Mechanism Of Bach2 Modulates Photoaging Through Autophagy In Fibroblasts

Skin photoaging is the accumulation of photodamage caused by long-term exposure to ultraviolet(UV)light.Improper development or treatment of photoaging leads to actinic keratosis(AK)and even skin tumors.Photoaging is linked to our health,aesthetics,psychology as well as the quality of our life.Therefore,the understanding of the mechanisms underlying photoaging is of paramount importance,because these can contribute to diminishing the adverse effects of photoaging.Bach2(BTB and CNC homolog 2)is a transcription factor with a basic leucine zipper structure(bZIP domain)involved in cell transcriptional regulation.Previous studies have focused on its involvement in the regulation of lymphocyte development,differentiation and immune tumorigenesis.Recently,it has been demonstrated that Bach2 is involved in the regulation of cellular senescence.This is because the knockdown of Bach2 promotes lymphocyte senescence and Bach2 is significantly down-regulated in UV-induced damaged and senescent cells in mouse embryonic fibroblasts(MEF).These finding suggest Bach2 as a potential target of transcription block in UV-induced DNA damage.Our previous study also found that Bach2 expression is lower in AK patients’skin tissues when compared to the normal skin counterparts,The latter suggests that Bach2may play an important role in UV-related photoaging and diseases,and it has been no reported in this area.Therefore,we systematically explored the function of transcription factor Bach2 in photoaging and its biological mechanism mediated by ultraviolet A(UVA)irradiation.It is also preliminarily clarified the role of Bach2 in UVA-mediated photoaging and related mechanisms,and provided a theoretical basis for photoaging treatment.This study includes:(1)Bach2 reduction in UVA induced aged fibroblasts.To explore the involvement of Bach2 in UV-mediated DNA damage,we analyzed transcriptomic data from UV irradiated MEF cells.The results suggested that Bach2expression level were decreased,whilst p21 and MMP3 expression levels were increased.In addition,we found that Bach2 expression level was lower in AK tissue compared to normal skin counterpart.To better understanding the change in transcription factors induced by UVA radiation,we mimicked UVA dosages of solar exposure received by skin during daily life exposure:i.e.a repeated low dose of UVA irradiation in primary fibroblasts mimicked a chronic UV exposure.In agreement with previous reports,UVA exposure in two primary fibroblasts in vitro,caused elevated senescence-associated release ofβ-galactosidase(SA-β-gal)activity.In addition,repeated UVA irradiations increased both mRNA expression and protein levels of p53,p21 and p16 in fibroblasts.We also examined the level of Bach2,Bach1 and Lamin B1in UVA-induced photoaging of fibroblasts.The results revealed that expression levels of Bach2,Bach1 and Lamin B1 were decreased in UVA-irradiated fibroblasts,and Bach2expression level was decreased further by the increasing of UVA dose.Taken together,these results suggested that Bach2 might be involved in the process of UVA-induced fibroblasts photoaging mediated by UVA exposure.(2)Bach2 decreased UVA-induced damage and photoaging in fibroblasts.To identify whether Bach2 is responsible for aging mediated by UVA,we depleted Bach2 with siBach2 adenovirus.When levels of Bach2’s mRNA and protein levels were decreased,Bach2 target genes such as MAFK were also reduced at the mRNA level.Depletion of Bach2 enhanced SA-β-gal staining activity and elevated the abundance of p16,whilst it also increased the mRNA levels of IL-1α,IL-1β,IL-6 and IL-8.Most importantly,Bach2 knockdown further reduced UVA-induced Bach2 reduction,and additionally increased of the abundance of p16 in fibroblasts.The results revealed that Bach2 depletion increased the sensibility of UVA-mediated cell senescence.In addition,we also over-expressed Bach2 with Flag-tagged vector and examined the function in fibroblasts by real time-quantitative PCR(qRT-PCR)and Western blotting.These results showed that overexpression of Bach2 reduced SA-β-gal activity,and markedly decreased expression levels of p16 and Rb mRNA,and protein levels of p16.Moreover,the early and late SASP factors IL-1αand IL-6 were also decreased,respectively.Furthermore,inducing Bach2 followed by repeated UVA irradiation reduced Bach2expression while no significantly change in p16 protein level when compared to the non-irradiated Bach2 overexpression group.Together,these results demonstrated that Bach2 increases the susceptibility of UVA-mediated photoaging in fibroblasts.