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In JoVE (1)
Other Publications (16)
- Transgenic Research
- The Journal of Biological Chemistry
- Investigative Ophthalmology & Visual Science
- Investigative Ophthalmology & Visual Science
- The Journal of Biological Chemistry
- Molecular Vision
- The International Journal of Developmental Biology
- The Journal of Biological Chemistry
- Biochemical and Biophysical Research Communications
- Molecular Vision
- Experimental Cell Research
- Developmental Biology
- Molecular and Cellular Biology
- Cell Adhesion & Migration
- Cellular and Molecular Life Sciences : CMLS
- Developmental Biology
Articles by Peggy S. Zelenka in JoVE
JoVE General
Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation
Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.
Other articles by Peggy S. Zelenka on PubMed
General Utility of the Chicken BetaB1-crystallin Promoter to Drive Protein Expression in Lens Fiber Cells of Transgenic Mice
Transgenic mouse technology has been very valuable for the study of lens fiber cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the alphaA-crystallin promoter. However, while lens-specific, transgenic lines made with the alphaA-crystallin promoter express the transgene at levels 100-300-fold lower than endogenous alphaA-crystallin. Here we propose an alternative, the chicken betaB1-crystallin promoter (-432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Weel, and betaB2-crystallin mRNA at levels comparable to the endogenous betaB1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken betaB1-crystallin promoter have expressed the transgene in the lens fiber cells, and the best lines express at levels close to endogenous betaB1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken betaB1-crystallin promoter directs high level RNA expression to lens fiber cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins.
Synergy of Epidermal Growth Factor and 12(S)-hydroxyeicosatetraenoate on Protein Kinase C Activation in Lens Epithelial Cells
12(S)-hydroxyeicosatetraenoic acid (12(S)HETE) is a bioactive metabolite of arachidonic acid synthesized by 12-lipoxygenase. The 12-lipoxygenase blocker, baicalein, prevents epidermal growth factor (EGF)-induced activation of protein kinase C (PKC) alpha and beta in lens epithelial cells, whereas supplementation with 12(S)HETE reverses this effect, suggesting that EGF and 12(S)HETE may work together to activate PKC. This study investigates the mechanism of PKCbeta activation by EGF and 12(S)HETE. 12(S)HETE alone directed translocation of PKCbeta through the C1 rather than the C2 domain, without activating phosphoinositide 3-kinase (PI3K) or MAPK signaling or increasing intracellular calcium concentration. In the presence of baicalein, EGF triggered an asymmetric phosphorylation of the EGF receptor initiating signaling through PI3K and MAPK, but not PLCgamma. Together, 12(S)HETE and EGF synergistically increased phosphorylation of PKCbeta in the activation loop and C terminus as well as PKCbeta-specific activity. PI3K inhibitors blocked phosphorylation, but MEK1 inhibitors did not. Microvesicles containing phosphatidylinositol 3,4,5-trisphosphate mimicked the action of EGF on PKCbeta activity in the presence of 12(S)HETE. Kinase-inactive PKCbeta mutations in either activation loop or C terminus were effectively translocated by 12(S)HETE, as was PKCbeta in the presence of chelerythrine or Gö-6983. These findings indicate that unphosphorylated PKCbeta is translocated to the membrane by 12(S)HETE and phosphorylated by EGF-dependent PI3K signaling, to generate catalytically competent PKCbeta.
Protein Kinase Cgamma Regulation of Gap Junction Activity Through Caveolin-1-containing Lipid Rafts
To demonstrate the interactions of PKCgamma with caveolin (Cav)-1 and connexin(Cx)43 in lipid rafts and its regulation of gap junctions.
GammaE-crystallin Recruitment to the Plasma Membrane by Specific Interaction Between Lens MIP/aquaporin-0 and GammaE-crystallin
Major intrinsic protein (MIP), also called aquaporin-0, is essential for lens transparency and is specifically expressed in the lens fiber cell membranes. The goal of the current study was to identify and characterize proteins that interact with MIP and to elucidate the role of these interactions in MIP functions.
Lens Major Intrinsic Protein (MIP)/aquaporin 0 Expression in Rat Lens Epithelia Explants Requires Fibroblast Growth Factor-induced ERK and JNK Signaling
Lens major intrinsic protein (MIP), exclusive to the vertebrate lens, otherwise known as MIP26 and Aquaporin 0, is abundantly expressed as a lens fiber membrane protein. Although relatively less efficient compared with other aquaporins, MIP is suggested to function as a water channel, as an adhesion molecule, and is required for lens transparency. Because MIP is specifically expressed in lens fiber cells, we investigated in this study the activation of Mip expression after triggering differentiation of rat lens epithelia explants by fibroblast growth factor (FGF)-2. Here, we show that Mip expression in the lens cells is regulated by FGF-2. Using Real time PCR we demonstrate that endogenous Mip levels in the explants were up-regulated upon FGF-2 stimulation, in a concentration-dependent manner. Up-regulation of Mip at the transcriptional level was simultaneous with the activation of the FGF down-stream signaling components, ERK1/2 and JNK. Specific inhibitors, UO126 for ERK1/2 and SP600125 for JNK, abrogated Mip expression in response to FGF-2 in the explants. This inhibition pattern was recapitulated in reporter assays for transfection of the rat lens epithelia explants, driven by the Mip promoter (-1648/+44). Our studies show that ERK1/2 and JNK signaling pathways are required for Mip expression in lens epithelia explants induced to differentiate by FGF-2.
