Transmethylation of phosphatidylethanolamine: an initial event in embryonic chicken lens fiber cell differentiation.

Agents that induce differentiation of lens epithelial cells into lens fiber cells in vitro transiently stimulate the transmethylation of phosphatidylethanolamine. Inhibition of transmethylation by 3-deazaadenosine results in a corresponding inhibition of the cell elongation that characterizes lens fiber formation, suggesting that phospholipid methylation plays an essential role in the differentiation of these cells.

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.

Phosphatidylcholine and phosphatidylethanolamine metabolism during lens fiber cell formation.

Phosphatidylinositol is metabolized with a half-life of about 5 h in lens epithelial cells of 6-day-old embryonic chickens. When these cells differentiate to form lens fiber cells, however, phosphatidylinositol turnover virtually ceases. The present study was undertaken to determine whether there is a similar change in the metabolism of phosphatidylcholine and phosphatidylethanolamine. [32P]Orthophosphate was injected into 6-day-old chicken embryos, and the incorporation of label into phosphatidylcholine and phosphatidylethanolamine was followed for 48 h. The specific activities of the precursors phosphorylcholine and phosphorylethanolamine were also measured during this time. The data were then analysed by means of a simple kinetic model to determine the rate of synthesis and the half-life of each phospholipid. The results showed that phosphatidylcholine is synthesized at a rate of about 1.2 X 10(-20) mol/s per cell in the lens epithelial cells, and 6.4 X 10(-20) mol/s per cell in the fiber cells. Phosphatidylethanolamine is synthesized at approximately 0.9 X 10(-2)) mol/s per cell in the epithelial cells, and 4.0 X 10(-20) mol/s per cell in the fiber cells. Both phospholipids are stable in both the epithelial cells and in the fiber cells, with half-lives of 48 h or greater. Thus, although phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol all experience an increase in synthesis following lens fiber formation, the previously observed decrease in phosphatidylinositol turnover accompanying differentiation is a specific effect.

Expression of alternatively spliced PCTAIRE-1 mRNA in PC12 cells and neonatal rat brain.

A rat PCTAIRE-1 cDNA clone was isolated by immunoscreening of a PC12 cDNA library, followed by 5' RACE (rapid amplification of cDNA ends) to determine the 5' end. The rat PCTAIRE-1 cDNA sequence is 96% identical to mouse PCTAIRE-1 and contains an alternatively spliced exon of 131 bp near the 5' end. Although a mouse cDNA containing this exon has been reported, examination of several mouse cell lines provided no evidence for expression of the corresponding mRNA (Okuda et al., 1992). In contrast, reverse transcription and polymerase chain reaction (RT/PCR) across this region using RNA from proliferating, differentiated, and apoptotic PC12 cells demonstrated that alternatively spliced forms of PCTAIRE-1 mRNA with and without this exon are expressed. Both forms of PCTAIRE-1 mRNA are also expressed in vivo in neonatal rat brain, although other tissues examined contained only the form lacking the alternatively spliced exon. In the absence of the alternatively spliced exon PCTAIRE-1 mRNA contains an open reading frame of 1488 bp, corresponding to a 55-kDa protein that is 97% identical to mouse PCTAIRE-1 protein. When the alternatively spliced exon is present, this open reading frame is terminated by a stop codon and a second open reading frame is initiated, predicting a second PCTAIRE-1 protein of 52 kDa. The two predicted PCTAIRE-1 proteins are identical downstream of the splice site, but share no homology at their N-terminal ends.

A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin.

