Perlecan expression influences the keratin 15‐positive cell population fate in the epidermis of aging skin

Perlecan expression in epidermal and capillary BMs in skin aging

We performed an immunohistochemical analysis of perlecan in a cohort of 38 human skin samples from donors ranging in age from 22 to 73 years. We detected perlecan expression with a monoclonal antibody (mAb) against perlecan domain III (Figure 1a). An analysis of the biopsies from donors that were 22 to 35 years old revealed continuous staining at the DEJ and around dermal capillaries (Figure 1b and c), consistent with previous studies [16,17]. Perlecan staining began to decrease in biopsies from donors aged 39 to 50 years. Perlecan staining again decreased in both intensity and area in biopsies from donors aged 54 to 70 years (Figure 1b and d). This was also observed in the capillary BMs (Figure 1c and e). An analysis of perlecan domains I, IV, and V revealed that all domains were expressed along the DEJ and around dermal capillaries, similar to the domain III expression pattern (Figure 1f). In aged skin, both the DEJ and dermal capillary BM showed reduced staining of each domain; this result suggested that the entire perlecan molecule was subject to expression changes over time.

To characterize the perlecan expression pattern in cultured keratinocytes, we first examined its localization in the ECM of young keratinocyte cultures (Figure 2a). When the anti-perlecan mAb was applied to confluent cells, the protein appeared to be regularly distributed over the entire support, which suggested that perlecan was present in the underlying ECM. At the individual cell level, we observed that the substrate immediately adjacent to the cells was fluorescently stained in regularly aligned patches, resembling adhesion contacts. Higher magnification revealed that the ends of actin cables often colocalized with perlecan, which suggested that perlecan may be involved in keratinocyte adhesion. Moreover, immunostaining of β1-integrin subunits revealed that these molecules often co-localized with perlecan. Although a direct interaction remains to be demonstrated, this finding indicated that an integrin that included a β1 subunit might be involved in keratinocyte adhesion to perlecan. In comparison, an analysis of aged keratinocytes revealed similar, though much weaker, perlecan staining. Moreover, at the point of perlecan co-localization with actin, the β1-integrin subunit appeared as a faint punctuation. These results showed that perlecan expressed in keratinocyte ECM was localized in close vicinity to the cell membrane. This location was reminiscent of the previously described “cellular perlecan”. Our results also suggested that this interaction weakened with aging.

Figure 2. Keratinocyte aging results in decreased perlecan in the ECM. (a) NHKs from young or aged donors were cultured, fixed, and stained with either polyclonal antibodies (pAb) against perlecan (red), phalloidin‐FITC (green) or monoclonal antibodies (mAb) against Δ1‐integrin (green), as indicated. The merged images show the juxtaposition of perlecan and β1‐integrin staining (arrows). Optical slices (0.8 μm) were acquired at the cell‐matrix interface. Scale bar = 50 μm. (b) Perlecan immunoblotting in the ECM. Ages are indicated, and the asterisk shows perlecan. (c) Perlecan quantification. NHKs (5 × 10³) from donors aged 22, 24, 26, 27, and 32 years or 52, 55, 56, 61, and 64 years were seeded in 96‐well plates, and perlecan immunodetection was performed over 4 days. Six assay points were performed per experiment. (d) Results from QPCR of NHK cDNA show perlecan gene transcription in 12 NHK strains from donors aged 24 to 64 years, as indicated. Each assay point was performed in triplicate (c, d). Data are presented as mean ± SD of 3 independent experiments conducted with keratinocytes from each individual donor.

