- Email: [email protected]
An Important Role for Protein Kinase C-δ in Human Keratinocyte Migration on Dermal Collagen
Experimental Cell Research 273, 219 –228 (2002) doi:10.1006/excr.2001.5422, available online at http://www.idealibrary.com on
An Important Role for Protein Kinase C-␦ in Human Keratinocyte Migration on Dermal Collagen Wei Li,* ,† ,1 Celina Nadelman,* Noah S. Gratch,* Weiquin Li,‡ Mei Chen,* Nori Kasahara,§ and David T. Woodley* ,† ,1 *Department of Medicine, Division of Dermatology, and the Norris Cancer Center, and §Institute for Genetic Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, 90033; †Greater Los Angles Veterans Administration Health System; and ‡Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007
Migration of human keratinocytes plays a critical role in the re-epithelialization of human skin wounds, the process by which the wound bed is resurfaced and closed by keratinocytes as it forms a new epidermis. While the importance of ECM components and serum factors in the regulation of keratinocytes motility is well established, the intracellular signaling mechanisms remain fragmentary. In this study, we investigated the role of protein kinase C␦ (PKC␦) signaling in the promotion of human keratinocyte migration by a collagen matrix and bovine pituitary extract. We found that pharmacological inhibition of the PKC␦ pathway completely blocks migration. Using a lentivirus-based vector system, which offers more than 90% gene transduction efficiency to human keratinocytes, we show that the kinase-defective mutant of PKC␦ (K376R) dramatically inhibits human keratinocyte migration. Furthermore, PKC␦ is activated in migrating human keratinocytes. These observations indicate for the first time that the PKC␦ pathway plays an important role in the control of human keratinocyte migration. © 2002 Elsevier Science (USA)
INTRODUCTION
The lateral migration of keratinocytes over the wound bed is an early and critical event in the reepithelialization of human skin wounds. Within hours after an acute wound, the basal keratinocytes at the margins of the wound and within the cut skin appendages (hair follicles, eccrine gland duct) enter a migratory mode and begin to migrate laterally across the provisional wound bed to resurface the wound [1–3]. The cells migrate on a provisional matrix composed of fibronectin, fibrin, and collagen fragments, which are formed by blood clot following tissue injury [3]. While both keratinocyte motility and division ultimately con1
To whom correspondence and reprint requests should be addressed. Fax: 323-224-7679. E-mail: [email protected]; [email protected].
tribute to the re-epithelialization of a skin wound, they are controlled by independent intracellular signaling mechanisms. First, keratinocyte migration occurs within 2–3 h in response to extracellular signals (ECM and growth factors/cytokines), while cell division does not take place until more than 24 h later [6]. Second, TGF inhibits the proliferative potential of human keratinocytes to virtually zero and yet the cells are fully capable of collagen-driven migration [4]. It is well established that the Ras–MEKK–MEK– ERK pathway mediates cell DNA synthesis and proliferation in response to extracellular signals such as EGF. In contrast, the intracellular signaling mechanisms that regulate human keratinocyte migration have just begun to be understood. Yurko et al. reported that, in highly migratory human keratinocytes, FAK (focal adhesion kinase) was heavily tyrosine phosphorylated throughout the 18-h period of the migration assay. Antisense oligonucleotides against FAK blocked human keratinocyte migration on collagen and fibronectin [7]. Zeigler et al. showed that hepatocyte growth factor (HGF) and EGF stimulate prolonged activation of ERKs and JNK in human keratinocytes. Blockade of the ERK activation by PD098059 inhibited HGF-stimulated cell migration and MMP-9 production in human keratinocytes [8]. Moreover, in the transformed keratinocyte cell line HaCaT, expression of MMP-13 and MMP-1 requires the p38 –MAPK 2 pathway [9]. We have recently reported that the p38 – MAPK pathway is involved in collagen-driven human keratinocyte migration [6]. Our results show that while the p38 –MAPK pathway is necessary for collagendriven migration, activation of this pathway alone is not sufficient to invoke keratinocyte migration on a collagen matrix [6]. This suggests that other parallel 2
Abbreviations used: PKC, protein kinase C; BMZ, basement membrane zone; ECM, extracellular matrix; TBS, Tris-buffered saline; PY, phosphotyrosine; DAG, diacylglyceral; MAPK, mitogenactivated protein kinase; GFP, green fluorescent protein; MI, migration index.
219
0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
220
LI ET AL.
signaling pathways are also needed and work in concert with the p38 –MAPK pathway. Protein kinase C (PKC) represents a superfamily of serine/threonine protein kinases, including three subgroups of PKCs. These subgroups are (i) the “conventional” PKCs that include ␣, I, II, and ␥; (ii) the “novel” PKCs that include ␦, , , and , and (iii) the “atypical” PKCs, , , and . There is also a fourth distinct group called PKC [14]. The differences between these subgroups are defined by their dependence or independence on calcium and phospholipids such as diacylglyerol (DAG) [10]. The human keratinocyte line, HaCaT, was reported to express four PKC family members, PKC␣, PKC␦, PKC, and PKC [11]. Mouse keratinocytes are detected to express the mRNAs of PKC␣, -␦, -, -, and - [12]. Recently, a great deal of attention has been paid to PKC␦ because (i) it has a wide range of expression in almost all types of cells and tissues studied, (ii) it is the only PKC that is a direct target for protein tyrosine kinases and (iii) it has been implicated to play an important role in growth control, cellular differentiation, and apoptosis [10]. The keratinocyte cell line, HaCaT, transfected with oncogenic H-Ras exhibited a down regulation of PKC␦. This effect was via an autocrine loop leading to an augmentation of TGF␣ expression by the cells. Evidence for this mechanism was the observation that the addition of TGF␣ to the HaCaT keratinocyte culture induced a selective down regulation of PKC␦ while three other PKC family members were unaffected [11]. With regard to keratinocyte differentiation, it has been shown that overexpression of PKC␦ in the epidermis of transgenic mice reduced tumor formation by phorbol ester, TPA [13]. In concordance with this observation, Szallasi and coworkers found that bryostatin inhibits TPA-induced differentiation of mouse keratinocytes while also inhibiting the TPA-induced down regulation of PKC␦ [14]. In this article, we report that the inhibition of PKC␦ dramatically inhibited human keratinocyte motility on collagen. We present both pharmacological and genetic evidence that activation of PKC␦ is required for human keratinocyte migration on a collagen matrix. MATERIALS AND METHODS Materials. Primary human keratinocytes were purchased from Clonetics (San Diego, CA) and cultured in keratinocyte growth medium (KGM; Clonetics) supplemented with bovine pituitary extract (BPE) as the source of growth factors in low Ca 2⫹ conditions, according to the manufacturer’s instruction. Native rat tail type I collagen was purchased from Collaborative Biomedical Products (Bedford, MA). Anti-PKC␦ antibody (P36520) and anti-PKC antibody kit (611421) were purchased from Transduction Laboratories (Lexington, KY). Rabbit anti-rat PKC␦ antiserum (No. UCG-88) was raised in our laboratories and specifically used for immunoprecipitations (see 71-3100, Zymed Laboratories, Inc., South San Francisco, CA). Anti-phosphotyrosine antibody (No. 72) was used as previously described [15]. Green fluorescent protein (GFP) gene in the lentiviral
vector pRRLsinhCMV was as described previously [16]. Gold chloride was purchased from Sigma. FTI and GGTI PP1 were gifts from Saı¨d Sebti (University of South Florida). PP1, Li ⫹, rottlerin, HA1004, wortmannin, SB202190, rapamycin, staurosporin, tyrphostin, SB203580, and LY294002 were purchased from Calbiochem (San Diego, CA). The potential cytotoxicity for human keratinocytes by these agents was examined by a trypan blue exclusion assay and a cell proliferation assay after drug withdrawal, as described [6]. Our criterion for the trypan blue exclusion assay was that after exposure to the agent, more than 90% of the cells excluded the trypan blue dye. The second criterion for the lack of cytotoxicity was that after exposure to the agent and its subsequent withdrawal, the cells were able to proliferate and double in culture at or above the 90% level of control unexposed cells. The effect of these inhibitors on human keratinocyte migration, as measured by the colloidal gold phagokinetic migration assay, was subsequently investigated. Subcloning and production of lentiviral stocks and infection. Human immunodeficiency virus (HIV, Lentivirus)-derived vector, pRRLsinhCMV, was digested with EcoRI and SalI and purified. Similarly digested and purified wild-type or the dominant negative mutant (K376R) of the bovine PKC␦ cDNA fragments from pCEV27 vector [17] was ligated into the pRRLsinhCMV vector. These constructs were used to cotransfect 293T cells together with two packaging vectors, pCMV⌬R8.2 and pMDG, the process called the three plasmid expression system [16]. Conditioned media were collected, filtered, and concentrated prior to being used for infecting human keratinocytes. Human keratinocytes were cultured in KGM with BPE, according to the manufacturer’s instruction. The third or fourth passage of keratinocytes in culture was used for experiments. For infection experiments, cells were plated 1 day prior to infection in six-well tissue culture plates and after the cells reached 40 – 60% confluence, the cultures were subjected to infection. Infection was carried out by following a previously published procedure [16]. Expression of the PKC␦ proteins was confirmed by immunoblotting the lysates of infected cells with anti-PKC␦ antibody (36520, Transduction Laboratories) (vide infra). Titration of the wild-type and mutant PKC␦ viral stocks was conducted in order to achieve equal expression of the wild-type and the mutant PKC␦ in human keratinocytes. Preparation of cell lysates, SDS–polyacrylamide gel electrophoresis (PAGE), and Western blot. Tissue culture dishes with human keratinocyte adherent cultures were washed with PBS buffer and solubilized in lysis buffer (0.3 ml/10 cm dish) as described by us [18]. Following measurement of the protein concentrations (Bio-Rad protein assay using known concentrations of BSA as the reference at 595 nm visible light (Hitachi 4000)), 50 g of the lysates was directly subjected to SDS–PAGE and Western immunoblotting analysis. In selected experiments, 500 g of the lysates was immunoprecipiated with ant-PKC␦ antibodies (UCG-88) prior to SDS–PAGE and Western blot analysis (vide infra). SDS–PAGE was carried out in MiniProtean II system (Bio-Rad). We used 10% SDS bis-acrylamide resolving gels (2 ml of bis-acrylamide solution (0.8:30), 1.5 ml of Tris– HCl buffer, pH 8.8, 2.5 ml of H 2O, 40 l of 10% ammonium persulfate, and 2.5 l of TEMED) and 1% stacking gel (0.5 ml of bis-acrylamide solution, 0.625 ml of Tris–HCl buffer, pH 6.8, 1.625 ml of H 2O, 7.5 l of 10% ammonium persulfate, and 2.5 l of TEMED) for all experiments. Electrophoresis was conducted in running solution (0.25 M of Tris, 1.92 M of glycine, and 1% SDS) under a fixed voltage (150 V) for 1 h. Electrical transfer to a nitrocellulose membrane was carried out in a Mini Trans-Blot Transfer cell system (Bio-Rad) containing transfer buffer (25 mM Tris– base, 0.2 M glycine, 0.05% SDS (w/v)) in an electrical field of a fixed 50 V for 2 h at RT. Transfer efficiency under these conditions was assessed by staining the membrane with 5% Ponceau’s dye and by staining the gel afterward with Coomassie brilliant blue. Under these conditions, we were constantly experiencing more than 95% transfer efficiency. Membranes were washed two times in TBS-T (Tris-buffered saline
