Differentiation Induction of Human Keratinocytes by ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 279, No. 31, Issue of July 30, pp. 32191–32195, 2004 Printed in U.S.A.

Differentiation Induction of Human Keratinocytes by Phosphatidylethanolamine-binding Protein* Received for publication, April 12, 2004 Published, JBC Papers in Press, May 20, 2004, DOI 10.1074/jbc.M404029200

Takehiko Yamazaki‡, Hajime Nakano§, Makoto Hayakari‡, Masanori Tanaka¶, Jun Mayama‡, and Shigeki Tsuchida‡§储 From the ‡Second Department of Biochemistry, §Department of Dermatology, and ¶Second Department of Pathology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan

Phosphatidylethanolamine-binding protein (PEBP)1 is a 21to 23-kDa basic protein that was originally purified from bovine * This work was supported in part by grants-in-aid from the Ministry of Health and Welfare of Japan and from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a grant for Medical Research from Aomori Bank. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 储 To whom correspondence should be addressed: Second Department of Biochemistry, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. Tel.: 81-172-39-5018; Fax: 81-172-39-5205; E-mail: [email protected]. 1 The abbreviations used are: PEBP, phosphatidylethanolaminebinding protein; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; MEK, MAPK kinase; NHEK, normal human epidermal keratinocyte; KGM, keratinocyte growth medium; RT, reverse transcription. This paper is available on line at http://www.jbc.org

brain (1) which shows preferential affinities for phosphatidylethanolamine (2), a component of the cell membranes for nucleotides like GTP and GDPs and for small GTP-binding proteins (3). Species orthologues of PEBP show a high degree of sequence similarity (4) and are expressed in many tissues, such as the testis, adrenal gland, liver, and kidney (5), suggesting that the proteins have an important and conserved biological function. PEBP is believed to be the precursor of hippocampal neurostimulatory peptide, a bioactive peptide which is involved in the development of the hippocampus (6). Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that play an important role in signal transduction from the cell surface to the nucleus. The mammalian MAPKs can be divided into extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. All MAPK pathways utilize a three-kinase cascade mechanism. Thus, each MAPK has upstream-activating kinases (MAPKKs) that are, in turn, regulated by MAPKK kinases (7). In response to a wide range of extracellular stimuli, the MAPK cascades determine cell fate, including cell growth, differentiation, and apoptosis (7). The Raf-1/MAPKK (MEK)/ERK signaling pathway is activated by Ras and other factors and commonly transmits cell proliferation signals to a variety of cell types (8, 9). Mutated and constitutively activated forms of Ras are found in 30% of human cancers (10); the pathway is activated in many cancer cells and is important for oncogenic transformation (11). PEBP has been demonstrated to bind to Raf-1 and MEK and thereby inhibits the ERK pathway, resulting in the suppression of cell proliferation (12). Recently, PEBP was also reported to bind to thrombin (13) and heterotrimeric G-protein (14). Thrombin exerts a mitogenic effect, and Raf-1 is phosphorylated upon binding of thrombin to a G protein-coupled receptor (15). Cell proliferation and differentiation are reciprocally regulated in many cell lines (16). In the skin, there is a balance between proliferation of mitotically active keratinocytes in the basal layer and differentiation of post-mitotic cells within the suprabasal layers of the epidermis (17). Under certain pathological conditions such as tumor, this equilibrium is disturbed. Epidermal growth factor and transforming growth factor-␤ are known as in vitro stimulators of keratinocyte proliferation (18, 19). On the other hand, elevation of extracellular calcium levels induces rapid cell-cycle arrest in keratinocytes (20, 21) and morphological changes accompanying increased expression of differentiation-associated proteins such as involucrin (22). Calcium treatment, however, is reported to induce transient activation of the Raf/MEK/ERK pathway (23), the pathway being associated with not only cell proliferation but also differentiation in certain cell lines (24, 25). Although PEBP is suggested to inhibit cell proliferation, it remains to be clarified whether it is also involved in differentiation induction. In the present

