Urocortin stimulates the ERK1/2 signaling pathway and the proliferation of HeLa cells via CRF receptor 1

Corticotropin‐releasing factor (CRF) stimulates adrenocorticotropic hormone (ACTH) secretion from the pituitary gland and is an essential regulator of the hypothalamic–pituitary–adrenocortical axis. Isoforms of CRF receptor are known to mediate the effects of urocortin stress ligands on the regulation of stress responses, anxiety, and feeding behavior; however, urocortin stress ligands also influence cell proliferation. In view of the tumor‐promoting capacity of prolonged stress, here we investigated (a) the effect of urocortin on cell proliferative signaling via extracellular signal‐regulated kinase 1/2, (b) the expression and cellular distribution of the specific CRF receptor isoforms, and (c) the intracellular localization of phosphorylated ERK1/2 in HeLa cells. Stimulation of cell proliferation was observed in the presence of 10 nm urocortin. Our data also suggest that MAP kinase MEK, the transcription factors E2F‐1 and p53, and PKB/Akt are involved in this process. These findings may have therapeutic relevance for the targeted treatment of various malignancies.

effects [5]. CRF1 is expressed in the central nervous system (CNS) and the anterior pituitary gland, while CRF2 has three splice variants, CRF2-a, b and c, the latter being only expressed in the human brain [4] CRF2-a is expressed throughout the CNS, while CRF2-b is preferentially expressed in peripheral tissues, such as the heart, skin, and skeletal muscle [6]. CRF receptors are also present in various human cancers, such as melanomas, small-cell lung cancer, and neuroblastoma [3].
In 1995 a new mammalian member of the CRF family was described by Vaughan et al. [7]. The peptide named urocortin is related to CRF and urotensin. Three main types of urocortin are known, urocortin1 (UCN1), urocortin2 (UCN2, also known as stresscopin-related peptide), and urocortin3 (UCN3, or stresscopin). These neuropeptides are synthesized in the central nervous system, they have varying distribution patterns and are also detectable in numerous peripheral tissues [8]. The in vitro binding affinities of UCN1 to CRF1 and CRF2 are essentially equal and it is up to 10-fold higher to CRF1 than the binding of CRF itself to this receptor isoform [7,9]. In addition, CRF, UCN2, and UCN3 all bind with basically equal affinities to CRF2 the latter two ligands also being selective for this receptor isoform [9,10]. In vitro binding affinity, however, cannot be regarded as a direct indicator of in vivo efficacy as also demonstrated, for example, by Hasdemir et al. [11]. In addition, CRFbinding protein (CBP) is an important regulator of CRF and UCN1 activity. It is commonly regarded as an endogenous buffer for CRF and UCN, regulating ligand availability for CRF receptors [8]. Members of the GPCRs' families can bind to various heterotrimeric G-proteins leading to rather promiscuous signaling patterns [5]. Ligand binding of CRF1 and CRF2 stimulates adenylate cyclase activity, which increases cAMP production that in turn activates Protein Kinase A (PKA) [12,13]. Numerous studies suggest that members of the CRF family exert their effects through Gsa leading to increased cAMP production, but other second messengers and signaling components, such as MAPK pathways are also implicated [14].
In this study, we investigated the effects of human urocortin1 (hUCN1) in culture on the signaling of HeLa cells with special emphasis on the expression and cellular distribution of the CRF receptors, the activating phosphorylation, and intracellular localization of the extracellular signal-regulated kinases (ERK1 and 2). The involvement of Protein Kinase B (PKB)/Akt, and that of p53 was also examined, together with possible biological responses of the cells, like changes in proliferation. In comparison to our previous observations in MCF7 human breast cancer cells [15] we could detect both similarities and important differences in the signaling patterns induced by hUCN1 in HeLa cells, also accompanied by important differences in the elicited biological responses.

