Aspartase/fumarase superfamily: a common catalytic strategy involving general base-catalyzed formation of a highly stabilized aci-carboxylate intermediate.

Members of the aspartase/fumarase superfamily share a common tertiary and quaternary fold, as well as a similar active site architecture; the superfamily includes aspartase, fumarase, argininosuccinate lyase, adenylosuccinate lyase, δ-crystallin, and 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE). These enzymes all process succinyl-containing substrates, leading to the formation of fumarate as the common product (except for the CMLE-catalyzed reaction, which results in the formation of a lactone). In the past few years, X-ray crystallographic analysis of several superfamily members in complex with substrate, product, or substrate analogues has provided detailed insights into their substrate binding modes and catalytic mechanisms. This structural work, combined with earlier mechanistic studies, revealed that members of the aspartase/fumarase superfamily use a common catalytic strategy, which involves general base-catalyzed formation of a stabilized aci-carboxylate (or enediolate) intermediate and the participation of a highly flexible loop, containing the signature sequence GSSxxPxKxN (named the SS loop), in substrate binding and catalysis.

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland.

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.

BMP-induced L-Maf regulates subsequent BMP-independent differentiation of primary lens fibre cells.

Bone morphogenetic protein (BMP) signals are essential for lens development. However, the temporal requirement of BMP activity during early events of lens development has remained elusive. To investigate this question, we have used gain- and loss-of-function analyses in chick explant and intact embryo assays. Here, we show that BMP activity is both required and sufficient to induce L-Maf expression, whereas the onset of δ-crystallin and initial elongation of primary lens fibre cells are BMP-independent. Moreover, before lens placode formation and L-Maf onset, but not after, prospective lens placodal cells can switch to an olfactory placodal fate in response to decreased BMP activity. In addition, L-Maf is sufficient to up-regulate δ-crystallin independent of BMP signals. Taken together, these results show that before L-Maf induction BMP activity is required for lens specification, whereas after L-Maf up-regulation, the early differentiation of primary lens fibre cells occurs independent of BMP signals.

Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress.

δ-Crystallin is a taxon-specific eye lens protein that was recruited from argininosuccinate lyase (ASL) through gene sharing. ASL is a metabolic enzyme that catalyzes the reversible conversion of argininosuccinate into arginine and fumarate and shares about 70% sequence identity and similar overall topology with δ-crystallin. ASL has a lower thermal stability than δ-crystallin. In this study, we show that the small heat shock protein, αA-crystallin, functions as a molecular chaperone, and enhanced thermal stability of both δ-crystallin and ASL. The stoichiometry for efficient protection of the two substrate proteins by αA-crystallin was determined by slowly increasing the temperature. N- or C-terminal truncated mutants of δ-crystallin co-incubated with αA-crystallin showed higher thermal stability than wild-type enzyme, and the stoichiometry for efficient protection was the same. Thermal unfolding of δ-crystallin or ASL in the presence of αA-crystallin followed a similar three-state model, as determined by circular dichroism analyses. A stable intermediate which retained about 30% α-helical structure was observed. Protection from thermal denaturation by αA-crystallin was by interaction with partly unfolded ASL or δ-crystallin to form high molecular weight heteroligomers, as judged by size-exclusive chromatography and SDS-PAGE analyses. Aggregate formation of ASL was significantly reduced in the presence of αA-crystallin. The extent of protection of ASL and δ-crystallin at different ratios of αA-crystallin were described by hyperbolic and sigmoidal curves, respectively. These results suggest the preferential recognition of partly unfolded ASL by αA-crystallin. In contrast, unstable δ-crystallin might trigger a cooperative interaction by higher stoichiometries of αA-crystallin leading to fuller protection. The different interactions of αA-crystallin with the two homologous but functionally different substrate proteins show its behavior as a chaperone is variable.

The interaction of Glu294 at the subunit interface is important for the activity and stability of goose delta-crystallin.