Differential Expression of Splice Variants of Chemokine CCL27 MRNA in Lens, Cornea, and Retina of the Normal Mouse Eye
Constitutive expression of RNA sequences complementary to the chemokine CCL27 mRNA has been found in the normal mouse eye. This study examines the nature and location of these endogenous RNAs in ocular tissues.
Regulation of Cell Adhesion and Migration in Lens Development
Cell movements during lens development and differentiation involve dynamic regulation of cell-matrix and cell-cell adhesion. How these processes are regulated depends on the particular array of matrix components and adhesion proteins that are expressed, as well as the signaling pathways that affect them. This review examines what is known about adhesion proteins and their regulation in the lens in light of recent findings about the mechanism of cell migration. The characteristic shape and organization of the lens depends on highly regulated cell movements during development and differentiation. Epithelial cells at the equator migrate posteriorly, bringing them into contact with factors in the vitreous humor and initiating differentiation. Elongation of the differentiating fiber cells is coupled with directed migration, posteriorly along the capsule and anteriorly along the fiber cell-epithelial interface, to generate a symmetrically organized fiber cell mass with aligned suture planes. To make these movements, cells systematically create and dissolve cell-cell and cell-matrix adhesions, form connections between these adhesions and the cytoskeleton, and generate contractile force. Since errors in cell migration may lead to aberrant lens shape or misplacement of the lens sutures, precise regulation of each step is essential for the optical quality of the lens. Recent advances in cellular developmental biology have begun to shed light on the molecular mechanisms underlying cell movements and the changes in adhesion that make them possible. This review will summarize those findings and relate them to relevant studies of the lens to provide an outline of the cellular events that lead to lens morphogenesis.
A Specific Interaction Between Muskelin and the Cyclin-dependent Kinase 5 Activator P39 Promotes Peripheral Localization of Muskelin
Previous studies implicate cyclin-dependent kinase 5 in cell adhesion and migration of epithelial cells of the cornea and lens. To explore molecular interactions underlying these functions, we performed yeast two-hybrid screening of an embryonic rat lens library for proteins that interact with cyclin-dependent kinase 5 and its regulators, p35 and p39. This screen identified a specific interaction between p39 and muskelin, an intracellular protein known to affect cytoskeletal organization in adherent cells. Immunohistochemistry detected muskelin in the developing lens and in other tissues, including brain and muscle. Glutathione S-transferase pull-down experiments and co-immunoprecipitations confirmed the specificity of the p39-muskelin interaction. Deletion analysis of p39 showed that muskelin binds to the p39 C terminus, which contains a short insertion (amino acids 329-366) absent from p35. Similar analysis of muskelin mapped the interaction with p39 to the fifth kelch repeat. Co-expression of p39 and muskelin in COS1 cells or lens epithelial cells altered the intracellular localization of muskelin, recruiting it to the cell periphery. These findings demonstrate a novel interaction between muskelin and the cyclin-dependent kinase 5 activator p39 and suggest that p39 may regulate the subcellular localization of muskelin.
The CDK5 Activator, P39, Binds Specifically to Myosin Essential Light Chain
Cyclin-dependent kinase 5 (Cdk5) has been shown to regulate adhesion and migration of lens and corneal epithelial cells. To explore protein-protein interactions that may mediate these functions, we performed yeast two-hybrid screening on an embryonic rat lens library using Cdk5 and its regulators, p35 and p39 as baits. This screen identified an interaction between p39 and non-muscle myosin essential light chain (MLC(17)). GST pull-down experiments demonstrated that p39 binds directly to MLC(17) through a strong binding site in the N-terminal 109 amino acids of p39. Immunoprecipitation of proteins from Cos1 cells co-transfected with GFP-MLC(17) and HA-p39 confirmed that these proteins interact intracellularly. Immunofluorescence microscopy of co-transfected lens epithelial cells showed that GFP-MLC(17) and HA-p39 co-localize along cytoskeletal fibrils. Moreover, endogenous rat lens p39 co-immunoprecipitated with MLC(17) and myosin heavy chain II (MHC II), demonstrating that the interaction is physiological and serves to link p39 to the cytoskeleton.
The Cdk5 Inhibitor Olomoucine Promotes Corneal Debridement Wound Closure in Vivo
To investigate the effect of the Cdk5 inhibitor olomoucine on corneal debridement wound healing in vivo.
