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(The FASEB Journal. 2003;17:610-620.)
© 2003 FASEB

In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis

JIRO UMEDA, SHIGETOSHI SANO1, KAZUHIKO KOGAWA{dagger}, NOBORU MOTOYAMA{dagger}, KUNIHIKO YOSHIKAWA, SATOSHI ITAMI, GEN KONDOH{ddagger}, TAKESHI WATANABE{dagger} and JUNJI TAKEDA{ddagger}

Department of Dermatology,
{ddagger} Department of Social and Environmental Medicine, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; and
{dagger} Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan

1Correspondence: Department of Dermatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Osaka 565-0871, Japan. E-mail: sano{at}derma.med.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the function of Bcl-xL in the skin, we established keratinocyte-specific Bcl-x gene-targeted mice under the keratin 5 promoter (K5). K5.Bcl-xL-/- mice were viable, devoid of alteration in the development of skin or appendages. However, they harbored spontaneous apoptotic keratinocytes in the epidermis. Bcl-xL-deficient keratinocytes cultured in vitro readily underwent apoptosis in the absence of growth factors, but the addition of HGF or EGF resulted in restoration of cell survival, which was reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K). Topical treatment of K5.Bcl-xL-/- mice with wortmannin sensitized the skin for apoptosis induced by UV (UV) B, although wild-type epidermis was only marginally affected by this treatment, suggesting that the resistance to UVB largely depended on PI3K-Akt signaling in Bcl-xL-deficient mice but not in wild-type mice. Furthermore, UVB irradiation resulted in redistribution of phosphorylated Akt from the basal layer to the suprabasal layer, indicating that Akt could spatially cooperate with Bcl-xL upon UVB exposure in the upper epidermis where Bcl-xL is normally localized. These results suggest that Bcl-xL and the PI3K-Akt pathway form a cooperative, intercompensatory axis for the protection of epidermal keratinocytes from apoptosis in vivo.—Umeda, J., Sano, S., Kogawa, K., Motoyama, N., Yoshikawa, K., Itami, S., Kondoh, G., Watanabe, T., Takeda, J. In vivo cooperation between Bcl-xL and the phosphoinositide 3-kinase-Akt signaling pathway for the protection of epidermal keratinocytes from apoptosis


Key Words: Cre-loxP • UVB irradiation • sunburn cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THEBcl-2 FAMILYof proteins constitutes the central regulators for cell survival and programmed cell death or apoptosis (1 2 3 4) . This family comprises both pro- and antiapoptotic cytoplasmic proteins, all of which possess at least one of four conserved motifs known as Bcl-2 homology domains (BH1 to 4). The various members of the Bcl-2 family fall into three distinct groups in terms of their structure of the BH domains: the Bcl-2, Bax, and BH3 subfamilies. The Bcl-2 subfamily promotes cell survival while the other two subfamilies induce cell death. Pro- and antiapoptotic family members heterodimerize to limit one another's function, although heterodimerization is not required for the pro-survival function. Studies with the 3-dimensional structure of Bcl-xL revealed that formation of the {alpha} helices in its BH1, BH2, and BH3 regions results in generation of an elongated hydrophobic cleft, to which a BH3 of apoptogenic protein can bind to keep Bcl-xL inactive (5) . Many lines of evidence from biochemical and gene knockout approaches have demonstrated that most of the apoptotic pathways are required for cytochrome c release (6 , 7) . Cytochrome c is an essential cofactor for apoptotic protease-activating factor (Apaf) -1 that associates with procaspase-9, thereby triggering caspase-9 activation and initiating the subsequent caspase-dependent cell death. Both Bcl-2 and Bcl-xL prevent all mitochondrial changes, including cytochrome c release and a decrease of the mitochondrial transmembrane potential. By contrast, proapoptotic proteins such as tBid and Bax can heterodimerize with Bcl-xL, resulting in release of cytochrome c from mitochondria. Thus, anti-and proapoptotic molecules of the Bcl-2 family converge at the mitochondrial membrane and determine a cell's fate through regulation of downstream signaling (2 , 8) .

Since the skin is the outermost organ of the body, epidermal keratinocytes are continuously exposed to environmental stresses, including infectious agents, chemicals, and UV irradiation, UVB in particular (9) . UVB irradiation provokes a variety of biological responses, known as UVB responses, that are associated with DNA damage of cells. UVB-mediated DNA damage stabilizes the tumor-suppressor protein p53, which activates the downstream checkpoint molecules in the nucleus to perform cell cycle arrest, during which DNA repair can take place if the damage is relatively small (10 , 11) . Concomitantly, p53 stimulates the transcription of proapoptotic members of the Bcl-2 family such as Bax or directly up-regulates the death domain proteins, Apaf-1, and others. It is assumed that if the damage appears irreversible, the cells undergo apoptosis and are eliminated, thereby circumventing cancer development (12) . It has also been reported that UVB-induced apoptosis is introduced by direct triggering of cell surface receptors such as Fas without the need for ligands (9) . Furthermore, stress-associated kinases such as JNK and p38 (13) or certain phosphatases (14) are known to be involved in UVB responses. Whatever the underlying mechanism, different apoptogenic signals are likely to converge at the mitochondria via different proapoptotic members of the Bcl-2 family, with the outcome being determined by the intensity of survival signaling in the target cells (3 , 8) . The integrity of epidermal homeostasis upon UVB irradiation thus depends on the coordinated balance between pro- and antiapoptotic signaling at the mitochondria of keratinocytes.

