Receptor-mediated induction of human dermal fibroblast ectoaminopeptidase N by glucocorticoids

Article abstract of DOI:10.1007/s000180050189

Aminopeptidase N was demonstrated in human dermal firbroblasts as an ectoenzyme. The enzyme has wide substrate specificity, with a K(m) of 0.63 mM and V(max) of 338 nmol min^-1 mg^-1. Addition of fetal calf serum to the culture medium

Full text of DOI:10.1007/s000180050189

CMLS, Cell. Mol. Life Sci. 54 (1998) 614–617
1420-682X/98/060614-04 $ 1.50+0.20/0
© Birkha¨user Verlag, Basel, 1998
Research Article
Receptor-mediated induction of human dermal fibroblast
ectoaminopeptidase N by glucocorticoids
V. Stefanovic´*, P. Vlahovic´ and M. Mitic´-Zlatkovic´
Institute of Nephrology and Hemodialysis, Faculty of Medicine, B. Taskovic´a 48, YU-18000 Nisˇ (Yugoslavia),
Fax +381 18 321 313
Received 3 April 1998; accepted 21 April 1998
Abstract. Aminopeptidase N was demonstrated in hu- dexamethasone for 3 days. Actinomycin D, a blocker of
man dermal fibroblasts as an ectoenzyme. The enzyme RNA synthesis, and cycloheximide, an inhibitor of
has wide substrate specificity, with a Km of 0.63 mM protein synthesis, did not alter basal aminopeptidase N
and Vmax of 338 nmol min−1 mg−1. Addition of fetal activity. However, they prevented stimulation by dex-
calf serum to the culture medium increased aminopep- amethasone. RU 38486, a glucocorticoid receptor antag-
tidase N activity up to 63% by 10% serum in a 48-h onist, suppressed the dexamethasone-induced increase in
culture. Treatment of fibroblasts by dexamethasone aminopeptidase N activity. This study shows that hu-
increased ectoaminopeptidase N activity in a dose- man dermal fibroblasts contain ectoaminopeptidase N
and time-dependent manner. Maximal increase of controlled by glucocorticoids through a receptor-medi-
aminopeptidase N occurred after treatment with 1 mM ated mechanism.
Key words. Dermal fibroblasts; aminopeptidase N; ectoenzyme; glucocorticoids.
Ectopeptidases play an important role in signal trans- Its presence on the surface of myeloid cells is thought to
duction and communication between cells [1].
Aminopeptidases have attracted much interest, and sev-
eral different types of these enzymes, N, A, B, P and W,
be immunoregulatory in nature [4]. Aminopeptidase N
is thought to participate in the degradation of regula-
tory peptides such as enkephalins [5].
have been described. Aminopeptidase N (APN;
L-h-
Human dermal fibroblasts function to synthesize and
degrade the extracellular matrix, as well as to secrete
diffusible factors that promote epidermal cell growth
[6]. Several membrane-bound proteases have been
demonstrated on human skin fibroblasts, and their
modulation in pathology has been suggested [7]. The
aim of this work was to study regulation of human
dermal fibroblast aminopeptidase N by cyclic adenosine
monophosphate (cAMP), growth factors and glucocor-
ticoids. Evidence is presented that fibroblast aminopep-
tidase N is upregulated by glucocorticoids through a
receptor-mediated mechanism.
aminoacylpeptide hydrolase, EC 3.4.11.2) acts with rel-
atively broad substrate specificity on peptides with an
N-terminal neutral amino acid. Aminopeptidase N is
widely distributed, with the highest expression in renal
cortex and intestinal microvilli [2]. Sequence analysis
showed aminopeptidase N to be identical to CD 13, a
150-kDa cell surface glycoprotein originally used as a
marker for subpopulations of haematopoietic cells [3].
* Corresponding author.