Distinct Functions of Cdk5(Y15) Phosphorylation and Cdk5 Activity in Stress Fiber Formation and Organization
Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.
Notch Signaling is Required for Lateral Induction of Jagged1 During FGF-induced Lens Fiber Differentiation
Previous studies of the developing lens have shown that Notch signaling regulates differentiation of lens fiber cells by maintaining a proliferating precursor pool in the anterior epithelium. However, whether Notch signaling is further required after the onset of fiber cell differentiation is not clear. This work investigates the role of Notch2 and Jagged1 (Jag1) in secondary fiber cell differentiation using rat lens epithelial explants undergoing FGF-2 dependent differentiation in vitro. FGF induced Jag1 expression and Notch2 signaling (as judged by the appearance of activated Notch2 Intracellular Domain (N2ICD)) within 12-24 h. These changes were correlated with induction of the Notch effector, Hes5, upregulation of N-cadherin (N-cad), and downregulation of E-cadherin (E-cad), a cadherin switch characteristic of fiber cell differentiation. Induction of Jag1 was efficiently blocked by U0126, a specific inhibitor of MAPK/ERK signaling, indicating a requirement for signaling through this pathway downstream of the FGF receptor. Other growth factors that activate MAPK/ERK signaling (EGF, PDGF, IGF) did not induce Jag1. Inhibition of Notch signaling using gamma secretase inhibitors DAPT and L-685,458 or anti-Jag1 antibody markedly decreased FGF-dependent expression of Jag1 demonstrating Notch-dependent lateral induction. In addition, inhibition of Notch signaling reduced expression of N-cad, and the cyclin dependent kinase inhibitor, p57Kip2, indicating a direct role for Notch signaling in secondary fiber cell differentiation. These results demonstrate that Notch-mediated lateral induction of Jag1 is an essential component of FGF-dependent lens fiber cell differentiation.
Cdk5-dependent Regulation of Rho Activity, Cytoskeletal Contraction, and Epithelial Cell Migration Via Suppression of Src and P190RhoGAP
Cdk5 regulates adhesion and migration in a variety of cell types. We previously showed that Cdk5 is strongly activated during stress fiber formation and contraction in spreading cells. Here we determine the mechanism linking Cdk5 to stress fiber contractility and its relevance to cell migration. Immunofluorescence showed that Cdk5 colocalized with phosphorylated myosin regulatory light chain (pMRLC) on contracting stress fibers. Inhibiting Cdk5 activity by various means significantly reduced pMRLC level and cytoskeletal contraction, with loss of central stress fibers. Blocking Cdk5 activity also reduced Rho-Rho kinase (ROCK) signaling, which is the principal pathway of myosin phosphorylation under these conditions. Next, we examined the effect of Cdk5 activity on Src, a known regulator of Rho. Inhibiting Cdk5 activity increased Src activation and phosphorylation of its substrate, p190RhoGAP, an upstream inhibitor of Rho. Inhibiting both Cdk5 and Src activity completely reversed the effect of Cdk5 inhibition on Rho and prevented the loss of central stress fibers, demonstrating that Cdk5 exerts its effects on Rho-ROCK signaling by suppressing Src activity. Moreover, inhibiting either Cdk5 or ROCK activity increased cell migration to an equal extent, while inhibiting both kinases produced no additional effect, demonstrating that Cdk5-dependent regulation of ROCK activity is a physiological determinant of migration rate.
Cdk5: A Regulator of Epithelial Cell Adhesion and Migration
Cell adhesion is a fundamental property of epithelial cells required for anchoring, migration and survival. During cell migration, the formation and disruption of adhesion sites is stringently regulated by integration of multiple, sequential signals acting in distinct regions of the cell. Recent findings implicate cyclin dependent kinase 5 (Cdk5) in the signaling pathways that regulate cell adhesion and migration of a variety of cell types. Experiments with epithelial cell lines indicate that Cdk5 activity exerts its effects by limiting Src activity in regions where Rho activity is required for stress fiber contraction and by phosphorylating the talin head to stabilize nascent focal adhesions. Both pathways regulate cell migration by increasing adhesive strength.
Cdk5 Targets Active Src for Ubiquitin-dependent Degradation by Phosphorylating Src(S75)
The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity.
Conditional Ablation of the Notch2 Receptor in the Ocular Lens
Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors have not been investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, denucleation defects, and cataracts. Notch2 mutants also had persistent lens stalks as early as E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed that upon loss of Notch2, there were elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2), and Trp63 (p63) that negatively regulates Wnt signaling, plus down-regulation of Cdh1 (E-Cadherin). Removal of Notch2 also resulted in an increased proportion of fiber cells, as was found in Rbpj and Jag1 conditional mutant lenses. However, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. We found that Notch2 normally blocks lens progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation.