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Phosphatidylethanolamine-binding protein (PEBP) has been demonstrated to bind to Raf-1 and mitogenactivated protein kinase kinase, components of the extracellular signal-regulated protein kinase (ERK) pathway, thereby inhibiting the pathway and resulting in the suppression of cell proliferation. In the present study, we examined whether PEBP is involved in differentiation induction of human keratinocytes. PEBP expression was immunohistochemically examined in normal human skin and skin cancers with different differentiation properties. PEBP was not expressed in the basal layer of the epidermis but was expressed in the spinous and granular layers of normal skin. The protein was expressed in differentiated but not in undifferentiated carcinoma. PEBP expression was also examined in cultured normal human epidermal keratinocytes in which differentiation was induced by calcium treatment. Involucrin was used as a differentiation marker for spinous and granular cells. Northern blotting analysis indicated that both PEBP and involucrin mRNAs were enhanced 6 h after treatment with 2.0 mM CaCl2. The protein amount of PEBP was also increased by this treatment. To investigate whether PEBP is involved in differentiation induction of keratinocytes, HaCaT keratinocytes were transfected with an expression vector. Fluorescent immunostain revealed that cells expressing PEBP exhibited enlarged and flattened cell shape, and induction of involucrin expression was demonstrated by immunoblot analysis. Although the protein amount of ERK was not altered, phosphorylated ERK levels were decreased and cell proliferation was partly inhibited by PEBP expression. These results indicate that PEBP not only inhibits cell proliferation but also induces differentiation of human keratinocytes.

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Keratinocyte Differentiation by Phosphatidylethanolamine-binding Protein

study, PEBP expression was immunohistochemically examined in normal human skin and squamous cell carcinomas with different differentiation properties. Its expression was also examined in cultured keratinocytes in which differentiation was induced by calcium treatment. These studies showed enhanced PEBP expression in keratinocyte differentiation, and transfection with expression vector revealed PEBP as a causative factor for differentiation induction. EXPERIMENTAL PROCEDURES


