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INTRODUCTION |
The skin is a dynamic organ consisting of the dermis and
epidermis, with the latter continuously undergoing regeneration to replace cells lost through normal exposure to the environment. The
epidermis is composed of several cell layers. The deepest layer,
located at the dermal-epidermal junction, is the basal layer,
consisting of the undifferentiated basal keratinocytes, which
continuously proliferate. As the cells migrate up through the
epidermis, keratinocytes undergo a distinct pattern of differentiation that is essential for the function of the skin as a protective barrier.
This pattern is characterized by growth arrest and expression of the
mature cytokeratins 1 and 10 in the first differentiated layer of the
epidermis, the spinous layer. Early differentiation in the spinous
layer is followed by late differentiation in the granular layer
accompanied by expression of proteins, including the enzyme
transglutaminase, that are essential for the formation of the cornified
envelope and corneocytes. The corneocytes are terminally differentiated
and constitute the outer layer of the epidermis, the cornified layer,
which gives skin its resistance to mechanical stresses (for reviews,
see Refs. 1 and 2).
Despite the importance of the keratinocyte differentiation program and
intense investigation, both the extracellular and the intracellular
signals controlling this process are largely unknown. Numerous studies
have pointed to a role for 1
,25-dihydroxyvitamin D3
(1,25(OH)2D3)1
in regulating keratinocyte differentiation (reviewed in Ref. 1). In
fact, this compound and its structural analogs have been used
successfully to treat psoriasis (reviewed in Ref. 3), a human disease
characterized by hyperproliferation, abnormal differentiation, and
inflammation of the skin. A physiologic role for
1,25(OH)2D3 in regulating keratinocyte
differentiation in the epidermis in vivo is suggested by
several lines of evidence: 1) keratinocytes express the
1
-hydroxylase, which converts the inactive 25-hydroxyvitamin
D3 to its active 1,25-dihydroxymetabolite (reviewed in Ref.
1); 2) receptors for 1,25(OH)2D3 are present in
the skin and in epidermal keratinocytes (references cited in Ref. 4);
and 3) 1,25(OH)2D3 triggers two major events in
keratinocytes in vitro: inhibition of proliferation and
induction of differentiation (reviewed in Ref. 1).
The mechanism by which 1,25(OH)2D3 inhibits
proliferation and stimulates differentiation is still unclear. This
hormone is thought to function through the vitamin D receptor, a
transcription factor affecting expression of genes possessing vitamin D
response elements. However, the keratin 1 gene is the only keratinocyte differentiation marker known to possess a
1,25(OH)2D3 response element (5, 6), and the
expression of this marker is inhibited by
1,25(OH)2D3 (7). In addition,
1,25(OH)2D3 was recently shown to enhance the
expression of several phosphoinositide-specific phospholipase C
isoenzymes (8), the activity of which generates diacylglycerol (DAG).
DAG, in turn, is known to regulate the activity of protein kinase C
(PKC), and numerous data suggest the involvement of PKC in the
regulation of keratinocyte growth and differentiation (reviewed in
Refs. 1 and 9).
Although PKC-activating DAG can be generated directly by
phosphoinositide turnover via phospholipase C, such DAG can also be
generated indirectly by an additional pathway. Diacylglycerol is
generated by the combination of phospholipase D (PLD), which hydrolyzes
phospholipids to generate phosphatidic acid (PA), and PA
phosphohydrolase, which dephosphorylates PA to yield DAG (reviewed in Ref. 10). Indeed, in several cell systems, PLD activity has been
shown to underlie at least a portion of agonist-induced sustained DAG
production (reviewed in Ref. 10), and it has been proposed that chronic
elevations in DAG content are the product of the combined activities of
PLD and PA phosphohydrolase (11).
Several lines of evidence support a role for PLD in differentiation of
keratinocytes. For example, the tetrasialo ganglioside GQ1b
induces PLD activation, a sustained elevation in DAG content, and
induction of keratinocyte differentiation (12, 13). Studies in our
laboratory also provide evidence for an involvement of PLD in
keratinocyte growth inhibition and differentiation. Specifically, incubation of epidermal keratinocytes with bacterial PLD results in
inhibition of proliferation and an increase in the activity of a marker
of differentiation, transglutaminase (14). In addition, staurosporine
and 12-O-tetradecanoylphorbol-13-acetate elicit sustained
PLD activation and also induce transglutaminase activity in primary
mouse keratinocytes.2
Finally, previous findings with two microbial toxins, epidermal cell
differentiation inhibitor derived from Staphylococcus aureus E-1 and exoenzyme C3 from Clostridium botulinum, also
support an involvement of PLD in keratinocyte differentiation. These
toxins inhibit the differentiation of human epidermal keratinocytes
through a common mechanism (15) involving ADP-ribosylation of small GTP-binding proteins of the Rho family. Since RhoA regulates PLD (reviewed in Ref. 16) and since in other systems C3 exoenzyme is known
to block PLD activation (17), it is possible that the ability of
epidermal cell differentiation inhibitor and C3 to inhibit keratinocyte
differentiation may be through their inhibition of the activity of PLD.
