Overexpression of H- and L-ferritin subunits in lens epithelial cells: Fe metabolism and cellular response to UVB irradiation.
OBJECTIVE
To determine the effect of changes in ferritin subunit makeup on Fe metabolism and the resistance of lens epithelial cells (LEC) to photo-oxidative stress.


METHODS
Cultured canine LEC were transiently transfected with pTargeT mammalian expression vector containing the whole coding sequence of H- or L-chain cDNA. The subunit composition of newly synthesized ferritin was analyzed by metabolic labeling and SDS-PAGE electrophoresis. Total ferritin concentration was measured by ELISA: Fe uptake and incorporation into ferritin was determined by incubating transfected cells with (59)Fe-labeled transferrin followed by native PAGE electrophoresis. The effect of UV irradiation was assessed by cell count after exposure of transfected cells to UVB radiation.


RESULTS
Transfected cells differentially expressed H- and L-ferritin chains from cDNA under the control of CMV promoter; overexpression of L-chain was much greater than that of H-chain. The expressed chains assembled into ferritin molecules under in vitro and in vivo condition. The ferritin of H-transfectants incorporated significantly more Fe than those of L-transfectants. The UVB irradiation reduced cell number of L-transfectants by half, whereas H-chain transfectants were protected.


CONCLUSIONS
Post-transfectional expression of ferritin H- and L-chains in LEC appears to be regulated differentially. Overexpression of L-chain ferritin did not have a major effect on cellular Fe distribution and did not protect LEC against UV irradiation, whereas overexpression of H-chain resulted in increased storage of Fe in ferritin and protected cells from UV damage.
Goralska
M
M
Department of Anatomy, Physiology, and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606, USA.
Holley
B L
BL
McGahan
M C
MC
D000595:Amino Acid Sequence; D000818:Animals; D001052:Apoferritins; D001483:Base Sequence; D002452:Cell Count; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism / Q000528:radiation effects*; D005293:Ferritins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007908:Lens, Crystalline / Q000378:metabolism / Q000528:radiation effects*; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D014162:Transfection; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D005293:Ferritins; D001052:Apoferritins; D007501:Iron