Involvement of human beta-defensin-2 in intracellular signaling: in vitro study
Aim: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. Materials and Methods: A431cells were cultured in the presence of 1 µg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PC...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2006
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| Цитувати: | Involvement of human beta-defensin-2 in intracellular signaling: in vitro study / D. Lytvyn, E. Zhuravel, N.V. Markeeva, M. Soldatkina, T. Dudchenko, P.V. Pogrebnoy // Experimental Oncology. — 2006. — Т. 28, № 3. — С. 198-202. — Бібліогр.: 25 назв. — англ. |
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irk-123456789-1375762018-06-18T03:06:27Z Involvement of human beta-defensin-2 in intracellular signaling: in vitro study Lytvyn, D. Zhuravel, E. Markeeva, N.V. Soldatkina, M. Dudchenko, T. Pogrebnoy, P.V. Original contributions Aim: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. Materials and Methods: A431cells were cultured in the presence of 1 µg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. Results: 30 min incubation of A431 cells with 1 µg/ml of hBD-2 didn’t influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment – in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment – in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro. Conclusion: Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells. Цель: проанализировать участие бета-дефенсина-2 человека (hBD-2) в механизмах передачи внутриклеточных сигналов в модели in vitro. Материалы и методы: клетки линии A431 культивировали в присутствии 1 µг/мл рекомбинантного hBD-2 и/или 10 нг/мл ЭФР. Экспрессию мРНК альфа- и бета-изоформ p70S6 киназы оценивали методом полуколичественного ОТ-ПЦР анализа. Экспрессию и активность p70S6K, фосфорилирование PDK1, ERK, JNK, p38 киназ и рецептора ЭФР (ЭФРР) исследовали методом Вестерн-блот анализа. Результаты: 30 мин инкубация клеток A431 с 1 µг/мл hBD-2 не оказывала влияния на уровень аутофосфорилирования ЭФРР, но препятствовала образованию димеров рецептора в присутствии ЭФР. В то же время 30 мин обработка клеток hBD-2 приводила к активации p70S6K, 12 ч — к значительному повышению уровня мРНК альфа- и бета-изоформ p70S6 киназы, а 24 ч — к повышению синтеза p70S6K на уровне белка. PDK1-киназа, фосфорилирующая p70S6K, также подвергалась фосфорилированию в присутствии экзогенного hBD-2 in vitro. Выводы: данные свидетельствуют об участии каскада PDK1-p70S6K в опосредовании действия hBD-2 в клетках A431. 2006 Article Involvement of human beta-defensin-2 in intracellular signaling: in vitro study / D. Lytvyn, E. Zhuravel, N.V. Markeeva, M. Soldatkina, T. Dudchenko, P.V. Pogrebnoy // Experimental Oncology. — 2006. — Т. 28, № 3. — С. 198-202. — Бібліогр.: 25 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/137576 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
| topic |
Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Lytvyn, D. Zhuravel, E. Markeeva, N.V. Soldatkina, M. Dudchenko, T. Pogrebnoy, P.V. Involvement of human beta-defensin-2 in intracellular signaling: in vitro study Experimental Oncology |
| description |
Aim: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. Materials and Methods: A431cells were cultured in the presence of 1 µg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. Results: 30 min incubation of A431 cells with 1 µg/ml of hBD-2 didn’t influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment – in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment – in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro. Conclusion: Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells. |
| format |
Article |
| author |
Lytvyn, D. Zhuravel, E. Markeeva, N.V. Soldatkina, M. Dudchenko, T. Pogrebnoy, P.V. |
| author_facet |
Lytvyn, D. Zhuravel, E. Markeeva, N.V. Soldatkina, M. Dudchenko, T. Pogrebnoy, P.V. |
| author_sort |
Lytvyn, D. |
| title |
Involvement of human beta-defensin-2 in intracellular signaling: in vitro study |
| title_short |
Involvement of human beta-defensin-2 in intracellular signaling: in vitro study |
| title_full |
Involvement of human beta-defensin-2 in intracellular signaling: in vitro study |
| title_fullStr |
Involvement of human beta-defensin-2 in intracellular signaling: in vitro study |
| title_full_unstemmed |
Involvement of human beta-defensin-2 in intracellular signaling: in vitro study |
| title_sort |
involvement of human beta-defensin-2 in intracellular signaling: in vitro study |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2006 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/137576 |
| citation_txt |
Involvement of human beta-defensin-2 in intracellular signaling: in vitro study / D. Lytvyn,
E. Zhuravel, N.V. Markeeva, M. Soldatkina, T. Dudchenko, P.V. Pogrebnoy // Experimental Oncology. — 2006. — Т. 28, № 3. — С. 198-202. — Бібліогр.: 25 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T02:36:58Z |
| last_indexed |
2025-07-10T02:36:58Z |
| _version_ |
1837225761281409024 |
| fulltext |
198 Experimental Oncology 28, 198–202, 2006 (September)
Human defensins — small antimicrobial pep‑
tides — are in the focus of numerous researches aimed
on evaluation of their structure, biological functions
and patterns of expression in vitro and in vivo in normal
state and upon pathology [1, 2]. Such interest to these
biomolecules is due to recently established involve‑
ment of defensins in a number of processes important
for cell behavior — proliferation, differentiation, growth
and death [3–6]. On multicellular level, these antimi‑
crobials are taking part in the protection of host from
invading pathogens, functioning of immune system,
tissue repair and tumorigenesis [1, 2, 5, 7–13].
