Báo cáo khoa học: Stimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERK pdf

Stimulation of fibroblast proliferation by neokyotorphin
requires Ca
2+
influx and activation of PKA, CaMK II and
MAPK/ERK
Olga V. Sazonova, Elena Yu. Blishchenko, Anna G. Tolmazova, Dmitry P. Khachin,
Konstantin V. Leontiev, Andrey A. Karelin and Vadim T. Ivanov
Regulatory Peptides Group, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
Neokyotorphin, a-globin segment (137–141), was ini-
tially isolated from bovine brain and characterized as
an analgesic peptide [1], the effect of which is not
associated with binding of the peptide to any mem-
brane receptor [2,3]. We have previously shown that
neokyotorphin, along with a number of other func-
tional protein fragments [4], is present in a variety of
mammalian tissues [5] and is secreted by human
erythrocytes [6]. The proliferative activity of neokyo-
torphin in normal and tumor cells has been widely
reported [7–9]. In fibroblasts, the effect depends on
culture conditions, being maximal in sparse culture
maintained in serum-deficient medium [8]. The mode
of proliferative action of neokyotorphin is not clear.
It is not known whether neokyotorphin is able to ini-
tiate the cell cycle, or, like some regulatory peptides,
e.g. neurotensin or substance P, enhances proliferation
induced by growth factors [10,11]. Some results point
to the ability of neokyotorphin to affect intracellular
Ca
2+
levels. In brown preadipocytes, neokyotorphin
has been shown to increase cytoplasmic Ca

2+
levels
[7], however, it is not clear whether Ca
2+
entered
cells from the medium or was released from intra-
cellular Ca
2+
stores. Neokyotorphin has also been
shown to stimulate Ca
2+
influx via L-type channels
in frog cardiocytes, although such an effect has not
been found in mammalian cardiocytes [12]. However,
it has been suggested that in nonexcitable mammalian
Keywords
hemoglobin fragment; intracellular Ca
2+
protein kinase; proliferation; tissue
homeostasis
Correspondence
O. V. Sazonova, Shemyakin-Ovchinnikov
Institute of Bioorganic Chemistry RAS,
Miklukho-Maklaya 16 ⁄ 10, 117997 Moscow,
Russia
Tel ⁄ Fax: +7 495 335 3200
E-mail:
(Received 21 September 2006, revised 2
November 2006, accepted 13 November
2006)

doi:10.1111/j.1742-4658.2006.05594.x
Neokyotorphin [TSKYR, hemoglobin a-chain fragment (137–141)] has pre-
viously been shown to enhance fibroblast proliferation, its effect depending
on cell density and serum level. Here we show the dependence of the effect
of neokyotorphin on cell type and its correlation with the effect of protein
kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4b-phor-
bol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative
effect of neokyotorphin was suppressed by the Ca
2+
L-type channel inhibi-
tors verapamil or nifedipine, the intracellular Ca
2+
chelator 1,2-bis(2-ami-
nophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid acetoxymethyl ester, kinase
inhibitors H-89 (PKA), KN-62 (Ca
2+
⁄ calmodulin-dependent kinase II) and
PD98059 (mitogen-activated protein kinase). The proliferative effect of
8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppres-
sion (downregulation with PMA or inhibition with bisindolylmaleimide XI)
did not affect neokyotorphin action. The results obtained point to a
cAMP-like action for neokyotorphin.
Abbreviations
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid acetoxymethyl ester; CaMK II, Ca
2+
⁄ calmodulin-dependent kinase II;
CREB, cAMP-response element binding protein; ERK, extracellular signal-regulated protein kinase; MAPK, mitogen-activated protein kinase;
MSK1, mitogen and stress-activated protein kinase 1; PKA, protein kinase A; PMA, 4b-phorbol 12-myristate, 13-acetate; S6K1, ribosomal S6
kinase 1.
474 FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS

cells expressing L-type Ca
2+
channels, the prolifera-
tive effect of neokyotorphin is due, at least in part, to
an increase in Ca
2+
influx. L-type Ca
2+
channels are
expressed in certain nonexcitable cells [13], expression
of the cardiac L-type Ca
2+
channel isoform being ele-
vated in some cancer cells [14]. Influx of Ca
2+
via
plasma membrane channels is usually required for cell
proliferation, although L-type channel inhibition does
not necessarily result in inhibition of the cell cycle
[15]. By contrast, there are data indicating a prolifera-
tive effect for L-type Ca
2+
channel activators in non-
excitable cells [13,16].
Taking the above data into account, we suggest that
neokyotorphin is a cell-penetrating peptide that acti-
vates L-type Ca
2+
channels. In this study, we show
the involvement of L-type Ca

