title: Induction of senescence pathways in Kindler syndrome primary keratinocytes.
abstract: BACKGROUND
  Individuals with Kindler syndrome (KS) have loss-of-function mutations in the FERMT1 gene that encodes the focal adhesion component kindlin-1. The major clinical manifestation of KS is epidermal atrophy (premature skin ageing). This phenotypic feature is thought to be related to the decreased proliferation rate of KS keratinocytes; nevertheless, molecular mediators of such abnormal behaviour have not been fully elucidated.
  
  
  OBJECTIVE
  To investigate how kindlin-1 deficiency affects the proliferative potential of primary human keratinocytes.
  
  
  METHODS
  We serially cultivated nine primary KS keratinocyte strains until senescence and determined their lifespan and colony-forming efficiency (CFE) at each serial passage. The expression of molecular markers of stemness and cellular senescence were investigated by immunoblotting using cell extracts of primary keratinocyte cultures from patients with KS and healthy donors. In another set of experiments, kindlin-1 downregulation in normal keratinocytes was obtained by small interfering RNA (siRNA) technology.
  
  
  RESULTS
  We found that KS keratinocytes exhibited a precocious senescence and strongly reduced clonogenic potential. Moreover, KS cultures showed a strikingly increased percentage of aborted colonies (paraclones) already at early passages indicating an early depletion of stem cells. Immunoblotting analysis of KS keratinocyte extracts showed reduced levels of the stemness markers p63 and Bmi-1, upregulation of p16 and scant amounts of hypophosphorylated Rb protein, which indicated cell cycle-arrested status. Treatment of normal human primary keratinocytes with siRNA targeting kindlin-1 proved that its deficiency was directly responsible for p63, Bmi-1 and pRb downregulation and p16 induction.
  
  
  CONCLUSIONS
  Our data directly implicate kindlin-1 in preventing premature senescence of keratinocytes.
authors.affiliation: Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata-IRCCS, via dei Monti di Creta 104, 00167 Rome, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Piccinni
authors.forename: G
authors.initials: G
authors.lastname: Di Zenzo
authors.forename: R
authors.initials: R
authors.lastname: Maurelli
authors.forename: E
authors.initials: E
authors.lastname: Dellambra
authors.forename: M
authors.initials: M
authors.lastname: Teson
authors.forename: C
authors.initials: C
authors.lastname: Has
authors.forename: G
authors.initials: G
authors.lastname: Zambruno
authors.forename: D
authors.initials: D
authors.lastname: Castiglia
mesh_terms: D000293:Adolescent; D000328:Adult; D001768:Blister / Q000235:genetics / Q000473:pathology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D002648:Child; D004820:Epidermolysis Bullosa / Q000235:genetics / Q000473:pathology*; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000172:deficiency* / Q000235:genetics*; D008875:Middle Aged; D009154:Mutation; D009363:Neoplasm Proteins / Q000172:deficiency* / Q000235:genetics*; D010510:Periodontal Diseases / Q000235:genetics / Q000473:pathology*; D010787:Photosensitivity Disorders / Q000235:genetics / Q000473:pathology*
chemical_list: C475301:FERMT1 protein, human; D008565:Membrane Proteins; D009363:Neoplasm Proteins