Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome
Aim. To determine 293 and U373 cell response and ERK1/2 activation profile after CHI3L1 or CHI3L2 treatment. Methods. Specific activation and localization of ERK1/2 kinases after CHI3L1 or CHI3L2 addition to unsupplemented cell medium were evaluated by Western blots, immunofluorescence and confocal...
Gespeichert in:
| Datum: | 2011 |
|---|---|
| Hauptverfasser: | , , , |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут молекулярної біології і генетики НАН України
2011
|
| Schriftenreihe: | Вiopolymers and Cell |
| Online Zugang: | https://nasplib.isofts.kiev.ua/handle/123456789/155649 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome / P.O. Areshkov, S.S. Avdieiev, A.V. Iershov, V.M. Kavsan // Вiopolymers and Cell. — 2011. — Т. 27, № 5. — С. 343-346. — Бібліогр.: 12 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-155649 |
|---|---|
| record_format |
dspace |
| spelling |
nasplib_isofts_kiev_ua-123456789-1556492025-02-09T21:10:24Z Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome Стимуляція короткотривалого vs довготривалого фосфорилювання кіназ ERK1/2 додаванням двох близькоспоріднених білків CHI3L1 і CHI3L2 корелює зі зміною локалізації відповідних кіназ та біологічною відповіддю активованих клітин Стимуляция кратковременного vs длительного фосфорилирования киназ ERK1/2 добавлением двух близкородственных белков CHI3L1 и CHI3L2 коррелирует с изменением локализации соответствующих киназ и биологическим ответом активированных клеток Areshkov, P.O. Avdieiev, S.S. Iershov, A.V. Kavsan, V.M. Aim. To determine 293 and U373 cell response and ERK1/2 activation profile after CHI3L1 or CHI3L2 treatment. Methods. Specific activation and localization of ERK1/2 kinases after CHI3L1 or CHI3L2 addition to unsupplemented cell medium were evaluated by Western blots, immunofluorescence and confocal microscopy. To determine whether CHI3L1 or CHI3L2 can enhance mitogenesis, [3H]thymidine incorporation in cellular DNA was measured. Results. The obtained results show that ERK1/2 phosphorylation was stimulated in both cell types (293 and U373 cells) following addition of CHI3L2 or CHI3L1 in dose- and time-dependent manner. Unexpectedly, in opposite to CHI3L1, the dose-dependent decreasing of measured mitogenesis parameters was observed in 293 and U373 cells. In both cell types the treatment with CHI3L2 gave more sustained than CHI3L1 MAPK-pathway activation with prolonged phospho-ERK1/2 nuclear accumulation in 293 cells. Conclusions. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 cell treatment, that leads to a proliferative signal, the activation of these kinases by CHI3L2 addition inhibits cell mitogenesis and proliferation in serum starved 293 and U373 cells. Keywords: chitinase 3-like 1 protein (CHI3L1), chitinase 3-like 2 protein (CHI3L2), MAP kinase, mitogenesis. Мета. Визначити клітинну відповідь та встановити профіль активації кіназ ERK1/2 після обробки клітин 293 та U373 білками CHI3L1 чи CHI3L2. Методи. Специфічну активацію і локалізацію кіназ ERK1/2 після внесення білків CHI3L1 або CHI3L2 до клітинного середовища, що не містить сироватки, проаналізовано Вестерн-блот аналізо та методами імунофлуоресценції і конфокальної мікроскопії. Для встановлення, чи зможе додавання білків CHI3L1 або CHI3L2 посилювати мітогенез, застосовано аналіз інкорпорації [3H]тимідину в клітинну ДНК. Результати. Отримані результати демонструють, що стимуляція фосфорилювання кіназ ERK1/2 після додавання CHI3L1 або CHI3L2 має дозо- та часозалежний характер. Виявилося, що, на відміну