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. 2022 Jan 21;16(3):421–432. doi: 10.1007/s12079-021-00654-y

Reduced expression of Collagen 17A1 in naturally aged, photoaged, and UV-irradiated human skin in vivo: Potential links to epidermal aging

Yaping Xiang 1,#, Yingchun Liu 1,#, Yan Yang 1, Yan Yan 1, Ava J Kim 1, Chunfang Guo 1, Gary J Fisher 1, Taihao Quan 1,✉
PMCID: PMC9411357  PMID: 35060094

Abstract

Collagen 17A1 (COL17A1) is a transmembrane structural component of the hemidesmosome that mediate adhesion of keratinocytes to the underlying membrane. Recent work in mouse showed that COL17A1 deficiency leads to premature skin aging. Although the role COL17A1 in skin aging is becoming recognized in mouse models, its connection to human skin natural aging/photoaging/ultraviolet (UV)-irradiated human skin has received little attention. To determine COL17A1 expression in naturally aged and photoaged as well as acutely UV irradiated human skin, skin samples were obtained from: (1) young (N = 10, 26.7±1.3 years) and aged (N = 10, 84.0 ± 1.7 years) sun-protected buttock skin; (2) photoaged extensor forearm and subject matched sun-protected underarm skin (N = 6, 56.0 ± 3.4 years); (3) solar-simulated UV-irradiated buttock skin (N = 6, 51.2 ± 3.6 years). COL17A1 levels were determined by immunohistology and RT-PCR, and the potential role of COL17A1 in epidermal aging was investigated by immunostaining of the marker for interfollicular epidermal stem cells and keratinocytes proliferation. We found that COL17A1 is specifically expressed in interfollicular epidermal stem cell niches, and that significantly reduced in naturally aged, photoaged, and acute UV-irradiated human skin in vivo. COL17A1 is identified as keratinocyte-specific collagen, and UV irradiation significantly downregulates COL17A1 expression in keratinocytes. Reduced expression of COL17A1 is positively correlated with impaired regeneration of keratinocytes and reduced dermal-epidermal junction as well as thin epidermis in aged human skin (epidermal aging). We also confirmed that keratinocyte-specific integrin β4 (ITGB4), which interacts with COL17A1, is reduced in aged human skin. Mechanistically, we found that matrix metalloproteinases (MMPs) are responsible for UV-mediated COL17A1 degradation in both in vitro keratinocytes and in vivo mouse skin. These data suggest the possible links between reduced expression of COL17A1 and epidermal aging in human skin.

Keywords: COL17A1, Skin natural aging, Photoaging, UV, Keratinocytes

Introduction

COL17A1 is a hemidesmosome structural component of the dermal-epidermal basement membrane (Natsuga et al. 2019), which connects two major layers of skin: the cell-rich epidermis and the collagen-rich dermis. COL17A1, a transmembrane collagen, is distinct from other fibrillar collagens, which are generally secreted into extracellular space. The major function of COL17A1 is to mediate the adhesion of keratinocytes to the underlying basement membrane, and thus plays an important role in maintaining the integrity of the epidermis. Mutations in COL17A1 causes loss of epidermal attachment from the basement membrane and results in blistering skin disease (Condrat et al. 2018; Nishie et al. 2007). Recent publications in mouse work demonstrate that knockout of COL17A1 causes aging of the hair follicle (hair greying and loss) (Matsumura et al. 2016; Tanimura et al. 2011) and epidermal atrophy/thin epidermis (Liu et al. 2019; Watanabe et al. 2017). These phenotypes of skin aging in COL17A1 deficient mice are largely explained by skin stem cells exhaustion caused by stem cell detachment from the basement membrane in the niches. In COL17A1 deficient mice, either hair follicle and epidermal aging can be recapitulated by COL17A1 deficiency and prevented by the forced maintenance of COL17A1, suggesting a critical role of COL17A1 in mouse skin epidermal aging process. As such, the pivotal role of COL17A1 in skin epidermal aging is becoming recognized in mouse model.

