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Molecular Cancer Research 1:12-24 (2002)
© 2002 American Association for Cancer Research

Angiogenesis, Metastasis, and the Cellular Microenvironment

CDK5 Regulates Cell Adhesion and Migration in Corneal Epithelial Cells

Chun Gao1, Sewite Negash1, Hong Tao Guo1, Dolena Ledee1, Hwai-Shi Wang2 and Peggy Zelenka1

1 National Eye Institute, NIH, Bethesda, MD; and
2 Yang Ming University, Taipei, Taiwan

Requests for reprints: Peggy S. Zelenka, NIH/NEI, Room 214, Building 6, 6 Center Drive MSC 2730, Bethesda, MD 20892-2730. E-mail: zelenkap{at}intra.nei.nih.gov


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
CDK5 and its activator, p35, are expressed in mouse corneal epithelium and can be coimmunoprecipited from corneal epithelial cell lysates. Immunostaining shows CDK5 and p35 in all layers of the corneal epithelium, especially along the basal side of the basal cells. Stable transfection of corneal epithelial cells with CDK5, which increases CDK5 kinase activity by approximately 33%, also increases the number of cells adhering to fibronectin and the strength of adhesion. CDK5 kinase activity seems to be required for this effect, because the kinase inactive mutation, CDK5-T33, either reduces adhesion or has no significant effect, depending on the level of expression. Using an in vitro scrape wound in confluent cultures of stably transfected cells to examine the effect of CDK5 on cell migration, we show that reoccupation of the wound area is significantly decreased by CDK5 and increased by CDK5-T33. These findings indicate that CDK5 may be an important regulator of adhesion and migration of corneal epithelial cells.

Key Words: CDK5 • p35 • adhesion • cytoskeleton • migration


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
CDK5 is a member of the cyclin-dependent kinase family of proline-directed protein kinases (1). Although most of the members of this family are involved in cell cycle regulation, CDK5 is preferentially expressed in terminally differentiated neurons of the developing and adult nervous system (2, 3) and has an essential role in regulating the complex migration program of postmitotic neurons during embryogenesis (4–7). Several other neuronal functions of CDK5 have also been identified, including cell-cell adhesion (8), cell-matrix adhesion (9), neurite extension (4), and cytoskeletal regulation (10, 11). A number of known CDK5 substrates in neurons are either cytoskeletal proteins or proteins involved in cytoskeletal regulation. Examples include the neurofilament proteins, NH and NM (10, 12, 13), the microtubule-associated proteins, {tau} and Map1b (14, 15), the protein kinase PAK1 (16), which regulates actin polymerization, and ß-catenin (8, 17), a multifunctional component of cadherens junctions. To be enzymatically active, CDK5 must form a complex with one of two known regulatory subunits, p35 and p39, both of which are expressed at high levels in neurons (5, 18–20). These activators of CDK5 have been shown to bind to cytoskeletal components such as actin filaments (16, 21), microtubules (11, 21), and the cytoskeletal scaffolding protein, Cables (22).

A number of observations suggest that CDK5 may also have important functions in non-neuronal cells, especially during development and differentiation. CDK5/p35 activity seems to be an essential factor in monocytic differentiation (23–25). Not only do peripheral monocytes express high levels of CDK5 activity, but overexpression of CDK5/p35 in HL60 cells is sufficient to force differentiation along the monocytic pathway. CDK5 kinase activity also has been associated with myogenic differentiation (26, 27), and seems to be required for expression of the muscle differentiation markers, myogenin and troponin T (26). Finally, blocking CDK5 kinase activity by injecting CDK5-T33 into one of the two dorsal cells of a four-cell Xenopus embryo leads to numerous abnormalities in the development of the eye, including microphthalmia, disruption of eye structure, and malformation of the lens (28). Because this phenotype can be rescued by concomitantly injecting wild-type CDK5, the developmental defects seem to be caused by inhibition of endogenous CDK5 activity. In keeping with the above findings, the CDK5 activating protein, p35, has been found in monocytes (24), muscle (29), lens (30), and retina (31).

Although CDK5 expression and function have been investigated in the lens and retina, the available information provides little insight into its involvement in eye development and differentiation. CDK5 expression has been observed in the retina, where it is correlated with developmental neuroplasticity (32). This finding is consistent with numerous studies indicating the importance of CDK5 for neuronal differentiation (4, 5, 7, 33). In addition, CDK5 has been reported to phosphorylate the regulatory subunit of cGMP phosphodiesterase (P{gamma}) in the rod outer segments, suggesting that it may regulate some aspect of the visual transduction cycle (31, 34). Work from our laboratory has shown that differentiating fiber cells of the rat lens contain active CDK5/p35 (30). The present study examines the expression of CDK5 and p35 in the corneal epithelium and tests the possibility that CDK5 may have a role in regulating cell-matrix adhesion of corneal epithelial cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
CDK5 and Its Activator, p35, Are Expressed in Mouse Corneal Epithelium
To examine the expression of CDK5 and its regulatory subunit, p35, in the corneal epithelium, corneal epithelial lysates were immunoblotted using antibodies specific for the COOH termini of CDK5 and p35. Anti-CDK5 antibody detected a single strong immunoreactive band of the correct molecular weight (Fig. 1A ) that comigrated with an immunoreactive band in the brain lysate (Fig. 1A). Antibody against p35 also detected a single immunoreactive band of approximately Mr 35,000 that comigrated with the corresponding immunoreactive band from the brain lysate (Fig. 1A). The specificity of the anti-p35 antibody was confirmed by complete neutralization of the immunoreactivity by the antigenic peptide (not shown; see Fig. 4B).



