G-protein-coupled receptors receive many different signals to activate different functions such as cellgrowth, proliferation, and migration. KiSS1 is a metastasis suppressor gene that has been shown to inhibit metastasis of human melanomas and breast carcinomas. The human KiSS1 gene encodes a COOH-terminally amidated active peptide, and this peptide is the ligand of a novel G-protein-coupled receptor. However, the mechanism of the antimetastatic actions of KiSS1 and its G-protein-coupled receptor has not been elucidated. In this study, we identified the mouse homologues of the KiSS1 peptide and its G-protein-coupled receptor and characterized the signaling pathways mediated by the activation of the KiSS1 receptor. Although human and mouse KiSS1 proteins share relatively low overall homology (52%), the active peptides (10-amino-acid residues) are highly conserved between mouse and human KiSS1 proteins, varying by only one conserved amino acid [Tyr (Y) to Phe (F)]. Activation of the receptor by KiSS1 peptide leads to the activation of G-protein-activated phospholipase C (PLC-β), which suggests direct coupling of the KiSS1 peptide to the Gαq-mediate PLC-Ca2+ signaling pathway. Furthermore, activation of the KiSS1 receptor inhibits cell proliferation and cell migration, key characteristics of tumor metastasis.
GPCRs 3 receive many of the neural, hormonal, and paracrine inputs to activate different types of cells, regulating intracellular second messengers and a variety of cellular functions, including cell growth and proliferation. Activation of GPCRs leads to the rapid activation of heterotrimeric G-proteins (binding of GTP) and G-protein-coupled signaling pathways (1) . The heterotrimeric G-proteins are composed of α, β, and γ subunits. The Gα proteins comprise four subfamilies, the Gs, Gi/o, Gq, and G12/13 (1) . Each of these groups functions to activate different subsets of effectors: the Gs and the Gi/o regulate intracellular cAMP concentration by activating and inhibiting adenylyl cyclases, respectively; the Gq subfamily mediates the function of PLC-β, a family of key enzymes essential for the regulation and generation of intracellular second messengers (phosphatidylinositol 4,5-bisphosphate, IP3, and Ca2+) and other cell growth and differentiation processes. Besides the traditional G-protein-coupled signaling pathways, G-protein-coupled receptors have also been shown to participate in the mitogen-activated protein kinase signaling pathways and the Rho/Cdc42/Rac-p21-activated kinase signaling pathways in different cells and tissues (2, 3, 4) , which suggests a direct role for GPCRs in cell growth and proliferation processes.
Recently, studies on constitutive active mutants of GPCRs and G-proteins in cancers and other disease states demonstrated that GPCRs and G-proteins play an important role in both normal and aberrant growth control (5, 6, 7, 8, 9) . Constitutive active mutations of GPCRs have been implicated in a number of human neoplasias, including thyroid adenomas, small cell lung carcinoma, colon adenomas and carcinomas, and gastric hyperplasia and cancers (7) . Furthermore, a number of DNA viruses, including human CMV, Herpesvirus saimiri (HVS), and Kaposi’s sarcoma-associated herpesvirus (KSHV), have also been found to encode functional GPCRs and behave as oncogenes (7) .