A western blot analysis of perlecan in the ECM of confluent cells from young and aged donors revealed a band of 800 kDa in all samples, which corresponded to the full-length perlecan with intact heparan sulfate (HS) chains (Figure 2b). Because the intensity appeared to slightly decrease with age, we quantified perlecan in 5 young and 5 aged keratinocyte strains (Figure 2c) as a function of cell density to circumvent the bias caused by differences in cell proliferation rates. To that end, we quantified perlecan expression in the ECM of normal human keratinocytes (NHKs) over 4 days with concomitant monitoring of cell proliferation (Additional file 1: Figure S1). The fluorescence intensity of perlecan increased with time in the group of NHKs from young donors, during both cell proliferative and confluent phases. This result showed that perlecan was continuously produced and accumulated in the ECM. In contrast, low levels of perlecan were detected in the NHKs from aged donors. Additionally, the fluorescence intensity detected in the ECM of the NHKs from aged donors on Day 4 was at least 60% lower than that detected in the NHKs from young donors. This finding suggested that less perlecan may be produced and/or retained in the ECM as cells age. To address whether an age-associated decrease in perlecan was linked to genomic breakdown, we generated cDNA from a large panel of primary NHKs obtained from skin donors aged 24 to 64 years old for real-time polymerase chain reaction (QPCR) analyses. As shown in Figure 2(d), the perlecan mRNA levels were reduced in the older age groups. A 75% decrease in perlecan mRNA expression was observed from 24 to 43 years of age, followed by an additional 12% decrease from 49 to 64 years of age. These reductions were consistent with our findings that the perlecan protein levels decreased in aging skin (Figure 1b). These results suggested that perlecan expression in keratinocytes was down-regulated at the transcriptional level without excluding a potential additional post-transcriptional regulation.

Perlecan regulates K15 expression in cultured keratinocytes

Next, we evaluated perlecan cell adhesion activity. We found that purified perlecan induced adhesion of aged keratinocytes in a dose-dependent manner and that adding antibodies that specifically blocked Δ1 integrin function inhibited the adhesion of keratinocytes to perlecan. This finding suggested that a Δ1 integrin was involved in the adhesion mechanism (Figure 3a).

Figure 3. Perlecan induces keratinocyte adhesion and regulates K15 gene (KRT15) expression. (a) Keratinocytes adhere to perlecan in a dose‐dependent manner (left). Adhesion to perlecan is inhibited by antibodies against the Δ1 integrin subunit (right). (b) Proliferation of aged and young keratinocyte strains over 24 h. The proliferation of cells plated on perlecan is expressed as a percentage of the proliferation observed in control conditions. (c) Real‐time PCR analysis of keratins (K1, K5, K10, K14, and K15) and involucrin gene expression normalized to GAPDH in keratinocytes from the 61‐year‐old donor plated on culture dishes either covered beforehand with perlecan or untreated. (d) Scheme of the epidermis showing the locations of the differentiation markers. (e) Real‐time PCR analysis of KRT15 in keratinocytes from 3 distinct aged donors (age 64, 61, 60), cultured on perlecan, laminin 332 or plastic. (f) Real‐time PCR analysis of KRT15 in keratinocytes from 3 distinct aged donors (age 64, 61, 60) cultured either on native perlecan or digested perlecan, which lacked heparin sulfate (HS) moieties. (g) ELISA results show expression levels of perlecan in the ECM of keratinocytes transfected with perlecan‐specific siRNA, compared to cells transfected with control‐siRNA. (h) Real‐time PCR analysis of KRT15 and Ki‐67 gene (MKI67) in a 52 year‐old keratinocyte strain transfected with perlecan‐specific siRNA. (i) K15 staining in perlecan‐siRNA‐transfected keratinocytes; quantification is expressed as a percentage of DAPI positive cells. Each slide was performed in triplicate; 400 cells were counted within 4 areas of each slide. (j) Western blot of K15 expression in cultured perlecan knock‐down keratinocytes. Quantification of K15 expression is relative to actin expression. Experiment was done in triplicate. (c, g, h) Data are presented as the mean ± SD of 9 independent experiments with keratinocytes from a 61 (c) or 52 (g, h) years old donor. (e, f) All performed independent experiments per donor are shown (age 64 ○, 61 □, 60 ◇) and were combined for statistical analysis. ^*p<0.025,^**p<0.005,^***p<0.0002 vs. control, Student's t‐test.