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY plus Triton X-100) and then two times in TBS, blotted for 2 h in TBS containing 5% nonfat milk, and incubated with a primary antibody in the same solution. The membranes were washed once with TBS, twice with TBS–Triton X-100, and once with TBS prior to incubation with HRP-conjugated secondary antibody for 1 h at RT. The membranes were subjected to the same washing procedure as above, and the results were visualized by ECL reaction. Cell motility colloidal gold phagokinetic assay. Cell migration on collagen was examined by using the migration track assay, as previously described by Albrecht-Buehler [19] and modified by Woodley for computer-assisted analysis [1]. Briefly, approximately 3000 cells were plated onto coverslips and allowed to migrate for different periods of time. At the end of the assay, cells were fixed and migration was examined under dark field optics and photographed. Fifteen randomly selected and nonoverlapping fields under each experimental condition were analyzed with an attached CCD camera (Model KP-MIU, Hitachi-Denshi Ltd.) and a computer using the NIH Image 1.6 program. The system calculated the percentage of the total field area viewed by the camera that was consumed with linear cell migration tracks. This percentage is termed a migration index (MI). Statistical analysis. The methodology to determine whether the difference in MIs between experimental sets of migrating keratinocytes exists has previously been published [20]. In brief, statistical analyses of the differences in MIs among triplicate sets of experimental conditions were performed using Microsoft Excel. Confirmation of a difference in migration as being statistically significant requires rejection of the null hypothesis of no difference between mean migration indices obtained from replicate sets at the P ⫽ 0.05 level with the Student t test. Activation of PKC␦. Human keratinocytes, starved in growthfactor-free medium overnight, were incubated in tissue culture plates precoated with either polylysine (nonmigratory) or type I collagen (migratory) at 37°C for different periods of time. The cells were then solubilized in lysis buffer as previously described [18], and the protein concentrations in the lysates were measured as described above. Equal amounts of cell extract protein (⬃500 g) were incubated with anti-PKC␦ antiserum (UCG-89, 1:500) overnight at 4°C. The immune complexes were precipitated with protein A agarose beads (25 l packed beads, Zymed). After being washed three times with lysis buffer, the beads were heated at 95°C for 5 min in SDS– gel sample buffer. The immunoprecipitates were subjected to SDS– PAGE and Western immunoblotting analysis using anti-phosphotyrosine (PY) antibody (No. 72). The labeled protein bands were visualized by ECL, as described above. The tyrosine phosphorylation correlates well with the growth factor stimulation of PKC␦ [14]. To further confirm PKC␦ activation, we carried out cellular fractionation analysis. Human keratinocytes from nonmigratory (polylysine-coated plates in the absence of BPE) and migratory (collagencoated plates in the presence of BPE) conditions were subjected to cellular fractionation by a previously described procedure of ours [37]. The Triton X-100 soluble extracts of the membrane and the nuclear fractions, and the cytosol fraction, were immunoprecipitated with anti-PKC␦ antibodies (UCG-88, raised by our laboratory). The immunoprecipitates were further analyzed by Western blot with an anti-PKC␦ antibody (P-36520, Transduction Laboratories).
RESULTS
Inhibition of PKC␦ Blocks Human Keratinocyte Migration on Collagen To investigate the intracellular signal transduction following the attachment and migration of human keratinocytes on type I collagen, we screened a panel of chemical inhibitors for their potential inhibitory effects
221
on the migration of human keratinocytes. We found that 3 of the 11 inhibitors tested, SB202190/SB202580, rottlerin, and PP1, potently blocked (⬎80%) cell migration on type I collagen without compromising cell viability. The rest of the inhibitors had either no effect or insignificant (⬍15%) inhibitory effects. It is known that SB202190/SB202580 (IC 50, 0.3 M), rottlerin (IC 50, 3– 6 M), and PP1 (IC 50, 5 M) inhibit the activation of p38 –MAPK, protein kinase C␦, and Src, respectively. In the current study, we focused on the role of the PKC␦ pathway in the regulation of collagen-driven human keratinocyte migration (the role of p38 –MAPK has been independently investigated, Ref. [6]). As shown in Fig. 1A, on an uncoated surface of immobilized gold, human keratinocytes showed little migration even in the presence of complete medium (Fig. 1Aa). Likewise, no migration occurred on tissue culture polystyrine plastic in an in vitro wound healing assay (“scratch assay,” data not shown). On a collagen-coated colloidal gold substratum, however, the cells made markedly linear migration tracks, leaving behind black gold particle-free tracks (Fig. 1Ab) (location of the cell inside each track is indicated by an arrow). In contrast, parallel cells incubated in medium containing different concentrations of rottlerin lost their migratory ability on collagen in a concentration-dependent fashion (Figs. 1Ac to 1Af) and completely stopped migrating in the presence of 5–15 M rottlerin (Figs. 1Ae and 1Af). As mentioned above, the inhibition of cell migration was not due to a toxic effect of rottlerin on the cells under these conditions, as tested by the trypan blue exclusion and cell proliferation assays (see Materials and Methods). To quantitate the migration, migration indices were measured as described under Materials and Methods. As shown in Fig. 1B, in the absence of rottlerin human keratinocytes migrated and produced MIs over 30. However, in the presence of rottlerin, the MIs were reduced dramatically in a concentration-dependent manner. These results suggested that rottlerin-sensitive cellular targets, specifically PKC␦, are involved in the collagen-driven signal tranduction leading to human keratinocyte migration. Identification of PKC Family Members in Human Keratinocytes We then investigated whether human keratinocytes express PKC␦ and, further, how many of the 13 identified PKC family members were expressed in human keratinocytes. To address these questions, we screened human keratinocytes and the immortalized human keratinocyte line, IKC, with a panel of anti-PKC antibodies, which is commercially available (Materials and Methods). Rat brain extract (supplied by Transduction Laboratories) and NIH3T3 cells were included as comparative controls. As shown in Fig. 2, the expression of
222
LI ET AL.
FIG. 1. Inhibition of PKC␦ blocks human keratinocyte migration on collagen. Human keratinocytes (HK), cultured in KGM with BPE in a tissue culture dish, were lifted off by trypsinization, pelleted by centrifugation, and resuspended in KGM medium without or with the indicated concentrations of rottlerin. Approximately 3000 cells were seeded on each preprepared either uncoated (⫺) or collagen-coated (⫹) gold salt covered coverslips in KGM complete medium. The incubation was stopped after 12 h by fixing the cells with 0.1% formaldehyde in PBS, and migration was analyzed. (A) Representative photographic images. (B) The migration indices (MIs) were measured as described under Materials and Methods. Similar results were observed from three independent experiments.
PKC (Fig. 2B), PKC␥ (Fig. 2C), and PKC (Fig. 2H) was detected in rat brains (lanes 1), but not in human keratinocytes or in IKC cells (lanes 2 and 3). A low level
of PKC␣ expression was detected in the IKC cells (Fig. 2A), but not in human keratinocytes (lane 3 vs lane 2). PKC␦ (Fig. 2D) and PKC (Fig. 2E), however, were
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY
223
“atypical” PKC subfamily that does not requires Ca 2⫹ or DAG for activation and is therefore insensitive to rottlerin. Interestingly, PKC (Fig. 2F) and PKC (Fig. 2G) were detected in IKC cells but not in human keratinocytes. The reason for this difference is unknown. We have not examined the expression of PKC-, -, and -. Antibodies against these proteins from Transduction Laboratories did not work in our hands, i.e., they failed to detect the antigens from the rat brain extracts (positive control). Dlugosz et al. reported that mouse keratinocytes express mRNAs encoding ␣, ␦, , , and , although whether the proteins of all these PKC isoforms are expressed in the cells was not clear from the paper [12]. The expression of PKC-, -, and - was not tested in the mouse keratinocytes. Nevertheless, PKC␦ is expressed in both human and mouse keratinocytes, suggesting that it plays an evolutionarily important role in keratinocytes. Rottlerin was reported to have certain specificity toward PKC␦ (IC 50, 3– 6 M) among six other DAGsensitive PKC kinases (IC 50 ⬎ 30 M) [24]. It does not inhibit casein kinase II, protein kinase A, and src tyrosine kinases. However, under the range of concentrations which inhibit PKC␦, rottlerin has also been reported to inhibit CaM-kinase III (IC50, 5.3 M) [21]. Therefore, the role of PKC␦ on collagen-driven cell migration had to be further examined using more specific approaches. High Gene Transduction Efficiency in Human Keratinocytes by a Lentiviral Vector
FIG. 2. Identification of PKC family members in HKs. Expression of PKC family members in HK, as well in IKC and NIN 3T3 cells, was analyzed by a panel of monoclonal antibodies against 9 of the 13 PKC members so far identified. Rat brain extracts were included as the positive control. Equal amounts (40 g/lane) of cell lysates were resolved in SDS gels, transferred to nitrocellulose membrane, and immunoblotted with each of the specific antibodies. The results were visualized by ECL detection (Materials and Methods). This experiment was done three times.
clearly expressed in both human keratinocytes and IKC cells, as well as in NIH3T3 cells. Between the latter two PKC family members, PKC␦ is the only one that belongs to the “novel” group of PKCs whose activation requires DAG, but not Ca 2⫹. PKC belongs to the
To further study the specific role of PKC␦ in the control of human keratinocyte motility, we overexpressed dominant-negative (kinase-deficient) PKC␦ (PKC␦-K376R) and tested whether the collagen-driven keratinocyte migration was altered. Prior to carrying out these experiments, we sought to establish a gene delivery system that offers high transduction efficiency in transient expression to primary human keratinocytes (it is not possible to establish PKC␦ stable cell lines from primary cells). Most of the previously used transfection techniques, including calcium phosphate precipitation, Lipofectimine/Lipofectin (Gibco), Superfect (Qiegene), Cytofectin (Bio-Rad), and Targefect (F2) (Targeting System), proved to have expression efficiency of no more than 25%. Even retroviral infection of LZRSpBMN-Z–GFP and pLXSN–GFP vectors did not improve the expression efficiency significantly in these cells. This relatively low range of efficiencies of exogenous DNA uptake has often been the reason why the dominant negative effect of the gene is unclear. To solve this technical problem, we tested viral infection by using a recently established HIV (lentivirus) type-1 based vector, pRRLsinhCMV. Previous studies have shown that this viral vector system improves transduc-
224
LI ET AL.
