Decrease of laminin-511 in the basement membrane due to photoaging reduces epidermal stem/progenitor cells

Daily sunlight exposure damages the epidermal basement membrane (BM) and disrupts epidermal homeostasis. Inter-follicular epidermal stem cells (IFE-SCs) regulate epidermal proliferation and differentiation, which supports epidermal homeostasis. Here, we examine how photoaging affects the function of IFE-SCs and we identify key components in their cellular environment (niche). We found that sun-exposed skin showed a decrease of MCSP-positive and β1-integrin-positive cells concomitantly with a decrease of laminin-511 at the dermal–epidermal junction (DEJ), as compared with sun-protected skin. Higher levels of laminin-511 were associated with not only increased efficiency of colony formation, but also higher expression levels of MCSP as well as other stem cell markers such as Lrig1, ITGB1, CD44, CD46, DLL1, and K15 in keratinocytes from skin of 12- to 62-year-old subjects. UVB exposure to cultured human skin impaired laminin-511 integrity at the dermal–epidermal junction and reduced MCSP-positive basal epidermal cells as well as K15-positive cells. Combined treatment with matrix metalloproteinase and heparanase inhibitors protected the integrity of laminin-511 and inhibited the reduction of MCSP-positive cells and K15-positive cells. These results suggest that photoaging may reduce the levels of MCSP-positive and K15-positive epidermal stem/progenitor cells in the epidermis via loss of laminin-511 at the dermal–epidermal junction.


Results
McSp-positive inter-follicular epidermal stem/progenitor cells were reduced with aging in sun-exposed human skin, compared with sun-protected skin. In human skin IFE-SCs are known to highly express MCSP 8 , β1 integrin 4,5 and keratin-15 and to be reduced with aging 12 . To investigate the agedependent changes of MCSP-positive cells, we performed immunofluorescence staining of MCSP in sun-exposed and sun-protected skin samples. Age-dependent reduction of MCSP-positive cells was more pronounced in sun-exposed skin than in sun-protected skin ( Fig. 1A-E). In addition, the gene expression level of MCSP in cultured keratinocytes from sun-protected skin of various ages decreased with aging ( Fig. 1F). Similarly, the signal intensity of β1 integrin decreased earlier in sun-exposed skin than in sun-protected skin as compared with β4 integrin's intensity ( Fig. 2A-J). The gene expression level of Itgb1 decreased with aging whereas Itgb4 showed no change with aging (Fig. 2K, L). Furthermore, keratin-15-positive cells also decreased in number in sun-exposed skin ( Fig.S1A-E), and the gene expression level of K15 was reduced with aging ( Fig.S1F), although the location of K15-positive cells might be different from that of MCSP-positive cells. Thus, our findings indicate that the aging-related decrease of IFE-SCs occurs earlier in sun-exposed skin than in sun-protected skin.
Laminin-511 was reduced age-dependently in sun-exposed skin. In the epidermis, undifferentiated stem/progenitor cells residing at the basal layer bind to the basement membrane via integrin-laminin interaction. We next investigated the integrin-laminin interaction of IFE-SCs by immunofluorescence staining of laminin α5 and laminin α3. We found that laminin α5 was clearly reduced in sun-exposed skin (Fig. 3A-E), whereas laminin α3 was not ( Fig. 3F-J). We examined the gene expression levels of LAMA5, LAMA3, LAMB3 and LAMB1 in cultured keratinocytes from sun-protected skin in subjects of various ages and compared them with the histological data. Consistent with the age-dependent reduction of laminin α5, we found that the expression levels of LAMA5 and LAMB1 were significantly reduced with aging ( Fig. 3K-L). However, the expression levels of LAMA3 and LAMB3 did not change with aging (Fig. 3M, N). Thus, our findings indicate that laminin-511 was decreased more in sun-exposed skin compared to laminin-332. In addition, the gene expression levels of LAMA5 and LAMB1 were unchanged whereas the gene expression of MMP-9 was increased in UVB-irradiated cultured keratinocytes ( Supplementary Fig. S2), suggesting that UVB irradiation might lead to the degradation of laminin-511. Scientific RepoRtS | (2020) 10:12592 | https://doi.org/10.1038/s41598-020-69558-y www.nature.com/scientificreports/ Epidermal stem cell population was maintained by laminin-511. Since decreased levels of laminin-511 and α6β1 integrin during aging in sun-exposed skin were associated with the reduction of MCSPpositive epidermal stem cells, we examined whether laminin-511 could reduce the loss of inter-follicular epidermal stem/progenitor cells by means of a colony formation assay on laminin-511-coated plates. Human keratinocytes from several aged donors showed higher colony numbers and greater colony sizes on the coated plates, compared with the controls (Fig. 4A-C). In addition, MCSP-positive cell populations (%) increased on plates coated with laminin-511 or iMatrix-511, which consists of E8 fragments of recombinant laminin-511 ( Fig. 5A-D). Moreover, when human keratinocytes from donors of several ages were grown on laminin-511-coated plates, the mRNA level of MCSP increased (Fig. 5E). Laminin-511 and iMatrix-511 were better matrix proteins than type I collagen and non-coated controls in neonatal keratinocytes (Fig. 5F). The mRNA levels of other stem cell markers, Lrig1, ITGB1, CD44, CD46, DLL1, and K15 were also increased in neonatal keratinocytes cultured on type I collagen, laminin-511, or iMatrix-511, as compared to non-coated controls (Fig. S3). In addition, the mRNA levels of these stem cell markers MCSP, Lrig1, CD46, CD44, and DLL1 were increased on iMatrix-511 and were higher in keratinocytes from a 22-year-old donor than a 54-year-old donor ( Supplementary Fig. S4).
