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Cell and Cancer Biology Department (A.M., T.W.M., M.J.M., F.C.), Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, 9610 Medical Center Drive, Rockville, Maryland 20850; Agricultural Research Service (T.H.E.), United States Department of Agriculture, Beltsville, Maryland 20705; Department of Pathology (C.M.), H. Lee Moffitt Cancer Center, Tampa, Florida 33612; and Department of Obstetrics and Gynecology (C.J.M.), Uniformed Services University of Health Sciences, Bethesda, Maryland 20814
Address all correspondence and requests for reprints to: Dr. Alfredo Martínez, Cell and Cancer Biology Department, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, 9610 Medical Center Drive, Room 300, Rockville, Maryland 20850. E-mail: martineza{at}bprb.nci.nih.gov
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Abstract |
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Introduction |
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AM has been demonstrated to be a pluripotent peptide having numerous physiological roles, which include vasodilation (4), renal homeostasis (5), hormone regulation (6, 7, 8), neurotransmission (9), and growth modulation (10, 11, 12, 13). Recently, we have determined that AM and its gene-related peptide, pro-AM 20 N-terminal peptide (PAMP), have antibacterial and antifungal properties (14), constituting a new family of the already broad class of antimicrobial peptides (15). For a full review on AM, see Ref. 16.
Because we previously have shown AM immunoreactivity in the skin of the toad Xenopus laevis (14) and of the developing mouse (17), we hypothesized that AM could play an important role in the skin, maintaining a defensive barrier against invading microbes. In addition, because of its growth regulatory capabilities, AM may be involved with the wound repair process, sustaining normal epidermal turnover, and influencing tumor initiation and proliferation. This study was devised to further characterize AM and AM-R expression in human skin, in an attempt to better understand its potential functions in the integument.
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Materials and Methods |
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Cell lines
Four cell lines established from human skin were obtained from
ATCC (Gaithersburg, MD): CRL-7922 (normal skin), CRL-7729
(epidermolysis bullosa simplex), CRL-7585 (skin melanoma from a
53-yr-old oriental male), and CRL-7686 (skin pigmented melanoma). These
cell lines were maintained in DMEM (Life Technologies, Gaithersburg,
MD) containing 10% FCS.
Immunohistochemistry
A previously characterized rabbit antibody to human AM (hAM)
(no. 2343) was used at a concentration 1:1000 (18). Antigen retrieval
was performed by subjecting the tissue sections to microwave energy
(1100 watts, 2 x 5 min) while in 1.8 mM citric acid,
8.6 mM sodium citrate solution (19). After this, the
avidin-biotin complex method was used as previously described (18). In
brief, the sections were incubated overnight with the antiserum against
AM, followed by incubations with biotinylated goat antirabbit Ig
(1:200, Vectastain, Burlingame, CA) and then with avidin-biotin
peroxidase complex (1:100, Vectastain). The bound antibodies were
visualized using diaminobenzidine (Sigma). Sections were lightly
counterstained with hematoxylin. Preincubation of the antiserum with 10
nmol/ml synthetic hAM (Phoenix Pharmaceuticals, Mountain View, CA) was
used as a negative control.
In situ hybridization
Detection of the AM-R was performed using in situ
hybridization for its messenger RNA (mRNA), as previously described
(17, 20). The full-length complementary DNA (cDNA) was ligated into
plasmid cDNA1 (2) and used to generate riboprobes. The plasmid was
linearized with EcoRV and BamHI and used as a
template to generate digoxigenin-labeled sense and antisense RNA
transcripts. Hybridization was performed in a moist chamber at 46 C for
20 h in a 20-µl vol containing the probe. After stringency
washes, visualization of digoxigenin was performed using the
digoxigenin detection kit (Boehringer Mannheim, Indianapolis, IN). The
sense probe was used as negative control.
RT-PCR, cloning, and sequencing
Total RNA from the above mentioned tumor cell lines was
extracted using Trizol (Life Technologies) and reverse transcribed with
the SuperScript Preamplification System (Life Technologies), following
the manufacturer’s protocol. Amplification for AM and AM-R was
performed as previously described (6, 17). Primer sets for AM detection
were as follows: sense (AM 250–270),
5'-AAG-AAG-TGG-AAT-AAG-TGG-GCT-3'; antisense (AM 521–540),
5'-TGT-GAA-CTG-GTA-GAT-CTG-GT-3'; and nested probe antisense (AM
428–448), 5'-TCT-GGC-GGT-AGC-GCT-TGA-CTC-3', with a predicted product
of 291 bp. For h AM-R amplification, the following rat primers were
selected from the published sequence (Kapas receptor; Ref.2): sense
(AM-R 476–497), 5'-AGC-GCC-ACC-AGC-ACC-GAA-TAC-G-3'; antisense (AM-R
923–946), 5'-AGA-GGA-TGG-GGT-TGG-CGA-CAC-AGT-3'; and antisense nested
probe (AM-R 788–811), 5'-GGT-AGG-GCA-GCC-AGC-AGA-TGA-CAA-3', yielding
a 471-bp product. A Perkin-Elmer 9600 Thermocycler (Norwalk, CT) was
used to amplify the samples for 35 cycles, with annealing temperatures
of 55 and 61 C, respectively, for the ligand and its receptor. PCR
products were analyzed electrophoretically using 1% agarose gels,
transferred onto nitrocellulose filters, and subjected to Southern blot
analysis with 32P-end-labeled probes.
