UVB and Proinflammatory Cytokines Synergistically Activate TNF-α Production in Keratinocytes through Enhanced Gene Transcription Muhammad M. Bashir,

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UVB and Proinflammatory Cytokines Synergistically Activate TNF-α Production in Keratinocytes through Enhanced Gene Transcription Muhammad M. Bashir, Meena R. Sharma, Victoria P. Werth Journal of Investigative Dermatology Volume 129, Issue 4, Pages 994-1001 (April 2009) DOI: 10.1038/jid.2008.332 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 IL-1α synergistically increases TNF-α mRNA induction by UVB-irradiated neonatal KCs. Cells were treated with sham, sham+IL-1α (10ngml−1), UVB (30mJcm−2), and UVB+IL-1α. RNA samples were collected at the indicated times and analyzed by real-time PCR. TNF-α mRNA cycle numbers were normalized to cyclophilin A (PPIA). Expression levels of TNF-α mRNA are indicated as “fold change” compared to control. UVB+IL-1α-treated cells showed synergistically increased TNF-α mRNA as compared to UVB or IL-1α alone (Figure 1a and b). The time-course study showed that there was no biphasic induction of TNF-α mRNA in keratinocytes up to 72hours. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Anti-TNF-α antibody has no effect on TNF-α mRNA synthesis in UVB-irradiated keratinocytes in the presence of IL-1α. Conditions included cells treated with sham, sham+IL-1α, UVB, and UVB+IL-1α. In the test group, UVB+ IL-1α-treated cells were also incubated with anti-TNF-α antibody (5, 10, 100ngml−1). RNA samples were collected after 3hours and analyzed by real-time PCR. Results were normalized to PPIA mRNA and compared to control groups. No significant difference in expression of TNF-α mRNA is observed between UVB+ IL-1α and UVB+ IL-1α+anti-TNF-α-treated keratinocytes. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IFN-α2b has no effect on TNF-α mRNA synthesis in sham- or UVB-irradiated keratinocytes in the presence or absence of IL-1α. Sham- or UVB-irradiated keratinocytes were treated with IFN-α2b (2,000Uml−1) or IL-1α (10ngml−1) alone or in combination with IFN-α2b+IL-1α. RNA sample were collected after 6hours and analyzed by real-time PCR. Results were normalized to PPIA mRNA and compared to control groups. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Exogenous rTNF-α increases TNF-α mRNA induction in UVB-irradiated neonatal keratinocytes. Cells were treated with sham, sham+rTNF-α (5ngml−1), UVB, and UVB+rTNF-α. RNA sample were collected 3hours after irradiation and analyzed by real-time PCR. Results were normalized to PPIA (housekeeping gene control). Expression levels of TNF-α mRNA are indicated as “fold change” compared to control. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Synergistic increase of TNF-α protein in keratinocyte medium corresponds to TNF-α mRNA increase due to UVB and IL-1α response. Cells were treated with sham, sham+IL-1α, UVB, and UVB+IL-1α for various time points. Supernatant samples were collected from sham, sham+IL-1α, UVB, and UVB+IL-1α-treated cells at the indicated time points. An equal amount of protein was used for TNF-α ELISA. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 No significant changes were observed in the membrane bound TNF-α protein after UVB and IL-1α treatment. Cells were treated with sham, sham+IL-1α, UVB, and UVB+IL-1α for various time points. Membrane fractions were eluted and an equal amount of protein was used for TNF-α ELISA. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 UVB irradiation and IL-1α enhances TNF-α gene transcription. Nuclei were isolated 3hours after sham- or UVB-irradiation and stimulated with or without IL-1α. Nuclear run-off assay was performed to examine TNF-α and 18S (control) transcription. Detectable bands were assessed by densitometric analysis. UVB irradiation induced TNF-α gene transcription five-fold. One of four experiments is shown illustrating 11-fold induction of UVB+ IL-1α cells when compared with sham irradiated cells. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 TNF-α mRNA stability is not induced after UVB irradiation and IL-1α stimulation. Keratinocytes were UVB- or sham-irradiated and stimulated with IL-1α for 3hours. Actinomycin D (10μgml−1) was added after 3hours. Total RNA was extracted at different time points and TNF-α mRNA levels were determined by northern analysis and real-time PCR. The half-life was calculated in UVB, and UVB+IL-1α-treated cells. Journal of Investigative Dermatology 2009 129, 994-1001DOI: (10.1038/jid.2008.332) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions