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Vol. 20, Issue 1, 319-327, January 1, 2009
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*Department of Neural and Behavioral Sciences, and Department of Medicine, The Pennsylvania State University College of Medicine, Hershey, PA 17033
Submitted July 3, 2008;
Revised September 30, 2008;
Accepted October 7, 2008
Monitoring Editor: Jonathan Chernoff
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). OGF is a constitutively expressed native opioid that is autocrine produced and secreted to inhibit the growth of both normal cells (Zagon and McLaughlin, 1987, 1991; Hauser et al., 1990; Stiene-Martin and Hauser, 1990; Hauser and Stiene-Martin, 1991; Isayama et al., 1991; Villiger and Lotz, 1992; Zagon et al., 1994, 1995a,b, 1996a, 1997a,b, 1998, 1999b, 2000b; McLaughlin, 1996; Vertes et al., 1996; McLaughlin and Wu, 1998; Blebea et al., 2000; Wilson et al., 2000; Kornyei et al., 2003; Robertson and Andrew, 2003; Malendowicz et al., 2005) and cancer cells (Zagon et al., 1996c; Cheng et al., 2007, 2008). The action of OGF is tonic, stereospecific, reversible, noncytotoxic, and nonapoptotic inducing, is not associated with differentiative, migratory, invasive, or adhesive processes, is independent of serum, anchorage-independent and occurs at physiologically relevant concentrations (Zagon et al., 2002, 2007a; Zagon and McLaughlin, 2003, 2004, 2005).
The action of OGF is mediated by interaction with the OGF receptor (OGFr). The gene for human OGFr is at least 9 kb in length, consists of seven exons and six introns, and encodes a 677-amino acid protein that includes 7 imperfect repeats of 20 amino acids each and at least one bipartite nuclear localization signal (Zagon et al., 2002). OGFr has an apparent mass of 62 kDa. The chromosomal location of the human OGFr is 20q13.3 (Zagon et al., 2002). Although OGFr has characteristics of a classical opioid receptor (recognizes opioids, naloxone reversibility, stereospecificity), there is no homology of OGFr with classical opioid receptors at the nucleotide or amino acid levels (Zagon et al., 2002). Antisense experiments with OGFr and continuous blockade of opioid receptors by the potent opioid antagonist naltrexone (NTX) support that the OGF–OGFr axis is a tonically active inhibitory system targeted to cell replication and homeostasis and is ligand-dependent for function (Zagon et al., 2002). Immunoelectron and confocal microscopy have shown that OGFr is localized to the outer nuclear envelope, nucleus, and perinuclear cytoplasm (Zagon et al., 2003, 2005).
The action of the OGF–OGFr axis in normal and cancer cells is targeted to DNA synthesis (Zagon and McLaughlin, 1987, 1991; Isayama et al., 1991; Zagon et al., 1994, 1995b, 2000a; McLaughlin, 1996; McLaughlin and Wu, 1998; McLaughlin et al., 1999; Wilson et al., 2000; Blebea et al., 2002). In squamous cell carcinoma of the head and neck, OGF activity has been shown to be dependent on one CKI, p16INK4a (Cheng et al., 2007), whereas in pancreatic cancer, which often has a mutation/deletion of p16INK4a, another CKI, p21WAF1/CIP1, is the target of OGF with respect to modulating the cell cycle (Cheng et al., 2008). Because OGF depresses DNA synthesis and subsequent cell/tissue growth in a wide variety of normal and developing cells in humans and animals, including ectodermal, mesodermal, and endodermal derivatives (Hauser et al., 1990; Hauser and Stiene-Martin, 1991; Isayama et al., 1991; Zagon and McLaughlin, 1991; Zagon et al., 1994, 1995b, 1996a,b, 1997, 1999b; McLaughlin, 1996; Vertes et al., 1996; McLaughlin and Wu, 1998; Blebea et al., 2000, 2002; Wilson et al., 2000; Kornyei et al., 2003), the question arises as to the mechanism of peptide action on the cell cycle in these cells. The present investigation examined the specific target(s) in the cell cycle for the OGF–OGFr axis in cells derived from four normal human tissues: umbilical vein endothelial cells (HUVECs), epidermal keratinocytes (NHEKs), dermal fibroblasts (NHDFs), and mesenchymal stem cells (hMSCs). In contrast to the cancer cells we have examined, our data reveal that the OGF's inhibitory action on cell proliferation in normal human cells is dependent on both the p16INK4a and p21WAF1/CIP1 signaling pathways.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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OGF and NTX were purchased from Sigma-Aldrich (St. Louis, MO), dissolved in sterile water, and used at a final concentration of 10–6 M.