Two isoforms of PLD, PLD-1 and PLD-2, have recently been cloned,
sequenced, and characterized (18-21). Both isoenzymes utilize phosphatidylcholine as a substrate and require phosphatidylinositol 4,5-bisphosphate as a cofactor (19, 22). Interestingly, two recent reports have observed changes in the expression and/or activity of PLD-1 associated with differentiation of C6 glioma (23) and
HL-60 cells (24). Furthermore, PLD-1 appears to represent the isoform
sensitive to microbial toxin inhibition, since this isoenzyme is
regulated by small GTP-binding proteins, such as Rho. On the other
hand, PLD-2 exhibits constitutive activity (22), and its function is
unknown (19, 22).
Because 1,25(OH)2D3 increases the expression of
phosphoinositide-specific phospholipase, which generates PKC-activating
DAG, and because PLD not only generates DAG but also may be an
important signaling enzyme during keratinocyte differentiation, we
hypothesized that 1,25(OH)2D3 may also exert
its effects on keratinocyte differentiation by modulating PLD
expression and/or activity. Therefore, we investigated the effects of
1,25(OH)2D3 on PLD-1 and -2 expression and PLD activity. We report that 1,25(OH)2D3 induces
PLD-1, but not PLD-2, expression. The current work establishes the time
course of the induction of PLD-1 expression in primary mouse epidermal
keratinocytes in response to 1,25(OH)2D3 and
demonstrates a concomitant enhancement of PLD activity. Furthermore,
the data correlate the activation of this signaling pathway with the
induction and progression of keratinocyte differentiation.
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EXPERIMENTAL PROCEDURES |
Materials--
Calcium-free minimal essential medium and
antibiotics were obtained from Biologos, Inc. (Maperville, IL). Bovine
pituitary extract, epidermal growth factor, and TriZOL reagent were
purchased from Life Technologies, Inc. ITS+ (6.25 µg/ml insulin, 6.25 µg/ml transferrin, 6.25 ng/ml selenous acid, 5.35 µg/ml linoleic
acid, and 0.125% bovine serum albumin) was supplied by Collaborative Biomedical Products (Bedford, MA). All radiolabeled chemicals were
purchased from NEN Life Science Products. Silica Gel 60 TLC plates with
concentrating zone were obtained from EM Science (Gibbstown, NJ).
Phosphatidic acid and phosphatidylethanol standards, as well as
phosphatidylcholine and phosphatidylethanolamine, were obtained from
Avanti Polar Lipids (Alabaster, AL). Phosphatidylinositol 4,5-bisphosphate was purchased from Calbiochem. GF/A filters were purchased from Whatman International, Ltd. (Maidstone, United Kingdom),
and support filters were obtained from Millipore Corp. (Bedford, MA).
In situ hybridizations were performed using a DIG/Genius RNA
labeling kit from Boehringer Mannheim. All other reagents were
purchased from Sigma.
Cell Isolation and Culture--
Epidermal keratinocytes
harvested from neonatal ICR mice according to the method of Yuspa and
Harris (25) were used to initiate primary cultures. Cells were plated
at a density of 25,000 cells/cm2 and were allowed to attach
overnight in the presence of keratinocyte plating medium composed of
calcium-free minimal essential medium supplemented with 2% dialyzed
fetal bovine serum, 50 µM calcium chloride, 5 ng/ml
epidermal growth factor, 2 mM glutamine, ITS+, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml fungizone.
Following attachment, the cells were refed with keratinocyte growth
medium composed of the above medium in which serum was deleted and 90 µg/ml bovine pituitary extract was added.
