title: PKC-ζ Regulates Thrombin-Induced Proliferation of Human Müller Glial Cells.
abstract: OBJECTIVE
  To investigate the effect of thrombin on the proliferation of human Müller glial cells (MCs) and define the possible signaling mechanisms involved in this process.
  
  
  METHODS
  Protease-activated receptor (PARs 1-4) expression was analyzed using RT-PCR and Western blot in the MIO-M1 Müller cell line (MC). Müller cell proliferation was assessed by the MTS reduction method. Wound healing and immunoreactivity to Ki67 antigen were used to dissociate proliferation and migration. Cell migration was examined using transwell migration assays. The involvement of extracellular signal-regulated kinase (ERK1/2) phosphorylation/activation in thrombin-induced human MC proliferation was determined by Western blot. Intracellular pathways involved in ERK1/2 activation were analyzed by pharmacologic inhibition.
  
  
  RESULTS
  We first demonstrated that human MCs express PARs 1 to 4. Our results show that thrombin dose-dependently stimulates MC proliferation by 44%, with a calculated Ec50 of 0.86 nM. Müller cell maximal proliferation required sustained thrombin treatment for 72 hours, in contrast to our previous findings in RPE cells showing maximal thrombin-induced proliferation at 24-hour stimulation. We demonstrate that thrombin induces MC cell proliferation through the Ras-independent activation of the Raf/MEK/ERK cascade, under the control of protein kinase C (PKC)-ζ.
  
  
  CONCLUSIONS
  The breakdown of blood-retina barrier (BRB) exposes MCs to thrombin contained in serum. Our findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.
authors.forename: Irene
authors.initials: I
authors.lastname: Lee-Rivera
authors.forename: Edith
authors.initials: E
authors.lastname: López
authors.forename: Miriam Gabriela
authors.initials: MG
authors.lastname: Carranza-Pérez
authors.forename: Ana María
authors.initials: AM
authors.lastname: López-Colomé
mesh_terms: D000704:Analysis of Variance; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D063928:Ependymoglial Cells / Q000187:drug effects / Q000201:enzymology*; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology*; D018630:Vitreoretinopathy, Proliferative / Q000201:enzymology / Q000209:etiology; D014945:Wound Healing / Q000502:physiology; D000099221:Protein Kinase C zeta
chemical_list: D006490:Hemostatics; D011493:Protein Kinase C; D012333:RNA, Messenger; D044462:Receptors, Proteinase-Activated; D013917:Thrombin; D000099221:Protein Kinase C zeta