Cell culture
HeLa cervical adenocarcinoma cells (purchased from ATCC and generously gifted by Professor J ozsef Szeber enyi) were regularly maintained under standard circumstances (37°C, 5% CO 2 in a humidified atmosphere) in Dulbecco's Modified Eagle's Medium (DMEM) complemented with 10% heat-inactivated fetal bovine serum for 24 h to achieve sufficient adhesion as described earlier in an article published by our research group [15]. The cells were cultured on plastic Petri dishes or plastic Thermanox (Nalgene Nunc International, Rochester, NY, USA) coverslips. The media were routinely replaced by 0.5% heatinactivated horse serum-containing DMEM 1 day before the applied treatments to silence serum-stimulated mitogenic signaling.
Reagents hUCN1 was dissolved in 10% acetic acid according to the instructions of the manufacturer. The MEK inhibitor U0126 and the CRF1-specific inhibitor Antalarmin were dissolved in DMSO, while the CRF2-specific inhibitor Astressin 2b was in distilled water. We also confirmed that the vehicle of the applied agents had no effect on the examined parameters of the experiments, as published in a former article [15]. All chemicals were purchased from Sigma-Aldrich (a member of Merck Group, Budapest, Hungary) unless otherwise stated.

Western blotting
HeLa cells were treated with 10 nM final concentration of hUCN1 for the indicated times or were left untreated as control. The applied inhibitors were added to the culturing media 30 min before the start of hUCN1 treatments at the following final concentrations: U0126 at 20 lM, Antalarmin at 100 nM, Astressin 2b at 100 nM. Upon completion of treatments, the cultures were harvested into ice-cold lysis buffer (50 mM Tris-base, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EGTA, 1 mM Na-orthovanadate, 5 lM ZnCl 2 , 100 mM NaF, 10 lgÁmL À1 aprotinin, 1 lgÁmL À1 leupeptin, 1 mM PMSF, 1% Triton X-100) and were frozen for storage. Subsequent processing included thawing and homogenization by vortexing for 20 s, then the samples were centrifuged at 13 500 9 g and at 4°C for 20 min and the protein content of the supernatant was measured using a Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins (30 lg) were mixed with 49 Laemmli buffer (25 mL 1 M Tris-HCl, pH 6.8, 40 mL glycerol, 8 g SDS, 10 mL 100 mM EDTA, 10 mL 100 mM EGTA, and 1 mL 1% bromophenol blue brought up to 100 mL with distilled water) and boiled briefly to denature proteins. Protein samples were loaded onto 10% polyacrylamide gels and separated according to molecular weight. The proteins were electro-blotted onto PVDF membranes (Hybond-P, GE Healthcare, Buckinghamshire, UK) by using a Transfer Blot Turbo System (Bio-Rad). After the transfer of the proteins, we blocked the membranes in 5% nonfat dry milk (or in 3% bovine serum albumin in the case of phospho-Akt detection) dissolved in TBS-Tween (10 mM Tris-base, 150 mM NaCl, 0.2% Tween-20, pH 8.0). The primary antibodies were also diluted in the blocking solution (anti-CRF1, Fine Biotech, PRC; anti-CRF2, Sigma-Aldrich; phospho-ERK1/2, ERK1/2, pAKT, Akt, E2F-1, and beta-actin, Cell Signaling Technology, Beverly, MA, USA or p53, Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1 : 1000 and incubated overnight. Methodical controls prepared by the omission of the primary antibodies or the use of nonimmune sera of the appropriate species resulted in no signal production as determined in pilot experiments (latter data not shown). To remove unbound antibodies, we washed the membranes five times in TBS-Tween, then incubation followed with a horseradish-peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology) dissolved 1 : 5000 in the blocking solution. Five washes in TBS-Tween removed the unbound antibodies. Enhanced chemiluminescence detection of the target proteins followed (Immobilon Western, Millipore Corporation, Billerica, MA, USA) using a G-box gel documentation system (Syngene, Cambridge, UK). Then, densitometry was performed using the IMAGEJ software (National Institutes of Health, Bethesda, MD, USA).