Cell Growth and Flow Cytometry
For growth curves, cells were seeded in six-well plates at an initial density of 
2 x 105 cells/well. All cultures were treated in duplicate. Fresh media and OGF were added 24 h after initial seeding, and media and OGF were replaced daily. At appropriate times, the cells were washed with PBS, trypsinized with 0.25% trypsin-EDTA (Mediatech, Herndon, VA), and viable cell numbers were counted by trypan blue exclusion using a hemacytometer.
For flow cytometry, cells were treated with 10–6 M OGF for the indicated hours; this dosage of OGF was selected because it significantly inhibits cell proliferation of human cancer cell lines (McLaughlin et al., 1999) and does not induce apoptosis or necrosis (Zagon and McLaughlin, 2003), differentiation (Zagon and McLaughlin, 2005), or migration, invasion, adhesion, or chemotaxis (Zagon et al., 2007a). Cells were harvested and fixed with 70% ethanol at –20°C for up to 7 d before DNA analysis. DNA content was obtained by incubating cells in PBS containing propidium iodide (0.1 mg/ml) and RNase A (0.02 mg/ml) for 15 min at 22°C. Fluorescence was measured and analyzed using a Becton-Dickinson Biosciences FACScan flow cytometer (San Diego, CA) and Modfit software (Verity Software House, Topsham, ME).
Small Interfering RNA Knockdown of OGFr
The OGFr-targeted small interfering RNAs (siRNAs; antisense: 5'-uagaaacucagguuuggcg-3'; sense: 5'-cgccaaaccugaguuucua-3') were designed and obtained as ready-annealed, purified duplex probes from Ambion (Austin, TX). Before experimentation with siRNAs, transfection efficiency was determined by treating cells with a commercially available fluorescein-labeled negative control siRNA (Ambion). Cells were transfected for 24 h at a final concentration of 20 nM and counterstained with Hoechst stain for 5 min. Transfection efficiency was calculated as the percentage of fluorescently labeled cells. Transfection efficiencies ranged from 63 to 75%.
For transfection with OGFr-siRNA, 2 x 105 cells per well were seeded in six-well plates with 1 ml of serum-containing media without antibiotics. In each well, 20 nM OGFr-siRNA or control siRNA (Ambion) solutions in serum-free media were added. Cells were incubated for 4 h at 37°C before the addition of OGF. Cultures were incubated an additional 20 h, and then 1 ml fresh complete media either lacking or containing OGF was added. At 72 h cells were collected for computing growth. Two independent experiments were conducted.
Western Blot Analysis
Cells (2 x 106) from each treatment were solubilized in 200 µl RIPA buffer (1x PBS, 10 µM IGEPAL, and 1 mg/ml SDS), containing a cocktail of protease inhibitors. Total protein concentrations were measured using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (40 µg) were subjected to 10% SDS-PAGE followed by transfer of proteins on to polyvinylidene difluoride (Millipore, Billerica, MA) using standard protocols. The following antibodies were utilized: p15, p16INK4a, p18, and p19 (Santa Cruz Biotechnology, Santa Cruz, CA); p21WAF1/CIP1 and p27 (BD PharMingen, San Diego, CA); β-actin (Clone AC-15, Sigma-Aldrich). Membranes were probed with secondary anti-rabbit or anti-mouse horseradish peroxidase–conjugated antibodies (GE Healthcare-Amersham Biosciences, Piscataway, NJ), and developed using a chemiluminescence Western blotting detection system.