It is established also that in in vitro models produc‑
tion of defensin and/or exogenous addition of defen‑
sins to the cells results in elevation of their proliferation
rate [3, 4, 11]. At the same time the mechanisms me‑
diating defensin‑dependent cell proliferation remains
poorly studied yet as well as potential intracellular
targets of these antimicrobials.
Up to date the data on involvement of defensins in
intracellular signaling are scarce. One may speculate
that unique molecular properties of defensins (am‑
phipatic chain, high cationic charge, low molecular
weight) favor their interactions with subcellular com‑
ponents, plasma membranes, as well as with discrete
membrane‑bound and cytoplasmic proteins — com‑
ponents of intracellular signaling pathways. In few
publications the role of defensins in activation of intra‑
cellular signaling cascades in vitro has been analyzed.
It has been reported that alpha‑defensin may interact
with LRP receptor expressed in smooth muscle cells
(SMC), undergo internalization and specifically inhibit
protein kinase C [14]. In another research it was dem‑
onstrated that treatment of airway SMC with HNP1–3
leads to phosphorylation of extracellular signal‑regu‑
lated kinase (ERK) 1/2 [15]. In human keratinocytes,
hBD‑2, ‑3, and ‑4‑dependent secretion of IL‑18 was
shown to be mediated by p38 and ERK1/2 MAPK
pathways [16]. Human cathelicidin LL‑37 was shown to
activate MAPK/ERK pathway in airway epithelial cells
and transactivate EGFR via metalloproteinase‑medi‑
ated cleavage of membrane‑anchored EGFR‑ligands
[17]. In airway epithelial cells treatment with HNP1–3
caused activation of ERK1/2, and the process of
HNP‑dependent wound repair seems to be dependent
on EGFR activation [9].
Our previous studies have shown increase of cell
proliferation in vitro upon treatment of A431 cells with
exogenous hBD‑2 [4]. The present research was
aimed on identification of possible molecular targets of
hBD‑2 in A431 cells that could be involved in mediation
of its pro‑proliferative action.
Materials and Methods
Cell cultures and cultivation. A431 (human vulval
epidermoid carcinoma) cells were received from the
Institute of Molecular Biology of Russian Academy of
Sciences (Moscow, Russia).
A431 cells were cultivated in 6–well plates in
DMEM culture medium supplemented with 5% FBS
(Gibco BRL, UK) in atmosphere of 5%CO2 at 37 °C
until cells reached 70% confluence; then the medium
was replaced with DMEM without FBS and cultivation
continued for 24 h. Then the cells (5 х 105 cells per well)
were incubated with EGF (10 ng/ml) and/or recombi‑
nant hBD‑2 (1 µg/ml) [18] for 30 min, 12 h, and 24 h
for kinase assays, RT‑PCR analysis of expression of
p70S6K on mRNA and protein levels, respectively.
