A role for endogenous TGFalpha and associated signaling pathways in the differentiation of lens fiber cells.

TGFalpha is hypothesized to be an endogenous regulator of lens fiber terminal differentiation. With immunofluorescence, TGFalpha was localized to differentiating cells in the lens epithelium and superficial fiber cell mass of the adult chicken. A similar pattern of localization was also noted when differentiating epithelial cells were cultured. Immunoneutralization of endogenous TGFalpha inhibited the accumulation of filensin, a unique intermediate filament protein subunit restricted to developing vertebrate lens fibers. ELISA assays quantified the effects of TGFalpha on filensin expression. Surprisingly, inhibition of the TGFalpha receptors' tyrosine kinase activity with nanomolar concentrations of PD153035 increased the accumulation of differentiated characteristics in the presence or absence of ligand. Morphologically, PD153035-treated cells grew as aggregated masses and spread less well onto the substrate. Accompanying these morphologic changes was a complete inhibition of cell division. Post-receptor signaling events were examined with cAMP assays and Western blotting. TGFalpha did not affect cAMP levels while isoproterenol, an additional mediator of lens cell differentiation, caused significant increases in cAMP levels. Activation of ERK2 via dual phosphorylation was noted in response to TGFalpha but not isoproterenol. PD153035 reduced, but did not eliminate, ERK2 phosphorylation in response to TGFalpha. Phosphorylation of the CREB transcription factor was also observed in response to TGFalpha or isoproterenol. These data indicate that endogenous ligands can influence the expression of differentiated characteristics in cultured chick lens cells. A focus of multiple signaling pathways affecting filensin expression is the CREB transcription factor. While increased ERK2 activation may be involved in stimulating cell division, lower levels of persistent ERK2 activation promotes differentiation thus indicating some form of signaling pathway compartmentalization. This could also mean that additional receptors for TGFalpha not inhibited by PD153035 are responsible for promoting differentiation in chick lens cells.

Differentiation of chick lens epithelial cells: involvement of the epidermal growth factor receptor and endogenous ligand.

PURPOSE: To characterize the constitutively activated epidermal growth factor receptor in a lens epithelial cell population experiencing initial stages of lens fiber formation, the chick lens annular pad. METHODS: Phosphotyrosine levels of the receptor were examined with western blot analysis and immunoprecipitation after ligand stimulation. Endogenous receptor ligands were immunologically identified in whole cell lysates of freshly isolated cells. The expression of lens fiber-specific differentiation marker proteins was examined with western blot analysis and enzyme-linked immunosorbent assay (ELISA) in short-term primary cultures of annular pad cells exposed to ligand. RESULTS: The major phosphotyrosine-containing protein in annular pad cells comigrated with the epidermal growth factor receptor and increased its phosphotyrosine content after epidermal growth factor treatment. Both time- and dose-dependent responses were noted. The constitutive activation of the receptor was determined in the presence of phosphatase inhibitors. Endogenous transforming growth factor-alpha, but not epidermal growth factor, was detected in freshly isolated cells. Transforming growth factor-alpha (TGF-alpha) treatment produced greater increases in receptor phosphotyrosine levels than equimolar levels of epidermal growth factor. Finally, TGF-alpha treatment induced increased expression of the beaded filament protein filensin when compared with control cells. Filensin expression was increased further when cells were costimulated with TGF-alpha and cAMP analogs. CONCLUSIONS: At least in the postnatal lens, endogenous TGF-alpha may affect overall growth patterns by modulating differentiation-specific protein expression. Furthermore, signaling pathways elicited by TGF-alpha and cAMP analogs converge to cooperatively enhance lens fiber differentiation.

Up-regulation of novel intermediate filament proteins in primary fiber cells: an indicator of all vertebrate lens fiber differentiation?

