UVB-mediated DNA damage induces matrix metalloproteinases to promote photoaging in an AhR- and SP1-dependent manner

It is currently thought that UVB radiation drives photoaging of the skin primarily by generating ROS. In this model, ROS purportedly activates activator protein-1 to upregulate MMPs 1, 3, and 9, which then degrade collagen and other extracellular matrix components to produce wrinkles. However, these MMPs are expressed at relatively low levels and correlate poorly with wrinkles, suggesting that another mechanism distinct from ROS and MMP1/3/9 may be more directly associated with photoaging. Here we show that MMP2, which degrades type IV collagen, is abundantly expressed in human skin, increases with age in sun-exposed skin, and correlates robustly with aryl hydrocarbon receptor (AhR), a transcription factor directly activated by UV-generated photometabolites. Through mechanistic studies with HaCaT human immortalized keratinocytes, we found that AhR, specificity protein 1 (SP1), and other pathways associated with DNA damage are required for the induction of both MMP2 and MMP11 (another MMP implicated in photoaging), but not MMP1/3. Last, we found that topical treatment with AhR antagonists vitamin B12 and folic acid ameliorated UVB-induced wrinkle formation in mice while dampening MMP2 expression in the skin. These results directly implicate DNA damage in photoaging and reveal AhR as a potential target for preventing wrinkles.


Introduction
UV radiation is a ubiquitous environmental carcinogen that induces numerous harmful effects in the skin. Comprising 95% of the photons that reach the Earth's surface (1), UVA radiation (range 320-400 nm) generates ROS in both the dermis and epidermis, leading to the activation of various oxidative stress pathways. The MMPs that are upregulated as a result are thought to drive extrinsic aging (or photoaging) of the skin, degrading type I and type III collagen in the dermis to form fine and coarse wrinkles (2,3). UVB radiation (range 280-320 nm) is another important component of UV primarily penetrating the epidermal layers, which not only contributes ROS to the skin like UVA, but also directly damages DNA, characteristically introducing bulky adducts like cyclobutane pyrimidine dimers (CPDs) and double-stranded breaks (DSBs) (4). While the central role of DNA damage is already readily appreciated in skin carcinogenesis (5), it is still unclear whether UVB-induced DNA damage holds any nonredundant roles in inducing MMP expression and driving photoaging.
Among the MMP genes that are induced by UV, MMP1, MMP3, and MMP9 are often considered to be the primary mediators of photoaging. It is thought that UV-induced ROS activates MAPK pathways that subsequently activate activator protein-1 (AP-1) to upregulate MMP1, MMP3, and MMP9 (6). Conversely, retinoids are believed to reverse the effects of photoaging -clinically effacing wrinkles -by transrepressing the AP-1 pathway (7), thereby suppressing MMP expression and restoring lost collagen (8,9). The apparent absence of AP-1 binding sites in the proximal promoter regions of other MMP genes has further honed the field's intense focus on this trio of MMPs. There is some evidence, however, that the role of these MMPs in the skin may be overstated, particularly in the case of MMP1 and MMP3. In addition to MMP1/3/9 only being variably expressed across melanoma cell lines (10), type I collagen degradation activity (associated with MMP1) and MMP3 levels are not elevated in UV-exposed, wrinkle-bearing skin (11), raising the question of which MMPs are more relevant for photoaging.
It is currently thought that UVB radiation drives photoaging of the skin primarily by generating ROS. In this model, ROS purportedly activates activator protein-1 to upregulate MMPs 1, 3, and 9, which then degrade collagen and other extracellular matrix components to produce wrinkles. However, these MMPs are expressed at relatively low levels and correlate poorly with wrinkles, suggesting that another mechanism distinct from ROS and MMP1/3/9 may be more directly associated with photoaging. Here we show that MMP2, which degrades type IV collagen, is abundantly expressed in human skin, increases with age in sun-exposed skin, and correlates robustly with aryl hydrocarbon receptor (AhR), a transcription factor directly activated by UV-generated photometabolites. Through mechanistic studies with HaCaT human immortalized keratinocytes, we found that AhR, specificity protein 1 (SP1), and other pathways associated with DNA damage are required for the induction of both MMP2 and MMP11 (another MMP implicated in photoaging), but not MMP1/3. Last, we found that topical treatment with AhR antagonists vitamin B 12 and folic acid ameliorated UVB-induced wrinkle formation in mice while dampening MMP2 expression in the skin. These results directly implicate DNA damage in photoaging and reveal AhR as a potential target for preventing wrinkles.