Therefore, we used purified perlecan as an adhesion substrate to analyse its potential impact on keratinocyte proliferation and differentiation. First, we observed that culturing keratinocytes for 24 h on immobilized perlecan did not impact their proliferation compared to control cells plated directly on the culture dishes (Figure 3b). Moreover, we found that plating aged keratinocytes on perlecan for 24 h resulted in significantly reduced mRNA expression of the early and intermediate differentiation genes K1, K10, and involucrin; however, mRNA expression of the basal keratinocyte keratins, K5 and K14, remained unchanged (Figures 3c and 3d). Most interestingly, a significant increase in the K15 mRNA level was detected in aged cells plated on perlecan, compared to controls (Figure 3c). The experiment was repeated with strains of three distinct donors, and similar results were produced (Additional file 2: Figure S2). In contrast, plating cells on the BM protein, laminin 332, did not affect the K15 mRNA expression level (Figure 3e).

In the epidermis, K15-expressing cells belong to the stem cell population known to preferentially express the β1-integrin subunit [Figure 3d; 18, 19]; therefore, we also evaluated β1-integrin mRNA expression under the same conditions (Additional file 3: Figure S3). We found that culturing four aged keratinocyte strains on perlecan for 24 h resulted in a significant increase in β1- integrin mRNA levels; this result suggested that perlecan promoted enrichment of the cell population that expressed keratin 15 and β1-integrin. Digestion of perlecan to remove the HS moieties prior to the experiment abolished the rise in K15 mRNA expression (Figure 3f). This result suggested that the HS played a role in this enrichment mechanism. We further assessed the direct role of perlecan in K15 expression by blocking perlecan expression with a combination of three siRNA oligonucleotides specific for human perlecan. Transfection of NHKs with this siRNA mixture inhibited perlecan expression by approximately 80%, based on ELISA detection of perlecan protein expression (Figure 3g). Consistent with our previous results, we also found a significant reduction in K15 mRNA expression levels in cells transfected with perlecan-specific siRNA (Figure 3h). As expected, and as revealed by the unchanged expression of the Ki-67 proliferation marker, the perlecan deficiency did not affect keratinocyte proliferation. These results were confirmed by the reduced number of K15-positive cells found in the population of perlecan-deficient keratinocytes. After knocking down perlecan expression, the ratio of K15-positive keratinocytes:total NHK cells dropped from 36.6% (control cultures) to 18.8% (perlecan-deficient cultures; Figure 3i); this result was reinforced by western blot results, which showed a similar pattern in K15 protein expression (Figure 3j). Taken together, our results revealed a link between perlecan and K15 expression in NHKs. Moreover, the finding that this link depended on perlecan HS moieties suggested the involvement of a heparin-binding partner.

Perlecan is known to bind to many growth factors, including FGF7, which plays an important role in keratinocyte survival [10]. Therefore, we analysed FGF7, and other selected growth factors potentially involved in epidermal differentiation, to determine whether they impacted K15 expression. Simultaneously, we also verified their expected impact on keratinocyte proliferation. As previously reported, we found a slight, but significant, reduction in K15 mRNA expression levels in cells treated with either FGF7 or transforming growth factor beta 1 (TGF-Δ1) (Figure 4a) [20]. Among the other tested growth factors, we found a reduction or no change in K15 gene expression after treating with insulin growth factor (IGF)-1 or -2, respectively, and a very significant increase after treating with heparinbinding epidermal growth factor (HB-EGF). To determine whether perlecan was involved in HB-EGFinduced K15 expression, we treated perlecan knockdown cells with HB-EGF. Western blotting (Figure 4c) experiments confirmed that K15 expression was increased in control-siRNA transfected cells, and K15 expression was decreased in perlecan-siRNA transfected cells. The QPCR analysis, which confirmed these results, also drew our attention to the changes induced by HB-EGF treatment. We found that HB-EGF promoted diametrically opposite keratinocyte proliferation signals, depending on perlecan expression (Figure 4d, Additional file 4 : Figure S4). These results revealed that perlecan played a role in HB-EGFmediated regulation of proliferation and K15 expression in basal keratinocytes.

