|Molecular Vision 2001;
Received 12 April 2001 | Accepted 22 June 2001 | Published 25 June 2001
PKCa and PKCg overexpression causes lentoid body formation in the N/N 1003A rabbit lens epithelial cell line
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, 104 Willard Hall, Kansas State University, Manhattan, KS, 66506; Phone: (785) 532-7009; email: firstname.lastname@example.org
Purpose: The overexpression of PKCa or PKCg for extended periods of time causes the formation of lentoid bodies in the N/N 1003A rabbit lens epithelial cell line. To determine how differentiated the lentoid bodies are, we have looked for aA-, aB-, b-, and g-crystallin levels in lentoid bodies after 4 and 8 weeks of lentoid body development.
Methods: Cells overexpressing PKCa or PKCg were plated in 6 well plates and were allowed to form lentoid bodies for up to 8 weeks. Lentoid bodies were fixed and stained with PKCa or PKCg antibodies along with either aA-, aB-, b-, or g-crystallin antisera and viewed under a confocal microscope. Lentoid bodies were harvested in lysis buffer and homogenized. Fifty micrograms of protein per lane was loaded onto an SDS-PAGE gel and the bands transferred onto nitrocellulose. The blot was probed with either aA-, aB-, b-, or g-crystallin antibodies for 12 h. Total RNA from lentoid bodies was isolated and 5 mg of total RNA was transcribed to first-strand cDNA. The PCR products were analyzed by 2% agarose gel electrophoresis.
Results: aB-crystallin was present in normal N/N 1003A cells and the lentoid bodies formed from PKCa and PKCg overexpression. aA-crystallin was only detectable in lentoid bodies after PKCa or PKCg overexpression. RT-PCR was able to detect b-crystallin expression while the Western blot analysis and immunocytostaining detected small amounts of b-crystallin protein. No g-crystallin expression was noted in these lentoid bodies.
Conclusions: Overexpression of PKCa or PKCg in the N/N 1003A cell line induced lentoid body formation. These lentoid bodies expressed not only aB-crystallin but aA- and b-crystallin. These results suggest a role for PKCs in lens epithelial cell differentiation to a fiber cell.
The lens contains two major cellular types: the lens epithelium, limited to the anterior region of the lens, and the lens fiber cells which make up the rest of the lens. The lens epithelial cells synthesize specific lens proteins, the a-crystallins. As the epithelial cells begin to differentiate at the bow region of the lens, b-crystallins begin to be synthesized. Fully differentiated lens fiber cells also synthesize the g-crystallins [1,2].
In culture, lens epithelial cells do not differentiate into fiber cells but remain as a simple monolayer. It has been demonstrated that cultured lens epithelial cells will develop into lentoid bodies under a number of stimuli including the presence of the lens capsule [3-6]. The developing lentoid body undergoes a multiphase process which includes cellular replication, aggregation, and differentiation. Lentoid bodies have characteristics which are similar to the embryonic lens in vivo [7,8]. The morphogenesis of the lentoid body in cell culture provides a system that can be analyzed for cellular interactions, lens development, and differentiation. The synthesis of lens crystallins and other lens-specific proteins can be monitored as the lentoid body develops.
Protein kinase C (PKC) has been implicated in the control of cellular proliferation and differentiation in many types of cells [9-11]. The use of PKC activators such as phorbol esters and bryostatin have provided important insight into PKC's role in proliferation and differentiation. However, these activators give a too general indication as to which particular PKC isoform plays a direct role in a given cellular response. Overexpression of a certain PKC isoform is a more direct way to confirm isoform-specific function. PKCa overexpression in a number of cell types has narrowed down the function of PKCa as a major isoform responsible for differentiation [10-16]. Previously in our laboratory, the short term effects of PKCa overexpression were studied. It was demonstrated that PKCa overexpression caused N/N 1003A lens epithelial cells to elongate after 4 days of overexpression . Not only did the cells elongate but they also began to express aA-crystallin, a crystallin not normally expressed in N/N 1003A cells. This was indicative of a more differentiated cell type than normal lens epithelial cells [17,18].