FIG. 3. Lentiviral infection and activation of PKC␦ in migratory HKs. PKC␦ cDNAs or GPF gene were subcloned into HIV type-1-derived lentiviral vector pRRLsinhCMV and the constructs were transfected into 293T cells for production of replication-incompetent virus stocks. (A) HK cells were either infected with pRRLsinhCMV–GFP or transfected with pCDNA3–GFP by the five other listed reagents (calcium phosphate precipitation/Ca 2⫹, Lipofectimine/Lipof, Superfect/Superf, Cytofectin/Cytof. (Bio-Rad), and Targefect/F2 (Targeting System)). The transduction efficiencies were analyzed under fluorescent microscopy (top) and by fluorescence-activated cell sorting (% as given underneath each panel). (B) HKs, infected with either pRRLsinhCMV–WT–PKC␦ or pRRLsinhCMV–PKC–DN, were lysed and the expression of PKC␦ proteins was analyzed by anti-PKC␦ antibody immunoblotting (top). Nck expression was included as the control for sample loading (bottom). (C) Activation of the endogenous PKC␦ was analyzed in nonmigratory versus migratory HKs. Lysates of human keratinocyte cultures from either polylysine-coated without growth factors or collagen-coated with the presence of growth factors were immunoprecipitated with anti-PKC␦ antibodies. The immune complexes were resolved in SDS gels, transferred to nitrocellulose membranes, and immunoblotted either with an anti-phosphotyrosine antiserum (top) or anti-PKC␦ antibodies (bottom). (D) Cellular fractionation of the nonmigratory and migratory human keratinocytes was carried out [37]. Anti-PKC␦ antibody (UCG-88) immunoprecipitates of the Triton X-100 soluble membrane (memb.), cytosol (cyto.), and nuclear (nucl.) fractions were subjected to Western blot with a monoclonal anti-PKC␦ (P-36520). Results were visualized by ECL. (A) was done five times, (B) was done three times, (C) was done four times, and (D) was done two times. FIG. 4. Dominant-negative PKC␦ blocks HK migration on collagen. HK cells (40% confluence) were infected with pRRLsinhCMV–GFP, pRRLsinhCMV–WT–PKC, or pRRLsinhCMV–DN–PKC vector for 6 h, changed with fresh medium, and incubated for an additional 72 h. Following trypsinization, approximately 3000 cells were seeded on either uncoated (⫺) or collagen-coated (⫹) gold salt coverslips in complete SFM medium. Cells were fixed after 20 h and migration was measured as described under Materials and Methods. (A) Photographic images of representative migration tracks under each of eight conditions; (B) Quantitation of the migration by measuring the migration indices (Materials and Methods). This experiment was repeated for four times.
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY
225
226
LI ET AL.
tion efficiency up to 20-fold, and, most encouragingly, it does so in both dividing and nondividing cells [16]. Using the pRRLsinhCMV–GFP construct as the readout, as shown in Fig. 3A, more than 90% of the cells expressed GFP by 72 h following the infection (Fig. 3A1). In contrast, five other transfection techniques as listed in Fig. 3A offered a much lower range of gene transduction efficiency, from 6 to 23% (Figs. 3A2 to 3A6). Therefore, we tested the expression of the wild-type and mutant PKC␦ under the pRRLsinhCMV vector in human keratinocytes. As shown in Fig. 3B, expression of both the wild-type PKC␦ (top, lane 1) and the PKC␦K376R mutant (top, lane 2) in infected human keratinocytes was 5-fold higher, based on scanning densitometry analyses, than the endogenous PKC␦ in uninfected cells (top, lane 3). Increasing concentrations of the viruses for infection could reach an expression level 15-fold higher than the expression of the endogenous PKC␦ (data not shown). Similar levels of expression of the adapter protein Nck were included as the control for sample loadings (Fig. 3B, bottom). These results show that we would be able to introduce the dominant-negative PKC␦ gene (PKC␦-K376R) into more than 90% of the human keratinocyte population and study its effect on cell migration. Prior to the migration assay, we also sought to determine whether PKC␦ is a signaling target in collagen-driven migratory keratinocytes. As a marker for the activation of PKC␦, we used the well-established property of PKC␦, tyrosine phosphorylation in ECM and growth-factor-stimulated cells [10, 35, 36], as the readout. Growth-factor-starved human keratinocytes were plated on either polylysine-coated (nonmigratory) or collagen-coated (migratory) tissue culture plates for 1 h (the earliest time for the majority (⬎90%) of cells to attach or bind). The total cellular PKC␦ was recovered from the lysates of the nonmigratory and migratory cells by anti-PKC␦ antibody immunoprecipitation. The immunoprecipates were subjected to SDS–PAGE and Western analysis for tyrosine phosphorylation using an anti-phosphotyrosine antibody. Figure 3C demonstrates that binding to collagen (lane 2), but not to polylysine (lane 1), induced strong tyrosine phosphorylation of PKC␦. Similar to most of the ligand-induced protein tyrosine phosphorylation, this induced tyrosine phosphoryaltion was transient and declined by 3 h (data not shown). In these experiments, similar amounts of PKC␦ proteins were recovered by immunoprecipitation (Fig. 3C, bottom). To further confirm the above finding, we carried out cellular fractionation studies [37]. The membrane, cytosol, and nuclear fractions of human keratinocytes under either migratory (collagen plus BPE) or nonmigratory (polylysine) conditions were analyzed by Western blot with anti-PKC␦ antibodies. It is shown in Fig.
3D that there was a significant increase in the membrane-associated PKC␦ from the cells on collagen plus BPE (lane 1 vs lane 2). Consistently, there was a decrease in the cytosol-associated PKC␦ from the cells on collagen plus BPE (lane 3 vs lane 4). No significant amount of PKC␦ was detected in the nucleus (lanes 5 and 6). These data suggested that there was a collagendriven translocation of PKC␦ from the cytosol to the membrane. These results indicate that the PKC␦ pathway is activated in migratory but not stationary human keratinocytes. Dominant-Negative PKC␦ Blocks Collagen-Driven Migration Finally, human keratinocytes were infected with the lentiviral–PKC␦ constructs and subjected to migration assays. As shown in Fig. 4A, in the absence of collagen neither the cells with vector alone infection nor the wt or mutant PKC␦-infected cells showed significant migration (Figs. 4Aa, 4Ac, 4Ae, and 4Ag). On a collagencoated surface, however, the vector alone and wt PKC␦infected cells migrated as the parental HK cells (Figs. 4Ad and 4Af versus 4Ab). Interestingly, the collagendriven migration of human keratinocytes was dramatically inhibited in cells infected with the PKC␦-K376R mutant (Fig. 4Ah), which presumably competed with and prevented the endogenous PKC␦ from activation. Quantitation of these effects is shown in Fig. 4B. It is clearly shown that the MI of collagen-driven migration of the vector alone infected cells (MI 27 ⫾ 2.4) was reduced to 8 ⫾ 2.1 by overexpression of the PKC␦K376R mutant. Although the inhibition by the PKC␦K376R mutant was slightly less than that produced by rottlerin (Fig. 1), it is likely due to the fact that inhibition by a dominant-negative gene depends upon both the gene transduction efficiency and the expression levels of the mutant gene, both of which may be less than optimal in these experiments. Taken together, these results confirmed the finding that PKC␦ signaling is required for collagen-driven migration of human keratinocytes. DISCUSSION
In this study, we investigated the role of PKC␦ signaling pathway in type I collagen-driven migration of human keratinocytes. We found that PKC␦ is one of at least two PKC family members (but not including PKC␣, PKC, and PKC, as previously reported [11]) expressed in primary human keratinocytes. PKC␦ became tyrosine phosphorylated and translocated to the membrane in response to extracellular migratory signals. Inhibition of PKC␦ by rottlerin, a specific inhibitor for PKC␦, completely blocked keratinocyte migration on a collagen matrix in the presence of growth
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY
factors. To further confirm this finding, we used a HIV (lentivirus) type-1 based vector, which offers more than 90% gene transduction efficiency in primary human keratinocytes, to deliver and overexpress a loss-offunction mutant of PKC␦. We show that overexpression of a dominant-negative form of PKC␦ dramatically blocked human keratinocyte migration. We conclude that the PKC␦ pathway in human keratinocytes is necessary for migratory signal transduction. A great deal of attention has recently been paid to the function and mechanisms of action of PKC␦, largely because it is the only PKC family member which serves as a direct target for protein tyrosine kinases in response to growth factors such as PDGF-bb and stresses such as H 2O 2 (H 2O 2 also causes tyrosine phosphorylation of other PKC family members) [10, 22]. The effect of tyrosine phosphorylation on PKC␦ activity can be either positive or negative depending upon the cell type or downstream pathways [10, 22]. The reported biological functions of PKC␦ include transformation of NIH3T3 cells [23], differentiation of murine erythroleukemia cells [24] and murine myeloid 32D cells [22, 28], growth inhibition upon its overexpression in CHO [26], smooth muscle cells [30], NIH3T3 cells [31], human glioma cells [32], and capillary endothelial cells [30], and apoptosis in human myeloid leukemia cells [31, 32]. In human keratinocytes, Geiges et al. showed that expression of PKC␦ is markedly reduced upon ras transformation [11]. Moreover, skin tumor formation is significantly reduced in transgenic mice overexpressing PKC␦ in the epidermis [13]. Thus, these studies suggest that PKC␦ carries out multiple cellular functions under different cell types and cellular contexts. The signaling mechanism by which the binding of serum factors and collagen to their cell surface receptors leads to activation of PKC␦ remains to be studied. It is possible that that the growth factor receptor tyrosine kinases and/or integrin-activated protein tyrosine kinases such as FAK and Src phosphorylate and activate PLC␥, which produces IP3 and DAG. DAG binds PKC␦ and relocates it to the plasma membrane. Recently, we have found that FAK phosphorylation is tightly correlated with the migratory state of the keratinocyte and the degree to which the ECM upon which the cell is juxtuaposed induces motility [7]. Alternatively, Src may also directly phosphorylate PKC␦ on tyrosine, leading to the full activation of PKC␦. Indeed, it has been shown that Src is the likely tyrosine kinase that phosphorylates PKC␦ in vitro and in vivo [33, 34]. Consistent with this hypothesis, we also found that PP1, an inhibitor for Src tyrosine kinases, potently inhibits human keratinocyte migration on collagen [6], suggesting that Src tyrosine kinase is also involved. How the PKC␦ pathway contributes to human keratinocyte migration remains to be further studied. One possible mechanism is that PKC␦ activation increases
227
the transcriptional activation of genes whose products are involved in the regulation of cell motility. For instance, it is known that secretion of metalloproteases by keratinocytes and synthesis of lamellipodia-associated proteins (ezrin, radixin, and moesin) correlate with increased cell motility [5]. It would be of great interest to test whether the PKC␦ pathway mediates the collagen-induced secretion of metalloproteases and increased synthesis of the lamellipodia-associated proteins in human keratinocytes. This study was supported in part by NIH Grants R01 AR46538-01 to D. T. W. and R01 CA65567-05 to W. L. We thank Fritz Costa and Airie Kim for excellent technical assistance. The usage of the USC Norris Cancer Center FACS facility and the help from Hal Saucier are appreciated.
