Eisaku Ogawa

High Commitment of Embryonic Keratinocytes to Terminal Differentiation through a Notch1-caspase 3 Regulatory Mechanism

Embryonic cells are expected to possess high growth/differentiation potential, required for organ morphogenesis and expansion during development. However, little is known about the intrinsic properties of embryonic epithelial cells due to difficulties in their isolation and cultivation. We report here that pure keratinocyte populations from E15.5 mouse embryos commit irreversibly to differentiation much earlier than newborn cells. Notch signaling, which promotes keratinocyte differentiation, is upregulated in embryonic keratinocyte and epidermis, and elevated caspase 3 expression, which we identify as a transcriptional Notch1 target, accounts in part for the high commitment of embryonic keratinocytes to terminal differentiation. In vivo, lack of caspase 3 results in increased proliferation and decreased differentiation of interfollicular embryonic keratinocytes, together with decreased activation of PKC-delta, a caspase 3 substrate which functions as a positive regulator of keratinocyte differentiation. Thus, a Notch1-caspase 3 regulatory mechanism underlies the intrinsically high commitment of embryonic keratinocytes to terminal differentiation.

p53 homologue, p51/p63, maintains the immaturity of keratinocyte stem cells by inhibiting Notch1 activity.

p53 homologue, p51/p63, predominantly expressed in keratinocyte stem cells, is indispensable for the formation of epidermis. Notch1, another such gene indispensable for the process, induces growth arrest and differentiation in keratinocytes. We found that exogenous expression of ΔNp51B (ΔNp63α), one of the isoforms of p51 specifically expressed in basal keratinocytes, blocked Notch 1-dependent growth arrest and differentiation in mouse keratinocytes by inhibiting p21 expression and maintaining integrins expression. Furthermore, ΔNp51B by itself was found to have ability to induce expression of integrin α6β4, which promotes attachment of basal cells to basal membrane thereby keeping the cells in immature state. Therefore, we conclude that ΔNp51B expression warrants integrin expression even under the influence of Notch1 and that ΔNp51B is a long-sought factor required to maintain basal cell keratinocytes immaturity by inhibiting Notch1 activity. We will postulate a plausible model explaining the maintenance of the squamous epithelium architectures as well as offering mechanistic explanations for pathological features of skin diseases, including cancers, psoriasis along with physiological wound healings.

Eye-open at birth phenotype with reduced keratinocyte motility in LGR4 null mice.

We observed a consistent eye‐open at birth (EOB) phenotype in mouse pups homozygous for a leucine‐rich repeat containing G‐protein coupled receptor 4 (Lgr4) allele deleting the whole transmembrane domain coding region. An in vitro wound‐healing scratch assay showed notably reduced keratinocyte motility in the null mice. Phalloidin staining of F‐actin in the eyelid epidermis was also reduced. We also generated keratinocyte‐specific Lgr4 deficient mice, circumventing the embryonic/neonatal lethality and kidney abnormalities. Most of the conditional Lgr4 knockout mice showed the EOB phenotype. Thus, Lgr4 might be a novel gene class regulating cell motility.

Epidermal FABP (FABP5) Regulates Keratinocyte Differentiation by 13(S)-HODE-Mediated Activation of the NF-κB Signaling Pathway

Fatty acid-binding proteins (FABPs) are postulated to serve as lipid shuttles that solubilize hydrophobic fatty acids and deliver them to appropriate intracellular sites. Epidermal FABP (E-FABP/FABP5) is predominantly expressed in keratinocytes and is overexpressed in the actively proliferating tissue characteristic of psoriasis and wound healing. In this study, we found decreased expression of the differentiation-specific proteins keratin 1, involucrin, and loricrin in E-FABP(-/-) keratinocytes relative to E-FABP(+/+) keratinocytes. We also determined that incorporation of linoleic acid was significantly reduced in E-FABP(-/-) keratinocytes. Although linoleic acid did not directly affect keratinocyte differentiation, keratin 1 expression was induced by the linoleic acid derivative 13(S)-hydroxyoctadecadienoic acid (13(S)-HODE), and this induction was concomitant with increased NF-κB activity. In E-FABP(-/-) keratinocytes, the expression of 13(S)-HODE and the subsequent induction of NF-κB activity was lower than in wild-type keratinocytes. The reduction of linoleic acid in E-FABP(-/-) keratinocytes led to decreased cellular 13(S)-HODE content, resulting in decreased keratin 1 expression through downregulation of NF-κB activity. The regulation of fatty acid metabolism by E-FABP during keratinocyte differentiation suggests that E-FABP may have a role in the pathogenesis of psoriasis.