These results indicate that laminin-511 may play an important role in supporting epidermal stem/progenitor cell populations.
Blocking loss of laminin-511 in UVB-exposed and UVB-unexposed organotypic human skin maintained McSp-positive cell levels. We previously reported that UVB exposure induced and activated MMP-9 and heparanase in the epidermis, and damaged the structure of the basement membrane at the DEJ, leading to a reduction of proliferative keratinocytes and barrier disruption 18,19,30 . We also showed that MMP and heparanase inhibitors suppressed the degradation of the basement membrane and maintained proliferative keratinocytes and epidermal barrier function in UVB-exposed organotypic human skin 29,30 . Thus, we next performed ex vivo human skin cultures in the presence of MMP inhibitor CGS27027A, heparanase inhibitor BIPBIPU, and bifunctional inhibitor HEI after UVB exposure, in order to investigate the mechanisms underlying the effect of laminin-511 on epidermal stem/progenitor cells. Histological analysis showed that the UVBinduced decrease of laminin-α5 (Fig. 6A, B) was inhibited in presence of the MMP inhibitor and the heparanase Together, these results indicate that preventing the loss of laminin-511 may be essential for the maintenance of inter-follicular epidermal stem/progenitor cells in UVB-exposed organotypic human skin.

Discussion
Stem cell exhaustion is a well-established characteristic of aging 31,32 , leading to reduced regenerative potential of tissues, including mouse forebrain 33 , bone 34 , and muscle fibers 35 . In skin, epidermal stem cells are also reduced with aging in the bulge region 36 , and inter-follicular epidermis 12,13 . Our findings here show that MCSP-and K15-positive epidermal cells are reduced with aging at an earlier stage in sun-exposed facial skin, as compared with sun-protected skin. IFE-SCs are known to generate daughter cells continuously to maintain the rete ridge height 12 , and our findings are consistent with a previous report showing that rete ridge height is more markedly reduced with aging in photoaged skin than in sun-protected skin 12,17,[37][38][39][40] .
Our findings also show that the reduction of MCSP-positive epidermal stem cells during aging was associated with a decreased level of laminin-511, but not with a change of laminin-332. Laminin-511 at the basement membrane is known to serve to maintain hair follicle stem cells 15 . Recently laminin-511 was reported to be a useful matrix to culture iPS/ES cells and keratinocytes without the need for a feeder layer [41][42][43] . β1 Integrin, a stem cell marker, interacts with laminin-511 at the basement membrane 44 and asymmetric cell division in the epidermis is disrupted in β1 integrin KO mice, but not in β4 integrin KO mice 45 . Moreover, laminin-511 in the embryo and uterus is involved in the progression of implantation 46 . Although laminin-332 is also an important matrix component at the basement membrane, as it interacts with α6β4 integrin in the hemidesmosomes connecting keratinocytes to the underlying dermis 47 , it mainly functions in regulating epidermal differentiation 20 . Therefore, laminin-511 may play a more important role than laminin-332 in maintaining MCSP-positive epidermal stem/progenitor cells.
The location of human quiescent epidermal stem cells within the basal layer is still under debate, and two different hypotheses have been proposed. One is that cells with the highest expression of β1 integrin, MCSP and LRIG1 cluster at the top of the rete ridges and harbor the highest stemness of all basal cells 8,9,40 . The other is that cells with the highest expression of α6 integrin and keratin-15 are located at the bottom of the rete ridges and are relatively quiescent, displaying a higher clonogenic potential than the more proliferative and Age-dependent change of integrins. Immunofluorescence staining of β1 integrin (green) and α6 integrin (red) in 30s sun-protected Caucasian skin (A), 60s sun-protected Caucasian skin (B), 30s sun-exposed Caucasian skin (C) and 60s sun-exposed Caucasian skin (D) [numbers indicate the age range (decade) of the skin donors]. Immunofluorescence staining of β4 integrin (green) and α6 integrin (red) in 30s sun-protected Caucasian skin (F), 60s sun-protected Caucasian skin (G), 30s sun-exposed Caucasian skin (H) and 60s sunexposed Caucasian skin (I). β1 integrin's signal intensity (E) and β4 integrin's signal intensity (J) were analyzed using WINROOF 2013 image analyzing software (Mitani, Fukui, Japan, https ://www.mitan i-visua l.jp/produ cts/ image _analy s_ismea surem ent/winro of/). mRNA expression levels of ITGB1 (K) and ITGB4 (L) were analyzed by qPCR. Data are expressed as mean ± SD from each of 5 donors in I and J. *p < 0.05, **p < 0.01. Bars: 100 μm. We found that the aging-related decline of laminin-511 was greater in sun-exposed facial skin than in sunprotected skin. Laminin-511 was reported to decrease significantly with aging in sun-protected human skin 21 . Laminin-511 is known to be associated with perlecan networks in basement membrane 49 by binding to heparan sulfate proteoglycan 50,51 . We previously reported that heparanase was activated in the epidermis of UVB-exposed skin, leading to degradation of heparan sulfate of perlecan in the basement membrane at the DEJ 29,40,52 . Furthermore, treatment with inhibitors of MMPs and heparinase not only protected the heparan sulfate staining but also blocked the loss of laminin-511 at the DEJ in organotypic human skin 29,30 . In addition, we found that UVB irradiation did not change the gene expression levels of LAMA5 and LAMB1 in cultured keratinocytes (Supplementary Fig. S2). All of these data suggest that laminin-511 in the epidermis may be destabilized with aging via the increased degradation of heparan sulfate chains due to the activation of heparanase.
