Sustained phospholipase D activation is associated with keratinocyte differentiation

EunMi Jung, Soraya Betancourt-Calle, RaShawn Mann-Blakeney, Richard D. Griner and Wendy Bollinger Bollag1

Program in Cell Signaling, Institute of Molecular Medicine and Genetics, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912-2630, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our previous results and data in the literature have suggested a potential role for phospholipase D (PLD) in the regulation of epidermal keratinocyte growth and differentiation. Therefore, we investigated the effect of agents reported to modulate keratinocyte growth and differentiation on PLD activation. The purported protein kinase C (PKC) `inhibitor', staurosporine (Stsp), has been reported to activate PKC in keratinocytes, eliciting many of the same effects as active tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Stsp also induces a programmed pattern of differentiation similar to that seen in keratinocytes in vivo; TPA, on the other hand, appears to preferentially elicit markers consistent with late (granular) differentiation. In contrast, bradykinin is reported to stimulate keratinocyte proliferation. We found that these three agents had different effects on PLD activation in primary mouse epidermal keratinocytes. TPA increased PLD activity acutely and in a sustained fashion. In contrast, Stsp did not acutely activate PLD and inhibited acute TPA-induced activation of PLD. However, treatment of keratinocytes with Stsp for longer time periods (3–5 h) induced sustained PLD activation and this long-term effect was additive with that of TPA. Bradykinin activated PLD acutely but transiently. Both TPA and Stsp increased transglutaminase activity, a marker of late differentiation, whereas bradykinin had little or no effect on either cell proliferation or transglutaminase activity. These results suggest that a sustained activation of PLD is associated with the induction of keratinocyte differentiation. We hypothesize that PLD activity mediates late keratinocyte differentiation through generation of diacylglycerol and activation of specific PKC isoforms. Furthermore, we propose that the profound and immediate TPA-induced stimulation of PLD activity `drives' the keratinocytes to late differentiation steps. However, the less efficacious (and more gradual) sustained activation of PLD by Stsp may allow a patterned differentiation more like that observed in skin.

Abbreviations: BSA, bovine serum albumin; DAG, sn-1,2-diacylglycerol; FBS, fetal bovine serum; IC50, half-maximal inhibitory concentration; ITS, insulin transferrin and selenious acid; ITS+, ITS + linoleic acid/BSA; MEM, minimum essential medium; PA, phosphatidic acid; PEt, phosphatidylethanol; PLD, phospholipase D; PKC, protein kinase C; SDS, sodium dodecyl sulfate; SFKM, serum-free keratinocyte medium; Stsp, staurosporine; TCA, trichloroacetic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several agents that affect the growth and differentiation of epidermal keratinocytes in vitro elicit changes in phosphoinositide turnover, diacylglycerol (DAG) production and protein kinase C (PKC) activation (reviewed in ref. 1). One such agent is bradykinin, which increases cellular inositol phosphates (25) and DAG (4) and can presumably activate PKC. We (6) and others have provided evidence that PKC activity, in turn, is involved in terminal differentiation of these cells (reviewed in 1). PKC can also be activated directly by tumor-promoting phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA), which has been shown to inhibit proliferation and stimulate differentiation of keratinocytes (79). Interestingly, the purported PKC inhibitor, staurosporine (Stsp) has also been demonstrated to elicit various markers of keratinocyte differentiation (e.g. induction of transglutaminase mRNA expression and of SPR-1, loricrin, profilaggrin and keratins 1 and 10 mRNA and protein expression as well as cornified envelope formation; reviewed in ref. 10) and to elicit translocation and down-regulation of PKC isoenzymes (11). Thus, it has been proposed that in keratinocytes Stsp functions as a PKC agonist, rather than as an inhibitor (12). However, in vivo Stsp (within a narrow dose range) can inhibit TPA-induced tumor promotion as well as papilloma cell-derived tumor formation in nude mice (13). The mechanism of this inhibition in mouse skin is unclear.

In several systems, phorbol ester-activated PKC has been demonstrated to increase phospholipase D (PLD) activity, although other mechanisms of activation have also been shown (reviewed in ref. 14). The isoform likely to be involved in this effect is PKC-{alpha}, since PLD can be activated by this PKC isoenzyme in vitro (15). The result of PLD activation is the hydrolysis of phospholipids to yield phosphatidic acid (PA) and the polar headgroup. The PA, in turn, can be dephosphorylated to yield DAG, which presumably, like the DAG derived from phosphoinositide hydrolysis, can activate PKC. However, because the DAG generated by PLD is often derived from phosphatidylcholine, these unique PLD-produced DAG species may activate different PKC isoforms than DAG originating from phosphoinositide turnover (reviewed in ref. 16).