2+
channels, protein
kinase A (PKA), Ca
2+
⁄ calmodulin-dependent kin-
ase II (CaMK II) and mitogen-activated protein kinas-
es (MAPK ⁄ ERK) in the effect of neokyotorphin. In
general, the action of neokyotorphin in L929 cells
appears be similar to that of cAMP.
Results
Effect of neokyotorphin in the absence of
growth factors
To test the ability of neokyotorphin to initiate the cell
cycle we compared the effect of 1 lm neokyotorphin in
serum-deficient and serum-free culture medium
(Table 1). The residual fetal bovine serum in serum-
deficient culture medium allowed for an increase in cell
number of 24 ± 6.5% (P<0.01), compared with
cells completely deprived of serum. The activity of
neokyotorphin was evaluated compared with the cor-
responding controls. In contrast to fetal bovine serum-
deficient medium, in which neokyotorphin stimulated
proliferation, it had no effect in medium containing no
fetal bovine serum. Thus, in L929 cells, neokyotorphin
does not induce mitosis, rather it acts by enhancing
the effect of serum growth factors.
Involvement of Ca
2+
influx in the action
of neokyotorphin

We used L-type channel blockers with different chem-
ical structures: nifedipine (dihydropyridine) and verap-
amil (phenylalkylamine) [17]. The IC
50
values depend
strongly on cell type: for nifedipine, IC
50
¼ 3.0 nm to
0.1 lm; for verapamil IC
50
¼ 60.0 nm to 0.5 lm [17];
the concentrations used in L929 fibroblasts were
10–1000 times higher. The effect of 1 lm neokyotor-
phin was suppressed in the presence of blockers,
whereas the blockers themselves did not have any sig-
nificant effect on cell proliferation (Table 2).
The effects of 1 lm neokyotorphin and 10% fetal
bovine serum were tested in L929 cells in the presence
of the intracellular Ca
2+
chelator 1,2-bis(2-aminophen-
oxy)ethane-N,N,N¢,N¢-tetraacetic acid acetoxymethyl
ester (BAPTA-AM; 1.0 or 2.5 lm). As shown in
Table 3, 10% fetal bovine serum-induced proliferation
was only partially inhibited by BAPTA-AM, confirm-
ing that only the Ca
2+
-dependent proliferative effect
of the growth factors was inhibited in the presence of
BAPTA-AM. The effect of neokyotorphin was sup-

pressed at both concentrations of BAPTA-AM.
These results indicate that Ca
2+
influx via the
L-type channel and an increase in intracellular Ca
2+
levels are involved in the action of neokyotorphin in
L929 fibroblasts.
Protein kinases involved in the action of
neokyotorphin
To study the involvement of non-MAPK protein kin-
ases in the action of neokyotorphin, we used stauro-
sporine, a broad-spectrum kinase inhibitor that
suppresses multiple forms of PKC, Src, PKA, kinase
of epidermal growth factor receptor, and CaMK II
[18,19]. It has been shown that 300 nm staurosporine
inhibits L-type Ca
2+
channels [20]; the concentrations
we used were 10 and 25 nm. Staurosporine itself
decreased L929 cell number by 20–25%, compared
with negative controls, without cytotoxic action
Table 1. Effect of neokyotorphin in the absence of serum growth
factors. Cells were seeded in 96-well assay plates (5000 cells per
well). After 18 h of subculture, the fetal bovine serum-supplied
medium was removed and replaced by a double volume of the
washing medium, as indicated in the column ‘Conditions’. After
30 min, the washing medium was replaced by fetal bovine serum-
free medium containing 1 l
M neokyotorphin (test samples) or fetal

bovine serum-free medium (controls). After 24 h, a cell count was
performed using a universal particle counter ⁄ analyzer Z2.
Conditions
Cell number (%)
a
Change in cell
number (%)
b
Control
1. l
M
neokyotorphin
No fetal bovine serum
c
100 ± 8 96 ± 8 ) 4±8
Fetal bovine serum
deficit
d
124 ± 6.5 164 ± 9 32 ± 9*
a
Compared with cells maintained in the absence of FBS and pep-
tide.
b
Induced by neokyotorphin, compared with the corresponding
controls.
c
Cells were preincubated with a double volume of fetal
bovine serum-free medium.
d
Cells were preincubated with double