Purification of PEBP—PEBP was purified from human kidney according to the method of Bernier and Jolles (1) with modifications employing S-hexylglutathione-Sepharose affinity column. Human kidney was homogenized in 5 volumes of 20 mM Tris-HCl (pH 7.5) and centrifuged at 20,000 ⫻ g for 40 min. The supernatant was further centrifuged at 120,000 ⫻ g for 40 min. The supernatant was applied to DE52-cellulose column (2.0 ⫻ 25 cm) (Whatman International Ltd.) equilibrated with 20 mM Tris-HCl (pH 7.5). The non-absorbed fractions were dialyzed against 20 mM ammonium acetate (pH 5.0), 1 mM EDTA, and then applied to a CM-Sephadex C-50 column (1.5 ⫻ 15 cm) equilibrated with the same buffer. After washing with the buffer, absorbed proteins were eluted in a stepwise manner with increasing concentrations of NaCl (0.08 M, 0.15 M, and 0.5 M) in the buffer. The fractions eluted with 0.5 M NaCl were dialyzed against 20 mM Tris-HCl (pH 7.5) and applied to an S-hexylglutathione-Sepharose column (26) (1.2 ⫻ 10 cm) equilibrated with the same buffer. After washing with the buffer, bound proteins were eluted with 20 mM Tris-HCl (pH 7.5), 50 mM NaCl. The eluted fractions were dialyzed against 25 mM triethylamine-HCl (pH 11.0) and submitted to chromatofocusing chromatography with a PBE 118TM column (1.0 ⫻ 28 cm) (Amersham Biosciences) equilibrated with 25 mM triethylamine-HCl (pH 11.0). Elution was carried out with 400 ml of PharmalyteTM (pH 8 –10.5), which was diluted 80-fold with deaerated water and adjusted to pH 7.0 with HCl. PEBP was eluted from the PBE 118TM column at pH 9.3. The final PEBP preparation exhibited only a band with subunit Mr ⫽ 23 kDa on SDS-PAGE. This band was stained by immunoblot analysis with anti-C-terminal peptide of PEBP antibody. N-Terminal amino acid sequence of this preparation was identical with that reported for human PEBP (6). N-Terminal amino acid sequencing was performed with 490 Procise protein sequencer (PerkinElmer Applied Biosystems, Urayasu, Japan). Production of Anti-PEBP Antibody—PEBP (100 ␮g protein) purified from human kidney was dialyzed against 12 mM potassium phosphate buffer (pH 7.2), 145 mM NaCl, emulsified with an equal volume of Freund’s complete adjuvant (Iatron Laboratories, Tokyo, Japan), and then injected subcutaneously three times into albino rabbits at 1-week intervals. On the seventh day after the last injection, PEBP without the adjuvant was injected subcutaneously. The rabbits were bled on the seventh day after the booster injection. The antibody was purified from the antiserum by 20 –33% saturated ammonium sulfate and then dialyzed against phosphate-buffered saline. A C-terminal 14-amino acid peptide of PEBP synthesized by a peptide synthesizer (model 432A, Applied Biosystems, Foster City, CA) was coupled to keyhole limpet hemocyanin with m-maleimidobenzoyl N-hydroxysuccinimide ester (27). This conjugate was similarly immunized to a rabbit as PEBP, and the anti-C-terminal peptide of PEBP antibody was prepared. Immunohistochemistry—Skin tissues were obtained at surgery in the Department of Dermatology, Hirosaki University Hospital from four patients with squamous cell carcinoma of the skin. Samples containing normal and carcinoma tissues were fixed in 10% formaldehyde and embedded in paraffin. Tissue sections (5 ␮m thick) were routinely passed through xylene and a graded alcohol series and stained for PEBP by the avidin-biotin-peroxidase complex (ABC) method (28) using the specific antibodies. Affinity-purified, biotin-labeled goat anti-rabbit immunoglobulin G and ABC complex (Vectastain ABC kit, PK4001) were obtained from Vector Laboratories Inc. (Burlingame, CA). The sites of peroxidase binding were determined by the diaminobenzidine method. Sections were then lightly counterstained with hematoxylin for microscopic examination. As negative controls, preimmune rabbit serum or antibody absorbed with PEBP were used instead of the antibody. Cell Culture—Normal human epidermal keratinocytes (NHEK) were purchased from Cambrex Bio Science Walkersville, Inc. (Walkersville, MD). Cells were plated in 60-mm plastic tissue-culture dishes using 0.03 mM calcium keratinocyte growth medium (KGM) (Cambrex Bioscience Walkersville, Inc.) and grown at 37 °C in a humidified incubator under a 5% CO2, 95% air atmosphere. The medium was changed every

