TLQP-21 (TLQPPASSRRRHFHHALPPAR), YATL-23 (YATLQ PPASSRRHFHHALPPAR), TLQP-62 (TLQPPASSRRRHFHHA LPPARHHPDLEAQARRAQEEADAEERRLQEQEELEN-YIEH VLLHRP), TLQP-8 (TLQPPASS), HFHH-10 (HFHHALPP AR), HHPD-41 (HHPDLEAQARRAQEEADAEERRLQEQEELE NYIEHVLLHRP), and LRPS-21 (LRPSHTRPAHQSFARP LHRPA) have been synthesized by us using conventional solid phase synthesis.

Cyclosporine A (CsA), thapsigargin (TG), U73122, 2-Aminoethyl diphenylborinate (2-APB), SKF-96365, YM-58483, and EGTA were purchased from Sigma–Aldrich (St Louis, MO, USA). Unless otherwise specified, all other reagents were from Sigma–Aldrich.

CHO cells were cultured in HAM’S F12 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS), 100 IU/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (all Euroclone, Pero, Italy) under standard cell culture conditions (at 37°C, in 5% CO₂).

CHO cells were plated at 20.000 cells/well into black walled, clear bottom 96-well plate (Corning, Germany) and cultured one day up to 80–90% of confluence. Prior to assay, cells were incubated in dark conditions with 100 μl of Hank’s Balanced Salt Solution (HBSS) containing 20 mM HEPES, 2.5 mM probenecid and 4.5 μM FLUO-4 NW (Molecular Probes, Eugene, OR, USA) at 37°C and 5% CO₂ for 45 min. Fluorescence emissions were measured with the multilabel spectrophotometer VICTOR³ (Perkin Elmer, MA, USA) at 485/535 nm (excitation/emission filters) every 0.5 s for the 20 s preceding and the 60 s following the stimulation. TLQP-21, TLQP-62, TLQP-8, HHPD-41, HFHH-10, and LRPS-21 were diluted in HBSS solution and injected into the wells by an automated injector system. The nature of Ca²⁺ stores involved in TLQP-21 action was investigated preincubating cells with TG (2 μM, 20 min), an inhibitor of the Ca²⁺-ATPase pump responsible for sequestering Ca²⁺ in the ER (Feng et al., 2008), or CsA (2 μM, 15 min), an inhibitor of the mitochondrial permeability transition pore (PTP) (Liantonio et al., 2007). To ascertain whether phospholipase C (PLC) and inositol trisphosphate receptors (IP₃R) were involved in TLQP-21 mechanism of action, cells were incubated with a specific PLC inhibitor (U73122, 10 μM for 10 min) and a IP₃R antagonist (2-APB, 75 μM for 15 min) before the injection of TLQP-21 (Macmillan and McCarron, 2010; Li et al., 2013). Depletion of Ca²⁺ from the ER leads to Ca²⁺ entry from outside the cell by activation of Store-Operated Channels (SOCs). Again, to assess the involvement of this pathway, CHO cells were incubated with selective antagonists of this pathway (SKF-96365 and YM-58483 10 μM for 20 min, EGTA 1 mM for 30 min) before the injection of TLQP-21 (Ishikawa et al., 2003; Li et al., 2013).