Using differential display and subtractive hybridization, Lee et al. (10) isolated a novel human cDNA, called KiSS1, from melanoma cells. The KiSS1 gene product is a 145-amino-acid protein (11) . Transfection and overexpression of the full-length KiSS1 cDNA into metastatic human cancer cell lines, MDA-MB-435, C8161, and MelJuSo, suppressed metastasis in athymic nude mice, which suggested that KiSS1 is a metastasis suppressor in these types of tumors (12) . Fluorescence in situ hybridization indicates that KiSS1 is mapped to chromosome 1q32-q41 (13) . Although the function of the protein encoded by KiSS1 is unknown, the KiSS1 gene product has recently been shown to repress Mr 92,000 type-IV collagenase (matrix metalloproteinases-9) expression by inhibiting nuclear factor-κB binding to the promoter (14) . Very recent studies indicated that the KiSS1 gene encodes a COOH-terminally amidated peptide with 54 amino acid residues, and this peptide is a ligand of a novel human GPCR (named AXOR12, hOT7T175, GPR54, respectively; Refs. 11 , 15 , 16 ). In hOT7T175-transfected Chinese hamster ovary cells and B16-BL6 melanoma cells, the peptide ligand inhibits chemotaxis and invasion and attenuates pulmonary metastasis of B16-BL6 melanomas in vivo (11) . These findings suggest that KiSS1 may act as a metastasis suppressor gene through the activation of the novel GPCR. Using in situ hybridization, Shirasaki et al. have shown that the expression of KiSS1 gene was lost during melanoma progression and is associated with loss of heterozygosity of chromosome 6q16.3-q23 (17) . However, the mechanism of the antimetastatic activity of KiSS1 has not been elucidated. In this study, we identified and characterized the mouse homologues of the KiSS1 peptide and its GPCR. The 10-amino-acid KiSS1 peptide ligand is very conserved between mouse and human with one conserved amino acid replacement [Tyr (Y) to Phe (F)], although other regions of the protein showed low homology. Expression of the KiSS1 peptide ligand is found in all tissues, whereas the expression level for the receptor is very low and hardly detected in our Northern blot analysis. Activation of the receptor by KiSS1 peptide activates the G-protein-mediated PLC, leading to the production of IP3 in the cell. Furthermore, activation of the receptor by the KiSS1 peptide inhibited cell proliferation and migration, an essential feature in cancer cell metastasis.
Materials and Methods
NIH3T3 and COS 7 cells were maintained in DMEM supplemented with 10% FBS. A retrovirus pLNCX-encoding mKiSS1 receptor and the vector itself were used in our experiments. Retroviruses were generated by transfecting these plasmids into 293T cells, and supernatant was collected and stored at −70°C. NIH3T3 cells were transfected with retrovirus in the presence of 6 μg/ml Polybrene. Forty-eight h later, cells were subject to G418 (0.5 μg/ml) for 1 week. The resistant cells were used for some of the following experiments. COS 7 cells were transient transfected because they were easier to observe and transfect compared with the NIH3T3 cells.
Cloning of mKiSS1 Receptor and mKiSS1 Peptide.
The mKiSS1 receptor was identified with a genomic search with the hKiSS1 receptor to the mouse genome. One match was found. Primers were designed 5′-cggaattctcagagtgaggcagtgcgtt-3′ and 5′-cgggatccggaagcatggccaccgag-3′ for PCR of a mouse brain library. The receptor was cloned into pCMV tag 2B vector (Stratagene) and used for experiments. mKiSS1 peptide was found in a similar manner. Scanning the mouse genome for a match to the hKiSS1 peptide was performed. Again one match was found, and a PCR of the mouse brain library was preformed with the following primers: 5′-ccgctcgagtcagccccgtgctgcccgcgc-3′ and 5′-ccggaattcatgatctcaatggcttcttggc-3′. This was also cloned into pCMV tag 2B vector.
Expression of mKiSS1 Protein and mKiSS1 Receptor.
A multiple tissue cDNA panel (Clontech) was used to determine the expression of mKiSS1 and its GPCR in various tissue types. The protocol from the company was followed using the following primers for mKiSS1 receptor: 5′-atgcctggctggttcccctgtttttcg-3′ and 5′-atgcagtgagcccagatcttgaccgcgtag-3′ and 5′-gccacctatggggagccgctg-3′ and 5′-caggccgaaggagttccagttgtaggtcg-3′ for the mKiSS1 protein. After PCR, samples were run out on a 1.5% agarose gel and visualized with ethidium bromide.
Cell Transfection and Immunocytochemistry.
COS-7 cells were transfected with the mKiSS1 receptor by LipofectAMINE (Invitrogen). Cells were then allowed to grow for 24 h. The hKiSS1 peptide was added at different times and then washed with PBS three times. Cells were fixed with paraformaldehyde, made permeable with 0.5% Triton X-100, and washed with PBS. Cells were then incubated with mouse monoclonal anti-flag antibody (Sigma) for 1 h. Cells were washed in PBS, and then, mouse anti-flag antibody conjugated to FITC was used as the secondary antibody for 1 h. The cells were washed three times with PBS and then stained for actin using rhodamine-conjugated phalloidin (Sigma) for 45 min. For nuclear staining, cells were stained with DAPI (Molecular Probes), mounted onto slides, and then examined under the microscope.