REFERENCES 1.
2. 3. 4.
5.
6.
7.
8.
9.
10. 11.
12.
Woodley, D. T. (1996). Re-epithelialization. In “The Molecular and Cellular Biology of Wound Repair,” 2nd ed. (R. A. F. Clark, Ed.), pp. 339 –350, Plenum, New York. Martin, P. (1997). Wound healing—Aiming for perfect skin regeneration. Science 276, 75– 81. Singer, A. J., and Clark, R. A. (1999). Cutaneous wound healing. N. Engl. J. Med. 341, 738 –746. Sarret, Y., Woodley, D. T., Grigsby, K., Wynn, K., and O’Keefe, E. J. (1992). Human keratinocyte locomotion: The effect of selected cytokines. J. Invest. Dermatol. 98, 12–16. O’Toole, E. A., Marinkovich, M. P., Peavey, C. L., Amieva, M. R., Furthmayr, H., Mustoe, T. A., and Woodley, D. T. (1997). Hypoxia increases human keratinocyte motility on connective tissue. J. Clin. Invest. 100, 2881–2891. Li, W., Nadelman, C., Fan, J., Muellenhoff, M., Medina, E., Henry, G., Gratch, N. S., Chen, M., Han, J., and Woodley, D. (2001). The p38 MAPK/SAPK pathway is required for human keratinocyte migration on dermal collagen. JID, in press. Yurko, M. A., O’Toole, E. A., and Woodley, D. T. (2001). Phosphorylation of focal adhesion kinase 9pp125FAK is increased in human keratinocytes induced to migrate by extracellular matrices. J. Cell. Physiol. 188, 24 –32. Zeigler, M. E., Chi, Y., Schmidt, T., and Varani, J. (1999). Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes. J. Cell. Physiol. 180, 271–284. Johansson, N., Ala-aho, R., Uitto, V.-J., Gre´nman, R., Fusenig, N. E., Lo´pez-Otı´n, C., and Ka¨ha¨ri, V.-M. (2000). Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J. Cell Sci. 113, 227–235. Gschwendt, M. (1999). Protein kinase C␦. FEBS Lett. 259, 555–564. Geiges, D., Marks, F., and Gschwendt, M. (1995). Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha. Exp. Cell Res. 219, 299 –303. Dlugosz, A. A., Mischak, H., Mushinski, J. F., and Yuspa, S. H. (1992). Transcripts encoding protein kinase C-alpha, -delta, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells. Mol. Carcinog. 5, 286 –292.
228 13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26.
LI ET AL. Reddig, P. J., Dreckschmidt, N. E., Zou, J., Bourguignon, S. E., Oberley, T. D., and Verma, A. K. (2000). Transgenic mice overexpressing protein kinase C epsilon in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion. Cancer Res. 60, 595– 602. Szallasi, Z., Denning, M. F., Smith, C. B., Dlugos, A. A., Yuspa, S. H., Pettit, G. R. and Blumberg, P. M. (1994). Bryostatin 1 portects protein kinase C-␦ from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol esterinduced differentiation. Mol. Pharmacol. 48, 840 – 850. Li, W., Hu, P., Scolnik, E., and Schlessinger, J. (1992). The SH2 and SH3 domains-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors. Mol. Cell. Biol. 12, 5824 –5833 Sakoda, T., Kasahara, N., Hamamori, Y., and Kedes, L. (1988). A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. J. Mol. Cell. Cardiol. 31, 2037–2047. Li, W., Yu, J. C., Shin, D. Y., and Pierce, J. H. (1995). Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J. Biol. Chem. 270, 8311– 8318. Li, W., Yeung, Y. G., and Stanley, E. R. (1991). Tyrosine phosphorylation of a 57-kDa protein in growth factor stimulated and transformed cells. J. Biol. Chem. 266, 6808 – 6814. Albercht-Buehler, G. (1977). the phargokinetic tracks of 3T3 cells. Cell 11, 395– 404. Chen, J. D., Kim, J. P., Zhang, K., Sarret, Y., Wynn, K. C., Kramer, R. H., and Woodley, D. T. (1993). Epidermal growth factor (EGF) promotes human keratinocyte locomotion on collagen by increasing the alpha 2 integrin subunit. Exp. Cell Res. 209, 216 –223. Gschwendt, M., Mu¨ller, H. J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G., and Marks, F. (1994). Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93–98. Li, W., and Pierce, J. H. (1994). Protein kinase C-delta, an important signaling molecule in the platelet-derived growth factor beta receptor pathway. Curr. Topics Microbiol. Immunol. 211, 55– 65. Li, W., Michieli, P., Alimandi, M., Lorenzi, M. V., Wu, Y., Wang, L. H., Heidaran, M. A., and Pierce, J. H. (1996). Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells. Oncogene 13, 731–773. Pessino, A., Passalacqua, M., Sparatore, B., Patrone, M., Melloni, E., and Pontremoli, S. (1995). Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells. Biochem. J. 312, 549 –554. Wang, Q. J., Acs, P., Goodnight, J., Giese, T., Blumberg, P. M., Mischak, H., and Mushinski, J. F. (1997). The catalytic domain of protein kinase C-delta in reciprocal delta and epsilon chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes. J. Biol. Chem. 272, 76 – 82. Watanabe, T., Ono, Y., Taniyama, Y., Hazama, K., Igarashi, K., Ogita, K., Kikkawa, U., and Nishizuka, Y. (1992). Cell division
Received July 6, 2001 Revised version received October 29, 2001 Published online January 10, 2002
arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies. Proc. Natl. Acad. Sci. USA 89, 10159 –10163. 27.
Fukumoto, S., Nishizawa, Y., Hosoi, M., Koyama, H., Yamakawa, K., Ohno, S., and Morii, H. (1997). Protein kinase C delta inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression. J. Biol. Chem. 272, 13816 –13822.
28.
Mischak, H., Goodnight, J. A., Kolch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M. G., Blumberg, P. M., Pierce, J. H., and Mushinski, J. F. (1993). Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J. Biol. Chem. 268, 6090 – 6096.
29.
Mishima, K., Ohno, S., Shitara, N., Yamaoka, K., and Suzuki, K. (1994). Opposite effects of the overexpression of protein kinase C gamma and delta on the growth properties of human glioma cell line U251 MG. Biochem. Biophys. Res. Commun. 201(1), 363–372.
30.
Harrington, E. O., Loffler, J., Nelson, P. R., Kent, K. C., Simons, M., and Ware, J. A. (1997). Enhancement of migration by protein kinase Calpha and inhibition of proliferation and cell cycle progression by protein kinase Cdelta in capillary endothelial cells. J. Biol. Chem. 272, 7390 –7397.
31.
Emoto, Y., Kisaki, H., Manome, Y., Kharbanda, S., and Kufe, D. (1996). Activation of protein kinase Cdelta in human myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine Blood 87, 1990 –1996.
32.
Ghayur, T., Hugunin, M., Talanian, R. V., Ratnofsky, S., Quinlan, C., Emoto, Y., Pandey, P., Datta, R., Huang, Y., Kharbanda, S., Allen, H., Kamen, R., Wong, W., and Kufe, D. (1996). Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis. J. Exp. Med. 184, 2399 –23404.
33.
Gschwendt, M., Kielbassa, K., Kittstein, W., and Marks, F. (1994). Tyrosine phosphorylation and stimulation of protein kinase C delta from porcine spleen by src in vitro. Dependence on the activated state of protein kinase C delta. FEBS Lett. 347, 85– 89.
34.
Shanmugam, M., Krett, N. L., Peters, C. A., Maizels, E. T., Murad, F. M., Kawakatsu, H., Rosen, S. T., and HunzickerDunn, M. (1998). Association of PKC delta and active Src in PMA-treated MCF-7 human breast cancer cells. Oncogene 16, 1649 –1654.
35.
Li, W., Mischak, H., Yu, J. C., Wang, L. M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994). Tyrosine phosphorylation of protein kinase C-delta in response to its activation. J. Biol. Chem. 269, 2349 –2352.
36.
Li, W., Yu, J. C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994). Stimulation of the plateletderived growth factor beta receptor signaling pathway activates protein kinase C-delta. Mol. Cell. Biol. 14, 6727– 6735.
37.
Chen, M., She, H., Kim, A., and Li, W. (2000). Nck regulates PDGF-stimulated actin polymerization in NIH 3T3 fibroblasts. Mol. Cell. Biol. 20, 7867–7880.