The role of PLD activity in PKC activation and cellular responses in keratinocytes is unclear. However, in these cells PLD activation has been reported to underlie the sustained increase in DAG elicited by the ganglioside GQ1b (17). This ganglioside has been shown to decrease proliferation and stimulate terminal differentiation of these cells (18), which suggests the possibility that PLD activity is involved in keratinocyte differentiation. Our previous results also suggest a role for PLD in mediating growth inhibition and differentiation induction in keratinocytes (19). We hypothesized that (i) bradykinin and TPA activate PLD through PKC to assist in inducing keratinocyte differentiation, (ii) Stsp elicits its differentiative effects, at least in part, through an activation of PLD, and (iii) PLD activation is associated with keratinocyte differentiation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell culture
Primary epidermal keratinocytes were prepared from 1- to 3-day-old neonatal CD1 mice and cultured as described (6). Briefly, after trypsinization of the skin, the epidermis was mechanically separated from the dermis, and epidermal cells were released by scraping. Epidermal cells were then purified by centrifugation through Ficoll and plated in 6-well dishes in a fetal bovine serum (FBS)-containing RPMI medium. After 4 h, the cells were washed with phosphate-buffered saline lacking calcium and magnesium (PBS) and re-fed with serum-free medium (SFKM), which consisted of calcium-free minimum essential medium (MEM) containing 25–50 µM calcium, 5 ng/ml epidermal growth factor, 2 mM glutamine, 90 µg/ml bovine pituitary extract, 0.05% bovine serum albumin (BSA), ITS (insulin, transferrin and selenious acid) or ITS+ (ITS + linoleic acid/BSA), 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml fungizone. In some experiments, keratinocytes released by scraping of the dermis and epidermis were collected by centrifugation and plated overnight in calcium-free MEM containing 2% dialyzed FBS, 50 µM calcium, 5 ng/ml epidermal growth factor, 2 mM glutamine, ITS+, 0.05% BSA, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml fungizone prior to re-feeding with SFKM. Cells were re-fed with fresh SFKM every 1–3 days, and experiments were performed on near-confluent to confluent cell cultures. For density determinations, a single-cell suspension was generated by repeated aspiration of trypsinized cells through an 18 gauge needle, and the cell number was determined using an electronic particle counter (Coulter Electronics, Hialeah, FL).

Measurement of phosphatidic acid and phosphatidylethanol production
PLD activation can be measured using a unique feature of the enzyme: PLD not only catalyzes the hydrolysis of phospholipids in aqueous solution to produce PA, but in the presence of small quantities of ethanol, PLD catalyzes the transphosphatidylation of phospholipids to form the novel lipid, phosphatidylethanol (PEt) (14). In cells pre-labeled in their phospholipids with [3H]fatty acid, PLD activation will result in increases in radiolabeled PA and PEt. Thus, cells were labeled in SFKM containing 2.5 µCi/ml [3H]oleic acid for 20–24 h. The medium was then aspirated and replaced with SFKM lacking radiolabel. After a 30 min pre-incubation period, cells were stimulated with the appropriate agonists and incubated for the indicated time periods in the presence of 0.5 or 1% ethanol as indicated. The cells were then solubilized in 0.2% sodium dodecyl sulfate (SDS) containing 5 mM EDTA and extracted for 1 to 2 h with ice-cold chloroform–methanol to which acetic acid (1:2:0.04 v:v:v) was added. Additional chloroform and 0.2 M NaCl were added to break phase, and the lower phases collected and dried under N2. Samples were resuspended in chloroform–methanol, which also contained 25–50 µg PEt and PA per sample and were spotted onto heat-activated silica gel 60 thin-layer chromatography plates (0.25 mm thickness aluminum backed with concentrating zone). Plates were developed in the upper phase of a solvent system of ethyl acetate–iso-octane–acetic acid–water (13:2:3:10) and were visualized with iodine vapor and with autoradiography using En3Hance. Spots corresponding to PA and PEt, identified by co-migration with authentic standards, were cut, placed in liquid scintillation fluid and quantified.

