Growth factor-independent proliferation of normal human neonatal keratinocytes: production of autocrine- and paracrine-acting mitogenic factors.
When normal human foreskin keratinocytes were cultured in the absence of polypeptide growth factors at densities above 5 x 10(3)/cells cm2, the cells proliferated continuously and the addition of IGF-I, EGF, TGF alpha, bFGF, or aFGF did not significantly alter growth rate. Heparin sulfate, TGF beta, or suramin inhibited keratinocyte growth factor-independent proliferation. The addition of EGF, TGF alpha, or aFGF reversed heparin-induced growth inhibition, while bFGF partially negated this effect. RIA of keratinocyte-derived conditioned medium (CM) indicated the presence of TGF alpha peptide at a concentration of approximately 235 pg/ml. In contrast, clonal growth of keratinocytes required the addition of growth factors to the basal medium. Keratinocyte-derived CM replaced EGF in stimulating keratinocyte clonal growth, and an anti-EGF receptor mAb inhibited CM-induced keratinocyte clonal growth. In addition to its effect on keratinocytes, keratinocyte-derived CM stimulated the incorporation of [3H]thymidine by quiescent cultures of human foreskin fibroblasts, mouse AKR-2B cells, and EGF-receptorless mouse NR6 cells. CM-stimulated [3H]thymidine incorporation into quiescent normal human fibroblasts was partially reduced in the presence of anti-EGF receptor mAb. Heparin sulfate partially inhibited CM-induced keratinocyte clonal growth and [3H]thymidine incorporation into quiescent AKR-2B cells. We hypothesize from these data that autocrine and paracrine-acting factors produced by keratinocytes mediated their effect through the activation of both EGF receptor-dependent and EGF receptor-independent mitogenic pathways and that some of these factors appear to be sensitive to inhibition by heparin.
Cook
P W
PW
Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 97201.
Pittelkow
M R
MR
Shipley
G D
GD
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D004261:DNA Replication; D004815:Epidermal Growth Factor / Q000502:physiology; D066246:ErbB Receptors / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology; D006133:Growth Substances / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D006497:Heparan Sulfate / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D012867:Skin / Q000166:cytology; D013498:Suramin / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000502:physiology
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D006133:Growth Substances; D006497:Heparan Sulfate; D013498:Suramin; D016212:Transforming Growth Factor beta