RT‑PCR‑analysis. For detection of expression
of p70S6Kα and p70S6Kβ semiquantative RT‑PCR
analysis was applied as described in [19]. The next
involveMent of huMan beta-defensin-2
in intracellular signaling: in viTRo study
D. Lytvyn, E. Zhuravel, N. Markeeva, M. Soldatkina, T. Dudchenko, P. Pogrebnoy*
R.E. Kavetsky Institute of Experimental Pathology,
Oncology and Radiobiology NAS of Ukraine, Kyiv, Ukraine
Aim: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. Materials and Methods: A431cells
were cultured in the presence of 1 µg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs
for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of
PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. Results: 30 min incubation
of A431 cells with 1 µg/ml of hBD-2 didn’t influence autophosphorylation level of EGFR, but resulted in activation of p70S6K,
12 h treatment – in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment – in
elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon
influence of exogenous hBD-2 in vitro. Conclusion: Our data point on the involvement of PDK1-p70S6K pathway in mediation of
action of hBD-2 in A431 cells.
Key Words: human beta-defensin-2, EGF, EGF receptor, p70S6 kinase, PDK1.
Received: May 5, 2006.
*Correspondence: E-mail: pogrebnoy@onconet.kiev.ua
Abbreviations used: EGF – epidermal growth factor;
EGFR – receptor of epidermal growth factor; hBD-2 — human
beta-defensin-2; PDK1 – phosphoinositide-dependent kinase 1;
RT-PCR – reverse transcription polymerase chain reaction.
Exp Oncol 2006
28, 3, 198–202
original contributions
Experimental Oncology 28, 198–202, 2006 (September) 199
primers were used: direct ones — 5`‑ttggggcattta‑
catcaaaaggg – for p70S6Kα and 5`‑ggatcgccgccctt‑
taccgca — for p70S6Kβ; reverse primer 5`‑cccag(a/g)
aag(a/g)cctg(a/g)ttggcact — for both genes.
30 cycles of amplification at regimen (94 °С – 20 s,
64 °С — 30 s, 72 °С — 45 s) were performed. Expres‑
sion of glyceraldehyde‑3‑phosphate dehydrogenase
(G3PDH) (direct primer — 5′‑tgaaggtcggagtcaacg‑
gatttggt, reverse primer — 5′‑catgtgggccatgaggtc‑
caccac) served as the control (25 cycles at the regimen
95 °С — 20 s, 64 °С — 30 s, 72 °С — 45 s). The products
of RT‑PCR were routinely analyzed by electrophoresis
in agarose gel.
Western blot analysis, autophosphorylation of
EGF‑receptor, kinase assays and cross‑linking
technique. Western‑blot analysis was performed by
standard procedure with the use of the next antibod‑
ies: MoAbs against full size molecule of p70S6Kα [20],
polyclonal antibodies against C‑terminal fragment of
p70S6Kβ (Institute of Molecular Biology and Genetics,
NAS of Ukraine, Kyiv, Ukraine), MoAbs against phos‑
phorylated thyrosine (PY20, Santa Cruz Biotechnology
INC, USA), MoAbs against EGFR (Sigma, USA), phos‑
pho‑PDK1 mAbs (Ser241), phospho‑ERK1/2 (Thr202/
Tyr204), phospho‑p38 MARK (Thr180/Tyr182), phos‑
pho‑MARK/JNK (Thr183/Tyr185) (CellSignaling Tech,
USA), and polyclonal antibodies against ribosomal S6
protein phosphorylated at the position Ser235/236
(CellSignaling Tech, USA). The data of Western‑blot
analysis were analyzed by densitometry using Total‑
Lab v1.10 programme (the results were recalculated
by loading control values). Each experiment was re‑
peated in triplicate.
Kinase assay was performed as follow: A431 cells
were cultivated in 6–well plates (5 х 105 cells per well)
for 24 h in DMEM without FBS; then cells were washed
with serum‑free medium and incubated in HEPES‑PBS
buffer with 10 ng/ml EGF and/or 1 µg/ml hBD‑2 for
30 min. Then buffer was aspirated and the cells were
immediately lysed by hot Laemmly sample buffer.
Then the samples were subjected to 10 or 7–22%
SDS‑PAGE gradient electrophoresis and Western‑blot
analysis was carried out by standard schedule using
abovementioned antibodies and developed using ECL
kit (Amersham, UK).
For cross‑linking experiment for evaluation of
formation of EGFR dimers, after incubation of cells
with hBD‑2 the medium was removed and replaced
with BS3‑containing buffer (Pierce, USA) for 30 min
at 37 °C according to the instructions of manufacturer.