G-Protein-mediated PLC (PLC-β) Assay.
COS-7 cells were transfected with mKiSS1 receptor cDNA by LipofectAMINE (Invitrogen). Cells were allowed to grow for 48 h, washed three times with PBS and then added DMEM plus FBS with 1 μCi/ml [3 H]inositol was added. After 24 h, medium was replaced with new medium containing FBS, 10 mm LiCl (Sigma), and 10 μm mKiSS1 peptide or 100 μm carbachol for an hour. LiCl was used to stop the hydrolysis of [3 H]phosphoinositol. Carbachol was used to stimulate the GPCRs. The reaction was stopped by removing the medium and washing three times with PBS. Measurement of phosphoinositol production was performed as described previously (18 , 19) . Briefly, 10 mm formic acid (Sigma) was added, and the cells were incubated on ice for 20 min. The amount of inositol phosphates were separated on a column containing AG 1-X Resin (Bio-Rad) and counted in a Beta counter.
Cell Migration Assays.
Two types of cell migration assays were performed using NIH3T3 cells. First, we examined cell migration in modified Boyden chambers, as described previously (20) . Briefly, NIH3T3 cells were stably transfected with the mKiSS1 receptor or vector. The outside of filters were coated with either 1 μg/ml fibronectin or collagen for 1 h and then washed three times with PBS. Filters were then incubated with DMEM with BSA for 1 h. Filters were then put into DMEM medium without FBS and with 0.5 ng of mouse bFGF. NIH3T3 cells expressing the receptor or vector were seeded at 20,000/well on top of the filter. Plates were incubated for 24 h. Excess cells that did not migrate through the filter were removed from the insides of the filters. Cells were then fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and then stained with crystal violet. Stained cells were examined under the microscope.
Scratch assays were also performed using NIH3T3 cells expressing the vector or mKiSS1 receptor. Cells were allowed to grow to confluency on plates coated with collagen and washed twice with PBS. Cells were then scratched with a pipette tip and washed five more times with PBS. Fresh DMEM was added with 0.5 ng of mouse bFGF. This was allowed to incubate for 24 h and pictures were taken using a Nikon digital camera.
Cell Proliferation Assay.
Proliferation studies were carried out using the CellTiter96 AQueous One solution cell proliferation assay (Promega). Briefly, cells were plated at 500 cells/well and allowed to adhere to the plate. Ten μm KiSS1 peptide were added at an arbitrary time 0. At indicated time points, the AQueous One solution was added to the samples and measured at 490 nm.
Cell Viability and Cell Counts.
Vector and mKiSS1 cells were plated at 25,000 cells/well and allowed to adhere overnight. KiSS1 peptide was added at time 0 and cells were counted using trypan blue (Sigma) at indicated time.
Results and Discussion
Identification of mKiSS1 and Its GPCR.
To understand the molecular mechanism of KiSS1 mediated inhibition of cancer cell metastasis, we identified the mKiSS1 gene by searching the mouse genomic database using hKiSS1. Reverse transcription-PCR was performed to clone the mouse cDNA that encodes the mKiSS1 protein. As shown in Fig. 1A ⇓ , the mKiSS1 and hKiSS1 proteins share about 54% sequence homology at the protein level. However, the active 10-amino-acid peptide ligand identified in the protein is highly conserved with only one amino acid (Y to F) replacement. The same approach was also used to identify the mKiSS1 GPCR. There is a high homology (∼85%) between the mKiSS1 receptor and the hKiSS1 receptor at cDNA level. The most divergent region is located at the COOH termini of the proteins (Fig. 1B) ⇓ . Both receptors contain the typical seven-transmembrane domains of GPCRs (Fig. 1B) ⇓ , which suggests that KiSS1 mediates the G-protein-coupled signal transduction pathways. The expression of mKiSS1 is found weakly in all tissue types with highest levels found in lung and 15- and 17-day mouse embryos (data not shown). For mKiSS1 receptor, the expression level was highest in the heart and 15- and 17-day embryos, and a low level of expression was detected in other tissues (data not shown).