CMLS, Cell. Mol. Life Sci. Vol. 54, 1998
Research Article
615
Table 1. Hydrolysis of various chromogenic substrates by human
fibroblast aminopeptidases.
Materials and methods
Tissue culture flasks, dishes and culture media were
obtained from Flow Laboratories (Irvine, UK); actino-
mycin D, alanine p-nitroanilide (p-NA), 8-bromo-
cAMP, cycloheximide, dexamethasone and phorbol-
12-miristate-13-acetate (PMA) from Sigma (St. Louis,
MO, USA). The glucocorticoid antagonist, RU 38486, Arginine p-NA
was a gift from Roussel-Uclaf (Paris, France).
Culture of dermal fibroblasts. Human fibroblasts were
Substrate
Aminopeptidase activity
nmol min−1 mg−1
Alanine p-NA
Leucine p-NA
Lysine p-NA
11098.6
92.393.8
48.296.6
47.195.6
6.291.5
1.490.4
Valine p-NA
Proline p-NA
isolated from fragments of skin taken during surgery in
the trunk area of normal adult subjects. The explant
culture was performed as described previously [7]. Cells
were cultured in RPMI 1640 medium buffered with 20 strates (table 1). Alanine p-NA was hydrolysed at a
mM Hepes and supplemented with 10% fetal calf
serum, 50 U/ml of penicillin, 50 mg/ml of streptomycin
higher rate than leucine p-NA, with a Km of 0.63 mM
and Vmax of 338 nmol min−1 mg−1
.
sulphate and 2 mM L-glutamine. Fibroblasts were used
Culture medium supplemented with 1 to 30% fetal calf
serum for 24 to 48 h increased aminopeptidase N activ-
ity (fig. 1). Maximal increase was obtained in a 48-h
culture with 10% fetal calf serum (PB0.01). In order to
determine the characteristics of stimulatory factors, fe-
tal calf serum was subjected to heating and dialysis.
Stimulatory activity was maintained after 24 h of dialy-
sis at 4 °C. In contrast, aminopeptidase N induction
was nearly completely suppressed by heating serum at
100 °C for 5 min (data not shown). cAMP treatment of
cell culture in concentrations from 10 to 500 mM for 72
h revealed no effect on aminopeptidase N activity (data
not shown). PMA treatment of cultured dermal fibrob-
lasts, in concentrations of 0.1 to 10 ng/ml for 24 and 48
h did not change surface aminopeptidase N activity
(data not shown).
for experiments between the 6th and 10th passage.
Aminopeptidase N activity. Surface aminopeptidase N
activity was determined on cultures of human dermal
fibroblasts in 24-well plates. Cells were rinsed three
times and incubated at 37 °C in phosphate-buffered
saline (pH 7.8) containing NaCl (140 mM), MgCl2 (1.0
mM), Na2HPO4 (9 mM), NaH2PO4 (9 mM), KH2PO4
(1.5 mM) and KCl (3 mM). Alanine p-NA (3.0 mM)
was used as substrate. The amount of p-NA formed was
read at an optical density of 405 nm. Cell-free and
substrate-free blanks were run in parallel. Cell protein
was estimated by the method of Lowry et al. [8], after
appropriate digestion with 1 M NaOH. Enzyme activity
was expressed as nanomoles of p-NA formed per
minute and per milligram of cell protein.
Results
Characterization of fibroblast aminopeptidase N.
Aminopeptidase N in these cells was considered as an
ectoenzyme on the following grounds: (i) the substrate
was hydrolysed by intact cells in culture, and the
product was released in the extracellular medium; (ii)
the amount of substrate hydrolysed over 3 to 15 min
was linearly related to the length of incubation, so it is
unlikely that significant hydrolysis of the substrate oc-
curred after entry into the cell; (iii) aminopeptidase N
activity of cells treated with the diazonium salt of sul-
phanilic acid (DASA) (0.01–3.5 mM), a nonpenetrating
agent that forms covalent bonds with many proteins
and lipids at the cell membrane, as described previously
[9], rapidly decreased to about 25% of the basal level at
3.5 mM DASA; (iv) aminopeptidase N activity in the
medium after 15 to 30 min incubation of cells was
negligible, demonstrating that the enzyme was not re-
leased from the cells.
Figure 1. Effect of serum factors on dermal fibroblast aminopep-
tidase N. Fibroblasts were cultured for 24 and 48 h with 0–30%
fetal calf serum. Enzyme activity is given as percent of basal value.
Means9SD of four determinations are given. Regression analysis
showed a significant effect of serum on aminopeptidase N activity
in both 24-h (PB0.01) and 48-h (PB0.001) cultures. *PB0.01
Substrate specificity of the enzyme was broad, as
demonstrated by hydrolysis of several chromogenic sub- vs. control.

