title: Optimization of filaggrin expression and processing in cultured rat keratinocytes.
abstract: BACKGROUND
  In normal mammalian epidermis, cell division occurs primarily in the basal layer where cells are attached to the basement membrane. Upon release from the basement membrane, these basal cells stop dividing and begin to differentiate and stratify producing cornified cells expressing differentiation markers, including the keratin bundling protein filaggrin, and cornified envelope proteins. Little is understood about the regulatory mechanisms of these processes. A rat epidermal keratinocyte cell line synthesizing and processing profilaggrin at confluence in a synchronous manner for 4-5 days provides a useful culture model for epidermal differentiation. Profilaggrin expression in this cell line however decreases with passaging, and its processing involves extensive nonspecific proteolysis.
  
  
  OBJECTIVE
  Our objective was to identify culture conditions that effect the decrease in profilaggrin expression with passaging and nonspecific proteolysis of profilaggrin in order to study epidermal differentiation more closely.
  
  
  METHODS
  The large amount of nonspecific proteolysis suggested autophagocytosis. To test this, cells were cultured in the presence of 3-methyladenine (3-MA). Two known gradients in epidermis are decreasing serum components and increasing calcium concentrations in the upper cell layers. To determine whether these gradients effected processing, cells were cultured in serum/DMEM or in serum-free KGM and under varying external calcium concentrations. Cells were also cultured in presence of aminoguanidine in an attempt to maintain profilaggrin expression with passaging.
  
  
  RESULTS
  Profilaggrin expression was enhanced in the presence of 3-MA, with optimum around 6mM. In the absence of aminoguanidine, profilaggrin expression decreased as a function of increasing passage number; in its presence, profilaggrin expression remained high in some, but not in all of the independently maintained cell lines. Thus, culturing in aminoguanidine was necessary, but not sufficient, for sustained ability to express profilaggrin at confluence. Production of filaggrin from profilaggrin was maximized in a serum-free medium with [Ca(2+)] at 5mM. Filaggrin associates with phospholipid vesicles in vitro forming aggregates similar to those seen in vivo, suggesting that filaggrin release induces vesicular aggregation and autophagocytosis.
  
  
  CONCLUSIONS
  We have used a keratinocyte cell line that synthesizes and processes profilaggrin after confluence as a culture model to study epidermal differentiation. In this system profilaggrin processing must be preceded by inhibition of autophagosome formation and/or modulation of vesicular trafficking, and these processes are regulated by epidermal calcium and serum factor gradients.
authors.affiliation: Department of Chemistry & Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA.
authors.forename: Sudeshna M
authors.initials: SM
authors.lastname: Chatterjea
authors.forename: Katheryn A
authors.initials: KA
authors.lastname: Resing
authors.forename: William
authors.initials: W
authors.lastname: Old
authors.forename: Wilas
authors.initials: W
authors.lastname: Nirunsuksiri
authors.forename: Philip
authors.initials: P
authors.lastname: Fleckman
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001343:Autophagy; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D046508:Culture Techniques / Q000379:methods*; D000091344:Filaggrin Proteins; D006146:Guanidines / Q000494:pharmacology; D007381:Intermediate Filament Proteins / Q000187:drug effects / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D051381:Rats
chemical_list: D000091344:Filaggrin Proteins; D006146:Guanidines; D007381:Intermediate Filament Proteins; C025946:3-methyladenine; D000225:Adenine; C004479:pimagedine; D002118:Calcium