PURPOSE: delta-Crystallin is a soluble structural protein in found in avian eye lenses; it shares high amino acid sequence identity with argininosuccinate lyase. E294 is the only residue located at the double dimer interface and it performs hydrogen bonding with the active site residues of H160 and K323 in the neighboring and diagonal subunits, respectively. H160 is reported to play an important role in catalysis due to its H-bond interaction with the fumarate moiety of the substrate. In order to clarify the function of E294 in either stabilization of the quaternary structure or in catalysis, we carried out site-directed mutagenesis and functional analysis. METHODS: The structure of both wild-type and mutant proteins were analyzed by circular dichroism (CD) spectroscopy, fluorescence spectra, and analytical ultracentrifugation. Structural stability was measured by CD and tryptophan fluorescence. A modeled structure of the E294L mutant was built and optimized with energy minimization. RESULTS: No gross structural changes were observed when E294 was substituted with leucine, as judged by circular dichroism, tryptophan fluorescence, ANS fluorescence, and sedimentation velocity analyses. However, this mutant enzyme had only about 10% of the activity of a wild-type enzyme and its secondary structure was more easily denatured by increased temperature than that of a wild-type enzyme. The mutant protein also underwent its first unfolding transition at a lower concentration of guanidinium-hydrochloride than the wild-type protein. CONCLUSIONS: These results indicate that the interactions offered by E294 in the dimer-dimer interface of delta-crystallin are required to maintain the hydrogen bonding network in the active site for catalysis. Disruption of the interaction had no significant effect on the conformation and quaternary structure of delta-crystallin but it did lead to instability in the double dimer structure.

Essential role of BMPs in FGF-induced secondary lens fiber differentiation.

It is widely accepted that vitreous humor-derived FGFs are required for the differentiation of anterior lens epithelial cells into crystallin-rich fibers. We show that BMP2, 4, and 7 can induce the expression of markers of fiber differentiation in primary lens cell cultures to an extent equivalent to FGF or medium conditioned by intact vitreous bodies (VBCM). Abolishing BMP2/4/7 signaling with noggin inhibited VBCM from upregulating fiber marker expression. Remarkably, noggin and anti-BMP antibodies also prevented purified FGF (but not unrelated stimuli) from upregulating the same fiber-specific proteins. This effect is attributable to inhibition of BMPs produced by the lens cells themselves. Although BMP signaling is required for FGF to enhance fiber differentiation, the converse is not true. Expression of noggin in the lenses of transgenic mice resulted in a postnatal block of epithelial-to-secondary fiber differentiation, with extension of the epithelial monolayer to the posterior pole of the organ. These results reveal the central importance of BMP in secondary fiber formation and show that although FGF may be necessary for this process, it is not sufficient. Differentiation of fiber cells, and thus proper vision, is dependent on cross-talk between the FGF and BMP signaling pathways.

Molecular analysis of endoderm regionalization.

We have engaged in a number of studies in our laboratory that have focused on the molecular mechanisms underlying gut formation, with particular attention being paid to the establishment of regional differences found in the entire gut and within each digestive organ. We have found from our analyses that the presumptive fate of the endoderm in the embryos of vertebrates is determined quite early during development, but the realization of this fate often requires molecular cues from the neighboring tissues such as the lateral plate mesoderm and the mesenchyme derived from it. The mesenchyme seems often to exert instructive or supportive induction effects and, in some cases, a completely inhibitory role during the differentiation of the endodermal epithelium. In addition, many reports on the formation of the stomach, intestine, liver and salivary gland in vertebrates, and of Drosophila gut, all indicate that the morphogenesis and cytodifferentiation of these organs are regulated by the regulated expression of genes encoding growth factors and transcription factors. We have further shown that the epithelium can regulate the differentiation of the mesenchyme into the connective tissue and the smooth muscle layers, thus demonstrating the occurrence of literally interactive processes in the development of the digestive organs.

PAX6 and SOX2-dependent regulation of the Sox2 enhancer N-3 involved in embryonic visual system development.