other day. After the 3rd passage, at 60 –70% confluence, cultures were switched to either 2.0 or 0.03 mM calcium KGM. After 48 h, cells were harvested. HaCaT cells (29) were cultured at 37 °C in a humidified atmosphere containing 5% CO2 in Eagle’s minimum essential medium without calcium (Invitrogen) containing 8% chelex-treated FBS. HaCaT cells at 70% confluence were transfected with 2 ␮g of pCY4B-PEBP cDNA or pCY4B vectors in Eagle’s minimum essential medium using LipofectAMINE Plus reagent (Invitrogen), according to the manufacturer’s instructions. After incubation at 37 °C for 3 h, cells were cultured in 0.03 mM calcium KGM for up to 48 h. Northern Blotting—Twelve ␮g of total RNA were electrophoresed and transferred to nitrocellulose. The filters were hybridized with 32Plabeled cDNA probes and exposed to Kodak XAR-5 film at ⫺80 °C. cDNA probes for PEBP (6), involucrin (30), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (31) were generated by reverse transcription (RT)-PCR. The nucleotide sequences of these cDNAs were confirmed by sequencing with a BigDye terminator kit (Applied Biosystems) and an ABI DNA sequencer Model 310 (Applied Biosystems). Equal loading of RNA was confirmed by comparing the intensity of the GAPDH mRNA band in each lane. Quantitation of the results was performed by scanning the x-ray film with Photoshop version 5.5 software (Adobe Systems, Inc.) followed by densitometry with NIH Image version 1.62. Western Blotting—Proteins (20 or 100 ␮g/track) were separated by reducing SDS-PAGE (32) on 12.5% (w/v) polyacrylamide gels and electroblotting to Hybond nitrocellulose membrane (33) (Amersham Biosciences). Blots were probed with anti-PEBP antibody, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000 (v/v), Bio-Rad). Enhanced chemiluminescence (Amersham Biosciences) was used as a detection reagent. In some experiments, anti-loricrin antibody (BAbCO), anti-involucrin monoclonal antibody, anti-ERK2 antibody, or anti-phosphorylated ERK antibody (sc-21748, sc-1647, and sc-7383, respectively, Santa Cruz Biotechnology, Inc.) were used as the first antibody. Construction of Expression Vector for PEBP—For construction of expression vector for PEBP, cDNA encoding the entire open reading frame was generated by nested RT-PCR using primer sets designed to contain the XhoI and NotI linkers. Reverse transcription and the first PCR were performed using BcaBest RNA-PCR kit (TaKaRa, Shiga, Japan). cDNA was reverse-transcribed from total RNA isolated from cultured normal human keratinocytes by incubation at 65 °C for 1 min, 30 °C for 5 min, 65 °C for 15 min, 98 °C for 5 min, and 5 °C for 5 min in a reaction volume of 20 ␮l containing 22 units of BcaBest polymerase, 1⫻ buffer, 500 ␮M of each dNTP, 2.5 ␮M of oligo dT primer, and 40 units of RNase inhibitor. The first PCR conditions were at 94 °C for 2 min, followed by 25 cycles of denaturing at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 50 s, containing the all reversetranscription mixture (2.5 units Bca-Optimized Taq, 1.5 mM MgCl2, 1⫻ buffer, 0.2 ␮M of the primers). The 5⬘ primer 5⬘-TTTCTCGAGGCCATGCCGGTGGACCTCAGCAAG-3⬘ and the 3⬘ primer 5⬘-ACAGTTCAGGTCCCCAAGCTA-3⬘ (34) were used for the first amplification. The second PCR was performed using KOD DNA polymerase (Toyobo Co., Ltd., Osaka, Japan). For the second amplification, first-round amplification mixture was diluted by 100-fold with water and 1 ␮l of the solution was used. The 5⬘ primer was 5⬘-TTTCTCGAGGCCATGCCGGTGGACCTCAGCAAG-3⬘ and the 3⬘ primer was 5⬘-TTTGCGGCCGCAAGCTAACCCCCTACTTCCCA-3⬘. PCR conditions were at 94 °C for 2 min, followed by 25 cycles of denaturing at 94 °C for 15 s, annealing at 60 °C for 2 s, and extension at 74 °C for 30 s, containing 2.5 units KOD DNA polymerase, 1⫻ buffer, 0.2 ␮M of primers, and 200 ␮M of each dNTP. Each amplified cDNA was digested with XhoI and NotI and the digest was fractionated on a 1% agarose gel. The 585-bp fragment containing the entire PEBP open reading frame was ligated downstream of a cytomegalovirus-IE enhancer and ␤-actin promoter in the plasmid pCY4B, which is derived from pCAGGS expression vector (35). pCY4B was kindly donated by Dr. Jun-ichi Miyazaki (Graduate School of Medicine, Osaka University, Osaka, Japan). The nucleotide sequence of PEBP cDNA was confirmed by DNA sequencing (36). Cell Growth—Cell growth was determined with a CellTiter96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI), according to the manufacturer’s instructions. Briefly, cells seeded at a density of 2 ⫻ 103/180 ␮l of culture medium in 96-well tissue culture plates were incubated at 37 °C in a humidified 5% CO2 atmosphere for up to 48 h. Then, 20 ␮l of substrate solution was added to each well, and the plates were further incubated for 3 h. Absorbance at 490 nm was measured with a microplate reader Model 550 (Bio-Rad). Fluorescent Immunostaining—For fluorescent immunostaining, HaCaT cells transfected with expression vector or control vector were