Age-related reduction of COL17A1 has been described recently (Langton et al. 2016; Liu et al. 2019; Watanabe et al. 2017). However, the expression of COL17A1 in sunlight-induced photoaging skin and UV-irradiated human skin has received little attention. Here we report that COL17A1 expression is reduced in in naturally aged and photoaged skin as well as acutely UV irradiated skin in vivo. Furthermore, reduced expression of COL17A1 is closely related with epidermal aging characterized by loss of dermal-epidermal junction and epidermal thickness. These data suggest the possible links between reduced expression of COL17A1 and epidermal aging in human skin.

Materials and methods

Procurement of skin specimens and UV irradiation

All human skin punch biopsies (4mm in diameter, full thickness) were obtained from healthy adult volunteers. Sun-protected buttock skin biopsies were obtained from young group (N = 6, mean age 26.7 ± 1.3 years) and aged group (N = 6, mean age 84.0 ± 1.7 years). Photoaged skin and subject matched sun-protected skin (N = 6, 56.0 ± 3.4 years) were taken from the extensor forearm and underarms, respectively. The presence of photodamage was determined based on clinical criteria, as described previously (Griffiths et al. 1992). For UV irradiation, sun-protected buttock skin (N = 6, 51.2 ± 3.6 years) was irradiated with 2 MED(minimum erythema dose) solar-simulated UV (SPEC 450 W xenon arc solar simulator)(Quan et al. 2004). Minimal erythema dose for each patient was determined 24 hours after UV irradiation. UV-irradiated and non-irradiated skin biopsies were obtained at the indicated time points after UV exposure. Ethical approval involving human subjects for this investigation was obtained from the University of Michigan Institutional Review Board and the study was conducted according to the Declaration of Helsinki Principles. All subjects participating in this study provided written informed consent before entering the study.

Immunohistology

Immunohistology was performed as described previously (Quan et al. 2006). Briefly, paraffin-embedded sections were deparaffinized and antigen retrieved by heating at 65°C in target retrieval solution (Dako, Santa Clara, CA) for overnight. The sections were blocked by incubation with PBS containing 0.1% Triton X-100 and 3% skim milk for one hour in room temperature. Subsequently, the slides were incubated for one hour at room temperature with antibodies against COL17A1 (cat#: ab186415, Abcam, Cambridge, MA), CD271, ITGB4 (Abcam, Cambridge, MA) and Kalinin/laminin-5 (Santa Cruz Biotech, CA) and then incubated with biotinylated goat anti-rabbit immunoglobulin G antibody (Vector Laboratories, Burlingame, CA) at room temperature for one hour. After washing, the sections were incubated with avidin-biotin peroxidase complex using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) at room temperature for 30 min. All sections were lightly counterstained with hematoxylin, and the images were obtained using Zeiss Axio Observer Inverted Microscopy.

Cell culture and UV irradiation

Human N/TERT keratinocyte cell line was gifted from J. Rheinwald laboratory (Harvard Medical School, Boston, USA). Cells were cultured in a 6-well plate in keratinocyte-serum free medium (K-SFM, Gibco, Gaithersburg, MD) until 80% confluence. Sub-confluent cells were irradiated with UVB (40 mJ/cm2) using an Ultralite Panelite lamp containing six FS24T12 UVB-HO bulbs (Quan et al. 2004). The UV irradiation intensity was monitored with an IL400A phototherapy radiometer and a SED240/UVB/W photodetector (International Light, Newbury, MA). A Kodacel filter was used in order to remove UVC (wavelengths below 290 nm).

