Peggy S. Zelenka

EXPRESSION OF CDK5, P35, AND CDK5-ASSOCIATED KINASE ACTIVITY IN THE DEVELOPING RAT LENS

We have investigated the expression of Cdk5 and its regulatory subunit, p35, in the developing rat lens from embryonic day 16 (E16) to postnatal day 8 (P8). Reverse transcription and polymerase chain reaction (RT/PCR) detected Cdk5 and p35 mRNA expression in lens epithelial cells and in differentiating lens fibers throughout this developmental period. Subsequent sequencing of the RT/PCR products confirmed their identifies. In sity hybridization with Cdk5 and p35 riboprobes showed especially high expression of both mRNAs in the newly formed lens fiber cells in the bow region of the lens. Immunocytochemistry at E18 showed that Cdk5 was present in the cytoplasm of lens epithelial cells and fiber cells, with especially strong immunostaining at the anterior ends of the fibers. Fiber cells in the final stages of maturation, immediately prior to nuclear degeneration, showed positive staining for Cdk5 in the nucleus. Immunoprecipitation of proteins with Cdk5 antibody followed by immunoblotting with either N-terminal specific or C-terminal specific p35 antibodies demonstrated that p35 is complexed with Cdk5 in lens epithelial cells and lens fibers. Immunoprecipitates of Cdk5 from epithelia and fibers showed kinase activity in vitro using histone H1 as a substrate. These findings demonstrate that p35/Cdk5 activity is not restricted to neurons and raise the possibility that this kinase may play a role in lens fiber cell differentiation.

Molecular weight and subunit structure of delta-crystallin from embryonic chick lens fibers

Abstract The molecular weight of purified native delta-crystallin (F.I.S.C.) obtained from lens fibers of 13–17-day-old chick embryos was determined to be approximately 200 000 daltons by gel filtration and sedimentation equilibrium centrifugation. The subunit molecular weight of delta-crystallin was found to be between 45 000 and 50 000 daltons when examined in the presence of sodium dodecyl sulfate by electrophoresis and at sedimentation equilibrium. Delta-crystallin was completely dissociated by sodium dodecyl sulfate without reduction, indicating that the subunits are not covalently bound by disulfide bridges. Thus, native delta-crystallin appears to consist of four polypeptides of the same size. Delta-crytallin was heterogeneous in sedimentation and electrophoretic experiments in the presence of 8 m -urea or 5 m -guanidine hydrochloride. This finding requires that previous experiments with urea concerned with the subunit composition of delta-crystallin be re-examined. Amino acid composition analyses showed that delta-crystallin has an unusually high proportion of leucine and isoleucine, namely 15% and 7·5%, respectively. Possibly the high leucine and isoleucine content contributes to the stability of delta-crystallin to urea and guanidine hydrochloride.

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.

Insulin and IGF receptors are developmentally regulated in the chick embryo eye lens

We have previously reported that insulin-like growth factor (IGF) receptors appear to predominate over insulin receptors in early stages of embryogenesis in the chick (days 2-3 whole embryo membranes). Overall, [125I]IGF I and II binding to specific receptors was maximal when the rate of brain growth is highest. In the present study we used the embryonic chick lens, a well-defined tissue composed of a single type of cell, to analyse whether changes of insulin and IGF I binding are correlated with changes in growth rate and differentiation state of the cells. We show that both insulin receptors and IGF receptors are present in the lens epithelial cells, and that each type is distinctly regulated throughout development. While there is a direct correlation between IGF-binding capability and growth rate of the cells, there is less relation to differentiation status and embryo age. Insulin receptors, by contrast, appear to be mostly related to the differentiated state of cells, decreasing sharply in fibers, irrespective of their developmental age.

δ-Crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of δ-crystallin synthesis in cultured cells

Abstract Increasing specialization for δ-crystallin synthesis is a prominent feature of the differentiation of chick lens epithelial cells into lens fiber cells and can be studied in cultured embryonic lens epithelia. Quantitation of δ-crystallin mRNA by molecular hybridizaton to a [ 3 H]DNA complementary to δ-crystallin mRNA demonstrates that differentiation, both in ovo and in tissue culture, is associated with the accumulation of δ-crystallin mRNA. In the cultures, there is an overall stimulation of protein synthesis, including δ-crystallin mRNA during the first 5 hr in vitro . Between 5 and 24 hr in vitro there is a differential stimulation of δ-crystallin synthesis and an accumulation of δ-crystallin mRNA that can quantitatively account for this stimulation.

Cdk5 regulates activation and localization of Src during corneal epithelial wound closure

Recent studies have shown that Cdk5, a member of the cyclin-dependent-kinase family, regulates adhesion and migration in a mouse corneal epithelial cell line. Here, we extend these findings to corneal wound healing in vivo and examine the mechanism linking Cdk5 to cytoskeletal reorganization and migration. Cdk5 was overexpressed in the corneal epithelium of transgenic mice under control of the ALDH3 promoter. Elevated Cdk5 expression retarded corneal debridement wound closure in these animals and suppressed remodeling of the actin cytoskeleton. Conversely, the Cdk5 inhibitor, olomoucine, accelerated debridement wound healing in organ cultured eyes of normal mice, caused migrating cells to separate from the epithelial cell sheet, and increased the level of activated Src(pY416) along the wound edge. To explore the relationship between Cdk5 and Src in greater detail, we examined scratch-wounded cultures of corneal epithelial cells. Src was activated in cells along the wound edge and blocking this activation with the Src kinase inhibitor, PP1, inhibited wound closure by 85%. Inhibiting Cdk5 activity with olomoucine or a dominant negative construct, Cdk5T33, increased the concentration of Src(pY416), shifted its subcellular localization to the cell periphery and enhanced wound closure. Cdk5(pY15), an activated form of Cdk5, also appeared along the wound edge. Inhibiting Src activity with PP1 blocked the appearance of Cdk5(pY15), suggesting that Cdk5 phosphorylation is Src dependent. Cdk5 and Src co-immunoprecipitated from scratch-wounded cultures, demonstrating that both kinases are part of an intracellular protein complex. These findings indicate that Cdk5 exerts its effects on cell migration during corneal epithelial wound healing by regulating the activation and localization of Src.