RNA Isolation and Northern Blot Analysis--
Near confluent
cultures of primary keratinocytes were refed with keratinocyte growth
medium supplemented with 250 nM
1,25(OH)2D3 or vehicle control. After the
indicated time periods, the cells were lysed with 1.0 ml of TriZOL
reagent, and total RNA was isolated according to the manufacturer's
instructions. Total RNA (15 µg) was separated on 1.2%
formaldehyde-agarose gels and transferred to GeneScreen (NEN Life
Science Products) membrane. Following prehybridization overnight, the
blots were probed overnight in Church-Gilbert buffer (0.5 M
sodium phosphate, 7% SDS, 1% bovine serum albumin, pH 7.6) at
65 °C with a random hexamer-primed [32P]dCTP-labeled
probe fragment. Blots were then washed four times for 5 min each at
room temperature in 2× SSC plus 0.1% SDS and two times for 30 min
each at 65 °C in 0.1× SSC plus 0.1% SDS (1× SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and
exposed to film for 6-10 days. Quantitation was performed using a
Bio-Rad model GS-700 imaging densitometer, and the density of each band was normalized to the density of the corresponding
glyceraldehyde-3-phosphate dehydrogenase band. Probe fragments
consisted of nucleotides 542-1390 of murine PLD-1 (20) and 397-1229
of murine PLD-2 (22). The probe for cytokeratin K1 was kindly provided
by Dr. Stuart Yuspa (NCI, National Institutes of Health, Bethesda, MD).
Measurement of Total Phospholipase D Activity in Intact
Cells--
Total PLD activity was assayed as detailed in Ref. 26 and
is described briefly. To determine an acute effect of
1,25(OH)2D3 on PLD activity, near confluent
cultures of primary keratinocytes (densities of 15,000-30,000
cells/cm2) were labeled for 20-24 h with
[3H]oleic acid and then refed with keratinocyte growth
medium supplemented with 250 nM
1,25(OH)2D3 or vehicle control in the presence
of 0.5% ethanol for an additional 30 min. To determine a chronic effect of 1,25(OH)2D3 on PLD activity,
keratinocytes were incubated for 24 h with 250 nM
1,25(OH)2D3 or vehicle control prior to the addition of approximately 5 µCi/sample of [14C]ethanol
in ethanol (final concentration = 0.5%) for an additional 1 h. The incubations were terminated by aspirating the medium and lysing
the cells by the addition of 0.2% SDS containing 5 mM
EDTA. The radiolabeled phosphatidylethanol was then extracted into
chloroform/methanol (2:1, v/v), separated by thin layer chromatography, visualized with autofluorography using En3Hance, and
quantified by liquid scintillation spectrometry.
Measurement of Phospholipase D-1 Activity in Vitro--
Rho- and
ADP-ribosylation factor (ARF)-stimulatable PLD-1 activity was assayed
in vitro essentially as in Ref. 27. Near confluent epidermal
keratinocytes were treated for 24 h with control vehicle (0.05%
ethanol) or 250 nM 1,25(OH)2D3.
Cells were rinsed once with phosphate-buffered saline and scraped from
the plate in 300 µl of buffer A (25 mM HEPES, pH 7.4, 100 mM KCl, 3 mM NaCl, 5 mM
MgCl2, 1 mM EGTA, 5 mM
dithiothreitol, and protease inhibitors (0.5 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride, 2.1 µM
leupeptin, 1.8 µg/ml aprotinin, 1 µg/ml pepstatin A with or without
2.5 µg/ml trypsin inhibitor and 1 µg/ml chymostatin)).
Keratinocytes were then homogenized using a pellet pestle (Kontes,
Vineland, NJ) and centrifuged to remove unlysed cells. The resulting
supernatant (40 µl) was assayed in vitro for PLD activity
in the presence and absence of 1 µM each of
GTP
S-loaded Rho and ARF in a final volume of 150 µl of 50 mM HEPES, pH 7.4, 3 mM EGTA, 80 mM
KCl, 1 mM dithiothreitol, 3 mM
MgCl2, and 2 mM CaCl2. The
substrate consisted of phospholipid vesicles composed of 320 µg of
phosphatidylethanolamine, 20 µg of phosphatidylcholine, 14 µg of
phosphatidylinositol 4,5-bisphosphate, and 50 µCi of
[choline-methyl-3H]dipalmitoylphosphatidylcholine
and prepared by sonication in 2.5 ml of 20 mM HEPES, pH
7.4. Reactions were initiated by the addition of the phospholipid
vesicle mixture (1.3 µCi/sample) and proceeded for 30 min at 37 °C
with shaking. Reactions were terminated, and unreacted
[3H]phosphatidylcholine was precipitated by the addition
of ice-cold 20% trichloroacetic acid and bovine serum albumin. Samples
were centrifuged at 4 °C, and [3H]choline released
into the supernatant was quantified by liquid scintillation
spectrometry. Blanks to which no cell lysate was added were performed,
and the determined radioactivity was subtracted from all samples.
