Author response: Type XVII collagen coordinates proliferation in the interfollicular epidermis

Article Figures and data Abstract eLife digest Introduction Results Discussion Material and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin. https://doi.org/10.7554/eLife.26635.001 eLife digest The skin is one of the largest organs of the body and is constantly confronted with a range of external stresses including germs, heat and scratches. The outermost part of the skin is called the epidermis and it acts as a barrier to the external environment and works to stop the body from losing water. An abnormally thin or thick epidermis can impair the skin's ability to perform these roles. As such, the ability of epidermal cells to proliferate (i.e. divide to make new cells) is tightly regulated, both when the animal first develops and when it ages. However, most of the underlying mechanisms that regulate these processes are unknown. Watanabe et al. have now identified type XVII collagen (called COL17 for short) as a key molecule that controls how often epidermal cells in skin from mice and humans divide. COL17 is a protein that is made in the deepest layer of the epidermis, and it prevents the epidermis from thickening in newborn mice by coordinating with the Wnt signaling pathway. This signaling pathway, amongst other things, controls how often some cells divide. Older mice have a thicker epidermis than their younger counterparts. Watanabe et al. revealed that the distribution of COL17 in the epidermis also changes dramatically with age in mice and humans. Further experiments with mice showed that introducing COL17 back into the epidermis helped the tissue retain a more youthful state even in animals that had reached an old age. Together these findings give scientists a better understanding of how the ability of epidermal cells to divide is regulated at various stages in a mammal's life. The new findings also point to COL17 as a promising component in future anti-aging strategies targeted at the skin. Yet first, further work will be needed to uncover how the production of COL17 is controlled in the epidermis. https://doi.org/10.7554/eLife.26635.002 Introduction Skin is a highly structured organ in which stem cell self-renewal, cell proliferation and differentiation are coordinated to maintain homeostasis. In addition to hair follicles and other skin appendages, the interfollicular epidermis (IFE; non-haired skin) comprises distinct cellular populations. IFE stem cells reside in the basal cell layer; these cells both self-renew and generate the terminally differentiated outer cell layers which function as barriers to the external environment and prevent loss of body fluids (Giangreco et al., 2008; Hsu et al., 2014; Jones et al., 2007; Natsuga, 2014). Organismal aging, or physical aging, is defined as tissue impairment arising from the accumulation of numerous intrinsic and extrinsic factors that induce cell damage chronologically. The relationship between organismal aging and stem cells is an inescapable bond, and the heterogeneity of stem cells in organs may be reduced with aging (Goodell and Rando, 2015). Human skin aging is exemplified by alterations in the dermis and skin appendages, such as the thinning of dermis, dryness, wrinkles, gray hair and hair loss (Rittié and Fisher, 2015). However, the influence of aging on IFE has been controversial. Although decreased epidermal proliferation has been reported in in vitro and in vivo studies using aged individuals and mice (Giangreco et al., 2008; Gilchrest, 1983; Grove and Kligman, 1983), several recent studies have reported contradictory results, showing sustained and increased proliferation in the aged epidermis (Charruyer et al., 2009; Stern and Bickenbach, 2007). Thus, how organismal aging affects the IFE and its stem cells has not been clarified (Keyes et al., 2013). The extracellular matrix proteins of the basement membrane zone (BMZ) are important components of the IFE stem cell niche and connect the dermis and epidermis functionally. Type XVII collagen (COL17) is a type II transmembrane protein that is located along the hemidesmosomes in the BMZ. The N-terminus of COL17 is localized in hemidesmosomes, and