation by HNPE-derived CM and illustrate a role for TGF-β1 in maintaining RGC-5 cell survival and promoting neurite outgrowth through p38 MAPK.
authors.affiliation: Department of Ophthalmology, Harvard Medical School, Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Walshe
authors.forename: L L
authors.initials: LL
authors.lastname: Leach
authors.forename: P A
authors.initials: PA
authors.lastname: D'Amore
mesh_terms: D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D002923:Cilia / Q000378:metabolism; D017077:Culture Media, Conditioned; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D020935:MAP Kinase Signaling System; D016501:Neurites / Q000502:physiology; D051381:Rats; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D051785:Smad Proteins / Q000502:physiology; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology / Q000502:physiology*
chemical_list: D015415:Biomarkers; D017077:Culture Media, Conditioned; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1