An Important Role for Protein Kinase C-␦ in Human Keratinocyte Migration on Dermal Collagen Wei Li,* ,† ,1 Celina Nadelman,* Noah S. Gratch,* Weiquin Li,‡ Mei Chen,* Nori Kasahara,§ and David T. Woodley* ,† ,1 *Department of Medicine, Division of Dermatology, and the Norris Cancer Center, and §Institute for Genetic Medicine, University of Southern California Keck School of Medicine, Los Angeles, California, 90033; †Greater Los Angles Veterans Administration Health System; and ‡Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007
Migration of human keratinocytes plays a critical role in the re-epithelialization of human skin wounds, the process by which the wound bed is resurfaced and closed by keratinocytes as it forms a new epidermis. While the importance of ECM components and serum factors in the regulation of keratinocytes motility is well established, the intracellular signaling mechanisms remain fragmentary. In this study, we investigated the role of protein kinase C␦ (PKC␦) signaling in the promotion of human keratinocyte migration by a collagen matrix and bovine pituitary extract. We found that pharmacological inhibition of the PKC␦ pathway completely blocks migration. Using a lentivirus-based vector system, which offers more than 90% gene transduction efficiency to human keratinocytes, we show that the kinase-defective mutant of PKC␦ (K376R) dramatically inhibits human keratinocyte migration. Furthermore, PKC␦ is activated in migrating human keratinocytes. These observations indicate for the first time that the PKC␦ pathway plays an important role in the control of human keratinocyte migration. © 2002 Elsevier Science (USA)
INTRODUCTION
The lateral migration of keratinocytes over the wound bed is an early and critical event in the reepithelialization of human skin wounds. Within hours after an acute wound, the basal keratinocytes at the margins of the wound and within the cut skin appendages (hair follicles, eccrine gland duct) enter a migratory mode and begin to migrate laterally across the provisional wound bed to resurface the wound [1–3]. The cells migrate on a provisional matrix composed of fibronectin, fibrin, and collagen fragments, which are formed by blood clot following tissue injury [3]. While both keratinocyte motility and division ultimately con1
To whom correspondence and reprint requests should be addressed. Fax: 323-224-7679. E-mail: [email protected]; [email protected].
tribute to the re-epithelialization of a skin wound, they are controlled by independent intracellular signaling mechanisms. First, keratinocyte migration occurs within 2–3 h in response to extracellular signals (ECM and growth factors/cytokines), while cell division does not take place until more than 24 h later [6]. Second, TGF inhibits the proliferative potential of human keratinocytes to virtually zero and yet the cells are fully capable of collagen-driven migration [4]. It is well established that the Ras–MEKK–MEK– ERK pathway mediates cell DNA synthesis and proliferation in response to extracellular signals such as EGF. In contrast, the intracellular signaling mechanisms that regulate human keratinocyte migration have just begun to be understood. Yurko et al. reported that, in highly migratory human keratinocytes, FAK (focal adhesion kinase) was heavily tyrosine phosphorylated throughout the 18-h period of the migration assay. Antisense oligonucleotides against FAK blocked human keratinocyte migration on collagen and fibronectin [7]. Zeigler et al. showed that hepatocyte growth factor (HGF) and EGF stimulate prolonged activation of ERKs and JNK in human keratinocytes. Blockade of the ERK activation by PD098059 inhibited HGF-stimulated cell migration and MMP-9 production in human keratinocytes [8]. Moreover, in the transformed keratinocyte cell line HaCaT, expression of MMP-13 and MMP-1 requires the p38 –MAPK 2 pathway [9]. We have recently reported that the p38 – MAPK pathway is involved in collagen-driven human keratinocyte migration [6]. Our results show that while the p38 –MAPK pathway is necessary for collagendriven migration, activation of this pathway alone is not sufficient to invoke keratinocyte migration on a collagen matrix [6]. This suggests that other parallel 2
Abbreviations used: PKC, protein kinase C; BMZ, basement membrane zone; ECM, extracellular matrix; TBS, Tris-buffered saline; PY, phosphotyrosine; DAG, diacylglyceral; MAPK, mitogenactivated protein kinase; GFP, green fluorescent protein; MI, migration index.
219
0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
220
LI ET AL.
signaling pathways are also needed and work in concert with the p38 –MAPK pathway. Protein kinase C (PKC) represents a superfamily of serine/threonine protein kinases, including three subgroups of PKCs. These subgroups are (i) the “conventional” PKCs that include ␣, I, II, and ␥; (ii) the “novel” PKCs that include ␦, , , and , and (iii) the “atypical” PKCs, , , and . There is also a fourth distinct group called PKC [14]. The differences between these subgroups are defined by their dependence or independence on calcium and phospholipids such as diacylglyerol (DAG) [10]. The human keratinocyte line, HaCaT, was reported to express four PKC family members, PKC␣, PKC␦, PKC, and PKC [11]. Mouse keratinocytes are detected to express the mRNAs of PKC␣, -␦, -, -, and - [12]. Recently, a great deal of attention has been paid to PKC␦ because (i) it has a wide range of expression in almost all types of cells and tissues studied, (ii) it is the only PKC that is a direct target for protein tyrosine kinases and (iii) it has been implicated to play an important role in growth control, cellular differentiation, and apoptosis [10]. The keratinocyte cell line, HaCaT, transfected with oncogenic H-Ras exhibited a down regulation of PKC␦. This effect was via an autocrine loop leading to an augmentation of TGF␣ expression by the cells. Evidence for this mechanism was the observation that the addition of TGF␣ to the HaCaT keratinocyte culture induced a selective down regulation of PKC␦ while three other PKC family members were unaffected [11]. With regard to keratinocyte differentiation, it has been shown that overexpression of PKC␦ in the epidermis of transgenic mice reduced tumor formation by phorbol ester, TPA [13]. In concordance with this observation, Szallasi and coworkers found that bryostatin inhibits TPA-induced differentiation of mouse keratinocytes while also inhibiting the TPA-induced down regulation of PKC␦ [14]. In this article, we report that the inhibition of PKC␦ dramatically inhibited human keratinocyte motility on collagen. We present both pharmacological and genetic evidence that activation of PKC␦ is required for human keratinocyte migration on a collagen matrix. MATERIALS AND METHODS Materials. Primary human keratinocytes were purchased from Clonetics (San Diego, CA) and cultured in keratinocyte growth medium (KGM; Clonetics) supplemented with bovine pituitary extract (BPE) as the source of growth factors in low Ca 2⫹ conditions, according to the manufacturer’s instruction. Native rat tail type I collagen was purchased from Collaborative Biomedical Products (Bedford, MA). Anti-PKC␦ antibody (P36520) and anti-PKC antibody kit (611421) were purchased from Transduction Laboratories (Lexington, KY). Rabbit anti-rat PKC␦ antiserum (No. UCG-88) was raised in our laboratories and specifically used for immunoprecipitations (see 71-3100, Zymed Laboratories, Inc., South San Francisco, CA). Anti-phosphotyrosine antibody (No. 72) was used as previously described [15]. Green fluorescent protein (GFP) gene in the lentiviral
vector pRRLsinhCMV was as described previously [16]. Gold chloride was purchased from Sigma. FTI and GGTI PP1 were gifts from Saı¨d Sebti (University of South Florida). PP1, Li ⫹, rottlerin, HA1004, wortmannin, SB202190, rapamycin, staurosporin, tyrphostin, SB203580, and LY294002 were purchased from Calbiochem (San Diego, CA). The potential cytotoxicity for human keratinocytes by these agents was examined by a trypan blue exclusion assay and a cell proliferation assay after drug withdrawal, as described [6]. Our criterion for the trypan blue exclusion assay was that after exposure to the agent, more than 90% of the cells excluded the trypan blue dye. The second criterion for the lack of cytotoxicity was that after exposure to the agent and its subsequent withdrawal, the cells were able to proliferate and double in culture at or above the 90% level of control unexposed cells. The effect of these inhibitors on human keratinocyte migration, as measured by the colloidal gold phagokinetic migration assay, was subsequently investigated. Subcloning and production of lentiviral stocks and infection. Human immunodeficiency virus (HIV, Lentivirus)-derived vector, pRRLsinhCMV, was digested with EcoRI and SalI and purified. Similarly digested and purified wild-type or the dominant negative mutant (K376R) of the bovine PKC␦ cDNA fragments from pCEV27 vector [17] was ligated into the pRRLsinhCMV vector. These constructs were used to cotransfect 293T cells together with two packaging vectors, pCMV⌬R8.2 and pMDG, the process called the three plasmid expression system [16]. Conditioned media were collected, filtered, and concentrated prior to being used for infecting human keratinocytes. Human keratinocytes were cultured in KGM with BPE, according to the manufacturer’s instruction. The third or fourth passage of keratinocytes in culture was used for experiments. For infection experiments, cells were plated 1 day prior to infection in six-well tissue culture plates and after the cells reached 40 – 60% confluence, the cultures were subjected to infection. Infection was carried out by following a previously published procedure [16]. Expression of the PKC␦ proteins was confirmed by immunoblotting the lysates of infected cells with anti-PKC␦ antibody (36520, Transduction Laboratories) (vide infra). Titration of the wild-type and mutant PKC␦ viral stocks was conducted in order to achieve equal expression of the wild-type and the mutant PKC␦ in human keratinocytes. Preparation of cell lysates, SDS–polyacrylamide gel electrophoresis (PAGE), and Western blot. Tissue culture dishes with human keratinocyte adherent cultures were washed with PBS buffer and solubilized in lysis buffer (0.3 ml/10 cm dish) as described by us [18]. Following measurement of the protein concentrations (Bio-Rad protein assay using known concentrations of BSA as the reference at 595 nm visible light (Hitachi 4000)), 50 g of the lysates was directly subjected to SDS–PAGE and Western immunoblotting analysis. In selected experiments, 500 g of the lysates was immunoprecipiated with ant-PKC␦ antibodies (UCG-88) prior to SDS–PAGE and Western blot analysis (vide infra). SDS–PAGE was carried out in MiniProtean II system (Bio-Rad). We used 10% SDS bis-acrylamide resolving gels (2 ml of bis-acrylamide solution (0.8:30), 1.5 ml of Tris– HCl buffer, pH 8.8, 2.5 ml of H 2O, 40 l of 10% ammonium persulfate, and 2.5 l of TEMED) and 1% stacking gel (0.5 ml of bis-acrylamide solution, 0.625 ml of Tris–HCl buffer, pH 6.8, 1.625 ml of H 2O, 7.5 l of 10% ammonium persulfate, and 2.5 l of TEMED) for all experiments. Electrophoresis was conducted in running solution (0.25 M of Tris, 1.92 M of glycine, and 1% SDS) under a fixed voltage (150 V) for 1 h. Electrical transfer to a nitrocellulose membrane was carried out in a Mini Trans-Blot Transfer cell system (Bio-Rad) containing transfer buffer (25 mM Tris– base, 0.2 M glycine, 0.05% SDS (w/v)) in an electrical field of a fixed 50 V for 2 h at RT. Transfer efficiency under these conditions was assessed by staining the membrane with 5% Ponceau’s dye and by staining the gel afterward with Coomassie brilliant blue. Under these conditions, we were constantly experiencing more than 95% transfer efficiency. Membranes were washed two times in TBS-T (Tris-buffered saline
