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 upregulated in psoriasis, suggesting a pathogenic role for these cytokines. The sequence of these events, however, has not been elucidated, Recently it has been reported that TGF-alpha induces IL-6 in thymocytes through posttranscriptional regulation; therefore, we were interested in whether TGF-alpha can also induce IL-6 in human keratinocytes, Thus, we stimulated the human keratinocyte cell line HaCaT with TGF-alpha and tested supernatants for IL-6 activity, TGF-alpha resulted in a significant induction of the release of IL-6, This was also confirmed by northern blot analysis, which revealed a transient increase in IL-6 mRNA, This increase was unlikely due to enhanced mRNA stability, because we could not observe induction of IL-6-specific transcripts by TGF-alpha in the presence of actinomycin D. To determine whether IL-6 induction by TGF-alpha is transcriptionally regulated, we transfected fragments of the IL-6 upstream region, subcloned into a plasmid just upstream of the chloramphenicol acetyl transferase coding region, into HaCaT cells, A 238-bp fragment and a 123-bp fragment, both containing nuclear factor (NF)-IL-6 and NF kappa B sites, exhibited significant induction of chloramphenicol acetyl transferase activity upon treatment with TGF-alpha. Because IL-6 transcription is known to be regulated by activation of NF kappa B and NF-IL-6, we analyzed the activation of these DNA-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 min, whereas NF kappa B binding activity was enhanced after 20 min and returned 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 kappa B and NF-IL-6.