High levels of Bcl-xL have been detected in the upper layers of the epidermis, but not in the basal cells, suggesting that Bcl-xL may have a fundamental role in the protection of keratinocytes from cell death induced by UV irradiation and other mutagens (15) . Transgenic mice overexpressing the Bcl-x gene under the control of the keratin 14 promoter demonstrated a dramatic increase in resistance to UVB irradiation or etoposide treatment (16) . In contrast, reduction of the Bcl-xL expression of keratinocytes by the introduction of an antisense inhibitor resulted in an increase in sensitivity to UVB irradiation or cisplatinum in vitro (17) . It is thus of paramount importance to identify the factors and signaling pathways that regulate Bcl-xL levels in the epidermis. Previous studies have reported that Bcl-xL expression in keratinocytes was down-regulated by blocking of the epidermal growth factor (EGF) receptor (R) (18 , 19) or by inhibition of substratum adhesion through forced suspension (20) , suggesting that signaling pathways initiated by activation of EGFR or adhesion receptors such as integrins converge on Bcl-xL as a shared target relevant to keratinocyte survival (20) . Other studies have demonstrated that Bcl-x gene expression is up-regulated by transcription factors including signal transducers and activators of transcription (Stats) (21 , 22) , NF-{kappa}B (23 , 24) , and members of the Ets family (25) . It is likely, however, that the regulation of Bcl-xL is multifactorial and depends on cell type, differentiation status, and the nature of the stimuli, and that cell survival is an outcome of the cooperation by Bcl-xL with other signaling for antiapoptosis (26) .

Despite accumulating evidence, the biological role of Bcl-xL in vivo remained unclear until Bcl-x gene-targeted mice became available. Germline knockout of the Bcl-x gene resulted in embryonic lethality around E13, suggesting that Bcl-xL is essential for the survival of immature cells during development (27) . To circumvent the embryonic lethality, we used the Cre-loxP system under the control of the keratin 5 promoter to generate keratinocyte-specific knockouts (28 , 29) . The Cre-loxP system is a powerful tool for gene targeting, not only because it allows us to disrupt an essential gene in a given cell lineage in which the Cre transgene is specifically expressed behind the lineage-specific promoter, but also because it avoids possible indirect effects from other cell lineages when disrupted in germline. We therefore used this system to investigate the role of Bcl-xL in the skin and to clarify the in vivo cooperation of Bcl-xL with another antiapoptotic pathway, PI3K-Akt, for prevention against apoptosis of epidermal keratinocytes in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Keratinocyte-specific Bcl-x gene targeting using the Cre-loxP strategy and PCR for screening
The target construct comprised a floxed Bcl-x gene at the exons 1 and 2 together with a floxed neo gene ligated downstream (Fig. 1 A). Gene targeting was performed in ES cells by homologous recombination, followed by removal of the neo resistant gene in the mice carrying the EIIa Cre transgene, by which Cre was expressed transiently as described previously (30) . This resulted in the generation of mice carrying the floxed Bcl-x gene. Generation of the keratin-5 promotor-driven (K5)-Cre transgenic mice was described previously (28) . K5-Cre transgenic mice carrying the heterozygous floxed Bcl-x gene (K5-Cre:Bcl-xflox/+) were mated with Bcl-xflox/flox mice. Pups (K5-Cre:Bcl-xflox/flox, K5-Cre:Bcl-xflox/+, Bcl-xflox/flox, Bcl-xflox/+) were used for further analyses. Allele-specific PCR was performed as illustrated in Fig. 1A . The primers, 'a'(5'-GTGTGAGTCCTGGCCCTGTCACTTA-3'), 'b'(5'-CTCAGAAGCCGCAATATCCCCTATT-3'), 'c'(5'-CCTTAGTGTTTTGGAAGCCACTGG-3'), and 'd'(5'-GTAACAAACGCCTACCACGACAGC-3') were used for screening. The 'a' and 'b' primers were used for PCR with 35 reaction cycles consisting of 1 min of denaturation at 93°C, 1 min of annealing at 55°C, and 2 min of elongation at 72°C. The 'c' and 'd' primers were used for PCR with 30 cycles of a reaction consisting of 1 min of denaturation at 95°C, 1.5 min of annealing at 60°C, and 1 min of elongation at 72°C. Primers 'a' and 'b' yielded PCR products of 276 and 310 bp specific for wild-type and floxed Bcl-x alleles, respectively. Primers 'c' and 'd' yielded PCR product of 600 bp specific for the disrupted Bcl-x allele.



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  		Figure 1. Keratinocyte-specific disruption of the Bcl-x gene by the Cre-loxP system. A) Targeting construct of Bcl-xflox allele before and after Cre-mediated recombination. The exons 1 and 2 of the Bcl-x gene were flanked by two identically oriented loxP sites (shaded triangles). "a" " b " and "c" " d " indicate primers used for the detection of the floxed and the disrupted Bcl-x allele, respectively. B) Keratinocyte-specific deletion of the Bcl-xL protein in K5-Cre:Bcl-xflox/flox mice. Lysates of epidermal keratinocytes from Bcl-xflox/flox (+/+), K5-Cre:Bcl-xflox/+ (+/-), and K5-Cre:Bcl-xflox/flox mice (-/-) were immunoblotted with antibodies to Bcl-xL, Bcl-2, and ß-actin (left panels). Signals of Bcl-xL in liver cells from K5-Cre:Bcl-xflox/flox mice were detected as much as in wild-type (right panels). ND, not determined. C) Semiquantitative evaluation of Bcl-xL (shaded bars) and Bcl-2 protein (black bars) in keratinocytes from Bcl-xL +/+, +/-, and -/- mice using a densitometer based on signals of immunoblotting normalized by ß-actin signal of each group. Arbitrary unit is based on the ratios (densitometric value of blotting of interest, that of ß-actin) to those from wild-type keratinocytes as controls.

Wound healing assay
These experiments were performed with strict adherence to institutional guidelines for minimizing distress in experimental animals. Under anesthesia with sodium pentobarbital, full-thickness skin excisions were made on the back of 6- to 8-wk-old mice using a 6 mm diameter biopsy punch. The diameter of the wound was measured daily with a caliper.