від CHI3L1, дозозалежне пригнічення інкорпорації [3H]тимідину констатовано у 293 та U373 клітинах. В обох типах клітин обробка білком CHI3L2 викликає тривалішу активацію каскаду МАРК, ніж обробка білком CHI3L1, з акумулюванням кіназ ERK1/2 у ядрі клітин 293. Висновки. На противагу активації фосфорилювання кіназ ERK1/2 внаслідок обробки клітин білком CHI3L1, що спричиняє проліферацію, стимуляція цих кіназ додаванням білка CHI3L2 пригнічує мітогенез у клітинах 293 та U373, що перебувають у стані сироваткового голодування. Ключові слова: хітиназа 3-подібний білок 1 (CHI3L1), хітиназа 3-подібний білок 2 (CHI3L2), MAP-кіназа, мітогенез. Цель. Определить клеточный ответ и профиль активации киназ ERK1/2 после обработки клеток 293 и U373 белками CHI3L1 или CHI3L2. Методы. Специфическую активацию и локализацию киназ ERK1/2 после внесения белков CHI3L1 или CHI3L2 в клеточную среду, где отсутствует сыворотка, проанализировано методами Вестерн-блот анализа, иммунофлуоресценции и конфокальной микроскопии. Для определения, будет ли добавление белков CHI3L1 или CHI3L2 увеличивать митогенез, использован анализ инкорпорации [3H]тимидина в клеточную ДНК. Результаты. Полученные результаты демонстрируют, что стимуляция фосфорилирования киназ ERK1/2 после добавления CHI3L1 либо CHI3L2 имеет зависящий от дозы и времени характер. Оказалось, что, в отличие от CHI3L1, дозозависимое угнетение инкорпорации [3H] тимидина констатировано в 293- и U373-клетках. В обоих типах клеток обработка белком CHI3L2 вызывает более длительную активацию каскада МАРК, нежели обработка белком CHI3L1, с аккумуляцией киназ ERK1/2 в ядрах клеток 293. Выводы. В отличие от активации фосфорилирования киназ ERK1/2 после обработки клеток белком CHI3L1, приводящей к пролиферации, стимуляция этих киназ добавлением белка CHI3L2 угнетает митогенез в клетках 293 и U373, находящихся в состоянии сывороточного голодания. Ключевые слова: хитиназа 3-подобный белок 1 (CHI3L1), хитиназа 3-подобный белок 2 (CHI3L2), MAP-киназа, митогенез. 2011 Article Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome / P.O. Areshkov, S.S. Avdieiev, A.V. Iershov, V.M. Kavsan // Вiopolymers and Cell. — 2011. — Т. 27, № 5. — С. 343-346. — Бібліогр.: 12 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00011F https://nasplib.isofts.kiev.ua/handle/123456789/155649 577.22 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| description |
Aim. To determine 293 and U373 cell response and ERK1/2 activation profile after CHI3L1 or CHI3L2 treatment. Methods. Specific activation and localization of ERK1/2 kinases after CHI3L1 or CHI3L2 addition to unsupplemented cell medium were evaluated by Western blots, immunofluorescence and confocal microscopy. To
determine whether CHI3L1 or CHI3L2 can enhance mitogenesis, [3H]thymidine incorporation in cellular DNA
was measured. Results. The obtained results show that ERK1/2 phosphorylation was stimulated in both cell types (293 and U373 cells) following addition of CHI3L2 or CHI3L1 in dose- and time-dependent manner. Unexpectedly, in opposite to CHI3L1, the dose-dependent decreasing of measured mitogenesis parameters was observed in 293 and U373 cells. In both cell types the treatment with CHI3L2 gave more sustained than CHI3L1
MAPK-pathway activation with prolonged phospho-ERK1/2 nuclear accumulation in 293 cells. Conclusions. In
contrast to the activation of ERK1/2 phosphorylation by CHI3L1 cell treatment, that leads to a proliferative signal, the activation of these kinases by CHI3L2 addition inhibits cell mitogenesis and proliferation in serum starved 293 and U373 cells.