UV exposure is known to be one of the factors that enhance skin aging. We have been studying the role of the basement membrane in the process of skin aging, especially the photoaging, which occurs in the sun (UV)exposed skin. We reported that basement membranes at the DEJ were damaged and the lamina densa became disrupted and/or multilayered in sun-exposed skin. Recently, Liu et al. reported that the UV-induced degradation of the hemidesmosome component collagen XVII at the basement membrane in the DEJ might lead to hemidesmosome instability and stem cell competition, and basal keratinocytes highly expressing collagen XVII dominate as interfollicular epidermal stem cells in aged epidermis 53 . We found that the combined use of matrix metalloproteinase and heparanase inhibitors protected basement membrane integrity and promoted the stability of collagen XVII localization 54 , and our present results show that this treatment also inhibited the reduction of stem/progenitor cells. Therefore, protection of the basement membrane from damage may be one of the best methods of controlling the skin aging process.

Materials and methods
Subjects. Sun-exposed facial and sun-protected human abdominal skin samples were obtained from Biopredic International, Rennes, France. Helsinki principles were adhered to, and participants gave written, informed consent to provide samples for research. Some sun-exposed facial skin samples were obtained with the approval of the ethics committees at Gunma University and Shiseido. The experimental protocols were also Figure 3. Age-dependent change of laminins. Immunofluorescence staining of laminin-α5 in 30s sun-protected Caucasian skin (A), 60s sun-protected Caucasian skin (B), 30s sun-exposed Caucasian skin (C) and 60s sunexposed Caucasian skin (D) [numbers indicate the age range (decade) of the skin donors].Immunofluorescence staining of laminin-α3 in 30s sun-protected Caucasian skin (F), 60s sun-protected Caucasian skin (G), 30s sun-exposed Caucasian skin (H) and 60s sun-exposed Caucasian skin (I). Laminin α5′s signal intensity (E) and  Supplementary Table S1. Skin samples were fixed in cold acetone (AMeX procedure) and embedded in paraffin for immunohistochemical observation using specific antibodies for Figs. 1, 2, 3 and S1.  Supplementary Table S2) was cut into 1.5 × 1.5 cm pieces, which were cultured in the presence or absence of 0.1 mg/mL HEI, BIPBIPU (10 −5 M) or CGS27023A (10 −5 M) after UVB irradiation (50 mJ/cm 2 ), as described previously 30 . At 5 days after the start of culture, samples were fixed in cold acetone (AMeX procedure) and embedded in paraffin for immunohistochemical staining for Fig. 6 and S5. . Sections were examined with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and images were captured with a DP72 controller digital camera (Olympus). The staining intensity in 5 randomly selected microscopic fields was quantified by using   Quantitative real-time Rt-pcR. Total epidermal RNA from cultured keratinocytes was isolated using a Qiagen Rneasy mini kit (Qiagen) and cDNA was synthesised using a SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). Expression of MCSP, B2M, CK15, LAMA5, LAMB1, LAMA3, LAMB3, CD46, Lrig1, DLL1 and CD44 genes in the epidermis was analyzed by a quantitative PCR method using Platinum SYBR Green qPCR superMix-UDG (Invitrogen Japan, Tokyo, Japan), as described 28  colony formation assay. 3T3 fibroblasts were cultured in DMEM containing 10% FBS, and subcultured with 1:4 split for several passages when they reached 80-90% confluence. The fibroblasts were killed by exposure to 5 µg/ml mitomycin C for 2 h, and then trypsinized and plated at 5 × 10 5 cells/well in 6-well culture plates as a feeder layer for colony formation assay. Human normal epidermal keratinocytes obtained from donors of various ages were pre-cultured in Humedia-KG2 (KURABO, Osaka, Japan) in laminin-511-coated or non-coated flasks. After the cells had reached 80% confluence, 5 × 10 2 keratinocytes/well were seeded into 3T3 fibroblastcultured 6-well plates 57 . Clonal cultures were maintained in Humedia-KG2 medium for 12 days, fixed with 4% paraformaldehyde and stained with 1% crystal violet.