JCI Insight 2022;7(9):e156344 https://doi.org/10.1172/jci.insight.156344 MMP2, encoding for 72 kDa gelatinase, represents a promising candidate. Induced by UVB irradiation in human skin (12), MMP2 targets type IV collagen present in the dermal-epidermal junction, much like MMP9, and is elevated in wrinkle-bearing skin (11). Moreover, MMP2 is uniformly elevated across melanoma cell lines and has also been associated with more invasive cancers and poorer prognoses (10). Despite these clinical associations, however, it is still unknown exactly how UV induces MMP2 expression.
Alongside the generation of ROS in the skin, UV produces various aromatic photometabolites (most notably 6-formylindolo [3,2-b]carbazole, FICZ) that directly bind and agonize the aryl hydrocarbon receptor (AhR) (13). Upon activation, AhR localizes to the nucleus, heterodimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT), and binds xenobiotic response elements (XREs) to upregulate classic target genes like Cyp1b1 (14). As part of its pleiotropic transcriptional response, AhR regulates a number of dermatologic processes including the tanning response (15) and maintenance of barrier immunity (16). Furthermore, past studies have demonstrated that treatment of melanoma cells with the AhR agonist 2,3,7,8-tetrachlorodibenzodioxin (TCDD) is sufficient to induce expression of MMP2 (17). In light of these studies, we hypothesized that UVB may be inducing MMP2 in an AhR-dependent fashion in keratinocytes.
In the present study, we unexpectedly found that AhR is required, but not sufficient, for UVB-mediated induction of MMP2 in HaCaT human keratinocytes. Through a series of mechanistic studies, we found that UVB-mediated induction of MMP2 and MMP11 (another MMP previously implicated in photoaging) requires the transcriptional factor SP1, DNA damage sensor ataxia telangiectasia mutated (ATM), and p38/JNK MAPK pathways. We further discovered that chemically induced DNA damage was sufficient to induce MMP2/11 but not MMP1/3. Underscoring the clinical relevance of this new model, topical treatment with vitamin B 12 or folic acid (FA) (naturally occurring antagonists of AhR) significantly ameliorated UVB-induced wrinkles and sunburns, while reducing epidermal expression of MMP2. Therefore, by potentially linking DNA damage to MMP expression, this AhR-and SP1-mediated mechanism not only expands the field of photoaging to include other potentially more relevant MMPs, but also provides, to our knowledge, novel molecular targets for treating symptoms of photoaging.

Results
Mmp2 and Mmp11 correlate with age, sun exposure, and AhR pathways in human skin. To begin assessing whether MMP2 might be a relevant mediator of photoaging, we first measured its mRNA expression in normal human skin using the Genome-Tissue Expression (GTEx) RNA-Seq data set. In addition to being the most abundantly expressed photoaging-associated MMP gene (6) ( Figure 1A), Mmp2 mRNA steadily increased with age in both male and female skin, but only in sun-exposed skin ( Figure 1B and Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.156344DS1). This pattern stands in stark contrast to the expression profiles of other MMP genes, which either did not have a meaningful relationship with age, as in the case of Mmp1 (Supplemental Figure 1C), or even decreased with age, as with Mmp3 (Supplemental Figure 1D). Although Mmp9 expression increased with age (Supplemental Figure 1E), it did not exhibit as dramatic a difference as did Mmp2 between sun-exposed and sun-protected skin ( Figure 1B).