The early embryonic development and expression patterns of the eye lens specific cytoskeletal proteins, CP49 and CP95, were determined for the chick and were found to be similar in both human and mouse. These proteins, as well as their homologs in other species, are obligate polymerization partners which form unique filamentous structures termed "beaded filaments." CP49 and CP95 appeared as protein products after 3 days of embryonic development in the chick during the elongation of primary fiber cells. Although limited data were obtained for human embryos at these early developmental timepoints, they were consistent with the interpretation that the up-regulation of these lens specific proteins began only after the initiation of lens vesicle closure. In situ hybridization with the mouse lens confirmed that message levels for beaded filament proteins were greatly elevated in differentiating primary fiber cells. Nuclease protection assays established that mRNA levels for CP49 remained relatively constant while CP95 mRNA levels increased once the process of secondary fiber formation was under way. Although present in relatively low abundance, the mRNA for a unique splice variant of CP49, CP49(INS), was also detected early in embryonic development and into adulthood. Peptide-specific antibodies directed against unique predicted sequences were able to confirm the protein expression of CP49(INS) in both embryonic and adult chick lens cells. These data present the first detailed study of the expression of CP49 and CP95 during early lens development. They suggest that the up-regulated expression of CP49 and CP95 could serve as pan-specific markers for all vertebrate lens fiber development.

Matrix metalloproteinase secretion is stimulated by TGF-beta in cultured lens epithelial cells.

PURPOSE: To determine if TGF-beta regulates the expression of metalloproteinases in chick lens annular pad cells. METHODS: The activity of secreted matrix metalloproteinases was examined with gelatin zymography in primary cultures exposed to TGF-beta. RESULTS: Metalloproteinases with electrophoretic mobilities corresponding to MMP2 and MMP9 were tentatively identified. Activated, processed forms of the two metalloproteinases were also observed. Plasminogen activators potentially capable of initiating metalloproteinase cascades were concomitantly elicited. Metalloproteinase secretion was shown to be specific for TGF-beta stimulation and independent of substrate composition. CONCLUSIONS: These results indicate that TGF-beta-mediated processes could be responsible for localized lens capsular heterogeneity, establishing a substrate suitable for cell migration or the release of matrix-bound factors which influence the terminal differentiation of lens cells.

Kinetics of cyclic AMP-dependent accumulation of novel intermediate filament proteins in cultured chick lens cells.

PURPOSE: To refine the parameters affecting the accumulation of cytoskeletal markers of lens fiber terminal differentiation. METHODS: Primary cultures of chick lens annular pad cells were treated with a lipid soluble cyclic AMP analog under various culture conditions. The accumulation of beaded filament proteins, unique markers of lens fiber terminal differentiation, was quantified with an ELISA assay. The incorporation of beaded filament proteins into macromolecular structures was followed with immunofluorescence microscopy. RESULTS: In a time- and dose-dependent manner, beaded filament protein levels were increased in cyclic nucleotide treated cells. The addition of serum to treated cells caused a further dose-dependent increase in beaded filament protein levels. The continuous presence of cyclic nucleotides for maximal beaded filament protein accumulation was also established. At the light microscopic level, cyclic nucleotide treatment produced much more extensive multilayering of cells and lentoid formation. Macromolecular structures containing beaded filament proteins also increased in both abundance and complexity after cyclic nucleotide treatment and were restricted to the multilayers/lentoids. CONCLUSIONS: These results indicate that multiple mechanisms (including cyclic AMP, serum factors, and the degree of cell-cell interactions) affect the accumulation of beaded filament proteins during the normal differentiation of lens fibers.

TGF-beta elicits fibronectin secretion and proliferation in cultured chick lens epithelial cells.

PURPOSE: To determine if the cataract forming influence of TGF-beta on lens cells is due to its effects on the ECM. METHODS: Primary cultures of chick lens annular pad cells were exposed to TGF-beta and various exogenously supplied components of the lens capsule. Proliferative response were measured through tritiated thymidine incorporation into DNA. Cell spreading accompanying increased matrix interactions and growth was monitored with phase contrast microscopy. ECM proteins were detected in culture media and as deposited matrices with Western blotting and silver staining. TGF-beta receptors were identified with Western blotting. RESULTS: Chick lens cells were shown to express type I and II TGF-beta receptors. TGF-beta stimulated cell growth and ECM production particularly with regard to fibronectin. Fibronectin was secreted into the culture medium and deposited onto plastic substrates. Plating cells on ECM components found in the lens capsule further increased their growth in response to TGF-beta. CONCLUSIONS: These results indicate that TGF-beta may have a normal function in the lens regulating capsular protein production. The potent stimulation of lens cell growth by TGF-beta may be due to mis-regulated production of lens capsular proteins not normally found in great abundance.

Quantification and regulation of mRNAs encoding beaded filament proteins in the chick lens.