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  		FIGURE 1. Expression of protein and mRNA for CDK5 and p35 in mouse corneal epithelium. A. Immunoblots of whole cell extracts. Protein extracted from adult mouse corneal epithelium was immunoblotted with antibody to CDK5 and p35. The protein bands identified by anti-CDK5 from corneal epithelium comigrated with CDK5 from the brain and the p35 band in the corneal epithelia also comigrated with p35 from the brain lysate. B. Coimmunoprecipitation experiments. Whole cell lysates from corneal epithelium and brain were immunoprecipitated using anti-CDK5 antibody. Immunoblotting was performed on immunoprecipitates using anti-CDK5 and p35 antibodies. Anti-CDK5 detected CDK5 on immunoprecipitates from both corneal epithelium and brain. A p35 immunoreactive band was detected in the CDK5 immunoprecipitates from the corneal epithelium, which comigrated with p35 from the brain. C. RT-PCR for p35 mRNA. RT-PCR was performed on total RNA extracted from the corneal epithelium and A6(1) cells. A single product for p35 with correct size (923 bp) was detected. Controls contained RNA from the corneal epithelium or the A6(1) cells, but no reverse transcriptase (-RT). D. RT-PCR for p39 mRNA. RT-PCR was performed on total RNA from brain, corneal epithelium, and the A6(1) cells. The assay did not detect p39 mRNA in corneal epithelium or A6(1) cells although it successfully detected p39 mRNA in the brain RNA included as a positive control. This PCR product was not present in a negative control containing brain RNA without reverse transcriptase (not shown).

 


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  		FIGURE 4. Stable transfection of A6(1) cells with CDK5 and CDK5-T33. A. A6(1), a corneal epithelial cell line, was stably transfected with pGFP only, pGFP-CDK5, and pGFP-CDK5-T33. The expression of pGFP-CDK5 and pGFP-CDK5-T33 was examined by immunoblotting using antibodies against CDK5. The single arrow points to exogenous CDK5, whereas the arrowhead indicates endogenous CDK5. B. Proteins from A6(1) cells stably transfected with pGFP only, pGFP-CDK5, and pGFP-CDK5-T33 were immunoblotted with antibody to p35. The level of expression of endogenous p35 was not affected by the expression of exogenous CDK5 (left panel, open arrow). The p35-immunoreactive band was obliterated by the presence of p35 blocking peptide (right panel). C. CDK5 kinase activity. CDK5 was immunoprecipitated from GFP-, GFP-CDK5-, and GFP-CDK5-T33-transfected A6(1) cell lysates. Kinase activity of the immunoprecipitated protein was assayed in vitro using a biotinylated peptide substrate (PKTPKKAKKL). The results of six independent measurements from three experiments were averaged and expressed as pmol ATP/min/mg of total A6(1) protein. The differences between the GFP-CDK5 and GFP only, and GFP-CDK5- and GFP-CDK5-T33-transfected cells are statistically significant (P < 0.03 and P < 0.01, respectively).

 
To determine whether CDK5 and p35 form an intracellular protein complex, proteins were immunoprecipitated from lysates of corneal epithelium or brain using anti-CDK5 antibody, then immunoblotted with anti-CDK5 and anti-p35 antibodies. Anti-CDK5 antibody detected CDK5 in the immunoprecipitates from both tissues (Fig. 1B), confirming that the immunoprecipitation was successful. Anti-p35 antibody also detected p35 in the CDK5 immunoprecipitates from both tissues (Fig. 1B), indicating that p35 and CDK5 form an intracellular protein complex in corneal epithelium as well as in the brain. As in the immunoblots of whole cell lysates, the p35 band in CDK5 immunoprecipitates from corneal epithelium comigrated with p35 from the brain, further confirming its identification.

As an additional assay for the expression of p35, we performed reverse transcription-PCR (RT-PCR), using oligonucleotides specific for p35 mRNA. A single RT-PCR product of the predicted size (923 bp) was detected (Fig. 1C) in total RNA from corneal epithelium and from A6(1) corneal epithelial cells. Partial sequencing of the isolated PCR product confirmed that it was derived from p35 mRNA. In contrast, RT-PCR did not detect any expression of p39 from total RNA in corneal epithelium or A6(1) cells, although the 160-bp RT-PCR product corresponding to p39 mRNA was observed in an equal amount of total RNA from the brain (Fig. 1D). Thus, corneal epithelial cells appear to express the CDK5 activating protein, p35, as judged by RT-PCR, partial nucleotide sequence, immunoreactivity, comigration with brain p35, and the ability to bind CDK5. However, these cells do not express detectable levels of p39.

CDK5 and p35 Colocalize in Mouse Cornea
To examine the distribution and subcellular localization of CDK5 and p35 in mouse cornea, immunocytochemical staining was performed on paraffin sections from 2-month-old normal mice. In general, CDK5 and p35 showed similar localization patterns. Positive staining for CDK5 (Fig. 2, A–C ) and p35 (Fig. 2, E–G) was observed in the cytoplasm of all layers of corneal epithelial cells, especially along the basal side of the basal cells (Fig. 2, C and G, arrows). Signals were strong in the central corneal epithelium, but immunoreactivity for both CDK5 (Fig. 2B, arrow) and p35 (Fig. 2F, arrow) was reduced in the limbus region. Interestingly, just beyond the limbus region, in the conjunctival epithelium, CDK5 and p35 staining became strongly positive again (Fig. 2, B and F, arrowhead). Corneal endothelium was also positively stained for CDK5 (Fig. 2C). In addition, the inner nuclear layer of the retina (Fig. 2, A and E), lens epithelial cells, and the cortical fiber cells of the lens stained positive for CDK5 (Fig. 2, A and B) and p35 (Fig. 2, E and F). No specific staining was observed in controls in which the antigenic peptides were included during incubation with the primary antibodies for CDK5 (Fig. 2D) and p35 (Fig. 2H).



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  		FIGURE 2. Immunohistochemical staining for CDK5 (A–D) and p35 (E–H) on paraffin sections from adult mouse cornea. Sections were incubated with antibody to CDK5 (A–D) or p35 (E–H). To determine the level of nonspecific staining, the corresponding blocking peptides were included during incubation with the primary antibody (D,H). Positive staining was seen for CDK5 (A–C) and p35 (E–G) in corneal epithelium, but the staining for both CDK5 (B, arrow) and p35 (F, arrow) was attenuated within the limbus region. CDK5 and p35 were also strongly stained in the conjunctival epithelium (B,F, arrowheads). CDK5 and p35 appear to be present in the cytoplasm of all layers of corneal epithelium, especially along the basal side of basal cells (C,G, arrows). Staining for both CDK5 and p35 was also positive in the epithelium and cortical fiber cells of the lens (A,B and E,F), and inner nuclear layer of the retina (A,E, arrow). Scale bar, 250 µm for A, D, E, and H; 125 µm for B,F; 25 µm for C,G. L, lens; R, retina; C, cornea; S, stroma.