Keywords: chitinase 3-like 1 protein (CHI3L1), chitinase 3-like 2 protein (CHI3L2), MAP kinase, mitogenesis. |
| format |
Article |
| author |
Areshkov, P.O. Avdieiev, S.S. Iershov, A.V. Kavsan, V.M. |
| spellingShingle |
Areshkov, P.O. Avdieiev, S.S. Iershov, A.V. Kavsan, V.M. Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome Вiopolymers and Cell |
| author_facet |
Areshkov, P.O. Avdieiev, S.S. Iershov, A.V. Kavsan, V.M. |
| author_sort |
Areshkov, P.O. |
| title |
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome |
| title_short |
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome |
| title_full |
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome |
| title_fullStr |
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome |
| title_full_unstemmed |
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome |
| title_sort |
stimulation of transient versus sustained erk1/2 phosphorylation by relative chitinase-like proteins chi3l1 and chi3l2 correlates with different kinase localization and biological outcome |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2011 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/155649 |
| citation_txt |
Stimulation of transient versus sustained ERK1/2 phosphorylation by relative chitinase-like proteins CHI3L1 and CHI3L2 correlates with different kinase localization and biological outcome / P.O. Areshkov, S.S. Avdieiev, A.V. Iershov, V.M. Kavsan // Вiopolymers and Cell. — 2011. — Т. 27, № 5. — С. 343-346. — Бібліогр.: 12 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
AT areshkovpo stimulationoftransientversussustainederk12phosphorylationbyrelativechitinaselikeproteinschi3l1andchi3l2correlateswithdifferentkinaselocalizationandbiologicaloutcome AT avdieievss stimulationoftransientversussustainederk12phosphorylationbyrelativechitinaselikeproteinschi3l1andchi3l2correlateswithdifferentkinaselocalizationandbiologicaloutcome AT iershovav stimulationoftransientversussustainederk12phosphorylationbyrelativechitinaselikeproteinschi3l1andchi3l2correlateswithdifferentkinaselocalizationandbiologicaloutcome AT kavsanvm stimulationoftransientversussustainederk12phosphorylationbyrelativechitinaselikeproteinschi3l1andchi3l2correlateswithdifferentkinaselocalizationandbiologicaloutcome AT areshkovpo stimulâcíâkorotkotrivalogovsdovgotrivalogofosforilûvannâkínazerk12dodavannâmdvohblizʹkosporídnenihbílkívchi3l1íchi3l2korelûêzízmínoûlokalízacíívídpovídnihkínaztabíologíčnoûvídpovíddûaktivovanihklítin AT avdieievss stimulâcíâkorotkotrivalogovsdovgotrivalogofosforilûvannâkínazerk12dodavannâmdvohblizʹkosporídnenihbílkívchi3l1íchi3l2korelûêzízmínoûlokalízacíívídpovídnihkínaztabíologíčnoûvídpovíddûaktivovanihklítin AT iershovav stimulâcíâkorotkotrivalogovsdovgotrivalogofosforilûvannâkínazerk12dodavannâmdvohblizʹkosporídnenihbílkívchi3l1íchi3l2korelûêzízmínoûlokalízacíívídpovídnihkínaztabíologíčnoûvídpovíddûaktivovanihklítin AT kavsanvm stimulâcíâkorotkotrivalogovsdovgotrivalogofosforilûvannâkínazerk12dodavannâmdvohblizʹkosporídnenihbílkívchi3l1íchi3l2korelûêzízmínoûlokalízacíívídpovídnihkínaztabíologíčnoûvídpovíddûaktivovanihklítin AT areshkovpo stimulâciâkratkovremennogovsdlitelʹnogofosforilirovaniâkinazerk12dobavleniemdvuhblizkorodstvennyhbelkovchi3l1ichi3l2korreliruetsizmeneniemlokalizaciisootvetstvuûŝihkinazibiologičeskimotvetomaktivirovannyhkletok AT avdieievss stimulâciâkratkovremennogovsdlitelʹnogofosforilirovaniâkinazerk12dobavleniemdvuhblizkorodstvennyhbelkovchi3l1ichi3l2korreliruetsizmeneniemlokalizaciisootvetstvuûŝihkinazibiologičeskimotvetomaktivirovannyhkletok AT iershovav stimulâciâkratkovremennogovsdlitelʹnogofosforilirovaniâkinazerk12dobavleniemdvuhblizkorodstvennyhbelkovchi3l1ichi3l2korreliruetsizmeneniemlokalizaciisootvetstvuûŝihkinazibiologičeskimotvetomaktivirovannyhkletok AT kavsanvm stimulâciâkratkovremennogovsdlitelʹnogofosforilirovaniâkinazerk12dobavleniemdvuhblizkorodstvennyhbelkovchi3l1ichi3l2korreliruetsizmeneniemlokalizaciisootvetstvuûŝihkinazibiologičeskimotvetomaktivirovannyhkletok |
| first_indexed |
2025-11-30T21:02:31Z |
| last_indexed |
2025-11-30T21:02:31Z |
| _version_ |
1850250680166514688 |
| fulltext |
Stimulation of transient versus sustained ERK1/2
phosphorylation by relative chitinase-like proteins
CHI3L1 and CHI3L2 correlates with different kinase