Measurement of transglutaminase activity
Transglutaminase activity was determined as in (19). Briefly, near-confluent keratinocytes were incubated for 18 h with the indicated concentrations of bradykinin, TPA or Stsp in SFKM. The cells were then washed with PBS, scraped into homogenization buffer (0.1 M Tris–acetate, pH 7.8, 2 µg/ml aprotinin, 2 µM leupeptin, 1 µM pepstatin A, 0.2 mM EDTA, 0.2 mM PMSF and 0.1% ß-mercaptoethanol) and pelleted by centrifugation. The supernatants were removed and the cell pellets subjected to one freeze–thaw cycle followed by sonication in homogenization buffer. Duplicate aliquots of the homogenate were incubated for 14 h at 37°C with 2 µCi/sample [3H]putrescine in a reaction mixture that contained 1% dimethylated Hammerstein casein, 0.1 M Tris–acetate (pH 8.5), 10 mM CaCl2, 0.1 mM EDTA, 5 mM dithiothreitol and 0.1% Triton X-100 (final concentrations). Reactions were terminated by adding trichloroacetic acid (TCA) to a final concentration of 5% and the samples filtered through glass fiber filters. After thorough washing with 5% TCA, the filters were counted by liquid scintillation spectrometry. Protein content of the samples was determined using the micro-BCA protein assay (Pierce, Rockford, IL) with BSA as standard.

Statistical analysis
The significance of differences between mean values was determined using ANOVA, as performed by the program Instat (GraphPad Software, San Diego, CA).

Materials
BSA (fatty acid-free), TCA, dimethylated Hammerstein casein, bradykinin, TPA and PMSF were obtained from Sigma (St Louis, MO). Methyl-[3H]thymidine (sp. act.: 40–60 Ci/mmol), [3H]oleic acid and [3H]putrescine were purchased from DuPont/NEN (Wilmington, DE). Trypsin inhibitor and epidermal growth factor were purchased from Gibco BRL (Gaithersburg, MD); calcium-free MEM was purchased from Specialty Media (Lafayette, NJ) or Biologos (Naperville, IL) and RPMI 1640 and PBS from Mediatech (Herndon, VA). Stsp was obtained from Biomol (Plymouth Meeting, PA). Aprotinin, leupeptin and pepstatin A were purchased from Boehringer Mannheim Biochemicals (Indianapolis, IN). Bovine pituitary extract was obtained from Gibco BRL, Upstate Biotechnology (P-Neurext; Lake Placid, NY) or Collaborative Research (Bedford, MA). ITS and ITS+ were purchased from Collaborative Research or Sigma. Dialyzed FBS was purchased from Sigma. Other tissue culture reagents were obtained from Hazleton Biologics (Lenexa, KS), Atlanta Biologicals (Norcross, GA) or Biologos. Thin-layer chromatography plates were purchased from EM Science (Gibbstown, NJ). All other materials were of reagent grade.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously obtained data to suggest a possible role of PLD activity in mediating keratinocyte growth inhibition and differentiation induction (19). We therefore investigated the ability of two agents to activate PLD. One (bradykinin) has been reported to stimulate proliferation (4) while the other (TPA) is known to induce differentiation (79). We found that both bradykinin and TPA were able to increase PLD activity acutely, as measured by elevations in radiolabeled PEt levels (Figures 1 and 2GoGo). The activation of PLD by bradykinin was somewhat unexpected in view of the reported ability of this agent to stimulate growth (4) and our results, which suggests an involvement of PLD in keratinocyte differentiation (19). However, we were unable to demonstrate any significant effect of bradykinin on [3H]thymidine incorporation over a wide range of concentrations (P > 0.05 for all concentrations) and different conditions (data not shown; see Discussion).



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Fig. 1. Bradykinin activates PLD transiently. (A) Primary cultures of mouse epidermal keratinocytes were labeled for 20–24 h with [3H]oleate and then re-fed with SFKM alone (open symbols) or SFKM containing 10 µM bradykinin (closed symbols) for the indicated time periods in the presence of 0.5% ethanol (with or without first aspirating the SFKM containing the [3H]oleic acid; since similar time courses were observed with either protocol, the data from both types of experiments are combined). After extracting cellular lipids and separating by thin-layer chromatography, the radioactivity found in PA (circles) and PEt (squares) was quantified and the results are presented as -fold over the appropriate control value. Radioactivity in control samples averaged 30 600 ± 2300 c.p.m./106 cells for PA and 11 100 ± 1000 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 26 100 ± 3000 cells/cm2. Values represent the means (± SEM) of eight samples from four separate experiments; *P <= 0.05, **P <= 0.001. (B) [3H]oleate-labeled keratinocytes were stimulated with 10 µM bradykinin for 55, 25, 10 or 0 min prior to the addition of 1% ethanol for 5 min or the readdition of 10 µM bradykinin (and 1% ethanol) for 5 min (55-5). Control samples were exposed to 1% ethanol for 5 min and all reactions were terminated at the same time by addition of 0.2% SDS containing 5 mM EDTA. Following separation of PA (light bars) and PEt (dark bars) and quantitation, values were expressed as -fold over control. Radioactivity in control samples averaged 23 100 ± 4500 c.p.m./106 cells for PA and 7000 ± 1500 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 35 200 ± 2500 cells/cm2. Values represent the means (± SEM) of at least six samples from at least two separate experiments; *P <= 0.001.

