Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat epidermal keratinocytes.
Ichthyosis vulgaris is an epidermal disorder in which profilaggrin expression is decreased or absent. To determine whether the ichthyosis vulgaris phenotype could be mimicked by eliminating profilaggrin expression, a rat epidermal cell line was transfected with a plasmid that directs the constitutive expression of an RNA that is antisense to normal profilaggrin mRNA. Non-transfected and neomycin-resistant cells not containing antisense plasmid that were grown in the neomycin analogue G418 served as controls. Immunoblot and immunofluorescence analysis showed that profilaggrin protein expression and processing to filaggrin were delayed by 3 to 4 d and decreased in transfected cells. Profilaggrin mRNA was detected in both control and transfected cells only after the cells reached confluence, whereas antisense RNA was detected in transfectants at all times, even prior to confluence. Ultrastructural examination revealed that keratohyalin granules were decreased in number, globular, and heterogeneous in appearance in transfected cells in-contrast to angular structures seen in control cells. Unexpectedly, stratification was impaired, intermediate filaments were noticeably reduced, and cornified cell envelope formation was delayed in transfectants. Unlike ichthyosis vulgaris keratinocytes, where keratin expression is unaffected, appearance of K1 and K10 was delayed and K1/K10 synthesis was delayed and decreased in transfected cells. The precipitous drop in 35S-methionine incorporation into cytoskeletal protein seen at confluence in control cells was delayed by 3 d in transfected cells. We conclude that, rather than producing the ichthyosis vulgaris phenotype, antisense profilaggrin RNA has more broad-reaching effects on in vitro differentiation of rat epidermal keratinocytes.
Haydock
P V
PV
Department of Oral Biology, University of Washington, Seattle 98195.
Blomquist
C
C
Brumbaugh
S
S
Dale
B A
BA
Holbrook
K A
KA
Fleckman
P
P
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002999:Clone Cells; D004352:Drug Resistance, Microbial; D000091344:Filaggrin Proteins; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D016112:Ichthyosis Vulgaris / Q000235:genetics; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000235:genetics*; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D007633:Keratins / Q000502:physiology; D009355:Neomycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics*; D016372:RNA, Antisense / Q000032:analysis*; D051381:Rats; D014162:Transfection
D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D011498:Protein Precursors; D016372:RNA, Antisense; D007633:Keratins; D009355:Neomycin