To determine equal loading of total protein samples, blots were reprobed with mAb against β-actin at a dilution of 1:2000. If necessary, membranes were processed in stripping buffer (62.5 mM Tris-HCl and 100 mM β-mercaptoethanol/2% SDS, pH 6.7) at 50°C before being reprobed.
Quantitation of Western Blots
To quantify expression levels, the optical density of each band was determined by densitometry and analyzed by QuickOne (Bio-Rad Laboratories). Each value was normalized to β-actin from the same blot. To report the changes due to OGF treatment, we calculated the fold increase at each time point by dividing the normalized value from the OGF-treated or sterile water–treated samples by the normalized value of 0-h control samples. Means and SE were determined from three or more independent experiments.
SiRNA Knockdown of p16INK4 and p21WAF1/CIP1
The p16INK4a-targeted siRNAs (antisense: 5'-acaccgcttctgccttttctt-3'; sense: 5'-gaaaaggcagaagcggtgttt-3') were obtained as ready-annealed, purified duplex probes (Invitrogen, Carlsbad, CA). The p21WAF1/CIP1-targeted siRNAs were obtained from Santa Cruz Biotechnology, and negative control siRNAs were purchased from Ambion. For transfection, 2 x 105 cells per well were seeded in six-well plates containing 1 ml of serum-free medium without antibiotics. In each well, 20 nM of p16INK4a-siRNA, p21WAF1/CIP1-siRNA, both p16INK4a- and p21WAF1/CIP1-siRNA, or control siRNA solutions in serum-free media were added. Cells were incubated for 4 h at 37°C before the addition of OGF. Twenty hours later, 1 ml of fresh complete media with or without OGF was added to the cultures; media and OGF were replaced daily. At the indicated times, cells were collected for growth curves. Three independent experiments were conducted.
Statistical Analysis
Values were assessed by one-way analysis of variance (ANOVA) and Newman Keul's post-multiple comparison tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the specificity of OGFr, knockdown experiments with OGFr-siRNA were conducted with HUVECs (Figure 1C). Exposure to 10–6 M OGF depressed the growth of sterile water– and control siRNA–treated HUVECs by 17 and 33%, respectively, whereas 10–6 M NTX increased the number of sterile water– and control siRNA–exposed HUVECs by 31 and 27%, respectively. HUVECs subjected to OGFr-siRNA had 31% more cells than control siRNA–treated cultures, as well as sterile water–treated cultures. In contrast to cells expressing OGFr (i.e., control siRNA), exposure to 10–6 M OGF or NTX had no further effects on the OGFr-siRNA–treated cultures.
To assess the specificity of OGF action by OGFr mediation in NHEKs, knockdown experiments with OGFr-siRNA were conducted (Figure 1C). Exposure to 10–6 M OGF depressed the growth of sterile water– and control siRNA-treated NHEKs by 30 and 46%, respectively, whereas 10–6 M NTX increased the number of sterile water– and control siRNA–exposed NHEKs by 26 and 28%, respectively. NHEKs subjected to OGFr-siRNA had 42% more cells than control siRNA–treated cultures, as well as sterile water–treated cultures. In contrast to cells expressing OGFr (i.e., control siRNA), exposure to 10–6 M OGF or NTX had no further effects on the OGFr-siRNA–treated cultures.
