Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation.
We have used antisense RNA technology to inhibit endogenous PTH-related peptide (PTHRP) production in an established human keratinocyte cell line, HPK1A, to assess the role of PTHRP as a modulator of cell differentiation. Initially to determine the specificity of any alterations in cell function that might be observed, HPK1A cells and Rat-2 fibroblasts (which do not synthesize PTHRP) were both infected with the same retrovirus (pYN) containing antisense PTHRP. In contrast to antisense-infected HPK1A cells (HPK1A-AS), which show accelerated growth indices when endogenous PTHRP production is blocked, antisense-infected Rat-2 cells (Rat-2-AS) displayed no increase in cell proliferation. Consequently, this alteration in HPK1A cell function appeared to be specific to the inhibition of PTHRP production. In HPK1A-AS cells, no PTHRP transcript was observed in cytoplasmic RNA, and none was sequestered in a nuclear RNA preparation. Therefore, hybridization with the antisense strand appears to destabilize PTHRP mRNA, leading to rapid disappearance of the sense-antisense heteroduplex. We then examined the effect of PTHRP inhibition on keratinocyte differentiation using three indices. PTHRP inhibition in HPK1A-AS cells resulted in reduced high mol wt keratin production, as assessed by immunocytochemistry. Expression of mRNA encoding transglutaminase and involucrin was decreased in HPK1A-AS cells compared to that in control cells under conditions of high ambient calcium. Involucrin protein levels were also diminished in HPK1A-AS cells in parallel with the reduced levels of involucrin gene expression. These data, therefore, show that interference with PTHRP production inhibits expression of maturation-specific keratinocyte indices and indicate that endogenous PTHRP acts to enhance differentiation in this keratinocyte model.
Kaiser
S M
SM
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Sebag
M
M
Rhim
J S
JS
Kremer
R
R
Goltzman
D
D
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008970:Molecular Weight; D010281:Parathyroid Hormone / Q000096:biosynthesis / Q000502:physiology*; D044162:Parathyroid Hormone-Related Protein; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000502:physiology*; D016372:RNA, Antisense; D051381:Rats; D011916:Rats, Inbred F344
C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011498:Protein Precursors; D011506:Proteins; D016372:RNA, Antisense; C032253:involucrin