localization and biological outcome
P. O. Areshkov, S. S. Avdieiev, A. V. Iershov, V. M. Kavsan
Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
chitotrio@gmail.com
Aim. To determine 293 and U373 cell response and ERK1/2 activation profile after CHI3L1 or CHI3L2 treat-
ment. Methods. Specific activation and localization of ERK1/2 kinases after CHI3L1 or CHI3L2 addition to un-
supplemented cell medium were evaluated by Western blots, immunofluorescence and confocal microscopy. To
determine whether CHI3L1 or CHI3L2 can enhance mitogenesis, [3H]thymidine incorporation in cellular DNA
was measured. Results. The obtained results show that ERK1/2 phosphorylation was stimulated in both cell ty-
pes (293 and U373 cells) following addition of CHI3L2 or CHI3L1 in dose- and time-dependent manner. Un-
expectedly, in opposite to CHI3L1, the dose-dependent decreasing of measured mitogenesis parameters was ob-
served in 293 and U373 cells. In both cell types the treatment with CHI3L2 gave more sustained than CHI3L1
MAPK-pathway activation with prolonged phospho-ERK1/2 nuclear accumulation in 293 cells. Conclusions. In
contrast to the activation of ERK1/2 phosphorylation by CHI3L1 cell treatment, that leads to a proliferative sig-
nal, the activation of these kinases by CHI3L2 addition inhibits cell mitogenesis and proliferation in serum star-
ved 293 and U373 cells.
Keywords: chitinase 3-like 1 protein (CHI3L1), chitinase 3-like 2 protein (CHI3L2), MAP kinase, mitogenesis.
Introduction. Despite the lack of chitin synthesis in
mammalians, their genomes encode a set of homologo-
us chitinase-like proteins (CLPs). In human there are
six proteins of this family [1] and CHI3L1 is the most
investigated protein of the group. CHI3L2 protein is
closely related to CHI3L1 and has significant sequence
homology with other mammalian CLPs. CHI3L2 was
identified as a protein that co-purified with CHI3L1
from conditioned medium from human articular carti-
lage chondrocytes primary culture [2]. Overexpression
of CHI3L1 and CHI3L2 genes was found in glioblas-
toma [3–5] and other tumors (reviewed in [6]). It was
reported that CHI3L1, similarly to IGF1, promotes the
growth of human fibroblasts by MAPK/ERK1/2 and
PI3K/AKT pathways activation [7]. An assumption
about CHI3L2 function was gained from the structural
similarity with CHI3L1 in size, nucleotide and amino
acid sequences, and 3D structure [8]. However, Western
blot analysis did not show simultaneous production of
CHI3L1 and CHI3L2 in gliomas, apparently indicating
their different functions [5].
The work described here demonstrates the possible
influence of MAPK signaling duration and the locali-
zation of phosphorylated ERK1/2 on the control of mi-
togenesis and proliferation in CHI3L1 or CHI3L2 trea-
ted human glial U373 and embryonic kidney 293 cells.
Materials and methods. 293 cells (Human emb-
ryonic kidney 293 cells, also often referred to HEK
343
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 5. P. 343–346
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2011
293, or less precisely as HEK cells) and U373 cells (Hu-
man glioblastoma-astrocytoma, epithelial-like cells)
were kindly provided by Prof. I. Gout (UCL, Cell Signa-
ling and Metabolic Regulation Dept., UK). Cells were
grown in DMEM supplemented with 10 % FBS and
100 µg/ml penicillin, 100 units/ml streptomycin in an
environment of 5 % CO2.
Native CHI3L1 was purified from conditioned MG-
63 cell medium as described by Harvey et al. [9]. Syn-
thesis of recombinant CHI3L2 was performed accor-
ding to our previous description [5]. Antibodies were
obtained from multiple sources and used at the specifi-
ed dilutions for immunofluorescence microscopy: p44/
42 MAP (ERK1/2) (L34F12) Mouse mAb («Cell Signa-
ling Technology», USA) 1:100 (anti-phospho-ERK);
ERK2 (K-23) Rabbit polyclonal IgG («Santa Cruz Bio-
technology», USA) 1:100 (pan-anti-ERK1/2); Fluo-
rescein Anti-Mouse IgG (H + L) as well as Texas Red®
Anti-Rabbit IgG («Vector Laboratories Inc.», USA)
1:400; and for immunoblots: pERK (E-4) Mouse
monoclonal IgG («Santa Cruz», USA) 1:2000 (anti-
phospho-ERK1/2); ERK2 (K-23) Rabbit polyclonal
IgG 1:3000 (pan-anti-ERK1/2); Anti-Mouse IgG (H +
L), HRP Conjugate and Anti-Rabbit IgG (H+L), HRP
Conjugate («Promega», USA) 1:20000 (HRP-anti-
mouse IgG), (HRP-anti-rabbit IgG).