Sox2 is universally expressed in the neural and placodal primordia in early stage embryos, and this expression depends on various phylogenetically conserved enhancers having different regional and temporal specificities. The enhancer N-3 was identified as a regulator of the Sox2 gene active in the diencephalon, optic vesicle, and after the contact of the vesicle with the ectoderm, in the lens placodal surface area, suggesting its involvement in embryonic visual system development. A 36-bp minimal essential core sequence was defined in the 568-bp-long enhancer N-3, which in a tetrameric form emulates the original enhancer activity. The core sequence comprises a SOX-binding sequence and a non-canonical PAX6 (Paired domain) binding sequence, and is activated by the synergistic action of SOX2 and PAX6 in transfected cells. The SOX and PAX6 binding sequences of the N-3 core are arranged with the same orientation and spacing as the DC5 sequence of the delta-crystallin enhancer previously demonstrated to be cooperatively bound by SOX2 and PAX6. The N-3 core sequence was also bound by these factors in a cooperative fashion, but with a higher threshold of these factors' levels than DC5, and the enhancer effect of the tetrameric sequence activated by exogenous SOX2 and PAX6 was less pronounced than that of DC5. The observations suggest that gene activation mechanisms that depend on the cooperative interaction of SOX2 and PAX6 but with different thresholds of the factor levels are crucial for the regulation of visual system development.

The effect of N-terminal truncation on double-dimer assembly of goose delta-crystallin.

Delta-crystallin is a soluble structural protein in avian eye lenses that confers special refractive properties. In the presence of GdmCl (guanidinium chloride), tetrameric delta-crystallin undergoes dissociation via a dimeric state to a monomeric molten globule intermediate state. The latter are denatured at higher GdmCl concentrations in a multi-state manner. In the present study, the X-ray structure of goose delta-crystallin was determined to 2.8 A (1 A=0.1 nm). In this structure the first 25 N-terminal residues interact with a hydrophobic cavity in a neighbouring molecule, stabilizing the quaternary structure of this protein. When these 25 residues were deleted this did not produce any gross structural changes, as judged by CD analysis, but slightly altered tryptophan fluorescence and ANS (8-anilino-1-naphthalenesulphonic acid) spectra. The dimeric form was significantly identified as judged by sedimentation velocity and nondenaturing gradient gel electrophoresis. This mutant had increased sensitivity to temperature denaturation and GdmCl concentrations of 0.3-1.0 M. This protein was destabilized about 3.3 kcal/mol (1 kcal=4.184 kJ) due to N-terminal truncation. After incubation at 37 degrees C N-terminal truncated proteins were prone to aggregation, suggesting the presence of the unstable dimeric conformation. An important role for the N-terminus in dimer assembly of goose delta-crystallin is proposed.

Recovery of argininosuccinate lyase activity in duck delta1 crystallin.

Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions.

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.

Lens differentiation and crystallin regulation: a chick model.

The vertebrate lens is a transparent polarized tissue that acts as the gateway for vision. The chick lens is an excellent model for studying tissue organogenesis, since it is both accessible and easily manipulated during embryonic stages. The chick lens consists of two morphologically discrete compartments, the epithelium and the fiber-cell mass. Evidence indicates that the early phases of lens development involve several sequential events, including tissue interactions, cell proliferation and differentiation. The morphological change during lens development is associated with the concurrent and distinct functions of numerous transcription factors. Diffusible molecules from the complementary neural tissue play vital roles during the entire process of lens development. Lens tissue is characterized by the ample production of crystallins, lens specific proteins which provide structural integrity and functional properties to the lens. Thus, the study of crystallin regulation should provide insight into the development of a functional lens during embryogenesis. This process has been shown to involve a complex and evolutionary conserved pathway supported by different regulatory proteins.

Interplay of Pax6 and SOX2 in lens development as a paradigm of genetic switch mechanisms for cell differentiation.