Values were then expressed as the difference between PLD activity
assayed in the presence of Rho and ARF versus in the absence
of these two small GTP-binding proteins. Controls in which human PLD-1-
or mouse PLD-2-overexpressing (baculovirus-infected) Sf9
membranes were assayed as above showed that PLD-1 activity was
stimulated in the presence of Rho and ARF, whereas PLD-2 activity was
unaffected (and/or slightly inhibited), as previously reported (22).
Purified recombinant, baculovirus-expressed human Rho and ARF and
PLD-overexpressing insect cell membranes were generous gifts of Dr.
Nancy Pryer (Onyx Pharmaceuticals, Richmond, CA).
Measurement of DNA Synthesis--
Near confluent cultures of
primary keratinocytes were refed with keratinocyte growth medium
supplemented with the indicated concentrations of
1,25(OH)2D3 or vehicle control. After the
indicated time periods, the cells were labeled with 1 µCi/ml
[3H]thymidine for approximately 1 h. Cells were
washed twice with phosphate-buffered saline, and reactions were
terminated using ice-cold 5% trichloroacetic acid. Cells were washed
sequentially with 5% trichloroacetic acid followed by deionized water
and were solubilized in 0.3 M NaOH. An aliquot of this NaOH
extract was counted in a liquid scintillation spectrometer.
Measurement of Cellular Transglutaminase Activity--
Near
confluent cultures of primary keratinocytes were refed with
keratinocyte growth medium supplemented with 250 nM
1,25(OH)2D3 or vehicle control. Cells were
harvested, and transglutaminase activity was measured according to the
method of Folk and Chung (28) with minor modifications. Specifically,
dimethylcasein was substituted for
-casein, and reactions were
permitted to continue overnight before isolating the
[3H]putrescine dimethylcasein product on filters with
ice-cold trichloroacetic acid. The radiolabeled product was then
counted in a liquid scintillation spectrometer and normalized to
protein content of the samples as determined using the micro-BCA
protein assay with bovine serum albumin as the standard.
In Situ Hybridization--
The expression of PLD-1 mRNA in
neonatal mouse skin was detected by in situ hybridization as
described by Wilkinson (29), with modifications for use with tissue
sections. Briefly, neonatal mouse skins were dissected and fixed in 4%
paraformaldehyde overnight at 4 °C. Fixed skins were then dehydrated
in ethanol and xylene and embedded in paraffin. Seven-µm-thick
sections were cut, deparaffinated, and rehydrated. The sections were
treated with proteinase K and post-fixed in 4% paraformaldehyde, 0.2%
glutaraldehyde prior to hybridization with a digoxigenin-labeled PLD-1
probe (nucleotides 542-1390, as above) at 70 °C overnight in a
solution containing 50% formamide, 5× SSC, 1% SDS, 5 mg/ml heparin,
and 50 mg/ml yeast tRNA. Following hybridization, the tissue sections
were washed sequentially in 2× SSC plus 1% SDS, 1× SSC plus 1% SDS,
and 0.5× SSC plus 1% SDS for 1 h each at 70 °C. The sections
were then incubated with alkaline phosphatase-conjugated
anti-digoxigenin antibody for 2 h and washed in TBST overnight.
The colorimetric reaction for alkaline phosphatase was performed using
nitro blue tetrazolium and X-phosphate.
Statistics--
The data are presented as means ± S.E.
Each keratinocyte preparation represents a separate experiment
(n = 1). Data were analyzed using analysis of variance
or Student's t test, as appropriate, to determine if
individual time points or treatments differed significantly from
control. The level of significance was p < 0.05.
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RESULTS |
Induction of Phospholipase D-1 Expression and Activity by
1,25(OH)2D3--
To determine the ability of
1,25(OH)2D3 to modulate the expression of PLD,
Northern blot analysis was performed. Near confluent cultures of
primary mouse keratinocytes were treated with 250 nM
1,25(OH)2D3 (a maximal growth-inhibitory
concentration, as shown in Fig. 4A) or vehicle for 24 h, and total RNA was isolated. RNA was analyzed by Northern
hybridization with probes for PLD-1 and PLD-2 as described under
"Experimental Procedures." PLD-1 mRNA levels were specifically
increased by 250 nM 1,25(OH)2D3, while PLD-2 levels were unchanged (Fig.
1). Subsequently, treatment of near
confluent cultures of primary mouse keratinocytes with 250 nM 1,25(OH)2D3 for various times
revealed that PLD-1 expression increased to 167% of control within
8 h and reached a peak increase of 289% of control at 24 h
(Fig. 2).

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Fig. 1.