The reagents for enhanced chemiluminescence (ECL)
from «Sigma-Aldrich Co.» (USA) and «Fluka» (Swit-
zerland) were used for the visualization of immunoreac-
tive bands on Western blots.
Mitogenic activity was assessed by determination
of DNA-synthesis rates of treated and untreated cell cul-
tures. 293 cells or U373 cells were seeded into 24-well
tissue-culture plates and allowed growing to near-con-
fluence in DMEM supplemented with 10 % FBS, follo-
wed by a 24 h serum-starvation period in unsupple-
mented DMEM, CHI3L1 and CHI3L2 were added at
the concentration of 100 ng/ml, followed by [3H]thymi-
dine (3 µCi/ml) («Amersham», UK) 2 h later. Cultiva-
tion was stopped after 24 h of exposure and the cell la-
yers were briefly washed twice by PBS and lysed in
0.5 M NaOH with 0.5 % SDS. DNA was collected on
glass-fibre filters («Whatman», UK) and washed with
5 % trichloroacetic acid. [3H]thymidine content was de-
termined by liquid scintillation spectroscopy using a
Perkin Elmer scintillation counter.
Investigation of ERK1/2 phosphorylation was per-
formed according to our previous description [10]. Bri-
efly, cells were seeded into 6-well tissue culture plates
in DMEM contained 10 % FBS and allowed to grow to
near-confluence, serum-starved for 24 h, followed by
exposure to CHI3L1 or CHI3L2 for time periods up to
2 h. At the end of the incubation periods, cell layers
were washed twice in ice-cold PBS and whole cell lysa-
tes were mixed with 2 × Laemmli sample buffer, boiled,
proteins resolved by SDS-PAGE and transferred to
nitrocellulose membrane. Membranes were blocked
with 5 % powdered skim milk, reacted with anti-phos-
pho-ERK, and then incubated with HRP-anti-mouse.
Blots were developed with an ECL detection system.
Membranes were treated in stripping buffer and incu-
bated with pan-anti-ERK. After incubation with HRP-
anti-rbbit IgG for 1 h, total ERK1/2 was detected with
ECL. Specific activation of MAP kinases was measu-
red by densitometric analysis of Western blot signals
using Scion Image 1.62c program (NIH ImageJ; NIH,
Bethesda, MD).
For immunofluorescence and confocal microsco-
py, cells were seeded on coverslips and allowed to
grow to near-confluence. Cells were then serum-starved
for 24 h, followed by exposure to CHI3L1 or CHI3L2
(100 ng/ml) for time periods up to 4 h. After treatment,
immunofluorescence analysis was performed as descri-
bed by Volmat et al. [11].
Results and discussion. A dose-dependent decrea-
se of mitogenesis was observed in the cells of tumor
(U373) and non-tumor (293) origin treated with CHI3L2
(Fig. 1). The simultaneous addition of both CHI3L1 and
CHI3L2 chitinase-like proteins showed the inhibition
of [3H]thymidine incorporation in DNA of both cell types
344
ARESHKOV P. O. ET AL.
18
15
·103
CHI3L1
6
3
0
0
9
CHI3L2 CHI3L1+CHI3L2
.
Fig. 1. Cell mitogenesis assay. [3H]thymidine incorporation in cells
(white column – U273; black – 293) treated with CHI3L1 or CHI3L2
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 5
Fig. 3. Time cour-
se of ERK1/2 phos-
phorylation and lo-
calization in activa-
ted and inactivated
cells. Immunofluo-
rescence and con-
focal microscopy
analysis of CHI3L1
or CHI3L2 treated
293 cells (A) and
U373 cells (B) with
phospho-ERK (gre-
en) and pan-ERK
(red) antibodies.