Activation of KiSS1 Receptor Changes Cell Morphology.
Expression of the KiSS1 receptor is localized to the cell surface membrane as shown in Fig. 2A ⇓ . All of the cells that express the receptor look like normal flattened out cells. When the cells were stimulated by the KiSS1 peptide and incubated for 30 min, cells overexpressing the receptor became rounded and began to lose their shape (Fig. 2B) ⇓ . Addition and incubation of the cells with the KiSS1 peptide for 60 min induced shape change in cells expressing the receptor, resulting in rounded and smaller cells compared with control cells (Fig. 2C) ⇓ . These data suggest that activation of the receptor by KiSS1 peptide changes cell morphology and actin filament reorganization. We are still in the process of understanding how activation of the KiSS1 receptor leads to cell morphology change and actin reorganization in the cell.
Activation of KiSS1 Receptor by KiSS1 Peptide Inhibits Cell Migration.
To examine the potential mechanism of how activation of the KiSS1 signaling pathway inhibits cell metastasis, we generated NIH3T3 cells stably transfected with the mKiSS1 receptor and a control vector. When the cells were placed in a modified Boyden chamber coated with collagen or fibronectin, more cells migrated across the Boyden filter in control cells expressing vector alone than in the cells expressing the receptor (Fig. 3A) ⇓ . The addition of KiSS1 peptide to the cells transfected with the receptor dramatically slowed the migration of the cells in either collagen- or fibronectin-coated chambers (Fig. 3A) ⇓ . These data show that just overexpressing the receptor can slow the migration of the cell, which suggests that some endogenous peptide may be excreted into the medium and has some inhibitory effect on cells overexpressing the receptor. Furthermore, the addition of the KiSS1 peptide into cells expressing the receptor further dramatically inhibited cell migration (Fig. 3A) ⇓ . Together, our data suggest that both the KiSS1 peptide and its receptor are required to significantly inhibit cell migration.
To further confirm that the activation of KiSS1 receptor by the peptide ligand inhibits cell migration, we performed the scratch assays using cells expressing the vector alone and cells expressing the KiSS1 receptor. As shown in Fig. 3B ⇓ , there is a dramatic difference in cell migration between vector-expressing cells and cells expressing the receptor when the KiSS1 peptide is added into the cells. In the vector-expressing cells, with or without the KiSS1 peptide, cells migrated into the scratched area and almost filled the scratched area in 24 h (Fig. 3B, a and b) ⇓ . However, in the cells expressing the mKiSS1 receptor (Fig. 3B, c and d) ⇓ , and stimulated by KiSS1 peptide (Fig. 3B, d) ⇓ , very few cells migrated into the scratched area 24 h after incubation (Fig. 3B, d) ⇓ . This once again demonstrates that the KiSS1 peptide can inhibit cell migration in cells expressing the receptor. Overexpression of the KiSS1 receptor shows some inhibitory effect on cell migration (Fig. 3B, c) ⇓ , consistent with results obtained in Fig. 3A ⇓ and probably attributable to the existence of endogenous KiSS1 peptide. Taken together, these data indicate that both the KiSS1 peptide and its receptor are required to have significant inhibitory effect on cell migration. The molecular mechanism of inhibiting cell migration by the KiSS1 receptor pathway is not clear. One possible pathway that is coupled to G-protein receptor and cell migration is the small G-protein (Rac/Cdc42) pathway. Cell migration is very important in secondary tumor metastasis and growth. Understanding how the activation of KiSS1 receptor inhibits cell migration will open a new line of research in our fight against tumor metastasis.
Activation of KiSS1 Receptor Inhibits Cell Proliferation.