Effect of 1,25(OH)2D3
on the expression of PLD-1 and -2 in primary cultures of mouse
keratinocytes. Near confluent cultures of primary mouse
keratinocytes were treated with 250 nM
1,25(OH)2D3 (D3) or vehicle
control (C) for 24 h, and Northern blot analysis was
performed. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bands
are shown to indicate approximately equal loading of 15 µg of total
RNA. Also shown are molecular size markers to indicate the approximate
size of each transcript.
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Fig. 2.
The time-dependent effect of
1,25(OH)2D3 on the expression of PLD-1 in
primary cultures of mouse keratinocytes. Near confluent cultures
of primary mouse keratinocytes were treated with 250 nM
1,25(OH)2D3 (D3) or vehicle
control (C) for the indicated time periods, and Northern
blot analysis was performed. Two bands are typically recognized by the
PLD-1 probe; the size differential of these two bands (the upper band
is 7.5 kilobase pairs, and the lower band is 5 kilobase pairs) suggests
that they cannot represent the two splice variants of PLD-1 described
previously, PLD1a and PLD1b, which differ by only 114 base pairs (20).
Although the precise identities of the two bands are unclear, the lower
band was used for quantitation, since the 5-kilobase pair size matches
that defined as the cDNA coding region (20). The density of the
PLD-1 band was normalized to the density of the
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band within
each lane, and the level of PLD-1 expression by treated keratinocytes
is expressed as a percentage of the control PLD-1 expression for each
time point. The compiled results of multiple experiments are expressed
graphically, and one representative blot containing a range of time
points is shown. Asterisks indicate that PLD-1 expression by
treated keratinocytes is significantly greater than the control PLD-1
expression at the given time point (means ± S.E.,
n = 3-5, p < 0.05).
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To establish that the increase in PLD-1 mRNA levels resulted in
increased enzyme activity, total PLD activity was measured in near
confluent cultures of primary mouse keratinocytes following both acute
(30-min) and chronic (24-h) exposures to 250 nM
1,25(OH)2D3. After 30 min, there was no
significant effect of 1,25(OH)2D3 on PLD
activity (Fig. 3A). However,
after 24 h the PLD activity was increased to 140% of control
(Fig. 3A). Thus, 1,25(OH)2D3
increased both PLD-1 expression and PLD activity within 24 h in
primary cultures of mouse keratinocytes.

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Fig. 3.
The acute and chronic effects of
1,25(OH)2D3 on total PLD and PLD-1 activity in
primary cultures of mouse keratinocytes. A, near
confluent cultures of primary mouse keratinocytes were treated with 250 nM 1,25(OH)2D3 or vehicle control
(Con) for the indicated time periods, and PLD activity was
assayed by measuring the production of phosphatidylethanol
(PEt). Values are expressed as percentages of control and
represent the means ± S.E. of three separate experiments
performed in duplicate. *, p < 0.05. B,
near confluent cultures of primary mouse keratinocytes were treated
with 250 nM 1,25(OH)2D3 or vehicle
control for 24 h, and cell homogenates were prepared. Rho- and
ARF-stimulatable PLD-1 activity in the homogenate was then measured
in vitro, and PLD-1 activity was calculated as described
under "Experimental Procedures." Values are expressed as a
percentage of control and represent the means ± S.E. of three
experiments performed in triplicate.
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Previous reports indicate that PLD-1 activity is enhanced by the small
GTP-binding proteins, Rho and ARF (reviewed in Ref. 16). To verify that
the observed increase in PLD activity represented PLD-1, we measured
Rho- and ARF-stimulatable PLD activity in vitro in
homogenates of cells preexposed for 24 h to control vehicle or 250 nM 1,25(OH)2D3. We observed an
approximate 3-fold increase in Rho- and ARF-stimulated activity in
1,25(OH)2D3-pretreated cell homogenates (Fig.
3B), consistent with our observations of an approximate
3-fold increase in PLD-1 expression, as well as enhanced total PLD
activity, in response to 1,25(OH)2D3.
Induction of Keratinocyte Growth Arrest by
1,25(OH)2D3--
To confirm that
1,25(OH)2D3 induces growth arrest of primary
cultures of mouse keratinocytes, we examined the ability of
1,25(OH)2D3 to inhibit proliferation by
examining the incorporation of [3H]thymidine into DNA.
1,25(OH)2D3 inhibited proliferation of primary mouse keratinocytes in both a concentration- and
time-dependent manner (Fig.
4, A and B).
Following a 24-h exposure, only 0.1 nM
1,25(OH)2D3 was required to significantly
inhibit [3H]thymidine incorporation, and the inhibition
was maximal between 100 and 250 nM (Fig. 4A).
Thus, 250 nM 1,25(OH)2D3 was
established as the concentration to be used in all subsequent
experiments. At this concentration, [3H]thymidine
incorporation was significantly decreased after only 8 h (75% of
control), and maximal inhibition of [3H]thymidine
incorporation was achieved by 24 h (18% of control) (Fig.