PURPOSE: To quantify the expression of beaded filament protein mRNA levels in regions of the chick lens and to examine the in vitro regulation of message and protein levels using cell culture techniques. METHODS: RNase protection assays and Northern blotting were used to quantify beaded filament protein mRNA levels in dissected lenses. Cultured cells were assayed for mRNA with RNase protection and for protein with Western blotting and ELISA techniques after treatment with cAMP analogs. RESULTS: Beaded filament protein message levels were greatly up-regulated in cortical fiber cells compared to annular pad cells. Full length messages were also detected in nuclear fiber cells. The presence of an unusual form of the CP49 message with a lamin-like insert, CP49INS, was also established. Both message and protein levels were subject to regulation in response to elevated intracellular cAMP levels. CONCLUSIONS: The accumulation of beaded filament protein levels during fiber cell development may be due to the increased cAMP-mediated transcription of message. The presence of CP49INS may lend new insight into mechanisms of intermediate filament assembly.

Phorbol esters affect cyclic nucleotide-mediated responses in cultured chick lens annular pad cells.

Cultured chick lens annular pad cells were treated with a lipid soluble cAMP analog, the phorbol ester TPA or a combination of the two compounds in order to assess their effects on mitotic activity, cell spreading and the accumulation of differentiation marker proteins. Both 8b-cAMP and TPA were individually able to inhibit mitotic activity in cells cultured in the presence of 5% serum. The combination of the two produced a greater degree of mitotic inhibition. Both compounds were also able to inhibit cellular spreading onto laminin coated surfaces. Opposite effects on the accumulation of differentiation marker proteins were observed for the two compounds. While 8b-cAMP increased levels of marker proteins, TPA or the combination of TPA and 8b-cAMP reduced levels of marker proteins. These data indicate that crosstalk between two distinct signal transduction systems in the lens is able to influence cell behaviors implicated in the development of secondary or posterior subcapsular cataract. In addition, these data demonstrate that both positive and negative regulatory influences affect the accumulation of differentiated characteristics.

Identification of G-protein alpha-subunits during distinct stages of lens cell differentiation.

PURPOSE: The purpose of this study was to identify alpha-subunits of heterotrimeric guanosine triphosphate-binding proteins in lens cell populations at various stages of terminal differentiation. METHODS: Crude cell membranes were isolated from the annular pad, cortical fibers, and nuclear fibers of adult chickens and subjected to cholera and pertussis toxin-mediated ribosylation reactions. Specific labeling of toxin substrates was visualized after SDS-PAGE and radioautography. In complementary experiments, cell membranes were first separated by SDS-PAGE, transferred to a nitrocellulose support membrane, and probed with a panel of commercially available antibodies that recognize various classes of G-protein alpha-subunits. RESULTS: A cholera toxin substrate was identified in cortical fibers whose labeling was dependent on a soluble factor. No cholera toxin substrates were labeled in annular pad cells. Two pertussis toxin substrates were seen in the relatively undifferentiated annular pad and the more differentiated cortical fibers. Relative abundance of the pertussis toxin substrates differed between the two cell types. Antibody staining revealed the presence of alpha-subunits belonging to the Gs, G(o), and G(i), families throughout the lens. Molecular weight differences of G alpha 2 polypeptides were noted between annular pad and cortical fiber cells. CONCLUSIONS: These results suggest that G-protein-based signal transduction pathways continue to function in lens cells during various stages of terminal differentiation. Differences noted between annular pad and cortical fibers provide additional evidence that dynamic alterations in receptor-mediated processes may be responsible for the accumulation of differentiated characteristics during fiber formation. This also indicates that ocular pharmacologic intervention could affect various aspects of lens physiology throughout the process of fiber formation.

Regulation of lens beta-adrenergic receptors by receptor occupancy and dexamethasone.

Beta-adrenergic binding sites in primary cultures of chick lens annular pad (CLAP) cells were characterized with dihydroalprenolol (DHAP). Binding site affinities and densities were similar to beta-adrenergic receptors (BARs) previously characterized on crude membranes from freshly isolated cells. In competitive displacement studies, the beta-blocker propranolol was shown to increase the number of available binding sites in a concentration dependent manner. Acute exposure of CLAP cells to propranolol prior to DHAP binding also resulted in an increase in the number of available binding sites. Finally, lens beta-adrenergic binding site levels could be modulated by dexamethasone treatment. These results indicate that lens BARs are subject to common regulatory mechanisms and further implicate ophthalmic pharmaceuticals as possible cataractogenic agents.