Intradermal injection of recombinant HGF
Two-day-old mice were intradermally injected at the dorsum with 0.1 mL of recombinant HGF (Becton Dickinson Bio sciences, Franklin Lakes, NJ, USA) at a concentration of 3 µg/mL in PBS daily for 3 days (a total of 900 ng), and 1 day after the last injection the back skin at the injected site was biopsied for the histological examination. PBS was used as a vehicle control.

Preparation of keratinocytes and culture in vitro
Full-thickness skin taken from newborn to 5-day-old mice was treated with 250 U/mL of dispase (Godo Shusei, Tokyo, Japan) overnight at 4°C, after which the epidermis was peeled off from the dermis and trypsinized to prepare single cells. These were suspended in MCDB153 medium (Kyokuto Pharmaceutical, Tokyo, Japan) supplemented with 0.1 mM monoethanolamine, 0.1 mM phosphoryl ethanolamine, 0.5 µM hydrocortisone at 37°C under an atmosphere of 5% CO2. After being cultured in dishes precoated with collagen type I (Iwaki Glass, Tokyo, Japan) for 5 h until the cells had become attached, any unattached cells were removed by washing with PBS. The attached cells were then further cultured in the media as described in the absence or presence of growth factors. Growth factors used in our study included HGF, EGF (Upstate Biotechnology, Lake Placid, NY, USA), IGF-I (Austral Biological, San Ramon, CA, USA), and insulin (Sigma, St. Louis, MO, USA). In some experiments, cells were pretreated 30 min with wortmannin (Nacalai Tesque, Kyoto, Japan), a specific inhibitor of PI3K, at a final concentration of 50 nM throughout the culture in the absence or presence of growth factors.

UVB irradiation
Newborn to 3-day-old mice were used for these experiments. They were restrained with vinyl tape across the upper back and irradiated under a UVB irradiator (Torex, FL20S /DMR, Toshiba, Tokyo, Japan) at a dose rate of 8.3 J/m2/s. The mice were killed after irradiation at the indicated time. The dorsal skin was excised and fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosin. Sunburn cells were counted with morphological examination of the specimens under light microscopy. Three to four mice per group were used and the sunburn cell count was performed for at least three sections of a preparation taken from each mouse. In some experiments, the mice were topically treated with wortmannin 3 h before UVB irradiation. Wortmannin in Me2SO stock was diluted with acetone to 5 µM; 50 µL was applied to the dorsal skin. Before treatment with wortmannin, the stratum corneum of the dorsal skin was removed to increase skin permeability by stripping with Scotch® tape. An equal volume of Me2SO was diluted with acetone and used as a vehicle control.

Western blot
Equivalent numbers of keratinocytes or liver tissue of equivalent wet weight was lysed with ice-cold lysis buffer. To evaluate the effect of wortmannin, cultured keratinocytes remained untreated or were pretreated with 50 nM of wortmannin for 30 min, then unstimulated or stimulated for 20 min with 50 ng/mL of HGF, washed with PBS, and lysed in lysis buffer. The lysis buffer contained 0.5% Nonidet P-40, 10 mM Tris-HCl (pH7.4), 150 mM NaCl, 1 mM EDTA, 0.2 mM phenyl methyl sulfonyl fluoride, 1 mM NaVO3, and 5 µg/mL aprotinin. Lysates were further diluted with an equal volume of 2 x sample buffer (4% SDS, 20% glycerol, 12.5 mM Tris (pH 6.8), 0.004% bromophenol blue, 10% 2-mercaptoethanol), followed by boiling for 5 min, separation on SDS-polyacrylamide gel, transfer to a nitrocellulose membranes, and blotting with antibodies. The antibodies used in this study included anti-Bcl-x (1:500, Transduction Laboratories, Lexington, KY, USA), Bcl-2 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ß-actin (1:1000, Sigma), Akt (1:1000, Cell Signaling Technology, Beverly, MA, USA), phospho-Akt (1:1000, Cell Signaling Technology), Stat3 (1:1000, C-20, Santa Cruz), phospho-Stat3 (1:1000, specific for 705Y, Cell Signaling Technology), Bad, phospho (Ser112)-Bad (1:500, Cell Signaling Technology), and phospho (Ser136)-Bad (1:500, Upstate Biotechnology, Lake Placid, NY), followed by treatment with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin (1:1000, Amersham, Buckinghamshire, UK) and visualization with an enhanced chemiluminescence (ECL) system (Amersham). In some experiments, amplified signals were scanned with a densitometer and analyzed on NIH Image software to evaluate the blotted proteins semiquantitatively.

Flow cytometry
Cultured cells were harvested and fixed with 70% ethanol for 4 h. The cells were suspended in 100 µL phosphate-citrate buffer (mixture of 0.2 M Na2HPO4 with 0.1 M citric acid at 24:1) for 30 min, washed, and suspended in a final volume of 0.3 mL of PBS. Propidium iodide (3 µL of 1 mg/mL stock solution, Calbiochem-Novabiochem, San Diego, CA, USA) and RNase (3 µL of 1 mg/mL stock solution, Sigma) were added to the cell suspensions. Propidium iodide signals were measured with a FACScan (Becton Dickinson, San Jose, CA, USA). The % of cells below the G1 peak (sub-G1 fraction) measured by CellQuest software (Becton Dickinson) was determined as the apoptotic cell population.