PURPOSE: To determine whether the 12-lipoxygenase pathway of arachidonic acid metabolism is present in the human lens and whether 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) plays a role in regulating proto-oncogene expression and DNA synthesis in human lens epithelial cells (HLECs). METHODS: Second- and third-passage primary cultures of HLECs were used for analysis. Human cataract epithelia were obtained from surgery. 12-lipoxygenase mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR product was sequenced. The 12-lipoxygenase protein was detected by immunoblotting. 12(S)-HETE was detected in HLEC-conditioned medium by radioimmunoassay. For studies of growth factor-induced mitogenesis, HLECs were serum starved, then stimulated with 15 ng/ml epidermal growth factor (EGF) and 1 microgram/ml insulin or with 0.3 microM 12(S)-HETE. The 12-lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10 microM) was used to block endogenous 12-lipoxygenase activity. Expression of c-fos mRNA was determined by RT-PCR, and DNA synthesis was measured by 3H-thymidine incorporation. RESULTS: 12-lipoxygenase mRNA and protein were detected in HLECs and in human cataract tissues. 12(S)-HETE was released into the medium by HLECs in the presence of EGF-insulin. Stimulation of c-fos mRNA expression and DNA synthesis by EGF-insulin was inhibited when the 12-lipoxygenase pathway was blocked by CDC. This inhibition was reversed completely by exogenously added 12(S)-HETE. However, exogenous 12(S)-HETE was unable to stimulate HLEC DNA synthesis in the absence of growth factors. CONCLUSIONS: The 12-lipoxygenase pathway of arachidonic acid metabolism is present in human lens epithelial cells. 12(S)-HETE does not stimulate HLEC DNA synthesis in the absence of growth factors but enables the cellular response to EGF and insulin.

Regulation of c-fos induction in lens epithelial cells by 12(S)HETE-dependent activation of PKC.

PURPOSE: 12(S)-Hydroxyeicosatetraenoic acid (12(S)HETE), a 12-lipoxygenase metabolite of arachidonic acid, is required for epidermal growth factor (EGF)-dependent DNA synthesis and c-fos induction in lens epithelial cells. The present study was undertaken to identify signal transduction events upstream of c-fos induction that may be regulated by 12(S)HETE. METHODS: The rabbit lens epithelial cell line, N/N1003A, was cultured in serum-free medium, with or without EGF. Activation of PKC and other selected enzymes was examined in the presence of the lipoxygenase inhibitor baicalein and/or exogenous 12(S)HETE. Relative abundance of PKC isoforms in subcellular fractions was determined by immunoblot analysis with isoform-specific antibodies. PKC activity in subcellular fractions was measured by peptide substrate phosphorylation, with and without pseudosubstrate peptide inhibitor. Phosphorylated enzymes were detected by immunoblot analysis. Relative levels of c-fos mRNA were determined by RT/PCR with internal standard. RESULTS: Baicalein blocked EGF-dependent translocation and activation of PKC, without affecting phosphorylation of Erk1/2. Of several PKC isoforms investigated (alpha, betaI, betaII, and gamma), only PKCalpha and betaII were significantly activated by EGF and inhibited by baicalein. 12(S)HETE, in combination with EGF, countered the effect of lipoxygenase inhibitors on PKC activation, and 12(S)HETE in the absence of EGF stimulated PKC translocation. Also of note, 12(S)HETE alone activated PKCgamma, an isoform that was not significantly activated by EGF. Inhibiting PKC activation with GF109203X blocked induction of c-fos by EGF but did not affect EGF-stimulated phosphorylation of Erk1/2, indicating that the effect of PKC on c-fos induction is independent of the Erk1/2 pathway. CONCLUSIONS: In lens epithelial cells, 12(S)HETE-dependent activation of PKCalpha and betaII acts in concert with other EGF-dependent signals to induce c-fos mRNA.

The rabbit lens epithelial cell line N/N1003A requires 12-lipoxygenase activity for DNA synthesis in response to EGF.