The PCR products were cloned by insertion into the pCR2.1 vector (Invitrogen TA cloning kit, San Diego, CA), the plasmids purified (Qiagen Plasmid Purification kit, Chatsworth, CA; and Promega Plasmid Clean-up kit, Madison, WI), and nucleotide sequencing was carried out by Sequetech (Mountain View, CA).
Western blot
Whole-cell lysates were generated as previously reported (10).
Cell pellets (
5 x 107 cells) were resuspended in 1
ml of cold PBS containing 1 µM final concentration of
each of the following protease inhibitors: Pefabloc (Centerchem Inc,
Stamford, CT), bestatin, and phosphoramidon (Sigma). The cell
suspension was homogenized, sonicated, clarified by centrifugation
(14,000 x g), and the final protein concentration
determined (BCA kit, Pierce, Rockford, IL). Cell lysates were diluted
in 2x Tricine sample buffer (Novex, San Diego, CA) to an approximate
protein concentration of 35 µg/50 µl, heated to 95 C for 3 min, and
loaded into the sample well.
A Novex Minigel apparatus was used to run the 10–20% Tricine gels (100 V, 2 h) with approximately 35 µg per lane. Transfer blotting was accomplished in the same apparatus with a titanium blotter onto polyvinyldifluoride (Immobilon P, Millipore Corporation, Bedford, MA) filter (30 V, 2 h). The filter was blocked overnight in 1% BSA/PBS. The following day a 1:5000 dilution of rabbit antihuman AM antibody was used in a 2-h incubation, washed with PBS, and then 1 x 106 cpm per blot of 125I-protein A was added. Absorption of the antibody was accomplished by the addition of 10 µg/ml hAM into the antibody stock, then put through a 0.2 µm filter. The blots were washed thoroughly with PBS, sealed in a bag, and exposed onto Kodak XAR5 film (Eastman Kodak Co., Rochester, NY) for varying time points (2–10 h).
RIA
Sweat was collected from healthy volunteers during their regular
athletic training routine and kept frozen until analysis. Blood samples
were collected from healthy volunteers, the serum extracted, and kept
frozen until analysis. Samples were thawed on ice, mixed with an equal
volume of 0.1% alkaline-treated casein (21), and extracted trough C-18
Sep-Pak 400-mg cartridges (Waters Corp., Milford, MA). The proteins
were eluted with 80% isopropanol and the recovered volume freeze dried
to eliminate the solvents. Extracts were reconstituted in 400 µl RIA
buffer (10 mM phosphate, 50 mM EDTA, 135
mM NaCl, 0.05% Triton X-100, 0.1% Tween 20, 1% BSA,
0.1% alkaline-treated casein, 20 mg/liter phenol red, pH 7.5), spun at
14,000 rpm for 10 min at 4 C to remove any solid matter, and three
100-µl aliquots from each sample were separated for analysis. The RIA
was performed using the Phoenix h AM RIA kit and following
manufacturer’s instructions. Briefly, 100 µl anti-AM antibody and
100 µl 125I-AM were added to each sample and the mixture
incubated at 4 C overnight. The following day, 500 µl of goat
antirabbit antibody (1:150 in 6% PEG 8000) and 100 µl of normal
rabbit serum (1:100 in RIA buffer) were added and incubated for 1
h at 4 C. After centrifugation at 3750 rpm for 30 min at 4 C, the
supernatant was discarded and the radioactivity in the pellets measured
in a 1277 Gammamaster instrument (Wallac, Gaithersburg, MD).
The radioactive counts were compared with a standard curve and the concentration of AM calculated by linear regression. Recovery in the assay was average (66%) and the variation between assays was less than 10%. The displacement of tracer obtained by increasing volumes of serum extracts was parallel with that observed with the standard curve.
Receptor-binding assay
Receptor binding was performed as previously described (10).