volume of medium supplied with 10% fetal bovine serum.
*P < 0.01 versus corresponding negative control.
O. V. Sazonova et al. Neokyotorphin proliferative action in fibroblasts
FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS 475
(Table 4). Neokyotorphin showed no activity in the
presence of the inhibitor, indicating that staurosporine-
suppressed kinase(s) are involved in neokyotorphin
activity.
To evaluate the possible involvement of PKCs and
PKAs in the action of neokyotorphin, we compared
the effects of the PKC activator 4b-phorbol 12-myri-
state, 13-acetate (PMA; activates classic and novel
PKCs) and the PKA activator 8-Br-cAMP with the
effect of neokyotorphin in three types of fibroblasts
(Table 5). The effect of activation of these kinases is
known to depend on cell origin [21–25]; the effect of
PKA activators in fibroblasts has also been shown to
depend on the culturing conditions [26]. In this study,
the effects were examined under conditions optimal for
manifestation of the proliferative effect by cAMP [26],
i.e. with nonconfluent cell culture and serum-deficient
medium. In L929 cells, both PK activators and neo-
kyotorphin induced a significant increase in cell num-
ber. In CV-1 fibroblasts, PKC activation resulted in
significant inhibition of proliferation, whereas 8-Br-
cAMP and neokyotorphin showed only modest and
poorly reproducible suppressive effects. Swiss 3T3
fibroblasts enhanced their proliferation upon PKC
activation and did not respond to 8-Br-cAMP and
neokyotorphin.

Neokyotorphin activity was also tested in neuron-
like PC-12 rat pheochromocytoma cells (Table 5), for
which PMA and cAMP effects have been widely
reported [27,28]. In these cells, neokyotorphin, like
cAMP [27,28], induced suppression of cell prolifer-
ation.
Based on the results obtained, we assumed that neo-
kyotorphin’s effect is mediated by PKA rather than
PKC, although both possibilities were investigated.
We studied the combined effect of 1 lm neokyotor-
phin and 8-Br-cAMP (30–100 lm) in L929 cells
(Table 6). The effect of 8-Br-cAMP was minimal at
30 lm (P ¼ 0.08 versus 50 and 100 lm). Addition of
neokyotorphin enhanced the effect of 8-Br-cAMP only
at 30 lm of the latter (P ¼ 0.06 versus 30 lm 8-Br-
cAMP alone). The proliferative effect produced by
30 lm 8-Br-cAMP ⁄ 1 lm neokyotorphin did not exceed
the maximal activity of 8-Br-cAMP or neokyotorphin
taken alone, suggesting their possible concurrency. By
contrast, combining 1 lm neokyotorphin and 100 lm
8-Br-cAMP had a modest proliferative effect, which
may result from desensitization of the PKA pathway
due to its extra activation.
Table 3. Effect of neokyotorphin in the presence of the intracellular
Ca
2+
chelator BAPTA-AM. Experimental design as in Table 2.
Sample
Cell number (%) compared with negat-
ive controls

Control
BAPTA-AM
1.0 l
M 2.5 lM
Control 100 ± 9 87 ± 8 83 ± 4
1.0 l
M neokyotorphin 125 ± 5*§ 97 ± 10§ 84 ± 8§
10% fetal bovine serum 144 ± 10* 134 ± 7*
Ù
107 ± 6
Ù
#
*P < 0.05 versus negative control;
Ù
P < 0.01 versus BAPTA-AM
alone; §P < 0.05 versus 1 l
M neokyotorphin alone; #P < 0.01 ver-
sus 10% fetal bovine serum alone.
Table 2. Effect of meokyotorphin in the presence of L-type Ca
2+
channel blockers nifedipine and verapamil. Cells were seeded in 96-well
assay plates (5000–8000 cells per well). After 18 h of subculture, fetal bovine serum-supplied medium was replaced by fetal bovine serum-
free medium containing test substances. In control samples, fetal bovine serum-free medium without test substances was added. After
24 h, a cell count was performed using a universal particle counter ⁄ analyzer Z2.
Sample series
Change in cell number compared with negative control (%)
Neokyotorphin
1 l
M
Verapamil Nifedipine

