|Molecular Vision 2001;
Received 12 January 2001 | Accepted 28 February 2001 | Published 7 March 2001
Protein kinase C a and g in N/N 1003A rabbit lens epithelial cell differentiation
Lynn M. Wagner,1
Dolores J. Takemoto2
1Department of Anatomy and Physiology, 2Department of Biochemistry, Kansas State University, Manhattan, KS
Correspondence to: Dolores J. Takemoto, Department of Biochemistry, Kansas State University, 104 Willard Hall, Manhattan, KS, 66506; Phone: (785) 532-7009; email: email@example.com
Purpose: To determine if Protein Kinase C (PKC) plays a role in the initiation of lens epithelial cell differentiation into a lens fiber cell.
Methods: PKCa or PKCg was overexpressed in N/N 1003A lens epithelial cells for up to 7 days. Phase contrast microscopy was used to observe morphological changes associated with PKCa or PKCg overexpression. Cell cycle changes in cells overexpressing PKCa or PKCg were measured using acridine orange staining and flow cytometry. Crystallin levels in cells overexpressing PKCa or PKCg were measured using Western blots and RT-PCR.
Results: Significant differences in cell cycling were observed between untransfected cells and those overexpressing PKCa or PKCg. Overexpression of PKCa and PKCg caused the cells to lose their epithelial-like appearance and elongate. aB-crystallin expression was detected in all the samples while aA-crystallin was detected only in cells after 7 days of PKCa or PKCg overexpression.
Conclusions: The observations that aA-crystallin is only found in N/N 1003A cells overexpressing PKCa or PKCg for 7 days along with the finding that a block in the G0/G1 phase of the cell cycle and the consequent morphological changes are observed, indicate that PKCa and PKCg may have a role in the initiation of differentiation in lens epithelial cells.
The mammalian lens is an avascular tissue which consists of a single layer of nucleated epithelial cells at the anterior surface. The epithelial cells of the lens are arranged into four morphologically different cell types . The four zones consist of the central epithelial zone located on the anterior surface, a zone of smaller epithelial cells which neighbors the central zone, a pre-elongation zone of epithelial cells, and an elongation zone where epithelial cells elongate and differentiate into fiber cells . The differentiation of a lens epithelial cell can be followed by the increased expression of, first, a-crystallin, followed by b-crystallin, then g-crystallin [2-5]. In the centrally located epithelial cells, aA-crystallin and aB-crystallin are expressed at a ratio of 1:3 while in the elongation zone the ratio of aA-crystallin to aB-crystallin becomes 3:1 . b-crystallins and g-crystallins are not expressed in the epithelial cells but are found only in fiber cells [1,2,6-9].
Exactly what causes lens epithelial cells to differentiate is not well understood; however, some progress has been made in determining the underlying molecular and cellular processes of lens epithelial cell differentiation. The differentiation of lens epithelial cells can be promoted by growth factors present in the ocular fluids . Some growth factors such as epidermal growth factor promote mitosis . Other growth factors such as basic fibroblast growth factor, insulin growth factor, and insulin promote cell migration and differentiation . Leenders et al.  found that 12-tetradecanoyl-13-phorbol-acetate (TPA), a potent activator of protein kinase C (PKC), could replace bFGF in the induction of differentiation. TPA's ability to induce differentiation in several tumor cell lines suggests a role for PKC in the regulation of this process [13-15].
Lens epithelial cells including the N/N 1003A cell line contain high basal levels of PKCa and PKCg. Although a specific role for PKCa in lens epithelial cell differentiation is not known, there is a general consensus that PKCa is associated with cellular differentiation . This information is based upon the evidence that TPA can initiate lens epithelial cell differentiation and the fact that certain PKC isoforms are thought to play a role in tumor cell differentiation. We have overexpressed PKCa or PKCg in the rabbit lens cell line N/N 1003A to determine if the activation of PKCa or PKCg is a critical step in the initiation of lens epithelial cell differentiation. Our hypothesis is that PKC plays an important role in the initiation of differentiation. We have examined cell cycle changes, morphological changes, and results of Western blots and RT-PCR analyses to determine crystallin expression in N/N 1003A cells overexpressing either PKCa or PKCg.
The N/N 1003A rabbit lens epithelial cell culturing was performed as described previously , using Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% heat inactivated fetal calf serum (Atlanta Biologicals, Norcross, GA) and 50 mg/ml gentamicin (Life Technologies, Grand Island, NY). The cells were grown at 37 °C in an atmosphere of 10% CO2 and 90% air. All experiments were conducted with cells near 80-90% confluency (6.0 x 106 cells/flask) except where stated differently.