Proliferative signaling has been traditionally associated with polypeptide growth factor receptors that possess an intrinsic protein tyrosine kinase activity, whereas GPCRs have been linked to tissue-specific, fully differentiated cellular functions. However, many GPCR ligands, including thrombin, endothelin, E2A, and lysophosphatidic acid, are known to activate mitogenic responses in a variety of cell types (7 , 9) . To understand the potential role of the KiSS1 peptide and its GPCR in cell proliferation and tumorigenesis, we examined cell proliferation in cells stably transfected with the mKiSS1 receptor and vector using the CellTiter 96 (Promega) assay. The addition of KiSS1 peptide into cells overexpressing the KiSS1 receptor greatly inhibited cell proliferation in both collagen- and fibronectin-coated plates compared with cells transfected with a control vector (Fig. 4A) ⇓ . These data suggest that activation of the KiSS1 signaling pathways can inhibit cell proliferation.
To further confirm that activation of KiSS1 receptor inhibits cell proliferation, we did cell count and viability assays. As shown in Fig. 4B ⇓ , the addition of KiSS1 peptide to the cells expressing the receptor leads to the decrease in the number of viable cells, suggesting an inhibition in cell proliferation. Together, these data support the idea that activation of the KiSS1 receptor pathways inhibits cell migration and proliferation.
KiSS1 and Its Receptor Activate G-Protein-mediated PLC (PLC-β) in the Cell.
To examine whether activation of KiSS1 and its receptor directly activate G-protein-coupled signaling pathways, we measured the activation of G-protein-activated PLC (PLC-β) in cells transfected with the mKiSS1 receptor. COS-7 cells were transiently transfected with cDNA encoding mKiSS1 receptor and were labeled with [3 H]inositol. After 24 h, the cells were treated with the KiSS1 peptide, and inositol phosphates released from the cells were measured. As shown in Fig. 5 ⇓ , addition of the KiSS1 peptide increased intracellular IP3 production, suggesting that activation of KiSS1 receptor by KiSS1 peptide stimulates the G-protein-mediated PLC-β pathway. Activation of PLC leads to the production of two key intracellular second messengers, IP3 and diacylglycerol, which in turn mediate intracellular Ca2+ release and activation of protein kinase C, respectively. An increase in intracellular Ca2+ has been found to inhibit cell proliferation and induce cell differentiation or apoptosis in cancer cells (21) . Therefore, activation of the PLC-Ca2+ signaling pathway in the cell by KiSS1 and its receptor could provide one possible mechanism for the inhibition of cell proliferation.
In this study, we identified and characterized the mKiSS1 protein and its GPCR. The mKiSS1 protein has only about 53% sequence homology compared with hKiSS1 protein, but the active peptide is very conserved with only one amino acid replacement (Y to F). We also demonstrated that once the peptide is added to cells, the cells change their morphology from spread out to more of a rounded shape. This could be caused by an actin depolymerization and/or actin rearrangement. Activation of the KiSS1 signaling pathway dramatically inhibited cell migration and cell proliferation, possibly by inhibiting the activation of the small GTPases, such as Rac/Cdc42 signaling pathways. Furthermore, we demonstrate that activation of the KiSS1 receptor leads to the activation of G-protein-coupled PLC and the production of intracellular IP3 and diacylglycerol, key second messengers in cell proliferation, differentiation, and apoptosis (21) .
We thank members of the Center for Cancer Biology and Nutrition (CCBN) for insightful discussion.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 Supported in part by grants from the NIH (NHLBI), the American Heart Association National, and the Prostate Cancer Program of the Department of Defense (Army; to M. L.).
↵2 To whom requests for reprints should be addressed, at Center for Cancer Biology and Nutrition, Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030. Phone: (713) 677-7505; Fax: (713) 677-7512; E-mail:
↵3 The abbreviations used are: GPCR, G-protein-coupled receptor; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; FBS, fetal bovine serum; CMV, cytomegalovirus; mKiSS1, mouse KiSS1; hKiSS1, human KiSS1; DAPI, 4′,6-diamidino-2-phenylindole; bFGF, basic fibroblast growth factor.
- Received March 27, 2002.
- Accepted August 15, 2002.
- ©2002 American Association for Cancer Research.