10 l
M 1 lM 10 lM 1 lM
Without neokyotorphin ) 14 ± 8 4 ± 7 ) 4±8 3±18 NA
With 1 l
M neokyotorphin ) 5 ± 6§ 7 ± 11 5 ± 14 8 ± 6§ 25 ± 6*
*P < 0.05 versus negative control; §P < 0.05 versus 1 l
M neokyotorphin alone.
Table 4. Effect of neokyotorphin in the presence of the nonselec-
tive PK inhibitor staurosporine. Experiment design as in Table 2.
Control
Cell number (%) compared with negative
controls
Control
Staurosporine
10 n
M 25 nM
Control 100 ± 8 80 ± 9* 75 ± 7*
1 l
M neokyotorphin 125 ± 5* 89 ± 14§ 78 ± 4*§
*P < 0.05 versus negative control; §P < 0.05 versus 1 l
M neokyo-
torphin alone.
Neokyotorphin proliferative action in fibroblasts O. V. Sazonova et al.
476 FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS
H-89 was used to inhibit PKA activation. It inhibits
PKA I and II (IC
50
¼ 135 nm), and also ribosomal S6
kinase 1 (S6K1) and mitogen- and stress-activated pro-
tein kinase 1 (MSK1); with 10 lm of H-89 the activity

of these kinases is suppressed by 97–100% [29]. The
concentrations used in the study varied from 1 to
10 lm. Taken alone, only 10 lm H-89 reliably and
reproducibly suppressed L929 cell proliferation
(P<0.05; Table 7). The activity of 1 lm neokyotor-
phin was inhibited over the whole concentration range
of H-89, confirming the involvement of PKA in the
effect of neokyotorphin. As for participation of the
other H-89-suppressed kinases, the involvement of
S6K1 seems probable, as it is PKA dependent [30].
MSK1 is a cAMP-response element binding protein
(CREB)-phosphorylating kinase [31]; however, CREB
phosphorylation by MSK1 in fibroblasts is induced by
stress factors (UV, anizomycine) rather than by mito-
genes [32], thus the involvement of MSK1 in the neo-
kyotorphin effect seems less probable.
To study the involvement of PKCs in the neokyotor-
phin effect in L929 cells, the activity of PKC was
downregulated or suppressed using a specific PKC
inhibitor.
PKC downregulation was achieved by preincubating
cells for 24 h with 5 lm PMA in the presence of 10%
fetal bovine serum. Under these conditions, the level
of PMA-activated PKC is decreased [33]. After PKC
Table 6. Effect of 8-Br-cAMP and neokyotorphin applied together. Experimental design as in Table 2. The cell count was performed using
fluorescent microscopy and image analysis software.
Sample series
Change in cell concentration (%) compared with negative control
Neokyotorphin
1 l

M
8-Br-cAMP
30 l
M 50 lM 100 lM
Without neokyotorphin 16 ± 4.5* 26 ± 6* 29 ± 8* –
With 1 l
M neokyotorphin 30 ± 8*
Ù
20 ± 6* 18 ± 4* 25 ± 6*
*P < 0.05 versus negative control;
Ù
P ¼ 0.06 versus 30.0 lM 8-Br-cAMP alone.
Table 7. Effect of neokyotorphin in the presence of PKA inhibitor H-89 Experimental design as in Table 2. The cell count was performed
using fluorescent microscopy and image analysis software.
Sample series
Change in cell concentration (%) compared with negative control
Neokyotorphin
1.0 l
M
H-89
1.0 l
M 2.5 lM 5.0 lM 7.5 lM 10.0 lM
Without neokyotorphin 4 ± 12 6 ± 12 5 ± 13 ) 6±12 ) 31 ± 8* –
With 1.0 l
M neokyotorphin 13 ± 2§ 14 ± 8 1 ± 16 ) 7 ± 10§ ) 30 ± 9*§ 25 ± 5*
*P < 0.05 versus negative control; §P < 0.05 versus 1 l
M neokyotorphin alone.
Table 5. Comparison of the effects of PKC activator PMA, PKA activator 8-Br-cAMP and neokytorphin in cells of differing origins. Cells were
seeded in 96-well assay plate at 5000 cells per well (fibroblasts) or 15 000 cells per well (PC-12). After 18 h of subculture, fetal bovine
serum-supplied medium was replaced by fetal bovine serum-free medium containing test substances. In control samples, fetal bovine