4B).

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Fig. 4.
The concentration- (A) and
time-dependent (B) effects of
1,25(OH)2D3 on [3H]thymidine
incorporation in primary cultures of mouse keratinocytes. Near
confluent cultures of primary mouse keratinocytes were treated with the
indicated concentrations of 1,25(OH)2D3 or
vehicle control for 24 h (A) or with 250 nM
1,25(OH)2D3 for the indicated time periods
(B), and radiolabel was added for an additional 1 h.
The data are expressed as a percentage of the
[3H]thymidine incorporation by treated keratinocytes
compared with the corresponding control at the same time point. The
asterisks indicate that [3H]thymidine
incorporation by treated keratinocytes is significantly less than the
control [3H]thymidine incorporation at the given time
point (means ± S.E., n = 3-7, p < 0.05).
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Induction of Keratinocyte Differentiation by 1,25(OH)2
D3--
Expression of cytokeratin K1, a marker of the
early (spinous) stage of keratinocyte differentiation, was measured by
Northern blot analysis in order to determine the ability of
1,25(OH)2D3 to induce cultured basal
keratinocytes to differentiate to spinous keratinocytes. Near confluent
cultures of primary mouse keratinocytes were treated with 250 nM 1,25(OH)2D3 or vehicle for the
indicated time periods, and total RNA was isolated. Cytokeratin K1
mRNA levels began to decrease after 8 h, and K1 mRNA
levels were significantly decreased by 12 h to 41% of control
(Fig. 5). Thus, 250 nM
1,25(OH)2D3 decreased the expression of a
spinous stage marker in primary mouse keratinocytes.

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Fig. 5.
The time-dependent effect of
1,25(OH)2D3 on the expression of K1 in primary
cultures of mouse keratinocytes. Near confluent cultures of
primary mouse keratinocytes were treated with 250 nM
1,25(OH)2D3 (D3) or vehicle
control (C) for the indicated time periods, and Northern
blot analysis was performed. The density of the K1 band was normalized
to the density of the corresponding glyceraldehyde-3-phosphate
dehydrogenase band in each lane, and the level of K1 expression by
treated keratinocytes is expressed as a percentage of the control K1
expression for each time point. The compiled results of multiple
experiments are expressed graphically, and one representative blot
containing a range of time points is shown. The asterisks
indicate that K1 expression by treated keratinocytes is significantly
less than the control K1 expression at the given time point (means ± S.E., n = 3-5, p < 0.05).
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Activity of transglutaminase, a late (granular) stage marker of
keratinocyte differentiation, was measured using a radiolabeled substrate in order to determine the ability of
1,25(OH)2D3 to induce cultured keratinocytes to
differentiate to the granular stage. Near confluent cultures of primary
mouse keratinocytes were treated with 250 nM
1,25(OH)2D3 or vehicle for the indicated time
periods, and transglutaminase activity was measured as described under
"Experimental Procedures." Transglutaminase activity was increased
to 160% of control by 1,25(OH)2D3 after
12 h, and after 24 h transglutaminase activity was maximally
increased to 214% of control (Fig. 6).
Thus, 1,25(OH)2D3 increased the activity of a
late stage marker of differentiation in primary cultures of mouse
keratinocytes. Furthermore, the increase in transglutaminase activity
closely followed the increase in expression of PLD-1.

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Fig. 6.
The time-dependent effect of
1,25(OH)2D3 on the activity of transglutaminase
in primary cultures of mouse keratinocytes. Near confluent
cultures of primary mouse keratinocytes were treated with 250 nM 1,25(OH)2D3 or vehicle control
for the indicated time periods, and transglutaminase activity was
assayed. For each experiment, the transglutaminase activity in treated
keratinocytes was normalized to the control transglutaminase activity
of the same time point, and the data are expressed as the means of the
percentage of control (means ± S.E., n = 4-7,
p < 0.05).
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Expression of PLD-1 by Neonatal Mouse Epidermis--
To establish
that PLD-1 expression is important for the the intermediate stages of
mouse keratinocyte differentiation in vivo, in
situ hybridization using sense and antisense probes for PLD-1 was
performed on neonatal mouse skins as described under "Experimental Procedures." The cells between the basal and cornified layers are in
the spinous and granular stages of differentiation, and this region
showed strong staining for PLD-1 (Fig. 7,
A and C). The undifferentiated basal cells and
the terminally differentiated and metabolically inactive cornified
cells exhibited relatively light staining. Hybridization with the
control probe showed light and diffuse staining throughout the layers
(Fig. 7, B and D). Thus, the importance of PLD-1
expression during the intermediate stages of differentiation of
cultured keratinocytes is supported by the expression of PLD-1 in the
actively differentiating cells of neonatal mouse epidermis.