its extracellular domain reaches the lamina densa (McMillan et al., 2003). Non-hemidesmosomal COL17 in keratinocytes and human skin has also been reported (Hirako et al., 1998). COL17 has been characterized as a target protein in the autoimmune blistering disease bullous pemphigoid (Nishie, 2014) and also as being the defective protein in junctional epidermolysis bullosa (JEB), a congenital blistering disease (Fine et al., 2014). Recently, COL17 has been shown to form a niche for hair follicle stem cells (HFSCs), as mice lacking the protein and human JEB patients with mutations in COL17A1, the open-reading frame encoding COL17, exhibit a premature aged skin phenotype, including gray hair and hair loss (Matsumura et al., 2016; Nishie et al., 2007; Tanimura et al., 2011). Additionally, reduced labeling of epidermal basement membrane proteins, including COL17, in human skin has been associated with aging (Langton et al., 2016). However, the role of COL17 in maintaining IFE and its stem cells is still unclear. In the present study, we explore the comprehensive role of COL17 in regulating IFE homeostasis and also characterize age-related IFE alterations associated with a modified BMZ, including COL17. We show that COL17 is indispensable for regulating IFE proliferation in neonatal mice through activating the Wnt pathway. The IFE hyperproliferation is induced by organismal aging and can be reversed by COL17 replenishment. Results COL17 deficiency leads to epidermal hyperproliferation in neonatal IFE We investigated the phenotype of paw skin to study IFE in Col17a1−/− mice (Nishie et al., 2007) in the absence of hair follicles. Neonatal IFE skin (P1, postnatal day 1) of Col17a1−/− mice showed transient epidermal hyperproliferation, as demonstrated by counting the epidermal layers and the numbers of epidermal cells and phospho-Histone H3 (PH3)-positive cells (Figure 1a–b). The numbers of proliferating cell nuclear antigen (PCNA)- and Bromodeoxyuridine (BrdU)-positive cells in the Col17a1−/− IFE basal cells at P1 were also increased compared with the controls (Figure 1c), indicating that COL17 deletion affects both the S and M phases in the cell cycle of IFE neonatal keratinocytes. The proliferative IFE phenotype of Col17a1−/− mice gradually waned postnatally, and the epidermal thickness and the number of PH3-positive cells became comparable with those of the controls at P20 (Figure 1a–b). Figure 1 with 2 supplements see all Download asset Open asset COL17 deletion induces transient IFE hyperproliferation in neonates. (a) Hematoxylin and eosin (H&E) staining and E-cadherin (E-cad) labeling (with PI nuclear counterstain) of Col17a1−/− and control IFE skin samples from Col17a1+/- or Col17a1+/+ littermates (Control) at P1 (n = 5) and P20 (n = 4). Scale bar: 20 μm. Quantitation of the number of epidermal layers and epidermal cell counts. The values are shown as relative ratios to the controls. (b) PH3 staining at P1 and P20. Scale bar: 20 μm. The number of epidermal basal cells positively labeled for PH3 per mm epidermis (n = 4). BM, basement membrane. (c) PCNA and BrdU labeling at P1. Scale bar: 20 μm. Quantitation of PCNA- (n = 5) and BrdU-positive basal cells (n = 4). The values are shown as relative ratios to the controls. (d) Quantitative RT-PCR (qRT-PCR) of Itga6, Itgb1, Tgm1, Ppl and Ivl mRNAs (n = 5). (e) Loricrin and cleaved caspase-3 staining (representative images from 3 mice). Scale bar: 20 μm. BM, basement membrane. (f) An in silico model of the epidermal cell proliferation upon the reduced adhesion of committed progenitor cells to the BMZ. The details are described in the Material and Methods. The data in all of the histograms are the means ± SE. *0.01<p<0.05, **0.001<p<0.01, ****p<0.0001. Student's t-tests. https://doi.org/10.7554/eLife.26635.003 We investigated whether the expression levels of markers of basal cells and differentiated cells were altered in the hyperproliferative IFE of Col17a1−/− mice at P1. The gene expression of Itga6, which might compensate for COL17 deficiency, was increased in Col17a1−/− IFE skin, whereas the expression of Itgb1, itgb4, lamb3 and lamc2 was not altered (Figure 1d, Figure 1—figure supplement 1a). The mRNA expression