serum-free medium without test substances was added. L929, Swiss 3T3 and PC-12 cells were incubated with test substances 24 h, CV-1
cells – for 48 h. The cell count was performed using a universal particle counter ⁄ analyzer Z2. NT, not tested.
Cell line
Change in cell concentration compared with negative control (%)
0.1 l
M PMA 50.0 lM 8-Br-cAMP 1.0 lM neokyotorphin
L929, murine tumor fibroblasts 50 ± 15* 26 ± 6* 25 ± 5*
CV-1, tumor fibroblasts from African
green monkey (Cercopithecus aethiops)
40 ± 6* 5 ± 7 2 ± 7
Swiss 3T3, murine embryonic fibroblasts ) 48 ± 4* ) 16 ± 10 ) 14 ± 10
PC-12 rat pheochromocytoma cells NT NT ) 27 ± 7*
*P < 0.05 versus negative control.
O. V. Sazonova et al. Neokyotorphin proliferative action in fibroblasts
FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS 477
suppression, we tested for the neokyotorphin effect
(Table 8). Preincubation with 5 lm PMA led to a
decrease in cell number of 28 ± 5% (P<0.05), com-
pared with the cells preincubated without PMA. In
both PMA-treated and nontreated cells, 1 lm neokyo-
torphin increased cell number, compared with the cor-
responding controls.
Bisindolylmaleimide XI (Ro 32-0432) is a highly
selective inhibitor of Ca
2+
-activated PKCs a and bI
(IC
100
¼ 10–100 nm) [34,35]. Bisindolylmaleimide XI
alone (0.5–2.0 lm) did not induce a reproducible

change in cell number (Table 9), this may be due to
the pleiotropicity of the role of the sensitive forms of
PKC in L929 cell proliferation [36]. In the presence of
bisindolylmaleimide XI, the effect of neokyotorphin
did not change. Based on these results, we believe that
PKCs do not participate in neokyotorphin-induced
enhancement of cell proliferation.
To investigate the involvement of MAPK ⁄ ERK,
the MAPK cascade inhibitor PD 98059 was used.
PD 98059 binds to MKK1, preventing its activation
by upstream kinases, e.g. Raf-1 or B-Raf [29]. To pre-
serve the ability of the cells to proliferate, we used
20 lm PD 98059. At that concentration PD 98059
induces suppression of proliferative effect of 10% fetal
bovine serum by 50%. Because neokyotorphin is pos-
tulated to promote PKA activation, 8-Br-cAMP activ-
ity was also tested in the presence of the MAPK
inhibitor. In the presence of PD 98059 (Fig. 1A), the
effect of neokyotorphin was suppressed, although the
cell number in neokyotorphin-treated samples was sig-
nificantly higher than in samples with inhibitor alone.
The effect of 8-Br-cAMP was completely inhibited by
PD 98059. According to the literature, in certain cell
types, PKA activates the MAPK cascade [22]. Because
the activity of neokyotorphin was not completely sup-
pressed in the presence of PD 98059, some additional
pathways, i.e. different from PKA ⁄ MAPK activation,
may be involved.
To inhibit the activity of CaMK II, we used KN-62
(IC

50
¼ 500 nm) [29]. Of all CaMKs, CaMK II is the
one primarily involved in the regulation of cell prolif-
eration and can be activated in response to Ca
2+
influx [37]. At the chosen concentration (10 lm),
Table 8. Effect of neokyotorphin in cells with downregulated PKC. Cells were seeded in 96-well assay plates (5000 cells per well) and prein-
cubated for 24 h with 5.0 l
M PMA in medium supplied with 10% of fetal bovine serum (FBS). In the reference samples, preincubation was
carried out without PMA. At hour 24, medium was removed from all the samples and changed for fetal bovine serum-free medium in the
control samples or fetal bovine serum-free medium containing 1 l
M neokyotorphin in the experimental samples. The cell count was per-
formed at hour 48 using a particle counter ⁄ analyzer Z2.
Sample series
Incubation
Change in cell number (%)
induced by neokyotorphin
1–24 h 24–48 h
Pre-incubation with PMA
Control – FBS-deficient medium
Experimental 5 l
M PMA +
medium with 10% FBS
1.0 lM neokytorphin,
FBS-deficient medium
26 ± 6*
Pre-incubation without PMA
Control
– FBS-deficient medium
Experimental Medium + 10% FBS 1.0 l

M neokytorphin,
FBS-deficient medium
23 ± 5*
*P < 0.05 versus corresponding negative control.
Table 9. Effect of neokytorphin in the presence of the PKC inhibitor bisindolylmaleimide XI. Experimental design as in Table 2. The cell count
was performed using fluorescent microscopy and image analysis software.
Sample series
Change in cell concentration (%) compared with negative control
Neokyotorphin
1 l
M
Bisindolylmaleimide XI
0.5 l
M 1.0 lM 2.0 lM
Without neokyotorphin 7 ± 15 16 ± 15 9 ± 6 NA
With 1.0 l
M neokyotorphin 29 ± 6* 29 ± 7* 28 ± 7*
Ù
25 ± 5*
*P < 0.05 versus negative control;
Ù
P < 0.05 versus bisindolylmaleimide XI alone.
Neokyotorphin proliferative action in fibroblasts O. V. Sazonova et al.
478 FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS
KN-62 inhibited the effect of 10% fetal bovine serum
by 60%. 8-Br-cAMP activity was also tested in the
presence of KN-62, because PKA is a known L-type
Ca
2+
channel coactivator [38], therefore CaMK II