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FIG. 1. Immunohistochemical staining for PEBP in normal skin (A) and differentiated (C) and undifferentiated (D) squamous cell carcinomas. Immunohistochemistry was performed with anti-PEBP antibody as described under “Experimental Procedures.” B, result of using antiPEBP antibody absorbed with purified PEBP. Original magnification, 200⫻.

RESULTS

PEBP Expression in Human Skin Keratinocytes—PEBP expression in normal human skin was examined by immunohistochemistry as described under “Experimental Procedures.” A typical staining pattern for PEBP is shown in Fig. 1A. PEBP was not stained in the basal layer but was stained in the cytoplasm of the spinous and granular layers. In addition, the granular layer was more heavily stained than the lower part of the spinous layer. Control staining using preimmune serum and antibody absorbed with PEBP proved negative (Fig. 1B). The expression of PEBP was also examined in squamous cell carcinoma of the skin. Differentiated carcinoma retained a positive staining result (Fig. 1C), whereas undifferentiated carcinoma showed negativity (Fig. 1D). Data presented in Fig. 1 were obtained with antibody against PEBP, and similar results were also obtained with antibody against the C-terminal peptide (data not shown). These results suggested that PEBP expression may be dependent upon the differentiation of the epidermis in vivo and is lacking in proliferating basal cells. PEBP Expression in Keratinocyte Differentiation Induced by Calcium—To examine the relationship between PEBP expression and cell differentiation, NHEK cells were treated with 2.0 mM CaCl2 to induce keratinocyte differentiation (22). On microscopic examination, cells incubated with 0.03 mM CaCl2 for 48 h retained an elongated shape and loose arrangement (Fig. 2A, a), whereas cells incubated with 2.0 mM CaCl2 were densely packed and exhibited polygonal shapes (Fig. 2A, b). Immunoblot analysis revealed that the protein level of PEBP was increased in cells treated with a high concentration of calcium, as compared with a low calcium case (Fig. 2B). The effects of 2.0 mM calcium treatment on the levels of PEBP mRNA and involucrin mRNA, a marker for keratinocyte differentiation (37), in NHEK cells were evaluated by Northern blotting; typical results are presented in Fig. 2C. Similar results were obtained in two other experiments. PEBP mRNA and involucrin mRNA levels were increased to 1.8-fold and 2-fold, respectively, 6 h after treatment with 2.0 mM CaCl2, and their increases were

