In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.
Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.
Barreca
A
A
Cattedre di Endocrinologia e di Fisiopatologia Endocrina-DiSEM, Università di Genova, Italy.
De Luca
M
M
Del Monte
P
P
Bondanza
S
S
Damonte
G
G
Cariola
G
G
Di Marco
E
E
Giordano
G
G
Cancedda
R
R
Minuto
F
F
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D003470:Culture Media; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D018969:Insulin-Like Growth Factor Binding Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology*; D008934:Mitogens / Q000494:pharmacology; D011863:Radioimmunoassay; D013002:Somatomedins / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology
D002352:Carrier Proteins; D003470:Culture Media; D018969:Insulin-Like Growth Factor Binding Proteins; D008934:Mitogens; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II