(3)Bach2 is involved in autophagy to decrease cell senescence in fibroblasts.We aimed to determine the mechanism by which Bach2 regulates photoaging in fibroblasts.Inhibition of the proteasomes with MG-132,a proteasome inhibitor,had no significant effect on Bach2 expression levels.Therefore,we checked the autophagy-related proteins LC3 B expression in Bach2 overexpressing cells.Immunofluorescence confirmed that cells expressing GFP-Bach2 had increased numbers of LC3 B puncta(Red),a fluorescent marker of autophagosomes.We also examined autophagy-related gene expression in Bach2 overexpressing cells.The results revealed that Bach2 overexpression enhanced Atg5 expression,while no significant effect was observed on Atg3,Atg7 and p62 in mRNA levels.On the protein level,expression of Atg3,Atg7,p62 and LC3 were markedly increased in Bach2overexpressing cells.These results suggested that Bach2 overexpression increases autophagy-related proteins.Atg3,Atg5 and p62 were markedly decreased in Bach2knockdown on mRNA levels.On the protein level,Atg7,Beclin-1,p62 and LC-3 were markedly decreased by depletion of Bach2 in cells,whereas a knockdown of Bach2prevented autophagy progression.These results suggest that autophagy induced by Bach2 might be a positive cell senescence regulator.Treatment of GFP-Bach2expressing cells with autophagy inhibitors 3-MA inhibited Atg3,Atg7 and LC3expression while it had no significant effect on Bach2 accumulation.Importantly,we found that p16 and p21 were increased by 3-MA treatment of Bach2 expressing cells.Furthermore,we also checked cell senescence in Atg5 KO or Atg7 KO MEFs and found that p16 and p21 were augmented,Bach2 was slightly decreased in Atg5 KO and Atg7KO MEFs.To examine Bach2 involvement in the mechanisms of autophagy in fibroblasts,we probed its subnuclear distribution.Bach2 accumulation in the cytoplasm and co-localization with Atg3 and LC3 was observed,but there wrre no signs for Bach2and p62 co-localization in fibroblasts.Then,coimmunoprecipitation experiments were performed to analyze whether exogenously expressed and endogenous expressed Bach2interact with autophagy-related proteins.Our results show that endogenous Bach2physically interacts with Beclin-1,Atg3,Atg7 and LC3 in fibroblasts.However,the interaction of Bach2 and p62 was not detected.In addition,exogenously expressed Bach2 also interacts with Beclin-1,Atg3,Atg5,Atg7 and LC3,but there were no interaction with p62.Taking together,these results suggest that Bach2 is involved in autophagy by interaction with autophagy-related proteins thereby decreasing cell senescence.(4)Bach2 reduction in UVA-induced photoaging in mice skinWe used a daily exposure dose of 20 J/cm~2 UVA to irradiate mouse skin for 30 and70 days,respectively,in order to establish a chronic photoaging model.We examined sham-irradiated mice and mice after consecutive UVA-irrasiated mice.The results suggest that in the mid-dorsal epidermis,thickness was decreased in UVA irradiated mice.With increasing irradiation time,the epidermis thickness was further decreased.In addition,confocal microscopy was used to examine p16 and p21 expression levels which were increased in UVA irradiated groups.Furthermore,mouse skin extraction revealed that p16 and p21 levels were significantly elevated in 30 and 70 days post-irradiation mice.These results suggest that 20 J/cm~2 UVA irradiation over 30 days or 70 days are able to induce mouse skin aging.To determine whether Bach2 responds to UVA-mediated aging in mice in vivo,we examined irradiated mice and found a reduction of Bach2 accumulation in the aged mice skin relative to the sham irradiated mice skin.Taken together,these results revealed that Bach2 might contribute to UVA-mediated photoaging both in mice and in humans.(5)Low-dose Aminolevulinic acid-basedphotodynamic therapy(PDT)treatment reduces UVA-mediated photoaging by up-regulating Bach2To explore the anti-photoaging effect of PDT in fibroblasts,UVA-induced photoaging fibroblasts were treated with a range of concentrations of ALA-PDT(0-5mM ALA).As the ALA-PDT concentration increased,the expression of p16 and p21were decreased.Based on these analyses,we chose 1.0 mM ALA-PDT for all further experiments,The dose of 1.0 mM ALA could significantly decrease p16 and p21expression levels,and increase Bach2 accumulation in UVA-induced photoaged fibroblasts.In addition,Bach2 level was increased in mice skin tissues with PDT treatment.Bach2 down-regulation induced cell senescence,and PDT treatment of Bach2si cells decreased SA-β-gal positive cells,however,it caused no significant change in Bach2 and p16 expression levels.These results suggest that low dose PDT decreases photoaging by reduction of Bach2 in fibroblasts.Taking together,current study explore the role of Bach2 in the regulation of photoaging as well as its underlying mechanisms from pathological,cellular,molecular and animal views.It offers guidance for clinical applications and provides new research perspectives and ideas for the treatment of photoaging and UV-related diseases.It might also provid theoretical basis and new targets for photoaging prevention.