RNA isolation and quantitative real-time RT-PCR

Total RNA was isolated using a commercial kit (RNeasy midi kit, Qiagen, Chatsworth, CA) according to the manufacturer’s protocol. mRNA levels were quantified by real-time RT-PCR, as previously described (Quan et al. 2004). COL17A1, MMPs, and CCN1 PCR primers were purchased from RealTimePrimers.com (https://www.realtimeprimers.com/prsy.html). PCR was performed in duplicate with 2 μl of cDNA s using TaqMan Universal PCR Master Mix kit (Applied Biosystems) and a 7700-sequence detector system (Applied Biosystems, MA). PCR procedures were performed with a robotic workstation (Biomek 2000; Beckman Coulter, Inc., Hialeah, FL) to ensure accuracy and reproducibility. COL17A1 mRNA levels were normalized to the 36B4 (housekeeping gene internal control). COL17A1 and 36B4 PCRprimers were purchased from RealTimePromers.com.

Mice, UV irradiation, and MMPs inhibitor treatment

Female C57Bl6/J mice, 6–8 wks old, were purchased from The Jackson laboratory (Stock No: 000664). Twenty-four hours before UV irradiation, a uniform area of dorsal skin of mice was shaved (8 cm2). For UV irradiation, mouse skin was irradiated with 100 mJ/cm2 UVB/A2 using an Ultralite Panelite lamp containing six FS24T12 UVB/A2B-HO bulbs (47% UVB/A2B, 18% UVB/A2A2, 9% UVB/A2A1, 26% visible light, filtered with kodcel to remove wavelength below 290nm) (Quan et al. 2004). Mice dorsal skin was topically treated with MMP inhibitor, GM6001, (Millipore, Bedford, MA, USA) or vehicle for one hour before UV irradiation. Topical solutions of GM6001 were prepared at concentrations of 40µg/ml in vehicle consisting of 50 mmol/l N-2-hydroxyethylpipcrazine-N'-2-cthane sulfonic acid (HEPES) butter. The UV irradiation was administered once daily for three consecutive days. During irradiation, skin changes such as erythema, scaling, erosion, blisters, and pigmentation, as well as skin elasticity and texture, were monitored closely. Animals were humanely killed, and skin specimens were collected for histology and MMPs activity.

Charts and statistics

Charts were generated with Excel 2010 and Adobe Illustrator. Bar graphs represent Means ± SEM. Statistical analyses were performed using one-way ANOVA with Bonferroni post-hoc multiple comparisons. All p values are two-tailed, and considered significant when <0.05 (depicted by asterisks on figures).

Results

Reduced expression of COL17A1 in naturally aged human skin in vivo

To determine Col17A expression in naturally aged human skin, the skin samples were obtained from young (N = 6, 26.7 ± 1.3 years) and aged (N = 6, 84.0 ± 1.7 years) sun-protected buttock skin. We first confirmed that COL17A1 is abundantly expressed in the basement membrane zone and epidermal basal keratinocytes in adult sun-protected skin (Fig 1A left). Interestingly, COL17A1 is profoundly reduced in aged, compared to young skin (Fig 1A), as reduced expression of COL17A1 is closely related with skin epidermal aging in mouse model of skin aging (Liu et al. 2019). Quantification indicated that COL17A1 is reduced in aged human skin approximately 63% (Fig 1B). COL17A1 mRNA analysis from same tissues resulted in similar pattern of reduction in aged human skin (Fig 1C, reduced approximately 42%). These data indicate that COL17A1 is reduced in naturally aged human skin.

Fig. 1.

