The Function And Molecular Mechanisms Of MiRs-103/107 In Regulating Autophagy In The Epidermis

Background and purpose:Autophagy is a means by which cells adapt to differing intrinsic and extrinsic cellular stress-related situations.Through this self-cannibalistic process,cytoplasmic material is engulfed within double-membraned autophagosomes(initiation)and delivered into lysosomes for degradation and recycling.End-stage autophagy is when autolysosomes are recycled to reform lysosomes via autophagic lysosome reformation(ALR).Autophagy regulates numerous physiologieal and pathological processes in which cellular components need to be degraded and recycled,and hence is critical for maintaining homeostasis in various tissues.Epidermis protects the organism from external insults and dehydration,which are vital for survival.Autophagy is critical for epidermal homeostasis,which maintains proper barrier function.For example,autophagy removes metabolic organelles(mitochondria,Golgi,endoplasmic reticulum)and nuclei,which is crucial for the proper terminal differentiation of epidermal keratinocytes.Disruption of autophagy is associated with nuclear retention in the epidermis,which is a characteristic of numerous skin diseases.Despite its importance in epidermal physiology,little is known about the molecular pathways implicated in the regulation of autophagy in the epidermis.MicroRNAs,represent a major class of regulatory noncoding small RNAs,that are emerging as important regulators of epithelial homeostasis and stress responses.miRs-103/107 belong to a miRNA family and have an identical seed sequence,differing in only one 3’ nucleotide outside the seed sequence.Thus,they frequently target common genes.Recently,we have demonstrated that the miRs-103/107 family is preferentially expressed in the stem cell-enriched limbal epithelium.In this context,miRs-103/107 regulate the cell cycle,proliferative capacity,and cell-cell communication in primary human limbal epithelial keratinocytes(HLEKs).Interestingly,we have recently found that miRs-103/107 are essential for maintaining autophagy in vitro via ensuring dynamin activity,which is critical for end-stage autophagy.Loss of miRs-103/107 increases phosphorylated dynamin(inactive form)resulting in the inhibition of end-stage autophagy.However,many questions remain.For example,do miRs-103/107 regulate end-stage autophagy in a similar manner in vivo?Are miRs-103/107 involved in autophagy in other stratified epithelia such as the epidermis?Here we show that miRs-103/107 positively regulate end-stage autophagy in mouse epidermis in vivo via modulating dynamin activity.Methods:1.In situ hybridization of miR-103 and miR-107 using 20 nM locked nucleic acid(LNA)modified digoxigenin labeled probes.2.Construct a GFP-LC3 transgenic mouse model that expresses the fusion of GFP and LC3.Skin samples of wild-type mice(BALB/c)were also dissected and immediately embedded in OCT frozen sections in order to visualize miR-103/107 expression in mouse skin.3.Dissolve skin samples in Qiazol to obtain mouse total cellular RNA.The skin was treated with antagomir-107 and an irrelevant antagomir,and the total cellular RNA was then isolated and purified by the miRNeasy kit for real-time qPCR detection.4.Intraperitoneal injection of chloroquine was dissolved in PBS to measure autophagic flux in vivo.5.Subcutaneous injection of mice using Antagomir and MicroRNA mimics.The injected site was collected for further experiments.6.Mouse skin OCT samples were sectioned,GFP-LC3 spots were observed microscopically,and GFP spots were counted using ImageJ.7.Immunostaining was used to visualize the p62 of mouse skin tissue,phospho-engineered proteins,mobilized proteins,LAMPI,keratin 10,keratin 15,and loricrin,collected and analyzed images,and measured the relative fluorescence density.8.Western blot was used to detect activation of signal pathway and expression of autophagy markers after miR-103/107 knockout or overexpression,such as LC3B,PLDI,PLD2,keratin 10,desmogleinl,Activate protein and p62.9.Observe and record the effect of knocking out or overexpressing miR-103/107 in vacuoles of HEK cells at room temperature using an inverted light microscope and calculate the number of vacuoles per cell.10.In vitro western blot and immunofluorescence assays were performed using HEK cells to study how miR-103/107 regulate end stage autophagy.11.Perform an unpaired t-test to determine the statistical significance.Data are shown as mean ±standard deviation(SD).Differences were considered significant when the p-value was<0.05.All experiments were repeated at least three times.Results:1.miRs-103/107 family positively regulates autophagy in epidermisPreviously,we have shown that miRs-103/107 play a positive role in regulating end-stage autophagy in cultured HLEKs.To further investigate the role of this microRNA family in autophagy,we conducted in situ hybridization to localize the expression of miRs-103/107 in the skin,and found that these miRNAs were expressed in epidermis and hair follicle,but not in the dermis.In epidermis,miRs-103/107 are highly expressed in basal and suprabasal layers but not in terninally differentiated layers.This expression pattern is similar to that observed in the limbal epithelium,where miRs-103/107 appeared concentrated in the basal and suprabasal layers.We utilized mice that transgenically express a green fluorescent protein(GFP)-labeled LC3(GFP-LC3),which is a well-established mouse model to monitor autophagy.In the early stages of autophagy,LC3 is conjugated to phosphatidylethanolamine and recruited to autophagosomal membranes,which is required for autophagosome formation.We subcutaneously injected GFP-LC3 mice with antago-107 to inhibit endogenous miRs-107,or an irrelevant antagomir(control).qPCR indicated that antago-107 depleted endogenous miR-107 in mouse epidermis at 1 and 2 days after injection.To examine the effect of such treatment