CDK Inhibitors p16INK4a and p21WAF1/CIP1 Expression Are Up-Regulated by OGF
Cell cycle progression depends on both positive and negative regulators. Expression of p16INK4a and p21WAF1/CIP1 were evaluated in nonsynchronized HUVECs after 3, 6, 9, 12, and 15 h of OGF exposure. Both p16INK4a and p21WAF1/CIP1 were significantly (p < 0.05) up-regulated only at 3 h of treatment with OGF relative to control levels (Figures 2, A and B). Expression of p16INK4a and p21WAF1/CIP1 were evaluated in NHEKs after 3, 6, 9 and 12 h of OGF exposure. p16INK4a was significantly up-regulated (p < 0.05) in OGF-treated cells relative to control cultures only at 3 h (Figure 2C). p21WAF1/CIP1 was significantly up-regulated (p < 0.05) in OGF-treated cells relative to control levels only at 12 h (Figure 2D). Variation in p21WAF1/CIP1 up-regulation needs to be taken within the context of the doubling times. Analysis (nonlinear fit) of growth curves of treated and nontreated NHEKs and HUVECs revealed that OGF extended the doubling time from 24 to 28 h, whereas OGF-treated NHEKs extended the doubling time from 37 to 57 h. p16INK4a is known as a tumor suppressor gene, functioning as a cell cycle inhibitor by forming heterotrimeric complexes with cyclin-dependent kinases (Cdks) and cyclins. p21WAF1/CIP1 can also inhibit forming heterotrimeric complex with Cdks and cyclins. Therefore, these data suggest that under the effect of OGF, p16INK4a and p21WAF1/CIP1 protein levels were up-regulated and mediated the cell cycle block.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, these previous studies do not directly demonstrate that OGF, which also recognizes other classical opioid receptors (Leslie et al., 1980), may elicit opioid agonist–sensitive antiproliferative signaling through these receptors instead of, or in addition, to OGFr. The logical extension of this query is that the effects of OGF on the cell cycle may be related to OGFr and/or other opioid receptors. Using OGFr knockdown experiments, the present report now shows that the specific and singular receptor for OGF action on the replication of HUVECs and NHEKs is OGFr. Cells with silenced OGFr are not altered in their growth properties by addition of OGF. Moreover, these cells with a knockdown of OGFr are not increased in cell proliferation by NTX, documenting that up-regulation of cell proliferation by this opioid antagonist is at the OGF–OGFr level of interfacing and that other opioid receptors are not involved with the effects of this general opioid receptor antagonist. These data are consistent with previous in vitro and in vivo studies reporting that treatment of normal cells with antisense RNA to OGFr (Zagon et al., 1999a, 2006) eliminates the inhibitory effects of OGF exposure. Thus, the elucidation of the target of OGF in this study is directly—and solely—related to OGFr. This study shows for the first time that the target of the negative growth regulator, OGF, in normal human cells consists of both CKIs p16INK4a and p21WAF1/CIP1. Using HUVECs and NHEKs that exhibited growth inhibition after exposure to OGF, and flow cytometry observations documenting that OGF impeded cells exiting G1, we now conclude that peptide action targets key regulators of the G1-to-S phase transition. Confirmation that both p16INK4a and p21WAF1/CIP1 were indeed the target of OGF was validated in siRNA studies whereby HUVECs or NHEKs exposed to p16INK4a siRNA or p21WAF1/CIP1 siRNA exhibited no change in growth after exposure to OGF. Thus, our study makes the novel finding that OGF action is directed to CKIs p16INK4a and p21WAF1/CIP1 of the cell cycle in normal cells.
This investigation also showed that the requirement of p16INK4a and p21WAF1/CIP1 pathways for OGF action extends beyond the two cell lines, HUVECs and NHEKs, initially investigated. Two other human cell types, a dermal fibroblast and mesenchymal stem cell, also utilize p16INK4a and p21WAF1/CIP1 as discovered with siRNA technology. Therefore, a total of four different cell types: fibroblast, keratinocyte, endothelial, and mesenchymal, representing ectodermal and mesodermal derivatives, have a similar signaling pathway for OGF action. Given this diversity of cells and although further study is required, p16INK4a and p21WAF1/CIP1 may serve as a common denominator of OGF's inhibition of cell proliferation in all normal human and animal cells.