continued by 48 h. The time course of alterations in involucrin mRNA level was similar to that for PEBP mRNA. These levels were not altered in cells treated with 0.03 mM CaCl2 (data not shown). These results indicate that PEBP expression was enhanced in keratinocyte differentiation induced by calcium. Differentiation Induction and Repression of Cell Proliferation in HaCaT Cells by PEBP Expression—To study whether PEBP expression can induce differentiation and repression of cell proliferation in keratinocytes, a PEBP expression vector was constructed by ligating a PEBP cDNA to pCY4B containing a cytomegalovirus enhancer and chicken ␤-actin promoter (35), as described under “Experimental Procedures.” A keratinocyte cell line, HaCaT, derived from human skin, was used for transfection experiments because of low basal PEBP protein level, which is about one-tenth that of NHEK cells (data not shown). HaCaT cells transfected with a PEBP expression vector were subjected to fluorescent immunostaining with anti-PEBP antibody to examine its expression in individual cells and transfection efficiency. 47% of cells exhibited positive staining for PEBP, and 95% of positive cells showed an enlarged and flattened shape (Fig. 3, A and C). PEBP-negative cells retained small and round or spindle shapes, exhibiting properties similar to cells transfected with an empty vector (Fig. 3, B and D). Positive staining was never observed in control transfectants. To study the effects of PEBP expression on differentiation induction, expression of involucrin was examined by immunoblotting (Fig. 4). Involucrin was increased in cells transfected with a PEBP expression vector, as compared with that in the empty vector case. Increased PEBP levels were confirmed in its


fixed with 4% paraformaldehyde for 20 min, permeabilized with 100% methanol for 1 min, and then incubated with anti-PEBP antibody for 2 h. After washing with 20 mM Tris-HCl (pH 7.4), 145 mM NaCl three times, the cells were incubated with secondary antibody (fluorescent isothiocyanate-conjugated pig anti-rabbit immunoglobulin antibody) (Dako Cytomation, Kyoto, Japan) for 1 h. After washing with the same buffer, the cells were examined under a fluorescent microscope (Olympus).

FIG. 2. Effects of a high calcium concentration on PEBP expression in normal human epidermal keratinocytes. A, phasecontrast microscopy of NHEK cells cultured for 48 h in 0.03 mM calcium KGM (a) or 2.0 mM calcium KGM (b). Original magnification, 200⫻. B, increased PEBP level in NHEK cells by calcium. Cytosol fractions from these cells were analyzed for PEBP protein levels by immunoblotting as described under “Experimental Procedures.” Lanes 1 and 2, extracts derived from NHEK cells cultured in 0.03 mM and 2.0 mM calcium KGM, respectively. Each lane contained 20 ␮g of protein. Lane M, molecular mass marker proteins stained for protein with Coomassie Brilliant Blue. C, increased PEBP and involucrin mRNA levels in NHEK cells by calcium. Total RNAs were extracted form NHEK cells cultured for up to 48 h in 2.0 mM calcium KGM; the levels of PEBP, involucrin, and GAPDH mRNAs were analyzed by Northern blotting. Each 12 ␮l of total RNA was electrophoresed and transferred to nitrocellulose filters. The filters were hybridized with 32P-labeled cDNA probes and exposed to Kodak XAR-5 film at ⫺80 °C. cDNA probes for PEBP (a), involucrin (b), and GAPDH (c) were used. Lanes 1– 8, RNA samples from cells cultured for 0, 0.5, 1, 3, 6, 12, 24, and 48 h, respectively.

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FIG. 3. Morphological changes in HaCaT cells due to PEBP expression. The PEBP expression vector (pCY4B-PEBP) was constructed by ligating PEBP cDNA into the plasmid pCY4B containing cytomegalovirus-IE enhancer and chicken ␤-actin promoter (33). Transfection of the vector into HaCaT cells was carried out as described under “Experimental Procedures.” HaCaT cells transfected with pCY4BPEBP vector (A and C) or control pCY4B vector (B and D) were cultured for 48 h in 0.03 mM calcium KGM. After fixing with 4% paraformaldehyde, these cells were subjected to examination with a phase-contrast microscopy (C and D) and then fluorescent immunostaining for PEBP expression. The cells were examined under a fluorescent microscope (A and B). Original magnification, 200⫻.

