Differentiation Induction of Human Keratinocytes by Phosphatidylethanolamine-Binding Protein Keratinocyte Differentiation by Phosphatidylethanolamine-Binding

Phosphatidylethanolamine-binding protein (PEBP) has been demonstrated to bind to Raf-1 and mitogen-activated 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 CaCl(2). 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.

4 mitogenic effect and Raf-1 is phosphorylated upon binding of thrombin to a G-proteincoupled 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 (EGF) and transforming growth factor-β 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 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 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 (Perkin Elmer Applied Biosystems, Urayasu, Japan).
6 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 to 33% saturated ammonium sulfate and then dialyzed against phosphate-buffered saline. 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 anti-C terminal peptide of PEBP antibody was prepared. Northern Blotting---Twelve µg of total RNA was electrophoresed and transferred to nitrocellulose. The filters were hybridized with 3 2 P-labeled cDNA probes and exposed to Kodak XAR-5 film at -80 °C. cDNA probes for PEBP (6) Fig. 1A. PEBP was not stained in basal layer but stained in the cytoplasm of the spinous and granular layers. In addition, granular layer was more heavily stained than the lower part of spinous layer.

PEBP Expression in Human Skin
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 positive staining result (Fig. 1C), while 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 on the differentiation of the epidermis in vivo and is lacking in proliferating basal cells.  Fig. 2 panel A), while cells incubated with 2.0 mM CaCl 2 were densely packed and exhibited polygonal shapes (b in Fig. 2 panel A). Immunoblot analysis revealed that protein level of PEBP was increased in cells treated with a high concentration of calcium, as compared with a low calcium case (Fig. 2 panel B) Forty seven % of cells exhibited positive staining for PEBP, and 95% of positive cells showed enlarged and flattened shape ( Fig. 3A and C). PEBP-negative cells retained small and round or spindle shapes, exhibiting the similar properties as cells transfected with an empty vector ( Fig. 3B and D). Positive staining was never observed in control transfectants.

PEBP Expression in Keratinocyte Differentiation Induced by Calcium----
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 empty vector case.
Increased PEBP level was confirmed in its 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. h, but thereafter absorbance was increased at a low rate and the value at 48 h was 60% of that for cells without expression (Fig. 5). In transfection experiment with PEBP expression vector, about a half of 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 due to PEBP expression, alteration in MEK activity was evaluated by measuring phosphorylated ERK level. Immunoblot analysis revealed that PEBP expression did not alter ERK level (Fig. 4C) but repressed phosphorylated ERK level (Fig. 4D). These results suggested 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 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 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. 2B and C). To rule out a possibility that PEBP expression is secondary to differentiation of NHEK cells following calcium treatment, and 4B) and a partial inhibition of cell proliferation (Fig. 5). The latter finding seemed to be due to 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 involucrin level but did not alter loricrin level, suggesting that PEBP differentiates keratinocytes to an early stage rather than late stages.
Transient activation of the Raf/MEK/ERK pathway by EGF is known to result in cell 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 and involucrin mRNA levels showed a similar time course and 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). Protein kinase C (PKC) is suggested to play a important role for calcium or TPA-induced keratinocyte differentiation and certain PKC isoforms (delta, epsilon 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-κB activation pathway (48), it remains to by guest on March 24, 2020 http://www.jbc.org/ Downloaded from be clarified whether PEBP affects p38 MAPK activity. Thus, since 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 following calcium treatment (data not shown). Recent study has revealed that some PKC isoforms (zeta, alpha, beta etc.) phosphorylate PEBP at the 153Ser residue and its phosphorylation causes release of PEBP from Raf-1, thereby enhancing signal of the Raf/MEK/ERK pathway (49). Furthermore, phosphorylated PEBP can associate with G-protein-coupled receptor kinase-2 to block its activity (50). Since 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 is suggested as 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 (132Cys and 167Cys) (34) and is reported to form 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 the 77--108th amino acid residues of PEBP (55), the proximity of 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