Next, given prior reports that AhR activation induces MMP2 in melanoma (17), we evaluated whether it might correlate with Mmp2 expression in these skin samples. We stratified samples by donor demographics (using both age and sex) and generated a correlation map comparing Ahr expression with MMPs that have been implicated previously in photoaging (6). Only Mmp2 and Mmp11 positively correlated with Ahr expression across all demographics and exclusively in sun-exposed skin ( Figure 1C), which may reflect its elevated content of AhR ligands from UVB-induced photometabolites like FICZ (13). To determine how MMPs might correlate with endogenous AhR ligands (which should be at comparable levels in sun-exposed and sun-protected skin), we repeated this analysis by comparing MMP expression with Kyat2, which is involved in the generation of kynurenic acid, a major endogenous ligand responsible for basal stimulation of the AhR pathway (18). As might be expected, Kyat2 expression positively correlated with Mmp2 and Mmp11 mRNA -but not other evaluated genes -across all demographics, in both sun-protected and sun-exposed skin ( Figure 1D). This clustering of Mmp2 and Mmp11 suggests that both MMP genes could be regulated by the same AhR-dependent pathway. Consistent with this idea, the expression of Mmp11 also steadily increased with age in sun-exposed skin (Supplemental Figure 1F), similar to Mmp2 but unlike Mmp1 and Mmp3. Therefore, we next characterized the UV-induced transcriptional response of both Mmp2 and Mmp11.  (20). This treatment abrogated the UVB-mediated induction of Mmp2 and Mmp11 (Figure 2A and Supplemental Figure 2A) and suppressed Cyp1b1 mRNA ( Figure 2B), confirming inhibition of AhR activity. Protein levels of MMP2 largely followed changes in its mRNA expression in both HaCaT keratinocytes ( Figure 2C) and primary human dermal fibroblasts (Supplemental Figure 2B).
To confirm the requirement of AhR through a genetic approach, we knocked down AhR and its canonical heterodimerization partner ARNT with siRNA (Supplemental Figure 2C) and found that this significantly attenuated the induction of Mmp2 and Mmp11 mRNA ( Figure 2D and Supplemental Figure 2, D and E) and protein (Supplemental Figure 2F). Knockdown of AhR did not alter the mRNA expression of Mmp1 or Mmp3 (Supplemental Figure 2, G and H) but significantly reduced that of Mmp9 (Supplemental Figure 2I). These data suggest that, first, Mmp2 and Mmp11 are regulated by a distinct AhR-dependent transcriptional mechanism and, second, that Mmp9 may be coregulated by this new AhR-dependent mechanism and transcription factor AP-1 (which also regulates Mmp1 and Mmp3).
Next, we investigated whether AhR activation is sufficient to induce Mmp2 and Mmp11 mRNA in HaCaT cells. Unexpectedly, treatment with 3 prototypical AhR agonists -TCDD, FICZ, and benzo(a) pyrene (BaP) -did not result in induction of Mmp2 and Mmp11 ( Figure 2E and Supplemental Figure  2J), despite strong upregulation of Cyp1b1 ( Figure 2F). This suggested there might be another pathway activated by UVB that is working with AhR to induce Mmp2 and Mmp11 mRNA. Consistent with this idea, treatment with TCDD in the presence of UVB irradiation further induced Mmp2 and Mmp11 mRNA (Supplemental Figure 2, K and L), despite no further changes in Cyp1b1 mRNA (Supplemental Figure 2M), indicating that Mmp2 and Mmp11 are not induced simply as AhR target genes.
SP1 is required for UVB-mediated MMP2/11 expression and acts downstream of AhR. SP1 is a ubiquitous transcriptional factor that has previously been shown to regulate the expression of both MMP2 and MMP11 in nonskin cells (21,22). Given the role of SP1 in responding to DNA damage (23, 24) -which would be present in both UVB-irradiated cells and cancer -we hypothesized that SP1 might directly mediate the induction of Mmp2 and Mmp11 mRNA by UVB. We found that knocking down SP1 with siRNA (Supplemental Figure 3A) abrogated the induction of Mmp2 and Mmp11 mRNA ( Figure 3A and Supplemental Figure 3B) and the MMP2 protein ( Figure 3B). Interestingly, as with AhR, SP1 knockdown did not suppress the induction of Mmp1 or Mmp3 (Supplemental Figure 3, C and D) but attenuated Mmp9 mRNA induction (Supplemental Figure 3E). Cyp1b1 mRNA was unchanged (Supplemental Figure 3F) with SP1 silencing, suggesting that SP1 might act downstream of AhR. Consistent with this idea, the knockdown of SP1, alongside the knockdowns of AhR and ARNT, abrogated the induction of Mmp2 and Mmp11 by TCDD in the context of UVB irradiation ( Figure  3C and Supplemental Figure 3G), confirming that the effect of AhR on irradiated cells is mediated by SP1.