Figure 4. Perlecan functions in K15 expression regulation in aging skin. (a) Real time PCR analysis of KRT15 and MKI67 in the 52‐year old keratinocyte strain after treatment with growth factors (FGF7, 15 ng/ml; IGF‐1, 50 ng/ml; IGF‐2, 80 ng/ml; HB‐EGF, 100 ng/ml; TGF‐Δ1, 1 ng/ml). (b,c) Western blot of K15 expression in control (b) and perlecan knock‐down (c) keratinocytes after HB‐EGF treatment (100 ng/ml). Quantifications of K15 expression in control‐siRNA and perlecan‐siRNA transfected cells are relative to actin expression. Experiment was done in triplicate. (d) Real time PCR analysis of KRT15 and (e) MKI67 in control and perlecan knock‐down keratinocytes after HB‐EGF treatment (100 ng/ml). (f) K15 (red) immunostaining and DAPI (blue) staining in 2 young and 2 aged skin biopsy sections. Red and blue cells were counted along the BM of 6 independent sections. Scale bars = 50 μm (top) and 20 μm (bottom). (a, d, e). Data are representative of at least 12 independent experiments with keratinocytes from the 52 years old donor. Mean ± SD; ^*p<0.025,^**p<0.005, ^***p<0.0005 vs. control, Student's t‐test.

K15 expression decreases in aging skin

We next investigated whether skin aging might affect K15 expression. We analyzed the K15-positive cell population in epidermis biopsies from four donors, aged 22, 24, 61, and 74 years. In younger specimens, as previously described, K15 was detected throughout the basal layer, and strong staining was observed in reteridges, where it was detected throughout the entire cell cytoplasm (Figure 4f) [19,21]. In contrast, in older specimens, where the epidermal rete ridges were missing, the K15 staining appeared punctuated; staining was located mainly on the basal side of cells (Figure 4f). In addition, we evaluated the density of K15-expressing keratinocytes. We found K15-positive cells at densities of 20 and 21 cells/100 μm in the epidermal rete-ridges of young specimens; the density dropped to 12 and 11/100 μm in the two tested aged specimens. These results suggested that K15 expression was decreased and disorganized in the basal keratinocytes of aged skin.

Exogenous perlecan restores the ability of aged keratinocytes to form a full thickness epidermis