Briefly, cells (5 x 104) were placed in 24-well
plates coated with fibronectin (20 µg/well). When a monolayer was
formed, the cells were washed 3 times in salinium, insulin, and
transferrin (SIT) medium (DMEM containing 3 x 10-8
M Se2O3, 5 µg/ml insulin, and 10
µg/ml transferrin), followed by incubation with receptor-binding
medium (SIT plus 1% BSA and 1 mg/ml bacitracin) with 0.2
nM 125I-AM (2200 Ci/mmol, Phoenix) in the
presence or absence of competitor. After 2 h at 4 C, free peptide
was removed by washing 3 times in receptor-binding medium. Peptide
bound to the cells was solubilized in 0.2 N NaOH and counted in a
-counter. Peptides used as competitors include: PAMP, AM 22–52,
CGRP, CGRP 8–37, and amylin amide (Peninsula, Belmont, CA). These
peptides were added at 1 µM concentration.
Receptor quantification
The binding of 125I-AM was investigated as a
function of radiolabeled peptide concentration. CRL-7922 cells
(0.6 x 106) in 24-well plates were incubated with
increasing concentrations of 125I-AM, and the amount bound
was determined in the absence or presence of 1 µM AM. The
difference between the two represents specific binding, B. The amount
of specifically bound AM (B) was divided by the free radiolabeled
peptide (F) and presented as a Scatchard plot. The data were best fit
with a straight line, indicating a single class of sites.
3H-thymidine uptake assay
Confluent skin cells were incubated with SIT medium containing
0.5% FBS. AM was added at different concentrations together with
3H-deoxyribose-thymidine (106 cpm). After
16 h, the cells were rinsed twice with PBS; then 5%
trichloroacetic acid was added for 5 min at 4 C. The trichloroacetic
acid-insoluble material was washed with ethanol-ether (2:1, vol/vol)
and the precipitate dried at 25 C. The cells were extracted with 0.2 N
NaOH; followed by 0.2 M HCl. The contents were combined,
scintillation fluid (Aquasol, Packard Instrument Co., Meridan, CT)
added, and the samples counted in a ß counter.
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Results |
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). In the epidermis, the keratinocytes exhibited a
strong immunocytochemical granular pattern in the cytoplasm, with the
cells situated in the stratum basale and stratum spinosum being more
immunoreactive than the ones in the stratum granulosum. The stratum
corneum was devoid of immunoreactivity (Fig. 1B). Preabsorption of the antibody with
its antigen totally precluded immunostaining, demonstrating the
specificity of the reaction (Fig. 1C). mRNA for the receptor was found
in all living cells of the epidermis (Fig. 1D).
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). The sebaceous glands
of the pilosebaceous unit revealed stronger immunoreactivity than that
detected in the keratinocytes. The cells in the periphery of the gland
that are actively involved in cell regeneration showed a more intense
positivity to the antibody than the central, more differentiated cells,
which synthesize the lipids (Fig. 3). The
sweat glands of the dermis also were immunoreactive in all their
length, including both the ducts and the secretory portions, and
although the eccrine variety was homogeneously positive for AM (Fig. 4), the apocrine glands were more
strongly stained (Fig. 5). Normal skin
cells in culture retained immunoreactivity for AM (Fig. 6).
AM and its receptor in skin tumors
Because previous work from our group had shown that AM was
expressed in a variety of human epithelial cancers and that it
functioned as an autocrine growth factor (10), we evaluated different
skin malignancies to ascertain whether a similar relationship was in
place.
Thirty-four specimens corresponding to the major histological types of
skin cancer were studied by immunocytochemistry and in situ
hybridization. All of them showed a positive staining for both AM and
AM-R (see
Figs. 7–11), although some differences were observed:
squamous cell carcinomas showed a homogeneous pattern of staining (Fig. 7), whereas basal cell carcinomas were
especially positive at the peripheral palisading layer (Fig. 8). Melanomas, on the other hand, showed
a patchy staining with intensely positive cells among moderately
stained and negative cells (Fig. 9).
Interestingly, plasma cells in the perineoplastic inflammatory
infiltrate surrounding melanomas were intensely immunoreactive for AM
(Fig. 10). In general, melanomas showed
a higher degree of heterogeneity in AM expression than other skin
cancers. For instance, some metastatic melanomas showed strong
expression of AM at the periphery of the metastasis, whereas the
majority of melanoma cells at the center showed little or no
expression. We also studied some cases of keratoacanthoma and observed
the same distribution pattern described for normal skin, although in
these cases, the epidermis was more convoluted (Fig. 11).