Immunohistochemical analyses, TUNEL staining
For staining of phosphorylated Akt, deparaffinized skin specimens in xylene and ethanol were incubated in 10 mM sodium citrate and subjected to microwaving for antigen unmasking, then treated with H2O2 to block endogenous peroxidase activity and washed with PBS. They were blocked with 5% rabbit serum (DAKO, Glostrup, Denmark), treated with anti-phospho Akt (1:50, Ser473, IHC specific; Cell Signaling Technology), incubated with a goat anti-rabbit horseradish peroxidase conjugate (DAKO), and visualized with diaminobenzidine. For staining of Bcl-2 and Bcl-xL, snap-frozen sections from the skin specimen were stained with anti-Bcl-2 (Santa Cruz Biotechnology) or anti-Bcl-xL antibody (Santa Cruz Biotechnology) and incubated with anti-mouse or anti-rabbit horseradish peroxidase conjugate, respectively, followed by the same procedure described above. Unimmunized rabbit serum or nonspecific mouse IgG was used as a negative control for the first antibody. TUNEL staining was performed according to the manufacturer's protocol (Apoptosis Kit II, MBL, Nagoya, Japan). Skin specimens were deparaffined, treated with proteinase, DNA nick-end labeled with biotinylated dUTP mediated by deoxynucleotidyl transferase, then stained with avidin-conjugated FITC.

Statistical analysis
Groups were compared using one- or two-tailed Student's t tests. P< 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of keratinocyte-specific Bcl-xL disrupted mice
Since deletion of the Bcl-x gene resulted in early embryonic lethality (27) , we decided to disrupt this gene selectively in keratinocytes to study its in vivo role in the skin. The floxed allele of the Bcl-x gene was constructed by introducing two tandem loxP sequences at 5' of the exon 1 and at 3' of the exon 2, as shown in Fig. 1A . Since the ATG start codon is present in the exon 2 (31) , no transcript of the Bcl-x gene was generated upon recombination by Cre (data not shown). Mice carrying the floxed Bcl-x gene in both alleles were mated with mice carrying the Cre transgene behind the keratin 5 (K5) promoter (28) and the floxed Bcl-x gene in one allele. Offspring with four genotypes were generated, specified as Bcl-xflox/+, Bcl-xflox/flox, K5-Cre: Bcl-xflox/+, and K5-Cre: Bcl-xflox/flox. The former two genotypes represented the wild-type (+/+), the third one the heterozygous (+/-), and the last one the disrupted homozygous (-/-) of the Bcl-x gene specifically in keratinocytes. Screening of genotype was performed by allele-specific PCR (data not shown). The numbers of pups with four genotypes were born in accordance with the Mendel's laws (Bcl-xflox/+, n=60; Bcl-xflox/flox, n=65; K5-Cre: Bcl-xflox/+, n= 56; K5-Cre: Bcl-xflox/flox, n=61 for a total of 242 mice), indicating that the ablation of the Bcl-x gene occurred exclusively in cells in which the K5 promoter was active, but did not affect embryonic differentiation or survival. K5.Bcl-xL-/- mice were born normal, and their skin and hair development showed a normal gross appearance (data not shown). Western blot analyses of keratinocytes revealed that the Bcl-xL protein level was reduced to ~47% of wild-type and completely absent in the Bcl-x heterozygous and-deficient mice, respectively (Fig. 1B, C , hatched bars). Similarly, immunohistochemical staining of skin sections with anti-Bcl-xL antibody revealed that Bcl-xL was absent in the epidermis of K5.Bcl-xL-/- mice (data not shown) but expressed in the suprabasal layer of the epidermis of wild-type mice (data not shown) as previously reported (15) . By contrast, as much Bcl-xL was observed in the liver cell lysates of K5.Bcl-xL-/- mice as in those of controls (Fig. 1B ), clearly indicating that the ablation of the Bcl-x gene occurred selectively in keratinocytes mediated by the K5 promoter-dependent Cre-loxP system. Bcl-2 protein in K5.Bcl-xL-/- keratinocytes was found to be increased up to 2.3-fold of controls (Fig. 1C , black bars), suggesting that Bcl-2 up-regulation occurred reciprocally to Bcl-xL deficiency.

No abnormalities of K5.Bcl-xL-/- mice in wound healing and hair cycle
We previously reported that keratinocyte-specific Stat3-/- mice demonstrated delayed wound healing and failure in hair cycling, resulting in the development of spontaneous wounds and alopecia (29 , 32) . Since Bcl-xL is a downstream target of Stat3 (21 , 26 , 33) , it was considered possible that wound healing and the hair cycle process might be affected by the ablation of the Bcl-x gene in keratinocytes, even though the gross appearance of the skin and hair development were normal. K5.Bcl-xL-/- mice, however, showed normal wound healing comparable to that of wild-type mice, by assessing the healing process of skin ulcers generated with a biopsy punch in the back (data not shown). Furthermore, histological examination of hair follicles from skin biopsy taken every 2 days for up to 40 days after birth revealed no significant differences in the hair cycling process between K5.Bcl-xL-/- mice and wild-type mice (data not shown). This result contrasts to that from a previous study of transgenic mice overexpressing Bcl-xL under control of the K14 promoter (34) . Those Bcl-xL-overexpressing transgenic mice demonstrated a decrease in the duration of the anagen and prolongation of the telogen. These findings suggest that Bcl-xL of keratinocytes is dispensable for wound healing and the hair cycle, both of which require Stat3 activation (29 , 32) .