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Fig. 7.
In situ hybridization of neonatal
mouse epidermis using sense and antisense probes for phospholipase
D-1. Freshly dissected neonatal mouse skins were fixed and
hybridized with an antisense probe recognizing the mRNA for PLD-1
(A and C) or a sense (control) probe capable of
recognizing the complementary strand (B and D).
Magnifications are as follows: × 20 (A and B)
and × 40 (C and D). B, basal
layer; S/G, spinous and granular layers; C,
cornified layer. Shown is the result of one experiment representative
of two.
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DISCUSSION |
The ability of 1,25(OH)2D3 to increase the
expression of phosphoinositide-specific phospholipase C isoforms (8)
led us to examine the effect of this sterol hormone on another
DAG-generating signaling enzyme, PLD. We found that
1,25(OH)2D3 specifically increased the
expression of the PLD-1 isoform of PLD without affecting PLD-2
expression (Fig. 1). The specificity of this stimulation suggests that
the observed increase in expression is not due to a global enhancement
of transcription and/or message stability.
The time course of PLD-1 expression (Fig. 2) is consistent with several
previous studies that have documented the effect of 1,25(OH)2D3 on the expression of inducible
genes. While the mRNA levels of some
1,25(OH)2D3-responsive genes increase within
1-2 h (e.g. tumor necrosis factor-
(30)), other genes
require longer time periods for their induction. For example, the
expression of transforming growth factor-
is increased after a 6-h
exposure of human keratinocytes to 1,25(OH)2D3
(31). Similarly, two genes that respond to
1,25(OH)2D3 at later time points are known to contain vitamin D response elements in their respective promoters (32).
Specifically, in mouse epidermal cells osteopontin expression is
increased within 4 h by 1,25(OH)2D3 (33),
and in vivo, 25-hydroxyvitamin D3 24-hydroxylase
is induced between 3 and 6 h in intestine and kidney (32).
The 1,25(OH)2D3-stimulated PLD-1 expression
translates into an increase in PLD activity as well. The hormone has no
acute effect on PLD activity (unlike hormones that stimulate cells
through tyrosine kinase or G-protein-coupled receptors) but does
activate PLD over 24 h (Fig. 3A), consistent with the
time course of enhanced PLD-1 expression. On the other hand, the
pronounced change in PLD-1 expression results in only a relatively
small increase in 1,25(OH)2D3-stimulated total
PLD activity (approximately 1.4-fold over control) at 24 h.
However, significant levels of PLD-2 mRNA are detected in the
keratinocytes (Fig. 1). Although PLD-2 expression is unaltered by
1,25(OH)2D3, PLD-2 is likely to contribute to the basal PLD activity, thus masking the extent of the
1,25(OH)2D3-induced change in PLD-1 activity.
In fact, when we measured Rho- and ARF-stimulatable PLD-1 activity in
1,25(OH)2D3-pretreated keratinocyte
homogenates, we found an approximate 3-fold increase in PLD-1 activity
(Fig. 3B), consistent with our observed ~3-fold increase
in PLD-1 message. Thus, the observed increase in PLD-1 mRNA is
translated into elevated PLD-1 activity.
To establish a potential participation of
1,25(OH)2D3-induced PLD-1 expression/activation
in the various stages of keratinocyte differentiation, we examined the
time course of changes in markers of each of three stages of
keratinocyte differentiation. If PLD-1 activity is to be involved in a
particular stage of keratinocyte differentiation, PLD-1
expression/activation should precede changes in the appropriate marker.
DNA synthesis is arrested in cells undergoing the basal to spinous
transition. A significant growth arrest of proliferating keratinocytes
is first induced by 1,25(OH)2D3 within
approximately 8 h of exposure (Fig. 5). The expression of PLD-1 is
first elevated at 8 h also. Since alterations in
1,25(OH)2D3-induced PLD-1 expression are
coincident with growth arrest rather than preceding it, our result
argues against a role for PLD-1 in the basal to spinous transition. We
should note that our observation of a requirement for an 8-h exposure
to 1,25(OH)2D3 conflicts with a previous report
in mouse keratinocytes in which 1,25(OH)2D3 inhibited [3H]thymidine incorporation into DNA within
3 h (34). These investigators used as their control DNA synthesis
at time 0. However, we have found changes in
[3H]thymidine incorporation in vehicle-exposed
keratinocytes over time and therefore have used
[3H]thymidine incorporation in cells incubated with
vehicle for the same period of time as the
1,25(OH)2D3-treated cells as the corresponding
control value.