p16INK4a and p21WAF1/CIP1 have been shown to be both necessary and sufficient to inhibit cyclin/Cdk activity, and play critical roles in the negative control of cell growth (Sherr and Roberts, 1999). Our results with OGF and normal human cells are consistent in finding that up-regulating p16INK4a and p21WAF1/CIP1 expression has a negative effect on growth. It is important to note that both p16INK4a and p21WAF1/CIP1 are required for OGF's inhibitory activity in these normal cells and that attenuation of either p16INK4a or p21WAF1/CIP1 is not compensated by the other CKIs. Although p16INK4a and p21WAF1/CIP1 also have been found to be utilized by cancer cells in regard to peptide activity, in neoplastic cells the up-regulation of either p16INK4a or p21WAF1/CIP1 was found to be sufficient for OGF action on the cell cycle (Cheng et al., 2007, 2008). Why both CKIs are required in normal cells but only one CKI is sufficient in cancer cells is unclear. In the case of normal cells that are dependent on both p16INK4a and p21WAF1/CIP1 for OGF action, it may be conjectured that the hypophosphorylation of retinoblastoma protein necessitates Ser807/811 by Cdk4-cyclin D, and Thr821 by Cdk2, and that only one CKI is not sufficient to prevent phosphorylation of Rb. Alternatively, p16INK4a and p21WAF1/CIP1 are known to interact in some situations (Han et al., 2005) and that the loss of either p16INK4a or p21WAF1/CIP1 in the knockdown experiments with normal cells disturbed this signaling pathway leading to hypophosphorylation of pRb and repression of transcription of E2F that would lead to cell cycle arrest in G1. In cancer cells, deficiencies in CKIs are well known (Gartel et al., 1996; Sherr, 2004), particularly p16INK4a and p21WAF1/CIP1, and therefore we would postulate that cellular pathways regulating the cell cycle related to OGF action have adapted by having a dependency on only one CKI.
In conclusion, our results support the notion that both p16INK4a and p21WAF1/CIP1 act as suppressors to mediate the growth inhibitory function of the OGF–OGFr axis in normal cells. The clinical ramifications of our findings merit further discussion. For example, drugs used to attenuate p16INK4a or p21WAF1/CIP1 and to decrease OGF–OGFr interfacing would have the net effect of increasing cell proliferation and accelerating processes dependent on cell production (e.g., wound healing). Indeed, topical and systemic application of the opioid antagonist naltrexone stimulates wound repair of ocular surface epithelium in normal and diabetic rats (Zagon et al., 2007b). Increasing both p16INK4a and p21WAF1/CIP1 would be predicted to have an additive effect in concert with the OGF–OGFr axis to slow down cell proliferation. This could have an impact in situations wherein retardation in the generation of cell number would be desirable. For example, in cases of hyperplasia such as endometrial hyperplasia or benign prostatic hyperplasia, cell proliferation would be decreased by increasing p16INK4a and p21WAF1/CIP1 in concert with activating the OGF–OGFr system.
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Footnotes |
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Address correspondence to: Ian S. Zagon (isz1{at}psu.edu).
Abbreviations used: CKI, cyclin-dependent kinase inhibitor; NTX, naltrexone hydrochloride; OGF, opioid growth factor; OGFr, opioid growth factor receptor; Rb, retinoblastoma; siRNA, small interfering RNA.
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 I. S. Zagon, K. A. Rahn, A. P. Turel, and P. J. McLaughlin Endogenous Opioids Regulate Expression of Experimental Autoimmune Encephalomyelitis: A New Paradigm for the Treatment of Multiple Sclerosis Exp Biol Med, November 1, 2009; 234(11): 1383 - 1392. [Abstract] [Full Text] [PDF]
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I. S. Zagon, R. N. Donahue, and P. J. McLaughlin Opioid growth factor-opioid growth factor receptor axis is a physiological determinant of cell proliferation in diverse human cancers Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2009; 297(4): R1154 - R1161. [Abstract] [Full Text] [PDF]
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F. Cheng, P. J. McLaughlin, M. F. Verderame, and I. S. Zagon Dependence on Nuclear Localization Signals of the Opioid Growth Factor Receptor in the Regulation of Cell Proliferation Exp Biol Med, May 1, 2009; 234(5): 532 - 541. [Abstract] [Full Text] [PDF]
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