Increased number of apoptotic cells in epidermis of K5.Bcl-xL-/- mice
Histological examination of the skin sections disclosed that K5.Bcl-xL-/- mice harbored an increased number of so-called sunburn cells, which are characterized by condensed nuclei and eosinophilic cytoplasm in hematoxylin-eosin staining, representing apoptotic epidermal keratinocytes (arrows in right panel of Fig. 2 A). By contrast, virtually no sunburn cells were found in the epidermis of wild-type mice (Fig. 2A , left panel). Similarly, the TUNEL assay of skin specimens from the mutant mice revealed an increase in the number of TUNEL-positive cells in the epidermis, whereas there were no TUNEL-positive cells in wild-type skin (Fig. 2B ). No sunburn cells or TUNEL-positive cells were found in Bcl-xL heterozygous mice as in wild-type mice (data not shown), although the Bcl-xL protein level in the keratinocytes of heterozygous mice was approximately half of wild-type by immunoblotting (Fig. 1C ). UVB irradiation onto the back of K5.Bcl-xL-/- mice resulted in further increase in number of epidermal sunburn cells in a dose-dependent manner, although the net increase was no greater than that found in wild-type mice (Fig. 2C ). These findings indicate that Bcl-xL plays an antiapoptotic role in the constitutive survival of epidermal cells in vivo, but it appeared less likely that Bcl-xL deficiency enhanced the sensitivity to UVB. This result appeared inconsistent with the previous reports demonstrating that Bcl-xL of epidermal cells played a critical role in the protection of skin from UVB-induced apoptosis (16 , 17) . Rather, we suggested an alternative prosurvival mechanism in Bcl-xL-/- keratinocytes against UVB exposure in addition to a relative abundance of Bcl-2 (Fig. 1C ).



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  		Figure 2. Apoptotic keratinocytes in the epidermis of K5.Bcl-xL-/- mice and effect of UVB irradiation. A) Histological appearance of skin specimen taken from the dorsum (hematoxylin-eosin staining). Sunburn cells, characterized by condensed nuclei with eosinophilic or vacuolated cytoplasms, are seen in the epidermis of K5.Bcl-xL-/- mice (arrows, right panel) whereas none are found in wild-type mice (left panel). Scale bar, 50 µm. B) TUNEL staining. Epidermis of K5.Bcl-xL-/- mice shows scattered signals of TUNEL stain probed by FITC (arrows, right panel); no TUNEL positive cells are found in the epidermis of wild-type mice (left panel). Yellow lines, interface between epidermis and dermis. Scale bar, 100 µm. C) Numbers of sunburn cells after UVB irradiation. Sunburn cells are counted per cm in the skin sections of wild-type (shaded bars) and K5.Bcl-xL-/- mice (black bars) 24 h after UVB irradiation at the indicated doses. Note that the net increase in sunburn cells by UVB irradiation in K5.Bcl-xL-/- mice is no greater than that in wild-type mice.

Increased apoptotic incidence of Bcl-xL-deficient keratinocytes when cultured in vitro in the absence of growth factors
Isolation of keratinocytes from the epidermis imposes an apoptotic pressure on them because of deprivation of the environmental advantages, which include not only cell–cell and cell–matrix attachment but also endogenous diffusible factors that stimulate them to survive. Consistent with the finding of spontaneous apoptotic keratinocytes in K5.Bcl-xL-/- mice, their keratinocytes in primary culture in the absence of growth factors readily underwent apoptosis at a much higher frequency than did wild-type keratinocytes (Fig. 3 A–D). Since attachment of freshly isolated K5.Bcl-xL-/- keratinocytes to dishes was reduced to ~70% of that of wild-type cells (data not shown), primary adherent cells were used in this assay. The observation under a phase-contrast microscope revealed that many more apoptotic cells were found following culture time in the K5.Bcl-xL-/- than wild-type keratinocytes. Apoptotic keratinocytes were detached from the dishes (Fig. 3A ) and underwent morphological changes such as cell ballooning, nuclear condensation, and bleb formation (Fig. 3A , inset). Similarly, FACS analysis using propidium iodide revealed that more apoptotic cells that fell into the sub-G1population were found in K5.Bcl-xL-/- than in wild-type keratinocytes (Fig. 3C ). Apoptotic cells were quantitatively evaluated by either direct cell counting based on morphological changes (Fig. 3B ) or by FACS analysis (Fig. 3D ). The results of the two methods showed good correlation. It is thus clear that Bcl-xL plays an antiapoptotic role in the survival of cultured keratinocytes deprived of growth factors. Whereas Bcl-xL-deficient keratinocytes showed a high incidence of apoptosis in vitro up to >50% at 48 h of culture (Fig. 3B , 3D ), far fewer spontaneous sunburn cells were found in vivo. This discrepancy could be attributed to the cutaneous microenvironment where a variety of diffusible factors are present and play a stimulatory role for keratinocyte survival in vivo.



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  		Figure 3. Apoptotic keratinocytes in primary culture deprived of growth factors. A) Morphological appearance of keratinocytes of wild-type (left panels) and K5.Bcl-xL-/- mice (right panels) cultured in the absence of growth factor under phrase-contrast microscopy. Viable keratinocytes are dark, polygonal cells and attached to culture dishes whereas apoptotic keratinocytes are small, bright cells with morphological changes, including nuclear condensation and bleb formation, being detached from the dishes (arrowheads in inset). Scale bar, 100 µm. B) Quantification of apoptosis based on morphological changes under phrase-contrast microscopy in the cultures of wild-type (shaded bars) and K5.Bcl-xL-/- keratinocytes (black bars) in the absence of growth factors. Calculation of the % of apoptosis: number of cells with apparent apoptotic changes/number of apparent viable cells + number of cells with apparent apoptotic changes x 100. Differentiated keratinocytes, which are seen as floating, bright cells without changes in their nuclei, are omitted for counting. Mean values with SD are based on cell counts of at least 200 cells each from 4 separate fields. The results are representative of at least 3 independent experiments with reproducible results. C) FACS analysis. Cultured keratinocytes are stained with propidium iodide as described in Materials and Methods. Percentages refer to the sub-G1 population that corresponds to the apoptotic population of wild-type (left panels) and K5.Bcl-xL-/- keratinocytes (right panels). D) Evaluation of apoptosis based on FACS analyses of wild-type (shaded bars) and K5.Bcl-xL-/- keratinocytes (black bars) cultured in the absence of growth factors. Mean values with SD from 3 independent experiments are shown.