 
Subcellular Distribution of CDK5 and p35
In the brain, CDK5 is found primarily in the cytosol, whereas p35 is mostly associated with the membrane fraction (35). To investigate whether CDK5 and p35 have similar subcellular distributions in mouse corneal epithelium, we prepared cytosolic, cytoskeletal, and membrane fractions of corneal epithelial cells. Immunoblotting showed that although CDK5 and p35 were present to some degree in all three fractions, the cytoplasmic fraction contained the bulk of the CDK5 (Fig. 3A ), whereas the membrane fraction was enriched with p35 (Fig. 3B). CDK5 and p35 antibodies recognized multiple bands in all three fractions (Fig. 3, A and B). These are likely due to phosphorylated forms (18, 19, 22, 36, 37), which are preserved due to the presence of phosphatase inhibitors in the fractionation buffer.



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  		FIGURE 3. Differential distribution of CDK5 (A) and p35 (B) in subcellular fractions from corneal epithelium. Corneal epithelia were separated into three different fractions: cytosol, cytoskeleton, and membrane by differential detergent extraction. Immunoblotting was then performed on equal amounts of protein from each fraction using antibodies against (A) CDK5 and (B) p35. The multiple bands for CDK5 and p35 in these preparations are likely due to post-translational modifications.

 
Stable Transfection of A6(1) Cells with CDK5
To examine the physiological function of CDK5 in corneal epithelial cells, a mouse corneal epithelial cell line, A6(1), was stably transfected with GFP-tagged constructs of CDK5 or the kinase inactive form, CDK5-T33, which contains a K to T substitution at amino acid 33 (4). If expressed at sufficiently high concentrations, CDK5-T33 can exert a dominant negative effect on endogenous CDK5 activity by sequestering the available p35 (4). GFP-CDK5 and GFP-CDK5-T33 were expressed at comparable levels in stably transfected A6(1) cells (Fig. 4A, arrow ), and their expression did not affect the levels of endogenous CDK5 (Fig. 4A, arrowhead) or p35 (Fig. 4B, open arrow). Expression of CDK5 and CDK5-T33 also had no observable effect on growth or survival of A6(1) cells (not shown). Although expression of GFP-tagged CDK5 and CDK5-T33 was relatively low as compared to endogenous CDK5 (Fig. 4A), more than 95% of the stably transfected A6(1) cells expressed the constructs, as judged by microscopic examination of GFP fluorescence (not shown).

CDK5 Kinase Activity
To determine whether stable transfection with CDK5 and CDK5T33 altered CDK5 kinase activity, CDK5 was immunoprecipitated from extracts of GFP-transfected control A6(1) cells, GFP-CDK5-A6(1) cells, and GFP-CDK5-T33-A6(1) cells, and in vitro kinase assays were performed using a biotinylated peptide substrate (Fig. 4C). For comparison, we also measured CDK5 kinase activity in immunoprecipitates from extracts of newborn rat brain and mouse corneal epithelium (not shown). The results showed that A6(1) cells contain a low level of endogenous CDK5 activity (approximately 0.24 pmol PO4 transferred/min/mg cellular protein). Under the same assay conditions, newborn rat brain contained about 20 times as much CDK5 activity (4.66 pmol PO4 transferred/min/mg cellular protein), whereas corneal epithelial extracts contained no detectable activity. Stable transfection with CDK5 increased the activity in A6(1) cells to 0.32 pmol PO4 transferred/min/mg cellular protein, an increase of 33%. This increase was statistically significant (P < 0.03). In contrast, stable transfection with CDK5-T33 produced a small, but not statistically significant, reduction in CDK5 activity (to 0.21 pmol PO4 transferred/min/mg cellular protein). Thus, CDK5 activity is elevated in A6(1) cells stably transfected with CDK5, and is not significantly changed in cells expressing an equivalent amount of CDK5-T33.

Localization of GFP-CDK5 and GFP-CDK5-T33
Because the biological effects of protein kinases may also be affected by their subcellular localization, we tested whether localization of GFP-CDK5 or GFP-CDK5-T33 corresponded to the normal subcellular localization of CDK5. The distribution of GFP, GFP-CDK5, and GFP-CDK5-T33 fluorescence in transiently transfected A6(1) cells was compared with the immunolocalization of CDK5 in control cells transfected with GFP alone and in untransfected A6(1) cells. In cells transfected with the GFP tag alone, GFP fluorescence was seen only in the cytoplasm, especially in the nuclear/perinuclear region (Fig. 5A ). In contrast, the GFP-tagged CDK5 constructs were localized not only in the cytoplasm, but also in filopodia, and at the edges of lamellipodia, where cells make focal contacts with the extracellular matrix (Fig. 5, B, C, E, and F). Endogenous CDK5 in control cells transfected with GFP only and in untransfected cells was similarly localized in the cytoplasm, in filopodia, and along the edges of lamellipodia (Fig. 5D). Thus, neither overexpression nor the presence of the GFP tag seems to alter the subcellular localization of CDK5.



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  		FIGURE 5. Immunolocalization of CDK5 in A6(1) cells transfected with GFP, GFP-CDK5, and CDK5-T33. A6(1) cells were transiently transfected with GFP (A,D), GFP-CDK5 (B,E), or GFP-CDK5-T33 (C,F). GFP fluorescence was viewed to identify transfected cells and to localize the GFP-tagged proteins (A–C). Immunofluorescence using anti-CDK5 antibody was used to localize endogenous CDK5 (D) as well as the GFP-tagged CDK5 wild-type and mutated fusion proteins (E,F). CDK5 immunostaining was seen in the cytoplasm, in filopodia (arrows in D–F), and in lamellopodia (arrowheads in D–F). Panel D shows a GFP-transfected cell and an untransfected cell in the same field.