may be a downstream mediator of the 8-Br-cAMP
effect. As seen in Fig. 1B, KN-62 completely inhibited
the proliferative effect of neokyotorphin; the effect of
8-Br-cAMP was suppressed only partially, because cell
number in the samples treated with the cAMP ana-
logue was significantly higher compared with KN-62
alone. Thus, CaMK II is essential for the action of
neokyotorphin L929 cells; cAMP-induced stimulation
involves CaMK II as well as CaMK II-independent
pathways.
In summary, the protein kinases established as neo-
kyotorphin-effect mediators in L929 cells, are PKA,
CaMK II and MAPK.
Discussion
The set of mediators involved in the neokyotorphin
proliferative effect (Ca
2+
L-type channels, intracellular
Ca
2+
, PKA, CaMK II and MAPK ⁄ Erk) is not unique;
the same set is utilized by glucagon-like peptide-1 sti-
mulating proliferation in pancreatic b-cells [39]. The
major difference in the action of those peptides is
the absence of binding with cell-surface receptors in
the case of neokyotorphin [2,3]. Our preliminary stud-
ies have confirmed the ability of a neokyotorphin-like
peptide (dansyl-labeled peptide TVLTSKYR) to penet-
rate cells (unpublished data). The nonreceptoric action
of neokyotorphin may lead to the nontypical cell spe-

cificity seen in the action of neokyotorphin, which, if
this is the case, should not be associated with the
expression of specific receptors for that peptide. The
effectiveness of neokyotorphin should rather depend
on intracellular pathways existing in target cells.
We showed the inability of neokyotorphin to initiate
proliferation in quiescent cells, thus, for its effect to be
realized, the preactivated state of the proliferative sign-
aling pathways is required. We believe that neokyotor-
phin is a modulator of cell proliferation acting in
accordance with a cellular state and other external
stimuli.
The intracellular mode of action of neokyotorphin
raises the question of its primary molecular target
inside the cell. Neokyotorphin may activate L-type
Ca
2+
channels, in which case, channel activation
should induce activation of all protein kinases shown
to contribute to the neokyotorphin effect. Both PKA
and CaMK II are known to be activated by Ca
2+
influx, in the case of CaMK II activation is due to a
general increase in intracellular Ca
2+
[37], in the case
of PKA it is due to potential- or calmodulin-dependent
adenylyl cyclases [40]. The MAPK ⁄ ERK cascade may
be trans-activated by one or both of these kinases
[22,37,41]. However, Ca

2+
-activated adenylyl cyclases
are rather exotic and are predominantly expressed in
Fig. 1. Effect of 1 lM neokyotorphin and 50 lM 8-Br-cAMP in L929
cells in the presence of MAPK or CaMK II inhibitors. Cells were
seeded in 96-well assay plates (5000 cells per well). After 18 h of
subculture, the fetal bovine serum (FBS)-supplied medium was
replaced by medium with 0.5% of fetal bovine serum (for improve-
ment of the inhibitors solubility), containing the test substances.
Samples containing medium with 0.5% of fetal bovine serum with-
out test substances were used as controls. After 24 h of incubation
the cell count was performed, using a universal particle counter
and analyzer Z2. (A) 20 l
M PD 98059 (MAPK ⁄ ERK inhibitor). (B)
10 l
M KN-62 (CaMK II inhibitor). *P < 0.05 versus negative control;
§P < 0.05 versus 1.0 l
M neokyotorphin alone;
Ù
P < 0.01 versus
inhibitor alone; –P < 0.01 versus 50 l
M 8-Br-cAMP alone;
#P < 0.01 versus 10% fetal bovine serum alone.
O. V. Sazonova et al. Neokyotorphin proliferative action in fibroblasts
FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS 479
the nervous system [40]. Activation of L-type channel
by PKA is more common in most cell types [38]. Based
on the correlation between 8-Br-cAMP and neokyotor-
phin action, neokyotorphin may target one of the ele-
ments of the PKA-signaling system. PKA may activate

