title: Mechanisms for PDGF, a serum cytokine, stimulating loss of corneal keratocyte crystallins.
abstract: OBJECTIVE
  As corneal stromal cells (keratocytes) become activated before transition to the fibroblastic repair phenotype in response to injury (in situ) or serum (in culture), the corneal crystallins, transketolase (TKT) and aldehyde dehydrogenase (ALDH1A1), are lost. The authors previously showed that the serum cytokine platelet-derived growth factor-BB (PDGF), but not transforming growth factor beta2 (TGF-beta2), stimulates TKT loss. The goal of this study was to further define the molecular mechanisms for PDGF-stimulated loss of crystallins to elucidate the pathway for keratocyte activation.
  
  
  METHODS
  Freshly isolated rabbit corneal keratocytes were plated in serum-free medium with or without PDGF and/or specific inhibitors of the PDGF-relevant signal pathway components, PDGF receptor, PI3K/AKT, or ras-initiated MAPK proteins. Intracellular TKT protein levels were quantified by immunoblotting. Ubiquitinated TKT levels were assessed by immunoprecipitation, and TKT messenger RNA (mRNA) levels were quantified by quantitative reverse transcription-polymerase chain reaction.
  
  
  RESULTS
  PDGF treatment at the same time as inhibition of PDGF receptor, Akt, JNK, and ubiquitin-proteasome pathway prevented PDGF-induced TKT protein loss. In contrast, treatment with PDGF did not affect TKT mRNA levels.
  
  
  CONCLUSIONS
  The results suggest that PDGF-stimulated TKT loss is mediated through cross talk between PI3K-independent Akt and JNK. This signaling pathway leads to the degradation of existing TKT protein but does not compromise the accumulation of TKT mRNA. Therefore, cells retain the potential to reaccumulate TKT protein that is enabled by PDGF removal. These findings suggest that targeting PDGF signaling could improve repair outcomes after surgical procedures in the cornea.
authors.affiliation: Department of Biological Sciences, College of Arts and Sciences, Florida Gulf Coast University, Fort Myers, FL, USA. alagier@grandview.edu
authors.forename: Adriana J
authors.initials: AJ
authors.lastname: LaGier
authors.forename: Gabriel M
authors.initials: GM
authors.lastname: Gordon
authors.forename: Lee R
authors.initials: LR
authors.lastname: Katzman
authors.forename: Vasilis
authors.initials: V
authors.lastname: Vasiliou
authors.forename: M Elizabeth
authors.initials: ME
authors.lastname: Fini
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D043925:Angiogenesis Inducing Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000818:Animals; D000077214:Becaplermin; D001769:Blood; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D047468:Immunoprecipitation; D020574:Proto-Oncogene Proteins c-sis / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014174:Transketolase / Q000235:genetics* / Q000378:metabolism
chemical_list: D043925:Angiogenesis Inducing Agents; D004791:Enzyme Inhibitors; D020574:Proto-Oncogene Proteins c-sis; D012333:RNA, Messenger; D000077214:Becaplermin; D000444:Aldehyde Dehydrogenase; D014174:Transketolase; D017479:Receptors, Platelet-Derived Growth Factor