PURPOSE: Cultured rat lenses and primary human lens epithelial cells (HLECs) express12-lipoxygenase (12-LOX) and require a 12-LOX metabolite of arachidonic acid for growth in response to EGF and insulin. This study seeks to identify an established cell line with these characteristics. METHODS: Immunoblotting was used to screen eight lens epithelial cell lines for 12-LOX expression: the human line, HLE-B3; mouse lines alphaTN4, 17EM15, 21EM15, and MLE6, and rabbit lines N/N1003A, LEP2 and B3. DNA synthesis was measured as incorporation of 3H-thymidine into DNA. Expression of c-fos mRNA was detected by RT-PCR. The involvement of 12-lipoxygenase metabolites was determined using the lipoxygenase inhibitors baicalein, cinnamyl 3,4-dihydroxy-alpha-cyanocinnamate (CDC), or nordihydroguiairetic acid (NDGA). RESULTS: 12-LOX was detected only in the rabbit lines N/N1003A, LEP2 and B3. N/N1003A cells were chosen for further study. 12-LOX inhibitors blocked DNA synthesis in response to EGF with or without insulin. Inhibition of EGF-stimulated DNA synthesis was reversed by 0.3 microM to 3 microM 12(S)hydroxyeicosatetraenoic acid (HETE), but not by equivalent concentrations of 5(S)HETE, 8(S)HETE, 15(S)HETE, or 12(R)HETE. Baicalein prevented EGF induction of c-fos mRNA. The transformed HLEC line, HLE-B3, showed little stimulation of DNA synthesis in response to EGF and was unaffected by the presence of 12-LOX inhibitors. CONCLUSIONS: N/N1003A cells, like primary cultured human lens epithelial cells or neonatal rat lenses, require 12-LOX activity for EGF dependent growth. This line will be useful for studies of the mechanism of action of 12(S)HETE.

Lens lipids.

Lens cells can synthesize, degrade, and remodel lipids. Endogenous lipid synthesis, in conjunction with uptake of exogenous cholesterol and certain fatty acids, leads to the formation of a plasma membrane that is especially rich in sphingomyelin, cholesterol, and long-chain saturated fatty acids. As a result of this unusual lipid composition, lens membranes have very low fluidity, which is restricted even further by lipid-protein interactions. The composition and metabolism of membrane lipids may affect the formation of various types of cataracts. Diets rich in vegetable oils offer some protection against the formation of osmotic cataracts and the hereditary cataract of the RCS rat, although the mechanism of this effect is not clear. Vitamin E also protects against the formation of several types of cataract in vivo and in vitro, suggesting that lipid peroxidation may play a role in cataractogenesis. Certain drugs which inhibit lipid synthesis or degradation are cataractogenic, and a deficiency in cataractogenic, and a deficiency in phosphatidylserine is associated with a loss of Na+/K+ ATPase activity in several types of cataract. Human senile cataracts show a marked loss of protein-lipid interactions, although the overall lipid composition is normal. This loss of protein-lipid interactions may be related to oxidative damage to membrane-associated proteins. Interestingly, the decrease in the fluidity of lens membranes with age would counteract the formation of aqueous pores in the membrane, which can result from the oxidative cross-linking of membrane-associated proteins. Certain pathways of lipid metabolism seem to have regulatory functions. Among these are phosphatidylinositol turnover, phosphatidylethanolamine methylation, and arachidonic acid metabolism. All of these pathways function in the lens. Phosphatidylinositol turnover is correlated with the rate of lens epithelial cell division, while phosphatidylethanolamine methylation seems to be related to the initiation of lens fiber cell formation. Both pathways are associated with the release and metabolism of arachidonic acid in other cell types. While it is not known whether phosphatidylinositol turnover or phosphatidylethanolamine methylation result in the release of arachidonic acid in the lens, recent work has shown that lens cells from a variety of species can metabolize arachidonic acid by both the cyclooxygenase and lipoxygenase pathways. The possible physiological significance of these metabolites to the lens is yet to be determined.

Coordinating cell proliferation and migration in the lens and cornea.

Migration is a complex process for epithelial tissues, because the epithelium must move as an intact sheet to preserve its barrier function. The requirement for structural integrity is met by coupling cell-to-matrix and cell-to-cell adhesion at the cellular level, and by coordinating cell proliferation and cell migration in the tissue as a whole. Proliferation is suppressed at the migrating cell front, allowing cells in this region to remain tightly packed while advancing rapidly. At the same time, proliferation is enhanced in a region behind the advancing cell front to expand the epithelial cell sheet. This review considers the extracellular signals and intracellular signaling pathways that regulate these processes in the lens and corneal epithelium, with emphasis on the commonalities that link these tissues.