(Scale bar: 25µm)
In human, a single gene encodes the cytoplasmic
and mitochondrial forms of lysyl -tRNA synthe-
tase by means of alternative splicing. The cyto-
plasmic isoform is produced from the mRNA
lacking exon 2. This isoform is targeted to the
MARS (Multi Aminoacyl-tRNA Synthetase) com-
plex. The mitoсhondrial species is produced by
translation initiation at the level of exon 2. Mito-
chondrial LysRS is specifically hijacked by the
GagPol precursor protein during the process of pa-
ckaging of HIV-1 particles. Association of
mLysRS with GagPol involves catalytic domain of
the synthetase and transframe (p6*) and integrase
domains of the Pol region of GagPol. The for-
mation of the tRNA(Lys, 3) packaging complex is
an essential process of the HIV-1 life cycle, since
this tRNA is required for initiation of reverse
transcription of the RNA genome of HIV-1. The
tRNA(Lys, 3) packaging complex is a potential
therapeutic target
Figures to article by P. O. Areshkov et al.
Figure to article by L. Kobbi et al.
indicating that proliferative influence of CHI3L1 was
inhibited with CHI3L2 addition. The obtained results
demonstrate that CHI3L1 and CHI3L2 possess antago-
nistic properties in spite of their quite similar structure.
Using Western blot analysis we showed, that simi-
larly to CHI3L1, the treatment of both U373 and 293
cell lines with CHI3L2 leads to phosphorylation of
ERK1/2 (Fig. 2). However, CHI3L2 and CHI3L1 me-
diated phosphorylation of ERK1/2 occurred in the time-
and place-dependent manner. In 293 and U373 cells, the
CHI3L1 mediated phosphorylation of ERK1/2 is tran-
sient. Even after 2 min of the treatment with CHI3L1
cells became brightly fluorescent, while in 60 min we
did not see any phosphorylation of ERK1/2. At the sa-
me time the treatment of cells with CHI3L2 leads to pro-
long ERK1/2 phosphorylation, which was maintained
for more than 4 h. Moreover, such event was entailed
by translocation of phosphorylated ERK1/2 to the cell
nucleus (Fig. 3, see inset). Previously, using the model
of cells PC-12 treated with NGF, it was shown that
translocated to the nucleus ERK1/2 could activate seve-
ral transcription factors leading to the inhibition of pro-
liferation and apoptosis or to differentiation of cells [12].
Acknowlegements. The work was supported by
grants N Ф40.4/018 «По шук і ха рак те рис ти ка онко -
генів і пух лин них генів-суп ре сорів, що при й ма ють
участь в ініціації та роз вит ку гліом» спільної украї -
но-російської про гра ми фун да мен таль них дослід-
жень «ДФФД-РФФД-011» and «Ство рен ня сис те ми
інгібу ван ня рос ту пух лин го лов но го моз ку на осно-
ві на но кон ’ю гатів ан ти сенс-оліго нук ле о тидів та ан-
титіл, спе цифічних дo oнкoбiлкiв, з при род ними бio-
поліме ра ми» Дер жав ної цільо вої на уко во-техніч-
ної про гра ми «На но тех но логії та на но ма теріали».