Given this requirement for SP1, we next investigated whether SP1 binds directly to the promoter regions of Mmp2 and Mmp11 with UVB irradiation. ChIP-PCR revealed that SP1, but not AhR, was significantly more enriched with irradiation in the promoter regions of Mmp2 and Mmp11 than in those of Mmp1, Mmp3, and Mmp9 ( Figure 3D). Interestingly, in addition to each promoter containing at least 1 validated SP1 binding site (which are heavily composed of G and C nucleotides) (21,22), the 200 bp promoter regions of Mmp2 and Mmp11, as a whole, feature an overall elevated guanosine-cytosine (GC) content (68% and 75%, respectively), which could represent a potential mechanism by which SP1 responsiveness is achieved via selection. Notably, this GC content was similar to that in the promoter region of another SP1 target gene Vegfa (74%) (Supplemental Figure 3, H and I) (25). In contrast, promoters of other evaluated MMP genes did not exceed 50% GC content (as might be expected by chance), whereas the MMP9 promoter had a more intermediate GC content (59%, Supplemental Figure 3, H and I). Finally, through mutation analysis of luciferase constructs containing the promoter regions of human Mmp2 and Mmp11, we found that the abrogation of validated SP1 binding sites in these 2 promoters eliminated their responsiveness to UVB irradiation ( Figure 3E). In sum, these results show that induction of Mmp2 and Mmp11 requires binding of SP1 to their promoters.
DNA damage and MAPK pathways mediate induction of MMP2/11. Next, we sought to identify how UVB might activate SP1. As mentioned previously, SP1 is characteristically activated with DNA damage, especially with DSBs (24,26). DSBs can arise indirectly with UVB irradiation when actively dividing cells like basal keratinocytes attempt to replicate DNA containing bulky photoproducts CPDs that are not properly removed by nucleotide excision repair (NER) (27)(28)(29)(30). To evaluate whether DNA damage drives UVB-mediated induction of Mmp2 and Mmp11, we treated HaCaT cells with either camptothecin (a topoisomerase I inhibitor that induces single-stranded breaks that can progress to DSBs; ref. 31) Figure 4, B, C, F, and G) -resembling the dichotomous patterns seen with AhR and SP1 knockdown experiments. Interestingly, Mmp9 mRNA was inducible with hydrogen peroxide (Supplemental Figure 4H), but not with camptothecin (Supplemental Figure 4D).
Given the role of AhR in suppressing NER and clearance of CPDs (33), we hypothesized that AhR activation by UVB could promote DSB accumulation (as a result of persistent CPDs), ultimately leading to SP1 activation and induction of Mmp2 and Mmp11 mRNA. Such a model would help explain why AhR activation alone, in the absence of CPD generation by UVB, is not sufficient for inducing Mmp2 and Mmp11 JCI Insight 2022;7(9):e156344 https://doi.org/10.1172/jci.insight.156344 but may still be required for MMP induction in the presence of CPDs. Consistent with this hypothesis, we found that treatment with small-molecule inhibitors of AhR decreased the abundance of phosphorylated H2AX, a common marker of DSBs ( Figure 4C). Furthermore, knockdown of the serine/threonine protein kinase ATM (Supplemental Figure 4I), a classic sensor of DSBs, attenuated the induction of Mmp2 and Mmp11 mRNA in irradiated keratinocytes ( Figure 4D and Supplemental Figure 4J). This requirement for ATM -alongside the requirements for AhR, ARNT, and SP1 -was replicated in normal primary human dermal fibroblasts (Supplemental Figure 4, K and L), suggesting that this model may be broadly relevant across the different cell types of the skin and in more primary, p53-sufficient contexts.
Next, we examined how UVB-mediated DNA damage might ultimately activate SP1. Classically associated with stress responses, p38 and JNK MAPK pathways are activated by both UVB (34) and DNA damage (35) and have previously been shown to induce transcription of genes via SP1 phosphorylation (36,37). Targeting the 3 major MAPK pathways with small-molecule inhibitors, we found that inhibition of p38 and JNK, but not ERK, abrogated the induction of Mmp2 and Mmp11 mRNA ( Figure 4E and Supplemental Figure 4M). These results are consistent with prior reports that anisomycin, a small molecule activator of p38 and JNK pathways, is sufficient to induce Mmp2 mRNA (38). Next, we found that knockdown of ATM, which acts upstream of MAPK pathways (39), decreased phosphorylation of SP1 at Thr-278 and Thr-739 ( Figure 4F), residues that are targeted by p38 and JNK pathways and also known to induce the transcriptional activity of SP1 (36, 37). These results implicate MAPK pathways and SP1 phosphorylation as possible downstream mediators of UVB irradiation. Treatment with vitamin B 12 and FA attenuates UVB-associated sunburn and wrinkles in mice. Last, to assess the clinical relevance of this new AhR-dependent mechanism, we irradiated mice with increasing doses of UVB over a period of 16 weeks and treated them topically with either B 12 or FA, immediately after each exposure. Sunburns are often associated with sun-induced DNA damage as they represent cell death resulting from excessive UVB irradiation (40). We first hypothesized that B 12 and FA treatment, by antagonizing AhR, might ameliorate the severity of sunburns -presumably by rescuing NER activity, decreasing persistent DNA damage, and ultimately preventing cell death. Supporting this hypothesis, at week 10, irradiated mice exhibited more severe erythema and scaling than did nonirradiated or vitamin-treated mice (Supplemental Figure 5A). Through the use of a skin probe, we verified that treatments with B 12 and FA not only reduced erythema but also decreased transepidermal water loss (TEWL) associated with UVB (Supplemental Figure 5, B and C).