 


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Fig. 2. TPA stimulates PLD activity in a concentration-dependent and sustained manner. (A) [3H]oleate-labeled keratinocytes were incubated with various concentrations of TPA for 30 min in the presence of 0.5% ethanol. After extracting cellular lipids and separating by thin layer chromatography, as described in Materials and methods, the radioactivity found in PA (light bars) and PEt (dark bars) was quantified and the results are presented as percent of the appropriate control value. Radioactivity in control samples averaged 47 000 ± 9000 c.p.m./106 cells for PA and 17 000 ± 6000 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 29 100 ± 4400 cells/cm2. Values represent the means (± SEM) of 4–6 samples from two separate experiments; *P <= 0.001. (B) Primary cultures of mouse epidermal keratinocytes were labeled for 20–24 h with [3H]oleic acid and then re-fed with SFKM ± 100 nM TPA for 55, 25, 10 or 0 min prior to the addition of 1% ethanol for the final 5 min. Control samples were exposed to 1% ethanol for 5 min and all reactions were terminated at the same time by addition of 0.2% SDS containing 5 mM EDTA, as described in Materials and methods. After extracting cellular lipids and separating by thin layer chromatography, as described in Materials and methods, the radioactivity found in PA ({circ}) and PEt ({blacksquare}) was quantified and the results are presented as a percentage of the control value. Radioactivity in control samples averaged 42 000 ± 7000 c.p.m./106 cells for PA and 15 000 ± 3000 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 28 200 ± 1200 cells/cm2. Values represent the means (± SEM) of four samples from two separate experiments; *P <= 0.01. A third experiment yielded qualitatively similar results although the TPA-elicited increase in PEt levels relative to control was greater (~10-fold).

 
The inability of bradykinin to inhibit growth despite its activation of PLD argued against a role for PLD in keratinocyte differentiation. However, when we examined the time course of bradykinin's activation of PLD, we found that this peptide elicited increases in radiolabeled PA and PEt formation, which peaked at 5 min and declined subsequently (Figure 1AGo). Similar time courses were observed in cells incubated with bradykinin in the continued presence of [3H]oleic acid or following aspiration of the radiolabel, which indicates that the observed decline in [3H]PA and PEt with time was not caused by radiolabeled substrate depletion but rather to metabolism of the PEt by keratinocytes, as has been observed in mesangial cells and hepatocytes (20,21). Because the rate of the breakdown of PEt was unknown, it was not clear whether PLD activation was transient, or whether a low level of activity was maintained after an initial substantial increase in activation. This question was addressed by the experiment shown in Figure 1BGo. With this protocol [3H]oleate-labeled keratinocytes were stimulated with bradykinin for 55, 25, 10 or 0 min prior to the addition of 1% ethanol for 5 min to all samples and 10 µM bradykinin to some samples. Control samples incubated with SFKM alone were exposed to 1% ethanol for 5 min and all reactions were terminated at the same time by addition of 0.2% SDS containing 5 mM EDTA, as described in Materials and methods. As illustrated, only the samples that were not pre-treated with bradykinin, with bradykinin added simultaneously with ethanol for a total exposure time to bradykinin of 5 min, exhibited an increase in PLD activity, as measured by an elevation in [3H]PEt levels (Figure 1BGo). This result indicates that bradykinin activated PLD only transiently in keratinocytes.

To determine whether the transience of the bradykinin response was the result of hormone degradation, the ability of a second bradykinin addition to stimulate PEt production after a 55 min pre-treatment with the hormone was also examined. [3H]oleate-labeled keratinocytes were pre-exposed to SFKM alone or SFKM containing 10 µM bradykinin for 55 min. Cells were then incubated for an additional 5 min in the presence or absence of bradykinin in the presence of 1% ethanol. As illustrated, a second 5 min exposure to 10 µM bradykinin added in conjunction with the ethanol elicited no increase in [3H]PEt levels (condition 55 + 5 of Figure 1BGo). Thus, the transience of the bradykinin response appears not to be the result of degradation of the hormone.