Finally, to analyze the impact of keratinocyte aging on perlecan expression in three-dimensional conditions, in vitro skin equivalent (SE) models were produced. These SEs were composed of a previously described dermal equivalent (DE) made of foreskin dermal fibroblasts seeded in a porous dermal substrate (collagen, glycosaminoglycans, and chitosan), on top of which NHK were seeded [22]. As shown in Figure 5a, when 24 year old donor's NHK were seeded, the DE supported epidermal proliferation giving rise to a full thickness and well organized, terminally differentiated epidermis at day 21 that displayed perlecan expression evenly along the DEJ. As previously documented [22], normal differentiation stages were indicated by expression of the early differentiation marker K10 in the supra-basal layers, the intermediate differentiation marker involucrin in the spinous layer, and the late differentiation markers filaggrin and loricrin in the stratum corneum. Seeding 61 years old donor's NHK over these DEs led to an obvious decrease in epidermal thickness (Figure 5b). Only little perlecan was found in the DEJ while laminin 332 was still expressed. The reduction in cell layers was accompanied by anomalies in the differentiation markers staining (Additional file 5: Figure S5), reflecting disorganization and abnormalities in the differentiation process. These data show that aged skin keratinocytes are unable to form a multi-layered well-differentiated epidermis in three-dimensional culture conditions. To address whether the defect in epidermal formation with aging is influenced by perlecan expression, we epidermalized SEs with the 61 years old donor's NHK in the presence of purified human perlecan. Perlecan was immunopurified from the conditioned medium of human coronary arterial endothelial cells as described previously [23] (Figure 5c and d). Epidermis made from aged skin NHK was thin and poorly organized, reaching a 50 μm depth at day 21 (Figure 5e and f). Exogenous addition of perlecan at 5 μg/ml (+ perlecan), restored the ability of aged NHK to form a multi-layered epidermis, which was already visible at day 14 and reached 100 μm thickness at day 21, where differentiation layers were readily distinguishable (Figure 5e and f). An additional application of perlecan (++ perlecan) further enhanced this phenomenon as evidenced by the 150-μm epidermal thickness obtained at day 21. Perlecaninduced improvement of the epidermal differentiation process was apparent after HPS staining (Figure 5h), and we analysed the differentiation markers expression at day 21 (Figure 5g). Interestingly, the K5 staining revealed that the basal keratinocyte layer was enlarged. The early differentiation marker K10 was expressed in all suprabasal layers, indicating that a normal differentiation process occurs in the perlecan-restored epidermis. Staining of the intermediate differentiation marker involucrin revealed a large multilayer zone containing well delineated and organized cells, in contrast to those found in the flat and packed epidermis of the control SE. Staining of filaggrin in the spinous layer and loricrin in the stratum corneum both revealed thicker sections containing healthy and actively producing cells compared to the thin and poorly organized layers obtained in the control SE. These results suggest that supplementing SE with perlecan allowed the aged skin keratinocytes to accomplish each differentiation phase, whereas they seemed incomplete and cell-deprived in the control SE. Staining of collagen VII and laminin 332 revealed a regular BM below the epithelium of both models, appearing thicker and less interrupted in the perlecan-treated SEs (Figure 5h). The addition of perlecan modified the β1 and α6 integrin patterns both in intensity and localization (Figure 5h); suggesting that perlecan favoured keratinocyte cohesion in the basal layer. The increase in K5-positive cells in perlecan-treated SEs suggested increased proliferation of basal cells. With Ki-67 staining, we found that the abundance of Ki-67-positive cells in the basal layer was 24% higher in the perlecan-treated SEs than in untreated SEs (Figure 5i). This result suggested that adding soluble perlecan enhanced the proliferation of basal cells. An examination of perlecan expression revealed stronger staining in the BM of treated SEs than in the BM of untreated SEs (Figure 5j). This result showed that some of the added perlecan reached the BM. However, a punctuated, circular staining pattern was observed throughout the epidermis, which suggested that some perlecan was trapped between keratinocytes. Analysis of K15 expression revealed that K15- expressing cells were present in the basal layer of perlecan-treated SE, but only a few were observed in untreated SE (Figure 5k). As previously documented, the presence of Ki-67-positive cells in the K15-positive basal layer indicated that this layer maintained a healthy, self-renewing, proliferative compartment [24].

Figure 5. Exogenous soluble perlecan improves epidermal thickness. (a, b) HPS staining and immunohistochemical analysis of paraffin‐embedded SEs epidermalized with (a) young or (b) aged keratinocytes. (c) Western blot of HUAEC culture medium before (lane 1) and after (lane 2) immunopurification. The eluted material (lane 3) contained the perlecan core protein. (d) Coomassie blue staining of purified perlecan. (e) SEs generated with aged keratinocytes without (control) or with perlecan (+ perlecan and ++ perlecan). SEs were collected 14 and 21 days post‐epidermalization and processed for histology. (f) Epidermal thickness. *** p<0.001 for + perlecan and ++ perlecan vs. control, Student's t‐test. (g, h) Paraffinembedded and frozen SEs at 21 days post‐epidermalization were immunolabelled to identify (g) differentiation markers and (h) adhesion/ECM components. Nuclei were stained with DAPI. (i) Ki‐67 staining in SEs. Quantification is expressed as the percentage of DAPI staining in the basal layer. Mean ± SD; *p<0.025. (j, k) Frozen SEs at 21‐days post‐epidermalization were immunostained to identify perlecan (j) and K15 (k). Scale bars = 50 μm (j), = 60 μm (k).