 
Cell-to-Matrix Adhesion
To test whether CDK5 might be involved in cell adhesion, stably transfected A6(1) cells were allowed to adhere to fibronectin for 2 h. The plates were then inverted and the cells were centrifuged to determine the force necessary to dislodge them. When subjected to gentle centrifugation (50 x g) to dislodge nonadherent or weakly adherent cells, twice as many CDK5-transfected cells remained attached as CDK5-T33-transfected or nontransfected A6(1) cells (Fig. 6 ), indicating that CDK5 enhances cell-to-matrix adhesion. Increasing the centrifugation force to 200 x g dislodged many adherent CDK5-T33-transfected cells and nontransfected cells, but had no effect on CDK5-transfected A6(1) cells. In fact, even at 450 x g, the number of adherent CDK5-transfected cells decreased only slightly, indicating that CDK5 overexpression increases the strength of adhesion. The differences between CDK5-transfected cells and CDK5-T33-transfected cells or control cells were statistically significant (P < 0.001) at all centrifugation forces tested.



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  		FIGURE 6. Effect of CDK5 on cell adhesion. Adhesion to fibronectin was examined by a centrifugation assay at 50, 200, and 450 x g using CDK5-transfected A6(1) ({circ}), control-transfected A6(1) cells (•), or CDK5-T33-transfected A6(1) cells ({blacktriangleup}). Centrifugation at 200 x g dislodged control cells and CDK5-T33-transfected cells, but had no effect on CDK5-transfected cells. The differences between CDK5-transfected cells and control cells, or CDK5-transfected cells and CDK5-T33-transfected cells were significant (P < 0.001) at each centrifugation force tested.

 
Association of Vinculin with the Cytoskeleton
Since cell-to-matrix adhesion is associated with formation of vinculin-containing adhesion plaques linked to the detergent-insoluble cytoskeletal fraction, we tested whether CDK5 affects the association of vinculin with the detergent-insoluble fraction. Stably transfected A6(1) cells were separated into a Triton X-100 soluble fraction (cytosol) and a 9M urea soluble fraction (cytoskeleton), and both fractions were immunoblotted with vinculin antibody. GFP-CDK5-transfected A6(1) cells had more vinculin in the cytoskeletal fraction than cells stably transfected with GFP-CDK5-T33 or control cells (Fig. 7, A and B ). Quantitative densitometry of six immunoblots from four separate experiments indicated that the association of vinculin with the cytoskeleton increased about 30% in CDK5-transfected cells (Fig. 7B). This finding suggests that CDK5 overexpression promotes focal adhesion formation and/or linkage to the cytoskeleton.



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  		FIGURE 7. Increased association of vinculin with the Triton X-100 insoluble cytoskeletal fraction in A6(1) cell transfected with CDK5. A6(1) cells stably transfected with vector only, pGFP-CDK5, and pGFP-CDK5-T33 were maintained at 37°C for 5 days in the absence of IFN-{gamma} to establish confluent, quiescent cultures. Cell lysates were separated into soluble and insoluble fractions. The soluble and insoluble proteins were immunoblotted with anti-vinculin antibody. A. Representative Western blot showing the immunoreactive band with correct molecular size of vinculin. B. Quantitative densitometry of six different immunoblots from four separate experiments. Vinculin in the insoluble fraction from CDK5-transfected cells was approximately 30% greater than in vector-only controls (P < 0.05) or cells transfected with CDK5-T33 (P < 0.03).

 
Adhesion in A6(1) Cells Infected with Recombinant CDK5 Adenovirus Vectors
As a further test of the physiological role of CDK5 in cell adhesion, we infected A6(1) cells with recombinant adenovirus constructs Adv-CDK5 or Adv-CDK5-T33. The cDNAs carried by the adenovirus vectors were not "tagged" by additional residues at the NH2 terminus to minimize possible differences in protein structure. Adenovirus-mediated cDNA transfer seemed to yield higher expression of CDK5 and CDK5-T33 relative to endogenous CDK5 than stable transfection (compare Figs. 4A and 8A). The total level of exogenous protein (CDK5 or CDK5-T33) and endogenous CDK5 in the infected A6(1) cells was about twice as high as endogenous CDK5 expression in noninfected, control cells (Fig. 8A ), indicating that the exogenous and endogenous proteins are present in approximately equal amounts.



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  		FIGURE 8. Adenovirus-mediated transfer of CDK5 and CDK5-T33. A. Immunoblots of CDK5 and CDK5-T33 in A6(1) cells following adenovirus-mediated gene transfer. Expression of CDK5 or CDK5-T33 in infected A6(1) cells is increased about 2-fold compared to CDK5 expression in noninfected cells. B. Effect of CDK5 on cell adhesion to fibronectin. Adhesion strips were used to examine the effect of CDK5 or CDK5-T33 on cell adhesion to fibronectin in A6(1) cells infected with recombinant CDK5 or CDK5-T33 adenovirus. Results represent the average of three to five independent measurements. The number of adherent cells was significantly increased by Ad-CDK5 and decreased by Ad-CDK5-T33 as compared to control, uninfected cells (P < 0.001).

 
Fibronectin-coated adhesion strips were used to examine the effect of CDK5 and CDK5-T33 on cell adhesion in the adenovirus vector-infected cells. Overexpression of CDK5 increased the number of adherent cells approximately 2-fold as compared to noninfected controls (P < 0.0005) (Fig. 8B), in good agreement with the results obtained using stably transfected cells and the centrifugation assay. Interestingly, however, with this method of cDNA transfer, CDK5-T33 overexpression significantly reduced the number of adherent cells as compared to controls (P < 0.001).

Migration in A6(1) Cells Stably Transfected with CDK5
Since cell adhesion is an important component of cell migration, the observed effect of CDK5 on corneal epithelial cell adhesion suggested that it might also affect cell migration. We tested this possibility using an in vitro scrape wounding assay (38). GFP-CDK5-, GFP-CDK5-T33-, and GFP-transfected control cells were maintained at 37°C for 5 days in the absence of IFN-{gamma} to establish confluence. A uniform scrape wound was made across the culture dish and the ability of the cells to migrate was assessed by monitoring reoccupation of the wound area (Fig. 9A ). After 48 h, the area reoccupied by GFP-CDK5-transfected A6(1) was 30% less than that reoccupied by control cells transfected with the vector only (P < 0.01), whereas the area reoccupied by GFP-CDK5-T33-transfected cells was 20% greater than control (P < 0.05) (Fig. 9B). BrdUrd labeling of scrape wounded cultures showed very low levels of proliferation during the 48-h period, with no significant difference among the three cell lines (not shown). Thus, we attribute the differences in reoccupation of the wound area to differences in cell migration.



