title: Induction of apoptosis in human corneal and HeLa cells by mutated BIGH3.
abstract: OBJECTIVE
  To determine the effects of overexpression of mutated BIGH3 in HeLa and human corneal epithelial (HCE) cells.
  
  
  METHODS
  Six mutations known to be responsible for autosomal dominant corneal dystrophies linked to chromosome 5 were generated in a BIGH3 expression vector and transfected in HeLa and HCE cells. The expression and secretion of the various BIGH3-EGFP fusion proteins were measured by Western blot analysis. Apoptotic cells were identified by Hoechst/propidium iodide and annexin V staining. Lactate dehydrogenase (LDH) activity was measured in the medium of transfected cells. Truncated BIGH3 protein and site-specific mutations were generated to determine the exact region that mediated apoptosis.
  
  
  RESULTS
  The overexpressed BIGH3 fusion protein was secreted regardless of its mutation status and was clearly observed in the culture medium. Overexpression of mutated BIGH3 induced apoptosis in both cell lines through activation of caspase-3. Although all the disease-causing mutations tested in this experiment induced apoptosis, the strongest effect was observed with the R124C and R555W mutations. Overexpression of a carboxyl-truncated BIGH3 protein did not induce apoptosis, suggesting that a region located in the C-terminal domain was necessary to mediate cell death. In addition, mutation of the Pro-Asp-Ile (PDI) site at 616-618 was sufficient to prevent induction of apoptosis.
  
  
  CONCLUSIONS
  Overexpression of mutated BIGH3 induces apoptosis in HeLa and HCE cells through activation of a pathway that uses the PDI domain of the fourth internal Fas domain and activation of caspase-3. Because DI is a known site of interaction with alpha 3 beta 1 integrins, it suggests that integrins play a role in mediating apoptosis in the system used in the current study. This work suggests that apoptosis is a key element in the pathophysiology of BIGH3-related corneal dystrophies.
authors.affiliation: Division of Medical Genetics, and Unit of Oculogenetics, Sion, Switzerland.
authors.forename: Sabine
authors.initials: S
authors.lastname: Morand
authors.forename: Valérie
authors.initials: V
authors.lastname: Buchillier
authors.forename: Fabienne
authors.initials: F
authors.lastname: Maurer
authors.forename: Christophe
authors.initials: C
authors.lastname: Bonny
authors.forename: Yvan
authors.initials: Y
authors.lastname: Arsenijevic
authors.forename: Francis L
authors.initials: FL
authors.lastname: Munier
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Schorderet
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics / Q000473:pathology; D019573:Epithelium, Corneal / Q000378:metabolism / Q000473:pathology*; D016326:Extracellular Matrix Proteins; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D011419:Propidium; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein
chemical_list: D017304:Annexin A5; D053148:Caspase 3; D020169:Caspases; D016326:Extracellular Matrix Proteins; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D011419:Propidium; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein; C505407:CASP3 protein, human