Data presentation of western blots
As described in detail in an article published earlier by our research group [15], all experiments were repeated at least three times. Data were normalized to the mean of the corresponding control (untreated) group. Significance of differences was determined by GRAPHPAD PRISM 9.5.0 (GraphPad Software, Boston, MA, USA) using one-way (for proteins possessing one isoform) or two-way (for proteins exhibiting two isoforms) ANOVA testing applying the appropriate corrections, as also indicated in the figure legends. P values ˂ 0.05 were considered significant. The relevant intensity differences are marked in the graphs and their corresponding P values are also written into the figure legends. Presented images are representative of series that produced similar results. Densitometric values are displayed by box + whiskers plots indicating median (central horizontal line), min/max values, and mean (with '+' within the box).

Confocal microscopy
HeLa cells were treated in the presence of 10 nM hUCN1 for the indicated times or left untreated as control. As described in our earlier publication [15], treatments were stopped by quickly rinsing in 37°C PBS (1.37 mM NaCl, 0.27 mM KCl, 0.43 mM Na 2 HPO 4 Á7H 2 O, 0.14 mM KH 2 PO 4 , pH 7.4) and then fixing in 4% paraformaldehyde dissolved in PBS (pH 7.4) for 1 h followed at room temperature. Three changes of PBS and three changes of TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) removed the excess fixative. Then, the samples were incubated in 5% nonfat dry milk dissolved in TBS-Triton (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) at 4°C for 1 h under gentle rocking to block nonspecific binding sites. Commercially available anti-CRF1 (Fine Biotech, Wuhan, China) and anti-CRF2 (Sigma-Aldrich) primary antibodies were diluted 1 : 100. The dilution was 1 : 300 in the case of phospho-ERK1/2 (Cell Signaling Technology). The diluent was always the corresponding blocking solution and incubation with the primary antibodies lasted overnight at 4°C under gentle rocking. Unbound antibodies were removed by five washes in TBS. Cy3-conjugated anti-rabbit secondary antibody (Jackson Immuno Research, Cambridgeshire, UK) dissolved in the blocking solution was pipetted onto the samples at 4°C for an hour at a dilution of 1 : 300 for CRF1 and 2 detections and at 1 : 600 to detect phospho-ERK1/2. The omission of the primary antibody served as methodical control resulting in no detectable signal at the applied microscope settings.

Data presentation of confocal micrographs
An Olympus FV-1000 laser scanning system was used for confocal imaging, with a 409, long distance, combined phase contrast-and fluorescence objective (NA:0.75) to capture single optical sections. The confocal aperture was set at 130 lm, excitation of the Cy3 dye conjugated to the secondary antibody happened at 543 nm, and the emission wavelength was 567 nm. Signal integration type was line Kalman. The micrographs shown were taken from series with similar results. All experiments have been repeated at least three times.

MTT assay
Ninety-six-well plates were used to culture HeLa cervical adenocarcinoma cells (as described above) and then treated with 10 nM hUCN1 alone, or also with the CRF1 inhibitor Antalarmin or the CRF2 inhibitor Astressin 2b for 24 h. At the end of the treatments, we added 10 lL of Thiazolyl Blue Tetrazolium Bromide (Merck) dye solution (5 mgÁmL À1 ) dissolved in sterile water to the cells' medium (100 lL per well). Incubation in the cell culture thermostat followed for an additional 4 h. Then, the supernatant was removed and the accumulated formazan product-the amount of which is proportional to the cells' metabolic activity-was eluted by adding a 1 : 1 mixture of 100 lL DMSO-ethanol. The absorbance was determined at 595 nm using an ELISA microplate reader (FLUOstar OPTIMA, BMG Labtech, Ortenberg, Germany).