Fig. 1

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Reduced expression of COL17A1 in naturally aged human skin in vivo. Full-thickness skin punch biopsies were obtained from young (N = 6, 26.7 ± 1.3 years) and aged (N = 6, 84.0 ± 1.7 years) sun-protected buttock skin. A Reduced expression of COL17A1 protein in aged skin: COL17A1 immunohistology. Boxed regions are shown enlarged in the lower panels. Representative images of six individuals. Arrowheads indicate positive staining area. Bars = 100µm. B Reduced expression of COL17A1 protein in aged skin. COL17A1 protein levels were quantified by Image J software. Mean ± SEM, *p < 0.05, N = 6. C Reduced expression of COL17A1 mRNA in aged skin. COL17A1 mRNA levels were quantified by real-time RT-PCR. mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Mean ± SEM, *p < 0.05, N = 6

Reduced expression of COL17A1 in photoaged human skin in vivo

To determine Col17A expression in photoaged human skin, the skin samples were obtained from sun-exposed forearm and subject matched sun-protected underarm skin (N = 6, 56.0 ± 3.4 years). Consistent with naturally aged human skin, COL17A1 expression is reduced in photoaged forearm human skin, compared to sun-protected underarm skin (Fig 2A). Quantification indicated that COL17A1 is reduced in photoaged forearm human skin by approximately 42% (Fig 2B). COL17A1 mRNA analysis from same tissues resulted in similar pattern of reduction in photoaged forearm human skin (Fig 2C, reduced approximately 41%). These data indicate that COL17A1 is reduced in photoaged human skin.

Fig. 2.

Fig. 2

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Reduced expression of COL17A1 in photoaged human skin in vivo. Full-thickness skin punch biopsies were obtained from sun-exposed forearm and sun-protected underarm skin (N = 6, 56.0 ± 3.4 years). A Reduced expression of COL17A1 protein in photoaged skin: COL17A1 immunohistology. Boxed regions are shown enlarged in the lower panels. Representative images of six individuals. Arrowheads indicate positive staining area. Bars = 100µm. B Reduced expression of COL17A1 protein in photoaged skin. COL17A1 protein levels were quantified by Image J software. Mean ± SEM, *p < 0.05, N = 6 C Reduced expression of COL17A1 mRNA in photoaged skin. COL17A1 mRNA levels were quantified by real-time RT-PCR. mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Mean ± SEM, *p < 0.05, N = 6

Reduced expression of COL17A1 in acute UV-irradiated human skin in vivo

Next, we investigated the effect of acute ultraviolet (UV) irradiation on COL17A1 expression in human skin in vivo. Skin from sun-protected buttock skin (N = 6, 51.2 ± 3.6 years) was irradiated 2 minimal erythema dose (MED) solar-simulated UV and the tissues were collected 24 hours post UV irradiation. COL17A1 is reduced by UV irradiation compared to non-irradiated skin (Fig 3A). Quantification indicated that COL17A1 is reduced by UV irradiation by approximately 38% (Fig 3B). COL17A1 mRNA analysis from same tissues resulted in similar pattern of reduction in UV-irradiated human skin (Fig 3C, reduced approximately 37%). These data indicate that COL17A1 is reduced by acute UV irradiation in human skin.

Fig. 3.

Fig. 3

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Reduced expression of COL17A1 in photoaged human skin in vivo. Skin from sun-protected buttock skin (N = 6, 51.2 ± 3.6 years) was irradiated 2 minimal erythema dose (MED) solar-simulated UV, and the tissues from control (CTRL-No UV) and UV-irradiated skin were collected 24 hours post UV irradiation. A Reduced expression of COL17A1 protein in UV-irradiated skin: COL17A1 immunohistology. Boxed regions are shown enlarged in the lower panels. Representative images of six individuals. Arrowheads indicate positive staining area. Bars = 100µm. B Reduced expression of COL17A1 protein in UV-irradiated skin. COL17A1 protein levels were quantified by Image J software. Mean ± SEM, *p < 0.05, N = 6 C Reduced expression of COL17A1 mRNA in UV-irradiated skin. COL17A1 mRNA levels were quantified by real-time RT-PCR. mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification. Mean ± SEM, *p < 0.05, N = 6

COL17A1, a keratinocyte-specific collagen, is reduced by UV irradiation, and elevated MMPs are responsible for COL17A1 degradation