The ability of bradykinin to elicit DAG generation (4) suggested that this peptide should activate PKC. In several cell systems PLD activation has been demonstrated to occur in response to PKC activity (reviewed in refs 22,23). Therefore, we investigated the possible involvement of PKC in PLD activation. [3H]oleate-pre-labeled keratinocytes were treated for 30 min with various concentrations of the PKC activator, TPA, in the presence of 0.5% ethanol. TPA dose-dependently activated PLD with a maximal ~9-fold increase in PEt production at a dose of 100 nM, and a half-maximal stimulatory concentration of ~10 nM (Figure 2AGo). PA levels were increased ~2-fold, with a half-maximal stimulatory concentration of ~3 nM. However, in contrast to the bradykinin-elicited effect, we found that TPA-induced PLD activation was sustained (Figure 2BGo and Table IGo). Thus, [3H]PEt production was increased up to 4 h after the addition of TPA. The ability of TPA to activate PKC and to elicit increases in PLD activity suggests that PLD activation may be mediated through the effects on PKC.


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Table I. Staurosporine-induced PLD activation is not inhibited by Ro 31-8220
 
To determine if, in fact, TPA- and bradykinin-induced PLD activation was mediated by PKC, we investigated the ability of the selective PKC inhibitor, Ro 31-8220, to prevent PLD activation in response to these agents. TPA elicited an ~8-fold increase in PEt production, and this increase was largely prevented by Ro 31-8220 with a half-maximal inhibitory concentration (IC50) of ~0.2 µM (Figure 3AGo). The TPA-elicited increase of ~2-fold in PA levels was also inhibited, with an EC50 of ~0.2 µM. Using maximal doses of Ro 31-8220 (1 and 2 µM), we found that this PKC inhibitor also prevented bradykinin-induced PLD activation (Figure 3BGo), with a dose of 1 µM eliciting a 59 and 47% inhibition and 2 µM effecting a 63 and 80% reduction in bradykinin-elevated [3H]PA and PEt levels, respectively. This degree of inhibition is comparable to the inhibition of TPA-induced PLD activation elicited by these doses of Ro 31-8220 (Figure 3AGo). The ability of Ro 31-8220, a selective PKC inhibitor, to prevent both the TPA- and bradykinin-elicited increase in PLD activity provides further evidence that PLD activation can occur through PKC.



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Fig. 3. TPA- and bradykinin-stimulated PLD activation is inhibited by the selective PKC inhibitor, Ro 31-8220. (A) [3H]oleate-labeled keratinocytes were pre-incubated with various concentrations of Ro 31-8220 for 15 min prior to the addition of 0.5% ethanol in the presence and absence of 100 nM TPA for 30 min. After extracting cellular lipids and separating by thin layer chromatography, as described in Materials and methods, the radioactivity found in PA ({circ}) and PEt ({blacksquare}) was quantified and the results are presented as a percentage of the control value. Radioactivity in control samples averaged 30 600 ± 6500 c.p.m./106 cells for PA and 11 000 ± 2000 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 36 100 ± 2600 cells/cm2. Values represent the means (± SEM) of 6–9 samples from three separate experiments; *P < 0.05; **P < 0.001. (B) [3H]oleate-labeled keratinocytes were pre-incubated for 15 min with 0.1% DMSO or 1 or 2 µM Ro 31-8220 (8220) and 0.5% ethanol prior to stimulation with and without 10 µM bradykinin for 5 min. After extracting and separating cellular lipids, the radioactivity found in PA (light bars) and PEt (dark bars) was quantified and the results are presented as -fold of the control value. Radioactivity in control samples averaged 20 500 ± 4500 c.p.m./106 cells for PA and 6400 ± 1200 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 34 700 ± 11 000 cells/cm2. Values represent the means (± SEM) of 6–9 samples from three separate experiments; *P < 0.001.

 
Another agent found to translocate, and presumably activate, PKC is Stsp (11), which appears to act in keratinocytes as a PKC agonist rather than an antagonist (12). We investigated the ability of Stsp to activate PLD and found that, unlike TPA, Stsp had no significant effect on radiolabeled PA or PEt levels over 45 min of stimulation at any concentration ranging between 1 nM and 1 µM (a maximal 1.06 ± 0.08-fold change over the control PEt levels of 1.00 ± 0.04 at the 1 µM concentration; P > 0.05, not significant at any concentration). However, previous reports suggest that Stsp's effect on PKC translocation/activation occurs after longer time periods (2–5 h) (11). Therefore, the effect of longer incubations with Stsp on PLD activation were examined. A 3 or 5 h incubation with 10 nM to 1 µM Stsp resulted in a concentration-dependent and significant (at concentrations >=100 nM) elevation in [3H]PEt levels (Figure 4A and BGo), suggesting that, at these later times, Stsp also activates PLD.



