In Vitro Activities of Kissorphin, a Novel Hexapeptide KiSS-1 Derivative, in Neuronal Cells

The primary products of the metastasis-suppressor KiSS-1 gene are the kisspeptin (KP) peptides that stimulate gonadotrophin-releasing-hormone (GnRH) release via GPR-54 receptor activation. Recent studies have suggested that the KP-10 peptide also activates neuropeptide FF (NPFF) receptors. The aim of the current study was to determine the activities of the KiSS-1 derivative kissorphin (KSO), which contains the first six amino acids of the KP-10 peptide, is C-terminally amidated, and shares amino acid similarities with the biologically active NPFF 3–8 sequence. The KSO peptide inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) production in ND7/23 neuroblastoma cells via an action that could be inhibited by the NPFF receptor antagonist RF9. Release of GnRH by LA-N-1 neuroblastoma cells was not altered by the KSO peptide. In ND7/23 neuroblastoma cells, the KSO peptide was able to reduce forskolin neuroprotection against H2O2 toxicity. The KSO peptide was also able to prevent prostaglandin E2-induced apoptosis in rat cortical neurons. The NPFF receptor antagonist RF9 could inhibit these actions of the KSO peptide in oxidative stress and apoptosis models. In conclusion, the kissorphin peptide, comprising the amino acid sequence Tyr-Asn-Trp-Asn-Ser-Phe-NH2, has NPFF-like biological activity without showing any GnRH releasing activity and inhibits forskolin-activated cAMP release.

The KP peptides themselves play a central role in the hypothalamic-pituitary-gonadal (HPG)-axis by acting on gonadotrophin-releasing hormone (GnRH) neurons to activate GnRH release [9]. The KP peptides are encoded by the metastasis-suppressor KiSS-1 gene and are ligands for the GPR-54 receptor [1,[10][11][12]. A range of KP peptides are found in vitro and in vivo with the largest 54 amino acid form corresponding toresidues 68-121 of the KiSS-1 encoded pre-pro-KP [10]. Shorter derivatives of KP comprising the Cterminal 14, 13, and 10 amino acids have also been found in tissues and corresponding to residues 108-121, 109-121, and 112-121 of the KiSS-1 encoded pre-pro-KP [10,13]. Biological activity of KP peptides requires the KP 112-121 sequence with an amidated C-terminus [10,13,14].
Cleavage of KP peptides to remove the amidated Cterminal tripeptide Leu-Arg-Phe-NH 2 of the KP sequence by matrix metalloproteinase (MMP) enzymes abolishes the activation of GPR-54 by KP peptides [26]. However, the resultant peptides have a C-terminal glycine residue that could be alpha-amidated [27] and C-terminal alpha amidation is a feature of many biologically active neuropeptides. As such the MMP cleaved KP peptides could have an alternative biological activity. In view of the similarities between KP and NPFF sequences it is possible that the hexapeptide fragment of KP-10 containing residues 1-6 with a C-terminally amidated phenylalanine residue could have NPFF-like activity. The aim of the study has been to determine if the KP derivative Tyr-Asn-Trp-Asn-Ser-Phe-NH 2 , termed kissorphin (KSO), has biological activity and to determine whether KSO peptides have NPFF-like activities in neuronal cell culture-based assays.

Materials.
Synthetic NPFF, KP, and KSO peptides were obtained from Bachem. Mouse neuroblastoma x rat neuron hybrid ND7/23 [28] and human LA-N-1 neuroblastoma [29] cell lines were obtained from the Health Protection Agency, Cell Culture Collection. Rat cortical neurons and cell culture media were obtained from Invitrogen. The cAMP EIA kits and other test chemicals were obtained from Sigma-Aldrich.

Inhibition of Forskolin Stimulated cAMP
Assays. ND7/23 cells were plated onto 24-well plates in normal growth medium and cultured overnight. The medium was replaced with serum-free medium containing 100 μM IBMX on the day of the experiment for 2 h prior to the addition of test compounds [3,34]. Cells were then treated with medium alone (control) or medium containing 10 μM forskolin and 100 μM IBMX and varying doses (10 nM-100 μM) of the synthetic NPFF, KP, or KSO peptides. Cells were then incubated at 37 • C for a total of 30 min, after which the medium was aspirated and replaced with a 1 ml of 0.1 M HCl and incubated for 15 min to stop the reaction and lyse the cells.
Cell lysates were centrifuged to remove cellular debris and cAMP levels in the supernatant determined. The ir-cAMP levels were determined using a direct enzyme immunoassay kit according to the manufacturers instructions. Samples and standards were acetylated prior to incubation with anti-cAMP antibody and cAMP-alkaline phosphatase conjugate.

GnRH Release Assay.
LA-N-1 cells were plated onto 24-well plates in normal growth medium and cultured overnight. On the day of experiment, cells were then treated with medium alone (control) or medium containing varying doses (10 nM-10 μM) of the synthetic NPFF, KP, or KSO peptides. Cells were then incubated at 37 • C for a total of 2 hours, after which the medium was aspirated for determination of GnRH levels. The immunoreactive (ir) GnRH was measured by EIA using synthetic biotinyl-GnRH-I, GnRH-I standards, primary anti-GnRH antibody coated immunoplates, and detection with streptavidin labeled HRP and TMB substrate.