Next, we explored the cellular expression of COL17A1. We analyzed the most widely expressed fifteen collagens in human skin ref including COL17A1 in two major cell types (McCabe MC, 2020), epidermal keratinocytes and dermal fibroblasts. Interestingly, we found that COL17A1 is exclusively expressed in keratinocytes and not in fibroblasts (Fig 4A), indicating that COL17A1 is a keratinocyte-specific collagen. Next, we investigated the effect of UV irradiation on COL17A1 expression in keratinocytes. We found that UV irradiation time-dependently reduced COL17A1 expression in both mRNA (Fig 4B) and protein (Fig 4C). UV irradiation reduced COL17A1 mRNA levels by 75% (Fig 4B) and protein levels by 82% (Fig 4C) after 24 hours of UV irradiation. Next, we investigated the potential mechanisms, in which UV irradiation reduces COL17A1 expression. The family of mammalian matrix metalloproteinases (MMPs) is significantly upregulated in naturally aged, photoaged, and UV-irradiated human skin (Fisher et al. 2009; Qin et al. 2017; Quan et al. 2009), and primarily responsible for degradation of the collagen-rich ECM. We examined whether inhibition of proteolytic activity by MMP inhibitor can prevent COL17A1 downregulation by UV irradiation. To test this possibility, we first confirmed that keratinocytes exposed to UV radiation induced multiple MMPs (Fig 4D), as observed in UV-irradiated human skin in vivo (Quan et al. 2009). We also confirmed UV-induced CCN1 since CCN1 is potently induced by UV irradiation and that mediates elevation of MMP-1 in human skin (Quan and Fisher 2015; Quan et al. 2006; Quan et al. 2010). Next, we confirmed that MMP inhibitor GM6001 significantly reduced MMP activity induced by UV irradiation in keratinocytes (Fig. 4E). Importantly, suppression of proteolytic activity by pre-treatment of cells with MMP inhibitor GM6001 significantly reversed UV-mediated loss of COL17A1 expression (Fig. 4F). We further confirmed above result in mouse skin; UV irradiation markedly reduced COL17A1 expression, and that was prevented by MMP inhibitor GM6001 (Fig. 4G). Together, these data demonstrate that COL17A1, a keratinocyte-specific protein, is reduced by UV irradiation, and MMPs are, at least in part, responsible for UV-induced COL17A1 degradation.

Fig. 4.

Fig. 4

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Keratinocyte-specific expression of COL17A1 is reduced by UV irradiation and that is prevented by MMPs inhibitor. A keratinocyte-specific expression of COL17A1. Collagen mRNA expression levels in keratinocytes and fibroblasts were determined by real-time RT-PCR. B, C Reduced expression of COL17A1 mRNA and protein in UV-irradiated keratinocytes. Keratinocytes were irradiated with UV (40mJ/cm2) and harvested at indicated times. COL17A1 mRNA levels were quantified by real-time RT-PCR and protein levels were determined by immunostaining and quantified by Image J software. D Elevated multiple MMPs and CCN1 in UV-irradiated keratinocytes. Keratinocytes were irradiated with UV (40mJ/cm2) and harvested twenty-four hours post UV irradiation. MMPs mRNA expression levels were determined by real-time RT-PCR. All mRNA levels were normalized to the housekeeping gene 36B4, as an internal control for quantification (B, D). (E, F) keratinocytes were pretreated with MMPs inhibitor GM6001 (4µg/ml) for one hour followed by UV irradiation (40mJ/cm2) for twenty-four hours. E MMP activity was determined by MMP Activity Assay Kit. F COL17A1 protein expression levels were determined by immunostaining and quantified by Image J software. G Mouse dorsal skin was topically treated with GM6001 (80µl of 40µg/ml) or vehicle for one hour before UV irradiation (100mJ/cm2 UVB/A2). The UV irradiation was administered once daily for three consecutive days. COL17A1 protein expression levels were determined by immunohistology and quantified by Image J software. Mean ± SEM, *p < 0.05, N = 4. Bars 100µm