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY plus Triton X-100) and then two times in TBS, blotted for 2 h in TBS containing 5% nonfat milk, and incubated with a primary antibody in the same solution. The membranes were washed once with TBS, twice with TBS–Triton X-100, and once with TBS prior to incubation with HRP-conjugated secondary antibody for 1 h at RT. The membranes were subjected to the same washing procedure as above, and the results were visualized by ECL reaction. Cell motility colloidal gold phagokinetic assay. Cell migration on collagen was examined by using the migration track assay, as previously described by Albrecht-Buehler [19] and modified by Woodley for computer-assisted analysis [1]. Briefly, approximately 3000 cells were plated onto coverslips and allowed to migrate for different periods of time. At the end of the assay, cells were fixed and migration was examined under dark field optics and photographed. Fifteen randomly selected and nonoverlapping fields under each experimental condition were analyzed with an attached CCD camera (Model KP-MIU, Hitachi-Denshi Ltd.) and a computer using the NIH Image 1.6 program. The system calculated the percentage of the total field area viewed by the camera that was consumed with linear cell migration tracks. This percentage is termed a migration index (MI). Statistical analysis. The methodology to determine whether the difference in MIs between experimental sets of migrating keratinocytes exists has previously been published [20]. In brief, statistical analyses of the differences in MIs among triplicate sets of experimental conditions were performed using Microsoft Excel. Confirmation of a difference in migration as being statistically significant requires rejection of the null hypothesis of no difference between mean migration indices obtained from replicate sets at the P ⫽ 0.05 level with the Student t test. Activation of PKC␦. Human keratinocytes, starved in growthfactor-free medium overnight, were incubated in tissue culture plates precoated with either polylysine (nonmigratory) or type I collagen (migratory) at 37°C for different periods of time. The cells were then solubilized in lysis buffer as previously described [18], and the protein concentrations in the lysates were measured as described above. Equal amounts of cell extract protein (⬃500 g) were incubated with anti-PKC␦ antiserum (UCG-89, 1:500) overnight at 4°C. The immune complexes were precipitated with protein A agarose beads (25 l packed beads, Zymed). After being washed three times with lysis buffer, the beads were heated at 95°C for 5 min in SDS– gel sample buffer. The immunoprecipitates were subjected to SDS– PAGE and Western immunoblotting analysis using anti-phosphotyrosine (PY) antibody (No. 72). The labeled protein bands were visualized by ECL, as described above. The tyrosine phosphorylation correlates well with the growth factor stimulation of PKC␦ [14]. To further confirm PKC␦ activation, we carried out cellular fractionation analysis. Human keratinocytes from nonmigratory (polylysine-coated plates in the absence of BPE) and migratory (collagencoated plates in the presence of BPE) conditions were subjected to cellular fractionation by a previously described procedure of ours [37]. The Triton X-100 soluble extracts of the membrane and the nuclear fractions, and the cytosol fraction, were immunoprecipitated with anti-PKC␦ antibodies (UCG-88, raised by our laboratory). The immunoprecipitates were further analyzed by Western blot with an anti-PKC␦ antibody (P-36520, Transduction Laboratories).
RESULTS
Inhibition of PKC␦ Blocks Human Keratinocyte Migration on Collagen To investigate the intracellular signal transduction following the attachment and migration of human keratinocytes on type I collagen, we screened a panel of chemical inhibitors for their potential inhibitory effects
221
on the migration of human keratinocytes. We found that 3 of the 11 inhibitors tested, SB202190/SB202580, rottlerin, and PP1, potently blocked (⬎80%) cell migration on type I collagen without compromising cell viability. The rest of the inhibitors had either no effect or insignificant (⬍15%) inhibitory effects. It is known that SB202190/SB202580 (IC 50, 0.3 M), rottlerin (IC 50, 3– 6 M), and PP1 (IC 50, 5 M) inhibit the activation of p38 –MAPK, protein kinase C␦, and Src, respectively. In the current study, we focused on the role of the PKC␦ pathway in the regulation of collagen-driven human keratinocyte migration (the role of p38 –MAPK has been independently investigated, Ref. [6]). As shown in Fig. 1A, on an uncoated surface of immobilized gold, human keratinocytes showed little migration even in the presence of complete medium (Fig. 1Aa). Likewise, no migration occurred on tissue culture polystyrine plastic in an in vitro wound healing assay (“scratch assay,” data not shown). On a collagen-coated colloidal gold substratum, however, the cells made markedly linear migration tracks, leaving behind black gold particle-free tracks (Fig. 1Ab) (location of the cell inside each track is indicated by an arrow). In contrast, parallel cells incubated in medium containing different concentrations of rottlerin lost their migratory ability on collagen in a concentration-dependent fashion (Figs. 1Ac to 1Af) and completely stopped migrating in the presence of 5–15 M rottlerin (Figs. 1Ae and 1Af). As mentioned above, the inhibition of cell migration was not due to a toxic effect of rottlerin on the cells under these conditions, as tested by the trypan blue exclusion and cell proliferation assays (see Materials and Methods). To quantitate the migration, migration indices were measured as described under Materials and Methods. As shown in Fig. 1B, in the absence of rottlerin human keratinocytes migrated and produced MIs over 30. However, in the presence of rottlerin, the MIs were reduced dramatically in a concentration-dependent manner. These results suggested that rottlerin-sensitive cellular targets, specifically PKC␦, are involved in the collagen-driven signal tranduction leading to human keratinocyte migration. Identification of PKC Family Members in Human Keratinocytes We then investigated whether human keratinocytes express PKC␦ and, further, how many of the 13 identified PKC family members were expressed in human keratinocytes. To address these questions, we screened human keratinocytes and the immortalized human keratinocyte line, IKC, with a panel of anti-PKC antibodies, which is commercially available (Materials and Methods). Rat brain extract (supplied by Transduction Laboratories) and NIH3T3 cells were included as comparative controls. As shown in Fig. 2, the expression of
222
LI ET AL.
FIG. 1. Inhibition of PKC␦ blocks human keratinocyte migration on collagen. Human keratinocytes (HK), cultured in KGM with BPE in a tissue culture dish, were lifted off by trypsinization, pelleted by centrifugation, and resuspended in KGM medium without or with the indicated concentrations of rottlerin. Approximately 3000 cells were seeded on each preprepared either uncoated (⫺) or collagen-coated (⫹) gold salt covered coverslips in KGM complete medium. The incubation was stopped after 12 h by fixing the cells with 0.1% formaldehyde in PBS, and migration was analyzed. (A) Representative photographic images. (B) The migration indices (MIs) were measured as described under Materials and Methods. Similar results were observed from three independent experiments.
PKC (Fig. 2B), PKC␥ (Fig. 2C), and PKC (Fig. 2H) was detected in rat brains (lanes 1), but not in human keratinocytes or in IKC cells (lanes 2 and 3). A low level
of PKC␣ expression was detected in the IKC cells (Fig. 2A), but not in human keratinocytes (lane 3 vs lane 2). PKC␦ (Fig. 2D) and PKC (Fig. 2E), however, were
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY
223
“atypical” PKC subfamily that does not requires Ca 2⫹ or DAG for activation and is therefore insensitive to rottlerin. Interestingly, PKC (Fig. 2F) and PKC (Fig. 2G) were detected in IKC cells but not in human keratinocytes. The reason for this difference is unknown. We have not examined the expression of PKC-, -, and -. Antibodies against these proteins from Transduction Laboratories did not work in our hands, i.e., they failed to detect the antigens from the rat brain extracts (positive control). Dlugosz et al. reported that mouse keratinocytes express mRNAs encoding ␣, ␦, , , and , although whether the proteins of all these PKC isoforms are expressed in the cells was not clear from the paper [12]. The expression of PKC-, -, and - was not tested in the mouse keratinocytes. Nevertheless, PKC␦ is expressed in both human and mouse keratinocytes, suggesting that it plays an evolutionarily important role in keratinocytes. Rottlerin was reported to have certain specificity toward PKC␦ (IC 50, 3– 6 M) among six other DAGsensitive PKC kinases (IC 50 ⬎ 30 M) [24]. It does not inhibit casein kinase II, protein kinase A, and src tyrosine kinases. However, under the range of concentrations which inhibit PKC␦, rottlerin has also been reported to inhibit CaM-kinase III (IC50, 5.3 M) [21]. Therefore, the role of PKC␦ on collagen-driven cell migration had to be further examined using more specific approaches. High Gene Transduction Efficiency in Human Keratinocytes by a Lentiviral Vector
FIG. 2. Identification of PKC family members in HKs. Expression of PKC family members in HK, as well in IKC and NIN 3T3 cells, was analyzed by a panel of monoclonal antibodies against 9 of the 13 PKC members so far identified. Rat brain extracts were included as the positive control. Equal amounts (40 g/lane) of cell lysates were resolved in SDS gels, transferred to nitrocellulose membrane, and immunoblotted with each of the specific antibodies. The results were visualized by ECL detection (Materials and Methods). This experiment was done three times.
clearly expressed in both human keratinocytes and IKC cells, as well as in NIH3T3 cells. Between the latter two PKC family members, PKC␦ is the only one that belongs to the “novel” group of PKCs whose activation requires DAG, but not Ca 2⫹. PKC belongs to the
To further study the specific role of PKC␦ in the control of human keratinocyte motility, we overexpressed dominant-negative (kinase-deficient) PKC␦ (PKC␦-K376R) and tested whether the collagen-driven keratinocyte migration was altered. Prior to carrying out these experiments, we sought to establish a gene delivery system that offers high transduction efficiency in transient expression to primary human keratinocytes (it is not possible to establish PKC␦ stable cell lines from primary cells). Most of the previously used transfection techniques, including calcium phosphate precipitation, Lipofectimine/Lipofectin (Gibco), Superfect (Qiegene), Cytofectin (Bio-Rad), and Targefect (F2) (Targeting System), proved to have expression efficiency of no more than 25%. Even retroviral infection of LZRSpBMN-Z–GFP and pLXSN–GFP vectors did not improve the expression efficiency significantly in these cells. This relatively low range of efficiencies of exogenous DNA uptake has often been the reason why the dominant negative effect of the gene is unclear. To solve this technical problem, we tested viral infection by using a recently established HIV (lentivirus) type-1 based vector, pRRLsinhCMV. Previous studies have shown that this viral vector system improves transduc-
224
LI ET AL.