levels of Tgm1, Ppl and Ivl were somewhat higher in Col17a1−/− IFE skin (Figure 1d), while loricrin-labeled granular cell layers were not expanded in these mice (Figure 1e). Dye-permeability and transepidermal water loss at day 18.5 of embryogenesis (E18.5) were comparable between the Col17a1−/− mice and the controls (Figure 1—figure supplement 1b–c), indicating that IFE keratinocyte differentiation was not greatly altered in Col17a1−/− skin. There was no increase in the number of cells positive for cleaved caspase-3, a marker of apoptosis, in the Col17a1−/− epidermis compared with the controls (Figure 1e). The electron microscopy results indicated that the dermo-epidermal junction of the Col17a1−/− IFE presented hypoplastic hemidesmosomes in accordance with previous observations on the back skin of Col17a1−/− mice (Nishie et al., 2007) (Figure 1—figure supplement 1d). There were no significant differences in the number of inflammatory infiltrates, including CD3+, F4/80+ and Ly-6G+ cells, in the dermis of Col17a1−/− mice and control mice, which excludes inflammation as a contributor to immature hemidesmosome formation (Figure 1—figure supplement 1e–g). To explain the transient epidermal hyperproliferation of the Col17a1−/− neonatal IFE, we exploited an in silico model (Kobayashi et al., 2016) to recapitulate the epidermal development. As COL17 serves as a linker between the epidermis and dermis and is expressed in basal cells, most of which are committed progenitor cells in the epidermis (Doupé and Jones, 2012; Lim et al., 2013), epidermal thickness was calculated upon the loosening of the attachment of committed progenitor cells to the BMZ. In the model with diminished adhesion, the epidermal thickness was transiently increased and gradually returned to the baseline (Figure 1f), which was compatible with the experimental observations of Col17a1−/− neonates and with the transient hypertrophy of Itga6- and Itgb1-null epidermis (Brakebusch et al., 2000; Niculescu et al., 2011). These data suggest that COL17 deletion induces epidermal thickening by the hyperproliferation of IFE keratinocytes, at least partially through the loosening of dermo-epidermal adhesion at the neonatal stage. The site specificity of the hyperproliferative phenotype in the neonatal paw epidermis of Col17a1/-/ mice was confirmed by the comparable expression of proliferation markers in the back skin IFE of Col17a1−/− mice and control mice (Figure 1—figure supplement 2a). The discordance between the paw epidermis and back skin IFE might be explained either by the influence of hair follicle development on the back skin IFE or by the distinct regulation of the IFE at each body site (Rompolas et al., 2016; Roy et al., 2016; Sada et al., 2016). We also investigated cell-intrinsic properties due to COL17 defects using cultured normal human epidermal keratinocytes (NHEKs). The cell proliferation rates of NHEKs treated with COL17A1 siRNAs were slightly decreased (Figure 1—figure supplement 2b–d), which is compatible with reduced proliferation of cultured keratinocytes derived from Col17a1−/− mice (Tanimura et al., 2011), and the colony-forming abilities of these cells were similar to those of control cells (Figure 1—figure supplement 2e–f). These data indicate that the proliferation potential of Col17a1−/− IFE is dependent on in vivo conditions. COL17 regulates neonatal IFE proliferation through Wnt-β-catenin signaling Various signaling molecules are involved in controlling hair follicle stem cells and epidermal homeostasis (Kretzschmar and Watt, 2014); the relationships between these signaling molecules and BMZ proteins are only partially understood (Margadant et al., 2010; Rognoni et al., 2014; Tanimura et al., 2011). To further explore the underlying mechanisms of the transient epidermal hyperproliferation phenotype of Col17a1−/− IFE, we first screened the gene expression profiles of the receptors, co-receptors, transcription factors and cofactors of the major signaling pathways, including Wnt, TGF-β/BMP, Notch, Hedgehog and FGF in neonatal Col17a1−/− and control skin samples. The screening data showed that the expression levels of Wnt-related molecules (Fzd4, Nfatc2, Nfatc4 and Tcf7) were significantly