Then 100 ml of the sample buffer were added, and the
proteins were analyzed by Western blot analysis.
results
influence hBD‑2 on activation of EGFR and
down‑stream kinases. The first task of the present
research was to analyze possible influence of hBD‑2 on
activation of EGFR receptor in vitro. For that purpose
we have analyzed the autophosphorylation level of EGF
receptor in A431 cells treated with recombinant hBD‑2
at the concentration causing maximal increase of cell
proliferation in vitro [4] and compared the content of
monomers and dimers of EGF receptor in A431 cells
upon influence of 1 µg/ml hBD‑2 and/or 10 ng/ml
EGF (used as positive control). Our data have shown
(Fig. 1) that 30 min treatment of A431 cells with hBD‑2
did not influence autophosphorylation level of EGFR,
but decreased the level of formation of EGFR dimers
in the presence of EGF, in particular if 15 min pretreat‑
ment with hBD‑2 was applied prior to addition of EGF
(Fig. 1, line 5).
As we have reported earlier [21], the treatment of
A431 cells with hBD‑2 does not cause activation of
EGFR‑dependent downstream cascade, typical for
classic EGFR ligand. However, in A431 cells incubated
with hBD‑2 for 30 min, a band of 3–4 phosphorylated
proteins with molecular weights of 40–60 kDa appears,
and accumulation of these products lasts till 24 h of
incubation. Using anti‑phospho‑ERK1/2, ‑MEK1/2,
‑JNK1/2, and ‑р38 mAbs, we have estimated that
incubation of A431 cells with hBD‑2 didn’t influence
phosphorylation of р38 and JNK kinases (data not
shown), but resulted in phosphorylation of MEK1/2
and ERK1/2 kinases (Fig. 2).
influence hBD‑2 on activation of PDK1‑p70S6K
pathway. We have find out that 12 h incubation of
A431 cells with 1 µg/ml hBD‑2 resulted in a marked
fig. 1. Western blot analysis of autophosphorylation of EGF
receptor and EGFR‑dimer formation in A431 incubated with
EGF and/or hBD‑2 (A, pY20 MoAbs). Expression of EGFR (B,
anti‑EGFR‑MoAbs) served as loading control. A431 cells (2 x
105/well, 24‑well plate) were cultured 24 h without FBS, then EGF
(10 ng/ml) and/or hBD‑2 (1 µg/ml) were added for 15–30 min. In
case (a) cells were treated with BS3 crosslinker. Lines: 1 — control
cells; 2 — + EGF, 30 min; 3 — + EGF for 15 min, then + hBD‑2 for
15 min; 4 ‑+ hBD‑2 for 30 min; 5 ‑+ hBD‑2 for 15 min, then + EGF
for 15 min; 6 — EGF + hBD‑2 simultaneously for 30 min
fig 2. Western blot analysis of phosphorylation of MEK1/2 (a)
and ERK1/2 (b) kinases in A431 cells incubated for 30 min with
1 µg/ml hBD‑2 (line 2), 10 ng/ml EGF (line 3) or hBD2+EGF (line
4). Line 1 — control A431 cells without inductors. β‑actin expres‑
sion (anti‑β‑actin‑MoAbs (Sigma, USA) (c) served as control
200 Experimental Oncology 28, 198–202, 2006 (September)
(nearly 2‑fold) increase of expression of mRNAs for
alpha‑ and beta‑isoforms of p70S6 kinase that was
close to that upon treatment of cells with 10 ng/ml
EGF (Fig. 3). Western blot analysis of expression of
p70S6K isoforms in A431 cells after 24 h incubation
of the cells with 1 µg/ml hBD‑2 has demonstrated
the similar pattern — defensin strongly influenced
production of alpha‑ and beta‑isoforms of p70S6 in
A431 cells (by 3‑fold and 2‑fold, respectively) and
elevation of the activity of the p70S6 kinase (Fig. 4,
Fig. 5, a). Moreover, PDK1 — the up‑stream kinase
phosphorylating p70S6K – is also found to be nearly
2‑fold higher phosphorylated in A431 cells incubated
with 1 µg/ml hBD‑2 for 30 min compared to that in
control cells (Fig. 5).