FIG. 3. Lentiviral infection and activation of PKC␦ in migratory HKs. PKC␦ cDNAs or GPF gene were subcloned into HIV type-1-derived lentiviral vector pRRLsinhCMV and the constructs were transfected into 293T cells for production of replication-incompetent virus stocks. (A) HK cells were either infected with pRRLsinhCMV–GFP or transfected with pCDNA3–GFP by the five other listed reagents (calcium phosphate precipitation/Ca 2⫹, Lipofectimine/Lipof, Superfect/Superf, Cytofectin/Cytof. (Bio-Rad), and Targefect/F2 (Targeting System)). The transduction efficiencies were analyzed under fluorescent microscopy (top) and by fluorescence-activated cell sorting (% as given underneath each panel). (B) HKs, infected with either pRRLsinhCMV–WT–PKC␦ or pRRLsinhCMV–PKC–DN, were lysed and the expression of PKC␦ proteins was analyzed by anti-PKC␦ antibody immunoblotting (top). Nck expression was included as the control for sample loading (bottom). (C) Activation of the endogenous PKC␦ was analyzed in nonmigratory versus migratory HKs. Lysates of human keratinocyte cultures from either polylysine-coated without growth factors or collagen-coated with the presence of growth factors were immunoprecipitated with anti-PKC␦ antibodies. The immune complexes were resolved in SDS gels, transferred to nitrocellulose membranes, and immunoblotted either with an anti-phosphotyrosine antiserum (top) or anti-PKC␦ antibodies (bottom). (D) Cellular fractionation of the nonmigratory and migratory human keratinocytes was carried out [37]. Anti-PKC␦ antibody (UCG-88) immunoprecipitates of the Triton X-100 soluble membrane (memb.), cytosol (cyto.), and nuclear (nucl.) fractions were subjected to Western blot with a monoclonal anti-PKC␦ (P-36520). Results were visualized by ECL. (A) was done five times, (B) was done three times, (C) was done four times, and (D) was done two times. FIG. 4. Dominant-negative PKC␦ blocks HK migration on collagen. HK cells (40% confluence) were infected with pRRLsinhCMV–GFP, pRRLsinhCMV–WT–PKC, or pRRLsinhCMV–DN–PKC vector for 6 h, changed with fresh medium, and incubated for an additional 72 h. Following trypsinization, approximately 3000 cells were seeded on either uncoated (⫺) or collagen-coated (⫹) gold salt coverslips in complete SFM medium. Cells were fixed after 20 h and migration was measured as described under Materials and Methods. (A) Photographic images of representative migration tracks under each of eight conditions; (B) Quantitation of the migration by measuring the migration indices (Materials and Methods). This experiment was repeated for four times.
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY
225
226
LI ET AL.
tion efficiency up to 20-fold, and, most encouragingly, it does so in both dividing and nondividing cells [16]. Using the pRRLsinhCMV–GFP construct as the readout, as shown in Fig. 3A, more than 90% of the cells expressed GFP by 72 h following the infection (Fig. 3A1). In contrast, five other transfection techniques as listed in Fig. 3A offered a much lower range of gene transduction efficiency, from 6 to 23% (Figs. 3A2 to 3A6). Therefore, we tested the expression of the wild-type and mutant PKC␦ under the pRRLsinhCMV vector in human keratinocytes. As shown in Fig. 3B, expression of both the wild-type PKC␦ (top, lane 1) and the PKC␦K376R mutant (top, lane 2) in infected human keratinocytes was 5-fold higher, based on scanning densitometry analyses, than the endogenous PKC␦ in uninfected cells (top, lane 3). Increasing concentrations of the viruses for infection could reach an expression level 15-fold higher than the expression of the endogenous PKC␦ (data not shown). Similar levels of expression of the adapter protein Nck were included as the control for sample loadings (Fig. 3B, bottom). These results show that we would be able to introduce the dominant-negative PKC␦ gene (PKC␦-K376R) into more than 90% of the human keratinocyte population and study its effect on cell migration. Prior to the migration assay, we also sought to determine whether PKC␦ is a signaling target in collagen-driven migratory keratinocytes. As a marker for the activation of PKC␦, we used the well-established property of PKC␦, tyrosine phosphorylation in ECM and growth-factor-stimulated cells [10, 35, 36], as the readout. Growth-factor-starved human keratinocytes were plated on either polylysine-coated (nonmigratory) or collagen-coated (migratory) tissue culture plates for 1 h (the earliest time for the majority (⬎90%) of cells to attach or bind). The total cellular PKC␦ was recovered from the lysates of the nonmigratory and migratory cells by anti-PKC␦ antibody immunoprecipitation. The immunoprecipates were subjected to SDS–PAGE and Western analysis for tyrosine phosphorylation using an anti-phosphotyrosine antibody. Figure 3C demonstrates that binding to collagen (lane 2), but not to polylysine (lane 1), induced strong tyrosine phosphorylation of PKC␦. Similar to most of the ligand-induced protein tyrosine phosphorylation, this induced tyrosine phosphoryaltion was transient and declined by 3 h (data not shown). In these experiments, similar amounts of PKC␦ proteins were recovered by immunoprecipitation (Fig. 3C, bottom). To further confirm the above finding, we carried out cellular fractionation studies [37]. The membrane, cytosol, and nuclear fractions of human keratinocytes under either migratory (collagen plus BPE) or nonmigratory (polylysine) conditions were analyzed by Western blot with anti-PKC␦ antibodies. It is shown in Fig.
3D that there was a significant increase in the membrane-associated PKC␦ from the cells on collagen plus BPE (lane 1 vs lane 2). Consistently, there was a decrease in the cytosol-associated PKC␦ from the cells on collagen plus BPE (lane 3 vs lane 4). No significant amount of PKC␦ was detected in the nucleus (lanes 5 and 6). These data suggested that there was a collagendriven translocation of PKC␦ from the cytosol to the membrane. These results indicate that the PKC␦ pathway is activated in migratory but not stationary human keratinocytes. Dominant-Negative PKC␦ Blocks Collagen-Driven Migration Finally, human keratinocytes were infected with the lentiviral–PKC␦ constructs and subjected to migration assays. As shown in Fig. 4A, in the absence of collagen neither the cells with vector alone infection nor the wt or mutant PKC␦-infected cells showed significant migration (Figs. 4Aa, 4Ac, 4Ae, and 4Ag). On a collagencoated surface, however, the vector alone and wt PKC␦infected cells migrated as the parental HK cells (Figs. 4Ad and 4Af versus 4Ab). Interestingly, the collagendriven migration of human keratinocytes was dramatically inhibited in cells infected with the PKC␦-K376R mutant (Fig. 4Ah), which presumably competed with and prevented the endogenous PKC␦ from activation. Quantitation of these effects is shown in Fig. 4B. It is clearly shown that the MI of collagen-driven migration of the vector alone infected cells (MI 27 ⫾ 2.4) was reduced to 8 ⫾ 2.1 by overexpression of the PKC␦K376R mutant. Although the inhibition by the PKC␦K376R mutant was slightly less than that produced by rottlerin (Fig. 1), it is likely due to the fact that inhibition by a dominant-negative gene depends upon both the gene transduction efficiency and the expression levels of the mutant gene, both of which may be less than optimal in these experiments. Taken together, these results confirmed the finding that PKC␦ signaling is required for collagen-driven migration of human keratinocytes. DISCUSSION
In this study, we investigated the role of PKC␦ signaling pathway in type I collagen-driven migration of human keratinocytes. We found that PKC␦ is one of at least two PKC family members (but not including PKC␣, PKC, and PKC, as previously reported [11]) expressed in primary human keratinocytes. PKC␦ became tyrosine phosphorylated and translocated to the membrane in response to extracellular migratory signals. Inhibition of PKC␦ by rottlerin, a specific inhibitor for PKC␦, completely blocked keratinocyte migration on a collagen matrix in the presence of growth
PKC␦ REGULATES HUMAN KERATINOCYE MOTILITY
factors. To further confirm this finding, we used a HIV (lentivirus) type-1 based vector, which offers more than 90% gene transduction efficiency in primary human keratinocytes, to deliver and overexpress a loss-offunction mutant of PKC␦. We show that overexpression of a dominant-negative form of PKC␦ dramatically blocked human keratinocyte migration. We conclude that the PKC␦ pathway in human keratinocytes is necessary for migratory signal transduction. A great deal of attention has recently been paid to the function and mechanisms of action of PKC␦, largely because it is the only PKC family member which serves as a direct target for protein tyrosine kinases in response to growth factors such as PDGF-bb and stresses such as H 2O 2 (H 2O 2 also causes tyrosine phosphorylation of other PKC family members) [10, 22]. The effect of tyrosine phosphorylation on PKC␦ activity can be either positive or negative depending upon the cell type or downstream pathways [10, 22]. The reported biological functions of PKC␦ include transformation of NIH3T3 cells [23], differentiation of murine erythroleukemia cells [24] and murine myeloid 32D cells [22, 28], growth inhibition upon its overexpression in CHO [26], smooth muscle cells [30], NIH3T3 cells [31], human glioma cells [32], and capillary endothelial cells [30], and apoptosis in human myeloid leukemia cells [31, 32]. In human keratinocytes, Geiges et al. showed that expression of PKC␦ is markedly reduced upon ras transformation [11]. Moreover, skin tumor formation is significantly reduced in transgenic mice overexpressing PKC␦ in the epidermis [13]. Thus, these studies suggest that PKC␦ carries out multiple cellular functions under different cell types and cellular contexts. The signaling mechanism by which the binding of serum factors and collagen to their cell surface receptors leads to activation of PKC␦ remains to be studied. It is possible that that the growth factor receptor tyrosine kinases and/or integrin-activated protein tyrosine kinases such as FAK and Src phosphorylate and activate PLC␥, which produces IP3 and DAG. DAG binds PKC␦ and relocates it to the plasma membrane. Recently, we have found that FAK phosphorylation is tightly correlated with the migratory state of the keratinocyte and the degree to which the ECM upon which the cell is juxtuaposed induces motility [7]. Alternatively, Src may also directly phosphorylate PKC␦ on tyrosine, leading to the full activation of PKC␦. Indeed, it has been shown that Src is the likely tyrosine kinase that phosphorylates PKC␦ in vitro and in vivo [33, 34]. Consistent with this hypothesis, we also found that PP1, an inhibitor for Src tyrosine kinases, potently inhibits human keratinocyte migration on collagen [6], suggesting that Src tyrosine kinase is also involved. How the PKC␦ pathway contributes to human keratinocyte migration remains to be further studied. One possible mechanism is that PKC␦ activation increases
227
the transcriptional activation of genes whose products are involved in the regulation of cell motility. For instance, it is known that secretion of metalloproteases by keratinocytes and synthesis of lamellipodia-associated proteins (ezrin, radixin, and moesin) correlate with increased cell motility [5]. It would be of great interest to test whether the PKC␦ pathway mediates the collagen-induced secretion of metalloproteases and increased synthesis of the lamellipodia-associated proteins in human keratinocytes. This study was supported in part by NIH Grants R01 AR46538-01 to D. T. W. and R01 CA65567-05 to W. L. We thank Fritz Costa and Airie Kim for excellent technical assistance. The usage of the USC Norris Cancer Center FACS facility and the help from Hal Saucier are appreciated.