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  		FIGURE 9. The effect of CDK5 on cell migration. A6(1) cells stably transfected with vector only, pGFP-CDK5, and pGFP-CDK5-T33 were maintained at 37°C for 5 days in the absence of IFN-{gamma} to establish confluent, quiescent cultures. A uniform scrape wound was made across the culture dish at 0 h. The ability of A6(1) cells to reoccupy the scraped area was compared between 0 h and after 48 h. A. A representative micrograph showing the effect of CDK5 and CDK5-T33 on cell migration. B. Quantification of the reoccupied area after 48 h by image analysis software. Six measurements were made from three separate experiments for each cell type. Reoccupation of the wound area by CDK5-transfected A6(1) cells was 30% slower than control (P < 0.01), whereas reoccupation by pGFP-CDK5-T33-transfected A6(1) cells was 20% faster (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The corneal epithelium must be constantly renewed to maintain a smooth optical refractive surface and an intact physical barrier for the eye (39–44). This renewal is accomplished by coordinated proliferation, differentiation, and migration of stem cells from the limbus, a junctional zone between the cornea and conjunctiva. In the limbus, the pattern of gene expression is different from that of central cornea (42). For example, the corneal differentiation marker, keratin 12, is not expressed in this region (43–45). The limbus region is enriched with a distinct subpopulation of stem cells, serving as a proliferative reserve (40). To generate new epithelial cells, limbal stem cells move centripetally into the basal layer of the corneal epithelium, where they divide, differentiate, detach from the basement membrane, and eventually slough off from the superficial layer. The present study shows that CDK5 and p35 expression is closely correlated with corneal differentiation. CDK5 and p35 are expressed to appreciable levels throughout the corneal epithelium, except for the limbus. In fact, expression of CDK5 and p35 correlates well with the differentiation marker keratin 12, when both are stained in parallel (not shown). Thus, as in other cell types that have been studied (24, 26, 30, 46, 47–50), expression of CDK5 and p35 in the corneal epithelium seems to be associated with cellular differentiation.

The possibility that CDK5 and p35 might play a role in cell-to-matrix adhesion in the corneal epithelium was first suggested by their immunolocalization along the basal aspect of the basal cells, where hemidesmosomes maintain the stable attachment of cells to the basement membrane (51). Cell fractionation studies confirmed that the bulk of the p35 in the corneal epithelium is associated with the membrane fraction. This localization is also consistent with the presence of an NH2-terminal myristoylation site in p35, which targets p35 to the membrane (35).

Adhesion studies performed on the mouse corneal epithelial cell line, A6(1), provided a second line of evidence supporting a role for CDK5 in cell adhesion. A6(1) cells are conditionally transformed by a temperature-sensitive SV40 T-antigen. When cultured at the nonpermissive temperature in the absence of IFN-{gamma} for 5 days, A6(1) cells exhibit some characteristics of differentiated corneal epithelial cells and express certain differentiation markers such as keratin 12 and transketolase (61, 62). However, there are also some important differences between these cells and cells of the corneal epithelium. Like most cultured cells, A6(1) cells attach to the matrix through integrin-mediated focal adhesion complexes rather than hemidesmosomes. Moreover, fibronectin is the most favorable ECM molecule for attachment of these cells, as for other corneal epithelial cells in culture (54, 55), although it is not a major ECM component in the corneal basement membrane in vivo (56).

Two different adhesion assays using two different cDNA transfer techniques indicated that CDK5 overexpression strongly promotes adhesion of A6(1) cells to fibronectin. Because these assays allow 2 h for cell attachment, they measure a relatively late stage of cell adhesion that includes assembly of focal adhesion complexes, cytoskeletal engagement, and formation of stress fibers (57, 58). Indeed, cell fractionation studies demonstrated that stable transfection with CDK5 significantly enhanced the association of the focal adhesion component, vinculin, with the detergent-insoluble cytoskeletal fraction. We also found that the kinase-inactive construct, CDK5-T33, suppressed adhesion to fibronectin, when expressed at high levels using an adenoviral vector. We attribute this to the ability of CDK5-T33 to exert a dominant negative effect on endogenous CDK5 activity when expressed in high enough concentrations to sequester the available p35. The ability of CDK5 to regulate adhesion to fibronectin is especially interesting in view of the fact that fibronectin is transiently expressed under the flattened, actively migrating cells at the leading edge of a corneal wound (56). Although we did not detect CDK5 kinase activity in the intact corneal epithelium, it is possible that this enzyme may be activated when the epithelium is wounded, to regulate adhesion to fibronectin during wound healing.

Cell migration studies also support a role for CDK5 in regulating cell-to-matrix attachment. Cell migration is a coordinated series of integrated events consisting of lamellipodial extension, formation of focal adhesion contacts at the front of the cell, cytoskeletal contraction, and release of cell-to-substrate attachments at the rear (59, 60). The ability of a cell to migrate is contingent on disengaging trailing focal adhesions and generating new ones at the leading edge (61, 62). As a result, the relationship between adhesion and migration is a bell-shaped curve (59), with the highest rate of migration occurring at some intermediate value of adhesive strength. The strong enhancement of cell-to-matrix adhesion produced by CDK5 overexpression in A6(1) cells could reduce cell migration by opposing the disassembly of the trailing adhesions. Conversely, since disassembly of trailing adhesions is usually considered to be the rate-limiting factor in migration (59, 61, 63), a slight decrease in CDK5 activity might increase migration rate by promoting this disassembly. At some point, however, any additional decrease in CDK5 activity would be expected to reduce adhesive strength below the minimum needed to form new attachments effectively, and the rate of migration would be decreased. This is consistent with the observation that neuronal migration is inhibited in mice with targeted disruptions of CDK5 or p35 (5–7). Thus, the present results and the available literature on the effect of CDK5 deficiency on cell migration are consistent with a role for CDK5 in regulating cell-to-matrix adhesion. Nevertheless, we do not rule out the possibility that CDK5 may also affect migration in ways that are independent of its effect on adhesion.