discussion
In the present research we aimed to evaluate pos‑
sible involvement of EGFR in hBD‑2 dependent cell sig‑
naling. Our previous data [4] have demonstrated that
in micromolar concentration range exogenous hBD‑2
promotes proliferation of cultured cells in vitro and
the rate of hBD‑2–dependent proliferation correlates
with the content of EGFR in given cells. Moreover, we
have observed that simultaneous stimulation of A431
cells with EGF and hBD‑2 resulted in the decrease of
incorporation of [3H]‑thymidine compared with that
for cells treated separately with hBD‑2 or EGF (un‑
published data). That’s why we tried to analyze the
autophosphorylation level of EGF receptor in A431 cells
treated with micromolar concentration of hBD‑2 and to
evaluate the content of monomers and dimers of EGF
receptor in A431 cells upon influence of hBD‑2 and/or
EGF. Our data have shown that short‑term treatment
of A431 cells with hBD‑2 did not influence the level of
autophosphorylation of EGFR, but seems too decrease
the level of formation of EGFR dimers in the presence
of EGF. We hypothesize that there could be at least two
possible explanations of such phenomenon: 1) hBD‑2
may penetrate/intercalate cell membrane due to well‑
fig. 3. RT‑PCR analysis of expression of mRNA for p70S6 kinase, isoform alpha (a) and p70S6 kinase, isoform beta (b) in A431
cells. G3PDH expression (c) served as control. А431 cells were grown in 6‑well plates (5 x 105 cells per well) till 70% monolayer,
than the medium was replaced by serum‑free one for 24 h. Then cells were treated with 10 ng/ml) EGF and/or 1 µg/ml hBD‑2 for
12 h. Lanes: 1 — control; 2 — + EGF; 3 ‑+ FBS; 4 — EGF + hBD‑2; 5 — hBD‑2; M — DNA 1 kb ladder
fig. 4. Western blot analysis of expression of p70S6 kinase, isoform alpha (a), p70S6 kinase, isoform beta (b). β‑actin expres‑
sion (anti‑β‑actin‑MoAbs (Sigma, USA) (c) served as control. А431 cells were grown in 6‑well plates (5 x 105 cells per well) till 70%
monolayer, than the medium was replaced by serum‑free one for 24 h. Then the cells were treated with 10 ng/ml EGF and/or 1 µg/ml
hBD‑2 for 24 h. Lanes: 1 — control; 2 — + EGF; 3 ‑+ FBS; 4 — EGF + hBD‑2; 5 — hBD‑2
fig. 5. Western blot analysis of phosphorylation level of ribosomal S6 protein (Ser235/236) (a) and PDK1 (Ser241) (b) in A431 cells.
β‑actin expression (anti‑β‑actin‑MoAbs (Sigma, USA)) (c) served as control. А431 cells were grown in 6‑well plates (5 x 105 cells per
well) till 70% monolayer, than the medium was replaced by serum‑free one for 24 h. Then the cells were treated with 10 ng/ml EGF
and/or 1 µg/ml hBD‑2 for 30 min. Lanes (a and c): 1 — control; 2 — + EGF; 3 ‑+ FBS; 4 — EGF + hBD‑2; 5 — hBD‑2; b: 1 — control;
2 — + EGF; 3 — hBD‑2 (15 min) + EGF for 15 min; 4 — EGF (15 min) + hBD‑2 for 15 min; 5 — hBD‑2
Experimental Oncology 28, 198–202, 2006 (September) 201
known membrane‑permebealizing properties of anti‑
microbial pepties thus preventing dimer’s formation;
2) hBD‑2 may non‑specifically bind to EGF‑binding
site of EGF receptor due to its steric homology with
EGF molecule [22], however, without further dimer‑
ization/activation of receptor. Our data have shown
that action of hBD‑2 in vitro is different from that of
HNP‑1 on airway epithelial cells [17], where transient
activation of EGFR realized via activation of metallopro‑
teinase‑mediated cleavage of membrane‑anchored
EGFR‑ligands has been observed.
Taking into account the data of other authors [15,
16] on possible involvement of MAP‑kinase pathway,
in particular ERK1/2 kinase, in mediation of action of
defensins in vitro, we carried out the research of the
phosphorylation state of some kinases of MAP‑kinase
pathway in A431 cells treated with hBD‑2 and have shown
hBD‑2‑dependent activation of MEK1/2 and ERK1/2 ki‑
nases. Mentioned kinases are evolutionary conservative
proteins that link surface cell receptors with key regula‑
tory molecules and control cell survival and adaptation.