Data presentation of MTT assay
One-way ANOVA testing applying Tukey's corrections was used to determine the significance of differences. All experiments were repeated at least three times with samples prepared in triplicates. P values ˂ 0.05 were considered significant, which is also indicated in the figure legends. The results are the mean + SD for experiments performed independently.

CRF receptor expression in HeLa cells
We examined the expression of CRF receptors in HeLa cells by western blotting using commercially available antibodies that specifically recognize CRF1 and 2 subtypes. As shown in Fig. 1, we could detect both CRF1-and CRF2-specific immunoreactivities at the expected positions on western blots (Fig. 1).

Localization of CRF1 and CRF2 in HeLa cells by confocal microscopy
CRFR-specific antisera were also used to detect the intracellular localization of CRF receptor isoforms in HeLa cells. The secondary antibody recognizing the primary was conjugated to Cy3 fluorophore.
Methodical control was prepared by the omission of the primary antibodies. As shown by Fig. 2A,B, both CRFR immunoreactivities were distributed throughout the cytoplasm.

Time kinetics of hUCN1-induced activating ERK1/ 2 phosphorylation in HeLa cells
To test the possible cell proliferation-regulating effect of hUCN1, we examined the activating phosphorylation of ERK1/2 in HeLa cells induced by the peptide. Based on our previously published findings [15] the final concentration of hUCN1 was adjusted to 10 nM. In timecourse experiments, 10 nM of hUCN1 could induce a marked, transient ERK1/2 phosphorylation peaking 15 min following the start of the treatment. After it, the signal fell back rapidly to around baseline (Fig. 3).

Dose-dependence of hUCN1-induced ERK1/2 phosphorylation in HeLa cells
To see whether 10 nM of hUCN1 was indeed the most effective concentration for the treatment of HeLa cells, dose-response experiments were performed with various dilutions of hUCN1. Compared with the control group 10 nM of human urocortin1 could induce the most robust ERK1/2 phosphorylation 15 min after the start of the treatment, while other dilutions of hUCN1 proved to be less effective (Fig. 4).

Inhibition of MEK in hUCN1-treated HeLa cells
In order to examine the role of MEK (mitogenactivated protein kinase/extracellular signal-regulated kinase that activates ERK1/2) in hUCN1-induced ERK1/2 phosphorylation, a MEK inhibitor chemical compound was used in Hela cells. Pretreatment of the cells with U0126 could completely abolish the ERK1/2activating effect of hUCN1. Compared with the control group the inhibitor alone could reduce even basal EKR1/ 2 phosphorylation to below detectable. Based on these results MEK appears to play a pivotal role in hUCN1induced ERK1/2 phosphorylation in HeLa cells (Fig. 5).

Intracellular localization of phosphorylated ERK1/ 2 (pERK1/2) in hUCN1-treated HeLa cells
We also investigated the intracellular localization of pERK1/2 by immunofluorescence and laser scanning confocal microscopy. In untreated control samples, we could detect a barely noticeable phospho-ERK1/2 signal in the cytoplasm of individual HeLa cells. Fifteen minutes after the start of 10 nM hUCN1 treatment, a stronger pERK1/2 signal was apparent compared with the control group and its localization was also cytoplasmic. By the end of the treatment day, a weaker, cytoplasmic pERK1/2 signal could still be detected matching the kinetics of the pERK1/2 signal detected on western blots (Fig. 6).

Selective inhibition of CRF1 and CRF2
The involvement of CRF receptors in hUCN1-induced ERK1/2 phosphorylation was examined by the addition of selective chemical inhibitors of CRFR isoforms. Pretreatment of the cells with the CRF1-specific inhibitor Antalarmin (Ant) could significantly reduce the activating phosphorylation of ERK1/2 induced by 10 nM hUCN1 (Fig. 7).
In contrast to the previous observation, the CRF2specific inhibitor Astressin 2b (A2b) was not able to reduce the ERK1/2 phosphorylating effect of hUCN1 to the degree of measurable significance (Fig. 8).
hUCN1 stimulates the expression of E2F-1 protein Human urocortin could also increase the expression of the E2F-1 transcription factor, a pivotal activator of S-phase genes. Twenty-four hours after the start of hUCN1 treatment, a marked elevation in the level of E2F-1 could be detected by western blotting, while administration of the MEK inhibitor U0126 significantly reduced this effect of hUCN1 (Fig. 9).