the Ca
2+
L-type channel, resulting in an increase in
the Ca
2+
influx, elevation of the intracellular Ca
2+
level and CaMK II activation. A generalized scheme
of the action of neokyotorphin is given in Fig. 2.
Regardless of the primary neokyotorphin target
enigma, the involvement of PKA in the peptide’s pro-
liferative effect is apparent. This explains the charac-
teristic features of the peptide action in cell cultures
observed in this and previous studies, i.e. the depend-
ence on cell type and culturing conditions [8].
The dual role of PKA in cell proliferation has been
widely reported [22]. The proliferative action of
cAMP ⁄ PKA is believed to be exerted by MAPK cas-
cade trans-activation via B-Raf [22,42]. There is accu-
mulating evidence that of all Raf isoforms, B-Raf
might be the more important physiological MEK acti-
vator; it appears to have considerably stronger kinase
activity than the other two isotypes [43]. B-Raf plays a
major role in embryogenesis [44], hematopoiesis [45]
and normal cell physiology, e.g. in murine embryonic
fibroblasts it maintains higher basal level of phospho-
Erk1 ⁄ 2 [43]. Stronger B-Raf expression and activation
due to mutation have been seen in multiple cell types
where cAMP increases proliferation [46,47], for exam-
ple, small cell lung cancer [48], melanocytes and melan-

omas [42,49], colorectal cancer [42], fibroblastoma [50],
thyroid primary cells and tumors [42].
In this and previous studies [8], we have shown the
proliferative effect of neokyotorphin in L929 tumor
fibroblasts, primary murine embryonic fibroblasts and
M3 murine melanoma cells. According to the literature,
such cells strongly express B-Raf and maintain high
basal activity. In primary cultures of adult murine bone
marrow and spleen cells, neokyotorphin supported cell
number in serum-deficient medium [8]. One of the func-
tions demonstrated for B-Raf is the establishment of a
proper number of myeloid progenitor cells; the number
of B-Raf
– ⁄ –
progenitor cells was strongly reduced [45].
Swiss 3T3 cells are known to increase proliferation
upon PKA activation [23]. The Swiss 3T3 cells used in
this study did not respond to 8-Br-cAMP and neokyo-
torphin. The cells used had features indicating their
spontaneous transformation during high-density culti-
vation [51], namely, proliferation cycle shortening,
poorer adhesion, and an absence of contact inhibition.
The absence of the response to PKA activators in the
spontaneously transformed fibroblasts may be due to
the changes in PKA and ⁄ or B-Raf expression patterns
in those cells.
In PC-12 cells, neokyotorphin suppressed prolifer-
ation. According to the literature [22], activated PKA
in those cells stimulates MAPK⁄ ERK, inducing differ-
entiation associated with pronounced inhibition of cell

proliferation. Such pathway is utilized by NGF, which
suppresses proliferation of PC-12 cells and induces
neurite outgrowth in those cells [52]. cAMP, but not
PMA, induces neurite outgrowth due to sustained acti-
vation of MAPK ⁄ ERK [27,28]. The ability of neokyo-
torphin to induce PC-12 cell differentiation remains
Fig. 2. Proposed mechanism of action of
neokyotorphin in L929 cells.
Neokyotorphin proliferative action in fibroblasts O. V. Sazonova et al.
480 FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS
uncertain, as only the short-term effect of the peptide
was tested. In summary, the cell-type specificity of neo-
kyotorphin action is apparently correlated with the
pattern characteristic of cAMP.
Neokyotorphin was inactive in L929 cells upon com-
plete exclusion of serum growth factors from the cul-
turing medium. A similar phenomenon has been
reported for cAMP in NIH 3T3 fibroblasts; MAPK
activation was not detected in cells that had been
grown to confluence and serum-deprived for 24 h [26].
Previously, we have shown the dependence of the neo-
kyotorphin effect on initial cell density in L929 cells and
M3 murine melanoma [8]. In L929 cells the maximal
effect of the peptide was observed in a sparse culture in
fetal bovine serum-deficient medium. In the case of high
cell density, the peptide was active only in serum-defici-
ent medium. Based on data from the literature, the con-
ditions optimal for development of the peptide effect are
for the most part similar to those in which maximal
expression ⁄ activation of B-Raf is observed. The expres-

