|Molecular Vision 2006;
Received 1 February 2006 | Accepted 7 August 2006 | Published 16 August 2006
Expression of K6W-ubiquitin inhibits proliferation of human lens epithelial cells
Qing Liu,1 Fu Shang,1
Xinyu Zhang,1 Wei Li,2 Allen Taylor1
1Laboratory for Nutrition & Vision Research, Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA; 2Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, FL
Correspondence to: Dr. Fu Shang, Laboratory for Nutrition & Vision Research, JMUSDA-HNRCA at Tufts University, Boston, MA, 02111; Phone: (617) 556-3158; FAX: (617) 556-3132; email: firstname.lastname@example.org
Purpose: The ubiquitin-proteasome pathway plays an important role in controlling the cell cycle. The purpose of this study was to examine if expression of a dominant negative form of ubiquitin can inhibit the proliferation of lens epithelial cells.
Methods: Dominant negative K6W-ubiquitin was expressed in cultured human lens epithelial cells via an adenoviral vector. Cell proliferation was monitored by both cell counting and flow cytometry analysis. Protein levels of cell cycle regulators were assessed by western blotting.
Results: Expression of K6W-ubiquitin in lens epithelial cells prevented cell proliferation and specifically caused cell cycle delay/arrest in the G2/M phase. Consistent with the cell cycle delay/arrest in the G2/M phase, typical substrates of the ubiquitin-proteasome pathway and also M phase regulators such as cyclin A, cyclin B, and securin were stabilized by expression of K6W-ubiquitin. Cell cycle-dependent degradation of G1 phase regulators, such as the Cdk inhibitor p27KIP, was also inhibited by the expression of K6W-ubiquitin.
Conclusions: These data demonstrate that the ubiquitin proteasome pathway plays an important role in regulating lens epithelial cell proliferation. Expression of dominant negative K6W-ubiquitin inhibits lens cell proliferation by inhibiting the degradation of cell cycle regulators.
The continuous proliferation and differentiation of lens epithelial cells throughout life may be essential for maintaining the function of the lens. However, enhanced proliferation of lens epithelial cells after extracapsular cataract extraction is causally associated with the development of posterior capsule opacification (PCO) [1-6]. Cell proliferation involves the orderly transition from G1, S, G2, and M phases of the cell cycle. The transition from phase to phase of cell cycle is controlled by a sequence of proteolytic events, particularly proteolysis of cell cycle regulators [7,8]. Progression of the cell cycle is positively regulated by cyclins, which activate cyclin-dependent kinases (Cdks). The "mitotic cyclins" include cyclin A and cyclin B, which are involved in control of G2/M transition and mitosis [9-11]. Elevated cyclin A is required for progression through S phase and passage into G2  but it must be degraded before cells entering the M phase. Thus, cyclin A levels fall precipitously upon completion of prometaphase . Cyclin B is also a critical regulator of mitosis. In mammalian somatic cells, ubiquitin-dependent degradation of cyclin B occurs near the end of mitosis, continues into G1, and ceases around the time of the G1/S transition . Securin is another important regulator of mitosis, which inhibits sister-chromatid separation by inhibiting the activity of separase. Cdk inhibitors, such as KIP family proteins, negatively regulate progression of the cell cycle by inhibiting the activity of cyclin-Cdk complexes. The KIP family proteins p27KIP and p57KIP suppress the activities of cyclin E/Cdk2 and mediate the exit from the cell cycle. The balance between positive and negative regulators of Cdks controls progression from one phase of the cell cycle to another. Protein levels of these and other critical cell cycle regulators, such as pRb, p53, etc., are primarily regulated by the ubiquitin-proteasome pathway (UPP) [10,14]. Therefore, the UPP plays an important role in regulating the cell cycle and proliferation of lens epithelial cells could be manipulated via altering the UPP activity.
In the UPP, protein substrates are covalently ligated to one or more monomers of ubiquitin by the sequential activities of three groups of enzymes: ubiqutin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s or Ubcs), and ubiquitin isopeptide ligases (E3s) [15,16]. The ubiquitin-protein conjugates are recognized and degraded by a large protease complex, the 26S proteasome. Prior to degradation, at least four, but often more, ubiquitins are added to the protein substrates. This results in formation of a ubiquitin oligomer or a chain of ubiquitins. Formation of high mass ubiquitin conjugates is a prerequisite to being recognized and degraded by the 26S proteasome. Concerted with degradation, the ubiquitin chain is disassembled and the ubiquitin is recycled.
