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: sazonova@mail.ibch.ru (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. 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Stimulation of fibroblast proliferation by neokyotorphin requires Ca 2+ influx and activation of PKA, CaMK II and MAPK/ERK Olga V. Sazonova,. 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