П. О. Арешков, С. С. Авдєєв, А. В. Єршов, В. М. Кав сан
Сти му ляція ко рот кот ри ва ло го vs дов гот ри ва ло го
фос фо ри лю ван ня кіназ ERK1/2 до да ван ням двох
близь кос порідне них білків CHI3L1 і CHI3L2 ко ре лює
зі зміною ло калізації відповідних кіназ та
біологічною відповіддю ак ти во ва них клітин
Ре зю ме
Мета. Виз на чи ти клітин ну відповідь та вста но ви ти профіль ак -
ти вації кіназ ERK1/2 після об роб ки клітин 293 та U373 білка ми
CHI3L1 чи CHI3L2. Ме то ди. Спе цифічну ак ти вацію і ло каліза-
цію кіназ ERK1/2 після вне сен ня білків CHI3L1 або CHI3L2 до клі-
тин но го се ре до ви ща, що не містить си ро ват ки, про а налізо ва но
Вес терн-блот аналізо та ме то да ми іму ноф лу о рес ценції і кон фо -
каль ної мікрос копії. Для вста нов лен ня, чи змо же до да ван ня білків
CHI3L1 або CHI3L2 по си лю ва ти міто ге нез, за сто со ва но аналіз
інкор по рації [3H]тимідину в клітин ну ДНК. Ре зуль та ти. Отри -
мані ре зуль та ти де мо нстру ють, що сти му ляція фос фо ри лю ван -
ня кіназ ERK1/2 після до да ван ня CHI3L1 або CHI3L2 має дозо- та
ча со за леж ний ха рак тер. Ви я ви ло ся, що, на відміну від CHI3L1, до-
зо за леж не при гнічен ня інкор по рації [3H]тимідину кон ста то ва -
но у 293 та U373 кліти нах. В обох ти пах клітин об роб ка білком
CHI3L2 вик ли кає три валішу ак ти вацію кас ка ду МАРК, ніж об -
роб ка білком CHI3L1, з аку му лю ван ням кіназ ERK1/2 у ядрі клітин
293. Вис нов ки. На про ти ва гу ак ти вації фос фо ри лю ван ня кіназ
ERK1/2 внаслідок об роб ки клітин білком CHI3L1, що спри чи няє
проліфе рацію, сти му ляція цих кіназ до да ван ням білка CHI3L2 при-
гнічує міто ге нез у кліти нах 293 та U373, що пе ре бу ва ють у стані
сиро ват ко во го го ло ду ван ня.
Клю чові сло ва: хіти на за 3-подібний білок 1 (CHI3L1), хіти на -
за 3-подібний білок 2 (CHI3L2), MAP-кіназа, міто ге нез.
П. А. Арешков, С. С. Авдеев, А. В. Ершов, В. М. Кав сан
Сти му ля ция крат ков ре мен но го vs дли тель но го
фос фо ри ли ро ва ния ки наз ERK1/2 до бав ле ни ем двух
близ ко ро дствен ных бел ков CHI3L1 и CHI3L2 кор ре ли ру ет с
из ме не ни ем ло ка ли за ции со от ве тству ю щих ки наз и
би о ло ги чес ким от ве том ак ти ви ро ван ных кле ток
Ре зю ме
Цель. Опре де лить кле точ ный от вет и про филь ак ти ва ции ки наз
ERK1/2 по сле об ра бот ки кле ток 293 и U373 бел ка ми CHI3L1 или
CHI3L2. Ме то ды. Спе ци фи чес кую ак ти ва цию и ло ка ли за цию ки -
наз ERK1/2 по сле вне се ния бел ков CHI3L1 или CHI3L2 в кле точ-
345
STIMULATION OF TRANSIENT VERSUS SUSTAINED ERK1/2 PHOSPHORYLATION
293 cells U373 cells
phospho-ERK1/2
phospho-ERK1/2
pan-ERK1/2
pan-ERK1/2
BSA 0 25 75 100 EGF BSA 0 10 50 100 EGF ng/ml
Fig. 2. Dose-dependent induction of
ERK1/2 phosphorylation. Western
blot analysis of total lysates of
CHI3L1 or CHI3L2 treated cells
(BSA – 100 ng/ml; EGF – 50 ng/ml)
ную сре ду, где от су тству ет сы во рот ка, про а на ли зи ро ва но мето-
дами Вес терн-блот ана ли за, им му ноф лу о рес цен ции и кон фо каль -
ной мик рос ко пии. Для опре де ле ния, бу дет ли до бав ле ние бел ков
CHI3L1 или CHI3L2 уве ли чи вать ми то ге нез, ис поль зо ван ана лиз
ин кор по ра ции [3H]ти ми ди на в кле точ ную ДНК. Ре зуль та ты. По -
лу чен ные ре зуль та ты де мо нстри ру ют, что сти му ля ция фос фо -
ри ли ро ва ния ки наз ERK1/2 по сле до бав ле ния CHI3L1 либо CHI3L2
име ет за ви ся щий от дозы и вре ме ни ха рак тер. Ока за лось, что, в
от ли чие от CHI3L1, до зо за ви си мое угне те ние ин кор по ра ции [3H]
ти ми ди на кон ста ти ро ва но в 293- и U373-клет ках. В об оих ти пах
кле ток об ра бот ка бел ком CHI3L2 вы зы ва ет бо лее дли тель ную
ак ти ва цию кас ка да МАРК, не же ли об ра бот ка бел ком CHI3L1, с
ак ку му ля ци ей ки наз ERK1/2 в яд рах кле ток 293. Вы во ды. В от ли -
чие от ак ти ва ции фос фо ри ли ро ва ния ки наз ERK1/2 по сле об ра -
бот ки кле ток бел ком CHI3L1, при во дя щей к про ли фе ра ции, сти-
му ля ция этих ки наз до бав ле ни ем бел ка CHI3L2 угне та ет ми то ге -
нез в клет ках 293 и U373, на хо дя щих ся в со сто я нии сы во ро точ -
но го го ло да ния.