Restoration of Bcl-xL-deficient keratinocytes in vitro through activation of the phosphoinositide-3 kinase-Akt signaling pathway
To examine whether growth factors could rescue the Bcl-xL-/- keratinocytes from apoptosis in vitro, we cultured them in the presence of growth factors. Notably, apoptotic Bcl-xL-/- cells were significantly reduced in number by stimulation with epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin, or insulin-like growth factor (IGF)-I, whereas the survival of wild-type cells was not markedly improved by these growth factors (Fig. 4 A and data not shown). This attenuation was canceled by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI3K) (Fig. 4B and data not shown). These results implied that Akt, which is a downstream molecule of PI3K, exerted an antiapoptotic effect and could compensate for the defect of Bcl-xL, whereas wild-type cells were resistant to wortmannin. This notion was further confirmed by the fact that HGF induced the phosphorylation of Akt, which was inhibited by wortmannin in Bcl-xL-/- as well as wild-type cells (Fig. 4C ). The first component of the apoptotic machinery found to be phosphorylated by Akt was the Bcl-2 family member Bad (35 , 36) . In fact, HGF stimulation of Bcl-xL-deficient keratinocytes as well as wild-type cells leads to phosphorylation of Bad at Ser-136 and Ser-112, and the wortmannin treatment also attenuated its phosphorylation at both sites (Fig. 4C ). This result indicated that activation of the PI3K-Akt pathway by growth factor stimulation induced Bad phosphorylation, thereby dissociated from antiapoptotic Bcl-2 family member, resulting in cell survival. Among antiapoptotic Bcl-2 members associated with Bad, Bcl-2 could be available only in Bcl-xL-/- keratinocytes, in which Bcl-2 was found to be up-regulated (Fig. 1B , 1C ). However, Bcl-2 expression in Bcl-xL-/- keratinocytes was not further elevated by HGF stimulation (data not shown). HGF also induced Stat3 phosphorylation in Bcl-xL-/- and wild-type cells but wortmannin did not affect the phosphorylation of Stat3 (data not shown).



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  		Figure 4. Rescue of K5.Bcl-xL-/- keratinocytes from apoptosis by stimulation with growth factors and involvement of the PI3K-Akt pathway. A) Survival of K5.Bcl-xL-/- keratinocytes (black bars) is restored by stimulation with 30 ng /mL EGF or 30 ng/mL HGF in culture for 24 h, whereas these growth factors do not further rescue wild-type keratinocytes (shaded bars). Apoptosis is assessed by FACS analysis. Mean values with SD are from 3 independent experiments. *P< 0.05, **P< 0.01. NS, not significant. B) Wortmannin treatment (50 nM) cancels the antiapoptotic effect of 30 ng/mL HGF on Bcl-xL-/- keratinocytes (black bars) but not on wild-type cells (hatched bars). Mean values with SD are shown from 3 independent experiments. **P< 0.01. C) Immunoblotting of lysates from wild-type (+/+) and Bcl-xL-deficient (-/-) keratinocytes with indicated antibodies after the indicated treatment. D) Effect of intradermal injection of HGF on the epidermis of K5.Bcl-xL-/- mice. Intradermal injection to 2-day-old K5.Bcl-xL-/- mice with 0.1 mL HGF at a concentration of 3 µg/mL in PBS daily for 3 days (a total of 900 ng) results in significant decrease of sunburn cells (**P=0.0007). Note that PBS injection alone attenuates apoptotic incidence to some extent although not statistically significant (NS, not significant; P=0.143). Results are averages in specimens from three mice of each group.

Decrease of sunburn cells in number by intradermal HGF injection into K5.Bcl-xL-/- mice
Being prompted by the above results, we next examined whether HGF administration in vivo could attenuate keratinocytes apoptosis in K5.Bcl-xL-/- mice. Intradermal injection at a total dose of 900 ng recombinant HGF significantly reduced the number of spontaneous sunburn cells of K5.Bcl-xL-/- mice (Fig. 4D ), suggesting that forced activation of the PI3K-Akt pathway in vivo resulted in the attenuation of apoptosis of Bcl-xL-deficient keratinocytes, as well as that shown in the cultured cells in vitro (Fig. 4A ). PBS injection alone could decrease the spontaneous sunburn cells to some extent, although the statistical significance was not evident (Fig. 4D ). It was possible that PBS injection caused a nonspecific cutaneous reaction that could stimulate the PI3K-Akt pathway in keratinocytes.

Topical application of wortmannin sensitizes the skin of K5.Bcl-xL-/- mice to UVB-induced apoptosis
We next examined the involvement of the PI3K-Akt pathway in UVB-induced apoptosis in vivo. Mice were topically treated with wortmannin on the dorsal skin before UVB irradiation to reduce the PI3K signaling in vivo. Wortmannin treatment markedly enhanced the sensitivity of keratinocytes to UVB in K5.Bcl-xL mice (Fig. 5 A), which developed numerous sunburn cells in the epidermis (Fig. 5B ). In contrast, wild-type mice were only marginally affected in their sensitivity to UVB by the wortmannin treatment (Fig. 5A ). This result suggested not only that the activation of Akt was required for the protection from UVB-induced apoptosis in the keratinocytes in which Bcl-xL was deficient, but that the dependency of Akt was small in wild-type epidermis where Bcl-xL is present. Immunohistochemical study of the skin sections suggested UVB response affected Akt phosphorylation. Before irradiation, phosphorylated Akt was confined to the basal layer of epidermis of wild-type and Bcl-xL mice (Fig. 5C , upper panels). However, upon UVB irradiation, the signal of phosphorylated Akt was apparently distributed over the suprabasal layer in both types of mice (Fig. 5C , lower panels). Signals throughout the epidermis when irradiated were not merely a background because unimmunized rabbit serum did not show any positive staining (data not shown). Whereas Bcl-2 was found to be up-regulated in Bcl-xL-/- keratinocytes (Fig. 1B , 1C ), UVB irradiation did not influence the expression and distribution of Bcl-2 (data not shown). Therefore, this result suggests that emergence of active Akt in the suprabasal layer may contribute to the protection of epidermal keratinocytes from cell death by UVB irradiation, which mainly affects the uppermost of epidermis. Because Bcl-xL is normally localized in the suprabasal layers (data not shown) (15) , Bcl-xL and Akt might cooperate spatially in the epidermis for antiapoptosis upon UVB irradiation. In conclusion, the present study using keratinocyte-specific Bcl-xL-deficient mice revealed that Bcl-xL cooperates with the PI3K-Akt pathway for keratinocyte survival in the skin.