Changes in phosphatidylinositol metabolism during differentiation of lens epithelial cells into lens fiber cells in the embryonic chick.

During the development and growth of the lens, lens epithelial cells differentiate to form lens fiber cells. The present study investigates phosphatidylinositol metabolism in these two lens cell populations in the 6-day-old embryonic chick in vivo, to determine whether changes in the metabolism of this phospholipid are associated with lens fiber differentiation. [32P]Orthophosphate was injected into the embryos in ovo, and the incorporation of label into phosphatidylinositol in the lens epithelia and lens fiber masses was followed for 42 h. The time course of the specific activity of the gamma-PO4 of ATP was also determined during this time period, and was shown to be approximately equal to the specific activity of CDP-diacylglycerol, the immediate precursor of phosphatidylinositol. Analysis of the data by means of a simple kinetic model yielded a value of about 2 x 10(-9) pmol/s/cell for the rate of phosphatidylinositol synthesis in the lens epithelial cells, and 6.4 x 10(-9) pmol/s/cell in the lens fiber cells; the corresponding half-lives of phosphatidylinositol were 5 h and 63 h in the epithelial cells and fiber cells, respectively. Thus, lens fiber formation in the 6-day-old embryonic chick is associated with increased synthesis and decreased turnover of phosphatidylinositol. This is the first report of changes in phosphatidylinositol metabolism associated with cell differentiation during embryonic development.

Proto-oncogenes in cell differentiation.

Proto-oncogene products may be multi-functional proteins with various roles in cell differentiation as well as cell proliferation. The molecular biology of the gene products of three well characterized proto-oncogenes (c-fos, c-myc and c-src) are described, and the roles of three other proto-oncogene products, involved in hormone and growth factor reception, are reviewed.

Induction of cyclin B and H1 kinase activity in apoptotic PC12 cells.

The present study examines whether cyclin B may be involved in apoptosis of neuronally differentiated PC12 cells following withdrawal of NGF. Cyclin B mRNA increased approximately 10-fold 4 days after NGF withdrawal, as indicated by competitive RT/PCR. Sequencing of the PCR product confirmed that it was derived from cyclin B mRNA. Cyclin B protein increased in parallel with cyclin B mRNA, as shown by immunoblotting. Immunoprecipitation with anti-cyclin B antibody demonstrated that cyclin B was associated with H1K activity, which reached a maximum 5 days after NGF withdrawal. When proteins immunoprecipitated with anti-cyclin B antibody were immunoblotted with anti-PSTAIR antibody, a protein with apparent molecular weight of 34 kDa was detected. This protein was identified as p34cdc2 on the basis of immunoreactivity with antibody against the C-terminal portion of mouse p34cdc2. Since cyclin B/p34cdc2 complexes are known to catalyze chromosomal condensation and nuclear envelope breakdown during mitosis, these results suggest that cyclin B/p34cdc2 may play some role in the nuclear changes accompanying apoptosis of PC12 cells.

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.

Insulin regulates expression of c-fos and c-jun and suppresses apoptosis of lens epithelial cells.

This study investigates whether insulin (a differentiation factor for lens epithelial cells) acts as a survival factor. In the absence of insulin, 6-day embryonic chicken lens epithelial explants undergo apoptosis as shown by changes in cell morphology, DNA fragmentation, and loss of trypan blue exclusion. Insulin inhibits these changes and promotes survival of the cells. Aurintricarboxylic acid suppresses the apoptosis of lens explants. In contrast to 6-day embryonic explants, 19-day embryonic explants survive in the absence of insulin, presumably due to an endogenous survival factor. To explore the mechanism of the action of insulin as a survival factor for 6-day embryonic lens explants, we compared the pattern of cell cycle markers (c-fos, c-jun, c-myc, p53, histone H3, thymidine kinase, and cyclin B) in both apoptotic and differentiating lens explants. In the presence of insulin, the expression of c-fos and c-jun was down-regulated after an initial induction. Expression of these genes was also induced in the absence of insulin, but mRNA levels remained elevated as the cells underwent apoptosis. In contrast, expression of c-myc, p53, histone H3, thymidine kinase, and cyclin B showed only minor differences in differentiating and apoptotic cells. Since c-fos and c-jun have been shown to play a role in apoptosis in other cell types, the ability of insulin to regulate expression of these genes may be central to its ability to act as a survival factor for lens epithelial cells.

