Role Of STING Pathway In UVB Induced Apoptosis And Inflammation In Human Epidermal HaCaT Cells

Ultraviolet B(UVB)from sunlight is one of the main environmental causes of skin damage,leading to complex changes in exposed skin cells,including DNA damage,oxidative stress,and ultimately inflammation and apoptosis.Protectives against UVB now mainly use physical sun protection,but their strength and stability are poor,so it is necessary to find effective drugs for protecting against UVB damage.However,since broad targets of UVB damage and the concrete mechanisms on regulating the bodies are difficult to clarify,there are great difficulties in developing anti-UVB drugs.DNA damage is a classical cellular injury caused by UVB.Our study also found that UVB irradiation causes nuclear DNA damage in human immortalized keratinocytes HaCaT cells.Damaged DNA easily leaks into the cytoplasm.The cytoplasmic DNA is a typical dangerrelated molecular pattern(DAMP)that can be recognized by the body’s corresponding sensory proteins triggering inflammation.Here we observed activation of a cytosolic DNA sensor,cGAS-STING signaling pathway,in HaCaT cells within 2-3 h after DNA damage.Silencing STING by siRNA weakens UVB-induced apoptosis,indicating that STTING pathway activate the apoptosis.NF-κB signaling pathway and interferon regulatory factor 3(IRF3)-IFNβsignaling pathways are two major downstream pathways of the cGAS-STING pathway.NF-κB signaling is activated in UVB-irradiated HaCaT cells,in which NF-κB translocates to the nucleus.But silencing of STING attenuates NF-κB franslocation.Inhibition of NF-κB with BAY blocks UVB-induced apoptosis.UVB irradiation increases the nuclear translocation of IRF3,accompanied by enhanced expression of IFNβ mRNA,but these are attenuated by STING silencing.Treatment with MRT67307,an inhibitor of TBK1-IRF3-IFNβ pathway,blocks UVBinduced apoptosis.These results indicate that UVB irradiation-induced DNA damage in HaCaT cells,activates the cGAS-STING signaling pathway and its downstream NF-κB pathways and IFNβ pathway,thereby inducing apoptosis.Mitochondria are important cellular organelle,which serve for intracellular energy supply,but the dysregulated mitochondria cause many pathological changes,including apoptosis,inflammation and senescence.Therefore,we investigated the amounts of mitochondria.We found that mitochondrial amount increases dramatically in UVB-irradiated HaCaT cells.Whether the increase in mitochondrial amount is beneficial or detrimental in UVB-irradiated cells aroused our interest.Further study found that UVB treatment results in the collapse of mitochondrial membrane potential(MMP),decrease in ATP levels and increased production of reactive oxygen species(ROS),suggesting the disorders in mitochondria of UVB-irradiated HaCaT cells.Considering the increased amounts of mitochondria in UVB-treated cells,these results illustrate that UVB irradiation leads to accumulation of dysfunctional mitochondria.Mitophagy mediated by PINK1 kinase and parkin ubiquitin ligase is a process specifically clears damaged mitochondria,and is critical for the handling of dysfunctional mitochondria.So next we examined the mitophagy level.Study found that both PINK1 and parkin are decreased in UVB-irradiated cells,indicating that mitophagy is inhibited.Silencing PINK1 or parkin expression by transfection of siRNA results in cellular damages similar to UVB irradiation,including increased mitochondrial amount,decreased MMP and ATP production,and increased apoptosis,demonstrating that PINK1/parkin-mediated mitophagy is protective for mitochondrial function.Further studies also found that silencing PINK1 or parkin also increased the protein levels of cGAS and STING,facilitated nuclear accumulation of NF-κB,and promoted the transcription of IFNβ,suggesting the activation of cGAS-STING pathway.Similar to the damage caused by UVB irradiation,PINK1/parkin silencing promotes apoptosis.In addition to regulating apoptosis by increasing mitochondrial outer membrane permeability and promoting cytochrome c release,Bax also promoted the release of other mitochondrial contents,such as mitochondrial DNA(mtDNA)and others,to mediate inflammation-related responses.Our study reveals that UVB irradiation upregulates Bax protein levels and increases mitochondrial membrane permeability,subsequently mediates mtDNA leakage into the cytoplasm and induces the activation of cGAS-STING-apoptosis pathway.Transfection of siRNA targeting Bax significantly inhibits the above changes.Our study also found that mtDNA leakage-cGAS-STING-apoptosis process activated by Bax accumulation is negatively regulated by PINK1/parkin-mediated mitophagy.These results suggest us that the decreased mitophagy play a key role in UVB-induced skin damage.Mitochondria are highly dynamic,and in addition to mitophagy,mitochondrial number and quality are also regulated by dynamic activities such as mitochondrial fission and fusion.UVB irradiation abnormally increases mitochondrial content but decreases mitochondrial volume.Investigation of fission and fusion key proteins reveals that UVB irradiation results in marked up-regulation of mitochondrial fission protein dynamin-related protein 1(DRP1)and downregulation of mitochondrial outer membrane fusion protein 1&2(MFN1&2)in HaCaT cells,showing a relative increase in mitochondrial fission.Inhibition of mitochondrial fission by treatments with a DRP1 inhibitor,mdivi-1,or with DRP1-targeted siRNA,efficiently prevents UVB-induced NLRP3/cGAS-STING-mediated inflammation or apoptosis in the HaCaT cells,whereas inhibition of mitochondrial fusion with MFN1 and 2 siRNA increases UVB-induced NLRP3/cGAS-STING-mediated inflammation or apoptosis.The above results indicate that mitochondrial fission/fusion dynamics are involved in the activation of NLRP3 inflammasome,cGAS-STING pathway and apoptosis.The enhanced mitochondrial fission and reduced fusion cause the up-regulation of reactive oxygen species(ROS).Application of an antioxidant,NAC,which scavenges excessive ROS,attenuates inflammatory responses through suppressing NLRP3 inflammasome and cGAS-STING pathway activation,and rescues cells from apoptosis caused by UVB-irradiation.These results show that the imbalance of mitochondrial fission fusion is involved in the regulation of NLRP3/cGAS-STING proinflammatory pathway and apoptosis through the production of ROS.In summary,this study identifies the activation of cGAS-STING signaling pathway by UVB irradiation,and its downstream NF-κB and IFNβ pathways are both involved in the associated cellular inflammation and apoptosis.STING pathway activation by UVB irradiation is dependent on the cytosolic leakage of mtDNA resulted from decreased mitophagy and imbalanced mitochondrial fission/fusion dynamics.Studies elaborate the mechanisms by which UVB irradiation damages epidermal cells and provide new therapeutic strategies,suggesting that the mitochondria-STING pathway serves as a target for the development of skin protective drugs.