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Transforming growth factor-alpha induces interleukin-6 in the human keratinocyte cell line HaCaT mainly by
Y Aragane1, H Yamada, A Schwarz
1Ludwig Boltzmann Institute for Cellbiology and Immunobiology of the Skin, Department of Dermatology, University Münster, Münster, Germany.
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 NFkappaB 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 NFkappaB and NF-IL-6, we analyzed the activation of these DNA-binding proteins by electrophoretic mobility shift assays. NF-IL-6 binding to a 32P-labeled NF-IL-6 binding sequence was enhanced 20 min after TGF-alpha stimulation and returned to basal levels within 90 min, whereas NFkappaB 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 NFkappaB and NF-IL-6.
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 NFkappaB 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 NFkappaB and NF-IL-6, we analyzed the activation of these DNA-binding proteins by electrophoretic mobility shift assays. NF-IL-6 binding to a 32P-labeled NF-IL-6 binding sequence was enhanced 20 min after TGF-alpha stimulation and returned to basal levels within 90 min, whereas NFkappaB 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 NFkappaB and NF-IL-6.