When the cloning era arrived, our first target for cloning was the delta1-crystallin gene of the chicken, the lens-specific gene expressed earliest following lens induction. We have investigated the regulation of this gene with the idea that the mechanism of its activation must reflect that of lens differentiation per se. We here summarize the investigation carried out in our group along this line over the past 20 years. The delta1-crystallin gene is regulated by an enhancer in the third intron, and the specificity of this regulation is governed by a DNA region (called DC5) of only 30 bp DNA bound by two transcription factors. These factors have been identified as SOX1/2/3 (Group B1 SOX proteins, SOX2 being the major player) and Pax6, and have been shown to bind cooperatively to DC5 and form a ternary complex having a robust potency for transcriptional activation. In the embryo, Pax6 is widely expressed in the head ectoderm before the lens is formed, and as the optic vesicle comes into contact with the ectoderm, SOX2/3 expression is induced in the contacted area of the ectoderm, thereby allowing Pax6 and SOX2/3 to meet in the same cell nucleus, where they can then activate a battery of genes for early lens development including delta1-crystallin. Thus, the cooperative action of Pax6 and SOX2 initiates lens differentiation. More broadly, SOX1/2/3 interact with various partner transcription factors, and participate in defining distinct cell states that depend on the partner factors: Pax6 for lens differentiation, Oct3/4 for establishing the epiblast/ES cell state, and Brn2 for the neural primordia. Thus, the regulation of SOX2 (and SOX1/3) and its partner factors, exemplified by Pax6, determines the spatio-temporal order of the occurrence of cell differentiation.

Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation.

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.

A duck delta1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity.

Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.

Structural studies of duck delta2 crystallin mutants provide insight into the role of Thr161 and the 280s loop in catalysis.

Delta crystallin, a taxon-specific crystallin present in avian eye lenses, is homologous to the urea cycle enzyme ASL (argininosuccinate lyase). Although there are two delta crystallin isoforms in duck lenses, ddeltac1 (duck delta1 crystallin) and ddeltac2 (duck delta2 crystallin), only ddeltac2 is catalytically active. Previous structural studies have suggested that residues Ser283 and His162 in the multi-subunit active site of ddeltac2/ASL are the putative catalytic acid/base, while the highly conserved, positively charged Lys289 is thought to help stabilize the carbanion intermediate. The strict conservation of a small hydroxy-containing residue (Thr or Ser) at position 161 adjacent to the putative catalytic base, as well as its proximity to the substrate in the S283A ddeltac2 enzyme-substrate complex, prompted us to investigate further the role this residue. Structures of the active T161S and inactive T161D ddeltac2 mutants, as well as T161D complexed with argininosuccinate, have been determined to 2.0 A resolution. The structures suggest that a hydroxy group is required at position 161 to help correctly position the side chain of Lys289 and the fumarate moiety of the substrate. Threonine is probably favoured over serine, because the interaction of its methyl group with Leu206 would restrict its conformational flexibility. Residues larger than Thr or Ser interfere with substrate binding, supporting previous suggestions that correct positioning of the substrate's fumarate moiety is essential for catalysis to occur. The presence of the 280s loop (i.e. a loop formed by residues 270-290) in the 'open' conformation suggests that loop closure, thought to be essential for sequestration of the substrate, may be triggered by the formation of the carbanion or aci-carboxylate intermediates, whose charge distribution more closely mimics that of the sulphate ion found in the active-site region of the inactive ddeltac1. The 280s loop in ddeltac1 is in the closed conformation.

Monomeric molten globule intermediate involved in the equilibrium unfolding of tetrameric duck delta2-crystallin.

Duck delta2-crystallin is a soluble tetrameric lens protein. In the presence of guanidinium hydrochloride (GdnHCl), it undergoes stepwise dissociation and unfolding. Gel-filtration chromatography and sedimentation velocity analysis has demonstrated the dissociation of the tetramer protein to a monomeric intermediate with a dissociation constant of 0.34 microM3. Dimers were also detected during the dissociation and refolding processes. The sharp enhancement of 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence at 1 M GdnHCl strongly suggested that the dissociated monomers were in a molten globule state under these conditions. The similar binding affinity (approximately 60 microM) of ANS to protein in the presence or absence of GdnHCl suggested the potential assembly of crystallins via hydrophobic interactions, which might also produce off-pathway aggregates in higher protein concentrations. The dynamic quenching constant corresponding to GdnHCl concentration followed a multistate unfolding model implying that the solvent accessibility of tryptophans was a sensitive probe for analyzing delta2-crystallin unfolding.

Stimulation of lens cell differentiation by gap junction protein connexin 45.6.