Increased PKB/Akt phosphorylation by hUCN1
In time-course experiments we also examined the activating phosphorylation of the serine/threonine-specific enzyme PKB/Akt, a central regulator of cell survival. One hour after the start of hUCN1 treatment a significant increase in PKB/Akt phosphorylation was detected, then the signal intensity fell rapidly back to around baseline (Fig. 10).

hUCN1 decreases the level of p53 in HeLa cells
We examined the possible contribution of the cell cycle regulatory protein p53 in hUCN1-stimulated signaling pathways in lysates of HeLa cells. As indicated in Fig. 11, 10 nM hUCN1 could induce a significant, sustained decrease in the level of p53 at various time points (5 0 -6 h), then the signal intensity returned to around baseline by the end of the treatment day (Fig. 11).

Increased proliferation of HeLa cells measured by MTT assay
The MTT cell viability and proliferation assay was used to examine whether hUCN1 had any effect on the proliferation of HeLa cells. The cultures were kept in their regular culturing medium as described above or in a medium supplemented with 10 nM hUCN1. In the presence of the peptide, a significant increase in MTT production was measured 24 h after the start of hUCN1 treatment when compared to values without hUCN1 treatment. This effect of 10 nM hUCN1 could be blocked using the CRF1-specific inhibitor Antalarmin, while Astressin 2b, a chemical inhibitor of CRF2, could not prevent the hUCN1-stimulated increase in cell proliferation (Fig. 12).