As illustrated in Figure 1A, ubiquitin contains 7 lysine (K) residues and some of them play a critical role in determining the inter-ubiquitin linkage within the ubiquitin chain, whereas others play roles in maintaining the structure of the ubiquitin conjugates. We recently demonstrated that the K6 residue of ubiquitin is the most readily modified by biotin. We further demonstrate that several K6-modified or mutated ubiquitins (K6-biotinylated ubiquitin, K6A, and K6W mutant ubiquitin) behave as dominant negatives, which inhibit ubiquitin-mediated degradation . Although K6W-ubiquitin is competent for formation of ubiquitin conjugates, the conjugates formed with K6W-ubiquitin are resistant to proteasomal degradation due to diminished interaction with the proteasome .
In this work, we tested the possibility of inhibiting lens epithelial cell proliferation by expression of K6W-ubiquitin. We found that expressing K6W-ubiquitin in human lens epithelial cells (HLEC) inhibited cell proliferation. The inhibition of proliferation was due to the delay of the cell cycle at the G2/M phase and the inhibitory effect appears to be mediated through impaired UPP-dependent degradation of cell cycle regulators. The data further demonstrated that ubiquitin-dependent proteolysis is required for lens cell cycle progression and that targeted expression of the K6W-ubiquitin in lens epithelial cells could be tested for preventing PCO, one of the complications of modern cataract surgery.
Cell culture and synchronization
HLEC (SRA 01/04)  were grown at 37 °C in the presence of 5% CO2/atmosphere and maintained in DMEM (Life Technologies, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum (Life Technologies). HLEC were synchronized at G0/G1 using contact inhibition by allowing the cells to grow to and remain at confluence for 72 h. The G0 arrested cells were induced to resume the cell cycle by replating at about 20% confluence . The effects of empty adenovirus and K6W-ubiquitin adenovirus on proliferation of HLEC cultures were assessed by counting the cell numbers at three different times (0, 24 h, and 48 h) with a hemocytometer.
For analysis of cell cycle by flow cytometry, the cells were trypsinized, centrifuged for 5 min at 600x g, washed with phosphate-buffered saline (PBS), and fixed with ice cold 70% (v/v) ethanol. After washing twice with PBS, the cells were treated with 25 units RNase (Sigma, St. Louis, MO) at room temperature for 10 min and then resuspended in PBS containing 50 μg/ml propidium iodide (Sigma). DNA fluorescence was measured with a FACS Calibur Flow Cytometry System (Becton-Dickinson immunocytometer systems, San Jose, CA). The FACS data of cell cycle distribution was analyzed using ModFit LT software (Verity Software House, Topsham, ME).
Generation of recombinant adenoviruses and infection with adenoviral vector
Adenoviruses expressing green fluorescent protein (GFP) along with His6-ubiquitin or K6W-ubiquitin were generated using the AdEasyTM adenoviral vector system (Stratagene, La Jolla, CA) and were cloned as described previously . The DNA fragments encoding the wild-type or K6W-ubiquitin were inserted into pShuttle-CMV vector using the EcoRV restriction site. The expression vector was linearized with PmeI and were cotransformed with supercoiled pAdEasy viral DNA into BJ5183 bacterial cells. Adenoviral amplification was performed as described . The titer of the adenovirus stock, as determined by the ABS260, was 3.5x109 plaque forming units (pfu)/ml. Viral stock was diluted with PBS and added directly to the cultures. HLEC were arrested in G0 by contact inhibition and infected with K6W-ubiquitin or empty adenovirus for 24 h. After replating at 20% confluency, samples were harvested at 0, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h, and the cell cycle profile was analyzed.
Cell extract preparation
Cell monolayers were washed with ice-cold PBS and harvested by scraping. The cells were homogenized with lysis buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM EDTA, 1% NP-40, 0.1% SDS, 20 mM N-ethylmaleimide, and 2 mM 4-(2-aminoethyl)-benzene-sulfonylfluoride. After incubation in wet ice for 20 min with occasional vortexing, the lysate was centrifuged at 13,000x g for 15 min at 4 °C and the supernatant was recovered. To detect p27KIP, the cells were lysed directly in SDS-PAGE loading buffer at the times indicated. Protein concentrations in the supernatant were determined by the Coomassie Plus Protein Assay (Pierce, Rockford, IL) using bovine serum albumin as the standard.