c-myc mRNA is elevated as differentiating lens cells withdraw from the cell cycle

Explants of the central region of lens epithelia from early chicken embryos differentiate in vitro to form lens fiber cells when cultured in the presence of chicken vitreous humor. Hybridization of a 32P-labeled v-myc viral oncogene DNA probe to RNA extracted from differentiating explants and immobilized on nitrocellulose filters indicates that levels of 2.5 kb c-myc mRNA are transiently elevated 5-10-fold in the differentiating cells. Increased levels of c-myc mRNA are observed within 30 min of the initiation of differentiation in vitro and persist for 8-9 h. Thymidine labeling of nuclei in differentiating explants indicates that entry of cells into S phase is inhibited during this period, as differentiating cells complete a final round of mitosis and withdraw from the cell cycle. Levels of c-myc mRNA are also elevated in the peripheral region of the lens epithelium, which contains cells undergoing differentiation in vivo, suggesting that the regulation of c-myc mRNA which occurs in vitro may also occur in vivo. c-myc mRNA, c-fos mRNA, and c-src mRNA showed distinct patterns of regulation associated with lens fiber formation in vivo, thus providing evidence that the regulation of c-myc mRNA is specific to this proto-oncogene. The finding that c-myc mRNA undergoes a specific, transient elevation in differentiating lens cells as they withdraw from the cell cycle contrasts with a large body of evidence linking enhanced c-myc expression with increased cell proliferation.

Expression of c-fos and c-jun mRNA in the developing chicken lens: relationship to cell proliferation, quiescence, and differentiation.

The in vivo developmental pattern of c-fos and c-jun mRNA expression has been examined in the embryonic chicken lens using a coupled reverse transcription/polymerase chain reaction assay. Levels of each mRNA were measured in the central epithelium, equatorial epithelium, and fiber cell mass at 6, 10, 14, and 19 days of development. The results showed that c-fos and c-jun mRNAs accumulated during development of the embryonic chicken lens epithelium as the proportion of proliferating cells decreased, suggesting that quiescent epithelial cells express high levels of both protooncogene mRNAs. Cells in the early stages of terminal differentiation near the lens equator also contained relatively high levels of c-fos and c-jun mRNA. As lens fiber cells matured, the number of copies of c-fos mRNA per cell decreased markedly, while c-jun mRNA increased. These findings demonstrate that c-fos and c-jun are differentially regulated during terminal differentiation of lens fiber cells and suggest that these protooncogenes are expressed in lens epithelial cells following cell cycle arrest.

Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction

We have used the program FOLD, which employs the Zuker folding algorithm, to identify regions of stable secondary structure in three chicken proto-oncogene mRNAs: c-src, c-myc, and c-fos. We have found that use of reverse transcriptase to synthesize a cDNA template for amplification by the polymerase chain reaction is successful only if the region chosen for amplification does not contain stem structures with calculated free energies less than -14 kcal/mol.

pRb and p107 regulate E2F activity during lens fiber cell differentiation.

During growth arrest and differentiation, activity of the E2F family of transcription factors is inhibited by interactions with pRb and the related proteins, p107 and p130. To determine which members of the E2F and pRb families may contribute to growth arrest as lens epithelial cells differentiate into fiber cells, we examined the expression of individual E2F species and characterized the E2F protein complexes formed in rat lens epithelia and fibers. RT/PCR detected all five known members of the E2F family in lens epithelial cells, but only E2F-1, E2F-3, and E2F-5 in fiber cells. Proteins extracted from lens epithelia of newborn rats formed at least two specific complexes with an E2F consensus oligonucleotide. Proteins from lens fiber cells formed three specific complexes, one of which comigrated with an epithelial cell complex. Incubation of epithelial and fiber cell extracts with an antibody specific for p107 demonstrated that two fiber cell complexes and one epithelial cell complex contained p107. Although the remaining fiber cell complex did not react with antibodies to pRb or p130 in this assay, a strong reaction with pRb antibody was observed when the electromobility shifted complexes were subsequently immunoblotted (shift/Western assay). Immunocytochemistry confirmed that pRb protein is present in the nuclei of both epithelial cells and fiber cells. Immunoblotting of whole cell extracts with pRb antibody showed multiple, phosphorylated forms of pRb in the epithelial cells, but predominantly hypophosphorylated pRb in the fiber cells. None of the complexes formed with E2F were recognized exclusively by the p130 antibody, although the previously identified p107 complexes reacted weakly. The absence of p130/E2F complexes was correlated with the presence of multiple ubiquitinated forms of p130, especially in the fiber cells. Thus, although p130/E2F complexes are implicated in the terminal differentiation of many cell types, in differentiating lens fiber cells pRb and p107 seem to be the primary regulators of E2F activity.