The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin

Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

p63/p51-induced Onset of Keratinocyte Differentiation via the c-Jun N-terminal Kinase Pathway Is Counteracted by Keratinocyte Growth Factor

p63/p51, a homolog of the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. Because it remains unclear how p63 regulates epithelial cell differentiation, we examined the function(s) of p63 in keratinocyte differentiation through the use of a keratinocyte culture system. ΔNp63α (ΔNp51B), a p63 isoform specifically expressed in basal keratinocytes, suppressed the differentiation of specific late-stage proteins, such as filaggrin and loricrin. In contrast, ΔNp63α induced keratin 1 (K1), which is expressed at the start of differentiation, via c-Jun N-terminal kinase (JNK)/AP-1 activation. However, p63 did not induce K1 expression in the basal layer in vivo, although basal keratinocytes had high levels of p63. This discrepancy was explained by the suppression of K1 expression by dermis-secreted keratinocyte growth factor. This suppression occurred via extracellular signal-related kinase (ERK) signaling, and counteracted the p63-mediated induction of K1. Thus, a precise balance between p63 and keratinocyte growth factor mediates the onset of epithelial cell differentiation, through JNK and ERK signaling. These data may provide mechanistic explanations for the pathological features of skin diseases, including psoriasis.

Decreased keratinocyte motility in skin wound on mice lacking the epidermal fatty acid binding protein gene

Fatty acids are shown to be important in various skin functions. Fatty acid binding protein (FABP) is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system. Among the FABP family proteins, epidermal-type FABP (E-FABP) is solely expressed in keratinocyte but its specific role in skin is not yet fully established. We found an elevated expression of E-FABP in regenerative keratinocytes of healing wounds. However, E-FABP null mice showed no marked differences compared to wild type mice in the process of wound closure, in vivo. On the other hand, in keratinocyte culture, E-FABP gene disruption decreased the cell motility, but did not affect the cell proliferation. E-FABP deletion may be compensated for in vivo by the microenvironment comprised of various cells such as fibroblasts and endothelial cells around the wound. Our analyses suggest that the E-FABP elevation may be necessary for the activation of cell motility within regenerative epidermis during wound healing.

Bach1-dependent and -independent Regulation of Heme Oxygenase-1 in Keratinocytes

Bach1 is a member of the basic leucine zipper transcription factor family, and the Bach1/small Maf heterodimer specifically represses transcriptional activity directed by the Maf recognition element (MARE). Because Bach1 is a repressor of the oxidative stress response, we examined the function(s) of Bach1 in keratinocytes subjected to oxidative stress. Oxidative stress induced by H2O2 led to an increase in MARE activity and expression of heme oxygenase-1 (HO-1), an inducible antioxidant defense enzyme. Bach1 depletion by small interfering RNAs or by deletion of Bach1 enhanced HO-1 expression in the absence of H2O2, indicating that Bach1 is a critical repressor of HO-1 in keratinocytes. Although Bach1-deficient or -reduced keratinocytes expressed higher levels of HO-1 than control cells in response to H2O2, Bach1 down-regulation did not attenuate the production of reactive oxygen species by H2O2. In contrast, Bach1 overexpression abolished HO-1 induction by H2O2, which led to increased reactive oxygen species accumulation. HO-1 was induced during keratinocyte differentiation, but MARE activity did not change during differentiation. Furthermore, Bach1 overexpression did not inhibit differentiation-associated induction of HO-1 expression, suggesting that HO-1 induction in differentiation is independent of Bach1. Thus, in response to oxidative stress, Bach1 regulates the oxidation state through the negative control of HO-1 expression prior to terminal keratinocyte differentiation. However, Bach1-mediated repression is negated during keratinocyte differentiation.

Influences of p53 deficiency on the apoptotic response, DNA damage removal and mutagenesis in UVB-exposed mouse skin

p53 suppresses the genomic instability provoked by genotoxic agents. Ultraviolet (UV) B induces skin cancers by producing DNA damage and mutations in the skin genome, whereas the skin tissue responds to the UVB insult with cell cycle arrest and apoptosis as well as damage exclusion by DNA repair. To address the p53 contribution to these skin responses in vivo, we analyzed the time course of DNA damage removal, apoptosis induction and hyperplasia in the skin after UVB irradiation in p53-knockout mice. We also examined UVB-induced mutations in the skin. We found that p53 deficiency does not abolish the UVB-induced apoptotic response in the epidermis but delays the process and the following hyperplasia 12-24 h. Regardless of the p53 genotype, 1 kJ/m(2) UVB induced a total replacement of the epidermal layer by destroying the damaged epidermis by apoptosis and rebuilding a new one through hyperplasia. We failed to detect a clear defect in removal of UVB-induced DNA photolesions from the genome of the p53-deficient skin except for a delay in the epidermis, which seemed to result from the delay in the apoptotic response. However, we found that p53 deficiency enhanced UVB-induced mutagenesis. Furthermore, in a genetic study using Xpa-knockout mice, we showed that the enhanced mutagenic response depends on the activity of nucleotide excision repair (NER), which was also supported by the mutation spectrum observed in the UVB-exposed p53-knockout mice. These results indicate that p53 protects the skin genome from the UVB genotoxicity by facilitating NER, whereas its contribution to the UVB-induced apoptosis is limited.

p51/p63 inhibits ultraviolet B-induced apoptosis via Akt activation

The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member ΔNp51B/ΔNp63α (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, ΔNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of ΔNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that ΔNp51B blocks keratinocyte apoptosis. Although the exogenous expression of ΔNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH2-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.