REFERENCES 1.
2. 3. 4.
5.
6.
7.
8.
9.
10. 11.
12.
Woodley, D. T. (1996). Re-epithelialization. In “The Molecular and Cellular Biology of Wound Repair,” 2nd ed. (R. A. F. Clark, Ed.), pp. 339 –350, Plenum, New York. Martin, P. (1997). Wound healing—Aiming for perfect skin regeneration. Science 276, 75– 81. Singer, A. J., and Clark, R. A. (1999). Cutaneous wound healing. N. Engl. J. Med. 341, 738 –746. Sarret, Y., Woodley, D. T., Grigsby, K., Wynn, K., and O’Keefe, E. J. (1992). Human keratinocyte locomotion: The effect of selected cytokines. J. Invest. Dermatol. 98, 12–16. O’Toole, E. A., Marinkovich, M. P., Peavey, C. L., Amieva, M. R., Furthmayr, H., Mustoe, T. A., and Woodley, D. T. (1997). Hypoxia increases human keratinocyte motility on connective tissue. J. Clin. Invest. 100, 2881–2891. Li, W., Nadelman, C., Fan, J., Muellenhoff, M., Medina, E., Henry, G., Gratch, N. S., Chen, M., Han, J., and Woodley, D. (2001). The p38 MAPK/SAPK pathway is required for human keratinocyte migration on dermal collagen. JID, in press. Yurko, M. A., O’Toole, E. A., and Woodley, D. T. (2001). Phosphorylation of focal adhesion kinase 9pp125FAK is increased in human keratinocytes induced to migrate by extracellular matrices. J. Cell. Physiol. 188, 24 –32. Zeigler, M. E., Chi, Y., Schmidt, T., and Varani, J. (1999). Role of ERK and JNK pathways in regulating cell motility and matrix metalloproteinase 9 production in growth factor-stimulated human epidermal keratinocytes. J. Cell. Physiol. 180, 271–284. Johansson, N., Ala-aho, R., Uitto, V.-J., Gre´nman, R., Fusenig, N. E., Lo´pez-Otı´n, C., and Ka¨ha¨ri, V.-M. (2000). Expression of collagenase-3 (MMP-13) and collagenase-1 (MMP-1) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase. J. Cell Sci. 113, 227–235. Gschwendt, M. (1999). Protein kinase C␦. FEBS Lett. 259, 555–564. Geiges, D., Marks, F., and Gschwendt, M. (1995). Loss of protein kinase C delta from human HaCaT keratinocytes upon ras transfection is mediated by TGF alpha. Exp. Cell Res. 219, 299 –303. Dlugosz, A. A., Mischak, H., Mushinski, J. F., and Yuspa, S. H. (1992). Transcripts encoding protein kinase C-alpha, -delta, -zeta, and -eta are expressed in basal and differentiating mouse keratinocytes in vitro and exhibit quantitative changes in neoplastic cells. Mol. Carcinog. 5, 286 –292.
228 13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
25.
26.
LI ET AL. Reddig, P. J., Dreckschmidt, N. E., Zou, J., Bourguignon, S. E., Oberley, T. D., and Verma, A. K. (2000). Transgenic mice overexpressing protein kinase C epsilon in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion. Cancer Res. 60, 595– 602. Szallasi, Z., Denning, M. F., Smith, C. B., Dlugos, A. A., Yuspa, S. H., Pettit, G. R. and Blumberg, P. M. (1994). Bryostatin 1 portects protein kinase C-␦ from down-regulation in mouse keratinocytes in parallel with its inhibition of phorbol esterinduced differentiation. Mol. Pharmacol. 48, 840 – 850. Li, W., Hu, P., Scolnik, E., and Schlessinger, J. (1992). The SH2 and SH3 domains-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors. Mol. Cell. Biol. 12, 5824 –5833 Sakoda, T., Kasahara, N., Hamamori, Y., and Kedes, L. (1988). A high-titer lentiviral production system mediates efficient transduction of differentiated cells including beating cardiac myocytes. J. Mol. Cell. Cardiol. 31, 2037–2047. Li, W., Yu, J. C., Shin, D. Y., and Pierce, J. H. (1995). Characterization of a protein kinase C-delta (PKC-delta) ATP binding mutant. An inactive enzyme that competitively inhibits wild type PKC-delta enzymatic activity. J. Biol. Chem. 270, 8311– 8318. Li, W., Yeung, Y. G., and Stanley, E. R. (1991). Tyrosine phosphorylation of a 57-kDa protein in growth factor stimulated and transformed cells. J. Biol. Chem. 266, 6808 – 6814. Albercht-Buehler, G. (1977). the phargokinetic tracks of 3T3 cells. Cell 11, 395– 404. Chen, J. D., Kim, J. P., Zhang, K., Sarret, Y., Wynn, K. C., Kramer, R. H., and Woodley, D. T. (1993). Epidermal growth factor (EGF) promotes human keratinocyte locomotion on collagen by increasing the alpha 2 integrin subunit. Exp. Cell Res. 209, 216 –223. Gschwendt, M., Mu¨ller, H. J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G., and Marks, F. (1994). Rottlerin, a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun. 199, 93–98. Li, W., and Pierce, J. H. (1994). Protein kinase C-delta, an important signaling molecule in the platelet-derived growth factor beta receptor pathway. Curr. Topics Microbiol. Immunol. 211, 55– 65. Li, W., Michieli, P., Alimandi, M., Lorenzi, M. V., Wu, Y., Wang, L. H., Heidaran, M. A., and Pierce, J. H. (1996). Expression of an ATP binding mutant of PKC-delta inhibits Sis-induced transformation of NIH3T3 cells. Oncogene 13, 731–773. Pessino, A., Passalacqua, M., Sparatore, B., Patrone, M., Melloni, E., and Pontremoli, S. (1995). Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells. Biochem. J. 312, 549 –554. Wang, Q. J., Acs, P., Goodnight, J., Giese, T., Blumberg, P. M., Mischak, H., and Mushinski, J. F. (1997). The catalytic domain of protein kinase C-delta in reciprocal delta and epsilon chimeras mediates phorbol ester-induced macrophage differentiation of mouse promyelocytes. J. Biol. Chem. 272, 76 – 82. Watanabe, T., Ono, Y., Taniyama, Y., Hazama, K., Igarashi, K., Ogita, K., Kikkawa, U., and Nishizuka, Y. (1992). Cell division
Received July 6, 2001 Revised version received October 29, 2001 Published online January 10, 2002
arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-delta subspecies. Proc. Natl. Acad. Sci. USA 89, 10159 –10163. 27.
Fukumoto, S., Nishizawa, Y., Hosoi, M., Koyama, H., Yamakawa, K., Ohno, S., and Morii, H. (1997). Protein kinase C delta inhibits the proliferation of vascular smooth muscle cells by suppressing G1 cyclin expression. J. Biol. Chem. 272, 13816 –13822.
28.
Mischak, H., Goodnight, J. A., Kolch, W., Martiny-Baron, G., Schaechtle, C., Kazanietz, M. G., Blumberg, P. M., Pierce, J. H., and Mushinski, J. F. (1993). Overexpression of protein kinase C-delta and -epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J. Biol. Chem. 268, 6090 – 6096.
29.
Mishima, K., Ohno, S., Shitara, N., Yamaoka, K., and Suzuki, K. (1994). Opposite effects of the overexpression of protein kinase C gamma and delta on the growth properties of human glioma cell line U251 MG. Biochem. Biophys. Res. Commun. 201(1), 363–372.
30.
Harrington, E. O., Loffler, J., Nelson, P. R., Kent, K. C., Simons, M., and Ware, J. A. (1997). Enhancement of migration by protein kinase Calpha and inhibition of proliferation and cell cycle progression by protein kinase Cdelta in capillary endothelial cells. J. Biol. Chem. 272, 7390 –7397.
31.
Emoto, Y., Kisaki, H., Manome, Y., Kharbanda, S., and Kufe, D. (1996). Activation of protein kinase Cdelta in human myeloid leukemia cells treated with 1-beta-D-arabinofuranosylcytosine Blood 87, 1990 –1996.
32.
Ghayur, T., Hugunin, M., Talanian, R. V., Ratnofsky, S., Quinlan, C., Emoto, Y., Pandey, P., Datta, R., Huang, Y., Kharbanda, S., Allen, H., Kamen, R., Wong, W., and Kufe, D. (1996). Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis. J. Exp. Med. 184, 2399 –23404.
33.
Gschwendt, M., Kielbassa, K., Kittstein, W., and Marks, F. (1994). Tyrosine phosphorylation and stimulation of protein kinase C delta from porcine spleen by src in vitro. Dependence on the activated state of protein kinase C delta. FEBS Lett. 347, 85– 89.
34.
Shanmugam, M., Krett, N. L., Peters, C. A., Maizels, E. T., Murad, F. M., Kawakatsu, H., Rosen, S. T., and HunzickerDunn, M. (1998). Association of PKC delta and active Src in PMA-treated MCF-7 human breast cancer cells. Oncogene 16, 1649 –1654.
35.
Li, W., Mischak, H., Yu, J. C., Wang, L. M., Mushinski, J. F., Heidaran, M. A., and Pierce, J. H. (1994). Tyrosine phosphorylation of protein kinase C-delta in response to its activation. J. Biol. Chem. 269, 2349 –2352.
36.
Li, W., Yu, J. C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994). Stimulation of the plateletderived growth factor beta receptor signaling pathway activates protein kinase C-delta. Mol. Cell. Biol. 14, 6727– 6735.
37.
Chen, M., She, H., Kim, A., and Li, W. (2000). Nck regulates PDGF-stimulated actin polymerization in NIH 3T3 fibroblasts. Mol. Cell. Biol. 20, 7867–7880.