transfectants, and endogenous PEBP levels were negligible in control cells. Loricrin expression was not detected in PEBP transfectants as well as control cells (data not shown). The effects of PEBP expression on proliferation of HaCaT cells were also analyzed. Cell proliferation was evaluated by a colorimetric method employing a tetrazolium substrate. Absorbance at 490 nm was linear in the range of 0.3–1.5, corresponding to 3000 –15,000 cells/well. PEBP expression repressed cell proliferation almost completely during the first 12 h, but thereafter, absorbance was increased at a lower rate: the value at 48 h was 60% of that for cells without expression (Fig. 5). In transfection experiments with PEBP expression vector, about half of the cells showed PEBP expression, and this may be responsible for partial inhibition of cell proliferation. To examine whether the MEK/ERK pathway is involved in growth suppression because of PEBP expression, the alteration in MEK activity was evaluated by measuring the phosphorylated ERK level. Immunoblot analysis revealed that PEBP expression did not alter the ERK level (Fig. 4C) but repressed the phosphorylated ERK level (Fig. 4D). These results suggest that PEBP blocked the MEK/ERK pathway. DISCUSSION

The ERK cascade, composed of Raf/MEK/ERK, is mainly involved in the stimulation of cell proliferation in a variety of

cells (38, 39). Addition of a high concentration of calcium to the culture medium is known to induce cell-cycle arrest and differentiation of normal human keratinocytes by blocking the ERK pathway (40). However, calcium-induced up-regulation of the differentiation marker involucrin is repressed by MEK inhibitors, PD98059 or U0126, suggesting that activation of the Raf/ MEK/ERK pathway may enhance differentiation-specific gene expression (23). To clarify the role of the ERK pathway in differentiation induction, we examined whether PEBP expression resulted in keratinocyte differentiation. The present study has demonstrated that PEBP is not expressed in the basal layer of the epidermis with a cell proliferation potential but is expressed in the spinous and granular layers consisting of differentiating keratinocytes (Fig. 1). The protein is expressed in differentiated carcinoma but not in undifferentiated carcinoma. PEBP expression is enhanced in NHEK differentiation induced by a high concentration of calcium (Fig. 2, B and C). To rule out the possibility that PEBP expression is secondary to differentiation of NHEK cells following calcium treatment, we conducted a PEBP-expression experiment. Transfection experiments have revealed that HaCaT keratinocytes expressing PEBP exhibited characters of differentiated cells (Figs. 3 and 4B) and a partial inhibition of cell proliferation (Fig. 5). The latter finding seems to be due to the incomplete transfection efficiency of the expression vector. PEBP expression repressed MEK activities in keratinocytes (Fig. 4D), confirming the function of PEBP reported in fibroblasts (12). Thus, decreased MEK activities were suggested to be responsible for suppression of cell proliferation by PEBP. These results also suggest that PEBP is involved in calcium-induced suppression of cell proliferation and differentiation. PEBP expression up-regulated the involucrin level but did not alter the loricrin level, suggesting that PEBP differentiates keratinocytes to an early rather than a late stage. Transient activation of the Raf/MEK/ERK pathway by epidermal growth factor is known to result in cellular proliferation of PC12 cells, whereas sustained activation of the pathway by nerve growth factor promotes differentiation of the cells into sympathetic-like neurons (16, 24, 41). Other studies, however, have reported that sustained ERK activation alone is not sufficient to induce differentiation, and the p38 MAPK pathway is also involved in its differentiation (42, 43). Transient activation of the Raf/MEK/ERK pathway within 10 –15 min is suggested to be responsible for calcium-induced differentiation of HaCaT keratinocytes (23). In the present study, alterations in PEBP


FIG. 4. Enhanced involucrin expression and depressed ERK phosphorylation in HaCaT cells transfected with PEBP expression vector. HaCaT cells transfected with pCY4B-PEBP expression vector or control vector were cultured for 24 and 48 h. Cytosol fractions from cells cultured for 24 h (lane 1) and 48 h (lane 2) were analyzed for PEBP (A), involucrin (B), ERK (C), and phosphorylated ERK (D) proteins by immunoblotting, as described for Fig. 2. Cytosolic fractions were also prepared from cells transfected with control vector (lanes 3 and 4, cultured for 24 and 48 h, respectively). Each lane contained 100 ␮g of protein.