PURPOSE: The present study was undertaken to explore the roles gap junctions play in lens epithelial cell differentiation. METHODS: Recombinant retroviruses expressing three chick lens connexins (Cx)-Cx43, Cx45.6, and Cx56-were prepared and used to infect isolated chick lens primary cultures. The expression and distribution of proteins was determined using immunoblots and confocal immunofluorescence microscopy. Intercellular couplings were assessed by single cell microinjection and scrape-loading dye transfer, and cell proliferation was evaluated by [(3)H]thymidine labeling. RESULTS: Of the three lens connexins, only the cultures overexpressing exogenous Cx45.6 displayed the advancement of lens epithelial-fiber cell differentiation. The lentoids, a unique morphologic structure that is an indicative of lens fiber formation, were formed earlier in Cx45.6 overexpressed cultures; however, the rate of lens cell proliferation was not affected. The expression of the lens differentiation marker proteins, major intrinsic protein (MIP) and delta-crystallin, was also increased in Cx45.6-overexpressing cells. The cells overexpressing Cx45.6 displayed similar levels of intercellular couplings as did the controls. Moreover, exogenously expressed connexins were mostly colocalized with their endogenous counterparts and the overexpression of Cx45.6 had no impact on the expression of endogenous Cx43 and Cx56. CONCLUSIONS: These results suggest that Cx45.6 plays an important role in stimulating lens cell differentiation and fiber formation, which is different from the other lens connexins, Cx43 and Cx56. This stimulatory effect is independent of gap junction-mediated intercellular communication and lens cell proliferation.

The stability of the lens-specific Maf protein is regulated by fibroblast growth factor (FGF)/ERK signaling in lens fiber differentiation.

Fibroblast growth factor (FGF) signaling is necessary for both proliferation and differentiation of lens cells. However, the molecular mechanisms by which FGFs exert their effects on the lens remain poorly understood. In this study, we show that FGF-2 repressed the expression of lens-specific genes at the proliferative phase in primary cultured lens cells. Using transfected cells, we also found that the activity of L-Maf, a lens differentiation factor, is repressed by FGF/ERK signaling. L-Maf is shown to be phosphorylated by ERK, and introduction of mutations into the ERK target sites on L-Maf promotes its stabilization. The stable L-Maf mutant protein promotes the differentiation of lens cells from neural retina cells. Taken together, these results indicate that FGF/ERK signaling negatively regulates the function of L-Maf in proliferative lens cells and that stabilization of the L-Maf protein is important for lens fiber differentiation.

Quantification of chick lens alphaA- and delta-crystallins in experimentally induced ametropia.

PURPOSE: The role of the lens in experimentally induced ametropia is not known. A recent study of the chick lens demonstrated optical quality deterioration with the induction of refractive errors, without alteration in lens morphology, size or shape. A change in lens gradient of refractive index (which is dependent on alpha-, beta-, and delta-crystallin concentration and arrangement), could underlie this observation. The purpose of this work was to quantify the concentrations of alphaA- and delta-crystallin in lenses from chick eyes with induced high myopia or hyperopia. METHODS: White Leghorn chicks were unilaterally fitted on the day of hatching either with translucent plastic goggles to induce form-deprivation myopia (n=21) or with +15 D defocus goggles to induce hyperopia (n=14). The ungoggled contralateral eyes were used as controls. The chicks were refracted twice, once on the day of hatching and again seven days later, using streak retinoscopy. On day 7 chicks were sacrificed, lenses decapsulated, and soluble proteins were isolated. Western blot assays were optimized and used to assess crystallin concentration. RESULTS: Analysis revealed no significant difference in alphaA- or delta-crystallin concentration in lenses from eyes induced with form-deprivation myopia and hyperopia as compared to their respective control eyes. Analysis of the difference in medians of delta-crystallin between the control and treated groups of the myopia and hyperopia experiments revealed significance (p=0.030). CONCLUSIONS: This study suggests that with the induction of ametropia, the increased lens spherical aberration previously noted is not due to a change in the absolute concentration of lens alphaA- or delta-crystallin. However, results suggest that the myopic and hyperopic treatments had different effects on lens delta-crystallin concentration. Further investigation is necessary to expand the current knowledge of the role played by the lens in experimental ametropia.