Binding of TLQP-21 to crude membranes (30,000 × g pellet) obtained from CHO cells was carried out using [¹²⁵I]-YATL-23 as ligand. YATL-23 was radioiodinated (specific activity, 2000 Ci/mmol) using the lactoperoxidase method (Muccioli et al., 1998; Muccioli et al., 2001) by Perkin Elmer (Milan, Italy) and purified by reverse phase high-performance liquid chromatography. For the single point binding assay, cell membranes (corresponding to 100 μg membrane protein) were incubated in triplicate at 23°C, unless otherwise noted, for 4 h under constant shaking with 0.5 nM [¹²⁵I]-YATL-23 in a final volume of 0.5 ml assay buffer (50 mM Tris, 2.5 mM EGTA, 0.002% bacitracin, 0.1% bovine serum albumin, titrated to a final pH of 7.4 with HCl). Non-specific binding was measured in parallel incubations with 1 μM unlabelled YATL-23 to displace the radioligand. Similar results were obtained using TLQP-21 to displace the radioligand. The binding reaction was terminated by the addition of ice-cold assay buffer, followed by rapid filtration through Whatman GF/B filters as previously reported (Muccioli et al., 1998) and the radioactivity bound to membranes was measured by a Packard auto-γ-counter. Specific binding-values were calculated as the difference obtained subtracting non-specific from total binding. Specific binding was expressed as a percentage of the total radioactivity added. For saturation binding studies, cell membranes were incubated with various concentrations of the radioligand (0.03–4 nM). Competition studies were performed by incubating cell membranes (150 μg/tube) with 1 nM [¹²⁵I]-YATL-23 with or without various concentrations (from 10 pM to 0.1 μM) of unlabelled YATL-23, TLQP-21, or LRPS-21. Data were plotted and curves fit using the GraphPad Prism software version 4 (GraphPad Software, San Diego, CA, USA) assuming that the binding was due to a single class of binding sites, thus allowing determination of the maximum binding capacity (B_max), dissociation constant (K_d), Hill slope and concentration of the competitor causing 50% inhibition (IC₅₀) of specific radioligand binding.

CHO cells were plated 24 h before time course experiments in 35 mm dishes at 70% confluence. After three washes with medium w/o serum, cells were serum-starved for 1 h. Time course experiments were performed and after quick removal of the medium the reaction was stopped by placing the dish on ice and adding 100 μl of ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA) containing a protease inhibitor cocktail and PhosphoStop inhibitor cocktail (Roche Diagnostic, Mannheim, Germany).

Cells were stored at –80°C until further processing. Cells were harvested and equivalent amounts of cell extracts (corresponding to approximately 200.000 cells) were run on NuPAGE precast 4–12% gradient gels (Invitrogen, USA) and transferred to a polyvinylidene difluoride (PVDF) membrane (Amersham). After staining with Ponceau S to verify uniformity of protein load/transfer, membranes were analyzed for immunoreactivity. Incubation with primary antibodies (anti-phospho-AMPK (Thr172); anti-phospho-AKT (Ser473); anti-phospho-ERK1/2 (Thr202/Tyr204); anti-phospho-PKC (Ser660); anti-phospho-PLA2 (Ser505); anti-phospho-JNK (Ser63); and anti-phospho-PLCγ1 (Tyr783) rabbit polyclonal antibodies; Cell Signaling Technology, Danvers, MA, USA) at 1:1.000 dilution was performed overnight at 4°C (Petrocchi et al., 2010). Incubation with peroxidase-coupled secondary antibodies (Amersham, Arlington Heights, IL, USA; now GE; 1:5.000) was performed for 1 h at room temperature. Immunoreactivity was developed by enhanced chemiluminescence (ECL system; Amersham GE Healthcare, UK). Two parallel gels were run for each experiment and with probes for different anti-phosphorylated protein antibodies as indicated, avoiding stripping protocols. The gels were normalized with anti-β-actin monoclonal antibodies (Sigma). β-actin was chosen since we have previously observed that its levels remained stable in time, wherease those of α-tubulin decreased over time (Petrocchi et al., 2010).

Values are expressed as mean ± SEM. The statistical significance of differences between groups was evaluated with Tukey–Kramer’s t-test for multiple comparisons, preceded by the analysis of variance (ANOVA). Where appropriate, F-values and degrees of freedom (DF) are indicated in the legend of figures. A P-value of less than 0.05 was considered significant.

CHO cells were incubated in vitro with increasing concentrations (1 nM–10 μM) of TLQP-21. TLQP-21 (0.1 μM–10 μM) evoked acute and significant increases (P < 0.05) in intracellular Ca²⁺ levels in CHO cells (Figure 1A); by comparison, LRPS-21 tested at the concentration interval of 1 nM–10 μM did not stimulate Ca²⁺ levels in CHO cells (Figure 1A) thereby confirming the specificity of TLQP-21 action.

In the next series of experiments, we investigated whether CHO cells could respond to repeated TLQP-21 stimulations given at 5 min intervals from each other. Levels of free intracellular Ca²⁺ increased sharply upon the first challenge with 10 μM TLQP-21, whereas 5 min later a second application of 10 μM TLQP-21 induced only a blunted increase in cell fluorescence; no effects were observed when 5 min later 10 μM TLQP-21 was applied a third time (Figure 1B). A final challenge with 10 μM ATP to check for cell viability was not affected (data not shown).