FIG. 5. Growth curves of HaCaT cells transfected with PEBP expression vector. 2.0 ⫻ 103 HaCaT cells transfected with pCY4BPEBP expression vector (●) or control vector (E) were seeded in 180 ␮l of 0.03 mM calcium KGM in individual wells of 96-well plates and cultured for up to 48 h. At each indicated time point, a viable cell number was evaluated by a colorimetric method employing a tetrazolium substrate. Absorbance was measured at 490 nm. The mean and S.D. values per triplicate wells were plotted.


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and involucrin mRNA levels showed a similar time course: induction of their expression occurred 6 h after calcium treatment (Fig. 2C). Calcium may trigger activation of multiple pathways to induce differentiation in keratinocytes (44). It has been suggested that protein kinase C (PKC) plays an important role in calcium or TPA-induced keratinocyte differentiation and that certain PKC isoforms (␦, ⑀, etc.) stimulate involucrin gene expression through p38 MAPK activation (37, 45, 46). The p38 MAPK pathway is also involved in keratinocyte differentiation (47). Although PEBP inhibits the Raf/MEK/ERK pathway and the nuclear factor-␬B activation pathway (48), it remains to be clarified whether PEBP affects p38 MAPK activity. Thus, because the MEK/ERK pathway is not a sole target of PEBP, other targets may be involved in PEBP-induced differentiation. A p38 MAPK inhibitor, SB203580, did not affect differentiation induction and PEBP expression in NHEK cells after calcium treatment (data not shown). A recent study (49) has revealed that some PKC isoforms (␨, ␣, ␤, etc.) phosphorylate PEBP at the Ser-153 residue, and its phosphorylation causes the release of PEBP from Raf-1, thereby enhancing the signal of the Raf/ MEK/ERK pathway. Furthermore, phosphorylated PEBP can associate with G protein-coupled receptor kinase-2 to block its activity (50). Because the kinase inhibits signal transduction of the G protein-coupled receptor-mediated pathway, phosphorylated PEBP recovers the pathway from inhibition by the kinase. Thus, both unphosphorylated and phosphorylated PEBP act as signal modifiers between the two pathways. Certain protein kinases involved in signal transduction bind to some proteins, and their kinase activities are regulated by complex formation. For example, JNK binds to glutathione S-transferase (51), and apoptosis-signaling protein kinase forms a complex with thioredoxin (52). Disulfide bond formation in glutathione S-transferase due to oxidative stress has been suggested to be a signal for the release of JNK from the complex, and the resultant free JNK exhibits a kinase activity (51). PEBP has two cysteine residues (Cys-132 and Cys-167) (34) and is reported to form a disulfide bond under oxidative conditions (53). In the present study, we found PEBP to bind to S-hexylglutathione-Sepharose, and this property was used for purification. Several proteins with reactive cysteine residues are reported to bind to the affinity matrix (54). Raf-1 is demonstrated to bind to the domain corresponding to amino acid residues 77–108 of PEBP (55), which is in proximity to the Cys residues. Thus, in addition to phosphorylation, disulfide bond formation in PEBP under oxidative conditions may be involved in the release and activation of Raf-1. In conclusion, PEBP is shown to be expressed in the spinous and granular layers of the epidermis and, in addition to inhibition of cell proliferation, PEBP can induce differentiation of human keratinocytes.

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Mechanisms of Signal Transduction: Differentiation Induction of Human Keratinocytes by Phosphatidylethanolamine-binding Protein Takehiko Yamazaki, Hajime Nakano, Makoto Hayakari, Masanori Tanaka, Jun Mayama and Shigeki Tsuchida J. Biol. Chem. 2004, 279:32191-32195. doi: 10.1074/jbc.M404029200 originally published online May 20, 2004

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