on autophagy status,we imaged and measured GFP-LC3 puncta in the epidermis.A dramatic accumulation of autophagosomes(more GFP-LC3 puncta)in the epidermal basal layer of antago-107-injected mice was noted,compared with controls.Furthermore,immunoblotting demonstrated that antago-107 treatment resulted in accumulation of:(ⅰ)GFP-LC3 protein in GFP-LC3 transgenic mice;and(ⅱ)endogenous LC3 protein in wild type mice.Autophagy is a dynamic cellular process and thus it is necessary to determine the degree of autophagy flux.p62 is a well-accepted substrate of autophagy and is negatively correlated with autophagy flux.Depletion of endogenous miR-107 significantly increased p62 levels in mouse epidermis,as detected by immunostaining and immunoblotting,suggesting an inhibition of autophagy flux.The LC3 turnover assay is another well accepted approach for determining autophagy flux.GFP-LC3 mice injected with antago-107 were treated with the lysosomal inhibitor chloroquine.In chloroquine-injected mice,antago-107 was unable to further increase GFP-LC3.This suggests that the accumulation of GFP-LC3 in epidermis lacking miR-107 is due to an inhibition of end-stage autophagy rather than a defect in the early(induction)stages of autophagy Consistent with our in vitro observations,these results indicate that the miRs-103/107 family is required for proper autophagy in vivoMiRNA mimics are chemically modified double-stranded RNAs that simulated endogenous miRNAs and introduction of such miRNA mimics upregulate miRNA activity.To perform a gain-of-function study,miR-107 mimic was subcutaneously delivered to the skin using an in vivo transfection approach.Consistently,treatment with the miR-107 mimic decreased GFP-LC3 puncta in basal layerand p62 immunofluorescence,suggesting that miR-107 is sufficient to enhance autophagy flux in epidermis.It has been demonstrated that autophagy plays an important role during terminal differentiation in the granular layers.Immunoblotting and immunostaining showed that transient treatments of antago-107 or miR-107 mimic have no significant effect on the expression of keratin 10,desmoglein 1 and loricrin.2.miRs-103/107 family positively regulate dynamin activity in epidermisIt has been shown that dynamin activity is essential for lysosome reformation as well as lysosome clearance.Phosphorylation of dynamin,which inactivates this protein,inhibits end-stage autophagy.We have demonstrated that in HLEKs,antagos-103/107 treatment increased phosphorylated dynamin(p-dynamin),resulting in a disruption of autophagy.Thus,we examined the phosphorylated state of dynamin(p-dynamin;inactive form)immunohistochemically in the epidermis following modulation of miR-107.Depletion of endogenous miR-107 by antago-107 injection unregulated p-dynamin but not total dynamin,indicating an inhibition of dynamin activity.Consistently,immunoblotting showed that loss of miR-107 in mouse epidermis significantly increased p-dynamin protein.Conversely,in vivo transfection of miR-107 mimic attenuated phosphorylation of dynamin in mouse epidermis but didn’t affect total dynamin levels.Collectively,these results suggest that the miRs-103/107 family has a positive(activating)effect on dynamin activity in vivo,which results in proper end-stage autophagy.3.miRs-103/107 family regulates end-stage autophagy via targeting PKC pathwayTo explore the mechanism of altered autophagy following manipulation of miR-103/107,we inhibited endogenous miRs-103/107 in cultured human epidermal keratinocytes(HEKs)by antagomir treatment.We have demonstrated that loss of miRs-103/107 in HLEKs inhibits end-stage autophagy leading to accumulation of hollow vacuoles.Consistently,antagos-103/107 treatment in HEKs also induced numerous hollow vacuoles.Autophagy is a process with multiple stages.Thus,it is necessary to understand which stage of autophagy is inhibited by loss of this microRNA family.We used chloroquine(Chlo)and bafilomycin A1(BafA1),which inhibit autophagy by disrupting autolysosome formation.Chlo and BafAI treatment diminished vacuoles in antagos-103/107-treated HEKs,suggesting that proper autolysosome formation is required for vacuole accumulation and hence loss of miRs-103/107 inhibited autophagy at the end stage.In HLEKs,miRs-103/107 regulate dynamin activity via PKC and CDK5 pathways and thus modulate end-stage autophagy.Therefore,we examined whether inhibition of PKC and CDK5 can rescue the vacuole accumulation in HEKs with antagos-103/107 treatment.Interestingly,treatment with Ro-32-0432 and Rottlerin,two PKC inhibitors,diminished vacuole formation in antagos-103/107-treated HEKs.However,Roscovitine(CDK5 inhibitor)treatment in antagos-103/107-treated HEKs showed no significant change in vacuole formation compared with HEKs treated with antagos-103/107 alone.Collectively,these data suggest that PKC but not CDK5 is involved in miRs-103/107-mediated end-stage autophagy in HEKs.4.miRs-103/107 family regulates dynamin activity via PLD1/2/PKC pathwayWe have shown that loss of miRs-103/107 in HLEKs upregulates PLD1 and PLD2 and thus activates PKC,resulting in increased phosphorylation of dynamin and disassociation of dynamin with autolysosomes.To examine whether this occurs in epidermis,HEKs were treated with antagos-103/107 and ir-antago(control).Consistently,loss of miRs-103/107 in HEKs:(ⅰ)upregulated their direct targets PLD1 and PLD2;(ⅱ)increased p-dynamin(inactive);and(ⅲ)decreased colocalization of dynamin and LAMP1(a lysosomal marker).To test whether miRs-103/107 regulate dynamin activity via PKC,HEKs were treated with two PKC inhibitors:Ro-32-0432 and Rottlerin.The pharmacological inhibition of PKC diminished p-dynamin in HEKs treated with antagos-103/107,suggesting that PKC is required for antagos-103/107-induced phosphorylation of dynamin.Conclusion:The miR-103/107 family have a critical role in regulating end-stage autophagy in mouse epidermis via PLD1/2/PKC/dynamin pathway.