616
V. Stefanovic´, P. Vlahovic´ and M. Mitic´-Zlatkovic´
Dexamethasone induction of fibroblast aminopeptidase N
protein synthesis. Stimulation of aminopeptidase N was
blocked by both inhibitors (table 2). Receptor occu-
pancy by RU 38486, a glucocorticoid receptor antago-
nist, also prevented induction of aminopeptidase N by
dexamethasone.
Discussion
In this study we present evidence that human fibroblast
aminopeptidase N is an ectoenzyme. Wide substrate
specificity was found, with a Km for L-alanine p-NA in
the millimolar range, as described in a previous study
[7]. Growth factors, present in the serum, increased
aminopeptidase N activity of dermal fibroblasts, in con-
trast with an increase in human mesangial cell
Figure 2. Effect of dexamethasone treatment on dermal fibroblast
aminopeptidase N. Fibroblasts were cultured with dexamethasone aminopeptidase N upon serum withdrawal [10]. The
in the concentration range from 0.01 to 100 mM in a medium
fact that serum stimulatory factor was not dialysable
and that its activity was suppressed by heating the
supplemented with 10% fetal calf serum for 1, 3 and 5 days.
Enzyme activity is given as percent of basal value. Means9SD of
four determinations are given. Regression analysis showed that serum at 100 °C for 5 min before addition to the culture
aminopeptidase N activity significantly correlated with the con-
suggest that this factor is probably a protein or peptide.
centration of dexamethasone (PB0.001) at all lengths – 1, 3 and
Fetal calf serum contains some growth factors which
5 – days of treatment.
could influence aminopeptidase N expression. Previ-
ously, we identified in serum epidermal growth factor
(EGF) and showed that aminopeptidase N is upregu-
lated upon treatment of human mesangial cells with
nanomolar concentrations of EGF [11]. Dermal fibrob-
last aminopeptidase N was unaffected following treat-
ment with cAMP in the concentration range from 10 to
500 mM. This is different from mesangial cell aminopep-
tidase N, which was stimulated with cAMP and cAMP-
stimulating agents [10]. PMA treatment had no effect
on dermal fibroblast aminopeptidase N; however, it
Human dermal fibroblasts were treated with dexa-
methasone in a concentration range from 0.01 to 100
mM for up to 5 days. Aminopeptidase N activity of
fibroblasts treated with dexamethasone increased sig-
nificantly (fig. 2). The effect was significant (PB0.05) at
1.0 mM dexamethasone after 1 day of treatment, but at
lower concentrations after 3 and 5 days of treatment.
The enzyme activity doubled after 3 days of treatment
with 0.1 and 1.0 mM dexamethasone. To evaluate
whether the effect of dexamethasone on aminopeptidase
N activity required synthesis of a new enzyme, fibrob-
lasts were treated with actinomycin D, an inhibitor of
caused marked induction of the enzyme in glomerular
mesangial and epithelial cells [9, 12]. Dexamethasone
treatment induced dermal fibroblast aminopeptidase N
but had no effect on the enzyme in glomerular mesan-
gial cells [10]. Taken together, these data show varied
RNA synthesis, and cycloheximide, an inhibitor of regulation of aminopeptidase N in human dermal
Table 2. Effects of cycloheximide, actinomycin D and RU 38486 on dexamethasone-induced aminopeptidase N activity in human
fibroblasts.
Treatment
Aminopeptidase N activity
(nmol min−1 mg−1
)
Control
Dexamethasone, 1 mM
Cycloheximide, 0.1 mg/ml
108912.4
180914.3
9198.6
101911.7
9699.4
10696.5
9599.7
99915.5
Dexamethasone, 1 mM+cycloheximide, 0.1 mg/ml
Actinomycin D, 0.1 mg/ml
Dexamethasone, 1 mM+actinomycin D, 0.1 mg/ml
RU 38486, 10 mM
Dexamethasone, 1 mM+RU 38486, 10 mM
Cells were preincubated with cycloheximide, actinomycin D or RU 38486 for 1 h before addition of dexamethasone and were then
incubated for a further period of 72 h. Values are means9SD of four determinations. Data were analysed using two-way analysis of
variance. The effect of dexamethasone was statistically significant (PB0.001). Interactions between dexamethasone and each of the
agents tested (cycloheximide, actinomycin D, RU 38486) were also significant (PB0.001).

CMLS, Cell. Mol. Life Sci. Vol. 54, 1998
Research Article
617
fibroblasts and glomerular mesangial cells by cAMP, effect was specific for glucocorticoids and was obtained
growth factors, mitogens and gluco-corticoids.
with concentrations (0.1 mM) of dexamethasone corre-
sponding to physiologic corticoid concentrations. Thus
aminopeptidase N expression in human dermal fibrob-
lasts may be influenced by both normal corticoid secre-
tion and pharmacological drug administration.
Dexamethasone treatment was found to induce dermal
fibroblast aminopeptidase N. This effect was time- and
dose-dependent. The increase in aminopeptidase N was
inhibited by actinomycin D and cycloheximide, suggest-
ing that enzyme protein synthesis is responsible for the
increased activity. RU 38486, a glucocorticoid receptor
antagonist, prevented the increase in aminopeptidase N
activity, showing that induction of aminopeptidase N is
glucocorticoid receptor-mediated. The effect of gluco-
corticoids is relatively specific, since only a few proteins
were induced when protein profiles from dexam-
ethasone-treated cells are analysed by gel electrophore-
sis [13].
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The physiological role of surface aminopeptidase N in
human dermal fibroblasts has not been established. Due
to its broad substrate specificity aminopeptidase N
cleaves various peptides, leading to activation or inacti-
vation of certain biologically active peptides. Amino-
peptidase N is thought to participate in the degradation
of neuropeptides, such as enkephalins, endorphins and
opioid peptides [5]. Apart from their degradative role,
the second major property of cell surface proteases is
their role as signal-transducing molecules. They transfer
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processes is not yet understood. However, dermal
fibroblast Arg- and Leu-aminopeptidase and dipep-
tidylpeptidase IV activities were found to be increased
in rheumatoid arthritis, psoriasis and lichen planus. A
potential role for these peptidases in the modulation of
matrix catabolism has been suggested [7].
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This study demonstrates that glucocorticoids control
the expression of fibroblast aminopeptidase N. The
.