Collection of skin specimens

Full-thickness UV unexposed skin biopsies were obtained from female patients undergoing mammary or abdominal reduction following ethical and safety guidelines according to French regulation donors (Declaration no. DC-2008-162 delivered to the Cell and Tissue Bank of Hospices Civils de Lyon). Thirty-eight subjects were enrolled in this study: 18 elderly donors (ages 50–73), 12 intermediate donors (ages 35–49) and 8 young donors (age 22-34). After surgery, tissues were embedded in Tissue-Tek OCT compound (Euromedex, Mundolsheim, France) and kept at −80°C, processed for paraffin embedding or handled for cell extraction.

Monolayer cell cultures

NHK cultures were established from skin specimens according to previously published procedures [58] and grown in supplemented keratinocyte growth medium containing 0.15 mM CaCl₂ (KBM-2 BulletKit, Lonza Biosciences, Basel, Switzerland). Fibroblasts were grown in Fibroblast Growth Medium (Promocell, Heildeberg, Germany). Keratinocytes used in SEs were isolated and cultured as described by Rheinwald and Green [59] on a feeder layer of irradiated human fibroblasts as previously described [22].

Preparation of skin equivalents

Fibroblasts (passage P7, 25 × 10⁴ cells/cm²) were seeded onto a dermal substrate and grown for 21 days as described previously [22]. Keratinocytes were seeded on the DE at a density of 25 × 10⁴ cells/cm² in a 3:1 DMEM:Ham's F12 (Gibco, Life Technologies SAS, Saint Aubin, France) supplemented medium [22]. After 7 days, the SE was elevated to the air–liquid interface and cultured in a differentiation medium. SEs were cultured for up to 42 days, and were harvested at 21, 35, or 42 days after fibroblast seeding. SEs were either fixed in neutral buffered formalin 4% (Diapath, Martinengo, Italy) for 24 h and embedded in paraffin or in OCT compound and frozen at −80°C. When indicated, 5 μg/ml of human purified perlecan was added to the SEs culture at different time points. The first application was on top of DEs before keratinocytes seeding. Following incubation at 37°C for 16 h, keratinocytes were seeded in medium containing 5 μg/ml perlecan. After 2 days, the medium containing 5 μg/ml perlecan was either renewed once (namely + perlecan) or twice (namely ++ perlecan) and the normal culture conditions were maintained thereafter. All SEs were produced in triplicate and each experiment was repeated twice.

Western blot analysis and perlecan purification

Keratinocyte ECM was obtained as described previously (Décline and Rousselle, 2001). Proteins were resolved on SDS-PAGE gradient gels (3-5%) and perlecan was immunoblotted as previously described [38]. Perlecan was purified by ion exchange and mAb affinity chromatography from human coronary arterial endothelial cell conditioned medium [23]. To ensure removal of growth factors bound to perlecan, the affinity chromatography column was extensively washed with 0.02 M Tris-HCl, 1M NaCl, pH 7.4, before perlecan elution. Prior to SDS-PAGE analysis, aliquots were TCA precipitated and analysed either by western blotting or by staining of the gel with Coomassie Brilliant Blue. HS digestion was performed as previously described using 8 mU/ml heparitinase I for 2 h at 25°C [60].