Discussion
Urocortin members of the CRF ligand family are expressed throughout the CNS and also in peripheral organs, such as the heart, placenta, and gastrointestinal tract [16]. The effects of CRF family neuropeptides mediated through specific CRF receptors are extremely diverse, ranging from the regulation of stress responses, or anxiety, via influencing feeding behavior, to the modulation of cardiovascular and immune reactions [16,17]. Various actions of CRF neuropeptides and their receptors can also be important regulators of tumor development (see references from [15]). Urocortin, CRF, and their receptors have been detected in various human tumors, such as in breast cancer, glioma cell lines, and others [17,18]. According to Wang and Li [19], urocortin can inhibit the growth of tumor cells by activating CRF1. The MAPK isoforms ERK1 and 2 are key regulators of cell proliferation both in healthy and malignant cells. CRF receptors are coupled to Gas that can stimulate adenylate cyclase, which in turn increases intracellular cAMP production [20]. In neuronal cells the activating phosphorylation of the MAP kinase cascade through CRF receptors occurs by the stimulation of the cAMP/PKA pathway [21], while in mouse cardiomyocytes and Chinese hamster ovary cells MAPK activation is PKA-independent [20], indicating a cell-typespecific process. Other proteins could also be involved in the activation of ERK1/2 MAPK, such as PKB/ Akt, PKC, and Raf-1 [22]. In human pregnant myometrial cells, urocortin stimulates the phosphorylation of ERK1/2 MAP kinase, but the activating phosphorylation can be prevented in the presence of the MEK inhibitor U0126 [23]. A similar pattern was also  . The inhibitor could significantly reduce both baseline and hUCN1-induced ERK1/2 phosphorylation. Reprobing of the membrane for total ERK1/2 served as loading control. The phosphorylated/total signal ratio was used to determine relative protein phosphorylation. The values shown are presented by box + whiskers plots, indicating median (central horizontal line), min/max values, and mean (with '+' within the box) of five independently performed experiments (n = 5). *P < 0.05 Control vs. hUCN1 (10 nM); #P < 0.05 hUCN1 (10 nM) vs. U0126 + hUCN1 (10 nM); @P < 0.05 Control vs. U0126 (two-way ANOVA, Tukey). described in our previous work [15] in MCF7 breast cancer cells and is supported by the present study likewise. Urocortin also protects the isolated rat heart against ischaemic and reperfusion injury in a MAPK-dependent manner [21] by activating the phosphorylation of ERK1/2 [22]. Others have reported that urocortins (1, 2, and 3) can protect the rat heart from reperfusion injury not only in vitro but also in vivo via upregulation of the MAPK EKR1/2 signaling pathway [24,25].
MAPKs are central regulatory elements of cell division signaling. In our current experiments, hUCN1 could maximally stimulate the activating phosphorylation of the MAPK ERK1/2 at a concentration as low as 10 nM 15 min after the start of the treatment, then the signal rapidly fell back to baseline. Active, phosphorylated ERK1/2 immunoreactivity could be detected predominantly in the cytoplasm by means of fluorescence microscopy. Transient cytoplasmic ERK1/2 activation is correlated with increased cell proliferation in PC12 rat pheochromocytoma tumor cells (for references see [15]). Pretreatment of HeLa cells with the MEK inhibitor U0126 could completely abolish the ERK1/2-phosphorylating effect of the peptide, pointing to a central role of MEK in hUCN1induced ERK1/2 activation of HeLa cells.
In the Ishikawa endometrial cell line, CRF1 mediates antiproliferative actions, while in peripheral blood lymphocytes, it supports apoptosis [18]. In CRF receptorexpressing HL-1 cardiac cells urocortin-induced ERK1/ 2 phosphorylation can be inhibited either by the CRF1specific chemical inhibitor Antalarmin or by the specific CRF2 antagonist antisauvagine-30 [26]. The ERK1/2- Fig. 7. The effect of CRF1 inhibition on hUCN1-induced ERK1/2 phosphorylation by western blotting. HeLa cells were pretreated with the CRF1-specific antagonist Antalarmin at 100 nM final concentration. Inhibition was started 30 min before the urocortin treatment and was continued during the last 15 min (altogether 45 min) in the simultaneous presence of 10 nM hUCN1 (to induce maximal ERK1/2 phosphorylation). Compared with the hUCN1-treated group Ant could markedly reduce hUCN1-stimulated ERK1/2-activating phosphorylation. Reprobing of the membrane for total ERK1/2 served as loading control. The phosphorylated/total signal ratio was used to determine relative protein phosphorylation. The values shown are presented by box + whiskers plots, indicating median (central horizontal line), min/max values, and mean (with '+' within the box) of three independently performed experiments (n = 3). *P < 0.05 Control vs. hUCN1 (10 nM), #P < 0.05 hUCN1 (10 nM) vs. Ant + hUCN1 (10 nM; two-way ANOVA, Tukey). Fig. 8. The effect of CRF2-inhibition onto hUCN1-induced ERK1/2 phosphorylation by western blotting. Pretreatment of cells with the CRF2-selective antagonist A2b at 100 nM final concentration was started 30 min before the hUCN1 treatment and continued during the last 15 min (altogether 45 min) in the simultaneous presence of 10 nM hUCN1 (to induce maximal ERK1/2 phosphorylation). A2b could not markedly reduce the ERK1/2-phosphorylating effect of 10 nM hUCN1, significantly increased ERK1/2 phosphorylation by hUCN1 was observable even in the presence of the inhibitor. Reprobing of the membrane for total ERK1/2 served as loading control. Relative protein phosphorylation was determined by the phosphorylated/total signal ratio. The values shown are presented by box + whiskers plots, indicating median (central horizontal line), min/max values, and mean (with '+' within the box) of three independently performed experiments (n = 3). *P < 0.05 vs. Control (two-way ANOVA, Tukey).
phosphorylating effect of urocortin can also be blocked by Astressin 2b, a chemical inhibitor of CRF2, as it was experienced in rat esophageal cells [27].