We also hypothesized that the inhibition of AhR could attenuate wrinkle formation by suppressing MMP2 induction. At week 13, UVB-irradiated mice exhibited coarser wrinkles in the lower dorsum than did nonirradiated and vitamin-treated mice ( Figure 5, A and B). Through fluorescence IHC of sections cut perpendicularly to the mice's wrinkles, we observed that MMP2 expression localized as bright discrete bands in the epidermis ( Figure 5D). Quantifying the fluorescence intensity of these bands, we found that B 12 -and FA-treated mice featured less intense bands than untreated UVB-irradiated mice ( Figure 5C and Supplemental Figure 5D). Furthermore, the band intensities strongly correlated with wrinkle scores (Supplemental Figure 5E), indicating that these bands of MMP2 expression might be associated with wrinkles. In summary, these data suggest not only that MMP2 may play a critical role in wrinkle formation but also that vitamins B 12 and FA may counteract photoaging by potentially suppressing AhR and MMP2.

Discussion
With the current focus on ROS and MMP1/3/9 in the photoaging field, we demonstrate here that another major matrix metalloproteinase, MMP2 (alongside MMP11), is induced by UVB DNA damage through a distinct mechanism that relies on AhR, SP1, ATM, and p38/JNK MAPK ( Figure 6). In brief, we propose first, that UVB irradiation causes DNA damage that is enhanced by AhR via suppression of NER; second, that ATM kinase senses the resulting DNA damage and subsequently activates p38 and JNK, which can phosphorylate SP1; and third, that activated SP1 binds to the promoter regions of Mmp2 and Mmp11 to induce their transcription. (Mmp9 expression may be coregulated by both this mechanism and AP1.) Furthermore, based on in silico and in vivo studies, we propose that MMP2 may be a relevant driver in photoaging of the skin and that therapies aimed at reducing MMP2 (such as AhR antagonism by vitamin B 12 and FA) can potentially counteract photoaging. What ultimately emerges from our work is a more comprehensive picture of photoaging in which both ROS and DNA damage resulting from UVB activate 2 distinct signaling pathways, leading to the induction of complementary batteries of MMP genes, which target both type I and type III collagen present in the dermis and type IV collagen present at the dermal-epidermal junction. With the inclusion of DNA damage, this new expanded model of photoaging also offers a more obvious link to carcinogenesis, whereby accumulating DNA damage in skin could conceivably lead to both wrinkles (by MMP induction) and cancer (which can later utilize MMPs to invade the underlying basement membrane).
While MMP2 was the primary focus of this paper and arguably the most promising candidate for photoaging given its expression pattern in human skin (Figure 1, A and B), we also found that Mmp11 mRNA behaved similarly to Mmp2 mRNA across all in vitro studies, indicating that there is a larger SP1 transcriptional program that is enacted with UVB. In addition to harboring validated SP1 binding sites, the proximal promoter regions of Mmp2 and Mmp11 were markedly elevated in their GC content, similar to another SP1 target Vegfa but in contrast to Mmp1 and Mmp3. Regardless of whether this elevated GC content is merely the result of, or the means to, accumulating SP1 binding sites, it is compelling that MMP9 appears to have an intermediate GC content between MMP2/11 and MMP1/3. This finding, alongside the general observation that MMP9 clustered with MMP2 and MMP11 in most of our mechanistic studies, suggests that MMP9 could also be regulated by a similar SP1-mediated mechanism, perhaps alongside the currently accepted AP-1-mediated mechanism of induction. Curiously, recent work by Ujfaludi et al. (41) found that MMP9 did not cluster with MMP1 and MMP3 expression after UVB irradiation in a novel keratinocyte cell line HKerE6SFM, raising the possibility that AP-1 may not necessarily be the primary mode of regulation for Mmp9 mRNA. Future work could further elucidate the role of SP1 and DNA damage in driving MMP9 induction.