decreased in Col17a1−/− neonatal IFE skin compared with controls (Figure 2—figure supplement 1a). Although the expression of some TGF-β−related genes was altered in Col17a1−/− IFE skin (Figure 2—figure supplement 1a), the TGF-β and p-Smad2 immunostaining in Col17a1−/− IFE keratinocytes was comparable with that in IFE cells in the controls, in contrast to the TGF-β and p-Smad2 reduction in Col17a1−/− HFSCs (Tanimura et al., 2011) (Figure 2—figure supplement 1b–c). The gene expression profiles for Notch, Hedgehog and the FGF pathway were not significantly affected upon COL17 deletion (Figure 2—figure supplement 1a). To validate these findings, we analyzed the gene expression profiles of specific Wnt-related molecules in the independent replication samples. In line with the screening qPCR data, the expression levels of the Wnt target genes (Tcf7 l1, Tcf7 l2, and Axin2), the receptor gene (Fzd4), and stimulatory Wnt genes (Wnt2, Wnt2b, Wnt5a) were significantly downregulated in Col17a1−/−mice, whereas expression of the inhibitory Wnt gene (Wnt4) was increased (Figure 2a) (Bernard et al., 2008; Mikels and Nusse, 2006). The changes in these genes were paralleled by a reduction of basal cells positive for LEF1, a nuclear Wnt mediator of Wnt signaling, in neonatal Col17a1−/− mice IFE (Figure 2b). This reduction was also transient, and the number of LEF1-positive basal cells was comparable between the Col17a1−/− mice and the control mice at P4, which might account for the transient epidermal hyperproliferation of the paw IFE in Col17a1−/− mice. As β-catenin binds to LEF1 in the nucleus in the Wnt-canonical pathway (Lien and Fuchs, 2014), we performed β-catenin immunostaining. The number of nuclear β-catenin-positive cells in Col17a1−/− mice IFE was diminished compared to the controls (Figure 2c). Figure 2 with 3 supplements see all Download asset Open asset COL17 deficiency destabilizes Wnt-β-catenin signaling in neonates. (a) qRT-PCR of Wnt-related molecules in Col17a1−/− and control IFE skin samples at P1 (n = 5). Student's t-test. (b) LEF1 staining of Col17a1−/− and control IFE skin at P1 (n = 5), P4 (n = 4) and P10 (n = 4). Inlet: higher magnification of LEF1-positive basal cells at P1. Scale bar: 20 μm. Quantitation of LEF1-positive basal cells as a percentage of all basal cells (%). Student's t-test. (c) β-catenin staining of Col17a1−/− and control IFE skin at P1. Nuclear β-catenin accumulation is indicated with arrows. The quantification of nuclear β-catenin-positive cells (n = 3). Student's t-test. Scale bar: 20 μm. (d) Wnt activity in STF293 cells expressing hCOL17 treated with Wnt3a CM (n = 3). One-way ANOVA test, followed by Tukey's test. (e) Wnt activities in the hindpaw IFE from ins-Topgal+ (Control: left) and ins-Topgal+:Col17a1−/− (Col17a1−/−: right) mice. Calculated areas devoid of hair follicles or sweat glands are indicated with squares in the representative figures. The results are quantified as the Wnt-activated area per unit (n = 4). Scale bar: 100 μm. Mann-Whitney test. (f) H&E, E-cad, PH3 and PCNA staining of IFE skin samples from K14-ΔNLef and littermate controls at P1. Scale bar: 20 μm. The numbers of epidermal layers, epidermal cell counts and PCNA- and PH3-positive basal cells (n = 4). Student's t-test. (g) Quantification of BrdU- and PH3-positive cells in WT paw skin IFE treated with Wnt inhibitors (IWP-2 (n = 6) vs DMSO (n = 5) or Wnt-C59 (n = 6) vs DMSO (n = 4)). The data are presented as the means ± SE. Student's t-test. *0.01<p<0.05, **0.001<p<0.01, ***0.0001<p<0.001, ****p<0.0001. https://doi.org/10.7554/eLife.26635.006 To further evaluate the relationship between Wnt signaling and COL17, we utilized SuperTopFlash 293 (STF293) reporter cells (Tsukiyama et al., 2015). Overexpression of human COL17 significantly upregulated Wnt activity in this cell line (Figure 2d). Additionally, to visualize Wnt signaling in vivo, we crossed ins-Topgal+ mice (Moriyama et al., 2007) with Col17a1−/− mice. The LacZ-positive area that was indicative of active Wnt signaling in the IFE was significantly diminished in the ins-Topgal+:Col17a1−/− mice (Figure 2e, Figure 2—figure supplement 2). These results suggest that COL17 expression stabilizes Wnt