Histological and immunohistological procedures on tissues

Paraffin sections were processed either for Haematoxylin – Phloxin – Saffron (HPS) staining or for immunohistochemical experiments. Sections were dewaxed, antigen retrieval was performed according to the manufacturer's instructions and immunolabelling experiments were performed, using antibodies against: perlecan (3G166 against domain III, US Biologicals, Swampscott, USA); keratin 10 (DE-K10, Dako), Δcatenin (E-5, Santa Cruz Biotechnology, distributed by Clinisciences, Montrouge, France); laminin 332 [61] and collagen VII (Santa Cruz Biotechnology). Other mAbs against perlecan (clone 6F165 against domain I, clone A7L6 against domain IV, and clone 6F166 against domain V from US Biologicals) were applied on 10 μm thick frozen sections after fixation in 4% paraformaldehyde in PBS and incubation in 20 mM sodium acetate, pH 7.0, 5 mM CaCl₂ containing 2 mIU/ml heparitinase I and 10 mIU/ml chondroitinase ABC for 30 min at 25°C (Seikagaku America, distributed by Coger, Paris, France). After incubation with 5% normal goat serum in PBS, peroxidaseconjugated antibodies (En Vision dual-link, Dako, Trappes, France) were used. Counterstaining was performed using Harris haematoxylin (25%, Sigma Aldrich, Saint-Quentin-Fallavier, France).

Frozen sections were fixed in pre-cooled acetone for 10 min and rehydrated with 10% FCS in PBS. After washing, sections were incubated overnight with Abs against keratin 5 (D5/16B4, Dako), filaggrin (15C10, Vector Labs, distributed by Clinisciences), involucrin (BT-600, BTI, Clinisciences), loricrin (15C10, Vector Labs), Δ1 (P4C10, Millipore, Molsheim, France) and α6 (GoH3, Millipore) integrin subunits mAbs or Ki-67 (clone MIB-1, Dako), and washed. For K15 staining, frozen section were fixed in ethanol 70% and blocked. Anti-K15 pAb [62] (EPR1614Y, Gene Tex distributed by tebu-bio SAS, Le Perray en Yvelines, France) was applied 1 h and rinsed. Cy3 (Jackson, Beckman Coulter, Paris) or alexa-488 (Molecular probes, Invitrogen) conjugated antibodies were applied for 1 h. Nuclei were stained with DAPI. Fluorescence was visualized using LSM700 laser scanning confocal microscope (Zeiss, Jena, Germany).

Image analysis and processing

Perlecan quantification for the skin cohort study and epidermal thickness measurements were performed using ImageJ software. Brightfield images of DAB-labelled perlecan and haematoxylin counterstaining were converted to normalized blue images that allow automated identification of positively stained tissue according to [63]. Epidermal thickness of HPS-stained sections was obtained by means of an Euclidean distance map. Pixels corresponding to the epidermis were isolated from other pixels. Images were converted to 8-bit binary images. Images corresponding to the area of interest were converted to 16-bit distance maps. To each epidermis pixel (nonzero) in the distance map binary image a value equal to its distance from the nearest background pixel (zero) was assigned. The basal epidermal line was selected and then applied on the distance map. The mean intensity of the basal line corresponds to the mean distance between the basal line and the stratum corneum. Twelve different fields per experimental condition were quantified. Data are expressed in μm.

Immunofluorescence studies on cells

NHEK from young and aged donors were grown on chambered coverglasses in supplemented KBM-2. After washing, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 for 2 minutes and incubated with 1% BSA / PBS. Anti-perlecan pAb (Santa Cruz) or anti-K15 [57], (EPR1614Y, Gene Tex distributed by tebu-bio SAS, Le Perray en Yvelines, France) were applied. Alexa Fluor 488- and/or Alexa Fluor 568-antibodies were applied, either alone or with fluorescein isothiocyanate-phalloidin (FITC; Sigma). Nuclei were stained with DAPI. Cells were observed with LSM700 laser scanning confocal microscope.

To quantify perlecan in cell cultures, NHEK were seeded in 96-well plates and grown until subconfluence. The same culture procedure as described above was applied except that wells were incubated in digestion buffer containing 2 mIU/ml heparitinase I and 10 mIU/ml chondroitinase ABC prior to the saturation step with BSA 1% and subsequent incubation with antiperlecan antibodies. Next, culture plates were washed with PBS and incubated with FITC-conjugated secondary antibodies for 1 h. After PBS washes, 20 mM NH4OH, Triton 0.5% was added to each well and the fluorescence intensity determined at 485-535 nm using a VICTOR X4 Multilabel plate reader (Perkin Elmer SAS, Courtaboeuf, France). A blank value corresponding to BSA-treated wells was subtracted and each assay point was measured 6 times. Proliferation assays were performed using Cell Proliferation Kit XTT (Roche Diagnostics).