Cell-cell interactions affect the accumulation of a cytokeratin-like protein during lens fiber development.

Extracellular matrix proteins were presented in culture to postmitotic, epithelial precursors of chick lens fiber cells as rigid, planar surfaces or malleable gels. Their ability to maintain and promote differentiated characteristics was judged by tritiated thymidine incorporation, immunologic detection of a cytokeratin-like protein (CP49) which accumulates during lens fiber development, and the formation of multicellular aggregates known as lentoids. Regardless of their composition, planar substrates stimulated the reentry into the cell cycle by promoting cell spreading. Laminin- and type IV collagen-coated surfaces facilitated monolayer growth and no appreciable accumulation of CP49. Thin films of Matrigel, which initially stimulated cell division, eventually promoted the formation of extensive lentoids, multicellular aggregates exhibiting many morphologic and biochemical properties of lens fibers. Malleable gels of Matrigel, however, inhibited cell division and immediately allowed the cells to begin lentoid formation. Culture conditions which favored lentoid formation also showed greatly enhanced levels of CP49 accumulation. In addition, lentoids were also shown to accumulate an integral membrane protein (MIP26) which is present in communicating junctions between neighboring fiber cells. These studies indicate that increased cell associations within forming lentoids may influence the progression of lens fiber terminal differentiation in vitro.

Beta-adrenergic mechanisms affect cell division and differentiation in cultured chick lens epithelial cells.

Primary cultures of chick lens annular pad (CLAP) cells were exposed to beta-adrenergic drugs and lipid soluble cAMP analogues. In dose-dependent manners, these agents inhibited re-entry into the cell cycle when these normally post-mitotic cells were cultured in the presence of serum. The inhibition of cell division was evidenced by a lack of DNA accumulation and greatly reduced tritiated thymidine incorporation into DNA. Treated cells were also largely inhibited from spreading onto the substrate. Instead, the epithelial monolayers originally placed into culture formed nearly spherical aggregates. Treated cultures continued to synthesize crystallins and exhibited increased levels of a 49-kDa cytoskeletal protein which accumulates in fiber cells. These results indicate that beta-adrenergic mechanisms may be involved in regulating cell division and terminal differentiation of lens fiber cells.

Cyclic AMP-mediated phosphorylation and insolubilization of a 49-kDa cytoskeletal marker protein of lens fiber terminal differentiation.

Terminal differentiation of chick lens fiber cells has been previously characterized by the accumulation, acidification via phosphorylation, and increased membrane association of a 49-kDa cytoskeletal protein. In these studies, we examine: (1) the subcellular distribution of the 49-kDa protein with regard to ageing and isoform composition; and (2) potential mechanisms regulating 49-kDa phosphorylation and insolubilization. With conventional Western blotting techniques, the 49-kDa protein is found exclusively in insoluble form within terminally differentiated nuclear fiber cells. Cortical fibers, on the other hand, exhibit a more widespread subcellular distribution of the 49-kDa protein. On two-dimensional gels, cortical 49-kDa isoelectric variants segregate according to their ease of sedimentation. After homogenization in detergent-containing buffers, the major isoform of the 49-kDa protein found in low speed pellets (40,000 g, 20 min) exhibits an acidic pI. The 40,000 g supernate and the high speed pellet (100,000 g, 2 hr) which is sedimented from this supernate are enriched in more basic isoforms of the 49-kDa protein. The 100,000 g supernate overlying the high speed pellet is dominated by the most basic isoform. With in vitro phosphorylation assays, the 49 kDa protein is shown to be a major substrate affected by endogenous cAMP-dependent mechanisms. Both the low and high speed pellets exhibit endogenous cAMP-dependent kinase activity. An inhibitor of cAMP-dependent protein kinase activity is also found in soluble lens fractions. Conversion of the 49-kDa protein into more acidic, phosphorylated isoforms increases its insolubility and ease of sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS)

The second messenger pathway for germ cell-mediated stimulation of Sertoli cells.