Potential role of COL17A1 expression in interfollicular epidermal (IFE) stem cells homeostasis in human skin

COL17A1 has been reported to serve as a niche for IFE stem cells and the loss of COL17A1 is responsible for the loss of IFE stem cells in mouse model of skin aging (Liu et al. 2019; Matsumura et al. 2016). This information let us explore the potential links between reduced expression COLA17A1 and age-related loss of IFE stem cells in human skin. Interestingly, within the basal layer, COL17A1 is predominantly expressed at the top ret ridge regions (indicated by arrowheads in Figs. 1, 2 and 3), in which IFE stem cells often reside (Akamatsu et al. 2016; Jensen and Watt 2006; Truzzi et al. 2015; Yamada et al. 2018). This data prompt us to investigate the possible links between COL17A1 expression and IFE stem cells homeostasis. We first stained young and aged skin with CD271, a marker of IFE stem cells (Fig. 5A)(Akamatsu et al. 2016; Truzzi et al. 2015; Yamada et al. 2018). Like Col17A expression (Fig 1 arrowheads), CD271 is predominantly expressed in the uppermost projections of the ret ridge regions in young human skin (Fig.5A left arrowheads), and that is significantly reduced in aged human skin (Fig.5A right, reduced 47%). Similarly, acute UV irradiation reduced CD271 expression in human skin (Fig.5B right, reduced 58%). These data suggest that age-related reduction of COL17A1 may destabilize IFE stem cell maintenance, which may reduce IFE stem cell abundance or self-renewal ability, a feature of aging epidermis (Giangreco et al. 2010). Consistent with these data, we observed that significant reduction of proliferating basal keratinocytes in aged human skin, (Fig 5C, reduced 54%), compared to young human skin, suggesting diminished regeneration rate of keratinocytes. We further assessed two most prominent features of human skin epidermal aging (Lavker et al. 1989; Lock-Andersen et al. 1997; Lorencini et al. 2014), flattened rete ridges/dermal and epidermal junction (Fig. 5E) and thinner epidermis (Fig. 5F), by the immunostaining of basement membrane proteins Kalinin/Laminin-5 (Fig.5D). Quantification indicated that in aged human skin both rete ridges/dermal and epidermal junction (Fig.5E) and epidermal thickness (Fig. 5F) is reduced by 54% and 41%, respectively, compared to young human skin. Finally, we assessed basal keratinocyte-specific integrinβ4 (ITGB4), which interacts with COL17A1 and is necessary for the stable attachment of hemidesmosomes to keratin intermediate filaments (Aumailley et al. 2006; Borradori and Sonnenberg 1999). ITGB4 was detected in a linear distribution at the basement membrane (Fig. 5G). Interestingly, a significant reduction in the intensity of immunostaining across the dermal epidermal junction was identified in the aged skin, compared to the young skin (Fig.5G right aged skin). Together, these data suggest the possibility that reduced expression of COL17A1 and ITGB4 in aged human skin leads to loss of IFE stem cells, which in turn contributes to epidermal aging.

Fig. 5.