Клю че вые сло ва: хи ти на за 3-по до бный бе лок 1 (CHI3L1), хи -
ти на за 3-по до бный бе лок 2 (CHI3L2), MAP-ки на за, ми то ге нез.
REFERENCES
1. Bussink A. P., Speijer D., Aerts J. M., Boot R. G. Evolution of
mammalian chitinase(-like) members of family 18 glycosyl
hydrolases // Genetics.–2007.–177, N 2.–P. 959–970
2. Hu B., Trinh K., Figueira W. F., Price P. A. Isolation and sequen-
ce of a novel human chondrocyte protein related to mammalian
members of the chitinase protein family // J. Biol. Chem.–
1996.–271, N 32.–P. 19415–19420.
3. Garifulin O. M., Shostak K. O., Dmitrenko V. V., Rozumenko V.
D., Khomenko O. V., Zozulya Yu. A., Zehetner G., Kavsan V. M.
The genes SOX-2 and HC gp-39 are overexpressed in astrocytic
gliomas // Biopolym. Cell.–2002.–18, N 4.–P. 324–329.
4. Shostak K., Labunskyy V., Dmitrenko V., Malisheva T., Shama-
yev M., Rozumenko V., Zozulya Y., Zehetner G., Kavsan V. HC
gp-39 gene is upregulated in glioblastomas // Cancer Lett.–
2003.–198, N 2.–P. 203–210.
5. Kavsan V., Dmitrenko V., Boyko O., Filonenko V., Avdeev S.,
Areshkov P., Marusyk A., Malysheva T., Rozumenko V., Zozulya
Y. Overexpression of YKL-39 gene in glial brain tumors // Scho-
larly Research Exchange.–2008.–doi: 10.3814/2008/814849.
6. Johansen J. S. Studies on serum YKL-40 as a biomarker in disea-
ses with inflammation, tissue remodelling, fibroses and cancer //
Dan. Med. Bull.–2006.–53, N 2.–P. 172–209.
7. Recklies A. D., White C., Ling H. The chitinase 3-like protein hu-
man cartilage glycoprotein 39 (HC-gp39) stimulates prolifera-
tion of human connective-tissue cells and activates both extracel-
lular signal-regulated kinase- and protein kinase B-mediated sig-
nalling pathways // Biochem J.–2002.–365, Pt 1.–P. 119–126.
8. Iershov A., Odynets K., Kornelyuk A., Kavsan V. Homology mo-
deling of 3D structure of human chitinase-like protein CHI3L2
// Central Eur. J. Biol.–2010.–5, N 4.–P. 407–420.
9. Harvey S., Weisman M., O’Dell J., Scott T., Krusemeier M., Vi-
sor J., Swindlehurst C. Chondrex: new marker of joint disease //
Clin Chem.–1998.–44, N 3.–P. 509–516.
10. Areshkov P. O., Kavsan V. M. The chitinase 3-like human pro-
tein 2 (CHI3L2) activates ERK1/2 signaling pathway in human
embryonic kidney cells (HEK293) // Biopolym. Cell.–2009.–
25, N 6.–P. 500–503.
11. Volmat V., Camps M., Arkinstall S., Pouyssegur J., Lenormand
P. The nucleus, a site for signal termination by sequestration and
inactivation of p42/p44 MAP kinases // J. Cell Sci.–2001.–114,
Pt 19.–P. 3433–3443.
12. Traverse S., Gomez N., Paterson H., Marshall C., Cohen P.
Sustained activation of the mitogen-activated protein (MAP)
kinase cascade may be required for differentiation of PC12
cells. Comparison of the effects of nerve growth factor and
epidermal growth factor // Biochem. J.–1992.–288, Pt 2.–
P. 351–355.
UDC 577.22
Received 10.06.11
346
ARESHKOV P. O. ET AL.
|