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Fig. 4. Staurosporine activates PLD after 3 and 5 h exposures. (A) Primary cultures of mouse epidermal keratinocytes were labeled for 20–24 h with [3H]oleic acid and then re-fed with SFKM containing various concentrations of Stsp or vehicle control (0.1% DMSO) for 3 h in the presence of 0.5% ethanol. The radioactivity found in PA (light bars) and PEt (dark bars) was quantified and is presented as -fold over the appropriate control value. Radioactivity in control samples averaged 15 000 ± 3000 c.p.m./106 cells for PA and 7000 ± 2000 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 42 300 ± 9400 cells/cm2. Values represent the means (± SEM) of six samples from three separate experiments; *P < 0.05, **P < 0.01. (B) [3H]oleate-pre-labeled keratinocytes were re-fed with SFKM containing various concentrations of Stsp or vehicle control (0.1% DMSO) for 4.5 h in the presence of 0.5% ethanol. At that time, cultures were pulsed with an additional 0.5% ethanol, to yield a final nominal concentration of 0.1%. Because of the extreme volatility of ethanol, this second addition was necessary to ensure adequate quantities of ethanol for the generation of PEt at later times. Reactions were terminated and radioactivity found in PA (light bars) and PEt (dark bars) quantified. Values, expressed as -fold over the control, represent the means (± SEM) of eight samples from three separate experiments; *P < 0.01, **P < 0.001. Radioactivity in control samples averaged 11 700 ± 400 c.p.m./106 cells for PA and 4300 ± 500 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 86 700 ± 19 400 cells/cm2.

 
The fact that PEt levels were elevated at both 3 and 5 h suggested that PLD activation in response to Stsp was sustained. We tested this assumption by treating [3H]oleate-pre-labeled keratinocytes for 3.5–4.5 h with 100 nM Stsp prior to adding 0.5% ethanol for the final 30 min of incubation. Using this protocol, a rate of PEt production over a 30 min time period can be determined, rather than measuring the total accumulation of PEt over the entire treatment period. We found that Stsp-induced [3H]PEt production between 4 and 5 h was essentially constant at ~1.5-fold over the control (1.41 ± 0.09-fold for a total incubation time of 4 h; 1.39 ± 0.11-fold for 4.5 h; and 1.54 ± 0.14-fold over the control level for 5 h; P <= 0.05). These results indicate that Stsp activated PLD in a sustained fashion within this time period.

The ability of Stsp to trigger PKC isoform translocation suggested that this agent might also activate PLD via a PKC-mediated mechanism. Therefore, we investigated the effect of Ro 31-8220 on the long-term activation of PLD in response to Stsp. In contrast to our results with TPA and bradykinin, we found that Ro 31-8220 did not inhibit Stsp-elicited PLD activation (Table IGo) at a dose (2 µM), at which the PKC inhibitor produced a significant decrease in acute TPA- and bradykinin-stimulated PLD activity (Figure 3Go), as well as TPA-elicited PLD activation at 4 h of exposure (data not shown). This concentration of Ro 31-8220 also did not itself activate PLD over the 4 h treatment period (Table IGo).

Our previous results have suggested a role for PLD activity in inhibiting keratinocyte growth and stimulating differentiation (19). The ability of Stsp to activate PLD indicated that this agent could potentially induce keratinocyte differentiation. Indeed, Yuspa's laboratory has reported that Stsp increases keratins 1 and 10 expression and other markers of various stages of keratinocyte differentiation (11). On the other hand, since bradykinin elicits only transient PLD activation, this hormone might be ineffective in inducing differentiation. We investigated the effect of all three agents, Stsp, TPA and bradykinin, on transglutaminase activity, a late marker of keratinocyte differentiation. As shown in Figure 5Go, TPA and Stsp, both of which trigger sustained PLD activation, also elicited a significant increase in transglutaminase activity. Bradykinin, on the other hand, had little or no effect on transglutaminase activity, which is consistent with its inability to inhibit proliferation. This result suggests that sustained PLD activity may be important for keratinocyte differentiation.



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Fig. 5. TPA and Stsp, but not bradykinin, increase transglutaminase activity. Near-confluent primary keratinocyte cultures were incubated for 18 h with 10 µM bradykinin (BK), 100 nM Stsp or 100 nM TPA (samples contained <0.01% DMSO as solvent) and transglutaminase activity was measured as [3H]putrescine incorporation into casein, as described in Materials and methods. Values represent the means ± SEM of 10 samples from five separate experiments; *P < 0.01.