Stimulation of CRF1 by its ligands CRF or UCN1 is an important initial step in stress conditions like anxiety, depression, or inflammatory diseases of the intestinal tract. Hence, the inhibition of the receptor's activity is often favorable in experimental models of these conditions, and deciphering the cellular and molecular reactions in the background is of potential medical relevance. We could detect the presence of CRF1 and 2 by western blotting and immunofluorescence microscopy in HeLa cells using commercially available CRFR isoform-specific antibodies. In our earlier study with MCF7 human breast carcinoma cells [15], like in the work of others [10,11] CRF1 immunoreactivity was localized mostly to and along the plasma membrane. The CRF2 immune signal, on the contrary, had a broader distribution pattern with a more granular appearance and not only along the cell surface but reaching deeper into the cytoplasm as well. In HeLa cells, at the same time, the immune signal of both CRF1 and 2 was detectable not just along the cell surface but also reaching deeper into the cytoplasm. In this cell type, the signal pattern contained bigger granules in the case of CRF1 and finer ones for CRF2. Since the culturing conditions and all steps of the methodology were identical during the fluorescence microscopic work with both cell lines (MCF7 and HeLa), the observed differences in signal distribution (CRF1 in MCF7 closer to the membrane while CRF1 in HeLa and CRF2 also deeper in the cytoplasm of both cell lines)-we attribute to cell-type-specific features. We have not examined the potential  heterodimerization of CRF1 and 2 receptors, which can also have a serious effect on their subcellular localization [10].
The general experience in stress signaling is that CRF1 mediates mainly stress-adaptive responses, while signals originating from CRF2 mostly dampen the effects of CRF1 receptor and that way ameliorate stress reactions of biological systems [28,29]. Pretreatment of HeLa cells with the CRF1 inhibitor Antalarmin could significantly decrease the ERK1/2phosphorylating effect of hUCN1, indicating the importance of CRF1 receptor in hUCN1-induced ERK1/2 phosphorylation in this cell type. On the contrary, we could not detect a statistically significant inhibitory effect in the presence of the CRF2 inhibitor Astressin 2b during hUCN1 treatment of HeLa cells. Furthermore, we examined the effect of 10 nM hUCN1 treatment on the proliferation of HeLa cells by MTT assay. Our results showed a significant increase in MTT production 24 h after the start of hUCN1 treatment. The cell proliferation-stimulating effect of hUCN1 could also be inhibited using Antalarmin, a specific inhibitor of CRF1, while pretreatment with CRF2 inhibitor Astressin 2b did not show such an effect-a striking difference compared with that seen earlier with MCF7 cells, where both receptor inhibitors could reduce MTT production [15]. The observed differences are pointing to important celltype-specific deviations in urocortin's signaling mechanism of action. The converted MTT tetrazolium compound is used to determine the number of healthy, viable cells. Based on our results we can conclude that 24 h of 10 nM hUCN1 treatment increased the number of live HeLa cells, which could be inhibited by  Antalarmin, indicating the significance of CRF1 in this signaling process induced by urocortin in HeLa cells.
We tried to decipher the molecular background of the observed increase in cell proliferation by measuring the expression of E2F-1, the pivotal activator of Sphase genes. We could detect a significant increase in the level of E2F-1 24 h after the start of hUCN1 treatment by western blotting. Meanwhile, the MEK inhibitor U0126 could inhibit the E2F-1-stimulating effect of hUCN1, suggesting the involvement of MEK in these urocortin-stimulated signaling steps.
Apoptosis is another phenomenon that is frequently mediated via CRF receptors. According to Tsatsanis et al. [30], urocortin can induce apoptosis in macrophages via CRF2, including the activation of central pro-apoptotic proteins, like Bad and Bax. On the contrary, in the RM-1 mouse prostate cancer cell line, the presence of urocortin did not support apoptosis, while CRF had the opposite effect [31]. The cardioprotective actions of urocortin2 against ischemia-reperfusion (I/ R) are well known, and at the same time, it decreases apoptosis in Wistar rats [32]. CRF also suppressed apoptosis in Y79 human retinoblastoma cells by inhibiting the proteolytic cleavage and activation of procaspase 3 [33].
Based on the above data, we tried to determine the contribution of the cell cycle-and cell survivalÀ/ apoptosis-regulator protein p53 to hUCN1-stimulated signaling events in HeLa cells. Data generated by western blots showed a significant decrease in p53 expression in the presence of 10 nM hUCN1, then the signal intensity rose back to around baseline 24 h after the start of the treatment. In an earlier study [15] treatment of MCF7 cells resulted in significantly increased expression of p53. The differing effects of hUCN1 on p53 expression again underline the importance of celltype-specific differences in urocortin signaling under the applied settings.
In this direction, we could also detect the activating phosphorylation of the key cell survival-regulator PKB/Akt 1 h after the start of hUCN1 treatment. hUCN1-induced activation of the enzyme and the decreased p53 level may both contribute to the increased survival of HeLa cells. An additional important factor worth considering is that HeLa human cervical carcinoma cells used in the current study similar to the MCF7 human ductal breast carcinoma cell line used in our previously published work [15] are both of female origin. Furthermore, in HeLa cells of cervical cancer origin, the E6 and E7 early proteins of human papilloma viruses interfere with the normal function of key signaling and cell cycle-controlling proteins like that of retinoblastoma and p53 [38][39][40][41]. Therefore we should keep sex [42] and the presence of potent signal-distorting components of viral origin as important possible factors in mind when interpreting our data and their potential pathological and therapeutic relevance.