The PKCa and PKCg plasmids were a kind gift from Dr. W. Anderson (NCI). The holo PKCa plasmid was derived from the bovine sequence and the holo PKCg plasmid was derived from the rodent sequence . In addition, the eMTH (empty vector) was used as a control in stable transfection experiments. The MTH vector is inducible at low levels by 20 mM zinc acetate for 18 to 24 h.
Transfection of N/N 1003A rabbit lens epithelial cells took place when the cells reached approximately 60% confluency. The cells were transfected using Lipofectamine (Life Technologies). Five mg of the DNA plasmid and 0.1 mg of Lipofectamine in 200 ml of serum free media was used for each transfection. The cells were incubated with the DNA plasmid and Lipofectamine for 12 h at 37 °C. After incubation, media containing twice the amount of fetal calf serum (20%) was added for 24 h. Following this incubation, the media was replaced with media containing 10% fetal calf serum. The transfected cells were selected with 750 mg/ml G418 (Life Technologies) for 6 weeks and grown in half that concentration of G418 thereafter.
Protein kinase C assay
A mixed micelle protein kinase C assay system (Life Technologies) was used to measure PKC activity according to the manufacturer's instructions. N/N 1003A cells (6.0 x 106) were lysed with 0.5 ml of extraction buffer (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 mg/ml aprotinin, and 25 mg/ml leupeptin). Protein concentrations were measured and 25 ml of the cell lysate containing 40 mg of protein was incubated for 20 min at room temperature with the 5X PKC substrate solution (250 mM Ac-MBP (residues 4-14), 100 mM ATP, 5 mM CaCl2, 100 mM MgCl2, 20 mM Tris, pH 7.5) or the 5X inhibitor solution (100 mM PKC (residues 19-36), 20 mM Tris, pH 7.5).To begin the reaction, PKC reaction mixture consisting of 3000 Ci/mmol of [g-32P] ATP was added for 5 min at 30 °C. A 25 ml aliquot of each reaction was spotted onto phosphocellulose discs and washed 3 times with 1% phosphoric acid for 5 min each and then washed 3 times with water. Radioactivity that was transferred to the substrate was measured by liquid scintillation counting. Results are expressed as total PKC activity in pmol of substrate phosphorylated/min ± standard error of the mean.
N/N 1003A cells (6.0 x 106 cells/flask) were harvested in cold 50 mM Tris, 20 mM MgCl2. A 12.5% SDS-PAGE gel containing 50 mg of total cell protein was transferred to a nitrocellulose membrane (OPTI TRAN, Midwest Scientific, St. Louis, MO). The membrane was incubated for 12 h in primary antibody in 3% powdered milk in water at room temperature. The membranes were then washed for 10 min, 3 times, in phosphate buffered saline (PBS). Anti-mouse IgG or anti-rabbit IgG (Promega, Madison, WI) at 1:2500 in 3% milk/PBS was added to the membrane for 3 h at room temperature. Membranes were then washed for 10 min, 3 times, in PBS. The membranes were then developed using SuperSignal Chemiluminescent Substrate (Pierce, Rockford, IL) and X-ray film (Molecular Technologies, Midwest Scientific, St. Louis, MO). After exposure, the X-ray films were scanned and quantitated (Un-Scan-It, Silk Scientific, Inc., Orem, UT). The antibodies specific against the C-terminal region of aA-crystallin at 1:500 (specific to amino acid residues 164-173) and the C-terminal region of aB-crystallin at 1:500 (specific to amino acid residues 167-174) were a kind gift of Dr. Larry Takemoto at Kansas State University . PKCa antisera was used at 1:1000 (Transduction Laboratories, San Diego, CA), and PKCg antisera at 1:2500 (Transduction Laboratories).
Cell morphology changes
N/N 1003A cells were grown in 6-well tissue culture plates at 5.0 x 102 cells per well (approximately 80% confluent). Live cells were viewed on a laser scanning confocal microscope, model LSM 410 (Ziess, Thornwood, NY) equipped with an Axiovert 100 inverted microscope, an Argon-Krypton 488/568/647 laser. A 63x objective with a pinhole of 18 was used to view the cells.
Acridine orange staining and flow cytometry
The N/N 1003A rabbit lens epithelial cells were stained with acridine orange to determine cell cycle changes following the procedure by Darzynkiewicz . Cells (200 ml,1.0 x 106 cells/flask) in PBS were permeabilized with 400 ml of a solution containing 1% Triton X-100, 0.15 N NaCl, and 0.08 N HCl for 15 s on ice. Immediately after permeabilization, cells were stained with 1.5 ml of acridine orange solution containing 0.6 mg/ml acridine orange, 1 mM EDTA-Na, 0.15 M NaCl, and 126 mM phosphate-37 mM citric acid buffer, pH 6.0 for 3 min on ice. Fluorescence was measured in a FACScan flow cytometer (Becton Dickinson, Cockeysville, MD) using an argon laser beam that emits at 488 nm and a combination of filters that detected green fluorescence for DNA (FL1) and red fluorescence for RNA (FL3). The LP 530/30 filter was used for DNA fluorescence and the LP 650 filter was used for RNA fluorescence. The data using the information from only RNA was analyzed using the ModFit program (Verity, Sunnyvale, CA) in order to determine percentages at each phase of the cell cycle.