Fig. 5

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Full-thickness skin punch biopsies were obtained from young (N = 6, 26.7 ± 1.3 years) and aged (N = 6, 84.0 ± 1.7 years) sun-protected buttock skin and acute UV-irradiated human skin (2 MED solar-simulated UV). A Reduced expression of CD271 protein in aged skin: CD271 immunohistology. B Reduced expression of CD271 protein in UV-irradiated human skin: CD271 immunohistology. C Reduced expression of Ki67 positive cells in aged skin. D Dermal-epidermal junction immunostaining by basement membrane proteins Kalinin/Laminin-5. E Reduced dermal epidermal junction. F Reduced epidermal thickness in aged skin. G ITGB4 was  detected at the basement membrane, and that was reduced in aged skin. CD271 (A, B), Ki67 (C), dermal-epidermal junction (D, E), epidermal thickness (F), and ITGB4 (G) were quantified by Image J software. Mean ± SEM, *p < 0.05, N = 6. Representative images of six individuals. Mean ± SEM, *p < 0.05, N = 6. Bars = 100µm. H A proposed model of epidermal aging. COL17A1 is predominantly expressed in uppermost projections of the ret ridge regions (left, young skin), which serves as a niche for epidermal stem cells. As the epidermis ages, it undergoes a series of structural modifications. COL17A1 is degraded by proteolysis during skin aging process and environmental insults, such as UV irradiation, by multiple proteases including matrix metalloproteases (MMPs). Reduced expression of COL17A1 in epidermal stem cell niches weakens stem cells adhesion from the basement membrane, which results in loss of a stem cell population. Loss of stem cells results in flattening of the ret ridge and thinning of the epidermis (right, aged), the characteristic features of aging epidermis (see more details for Discussion). Deep yellow lines indicate COL17A1, and green cells indicate IFE stem cells

Discussion

Here we report reduced expression of COL17A1 in naturally aged, photoaged, and acute UV-irradiated human skin in vivo. Reduced expression of COL17A1 may contribute to epidermal aging, which is characterized by loss of the finger-like rete ridges and thinning of the epidermis (Fig.5F) (Lavker et al. 1989; Lock-Andersen et al. 1997; Lorencini et al. 2014). COL17A1 expression pattern in ret ridge specific region is of notable interest for skin epidermal aging. It is important to note that COL17A1 is more abundant at the top ret ridges (uppermost projections of the rete ridges) in normal skin (arrowheads in Figs. 1, 2 and 3), and significantly reduced in naturally aged (Fig. 1), photoaged (Fig. 2), and acute UV-irradiated (Fig.3) human skin. In normal healthy skin the basal layer is not flat but undulates. Rete ridges are the epithelial extensions that project into the underlying dermis. Rete ridges are common structure of human skin and provides skin with both structural integrity and mechanical stability (Wu et al. 2013). Importantly, IFE stem cell niches often reside uppermost projections of rete ridges (Akamatsu et al. 2016; Jensen and Watt 2006; Truzzi et al. 2015; Yamada et al. 2018). Expression of COL17A1 in the uppermost projections of the rete ridges region might have an important implication in maintenance of IFE stem cells, as COL17A1 serves as a niche for IFE stem cells (Akamatsu et al. 2016; Liu et al. 2019; Matsumura et al. 2016). The functional implication of the reduced deposition of COL17A1 at ret ridge-specific region would be expected to weaken stem cells adhesion from the niches and eventually are eliminated from the skin surface through terminal epidermal differentiation (Liu et al. 2019). Consequently, loss of COL17A1 leads to lower keratinocyte renewal rates and diminished epidermal layers, the major morphological characteristic of the aging epidermis. As such, reduced expression of COL17A1 in epidermal stem cell niches (uppermost ret ridge region) could impose a significant impact to epidermal aging (Fig. 5F). Our human skin in vivo data are consistent with previous mouse work that showed reduced expression of COL17A1 , due to progressive depletion of stem cell population, which is a causative factor of aging of the hair follicle (Matsumura et al. 2016) and epidermal atrophy (Liu et al. 2019). As such, it has been postulated that the maintenance of both IFE and hairfollicle stem cells is dependent on the integrity COL17A1 around cells.

While  skin epidermis is continually replenished by keratinocytes, age-related decrease in stem cells cause an insufficient generation of keratinocytes, which results in flattening of the ret ridges and thinning of the epidermis, the characteristic features of aging epidermis (Fig. 5) (Lavker et al. 1989; Lock-Andersen et al. 1997; Lorencini et al. 2014). Flattening of the rete ridges is thought to diminish the exchange surface between epidermis and dermis, and also reduces epidermal resistance to shearing forces and thereby makes the epidermis more fragile (Lavker et al. 1989). As such, loss of epidermal thickness and flattening of the rete ridges could contribute substantially to increased skin fragility and impaired barrier function in the aged population. It is of note that due to the structure of mouse epidermis neither rete ridges nor thinning of the epidermis are present in mouse skin, and therefore cannot be assessed. As such, mouse skin might not be suitable model to study epidermal aging.