Antibodies and western blot analysis
Antibodies to E1 and ubiquitin were produced in this laboratory [21,22]. Antibodies to cyclin A, cyclin B, and p27KIP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to securin (PTTG) was purchased from Zymed laboratories (San Francisco, CA). For each experimental condition, 20 μg of total protein was resolved using SDS-PAGE as described previously  and transferred to nitrocellulose membranes. The protein loading and transfer efficiency were monitored by staining the membranes with 1% Ponceau S. The membranes were incubated with primary antibodies overnight at 4 °C in a solution containing TST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.02% Tween 20) and 2.5% milk proteins. After washing four times with TST, the membrane was incubated with an HRP-conjugated secondary antibody for 1 h. The membrane was then washed five times with TST and incubated with enhanced chemiluminescent detection reagents (Pierce, Rockford, IL) and exposed to film (Kodak, Rochester, NY). Ubiquitin-activating enzyme E1 was used as a house keeping protein for normalizing the protein load .
Expression of K6W-ubiquitin stabilizes ubiquitin conjugates
As previously described, we prepared a ubiquitin variant, K6W-ubiquitin, in which Lys6 was replaced by Trp (Figure 1A) . Adenoviral vectors were used to obtain high transfection efficiency for expression of wild-type or K6W-ubiquitin in HLEC. As monitored by expression of GFP, approximately 75% of the HLEC were infected by the adenoviral vector (data not shown). As compared to cells that were infected with equal amount of empty adenovirus (which does not carry the ubiquitin gene), over-expression of wild-type ubiquitin resulted in a 2 fold increase in levels of high molecular weight ubiquitin conjugates (Figure 1B; compare lane 2 with lane 1). In comparison, when equivalent amounts of K6W-ubiquitin were expressed, levels of high molecular weight ubiquitin conjugates increased 10 fold (Figure 1B; compare lane 3 with lane 1). These enhancements are similar to 2 and 10 fold increases in ubiquitin conjugates which were observed when wild-type ubiquitin and K6W-ubiquitin were expressed in HEK293 cells . As we demonstrated previously, infection with adenoviruses that encodes wild-type or K6W-ubiquitin resulted in a comparable increase (about 2 fold) in levels of free ubiquitin as compared to cells that were infected with empty virus (data not shown). These data indicate that the dramatic increase in levels of high molecular weight ubiquitin conjugates upon expression of K6W-ubiquitin is caused by the impairment of proteasomal degradation of the K6W-ubiquitin-containing conjugates.
Expression of K6W-ubiquitin in HLEC inhibits cell proliferation
Our previous experiments using human or rabbit lens epithelial cells indicated that expression of K6W-ubiquitin interfered with the response of cells to stress and rendered the cells more susceptible to oxidative stress [17,25]. It appeared that part of the defect in response to stress is related to the antiproliferative effects of K6W-ubiquitin. Because the UPP is an important control mechanism of the cell cycle, we studied the effects of expression of K6W-ubiquitin on lens cell proliferation. The proliferation rate of HLEC infected with empty adenovirus (which does not carry the recombinant gene) was indistinguishable from that of uninfected cells (Figure 2). In contrast, the rate of proliferation of cells in which K6W-ubiquitin was expressed was 50% of that observed in HLEC infected with an equal amount of the empty adenovirus (Figure 2).
Expression of K6W-ubiquitin delays HLEC in G2/M phase and stabilizes mitotic regulators
To investigate the mechanism by which expression of K6W-ubiquitin inhibits HLEC proliferation, we determined if a particular phase of the cell cycle was affected by expressing K6W-ubiquitin. HLEC infected with empty adenovirus showed patterns and timing of progression through the different phases of the cell cycle that were indistinguishable from HLEC which were not infected with the adenovirus (Figure 3), and compared well with the timing of the cell cycle which we previously documented . Similar to HLEC which were infected with empty adenovirus, HLEC infected with adenovirus encoding K6W-ubiquitin progressed unimpeded through G1 and S phases of the cell cycle. However, by 40 and 48 h, HLEC which expressed K6W-ubiquitin showed increases in the proportion of cells in the G2/M phase, along with proportional decreases in the fraction of cells which enter the next G1 phase. These data suggest that expression of K6W-ubiquitin delays or arrests the cell cycle of HLEC in the G2/M phase.
Completion of M phase of the cell cycle requires the timely degradation of mitotic cyclins and other mitotic regulators, including cyclin A, cyclin B, and securin, by the UPP. To determine the mechanism by which expressing K6W-ubiquitin delays cell cycle in the G2/M phase, we assessed the effects of K6W-ubiquitin on the stability of these mitotic regulators. In HLEC infected with empty adenovirus, levels of cyclins A and B, and securin decrease as the cells exit the mitosis phase of the first cell cycle (Figure 4), which occurs at approximately 38 h (Figure 4A; lanes 1-4). In contrast, levels of the mitotic regulators were stable even at 42 h in K6W-ubiquitin-expressing cells (Figure 4A; lanes 5-8). The increased stability of these cell cycle regulators is consistent with the G2/M phase arrest noted above. Taken together, these data indicate that the K6W-ubiquitin-induced delay in the timely degradation of mitotic regulators is directly related to the G2/M phase delay in these cells.