The N/N 1003A rabbit lens epithelial cell line was created by Reddan, et al from cultured lens explants . This cell line exhibits a stable epithelial morphology and it retains lens-specific functions in long-term culture. The cell line synthesizes aB-crystallin. For this study, the eMTH vector containing either holo PKCa or holo PKCg was stably transfected into N/N 1003A cells. The eMTH vector is inducible by 20 mM zinc acetate for 18-24 h. NIH 3T3 cells have been previously transfected with the eMTH vector containing either Raf-1 holo-enzyme or the Raf-1 catalytic fragment . In this study, there were no adverse reactions with the addition of 20 mM zinc acetate and furthermore, overexpression of the desired protein was achieved.
In this study, PKCa or PKCg has been overexpressed in N/N 1003A rabbit lens epithelial cells for prolonged time periods. The overexpression of either PKC isoform for extended periods of time promoted the formation of lentoid bodies in these cultured rabbit lens epithelial cells. Although the overexpression of both PKC isoforms promoted lentoid body formation, the lentoid bodies formed by the overexpression of PKCa were more highly differentiated and expressed higher levels of aA- and b-crystallin than lentoid bodies formed after the overexpressionof PKCg.
The N/N 1003A rabbit lens epithelial cell culturing was as described previously  using Dulbecco's Modified Eagles 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 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 micrograms of the DNA plasmid and 0.1 mg of Lipofectamine into 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.
Phase contrast confocal microscopy
Twenty micromolar zinc acetate was added for 2 to 8 weeks to N/N 1003A cells which contained either the PKCa or PKCg plasmid to induce overexpression. During this time lentoid bodies formed. Live cells were viewed with a Ziess (Thornwood, NY) laser scanning confocal microscope model LSM 410 equipped with an inverted microscope and an Argon-Krypton 488/568/647 laser. A 63x objective with a pinhole of 19 was used to view the cells.
Fluorescent confocal microscopy
Zinc acetate (20 mM) was added for 4 or 8 weeks to N/N 1003A cells which contained either the PKCa or PKCg plasmid to induce overexpression. Lentoid bodies were fixed with 2% paraformaldehyde, 0.2% glutaraldehyde. To label the PKCs and crystallins, the primary antisera was diluted in blocking buffer (3% BSA in PBS) and added to the lentoid bodies and incubated for 12 h at room temperature. The working dilution of the primary antisera was: anti-PKCa (Transduction Laboratories, San Diego, CA) 1:1000, anti-PKCg (Transduction Laboratories) 1:1000, and aA-crystallin, aB-crystallin, b-crystallin, or g-crystallin , 1:50. Lentoid bodies were then washed several times with blocking buffer and incubated with the secondary antisera which was attached to a fluorochrome. Two types of secondary antisera were used (working concentration 7 mg/ml): Alexa fluor 488 (Molecular Probes, Eugene, OR) which emits green (for PKCs) and Alexa fluor 568 (Molecular Probes) which emits red (for crystallins). Lentoid bodies were examined under a Zeiss laser confocal microscope using a 63x objective and a pinhole of 15. The excitation filter was KP 600, the emission filters were BP 515-540 for the green signal and LP 590 for the red signal.
N/N 1003A cells (6 x 106 cells/flask) were harvested in cold 50 mM Tris, 20 mM MgCl2 or lentoid bodies (approximately 80 lentoid bodies contained 25 mg of protein) were carefully removed from the bottom of the flask with forceps using a dissecting microscope. A 12.5% SDS-PAGE gel containing 25 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 distilled 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 dilution 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 dilution (specific to amino acid residues 164-173), the C-terminal region of aB-crystallin at 1:500 dilution (specific to amino acid residues 167-174), and the polyclonal antibodies to b-crystallin and g-crystallin were a kind gift of Dr. Larry Takemoto at Kansas State University [22-24].