Basal keratinocytes attach to the underlying dermal stroma through complex basement membrane zone. Kalinin/Laminin-5 is necessary for the attachment of basal keratinocytes to the basement membrane by interacting with the hemidesmosome to the underlying tissue. Kalinin/Laminin-5 immunostaining indicate that overall dermal epidermal junction seems to remain unchanged in aged skin compared to young skin (Fig. 5D). However, hemidesmosomes, such as COL17A1 and ITGB4, are reduced in aged human skin. The functional implications of age-related loss of hemidesmosomal proteins would be expected to weaken epidermal adhesion to dermis and protective mechanical function between the epidermis and dermis. As such, age-related loss of hemidesmosomal proteins contributes to skin conditions in the elderly, such as less resistance to shearing force and more vulnerable to environmental injury.

Our data are consistent with previous work that reduced expression of COL17A1 in aged human skin (Langton et al. 2016; Liu et al. 2019; Watanabe et al. 2017). COL17A1 is also reduced in photoaged and acute UV-irradiated human skin, suggesting that reduced expression of COL17A1 may contribute to both intrinsic natural skin aging and sunlight-induced extrinsic photoaging. COL17A1 is reduced in natural skin aging, photoaging, and UV-irradiated human skin at mRNA levels, suggesting that COL17A1 is regulated at transcriptional level. While little is known about COL17A1 transcriptional regulation, recent works suggest that YAP (Otsubo et al. 2017), a key transcription co-activator, and p53 (Yodsurang et al. 2017), a well-known tumor suppressor gene, directly binds to COL17A1 promoter to regulate COL17A1 gene expression (Natsuga et al. 2019). In mouse model of skin aging, COL17A1 is preferentially degraded by proteolysis during skin aging process by several different proteases including matrix metalloproteases (MMPs) (Matsumura et al. 2016). We previously reported elevated multiple MMPs are common features in naturally aged (Fisher et al. 2009; Qin et al. 2017) and photoaged (Quan et al. 2013) as well as UV-irradiated human skin (Quan et al. 2013; Quan et al. 2009). In UV-irradiated human skin in vivo, MMP-1, MMP-3, and MMP-9 are most potently induced (Quan et al. 2009), as observed in UV-irradiated keratinocytes (Fig 4D). We demonstrate that GM6001, a potent and broad spectrum MMPs inhibitor, significantly prevents UV-induced COL17A1 downregulation in both keratinocytes and mouse skin. These data suggest that age-related elevation of MMPs (MMP-1, MMP-3, and MMP-3) likely, at least in part, contribute to reduced deposition of COL17A1 by stimulating proteolytic degradation. We also previously reported that CCN1 is elevated in naturally aged and photoaged as well as UV-irradiated human skin and that plays an important role in upregulation of MMPs (Quan and Fisher 2015; Quan et al. 2006; Quan et al. 2010). These data suggest that CCN1 might be involved in COL17A1 degradation by induction of MMPs in human skin. Obviously, more work needs to be done to understand the precise mechanism underling reduced expression of COL17A1 in aged and UV-irradiated human skin in both transcriptional and post-transcriptional levels. Such effort will help us develop novel anti-epidermal aging technologies.

In summary, here we report reduced COL17A1 expression in naturally aged, photoaged, and UV-irradiated human skin, which raises the possibility that reduced COL17A1 expression contributes to epidermal aging in human skin.

Funding

None

Declarations

Conflict of interest

The authors state no conflicts of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yaping Xiang and Yngchun Liu have contributed equally to this work.

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