signaling. To examine whether these findings correlate with the phenotype of JEB patients with COL17 deficiency, we also performed immunostainings for LEF1, β-catenin and PH3 in JEB skin. In the JEB epidermis, the numbers of LEF1-positive cells and cells with nuclear β-catenin were decreased, while the number of PH3-positive cells was elevated (Figure 2—figure supplement 3); these findings were compatible with the data from the Col17a1−/− mice, although this result requires further investigation due to the small sample size. We next investigated whether a defect in Wnt signaling in the IFE could be responsible for the hyperproliferation phenotype and whether that phenotype could be reversed by the introduction of COL17. K14-deltaNLef1 mice express a Lef1 transgene that lacks a β-catenin-binding site under the control of the keratin 14 (K14) promoter and serve as a model of inactive Wnt signaling in the epidermis (Niemann et al., 2002). Neonatal K14-deltaNLef1 mice IFE exhibited epidermal thickening and a larger number of PH3-positive basal cells than the controls (Figure 2f). The number of PCNA-positive basal cells was also increased, albeit not significantly. To confirm that the abated Wnt activities were related to neonatal IFE proliferation in vivo, we intraperitoneally administered Wnt inhibitors (IWP-2 or Wnt-C59) into wild-type mouse neonates (Kuo et al., 2016; Carotenuto et al., 2017). The number of BrdU- and PH3-positive epidermal cells was increased in mice treated with these inhibitors at P1 compared with untreated control mice (Figure 2g). Transgenic rescue by the expression of human COL17 (hCOL17) under the K14 promoter in Col17a1−/− mice (Nishie et al., 2007) abrogated IFE hyperproliferation and restored LEF1- and nuclear β-catenin-positive basal cells (Figure 3a,c–d). The expression levels of Wnt-related genes that were altered in Col17a1−/− mice at P1 were restored by transgenic rescue with hCOL17 (Figure 3b). These data demonstrate that COL17 unambiguously contributes to the maintenance of neonatal IFE proliferation via its effect on Wnt signaling. Figure 3 Download asset Open asset Induction of human COL17 abrogates epidermal hyperproliferation and the expression of Wnt-β-catenin signaling molecules in neonatal Col17a1−/− IFE. (a) H&E, E-cad, PH3 and PCNA staining of IFE skin specimens from Col17a1+/+ or Col17a1+/- (as hCOL17-; CTL (control)) and hCOL17+; Col17a1−/− littermate mice at P1. Quantification of epidermal layers, epidermal cell counts, and PH3- and PCNA-positive cells (n = 4). Scale bar: 20 μm. (b) Gene expression of Wnt-related molecules in IFE skin samples from hCOL17-; CTL and hCOL17+; Col17a1−/− littermate mice at P1 (n = 4). (c) LEF1 staining of IFE skin samples from hCOL17-; CTL and hCOL17+; Col17a1−/− littermates at P1 (n = 4). Scale bar: 20 μm. (d) β-catenin staining of IFE skin samples from hCOL17-; CTL and hCOL17+; Col17a1−/− littermates at P1 (n = 4). Nuclear β-catenin is indicated with arrows. The number of nuclear β-catenin-positive cells. Scale bar: 20 μm. The data are the means ± SE. Student's t-tests. https://doi.org/10.7554/eLife.26635.010 Distribution of COL17 is altered with physical aging Because Col17a1−/− mice (3-month-old) clearly exhibited the premature aging phenotypes of gray hair and hair loss (Figure 4—figure supplement 1) (Nishie et al., 2007; Tanimura et al., 2011), we examined the effects of physical aging on the IFE and the relationship between aging and COL17. We compared wild-type (WT) mice to with aged mice to In the IFE of aged mice, the epidermis was and the numbers of BrdU- and PCNA-positive cells were increased (Figure This was specific to paw skin and was not in the back skin IFE (Figure 4—figure supplement as was the for the neonatal Col17a1−/− IFE. These data indicate that physical aging leads to paw IFE hyperproliferation. Figure with supplements see all Download asset Open asset Physical aging affects epidermal proliferation and COL17 (a) H&E, E-cad, BrdU and PCNA staining of IFE skin from and aged wild-type (WT) mice. Scale bar: 20 μm. The numbers of epidermal layers, epidermal cell counts, and BrdU- and PCNA-positive basal cells (n = 5). Student's t-test. (b) The gene expression levels of