The expression of the cytokeratin K1 is known to mark early (spinous)
keratinocyte differentiation. However, in our studies 1,25(OH)2D3 decreased K1 expression (Fig. 5);
thus, our results argue against a role for PLD-1 in mediating early
(spinous) differentiation, because K1 expression was not increased.
However, these findings may not accurately reflect the true involvement
of PLD-1 in mediating the events of early differentiation, because the
results of others indicate that cytokeratin K1 expression may be
decreased directly by 1,25(OH)2D3, and
therefore, any effect of PLD-1 on K1 expression may be masked.
Specifically, Su et al. (35) demonstrated K1 message
destabilization by 1,25(OH)2D3. In addition,
there exists a 1,25(OH)2D3 inhibitory element
in the 3'-untranslated region of the K1 gene (7). Further experiments
using lower concentrations of 1,25(OH)2D3 and
longer exposures may reveal a correlation of PLD-1 expression with
early keratinocyte differentiation.
Exposure to 1,25(OH)2D3 also increased
transglutaminase activity, a marker of late (granular) differentiation,
with maximal stimulation observed at 24 h (Fig. 6). Our result is
consistent with a previous report (35), in which the authors described a peak increase of approximately 2-fold after a 24-h
1,25(OH)2D3 treatment and a slight decline by
48 h (35). These data suggest that the mouse keratinocyte behaves
similarly to the human and that transglutaminase activity can serve as
a marker of differentiation in our primary mouse keratinocyte system.
Our time course is also consistent with a potential role for PLD
activity in regulating differentiation in that PLD-1 is expressed in
response to 1,25(OH)2D3 prior to the measured
increase in transglutaminase activity. Additional evidence for a role
for PLD-1 in differentiation is provided by our in situ
hybridization study (Fig. 7). The finding that little or no expression
of PLD-1 is detected in basal keratinocytes argues against its
involvement in growth arrest. Instead, expression is detected strongly
in the upper layers (the spinous and granular layers), consistent with
PLD-1 mediating keratinocyte differentiation.
There are numerous published data supporting a role for PKC in
regulating keratinocyte growth and differentiation (reviewed in Refs. 1
and 9). However, the exact role of PKC is controversial, since agents
that stimulate proliferation and those that induce differentiation have
each been reported to increase phosphoinositide turnover and DAG levels
(36-38) and/or activate PKC (13, 39-42). The explanation for these
conflicting results may reside in the fact that PKC is a family of
isoenzymes (reviewed in Ref. 11), with different PKC isoenzymes
involved in proliferative versus differentiative events.
Thus, two agents that both stimulate PKC yet produce two different
results may be activating two different PKC isoenzymes. In additional
studies, treatment of keratinocytes with phorbol esters yielded
paradoxical results of initial differentiation induction and subsequent
growth promotion (43). These results are probably due to the ability of
phorbol esters to activate multiple isoforms of PKC.
Because it is derived from phosphatidylcholine rather than from
phosphoinositides, the DAG species generated by PLD (and PA phosphohydrolase) is composed of different acyl groups than DAG generated by other phospholipid-hydrolyzing enzymes. Thus, DAG generated by PLD may activate distinct PKC isoforms (reviewed in Ref.
10), leading to the expression of specific markers of keratinocyte
differentiation. Numerous studies in other systems have suggested that
individual PKC isoenzymes regulate specific cellular processes
(reviewed in Ref. 11). For example, in a transfected rat keratinocyte
cell line, PKC-
has been shown to increase the transcription of
transglutaminase, a marker of late keratinocyte differentiation, more
effectively than other PKC isoforms (44). Thus, individual PKC
isozymes, activated by distinct DAG species, may be responsible for
determining the patterned expression of genes during keratinocyte
differentiation. Alternatively, DAG derived from PLD activity may be
differentially metabolized (45, 46), and/or the PA produced directly by
PLD activity may itself serve a second messenger function (reviewed in
Ref. 10).
Our present results are indicative of a role for
1,25(OH)2D3-induced PLD-1 expression/activity
in mediating keratinocyte differentiation. Specifically, our time
course studies, as well as our in situ hybridization data,
support an involvement of PLD-1 in the spinous to granular transition
in keratinocytes. Current experiments in our laboratory are directed
toward attempting to prove this hypothesis through other methods of
regulating PLD-1 expression and/or activity. Finally, our report is the
first to document a 1,25(OH)2D3-elicited increase in the expression of the important phospholipid-metabolizing enzyme PLD.