Quantitative real-time PCR analysis

Total RNA was isolated from monolayers using RNA isolation NucleoSpin RNA XS (Macherey-Nagel EURL, Hoerdt, France) according to the manufacturer's instructions. 500 nanograms of total RNA extracted from young and aged NHK cells were subjected to the first strand cDNA synthesis using a mix of oligo dT and random 6-mers using the PrimeScript RT reagent Kit (Perfect Real Time, TAKARA Biotechnology, distributed by OZYME, Montigny Le Bretonneux, France). QPCR was performed using the FastStart Universal SYBR Green Master kit (Roche Diagnostics, Mannheim, Germany). The primers used for the detection of gene expression are listed below. Reactions were performed using a Qiagen real-time PCR cycler Rotorgene. The presence of a single dissociation peak was verified by melt curve analysis. PCR end products were separated on a 2% agarose gel to confirm the amplification of single bands of the expected size. The specificity of target amplification was confirmed by sequencing. Relative quantification was determined using the comparative C(t) method [64] with normalization to the housekeeping gene GAPDH. Each assay point was performed in triplicate.

Cell adhesion assays

Perlecan was immobilized on multiwell plates (Greiner, Dutscher, Brumath, France) as indicated concentrations by overnight adsorption at 4°C. Plates were washed with PBS, and saturated with 1% BSA. Next, keratinocytes were detached with trypsin, suspended in supplement-free KBM-2 basal medium (0,15 mM CaCl₂) and seeded at 3 × 10⁴ cells/well. For cell adhesion inhibition experiments, the anti-Δ1 integrin antibodies (P4C10, Millipore, Molsheim, France) were added to the cells at the time of seeding. After 1 h, non-adherent cells were removed with a PBS wash. The extent of adhesion was determined by fixing adherent cells, then staining with 0.1% crystal violet and measuring absorbance at 570 nm as previously described [65]. A blank value was subtracted that corresponded to BSA-coated wells. Each assay point was derived from six measurements ± SD (6 wells per assay point).

Growth factor treatments

10⁵ keratinocytes were cultured in a 24 well plate for 16-20 hours in KBM-2. They were rendered quiescent by serum starvation for 16 to 20 h and subsequently treated with fresh supplement-free KBM-2 medium containing 10 ng/ml FGF7, 50 ng/ml IGF-1, 80 ng/ml IGF-2, 100 ng/ml HBEGF or 1ng/ml TGF β1 respectively (Pepro Tech France, Neuilly sur Seine, France), for 5 hours. Cells were harvested and counted or lysed with PBS 1% Triton prior to K15 western blotting experiments or for RNA isolation.

SiRNA experiments

For human perlecan knock-down experiments, a pool of 3 perlecan specific siRNAs was used (Perlecan siRNA, Santa-Cruz Biotechnology). Cell transfection was performed using Lipofectamin 3000 reagent (Invitrogen, Cergy Pontoise, France) with 100 nM duplex siRNA according to the manufacturer's recommendations. In all experiments, cells were analysed 48h after transfection and viability was assessed using trypan blue. A firefly luciferase GL2 sequence (CGUACGCGGAAUACUUCGA) was used as a negative control (100 nM), as described [66], and resulted in no reduction of perlecan expression.

Statistical analysis

Data were presented as mean ± SD and the Student's t-test (unpaired, two-tailed) was used for two-group comparisons. The exact sample size (n) for replicate measurements of a specific keratinocyte strain or of groups of keratinocytes from distinct donors are specified in each graph legend. Individual data values are shown when n < 6 according to [67]. Differences were considered statistically significant at a value of P <0.05.