To gain further insight into the biological activity of VGF-derived peptides, we stimulated CHO cells with different peptides derived from the C-terminal region of VGF (Table 1). Natural processing of TLQP-62 by prohormone convertases yields TLQP-21 and HHPD-41 (Table 1). The 1 μM concentration has been chosen to compare the effects of TLQP-62 and its fragments on intracellular Ca²⁺ levels since this concentration was close to the EC50 of TLQP-21 and could have allowed to measure whether a test compound was less or more active than TLQP-21. At concentrations as large as 1 μM TLQP-62 failed to stimulate any significant increase in intracellular Ca²⁺ levels (Figure 2). Similarly, HHPD-41 was also ineffective, whereas TLQP-21 significantly stimulated intracellular calcium mobilization (Figure 2). TLQP-21 itself is a substrate for prohormone convertases to yield TLQP-8 (8 amino acids at the N-terminal of TLQP-21) and HFHH-10 (10 amino acids at the C-terminal of TLQP-21). HFHH-10 effectively stimulated an intracellular Ca²⁺ increase in CHO cells; by comparison, TLQP-8 at 1 μM induced only a slight and non-significant increase in intracellular Ca²⁺. These results strongly suggest that the C-terminal region of TLQP-21 is the sequence primarily involved in the stimulation of its receptor on CHO cells (Figure 2).

The addition of Tyr-Ala (YA) amino acidic residues to TLQP-21 resulted in a peptide (YATL-23) with the same in vitro biological activity of TLQP-21 in CHO cells (data not shown).

Given the evidence of intracellular Ca²⁺ mobilization induced by TLQP-21 in CHO cells, we investigated the presence of specific TLQP-21 binding sites on CHO cell membranes by radioreceptor assay using [¹²⁵I]-YATL-23 as a ligand. The specific binding of [¹²⁵I]-YATL-23 to cell membranes varied with both incubation time and temperature. Specific binding increased with the duration of incubation and was greater at 23°C when compared to 4°C after reaching equilibrium at 4 h (Figure 3A). A study of specific binding as a function of membrane protein concentration indicated that the binding was proportional to protein content up to at least 100 μg/tube (Figure 3B). Specific binding of [¹²⁵I]-YATL-23 to cell membranes occurred over a relatively wide range of pH. The maximal binding was observed at pH 7.4 and declined to half-maximal value at pH 5.0 and 9.0 (Figure 3C). Therefore, all subsequent incubations were carried out at pH 7.4 for 4 h at 23°C with 100 μg membrane protein. Brief exposure of CHO cell membranes to high temperature (100°C × 1 min) or enzymes that disrupt protein structure such as trypsin (50 μg/ml × 5 min) caused the loss of the specific binding of [¹²⁵I]-YATL-23, suggesting that protein integrity is functionally important in the binding site (Figure 3D).

Experiments using various concentrations of [¹²⁵I]-YATL-23 indicated the presence of a saturable and specific binding associated with minor low non-specific binding (Figure 4A). Scatchard transformation of the specific binding data (Figure 4B) yielded a linear Scatchard plot with Hill slope close to 1 (Figure 4C), indicating the existence of a single class of binding sites. The calculated K_d and B_max values (means ± SEM of three independent experiments) were 0.55 ± 0.05 × 10^-9 M and 81.7 ± 3.9 fmol/mg protein, respectively, and the Hill slope was 1.07 ± 0.1.

Unlabelled YATL-23 and TLQP-21 competed in a concentration-dependent manner with [¹²⁵I]-YATL-23 for binding sites in CHO membranes (Figure 5). The IC₅₀ values, calculated according a one-site binding model (means ± SEM of three independent experiments), were 1.2 ± 0.06 × 10^-9 M for YATL-23 and 0.98 ± 0.06 × 10^-9 M for TLQP-21. The results of these competition binding studies also revealed that the binding of [¹²⁵I]-YATL-23 to CHO membranes was specific and was not inhibited by LRPS-21, the scrambled peptide made using the same amino acidic residues of TLQP-21 (Figure 5). These findings provide evidence that CHO cells contain significant amounts of TLQP-21 binding sites showing typical features of ligand-receptor interaction.