Since G1 regulators are also substrates of the UPP, we asked if expression of K6W-ubiquitin affects the degradation of G1/S transition regulators in HLEC. As shown in Figure 5A (lanes 1-4), the protein levels of p27KIP are highest in the G0 phase. When the cells started entering S phase (16 h after release from G0 arrest), p27KIP levels decreased markedly and was barely detectable by 24 h. In contrast, the p27KIP were significantly stabilized in K6W-ubiquitin-expressing cells (Figure 5A; lanes 5-8). These data show that expressing K6W-ubiquitin also interferes with the timely degradation of cell cycle regulators which govern G1/S transition.
Controlled proliferation and differentiation of lens epithelial cells plays an important role in lens development and maturation. However, enhanced proliferation and migration of lens epithelial cells after extracapsular cataract extraction is a key initial step of PCO . Therefore, demonstrating the mechanism of lens epithelial proliferation not only help to understand lens development, it could also lead to a strategy for preventing PCO development [27,28]. In this work we utilized the viral mediated expression of dominant negative K6W-ubiquitin in HLEC and demonstrated that the UPP plays an important role in control lens cell cycle. Since K6W-ubiquitin is a highly specific competitive inhibitor of ubiquitin-mediated protein degradation, these data unequivocally demonstrate that ubiquitin-mediated proteolysis plays an important role in controlling cell cycle regulators and the proliferation of lens epithelial cells. The data also complement prior observations that small molecular inhibitors of the proteasome, such as MG132 and lactacystin, stabilize p21WAF and p27KIP in cultured HLEC or lens explants and prevent proliferation of lens epithelial cells [24,29].
It is well documented that the UPP plays key roles in virtually all phases of the cell cycle. In previous reports we showed that inhibition of proteasome activity by lactacystin resulted in cell cycle arrest in both G1 phase and G2/M phase . The present work shows that although expression of K6W-ubiquitin inhibited the degradation of p27KIP, this was not associated with a delay of the cell cycle in the G1 phase. These data imply that stabilization of p27KIP is not sufficient to prevent lens cells from progressing through G1/S phase transition, and that proteasome-dependent degradation of other regulators as well as p27KIP appears to be involved in regulating the G1/S phase transition in these cells. The data further suggest that cells in G2/M phase are more susceptible than cells in G1 phase to the inhibition of the UPP. Consistent with this speculation, a Chinese hamster cell line harboring a temperature sensitive mutant E1, the first enzyme of the ubiquitination cascade, also arrests in G2/M phase rather than in G1 phase at non-permissive temperatures .
Several anti-mitotic drugs have been tested as inhibitors of the proliferation and migration of lens epithelial cells for preventing PCO. For example, anticancer drugs such as mitomycin C can effectively block the proliferation of lens epithelial cells [28,32]. However, the toxicity of these drugs to adjacent ocular tissues compromises their clinical use [33-35]. Recent developments in gene therapy make it possible to deliver suicide or growth-inhibiting genes into lens epithelial cells. Some success has been obtained using retrovirus  and adenovirus  delivery systems. Our work indicates that K6W-ubiquitin can be effectively delivered into HLEC via an adenoviral vector to inhibit cell proliferation in cell culture system. However, since the UPP is essential for many functions in most cells, off-target expression of the K6W-ubiquitin will have to be avoided in order to utilize this strategy safely. Lens cell targeted expression of K6W-ubiquitin could be achieved by constructing a viral vector with a lens specific promoter, such as the αA-crystallin promoter, to drive the expression of K6W-ubiquitin only in lens cells. The data presented here indicate that expression of K6W-ubiquitin in HLEC inhibits cell proliferation and could potentially be utilized for preventing PCO or other proliferative diseases. However, further studies using tissue-specific promoters to drive expression of K6W-ubiquitin in epithelial cells of capsule bags or in animal model of PCO is needed to establish the potential use of K6W-ubiquitin for preventing PCO.
We thank Dr. E. Dudek and Dr. E. Whitcomb for critical review of the manuscript. This work is supported partially by NIH grants EY13250 (to AT), EY11717 (to FS), an NEI core grant EY13078 and EY14083-01A2 (to AT), and the US Department of Agriculture, under agreement number 1950-51000-060-01A.
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