In addition, we note an interesting reciprocal relationship between CDK5 and pinin (DRS/memA), a protein associated with desmosomes of corneal epithelial cells (64). Overexpression of pinin seems to strengthen cell-cell adhesion and decrease cell-matrix adhesion (65), exactly opposite to the effects of CDK5 observed in this study. Moreover, overexpression of pinin suppresses CDK5 expression (38). Because conditions that favor cell-cell adhesion often reduce cell-matrix adhesion, and vice versa (65), this reciprocal relationship between pinin and CDK5 lends further support to the view that CDK5 may be an important regulator of corneal cell adhesion and migration.

The available evidence supports the interpretation that endogenous CDK5/p35 is active and regulates adhesion and migration in cultured corneal epithelial cells by phosphorylating protein substrates involved in these processes. Accordingly, we have found that overexpressing CDK5 increases both CDK5 activity and adhesive strength. Since the kinase-inactive form, CDK5-T33, does not affect adhesion, kinase activity seems to be required for this effect. The alternative explanation, that CDK5 binds to cellular proteins that fail to interact with CDK5-T33, seems unlikely in view of the overall structural similarity between these two proteins (16). Moreover, since we have relied on the endogenous activating protein, p35, to activate the exogenous CDK5, the observed 33% increase in kinase activity is likely to be within physiological limits, thus minimizing the phosphorylation of nonphysiological substrates. Finally, the simplest explanation for the observation that high levels of CDK5-T33 have the opposite effect on adhesion is that CDK5-T33 inhibits the endogenous CDK5 activity. Certainly, CDK5-T33 has the potential to be a highly specific inhibitor of endogenous CDK5 activity by sequestering p35. Thus, the results of this study support a model in which endogenous CDK5/p35 regulates corneal epithelial cell adhesion and migration by phosphorylating specific proteins involved in these processes. Since our data indicate that p39 is not expressed in the corneal epithelium or in A6(1) cells, CDK5/p39 is not an essential component of this regulatory pathway.

One possible mechanism for the observed effects of CDK5 on adhesion and migration is through phosphorylation of the Rac1 effector, PAK1. A previous report has demonstrated that CDK5/p35 specifically binds activated Rac1, thus targeting PAK1 for phosphorylation by CDK5 and suppressing its activity (16). PAK1 is a known regulator of cell adhesion and migration. Constitutively activated PAK1 reduces the number of focal adhesions and causes loss of stress fibers (66), increases cell contractility (67, 68), and promotes polarized cell movement (68, 69). Conversely, dominant negative PAK1 has been shown to decrease migration of human microvascular endothelial cells (68). Thus, inactivation of PAK1 by CDK5 would be expected to promote cell adhesion by increasing focal adhesions and stress fiber formation. At the same time, CDK5-dependent inactivation of PAK1 could interfere with cell migration by inhibiting the generation of contractile force needed to pull off the rear attachment during migration, or alternatively, by disrupting the coordinated nature of directed movement (68, 69). Additional studies will be needed to determine whether PAK1 phosphorylation is a component of the regulatory pathway affected by CDK5 in corneal epithelial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Isolation of Mouse Corneal Epithelia
All animal studies were performed in accordance with the NIH Guidelines for Care and Use of Laboratory Animals. Two-month-old mice were obtained from Charles River Farms (Charles River Breeding Laboratories, Kingston, NY) and euthanized by asphyxiation in 95% carbon dioxide. The eyes were enucleated and transected posterior to the corneal limbus under a dissecting microscope. Corneal tissues were washed in PBS and placed in Dispase II solution at 2.4 units/ml for 1.5–2 h at 37°C (Roche Diagnostics, Basel, Switzerland). Sheets of corneal epithelium were then carefully removed from the stroma with forceps.

RNA Extraction and RT-PCR
Corneal epithelia were harvested as described above and cytoplasmic RNA was isolated using RNAzol (TelTest, Inc., Friendswood, TX) (70). The RNA was treated with DNase I (amplification grade; Invitrogen Life Technologies, Inc., Carlsbad, CA), 1 unit/µg RNA for 15 min at room temperature, followed by heat inactivation for 10 min at 65°C. RT-PCR of p35 and p39 was performed according to the manufacturer's instructions (Gene Amp RNA PCR core kit; Perkin-Elmer Corp., Boston, MA). A total of 1 µg of RNA was used with the following oligonucleotides:

For p35:
Upstream: 5'-CGGCACGGTGCTGTCCCTGTCT-3'
Downstream: 5'-TCACCGATCCAGGCCTAGGAG-3'

For p39:
Upstream: 5'-GGCCGTCCGTGCTCATCTCGGCGCTCA-3'
Downstream: 5'-CGGCCCTTGCGGAGAAGGTTCTCGCGGTTGCG-3'

The PCR protocol was 5 min at 95°C, followed by 35 cycles of 1 min at 95°C, 1 min at 55°C, 1 min at 72°C, and a final extension of 10 min at 72°C.

Immunoblotting and Immunoprecipitation
Immunoblotting and immunoprecipitation were performed as previously described (30). Cellular proteins were lysed and extracted in PBSTDS (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS in PBS) containing one Complete-MiniTM protease inhibitor cocktail tablet/10 ml. Cell lysate containing 200 µg of protein was immunoprecipited using anti-CDK5 rabbit polyclonal IgG (C-8) (sc-173, Santa Cruz Biotechnology, Santa Cruz, CA). Control immunoprecipitation was performed with an equivalent amount of cell extract in the absence of primary antibody. Immunoprecipitated proteins or 25–50 µg of total cell extract were immunoblotted using anti-CDK5 mouse monoclonal IgG (DC-17; sc-249, Santa Cruz Biotechnology) or anti-p35 rabbit polyclonal IgG (C-19; sc-820, Santa Cruz Biotechnology). Where indicated, 2–3 µg of p35 blocking peptide (sc-820P, Santa Cruz Biotechnology) were added during incubation with primary antibody. Immunoreactive bands were detected using horseradish peroxidase-linked anti-rabbit IgG (Santa Cruz Biotechnology) by enhanced chemiluminescence (ECL-Plus; Amersham Life Science, Piscataway, NJ).