Total RNA from N/N 1003A lens epithelial cells was isolated using TRIzol (Life Technologies) and 5.0 mg of total RNA was transcribed to first-strand cDNA using the Thermoscript RT-PCR System kit (Life Technologies) according to the manufacturer's directions. The primer sequences for aA-crystallin were 5'-CCCAGCTCAGGACGAGGGTGC-3' for the forward primer and 5'-ACGACCTGCTGCCCTTCCTGTC-3' for the reverse primer. The temperature profile for aA-crystallin was 45 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. The primer sequences for aB-crystallin were 5'-GACAGCAGGCTTCTCTTCACGGG-3' for the forward primer and 5'-GGAGAGCACCTGTTGGAGTCTGACC-3' for the reverse primer . The temperature profile for aB-crystallin was 35 cycles of 30 s at 95 °C, 30 s at 61 °C, and 30 s at 72 °C. The PCR products were analyzed by 2% agarose gel electrophoresis. Positive controls were performed using cDNA from mouse lens for aA-crystallin and aB-crystallin. In negative controls, no DNA template was added to the reaction mixture. Also, cDNA from mouse heart was used as a negative control for aA-crystallin..
Data presentation and statistical analysis
Data are expressed as mean ± standard error of the mean.The data were statistically analyzed using Student's t-test for paired or unpaired values with a sample number of less than 30. Values of p<0.05 were considered to be statistically significant.
The PKCa or PKCg overexpression system in the N/N 1003A cell line
The eMTH vector containing either the holo PKCa or the holo PKCg is inducible with 20 mM zinc acetate. Stable transfectants of N/N 1003A cells expressing the PKCa or PKCg isoform plasmid were selected with 750 mg/ml G418 for 6 weeks. N/N 1003A lens epithelial cells overexpressed the desired PKC isoform by induction with 20 mM zinc acetate for 24 h (Figure 1). Cells overexpressing PKCa exhibited an 1.68 fold increase compared to basal levels while cells overexpressing PKCg exhibited an 1.94 fold increase compared to basal levels; however, basal levels of PKCa and PKCg are already high in untransfected N/N 1003A cells. To determine the effect of the overexpression of PKCa and PKCg upon PKC activity, a PKC activity assay was performed. Total PKC activity increased by 1.5 fold in cells overexpressing PKCa. Total PKC activity increased by 2.3 fold in cells overexpressing PKCg (Figure 2). The differences in vector-only control cells with or without zinc acetate and untransfected cells with or without zinc acetate were not significant (Figure 2). It is important to note that PKC activity can be controlled by using a lower concentration of zinc acetate. This is desirable as excess overexpression causes cells to enter apoptosis (data not shown).
Cell morphology changes following overexpression of PKCa or PKCg in the N/N1003A cell line
At day 4 of overexpression of PKCa and at day 6 of overexpression of PKCg, the cells began to undergo distinctive morphological changes. The uninduced cells appeared as a simple epithelial cell layer which was identical in appearance to normal N/N 1003A cells with and without the addition of zinc acetate (Figure 3A-C,E). Approximately 60% of the cell population became elongated upon the overexpression of PKCa while the rest of the dish appeared as a monolayer of epithelial cells (Figure 3D). Of the cells overexpressing PKCg, approximately 45% of the cells underwent a similar elongation as seen in cells overexpressing PKCa while the rest of the dish appeared as a monolayer of epithelial cells (Figure 3F).
Cell cycle changes as a result of PKCa or PKCg overexpression
The cell cycle profiles for untransfected N/N 1003A cells with or without zinc acetate and N/N 1003A cells overexpressing PKCa or PKCg for 7 days were analyzed. Table 1, Table 2, and Table 3 list the percentage of cells present in the different stages of the cell cycle for PKCa and PKCg overexpression and for the vector-only control, respectively.
In the control N/N 1003A cell population, 34.1% were in G0/G1 and the majority of the population were in S-phase (56.84%). The cell population overexpressing PKCa for 24 h had a slight increase in G0/G1 to 41.24% while the S-phase population decreased slightly. In the cell population overexpressing PKCa for 7 days, 89.63% were in G0/G1 and a significant decrease in cells cycling in S-phase was observed (to 8.05%). These results correlate with the drop in 3H-thymidine incorporation (data not shown).