Itga6, Itgb1, Tgm1, and in IFE skin samples from and aged WT mice (n = for Itga6, Itgb1, and = 3 for Ppl and Student's t-test. (c) COL17 staining to the in IFE skin samples from the and aged WT mice (n = 5), and aged normal human individuals (representative images from human and and littermates at (representative images from mice). Scale bar: 20 μm. The of membrane of IFE basal cells from and aged WT mice (n = 5). Mann-Whitney test. (d) COL17 labeling the of IFE skin from WT mice and human individuals (representative images from Scale bar: 20 μm. (e) The of skin from and aged WT IFE. The IFE cell membrane was with was for nuclear Scale bar: μm. The of COL17 in membrane of IFE basal cells from and aged WT mice (n = Mann-Whitney test. (f) The of COL17 and 1 and 2 in WT IFE using (representative images from mice). keratinocytes were by Scale bar: μm. BM, basement membrane. The data are the means ± SE. *0.01<p<0.05, **0.001<p<0.01, The gene expression levels of and were decreased in the aged mouse IFE, whereas that of not (Figure the expression levels of differentiation markers and were not greatly Although gene expression was the distribution of COL17 was dramatically altered in the IFE of aged mice and humans. The of COL17 was greatly reduced in the IFE of aged mice and as with the extracellular of COL17 (Figure and is in line with the recent on age-related alterations of COL17 in human skin (Langton et al., 2016). This was also in the IFE of mice, a premature aging model The of COL17 (Nishie et al., in the altered COL17 distribution was using several that the of COL17 (Figure 4—figure supplement 3). To confirm the of COL17 in the aged IFE, the were treated with to immunostaining (Hirako et al., 1998). In both mouse and human skin, IFE the of COL17 and the aged epidermis (Figure To further examine the reduction in the of COL17 in aged mice, we performed staining of the IFE (Figure In COL17 was to the basement membrane in the aged IFE, while basal cells of mice had COL17 that was associated with the and basal structured microscopy on WT mouse IFE (Figure COL17 was at the cell of basal keratinocytes not with proteins 1 and indicating that the COL17 in basal keratinocytes was not into which are highly In contrast to COL17 with aging, the of and were not modified by aging (Figure 4—figure supplement proteins and were also (Figure 4—figure supplement gene expression in IFE skin with the decreased expression of and with aging (Figure 4—figure supplement These results suggest that physical aging leads to paw IFE hypertrophy and COL17 distribution in the epidermis in a controls the distribution of COL17 in IFE To the mechanisms that the altered COL17 distribution that with physical aging, we the environment of aged IFE. Because distribution is in the aged epidermis et al., and the in the epidermis is in aged individuals than in individuals et al., IFE skin samples from mice were treated with to changes in in aged IFE. staining showed that COL17 in basal cells, while COL17 was present (Figure This that the the of other molecules by may the COL17 distribution in the IFE. Figure Download asset Open asset COL17 distribution is by (a) COL17 staining of IFE skin treated with The of COL17 in basal cells of IFE from control and treated (n = 4). Mann-Whitney test. were from WT mice at Scale bar: 20 μm. (b) labeling with and results from and aged WT IFE skin (n = 4). Mann-Whitney test. Scale bar: 20 of cell as to basement and cell in to basement in IFE. staining indicates the of the cell basement membrane. Scale bar: μm. of percentage of and in and aged IFE (n = 4). Student's t-test. The of 1 of DMSO as and of water as in IFE skin from WT mice, followed by COL17 The of COL17 in membrane of basal cells from control and 1 treated (d) and 1 (e) (n = 4). Mann-Whitney test. Scale bar: μm. BM, basement membrane. as and of 1 of DMSO as and of water as on epidermis. The relative of COL17 in membrane of basal cells was (n = 4). Mann-Whitney test. BM, basement membrane. Scale bar: 20 μm. The data are the means ± SE. *0.01<p<0.05, **0.001<p<0.01, the numerous cellular by we on atypical protein kinase C a key of et al., 2013). The express

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