Immunohistochemistry
Paraffin sections (10 µm) from 2-month-old mouse eyes on silanized slides (Digene, Gaithersburg, MD) were deparaffinized by Hemo-De (Fisher, Pittsburgh, PA) twice for 5 min each. After rehydration in a series of decreasing ethanol concentrations, samples were permeabilized in 0.25% Triton X-100 in PBS for 10 min and postfixed in Bouin's solution (Sigma Chemical Co., St. Louis, MO) for 15 min. The samples were incubated in PBS containing 3% hydrogen peroxide for 30 min to remove the endogenous peroxidase activity. Following several washes in PBS and blocking in 5% normal goat serum in PBS, sections were incubated with either anti-CDK5 (C-8, Santa Cruz, Biotechnology) or anti-p35 (C-19, Santa Cruz Biotechnology) rabbit polyclonal antibodies for 1 h. After extensive washing in PBS, samples were incubated for 30 min with secondary biotinylated antibodies, followed by avidin-biotinylated-peroxidase complex (ABC Kit, Vector Laboratories, Inc., Burlingame, CA). Finally, the slides were developed with Vector NovaRED and hydrogen peroxide substrate (SG) (Vector Laboratories, Inc.) according to the manufacturer's instructions. Samples were then washed in distilled water, mounted with Aqua Poly mount (18606, Polysciences, Inc., Warrington, PA), and viewed with a Zeiss Axioplan 2 photomicroscope. Images were captured with a CCD camera (OPELCO, Dulles, VA). For controls, the antigenic peptides for CDK5 and p35 were included during incubation with primary antibodies.

Immunofluorescence
GFP-transfected A6(1) cells were cultured on chamber slides, fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, extracted in 0.25% Triton X-100 for 5 min, and incubated for 1 h at room temperature in a blocking buffer consisting of PBS with 5% goat serum. Subsequent antibody incubations and washes were also performed in blocking buffer. Slides were incubated with anti-CDK5 (C-8, Santa Cruz Biotechnology) for 1 h at room temperature, washed three times, and incubated with rhodamine-conjugated goat anti-rabbit IgG (111-295-144, Jackson ImmunoResearch Laboratories, West Grove, PA). Samples were washed and mounted in Aqua Poly mount (18606, Polysciences, Inc.), and examined with a Zeiss Axioplan 2 photomicroscope equipped with epifluorescence. Images were captured with a CCD camera (OPELCO).

Cellular Fractionation
Cellular fractionation was performed as previously described (71–73) with minor modifications. In brief, mouse corneal epithelia were extracted on ice in 1% Triton X-100 buffer containing 10 mM imidazole, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, 0.5 mM NaF, 1 mM sodium vanadate, at pH 7.4, and one Complete-MiniTM protease inhibitor cocktail tablet/10 ml (Roche Diagnostics, Indianapolis, IN). Extracts were centrifuged at 12,000 x g for 10 min at 4°C. The Triton X-100-soluble supernatant ("cytoplasmic fraction") contains cytosolic proteins and weakly associated membrane proteins. The Triton X-100-insoluble pellet was washed with the 1% Triton X-100 buffer and further fractionated by extraction with RIPA buffer (150 mM NaCl, 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, and 50 mM Tris-HCl, pH 7.4) into a "cytoskeletal-associated fraction" (RIPA soluble) and a "membrane fraction" (RIPA insoluble) as follows. Pellets were solubilized in RIPA buffer, with one Complete-MiniTM protease inhibitor cocktail tablet/10 ml, briefly homogenized, incubated on ice for 15 min, and centrifuged at 12,000 x g for 10 min. The supernatant containing the RIPA-soluble protein was removed, and the Triton X-100-insoluble/RIPA-insoluble pellet was washed in RIPA buffer and solubilized in SDS-containing sample buffer, as above. Protein concentration was measured by the BCA method (Pierce Chemical Co., Rockford, IL) and equal amounts of protein from each fraction were analyzed by 12% SDS-PAGE.

For vinculin fractionation, the Triton X-100-insoluble pellet remaining after extraction of the soluble fraction (see above) was solubilized in 9 M urea, 4% NP40 and 10 mM DTT, incubated at room temperature for 15 min, and centrifuged at 12,000 x g for 10 min (74). The supernatant from this step and the Triton X-100-soluble fraction were immunoblotted with anti-vinculin antibody (Sigma, V-4505) as described above.

Cell Culture
A6(1) corneal epithelial cells were derived from corneal epithelia of the 14-day-old Immorto-Mouse (Charles River Breeding Laboratories). These cells are conditionally immortalized by a temperature-sensitive SV40 T-antigen under control of an IFN-{gamma} inducible promoter (75). Cells were cultured in Medium 500, supplemented with corneal epithelial growth supplement (Cascade Biologics, Inc., Portland, OR), IFN-{gamma} (5 units/ml), 20% fetal bovine serum, L-glutamine (60 µg/ml), penicillin (20 units/ml), and streptomycin (20 mg/ml) at the permissive temperature, 33°C, in a humidified atmosphere of 95% air and 5% CO2. For in vitro migration and adhesion assays, subconfluent cultures were moved to the nonpermissive temperature, 37°C, and cultured in the same medium in the absence of IFN-{gamma}. Experiments were initiated after 5 days or more at 37°C.

Constructs and Stable Transfection
A6(1) cells were transfected with 2 µg of pGFP, pGFP-CDK5, and pGFP-CDK5-T33 cDNA constructs (46) using FuGENE 6 reagent (Roche Diagnostics) according to the manufacturer's instructions. Cells carrying the neomycin-resistance marker were selected by addition of G418 at 350 µg/ml, 3 days after transfection. Stably transfected cells were maintained in the presence of G418 at the same concentration.