The cell population overexpressing PKCg for 24 h had only a small increase in cells in G0/G1 (to 36.93%). In the cell population overexpressing PKCg for 7 days, 67.47% were in G0/G1 while the S-phase population continued to decrease (to 25.41%). Like PKCa overexpression, this data also correlated with a drop in 3H-thymidine incorporation which was exhibited (data not shown). These data indicate that overexpression of PKCa or PKCg (to a lesser extent) in the N/N 1003A cell line caused a G0/G1 block.
Expression of a-crystallin determined by western blot analysis
aA-crystallin and aB-crystallin protein expression was determined by Western blot analyses. aB-crystallin which is normally expressed in the N/N 1003A cell line was detected in all the samples (Figure 4A). aA-crystallin which is not expressed in the N/N 1003A cell line was not detected in the untransfected cell line, the cells transfected with empty vector, and cells overexpressing PKCa or PKCg for 24 h (Figure 4B). However, aA-crystallin was detected in cells overexpressing PKCa for 7 days. A lower level of aA-crystallin expression was observed in cells overexpressing PKCg for 7 days, thus correlating with the elongation of the lens epithelial cells observed at day 4 and day 6.
Analysis of a-crystallin by RT-PCR
aB-crystallin and aA-crystallin transcripts were measured in 5.0 mg of total RNA by RT-PCR. RT-PCR demonstrated that aA-crystallin was not expressed in normal untransfected N/N1003A cells (Figure 5B) while aB-crystallin was expressed in these cells(Figure 5A). RT-PCR confirmed the data from Western blots that aA-crystallin was expressed in low levels in the cells overexpressing PKCa for 7 days. Lower levels of expression of aA-crystallin were noted for PKCg overexpressing cells after 7 days (Figure 5B). aA-crystallin expression was not observed in the rest of the samples while aB-crystallin was expressed in all of the samples.
The results of the current study support the hypothesis that PKCa or PKCg overexpression in the lens epithelial cell line N/N 1003A could play a role in early differentiation. Significant differences in cell cycling were determined between normal N/N 1003A cells and those overexpressing PKCa and PKCg. It is known that once lens epithelial cells reach the elongation zone of the lens, they stop dividing and elongate . The present study also demonstrates changes in cell morphology after extended overexpression of PKCa or PKCg. Over half of the cells begin to elongate by day 7 while the rest remain as a monolayer of epithelial cells. These cellular shape changes are significant because without adding any type of exogenous growth factor except for what is contained in the FBS, the cells were driven to change their epithelial-like appearance and elongate compared to untransfected cells in the same media. The initial differentiation marker aA-crystallin which is not normally expressed in the N/N 1003A cell line, was detected in cells overexpressing PKCa and g for 7 days.
In 1986, Reddan developed the rabbit lens cell line N/N 1003A . The cell line is an immortalized population of morphologically undifferentiated lens epithelial cells. It is uncertain from previous studies whether the N/N 1003A cell line expresses aA-crystallin [21,23-25]. Previous studies have demonstrated by Western blot that aA- and aB-crystallin were present in the N/N 1003A cell line [23,24]. However, later studies have shown that the cell line does not produce detectable levels of aA-crystallin [21,25].
In this current study, aA-crystallin, which is expressed in higher levels at the bow region of the lens than aB-crystallin, was not detected in untransfected N/N 1003A cells, the cells transfected with empty vector, or the cells overexpressing PKCa or PKCg for 24 h by Western blot or RT-PCR. However, low levels of aA-crystallin were detected after 7 days of PKCa or PKCg overexpression. The morphological changes and the expression of aA-crystallin strongly suggest that PKCa, and to certain extent PKCg, play a role in the initiation of differentiation; however, it is important to bear in mind that the overexpression of PKCa or PKCg may lead to the phosphorylation of non-physiological substrates, therefore producing effects that are unrelated to the kinase activity of the enzyme. The fact that the morphological changes take longer in cells overexpressing PKCg along with the lower level of aA-crystallin expression, indicates that PKCg may play a lesser role in the differentiation of lens epithelial cells to lens fiber cells. It has been found that the overexpression of PKCg inhibited gap junctional intercellular communication while PKCa overexpression did not . Thus, it is PKCg and not PKCa which regulates gap junctions in lens epithelial cells. PKCa could be the major PKC isoform that initiates differentiation while PKCg may have a role in differentiation through its primary role in gap junction regulation.
The authors thank Dr. John Reddan for the gift of the N/N 1003A cell line, Dr. Wayne Anderson for the gift of the PKCa and PKCg plasmids, and Dr. Larry Takemoto for the gift of the aA- and aB-crystallin antisera. Thanks also to Dr. Peggy Zelenka and Dr. Daniel Boyle for the helpful discussions.
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