Correlation between phosphatidylinositol degradation and cell division in embryonic chicken lens epithelia.

Differentiation of embryonic chicken lens epithelial cells to form lens fibers is associated with a marked decrease in both the rate of phosphatidylinositol degradation and the rate of cell division. In cells of the central region of the lens epithelium, the rate of cell division also declines with developmental age. The present study measures phosphatidylinositol degradation in cultured explants of the central lens epithelium of chicken embryos of different ages to determine the extent of the correlation between phosphatidylinositol degradation and cell division in this tissue. The results show that the rate of phosphatidylinositol degradation also decreases during development and is proportional to the rate of cell division throughout the period from 6 to 19 days of development. Furthermore, stimulating cell division in central explants of lens epithelia of 19-day-old chicken embryos by culturing them in the presence of fetal calf serum produces a proportional increase in the rate of phosphatidylinositol degradation. These findings indicate that cell division and phosphatidylinositol degradation are tightly coupled in this tissue, and raise the possibility that phosphatidylinositol metabolism may regulate some aspect of the cell cycle.

Calpactin I in the differentiating embryonic chicken lens: mRNA levels and protein distribution.

Calpactin I, one of the EDTA-extractable proteins of the lens membrane, binds phospholipid and actin in a calcium-dependent manner. It is also known substrate of the pp60arc kinase. Analysis of embryonic chicken lens RNA with a bovine calpactin I-specific cDNA probe revealed the presence of a approximately 1.8 Kb calpactin mRNA in the lens cells. Six-day embryonic chicken lenses were microdissected into central epithelium, equatorial epithelium, and fiber cells. Total cytoplasmic RNA was isolated from these samples and calpactin I mRNA levels were determined by the polymerase chain reaction (PCR) following reverse transcription (RT). Quantitative PCR indicates that the calpactin I mRNA levels in the equatorial epithelium are greater than in the central epithelium by a factor of 12.7 +/- 2.7. Calpactin I mRNA in fiber cells is an additional 3.5 +/- 1.5 times greater than in the equatorial epithelium. Whole mounts of embryonic chicken lens epithelia and histological sections of whole lenses were also examined with an antibody directed against chicken calpactin I. Calpactin I was predominantly localized in a punctate distribution in equatorial epithelial cells and near the plasma membrane of elongate fiber cells. The elevated levels of calpactin I mRNA observed in the equatorial epithelium and fiber cells and the immunological localization of the protein suggest a possible role of calpactin I in the elongation of fiber cells during lens differentiation.

Cyclins, cyclin-dependent kinases and differentiation.

Cyclin-dependent kinases and their regulatory subunits, the cyclins, are known to regulate progression through the cell cycle. Yet these same proteins are often expressed in non-cycling, differentiated cells. This review surveys the available information about cyclins and cyclin-dependent kinases in differentiated cells and explores the possibility that these proteins may have important functions that are independent of cell cycle regulation.

Cyclin B, p34cdc2, and H1-kinase activity in terminally differentiating lens fiber cells.