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, A and B). A major band of 291 bp
was obtained for AM. Sequence analysis of this band demonstrated that
it was true hAM. An additional band of 524 bp was seen on all
specimens. This band has been previously reported (10, 18) and may
represent an alternatively spliced product of the AM gene. A single
band of 471 bp was obtained for the receptor (Fig. 12B).
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). Three
major immunoreactive bands of about 18, 14, and 6 kDa, respectively,
were found in the 4 cell lines. These 3 bands have been reported
previously in other cell line extracts (10, 17, 22) and may correspond
to the preprohormone (18 kDa), an intermediate product (14 kDa), and
the final mature peptide (6 kDa) (which migrates to the same position
as the synthetic peptide control). Preabsorption of the antibody with
the antigen quenched immunoreactivity (Fig. 12D).
Detection of AM in sweat
The presence of AM immunoreactivity in the sweat glands (Figs. 4 and 5) suggested that the peptide may be secreted into the sweat, and
to test this hypothesis, we performed RIA in sweat samples and compared
the values obtained with AM levels in blood serum (Fig. 13). Surprisingly, the values obtained
for AM in the sweat were very variable (87.93 ± 88.48 fmol/ml)
but, in general, were much higher than the values obtained in the blood
samples (16.83 ± 2.52 fmol/ml). These data confirm that AM is
secreted into the sweat in large amounts. The variation in AM levels
may reflect differences in exertion or in sweat secretion rates.
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). This binding was displaced by cold AM
but not by PAMP, AM 22–52, CGRP, CGRP 8–37, or amylin amide (Table 2), indicating that an AM-specific
receptor was present in these cell lines. Scatchard analysis on cell
line CRL-7922 (Fig. 14) demonstrated
that there is a single class of binding sites and that the number of
receptors per cell is approximately 14,000 with a Kd of 9
nM.
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Discussion |
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Both AM and the receptor were localized in the same cell types of healthy and malignant skin. This fact has been reported previously in other settings, including rodent development (17), breast tissue (22), and tumor cell lines of other origins (10). This frequent colocalization of the regulatory peptide and its receptor points to an autocrine and/or paracrine mechanism in the physiology of this system.
The role of AM in the skin could be related to different physiological functions already described for this peptide. For example, AM has been recently demonstrated to be involved in the defense of epithelial surfaces against microorganisms (14). Given that the skin is a critical protective interphase between external and internal environments, it is very plausible that the epidermis would express chemical substances to deter microorganism penetration, in addition to the physical barrier provided by keratin. This concept is well established in the skin of amphibians, where a large number of antimicrobial peptides has been found (23, 24). The amphibian skin needs to be very thin to allow gas exchanges and, as a consequence, possibly needs a larger battery of these defensive peptides than the mammalian skin. Nevertheless, our study shows the human skin as a rich source of this defensin-like peptide (14). To our knowledge, no antimicrobial peptides have been located previously in the skin of mammals (for a review, see Ref.15), but some of them have been found in similar epithelial surfaces; for instance, the lingual antimicrobial peptide has been demonstrated in the tongue epithelium (25).
The other function in which AM may be implicated in skin physiology is growth regulation. The skin is one of the organs in which a continuous renewal is needed, because of the exposure to the external milieu. The stratum basale of the epidermis is in constant proliferation, to replace the cells that are lost in the outer layers. The hair follicles incessantly produce more keratin for hair growth, and continuous replacement of gland cells is needed, in particular, the sebaceous gland cells, with their holocrine mechanism of secretion. If we add the frequent wounds inflicted upon the skin, it becomes clear that this is an organ in which proliferation is ubiquitous and where it has to be very tightly regulated. It has been demonstrated previously that AM increases growth in tumor cells (10) and in fibroblasts (11), albeit in other systems, it may be a growth deterrent (12, 13). In this study, we demonstrated that AM, acting through specific receptors, increases the incorporation of thymidine in skin cells, therefore driving cell proliferation. Considering that the receptor for AM has been located in the epidermis, the hair follicles, and the skin glands, all these structures may be growth-regulated by AM. In addition, all skin tumors expressed both AM and AM-R; therefore, they have a potential autocrine system capable of driving proliferation, which may be decisive in the initiation and subsequent progression of carcinogenesis. A deeper knowledge of the implications of the AM/AM-R system in skin proliferation may offer new opportunities to regulate wound repair, hair growth, and tumor progression.
In conclusion, we have shown that AM and its receptor are a main component of the skin epithelium in both normal and malignant conditions and that AM is present in the sweat. In addition, AM was shown to induce proliferation of a skin cell line upon binding to its AM-specific receptor. Although the physiological implications have to be investigated further, using the appropriate model systems, AM seems to play an important role in skin homeostasis and carcinogenesis.
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Note Added in Proof |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Received July 1, 1997.
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