Effect of KSO Peptides on NPFF-Like
Biological Activity. The NPFF-like biological activity was determined by measurement of forskolin-stimulated ir-cAMP release in response to KSO, KP, and NPFF peptides. The KSO peptides containing residues 1-6 and 3-6 significantly inhibited forskolin stimulated ir-cAMP release from ND-7/23 cells (Figure 1), whilst the KSO 1-3 fragment had no effect. The NPFF, KP-10, and KP-13 peptides also significantly inhibited forskolin stimulated ir-cAMP release, which could be antagonized by the NPFF receptor antagonist RF-9 [4] but not the GPR-54 receptor antagonist KP234 [39]. The RF-9 and KP234 had no effect on forskolin-stimulated ir-cAMP release.

Effect of KSO Peptides on KP-Like Biological Activity.
The KP-like biological activity was determined by measurement of irGnRH release in response to KSO, KP, and NPFF peptides. The KSO peptides containing residues 1-6, 1-3 and 3-6 had no effect on irGnRH release from LA-N-1 neuroblastoma cells ( Figure 2). The KP-10 and KP-13 peptides both stimulated a significant increase in irGnRH release, which could be antagonized by the GPR-54 receptor antagonist KP234 [39] but not the NPFF receptor antagonist RF-9 [4]. The KSO peptides had no effect on KP stimulated irGnRH release. NPFF, RF-9, and KP234 had no effect on irGnRH release.

Effect of KSO Peptides on H 2 O 2 and PGE 2 Neurotoxicity.
The effects of KSO peptides were tested in two models of neurotoxicity. In the H 2 O 2 -stimulated-toxicity model the KSO 1-6 and 3-6 peptides, plus NPFF and KP-10 peptides, inhibited the forskolin protection against H 2 O 2 toxicity in ND-7/23 cells (Figure 3). These effects could be reversed by the NPFF receptor antagonist RF-9 but not the GPR-54 antagonist KP234. In the PGE 2 -induced-toxicity model the KSO 1-6 and 3-6 peptides, plus NPFF and KP-10 peptides, were able to reduce the PGE 2 toxicity (Figure 4). These effects could be reversed by the NPFF receptor antagonist RF-9 but not the GPR-54 antagonist KP234.

Discussion
The results show that the KSO peptide has biological activity in neuronal cell models that can be antagonized by the NPFF receptor antagonist RF-9 [4] but not the GPR-54 receptor antagonist KP234 [39]. The actions of KSO involve inhibition of stimulated cAMP release and are similar to documented effects for both NPFF [23,40] and KP peptides [1][2][3] in vitro. The NPFF receptors respond to a number of RF-amide containing peptides [41], but this is the first demonstration of a response to an SF-amide peptide. The contribution of NPFFR1 and NPFFR2 receptors to the observed effects has not been tested. Both these receptors react with NPFF [42] and KP peptides [2,3], and it is likely that KSO peptides will act on both receptor types. The cross-reactivity of NPFF with some KP antibodies [43] suggests shared tertiary structure similarity as well as sequence similarity exists. The ability of NPFF, KP, and KSO peptides to act in similar neuronal models suggests that the KSO sequence represents the region of KP with NPFF similarity. The distribution of NPFF receptors [23,[44][45][46][47] suggests that the main effects of KSO peptides is likely to be restricted and there may also be species differences. The specificity of the NPFF antagonist RF-9 [3,4]  However, the actions of KSO are shared with NPFF and KP peptides, suggesting that a common feature that RF-9 can block mediates the effects. Cell lines overexpressing NPFFR1 and NPFFR2 receptors have been used to determine receptor-binding affinities and cAMP inhibition by NPFF related peptides [6,24,42] and could be employed to confirm KSO interactions with NPFF receptors. The ability of MMP enzymes to cleave KP-10 in vitro [26] to yield the KSO sequence, with an extra C-terminal Gly residue that can be amidated [27], raises the possibility that KSO-like peptides could be generated both in vitro and in vivo. The observations that KP peptides down regulate the expression of MMP enzymes [48] suggest that this action may be limited by release of KP peptides. However, there are conditions where MMP enzymes are elevated, and since the effects of KP peptides are at the level of protein expression rather than activity, there may be conditions where KSO-like peptides could be generated. The KSO sequence and MMP cleavage sites in the KiSS-1 precursor protein are shared between the human [10,13] and rat forms [49]. It cannot be excluded that the actions of KP-10 and KP-13 in the cell models tested are due to generation of KSO-like peptides.
The ability of KSO peptides to prevent PGE 2 toxicity in rat cortical neurons raises the possibility that KSO peptides could be neuroprotective; however, the reversal of forskolin neuroprotection by KSO peptides suggests that any neuroprotective properties may be model specific. The expression of appropriate NPFF or related receptors that KSO peptides can bind to and activate responses from may Journal of Amino Acids 5 also be key for neuroprotective activity. In a recent study the KP peptides including the KSO 1-6 fragment were shown to be neuroprotective against amyloid peptides via a receptor-independent mechanism [50]. The results with the reversal of forskolin neuroprotection suggest that in vivo the KSO peptides could have multiple actions and therefore not show measurable neuroprotective properties. The requirement for doses in the μM range suggests that the effects being observed may not be seen at physiological concentrations of endogenous KSO peptides.
The lack of GnRH releasing activity of the KSO peptides is in agreement with previous studies [26]. The KSO peptide used in the present study has an amidated C-terminus instead of the glycine residue used in the Takino et al. [26] study, however, this has not added a GnRH releasing bioactivity.

Conclusions
The present study has identified a biological activity as an inhibitor of stimulated cAMP release for the KSO peptides with the sequences Tyr-Asn-Trp-Asn-Ser-Phe-NH 2 and Trp-Asn-Ser-Phe-NH 2 , which can be antagonized by an NPFF receptor antagonist. These actions are seen in neuronal cell models based on human, mouse, and rat cells, suggesting that the action is not species specific.