Terminal differentiation of lens fiber cells is marked by chromatin condensation, abrupt dissolution of the nuclear lamina, vesicularization of the nuclear membrane, and complete degradation of the nucleus and other organelles. Since these events resemble the chromosomal condensation and nuclear envelope breakdown associated with mitosis, we investigated whether a similar biochemical mechanism might be involved by testing for the presence of cyclin B/p34cdc2 complexes and p34cdc2-associated histone kinase activity in differentiating lens fiber cells. A coupled reverse transcription/polymerase chain reaction using RNA from E7, E15, or E20 embryonic chicken lens fibers amplified a cyclin B product of the expected size, whose identity was confirmed by sequencing. In situ hybridization showed that cyclin B mRNA was present in nucleated lens fiber cells at E19. Immunoblotting of proteins isolated from E6 or E15 lens fibers by p13-agarose affinity chromatography with anti-cyclin B antibody detected the 45-kDa cyclin B protein, while immunoblotting with anti-PSTAIRE antibody detected a single, 34-kDa band, identified as p34cdc2. The p13-affinity purified fraction from E6 or E15 lens fibers showed histone H1 kinase activity in vitro. Immunocytochemistry of E6 lenses with anti-chicken cyclin B antiserum showed positive staining in the nuclei of postmitotic annular pad and fiber cells. These results demonstrate that cyclinB/p34cdc2 complexes and p34cdc2-associated histone kinase activity are present in postmitotic, differentiating lens fiber cells and support the possibility that phosphorylation of specific nuclear substrates by p34cdc2 may play a role in the denucleation of lens fiber cells.

Differential expression of the two delta-crystallin genes in lens and non-lens tissues: shift favoring delta 2 expression from embryonic to adult chickens.

Chicken argininosuccinate lyase (ASL)/delta-crystallin, a lens enzyme-crystallin, is encoded in two linked genes (delta 1 and delta 2); only the delta 2 polypeptide contains ASL activity. Here we have quantified delta 1- and delta 2-crystallin mRNA in the lens, cornea, neural retina, heart, and brain at different stages of embryonic development and in 1-wk-old and 1-yr-old chickens by the polymerase chain reaction using internal delta 1 and delta 2 RNA standards. The delta 1/delta 2 mRNA ratio differed for every tissue and was regulated during development. In the embryo there was more delta 1 than delta 2 mRNA in the lens (50-100 times), cornea (3-4 times), and neural retina (2-20 times), about equal amounts of delta 1 and delta 2 mRNA in the heart, and more delta 2 mRNA in the brain (15 times). delta 1-Crystallin mRNA differentially decreased in every tissue after hatching; by contrast, the delta 2 mRNA remained about the same except for the lens, where it decreased 50-fold between 1 wk and 1 yr after hatching. In the 1-yr-old chicken, the delta 2/delta 1 mRNA ratios were 7 in the lens, 175 in the cornea, 22 in the neural retina, 107 in the heart, and 136 in the brain, indicating that delta 2-crystallin is strongly favored in all adult tissues of the chicken. The excess of delta 1 to delta 2 mRNA in the embryonic lens, cornea, and neural retina is intriguing, and suggests some connection with developing transparent eye tissues. Finally, we raise the possibility that expression of both delta-crystallin genes may create tetrameric ASL isoenzymes (perhaps with different specific activities). The unexpected predominance of delta 2 mRNA in the 1-yr-old lens suggests that both the enzymatic and refractive functions of ASL/delta-crystallin are operative and spatially separated, with the enzymatic role present in the cortical fibers and the refractive role in the center of the lens.

Effects of c-Jun and a negative dominant mutation of c-Jun on differentiation and gene expression in lens epithelial cells.

We have used a retroviral vector (RCAS) to overexpress wild-type chicken c-Jun or a deletion mutant of chicken c-Jun (Jun delta 7) lacking the DNA binding region to investigate the possible role of c-Jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c-Jun increased the rate of cell proliferation and greatly delayed the appearance of "lentoid bodies," structures which contain differentiated cells expressing fiber cell markers. Excess c-Jun expression also significantly decreased the level of beta A3/A1-crystallin mRNA, without affecting alpha A-crystallin mRNA. In contrast, the mutated protein, Jun delta 7, had no effect on proliferation or differentiation but markedly increased the level of alpha A-crystallin mRNA in proliferating cell cultures. These results suggest that c-Jun or Jun-related proteins may be negative regulators of alpha A- and beta A3/A1-crystallin genes in proliferating lens cells.