 
Finally, since Stsp is reported to act in other systems as a PKC antagonist and has been shown to block TPA-elicited tumor promotion in vivo (13), we examined the ability of Stsp to inhibit acute TPA-induced PLD activation (Figure 6Go). After a 30 min treatment, TPA again elevated PEt levels ~8-fold, and this increase was prevented by Stsp with an IC50 of ~0.2 µM. The TPA-elicited increase of ~2-fold in PA levels was also inhibited, with an IC50 of ~1 µM. On the other hand, when TPA and Stsp were incubated in combination for a prolonged period of time, their effects on PLD activity were additive (Table IIGo). Thus, while in many respects Stsp acts as a PKC agonist in keratinocytes, this agent also can prevent some acute TPA-induced, PKC-mediated effects. Nevertheless, long-term effects of TPA on PLD activity are not inhibited by Stsp.



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Fig. 6. Stsp inhibits acute TPA-induced PLD activation. [3H]oleate-labeled keratinocytes were pre-incubated with various concentrations of Stsp for 15 min prior to the addition of 0.5% ethanol in the presence and absence of 100 nM TPA for 30 min. After extracting cellular lipids and separating by thin layer chromatography, as described in Materials and methods, the radioactivity found in PA ({circ}) and PEt ({blacksquare}) was quantified and the results are presented as a percentage of the control value. Radioactivity in control samples averaged 20 000 ± 4000 c.p.m./106 cells for PA and 9000 ± 3000 c.p.m./106 cells for PEt, with an average cell density (± SEM) of 46 000 ± 8100 cells/cm2. Values represent the means (± SEM) of six samples from three separate experiments; *P < 0.05; **P < 0.001.

 

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Table II. The sustained effects of staurosporine and TPA on PLD activity are additive
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our experiments we found no effect of bradykinin on [3H]thymidine incorporation. This finding is in agreement with results of Johnson et al. (5), who found no significant effect of bradykinin on [3H]thymidine incorporation in human keratinocytes, and with our data showing no significant enhancement of transglutaminase activity in response to bradykinin (Figure 5Go). Talwar et al. (4), on the other hand, observed a bradykinin-induced growth stimulation in these cells over 6 days in culture, using an essentially growth factor-free medium. We have examined the ability of bradykinin to alter cell number over a 4 day period or [3H]thymidine incorporation in a low growth-factor medium and found no consistent effect (unpublished data), which indicates that the basis of the discrepancy is unlikely to be the length of exposure to the hormone or the culture conditions. We are unclear as to the reason for this disparity, other than possibly a species difference [mouse in our study versus human (4)].

The bradykinin-induced activation of PLD suggested that this hormone should have inhibited DNA synthesis and stimulated differentiation, since in our previous study the products of (exogenous) PLD activity decreased [3H]thymidine incorporation and increased transglutaminase activity (19). However, we found that bradykinin stimulated PLD activity for only the first 5 min of exposure (Figure 1Go), and we believe that the lack of effect of bradykinin on growth and differentiation is likely the result of the transience of enzyme activation. Apparently, this short generation of PA is insufficient to affect proliferation or differentiation in the long term. Our observation appears to be the first in keratinocytes of a transient PLD activation, although such transience has been observed in other cell systems in response to hormonal activation of PLD, and has been proposed to occur through both homologous and heterologous desensitization (21,24). This transience appears not to be the result of rapid metabolism of the hormone (Figure 1Go). Instead, the transient response to bradykinin may be explained by the results of Munoz and Leeb-Lundberg (24) who found that the bradykinin receptor becomes desensitized after a short (15 min) pre-treatment with the hormone. Furthermore, these investigators demonstrated that this effect could be mimicked by agents, such as TPA and norepinephrine, which stimulate PKC activity (24). Our findings concerning the ability of TPA to activate PLD (Figure 2Go) and of the PKC inhibitor Ro 31-8220 to inhibit this activation (Figure 3Go) suggest a role for PKC also in mediating activation of PLD in keratinocytes. Thus, we postulate that an initial bradykinin-induced phosphoinositide turnover (25) stimulates (through the generation of DAG) PKC activity, which activates PLD (presumably via the PKC-{alpha} isoform) but also desensitizes the bradykinin receptor to terminate the activation of PKC and its effect on PLD.