It is known that phosphorylation of MEK1/2 and ERK1/2
leads to activation of different transcriptional factors (in
particular АР‑1 and NF‑kB), and initiation of prolifera‑
tion, growth and differentiation of cells [23, 24]. Our data
points on involvement of MEK1/2 and ERK1/2 in media‑
tion of action of hBD‑2 in A431 cells. Unfortunately, we
are unable yet to determine up‑stream kinases mediating
hBD‑2‑dependent phosphorylation of ERK 1/2.
Mitogenic properties of hBD‑2 make it reasonable
to research its influence on expression patterns and
activity of p70S6 kinase — key enzyme of the system
of protein biosynthesis, that is hyperactivated upon
mitogenic influence of growth factors in vitro. We have
find out that incubation of A431 cells with micromolar
concentrations of hBD‑2 resulted in a marked increase
of expression of mRNAs and respective protein prod‑
ucts of alpha‑ and beta‑isoforms of p70S6 kinase as
well as activity of the kinase. Such up‑regulation of
p70S6 kinase expression and activity may be in part
explained by the detected defensin‑dependent el‑
evation of phosphorylation of PDK1 — the up‑stream
kinase that phosphorylates p70S6K [25].
As far as we know it is a first report on involve‑
ment of defensins in regulation of enzymes control‑
ling protein biosynthesis. PDK1 is playing a central
role as activator of multiple signaling pathways, that
may be affected upon carcinogenesis, in particular
protein synthesis. The obtained data do not allow us
yet to propose the exact mechanism of interaction
between hBD‑2 and PDK1‑signaling cascade. How‑
ever, one may speculate that such effect possibly may
relay in direct interaction between plasma‑membrane
bound PDK1 and defensin — according to data‑base
(www.scansite.mit.edu) there is extremely high affinity
between hBD‑2 and PDK1 molecules. Our preliminary
data based on pull‑down technique for PDK‑1‑hBD‑2
(data not shown) also point on the possibility of such
interactions. We hope that new studies with the use
of recombinant defensins will put new insight on the
role of these multifunctional antibiotics in cell signal‑
ing and behavior.
acKnoWledgeMent
This research was in part supported by INTAS grant
No: 05‑1000004‑7755. Authors wish to thank Dr. Svet‑
lana Mikhalap (IEPOR NASU, Kyiv, Ukraine) for brilliant
advices in the performing kinase assays.
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участие бета-дефенсина-2 человека в передаче
внутриклеточных сигналов in viTRo
Цель: проанализировать участие бета-дефенсина-2 человека (hBD-2) в механизмах передачи внутриклеточных сигналов
в модели in vitro. Материалы и методы: клетки линии A431 культивировали в присутствии 1 µг/мл рекомбинантного hBD-2
и/или 10 нг/мл ЭФР. Экспрессию мРНК альфа- и бета-изоформ p70S6 киназы оценивали методом полуколичественного
ОТ-ПЦР анализа. Экспрессию и активность p70S6K, фосфорилирование PDK1, ERK, JNK, p38 киназ и рецептора
ЭФР (ЭФРР) исследовали методом Вестерн-блот анализа. Результаты: 30 мин инкубация клеток A431 с 1 µг/мл hBD-2
не оказывала влияния на уровень аутофосфорилирования ЭФРР, но препятствовала образованию димеров рецептора
в присутствии ЭФР. В то же время 30 мин обработка клеток hBD-2 приводила к активации p70S6K, 12 ч — к значительному
повышению уровня мРНК альфа- и бета-изоформ p70S6 киназы, а 24 ч — к повышению синтеза p70S6K на уровне белка.
PDK1-киназа, фосфорилирующая p70S6K, также подвергалась фосфорилированию в присутствии экзогенного hBD-2 in vitro.
Выводы: данные свидетельствуют об участии каскада PDK1-p70S6K в опосредовании действия hBD-2 в клетках A431.
Ключевые слова: бета-дефенсин-2 человека, ЭФР, рецептор ЭФР, p70S6 киназа, PDK1.
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