sion and 14-3-3 protein-dependent activation of this kin-
ase both depend on the cell density and the supply of
growth factors, being maximal in nonconfluent culture
and with a growth factor deficit [42,47,53].
In summary, the main features of neokyotorphin
action were shown to correlate with those of cAMP.
cAMP is commonly regarded as a major intracellular
factor that, in combination with external signaling mol-
ecules, produces a cell-specific response. In other words,
cAMP-dependent signaling results in cell- and ⁄ or tis-
sue-specific reactions, contributing to the maintenance
of tissue homeostasis. Previously, we postulated that
components of tissue-specific peptides pools, of which
neokyotorphin is a frequently found example, are also
involved in the regulation of tissue homeostasis [4]. We
have shown that the release of neokyotorphin by
erythrocytes is an active and energy-dependent process
[54], which confirms its physiological importance. The
processing of hemoglobin, leading to the release of act-
ive peptides has been shown to be carried out by tissue
macrophages [55]. In the both cases, we believe that the
release of neokyotorphin is associated with the physio-
logical state of the tissue ⁄ organism. Thus, neokyotor-
phin may be a link between cellular response and
tissue ⁄ organism state, promoting cooperation between
those levels in the regulation of homeostasis.
Experimental procedures
Cell culture
L929, CV-1 and spontaneously transformed Swiss 3T3 cells
were maintained at 37 °C in RPMI-1640 culture medium

(Sigma, St Louis, MO) containing 10% of fetal bovine
serum (Sigma), 2 mml-glutamine, 10% standard vitamins
solution, 100 lmÆmL
)1
of penicillin G, 0.1 mgÆmL
)1
of
streptomycin sulfate and 0.25 lgÆmL
)1
of amphotericin B
(all Sigma), in a humidified atmosphere containing 5%
CO
2
. PC-12 cells were cultured under similar conditions
in medium supplied with 15% of fetal bovine serum
(Sigma). All laboratory plastic ware was from Corning
(Acton, MA).
Chemicals
Peptide with the TSKYR sequence (neokyotorphin) was
kindly provided by A. Y. Surovoy (Laboratory of Peptide
Chemistry, Shemyakin-Ovchinnikov Institute of Bioorganic
Chemistry RAS). Verapamil, nifedipine, BAPTA-AM,
staurosporine, 8-Br-cAMP, PMA, H-89, PD 98059 and
KN-62 were from Sigma.
Cell count
The design of experiments is described in the corresponding
table ⁄ figure legends. Visual cell count was performed as
described previously [8]. For cell count using the universal
particle counter ⁄ analyzer model Z2 (Beckman Coulter,
Fullerton, CA), cells were suspended in 10 mL of counting

buffer ISOTON II (Beckman Coulter). One milliliter of the
suspension was analyzed. The number of cells in one
well of a 96-well assay plate was calculated as: N
well
¼
N
counted
· 10, where N
well
is the number of cells in a well
and N
counted
is cell count returned by the counter.
The suspended cells for the cell count with fluorescence
microcopy were loaded with 0.5 mgÆmL
)1
of 6-carboxyfluo-
resceine diacetate and 0.5 mgÆmL
)1
of propidium iodide
dissolved in dimethylsulfoxide and placed in a Goryaev
chamber. Cells were visualized in the dark field using a
fluorescent DMLS microscope (Leica Microsystems, Wetz-
lar, Germany), with blue excitation light (k ¼ 450–490 nm).
Images were captured using a PC-operated digital camera
(DC300F; Leica Microsystems). The image square was
7.92 mm
2
. Images were fragmented and the objects were
counted using image fragmentation and analysis software

(MECOS, Moscow, Russia). Cell concentration was calcu-
lated as: [C], cellsÆmL
)1
¼ N
sample
⁄ 7.92 · 10 000, where
N
sample
is the averaged cell number obtained for two images
corresponding to the sample.
Statistical processing of the results
Between five and six replicates corresponding to the control
and 3–4 corresponding to each experimental series were
analyzed in each independent experiment. Data obtained
from 3–5 independent experiments were averaged. The
effect was calculated for each data point using Eqn (1):
O. V. Sazonova et al. Neokyotorphin proliferative action in fibroblasts
FEBS Journal 274 (2007) 474–484 ª 2006 The Authors Journal compilation ª 2006 FEBS 481
Effect ð%Þ¼½N
sample
À N
control
=N
control
 100 ð1Þ
where N
sample
¼ number of cells in the experimental sample
and N
control

¼ average number of cells in negative control
samples.
The statistical significance of the data was determined
using nonpaired Student’s t-test.
Acknowledgements
We thank Ms D. V. Serebryanaya (Institute of Experi-
mental Cardiology of Russian Cardiology Research
Center, Moscow, Russia) and Prof A. V. Zelenin
(Engelhardt Institute of Molecular Biology RAS, Mos-
cow, Russia) who generously provided the cell cul-
tures; and R. Kh. Ziganshin (Laboratory of Peptide
Chemistry, Shemyakin-Ovchinnikov Institute of Bio-
organic Chemistry RAS) who generously provided
L-type channel blockers. This work was supported by
Presidium of Russian Academy of Sciences, grant
‘‘Molecular and Cell Biology’’.
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