We also learned from our studies that both natural and synthetic AhR inhibition attenuated double-stranded DNA damage resulting from 200 mJ/cm 2 UVB irradiation ( Figure 4C). Interestingly, it was reported earlier that, while AhR inhibition restores NER and clearance of CPDs, that same AhR inhibition actually introduces more DSBs in an NER-independent manner when keratinocytes are irradiated with 20 mJ/cm 2 UVB (33), a significantly smaller dose than that used in the present study. Juxtaposed with our own experiments, then, we find it plausible that AhR inhibition may have opposing effects on DSBs -one mediated indirectly through enhanced CPD clearance, as suggested by work in NER-deficient XPB cells (42), and another that more directly promotes DSB, possibly via CHK1 inhibition (43). We further propose that these 2 effects are differentially highlighted depending on the UVB dose. At very low doses of UVB (when CPD burden is more modest, perhaps), the NER-restoring benefits of AhR antagonists may be limited, allowing the direct DSB-promoting effects to dominate. With higher doses of UVB (and many more CPDs that could progress to DSBs), however, the NER-restoring benefits of AhR inhibition may become more apparent and outweigh the direct DSB-promoting effects, leading to a net decrease in DSBs and H2AX phosphorylation. Consistent with this complex relationship between AhR and DSBs, we unexpectedly observed that the genetic knockdown of AhR and ARNT had a more modest suppression of Mmp2 and Mmp11 expression ( Figure 2D and Supplemental Figure 2D) compared with small molecule inhibitors (Figure 2A and Supplemental Figure 2A), despite decreasing AhR activity substantially below baseline levels (Supplemental Figure 2E). Collectively, these results suggest that there is likely some intermediate level of AhR activity that optimally minimizes the net production of double-stranded DNA damage from UVB and the subsequent expression of MMP2/11. Finally, our in vivo work with vitamin B 12 and FA offer preliminary evidence that AhR antagonism could prove a useful clinical strategy in ameliorating some harmful effects of UV irradiation. We first observed that vitamin-treated mice exhibited less severe sunburns (Supplemental Figure 5, A-C), which is consistent with our model that AhR antagonism decreases UVB-induced DNA damage ( Figure 4E). We also observed that the vitamin treatments improved the appearance of wrinkles and concomitantly decreased MMP2 expression in the epidermis. This finding could lend insight into how various natural extracts or even prenatal vitamins, which contain high levels of B 12 or FA, might exert their long-purported (albeit often anecdotal) "anti-aging" effects. Additionally, given that vitamin B 12 and FA are sensitive to photolysis (44,45), topical delivery immediately after UV exposure (as in our mouse experiments) may prove a particularly effective strategy in directly repleting cutaneous stores of these vitamins and counteracting induction of MMPs.
Curiously, we observed that epidermal MMP2 was expressed in an almost binary manner, with sporadic "bands" (spanning all layers of the epidermis) in an "ON" state and the vast majority of cells in an "OFF" state ( Figure 5D). While the epidermal expression of MMP2 has been described before in human and mouse skin (46)(47)(48), this particular "banding" pattern has not been previously described, to our knowledge. Especially considering that these IHC sections were cut perpendicularly to the wrinkles, the discrete, sporadic nature of these MMP2 bands and close association with wrinkle scores (Supplemental Figure 5E) provide strong correlative evidence that these bands could somehow spatially mirror the wrinkles themselves. Given prior work demonstrating that MMP2 is enzymatically active at the dermal-epidermal junction in UVB-induced wrinkles and degrading type IV collagen (11), we find it plausible that these MMP2 bands could represent longitudinal "fault lines" of sorts -with underlying compromise in structural integrity that could cause the skin to buckle upon mechanical shear. To be clear, this model of epidermal MMP2 forming wrinkles by targeting type IV collagen in the basement membrane is not mutually exclusive to the current model of UVA inducing MMPs in the dermis causing degradation of type I collagen. Rather, we see the 2 models as potentially synergistic with one another, degrading collagen at different layers of the skin and leading to the multifaceted changes characteristic of photoaging.