Conclusions
Our results showed the presence of both CRF1 and 2 immunoreactivities in the cytoplasm of HeLa cells using isoform-specific antisera and laser scanning confocal microscopy. We could detect transient ERK1/2 phosphorylation peaking at 15 min in the presence of 10 nM hUCN1. This effect of hUCN1 could be prevented by the MEK inhibitor U0126 suggesting the pivotal role of the MAPK kinase in hUCN1-induced ERK1/2 activation. We also demonstrated that the ERK1/2 phosphorylating effect of 10 nM hUCN1 was initiated via CRF1, while the participation of CRF2 in this process is not confirmed by our data. hUCN1 treatment increased the expression of the E2F-1 transcription factor, a key regulator of S-phase genes supporting cell proliferation. This phenomenon could be blocked by U0126, the chemical inhibitor of MEK emphasizing the importance of the enzyme in hUCN1stimulated signaling pathways. At the same time, urocortin increased the overall production of MTT formazan crystals in HeLa cells, which could be inhibited by the CRF1-specific inhibitor Antalarmin, but not by Astressin 2b the inhibitor of the other main CRF receptor isoform. Interestingly, in MCF7 cells [15] both receptor isoform-specific inhibitors could reduce MTT production to a significant degree. The elevated expression of E2F-1 and the increased production of formazan dye in the presence of urocortin are both suggestive of increased HeLa cell proliferation. While the activating phosphorylation of PKB/Akt and the decreased expression of p53 upon hUCN1 treatment support increased cell survival in the same cell type. In view of our data, we can conclude that the effect of hUCN1 in HeLa cells is mediated via CRF1 and MEK towards ERK1/2 while MEK is also critical for the induction of increased E2F-1 expression, supporting increased cell proliferation. We believe that our results can help a better understanding of how urocortins exert their cell proliferation-regulating effects in a celltype-specific manner and it also sheds light onto their potential role in cancer formation. The above-revealed cell-type-specific aspects of urocortins' signaling may also have therapeutic relevance to targeted treatments of various malignancies in the future.