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  		Figure 5. Enhanced UVB sensitivity of K5.Bcl-xL-/- mice by topical treatment with wortmannin. A) Numbers of apoptotic keratinocytes in K5.Bcl-xL-/- mice (black bars) are greatly increased after UVB irradiation (1000 J/m2) by pretreatment with wortmannin (5 µM, 50 µL) vs. those in wild-type keratinocytes (shaded bars). *P< 0.05, **P< 0.01. B) Histological appearance of UVB-irradiated skin from wild-type mice (+/+, left panels) and K5.Bcl-xL-/- mice (-/-, right panels) after topical treatment with vehicle only (top panels) and wortmannin (bottom panels). Arrowheads, sunburn cells. H-E stain. Scale bar, 50 µm. C) Immunohistochemical study with anti-phospho-Akt antibody of untreated (upper panels) and UVB-treated (bottom panels, 1000 J/m2, 3 h) skin of wild-type (+/+, left panels), and K5.Bcl-xL-/- mice (-/-, right panels). B, basal layer; Sp, suprabasal layer. Scale bar, 66 µm. Note that no signal is found when unimmunized rabbit serum is used as control for the first antibody (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To maintain epidermal integrity and homeostasis, antiapoptotic molecules in keratinocytes play a critical role in the protection from environmental insults, including UV and genotoxic chemicals (12 , 37) . Two members of the antiapoptotic Bcl-2 family, Bcl-2 and Bcl-xL, are present in the epidermis (15 , 38) . The antiapoptotic role of Bcl-2 in keratinocytes has been demonstrated in transgenic mice overexpressing Bcl-2 under the keratin 1 promoter (39) . Their keratinocytes were found to be resistant to apoptotic stimuli including UVB irradiation and chemical mutagens. However, germline targeting of the Bcl-2 gene yielded viable mice whose epidermis was not subject to apoptosis and that were indistinguishable from wild-type littermates except for failure in melanin synthesis, resulting in the appearance of gray hair (40) . This discrepancy implies that Bcl-2 might not play a major role in antiapoptosis of the epidermis and that Bcl-2 might be a functional redundant in the presence of Bcl-xL. In addition, Bcl-2 expression is restricted to the basal layer of the epidermis (38) whereas Bcl-xL is preferentially expressed in the suprabasal layer (data not shown) (15) . These findings suggest that Bcl-xL may have a fundamental role in "the first-line protection" of epidermis against external stimuli. We found that Bcl-xL-deficient keratinocytes showed an increased level of Bcl-2 protein, although the underlying mechanism remains unclear. It may be due to a reciprocal gene regulation between Bcl-x and Bcl-2, which could compensate for Bcl-xL deficiency, being consistent with the notion of mutually exclusive expression of these molecules within the epidermis. However, the present study strongly suggests that Bcl-xL has a significant antiapoptotic role under growth factor-deprived condition that could not compensated for by the partial up-regulation of Bcl-2.

Since germline knockout of Bcl-x gene lead to embryonic lethality, we disrupted it under the control of the keratin 5 promoter with the aid of the Cre-loxP technology to delete Bcl-xL in the epidermis. K5 promoter-dependent as well as K14 promoter-dependent gene ablation occurred in both interfollicular epidermal keratinocytes and outer root sheath (ORS) cells (29 , 32) . K5.Bcl-xL-/- mice were viable and exhibited normal skin appearance in gross. Epidermal differentiation and the hair cycle were not affected in these mice. Pena et al. have demonstrated that K14.Bcl-xL overexpression resulted in a shortened anagen and prolongation of the telogen phase (34) . Whereas they suggested that overexpressed Bcl-xL perturbed the hair-growth cycle through forced survival of ORS cells, our study indicated that Bcl-xL is dispensable for the hair cycle. However, we do not exclude the possibility that Bcl-2 could compensate for Bcl-xL deficiency in the hair cycle. Wound healing of skin was not affected in K5.Bcl-xL-/- mice, although it was reported that Bcl-xL was involved in wound repair (16) . These results contrast to the phenotype of K5.Stat3-/- mice in which wound healing and hair cycling were impaired because of the failure in keratinocyte migration (29 , 32) , indicating that the role of Stat3 in the keratinocyte migration was not mediated by Bcl-xL, a downstream molecule of Stat3 (26) . Indeed, Bcl-xL-deficient keratinocytes did not demonstrate impaired migration in vitro (data not shown).