In conclusion, our data highlight MMP2 as a potential driver of UV-mediated photoaging, expanding the scope of the field beyond just ROS or MMP1/3/9. Implicating UVB-induced DNA damage, our proposed model not only complements the current model of photoaging that has heavily focused on ROS production but also provides exciting new avenues for rational drug design in the treatment of photoaging ( Figure 6).
GTEx analysis. Transcripts per million (TPM) data from GTEx v8 were downloaded from https://gtexportal.org. TPM counts for genes of interest were extracted using R and transformed into log 10 (TPM+1) values for further analysis. Expression analysis was performed with Microsoft Excel, GraphPad Prism, and R. The following packages were used in R: ggplot2, ggpubr, dplyr, ComplexHeatmap, CePa, corrplot, and RColorBrewer. For promoter GC content analysis, promoter regions of MMP genes were downloaded from the NCBI website and were analyzed by R.
In vitro experiments. Cells were grown at 37°C incubator at 5% CO 2 in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. For all in vitro experiments, cells were plated with supplemented media and grown to 80% confluence. Cells were then serum-starved in MEM supplemented only with 1% penicillin-streptomycin for 48 hours. For siRNA knockdown experiments, siRNA was transfected with Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778075) at the start of serum starvation. For luciferase experiments, luciferase constructs were transfected using Lipofectamine 3000 (Thermo Fisher Scientific, L3000008) 24 hours after start of serum starvation. Prior to irradiation, serum-starved media JCI Insight 2022;7(9):e156344 https://doi.org/10.1172/jci.insight.156344 were aspirated and wells were washed twice in prewarmed PBS. In a small volume of PBS, cells were then irradiated in ventilated cabinets equipped with UVB lamps at the indicated dose. Afterward, PBS was replaced with MEM supplemented with 10% FBS and 1% penicillin-streptomycin, and cells were returned to 37°C for 24 hours. Cells were then lysed for downstream analyses. All cellular experiments were repeated with at least 3 biological replicates.
In vivo experiments. Male 16-week-old SKH1 hairless mice (Charles River Laboratories) were irradiated 3 times a week in the aforementioned UVB cabinets, as described previously by Inomata et al. with slight modifications (11). Briefly, mice received 36 mJ/cm 2 in the first week and were increased to 54, 72, 108, 144, 162, 180, and 198 mJ/cm 2 at 1-week intervals. Mice were given 216 mJ/cm 2 UVB at weeks 9 and 10 and 180 mJ/cm 2 for all subsequent weeks. Immediately after each irradiation, mice were topically treated with 400 μL of corn oil, 250 μg/mL B 12 , or 100 μg/mL FA (dissolved in corn oil) across the entire dorsal surface. At week 10, mice were photographed and evaluated for sunburns. Erythema and TEWL were also quantified by DermaLab skin probes (Cortex Technologies). At week 13, mice were photographed and evaluated for wrinkles. Blinded investigators graded photographs of wrinkles on the lower dorsum of mice using the scale previously described by Inomata et al. (11). All mice were maintained under specific pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility at Johns Hopkins University and were housed according to procedures described in the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).
IHC. Mice were anesthetized 48 hours after the last irradiation at week 16. Formalin-fixed, paraffin-embedded sections were obtained from the lower dorsum of mice, oriented perpendicularly to the wrinkles. After deparaffinization using Trilogy buffer (Cell Marque), slides were washed 3 times with PBS and blocked with 10% normal goat serum diluted in PBS with 0.05% Tween 20 (PBST) for 1 hour. Samples were then incubated with primary antibody diluted in PBST (anti-MMP2, 1:200) overnight at 4°C. Following 3 washes with PBST on an orbital shaker, samples were incubated with a secondary antibody (goat anti-mouse-A488; diluted 1:1000 in PBST) for 1 hour at room temperature on an orbital shaker, protected from light. After 3 more washes with PBST, samples were stained with DAPI (diluted 1:1000 in PBS) for 2 minutes. Samples were quickly washed 3 times in PBS, and glass cover slips were mounted using ProLong Gold Antifade Mountant (Thermo Fisher Scientific). After 48 hours of drying while protected from light, slides were imaged using a fluorescence microscope at 200× original magnification (Leica, DFC365FX).