TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes
As the outermost physical barrier of an organism, the skin is diurnally exposed to UV radiation (UVR). Recent studies have revealed that the skin exhibits a circadian rhythm in various functions, and this oscillation is disturbed and reset via a strong environmental cue, the UVR. However, a molecular link between circadian perturbation by UVR and UVR-induced cellular responses has not been investigated. We identified tissue inhibitor of metalloproteinase (TIMP)-3 as a novel circadian locomotor output cycles kaput (CLOCK)–dependent diurnal gene by using a CLOCK-knockdown strategy in human keratinocytes. Among dozens of identified transcripts down-regulated by CLOCK knockdown, TIMP3 displayed a rhythmic expression in a CLOCK-dependent manner, in which the expression of matrix metalloproteinase (MMP)-1 and inflammatory cytokines, such as TNF-α, chemokine (C-X-C motif) ligand (CXCL)-1, and IL-8, were inversely regulated. Upon UVB exposure, the expression of CLOCK and TIMP3 was down-regulated, which led to an up-regulation of secretion of MMP1 and TNF-α proteins and in the transcription of CXCL1 and IL-8via CCAAT-enhancer binding protein (C/EBP)-α. UVB-induced TNF-α secretion increased further or decreased by knockdown or overexpression of TIMP3, respectively, as well as by CLOCK. As a novel CLOCK-dependent diurnal gene, TIMP3 inhibits the expression of inflammatory cytokines that are up-regulated by UV irradiation in human keratinocytes. Thus, our work suggests a molecular link between circadian perturbation by UVR and UVR-induced inflammation.—Park, S., Kim, K., Bae, I.-H., Lee, S. H., Jung, J., Lee, T. R., Cho, E.-G. TIMP3 is a CLOCK-dependent diurnal gene that inhibits the expression of UVB-induced inflammatory cytokines in human keratinocytes.
[…] The cells were transfected with scrambled, CLOCK, or BMAL1 siRNAs for 8 h and incubated overnight in growth medium. The cells were then synchronized with serum-rich medium for 2 h and harvested at ZT 32 and 44. Total RNAs were extracted with Trizol reagent (Thermo Fisher Scientific), and poly-A-containing mRNAs were purified and converted into a cDNA library for subsequent cluster generation and DNA sequencing, according to the instructions of the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA). Quality control checks on raw sequence data were performed by the FastQC program. For the data analysis, TopHat, Cufflinks, BWA, SamTools, Annovar, and DeFuse programs were used. […]