It is known that Ca²⁺ stores, such as ER and mitochondria, dynamically participate in generation of cytoplasmic Ca²⁺ signals (Verkhratsky and Petersen, 1998). We therefore studied the possible implication of these intracellular organelles in the increase of intracellular Ca²⁺ levels induced by TLQP-21. The involvement of the ER was investigated using TG, which inhibits the Ca²⁺-ATPase pump responsible for sequestering Ca²⁺ in the ER and depletes the store by irreversibly preventing its refilling. As shown in Figure 6A, TG reduced the TLQP-21-mediated increase of intracellular Ca²⁺, causing a 56% reduction after 20 min preincubation time. To rule out the possibility that Ca²⁺ release from the mitochondria could also be involved, we evaluated the effect of TLQP-21 in the presence of CsA, an inhibitor of the mitochondrial PTP (Chernyak, 1997). The incubation of CHO cells with 2 μM CsA did not modify the basal levels of intracellular Ca²⁺ and a subsequent stimulation with 1 μM TLQP-21 induced a significant increase in intracellular Ca²⁺ levels (Figure 6A). These results indicate that TLQP-21 stimulated Ca²⁺ release primarily from the ER store, whereas release of Ca²⁺ from the mitochondria appeared not involved in this mechanism of action.

In many cellular systems, PLC activation and subsequent IP₃ production is the transduction pathway regulating the release of Ca²⁺ from ER. U73122 is an aminosteroid that is reported to act as a specific inhibitor of PLC (Bleasdale et al., 1990) and it is widely used as a quick test for the involvement of PLC in a signaling pathway. To test whether TLQP-21 induces Ca²⁺ mobilization through a PLC-dependent mechanism, CHO cells were incubated for 10 min with 10μM U73122. Results demonstrate that U73122 induced a dramatic reduction, about 82%, of TLQP-21 stimulation of intracellular Ca²⁺ mobilization (Figure 6B). As a further confirmation, CHO cells were pre-incubated for 15 min with 75 μM 2-APB, which rapidly inhibits IP₃R-mediated Ca²⁺ release. Treatment with 2-APB strongly affect TLQP-21 activity, causing a decrement of intracellular Ca²⁺ mobilization of about 79% (Figure 6B). As previously reported, depletion of Ca²⁺ from the ER causes the activation of stromal interaction molecule (STIM) proteins that, translocating into junctions formed between the ER and the plasma membrane (PM), activate the highly calcium-selective Orai channels to homeostatically balance intracellular calcium (Soboloff et al., 2012). To assess the role of this pathway, CHO cells were pretreated with a STIM-mediated Ca²⁺ inhibitor (SKF-96365), an inhibitor of Orai channels (YM-58483) or an extracellular Ca²⁺ chelator (EGTA) before stimulation with 1 μM TLQP-21. We observed a statistically significant decrement in TLQP-21-mediated intracellular Ca²⁺ mobilization (Figure 6C), of about 36, 27, and 45% after treatment with SKF-96365, YM-58483, and EGTA, respectively.

Next, we measured the effects of TLQP-21 treatment on phosphorylation of intracellular signaling effectors. After 1 h of serum starvation, cells were exposed to 10 μM TLQP-21 for 0–30 min (Figure 7). The 10 μM concentration was chosen because it elicited a stimulation near to maximal on intracellular Ca²⁺ mobilization (Figure 1). TLQP-21 induced a prompt increase of phospho-AKT (1–5 min) (Figure 7A) that remained significantly higher than basal until 15 min after stimulation (Figure 7A). A rapid and transient dephosphorylation of phospho-AMPK and phospho-PLCγ1 occurred between 1 and 10 min after stimulation with TLQP-21(Figures 7B,C). A similar trend was seen also for phospho-PLA2, but dephosphorylation reached statistical significance only at 2 min (Figure 7D). Interestingly, PKC phosphorylation increased after 2 min and lasted until 15 min from stimulation with TLQP-21 (Figure 7D). ERK1/2 phosphorylation increased significantly at 2 min, reached a peak at 5 min and thereafter returned to basal (Figure 7F). No effects were induced on phospho-JNK levels (data not shown). A possible limitation of these determinations is that sample loading was normalized using the corresponding β-actin levels and not the levels of the unphosphorilated protein.