A previous study was able to delineate the role of Bcl-xL in keratinocytes by finding that treatment with an antisense inhibitor to Bcl-xL increased the sensitivity of cultured keratinocytes to UVB irradiation (17) . In turn, skin from Bcl-xL-overexpressing transgenic mice has been shown to be much more resistant to UVB irradiation (16) in the same manner as mice overexpressing Bcl-2 (39) . In our study, K5.Bcl-xL-/- mice were found to harbor spontaneous sunburn cells that were not in wild-type mice. Unexpectedly, however, K5.Bcl-xL-/- mice did not show an enhanced sensitivity to UVB irradiation, since the increment of sunburn cell number in response to increasing doses of UVB was no greater than that of wild-type mice. This result appeared inconsistent with previous studies, but may be due to the difference in the experimental condition between in vitro and in vivo. Bcl-xL-deficient keratinocytes generated in vitro by introduction of antisense oligonucleotides to Bcl-x (17) could not take advantage of alternative signals from the cutaneous microenvironment, which we suggest is critically important for antiapoptosis in K5.Bcl-xL-/- mice. The apoptotic incidence in Bcl-xL-deficient keratinocytes in culture was much greater than that found in vivo. Stimulation of cultured cells with growth factors such as HGF, EGF, insulin, or IGF-I resulted in attenuation of their apoptosis. Contribution of the PI3K-Akt signaling pathway to the protection of Bcl-xL-deficient keratinocytes from apoptosis was clearly demonstrated by in vitro inhibition experiments using wortmannin. The finding that intradermal injection of HGF significantly reduced the number of sunburn cells in these mice suggests that activated Akt could exert an antiapoptotic effect on Bcl-xL-deficient keratinocytes in vivo.

Upon activation by PI3K, Akt induces full activation of Akt kinase activity, leading to phosphorylation of Bad at Ser 136 (35 , 36 , 41) . Bad phosphorylation results in sequestration in the cytoplasm in association with 14–3-3 proteins leaving from a mitochondrial location after dissociation with antiapoptotic Bcl-2 members (42) . Not only wild-type but also Bcl-xL-deficient keratinocytes demonstrated Bad phosphorylation at Ser-136 and Ser-112 upon HGF stimulation, which was sensitive to wortmannin treatment, again suggesting that the PI3K-Akt pathway plays an antiapoptotic role in restoration of Bcl-xL-deficient keratinocytes through sequestration of Bad from Bcl-2 at the mitochondrial membrane. Alternatively, besides Bad phosphorylation, recent studies have found that Akt can directly regulate caspase activation either at a premitochondrial level (43) or at a postmitochondrial level downstream of cytochrome c release and before activation of caspase-9 (44) . A recent report demonstrated that the PI3K-Akt pathway was required for keratinocyte survival independent of Bcl-xL expression (45) , and may support our findings. Epicutaneous treatment with wortmannin of K5.Bcl-xL-/- mice resulted in a marked sensitization to UVB irradiation; control mice were not significantly affected by this treatment, suggesting that dependency on PI3K-Akt was reciprocal to Bcl-xL expression. UVB irradiation resulted in translocation of phosphorylated Akt from the basal cell layer to throughout epidermis in wild-type and K5.Bcl-xL-/- mice, although the underlying mechanism remains to be elucidated. Since Bcl-xL is expressed predominantly in the suprabasal keratinocytes, the redistribution of active Akt over the suprabasal layer might represent the spatial compensation for Bcl-xL deficiency upon UVB irradiation. Thus, these data provide compelling evidence that Akt can compensate for Bcl-xL deficiency to form a " fail-safe" system against apoptotic stimuli.

Upstream signals that regulate Bcl-xL levels have been reported to be EGFR (18 , 19 , 45) , Stats (21 , 22 , 26) , NF-{kappa}B (23 , 24) , Ets (25) , and adhesion signal to matrix (20) . Earlier studies have reported that EGFR-dependent keratinocyte survival was associated with Bcl-xL (46) . As EGFR activation induced by ligands resulted in up-regulation of Bcl-xL through activation of Stat3, EGFR blockade by a specific tyrosine inhibitor or monoclonal antibody against EGFR resulted in down-regulation of Bcl-xL (18) . Therefore, forced expression of Bcl-xL rescued keratinocyte survival when EGFR was blocked (19) . We previously generated keratinocyte-specific Stat3-/- mice (29) in which the epidermis was susceptible to apoptosis; this could be attributed to down-regulation of Bcl-xL (S. Sano, et al., unpublished results). It was also reported that Stat3 can function as an oncogene when activated constitutively, leading to up-regulation of Bcl-xL and cell transformation both in vitro and in vivo (21) , whereas an increase in EGFR signaling was found to correlate with a constitutive active state of Stat3 and higher Bcl-xL levels in squamous cell carcinoma (47) . Moreover, in vivo treatment with an antisense inhibitor to Stat3 increased tumor cell apoptosis and decreased Bcl-xL expression in a xenograft model (33) . Thus, constitutively active or enhanced antiapoptotic signals, including Bcl-xL, may possibly lead to cancer development (48 , 49) . In this context, K5.Bcl-xL-/- mice will become an animal model to delineate the relation between Bcl-xL and skin oncogenesis induced by either chemical mutagen or UV irradiation.

In this study we investigated the in vivo role of Bcl-xL using keratinocyte-specific Bcl-xL-deficient mice and managed to clarify the intricate cooperation and intercompensatory action of Bcl-xL and another antiapoptotic molecule, Akt, for the prevention of keratinocytes from apoptosis; we have depicted these signal cross-talks in Fig. 6 .



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  		Figure 6. Cooperative, intercompensatory action of Bcl-xL and the PI3K-Akt pathway for the protection of keratinocytes from apoptosis.


   ACKNOWLEDGMENTS
 
The first two authors contributed equally to this work. We would like to thank Ms. M. Nagai for her outstanding technical assistance with histological work and Dr. S. Shimizu for helpful discussions. This work was supported by grants of the Ministry of Education, Science, Sports, and Culture of Japan, and by Shiseido Research Fund for Skin Aging.

Received for publication June 24, 2002. Accepted for publication December 27, 2002.


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 RESULTS
 DISCUSSION
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