Protein Kinase Assay
CDK5 kinase activity was determined using SignaTECT protein kinase assay system (#V6430, Promega, Madison, WI). Briefly, GFP-, GFP-CDK5-, and GFP-CDK5-T33-transfected A6(1) cells were allowed to grow to 80% confluence, medium was removed, and cells were treated for 10 min with Ser/Thr/Tyr phosphatase inhibitor cocktail diluted 1:100 in 1x PBS (#17-317, Upstate Biotechnology, Inc., Lake Placid, NY). Cells were then harvested and homogenized in lysis buffer: 50 mM Tris-HCl, pH 7.4; 250 mM NaCl, 1 mM EDTA, 0.1% Triton X-100 containing the same phosphatase inhibitor cocktail (1:100 dilution) and one Complete-MiniTM protease inhibitor cocktail tablet/10 ml (Boehringer Mannheim). CDK5 was immunoprecipitated from cell lysates as previously described (30) using CDK5 antibody (c-8, Santa Cruz Biotechnology). Following three washes in the lysis buffer, the kinase activity of the immunoprecipitates was assayed using a biotinylated peptide substrate (PKTPKKAKKL) (Promega) according to the SignaTECT protocol provided by the manufacturer.

Preparation of Recombinant Adenovirus and Cell Infection
The shuttle vectors pGEM-CMV-CDK5 and pGEM-CMV-CDK5-T33 were constructed by inserting CDK5 or CDK5-T33 cDNA into the pGEM-CMV vector, which contains a human cytomegalovirus immediate early promoter, enhancer, and polyadenylate sequence (kindly provided by Dr. Terete Borras). Adenovirus DNA was obtained from Ad-CMV-ß-gal (also provided by Dr. Borras) by digestion with XbaI and ClaI. Recombinant adenoviruses, Adv-CDK5 and Adv-CDK5-T33, were constructed by cotransfection of the shuttle vectors and adenovirus DNA into the human embryonic kidney cell line 293 (ATCC 11573) by calcium phosphate precipitation (76). The recombinant adenoviral vectors were screened for the presence of CDK5 or CDK-T33 sequences and the absence of adenovirus E1A sequence, and purified three times by plaque assay in 293 cells. For large-scale purification of high-titer recombinant adenovirus, the virus was purified twice by cesium chloride density gradient centrifugation, dialyzed for 12 h at 4°C against 10 mM Tris-HCl, 1 mM MgCl2, 10% glycerol, pH 7.5, and stored at -80°C. Adenoviral vectors were titered by plaque assay with 293 cells. The titer range was at between 1 x 1010 and 1 x 1011 pfu/ml (77).

For infection of target cells by adenovirus vectors, A6(1) cells were plated at 5 x 106 cells/60-mm Petri dishes, incubated at 37°C overnight, then infected with Adv-CDK5 or Adv-CDK5-T33 (100 pfu/cell). The cells were continuously cultured at 37°C for an additional 48–72 h. The level of expression of CDK5 and CDK5-T33 proteins was determined by immunoblotting.

Cell Adhesion Assay
Adhesion assays were carried out as previously described (78). Cultured cells, at 70–80% confluence, were dissociated using 2 mM EDTA, and the cell suspension was brought to a density of 5 x 105 cells/ml in PBS. Flat-bottom polyvinyl chloride micro-titer plates (BD Biosciences, Bedford, MA) were coated with 10 µg/ml fibronectin (Invitrogen Life Technologies, Inc.), rinsed with PBS to remove unbound substrate, and kept on ice. Cells (5 x 104 cells/well) were added to each well and the wells were filled with tissue culture medium without serum. The cells were then forced into contact with the substrate by centrifuging the plates for 3 min at 35 x g at 4°C in a low-speed centrifuge with a micro-titer plate carrier. Plates were incubated at 37°C for 2 h, inverted, and centrifuged at 50, 200, or 450 x g for 5 min to remove weakly bound cells. The remaining cells were stained with 0.2% crystal violet in 10% ethanol. The stain was solubilized using a 50:50 mixture of ethanol and 0.1 M NaH2PO4, pH 4.5. Absorbance was measured at 540 nm on a microplate reader. Adhesion of transfected A6(1) cells at each centrifugation force was normalized to that of pGFP-transfected A6(1) cells at 50 x g and results were expressed as relative adhesion (percentage of the control). Statistical analysis was performed using SigmaStat 2.03.

Alternatively, cell adhesion was measured using CytoMatrixTM Cell Adhesion Strips (Chemicon International, Inc., Temecula, CA), according to manufacturer's recommendations. Cells obtained as above were plated on microstrip precoated wells at 5 x 104 cells/well, and incubated for 2 h. At the end of incubation, the strips were washed gently with PBS to remove unattached cells. Then, the remaining bound cells were stained, solubilized, and quantified as described above.

As negative control for both assays, cells were tested for adhesion to wells coated with BSA (40 mg/ml). All the substrate-containing wells were also treated with BSA to block nonspecific binding.

In Vitro Scrape Wounding
A6(1) cells stably transfected with pGFP, pGFP-CDK5, and pGFP-CDK5-T33 were cultured to 70–80% confluence at 33°C, then switched to 37°C and cultured to confluence in the same medium in the absence of IFN-{gamma} for 5 days. A central, 2-mm wide, linear scrape wound was made with a plastic pipette tip and the wound area was marked with three black ink dots for reference. Cultures were rinsed with PBS and incubated in fresh medium for 48 h. Migration of cells into the wound area was monitored during this period by phase-contrast microscopy of the area marked by the reference dots. After the final phase, contrast image was taken at 48 h, the culture dishes were fixed and stained with hematoxylin QS to increase the visibility of cells (Vector Laboratories, Inc.) and the total wound area reoccupied was quantified by Image Pro Plus morphometric software. In total, three different cultures were wounded and six different areas from those three cultures were sampled for each transfected cell types. Statistical analysis was performed using SigmaStat 2.03.


   Acknowledgements
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. L-H. Tsai for the CDK5 and CDK5-T33 cDNA clones, Dr. J. Piatigorsky for A6(1) cells, Dr. Terete Borras for the pGEM-CMV shuttle vector and Ad-CMV-ßGal recombinant adenovirus, and Drs. J. Piatigorsky and M. A. Stepp for critical reading of the text.

Received February 26, 2002; revised June 24, 2002; accepted July 22, 2002.


   References
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 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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