Total RNA from N/N 1003A lens epithelial cells or from lentoid bodies 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 primer sequences for b-crystallin were 5'-ATGGCCTCAGACCACCAGAC-3' for the forward primer and 5'-TGTCCTTGTAATCCCCCTTCTCCAGC-3' for the reverse primer . The temperature profile for b-crystallin was 40 cycles of 30 s at 95 °C, 30 s at 49 °C, and 30 s at 72 °C. The primer sequences for g-crystallin were 5'-CGAGGCTTCCAGGGCCGC-3' for the forward primer and 5'-CATCTCATAGAGGACCCAGCAGCCCTG-3' for the reverse primer . The temperature profile for g-crystallin was 40 cycles of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C. The PCR products were analyzed by 2% agarose gel electrophoresis. The bands on the agarose gels were quantitated using Un-Scan-It which measures the pixel intensity of the bands (Un-Scan-It, Silk Scientific, Inc.). Positive controls were performed using cDNA from mouse lens for aA-crystallin, aB-crystallin, b-crystallin, and g-crystallin. In negative controls, no DNA template was added to the reaction mixture. As a negative control for aA-crystallin, b-crystallin, and g-crystallin, cDNA from mouse heart was used.
For band intensity measurements in Western blots and RT-PCR using Un-Scan-It and for confocal microscopy measurements, the intensities and thicknesses respectively were statistically analyzed using Student's t-test for paired or unpaired values with a sample of less than 30. An a level of 0.05 was chosen for statistical significance.
Lentoid body formation following PKCa or PKCg overexpression
Lens epithelial cells which were plated in 6 well culture dishes reached 100% confluency within 2 days. Twenty micromolar zinc acetate was added to the media to induce PKCa or PKCg overexpression, and the cells maintained a normal epithelial-like appearance until 4 days after PKCa overexpression and 7 days after PKCg overexpression, at which time the cells began to elongate . These elongated epithelial cells began to differentiate into lentoid bodies beginning 2 weeks after PKCa overexpression (Figure 1A) and 3 weeks after PKCg overexpression (Figure 1B). After 8 weeks of differentiation, confocal microscopy was used to measure the thickness of lentoid bodies. These were approximately 33 mm ± 10 mm (n=28) for PKCa overexpression (Figure 1C) and 21 mm ± 7.3 mm (n=29) for PKCg overexpression (Figure 1D). From the appearance of the lentoid bodies and from measuring the thickness of the lentoid bodies, the lentoid bodies formed from PKCg overexpression did not appear as highly differentiated as those formed from PKCa overexpression (p=0.0018).
Immunofluorescent staining for crystallins
Immunofluorescent staining for aA-, b-, and g-crystallin confirmed that the N/N 1003A cell line does not express these crystallins while aB-crystallin was present in all cell samples (data not shown). However, as the cells began to differentiate into lentoid bodies and as the lentoid bodies began to become more highly differentiated, not only was aB-crystallin expressed but also aA- and b-crystallin. aB-crystallin was found throughout the differentiation process during the overexpression of both PKC isoforms (Figure 2A,B). As the differentiation process proceeded, aA-crystallin was expressed beginning at about 2 weeks and at about 3 weeks for cells overexpressing PKCa (Figure 2C) and PKCg (Figure 2D), respectively and was quickly followed by b-crystallin expression at about 3.5 weeks (PKCa overexpression, Figure 2E) and about 5.5 weeks (PKCg, Figure 2F). The lentoid bodies formed during the overexpression of either PKC isoform did not exhibit any detectable staining with g-crystallin antibody even at 10 weeks (Figure 2G,H).