Stsp has been proposed to act as a PKC activator in keratinocytes (12) and, in fact, we found that this agent stimulates PLD activity. This finding is in agreement with results in other systems in which Stsp activates PLD (25,26). However, unlike these other systems (25,26), Stsp-induced PLD activation occurs over hours (Figure 4Go) rather than minutes. The late time frame of PLD activation is consistent with the observations of others, who demonstrated PKC isoenzyme activation/translocation after 2–5 h of stimulation with Stsp (11), as well as inhibition of Stsp-induced differentiation marker expression by the PKC inhibitors bryostatin-1 (12) and GF 109203X (11) over several hours. These data suggest that Stsp-stimulated PLD (in conjunction with phosphatidate phosphohydrolase) may generate DAG to activate one or more PKC isoenzymes involved in Stsp-induced differentiation. In contrast to TPA and bradykinin, however, PKC does not appear to be involved in the activation of PLD in response to Stsp (Table IGo). Interestingly, Stsp can also inhibit acute TPA-elicited PLD activation (Figure 6Go), which indicates that under certain conditions this compound can act as a PKC inhibitor, as well as an activator, in keratinocytes.

The physiological relevance of PLD activation in epidermal keratinocytes is unknown. One study suggests that sustained DAG generation in response to ganglioside GQ1b is at least partially mediated by PLD (17). Furthermore, this agent inhibits growth and enhances proliferation (18), which suggests a potential role for PLD in such processes. Our data also indicate that the products of PLD activity, as generated through the action of an exogenous PLD, can inhibit DNA synthesis and stimulate transglutaminase activity (19). However, our results argue against a role for PLD in Stsp-induced growth inhibition, since we observed significantly decreased [3H]thymidine incorporation into DNA within 1 h of the addition of Stsp (unpublished data), before PLD has been activated by this compound. [Presumably, Stsp inhibits growth in this case by inhibiting tyrosine kinase activity (27).] Thus, we postulate that PLD activation may be a signaling event involved in the stimulation of (late) differentiation, but such long-term processes are only affected if the increase in PLD activity is sustained. Indeed, both TPA and Stsp elicit sustained increases in PLD (Figures 2 and 4GoGo and Table IIGo) and transglutaminase (Figure 5Go) activity, whereas bradykinin induces only transient PLD activation (Figure 1Go) and does not trigger differentiation (Figure 5Go). Moreover, the small, long-term effect of Stsp on PLD activation is reminiscent of the action of the physiological agent, 1,25-dihydroxyvitamin D3, on PLD activity (28), which suggests that this pattern of enzyme activation is more physiologically relevant than the acute and exaggerated increase in PLD activity produced by TPA. Since TPA appears to drive keratinocytes towards late (granular) differentiation marker expression (29), our results provide evidence for a potential role of PLD signaling in a later step in the patterned program of keratinocyte differentiation.

In conclusion, our results demonstrate several important points concerning PLD activation in epidermal keratinocytes. First, our data indicate that PLD activity can be regulated by PKC activity in these cells, as in other cells (reviewed in ref. 14), although other mechanisms must also exist and be utilized by Stsp. Agents that activate PLD increase PA formation, and this PA could then itself function as a messenger (16) or be converted to DAG through the action of phosphatidate phosphohydrolase. However, the results presented argue that only sustained PLD activity is associated with the induction of differentiation, since bradykinin, which activates PLD only transiently, does not trigger differentiation. The disparity in the time course and magnitude of PLD activation by TPA versus Stsp suggests that a differential effect of these compounds on differentiation should be observed, as has been reported. Thus, Stsp elicits a sequential program of keratinocyte differentiation, inducing markers of early (keratins 1 and 10) as well as late differentiation (e.g. filaggrin and loricrin) (11). On the other hand, TPA seems to preferentially promote late differentiation steps (29). The ability of Stsp to inhibit TPA-induced PLD activation suggests that this compound acts, at least in part, as a partial PKC antagonist, and may explain its ability to block TPA-induced tumor promotion in vivo (13). Thus, we propose that through its ability to induce gradual, long-term PLD activation, Stsp triggers the progression of differentiation in a more physiologically relevant fashion. Presumably, it does so by causing the gradual accumulation of phosphatidylcholine-derived DAG and activation of one or more specific isozymes of PKC. Moreover, because Stsp does not immediately `push' normal keratinocytes into a late terminal differentiation program, this agent does not cause clonal selection and expansion of differentiation-resistant keratinocytes as does TPA (8). In fact, Stsp can also induce differentiation of TPA-resistant neoplastic keratinocytes (10). Therefore, our results implicate an association between sustained PLD activity in late keratinocyte differentiation steps and suggest that agents such as Stsp, which are able to interfere with acute TPA-induced PLD activation without affecting long-term PLD activity may be useful in preventing tumor formation.


    Acknowledgments
 
Ro 31-8220 was a generous gift of Dr D.Bradshaw (Roche Research Center, Hertfordshire, UK).


    Notes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 22, 1998; revised November 23, 1998; accepted December 1, 1998.