TRANSFORMING GROWTH-FACTOR-ALPHA INDUCES INTERLEUKIN-6 IN THE HUMAN KERATINOCYTE CELL-LINE HACAT MAINLY BY TRANSCRIPTIONAL ACTIVATION

There is ample evidence that several cytokines, including transforming growth factor-alpha (TGF-alpha), interleukin (IL)-6, and IL-8 are upr egulated in psoriasis, suggesting a pathogenic role for these cytokine s. The sequence of these events, however, has not been elucidated, Rec ently it has been reported that TGF-alpha induces IL-6 in thymocytes t hrough posttranscriptional regulation; therefore, we were interested i n whether TGF-alpha can also induce IL-6 in human keratinocytes, Thus, we stimulated the human keratinocyte cell line HaCaT with TGF-alpha a nd tested supernatants for IL-6 activity, TGF-alpha resulted in a sign ificant induction of the release of IL-6, This was also confirmed by n orthern blot analysis, which revealed a transient increase in IL-6 mRN A, This increase was unlikely due to enhanced mRNA stability, because we could not observe induction of IL-6-specific transcripts by TGF-alp ha in the presence of actinomycin D. To determine whether IL-6 inducti on by TGF-alpha is transcriptionally regulated, we transfected fragmen ts of the IL-6 upstream region, subcloned into a plasmid just upstream of the chloramphenicol acetyl transferase coding region, into HaCaT c ells, A 238-bp fragment and a 123-bp fragment, both containing nuclear factor (NF)-IL-6 and NF kappa B sites, exhibited significant inductio n of chloramphenicol acetyl transferase activity upon treatment with T GF-alpha. Because IL-6 transcription is known to be regulated by activ ation of NF kappa B and NF-IL-6, we analyzed the activation of these D NA-binding proteins by electrophoretic mobility shift assays, NF-IL-6 binding to a P-32-labeled NF-IL-6 binding sequence was enhanced 20 min after TGF-alpha stimulation and returned to basal levels within 90 mi n, whereas NF kappa B binding activity was enhanced after 20 min and r eturned to normal 60 min after stimulation, We conclude that TGF-alpha induces IL-6 in HaCaT cells and, in contrast to thymocytes, may do so by transcriptional activation, possibly through activation of NF kapp a B and NF-IL-6.