Western blot analysis and RT-PCR to detect crystallin proteins and mRNA
Western blot analysis and RT-PCR demonstrated that in N/N 1003A cells, aA-, b-, and g-crystallin are not detectable in cultures before the formation of lentoid bodies, while aB-crystallin, which is normally expressed in this cell line, is detected by both methods (Figure 3, Figure 4, Figure 5, and Figure 6). Four and 8 week old lentoid bodies were used to detect crystallin expression. aB-crystallin is expressed in both 4 and 8 week old lentoid bodies after PKCa or PKCg overexpression (Figure 3A,B). This amount of expression for both time periods is not significantly different nor are the amounts of expression significantly different between the overexpression of the two PKC isoforms (Figure 3C, using Un-Scan-It). aB-crystallin was also detected in the vector-only control cells with the addition of 20 mM zince acetate. aA-crystallin was not detected in untransfected cells and in the vector-only control cells with the addition of 20 mM zinc acetate. aA-crystallin was expressed in both 4 and 8 week old lentoid bodies, however, Western blots and RT-PCR detected lower levels of expression in 4 week old lentoid bodies compared to 8 week old lentoid bodies (Figure 4A-C, using Un-Scan-It). The expression level of aA-crystallin was lower for both time periods in lentoid bodies formed after the overexpression of PKCg compared to that observed after PKCa overexpression (Figure 4C, using Un-Scan-It). b-Crystallin was not detected in untransfected cells and in the vector-only control cells with the addition of 20 mM zinc acetate. b-crystallin expression levels were very low in 4 week old lentoid bodies and the levels were slightly higher in 8 week old lentoid bodies (Figure 5A-C, using Un-Scan-It). Lentoid bodies formed after PKCa overexpression had significantly higher levels of b-crystallin than those formed in PKCg overexpressing lentoid bodies at 8 weeks. g-Crystallin was not detected in untransfected cells and in the vector-only control cells with the addition of 20 mM zinc acetate. Western blot analysis and RT-PCR did not detect any significant expression levels of g-crystallin for cells overexpressing PKCa or PKCg in 4, 8, or 10 week old lentoid bodies (Figure 6A,B).
The data reported herein describe lentoid body formation as a result of the overexpression of PKCa or PKCg in cultured N/N 1003A lens epithelial cells. Lentoid structures began to form within 2 weeks for PKCa overexpression and 3 weeks for PKCg overexpression. As the lentoid structures differentiated further, different lens crystallins were expressed. The N/N 1003A cell line normally expresses only aB-crystallin [25,26]. However, this study demonstrates that aA-crystallin is expressed in early lentoid bodies and b-crystallin is expressed later in lentoid body development.
Although the morphogenetic differentiation observed in the lentoid structures does not reproduce all of the same crystallin synthesis profile as that in the developing lens, the data indicate that some of the changes in lens crystallin syntheses are similar. It has been shown that the proportion of crystallins in lentoid structures does not resemble those of embryonic lens fibers . However, the cell culture system described herein permits a more detailed analysis of the cellular and biochemical changes taking place at the cellular level using this model system.
The lentoid structures which formed expressed the same lens crystallins as those observed during lens fiber formation in vivo. It has been demonstrated previously that lentoid-rich cultures show very similar quantitative changes in the proportions of a- and b-crystallin expression as those which occur in vivo . In this study, aB-crystallin is expressed throughout lentoid development. aA-crystallin is expressed at the initial formation of lentoid structures which occurred after 2 weeks for PKCa overexpression and 3 weeks after PKCg overexpression. b-crystallin expression appeared at 3.5 weeks and at 5.5 for PKCa and PKCg overexpression, respectively.
The overexpression of PKCa or PKCg was the only stimulus used to induce lentoid body formation. Previously, it was shown in our laboratory that the overexpression of PKCa or PKCg caused N/N 1003A lens epithelial cells to elongate after 4 days of PKCa overexpression and 7 days of PKCg overexpression . The development of lentoid structures resulted after prolonged overexpression. Lentoid bodies formed from PKCg overexpression developed slower and the expression of crystallins was lower when compared to that of PKCa overexpressing cells. Our previous studies suggest that PKCg has a direct role in controlling gap junction activity . In contrast, PKCa has no effect on gap junctions but it does cause changes in cell growth and differentiation [17,28]. Thus, these two major lens PKC isoforms appear to have unique targets. The protein(s) which is specifically phosphorylated by PKCa has not been identified but could include structural proteins involved in cell shape and/or differentiation.
The authors would like to 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. Daniel Boyle for helpful discussions on confocal microscopy.
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