Treatment of cultured rat Sertoli cells with FSH or dibutyryl cAMP for 30 min resulted in phosphorylation of the same Sertoli cell proteins. Different Sertoli cell proteins were phosphorylated after calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. A23187 stimulated the phosphorylation of hsp27, while TPA alone had no effect. TPA plus A23187 resulted in phosphorylation of a 14 kDa protein, in addition to hsp27. The effect of TPA plus A23187 was identical to that of germ cells on Sertoli cell protein phosphorylation. FSH-stimulated cAMP production by Sertoli cells was reduced by prior exposure of Sertoli cells to germ cells. The results indicate that germ cells stimulate Sertoli cells by the inositol trisphosphate/diacylglycerol mediated second messenger pathway. The results also suggest that the germ cell-activated pathway interacts within Sertoli cells to modulate Sertoli cell response to FSH.

Lens beta-adrenergic receptors. Functional coupling to adenylate cyclase and photoaffinity labeling.

Beta-adrenergic drugs affect lens epithelial and fiber cells. The regulation and cellular integration of lens beta-adrenergic responses are largely unknown. These studies further characterize beta-adrenergic receptors in lens cells with respect to cyclic adenosine monophosphate (cAMP) production and identification of receptor polypeptides. Stimulation of beta-adrenergic receptors in organ-cultured chick lenses resulted in dose-dependent increases in intracellular cAMP levels. Isoproterenol-elicited cAMP accumulation was found in both epithelial/superficial fiber cells and cortical fiber cells. Hormonal stimulation also apparently initiated additional mechanisms involved in the regulation of cAMP levels (ie, phosphodiesterase activation/receptor desensitization). Individual receptor polypeptides were identified in epithelial and fiber membranes with the photoaffinity probe 125I-iodocyanopindolol diazarine. The probe specifically labeled distinct populations of receptor polypeptides in the two cell types. Lens beta-adrenergic receptors were also shown to bind (-) stereoisomers of adrenergic ligands preferentially. These results indicate that differentiating fiber cells are hormonally sensitive to beta-adrenergic stimulation and that epithelial and fiber cells may respond differentially to beta-adrenergic drugs, at least in part, because of their distinct receptor polypeptides.

Ultrastructural, biochemical, and immunologic evidence of receptor-mediated endocytosis in the crystalline lens.

The lens epithelium is essentially the basal layer of the crystalline lens of the eye, an uncommon stratified epithelium. Ions and metabolites present in the aqueous humor gain access to the lens epithelium by diffusion through the lens capsule (the basement membrane of the lens epithelium). Then, it is presumed, the underlying lens fiber cells obtain necessary ions, metabolites, and nutrients through gap junctions conjoining the apical surfaces of the lens epithelial cells from the basal layer with the apical surfaces of elongating fiber cells from upper strata. In this report, correlative morphologic, biochemical, and immunochemical evidence is presented that both lens epithelial and fiber cells use endocytotic and/or transcytotic processes rather than being solely dependent on gap junctions for metabolic cooperation. Freeze-fracture analysis of the apicoapical interface between lens epithelial and elongating fiber cells (epithelial-fiber cell interface [EFI]) revealed protrusions and pits of two distinct sizes (average diameters, 46 and 126 nm). Gap junctions with tight particle packing were only rarely observed at the EFI. Gap junctions with loose particle packing were never observed at the EFI. "Orthogonal arrays" of intramembrane particles (OAPs) were not uncommon at the EFI. Thin-sections taken perpendicular to the EFI confirmed the existence of micropinocytotic and clathrin-coated vesicles in both lens epithelial and elongating fiber cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of separate preparations of lens epithelial and fiber cells, specifically enriched for clathrin-coated vesicles, showed a 180-kD protein. Western blot analysis of this protein revealed selective cross-reactivity with polyclonal anticlathrin antibodies. These results strongly suggest that transcytotic processes provide a primary route for the entry and egress of macromolecules in the lens.

Initial characterization of lens beta-adrenergic receptors.

Crude plasma membranes were prepared from chick lens epithelial and fiber cells and assayed for specific binding of the beta-adrenergic antagonist dihydroalprenolol. Both membranes specifically bound the ligand, with the epithelial membranes exhibiting a greater number of higher affinity sites. This is the first demonstration of lens beta-adrenergic receptors using this type of assay and the first direct demonstration of beta-adrenergic receptors in lens fiber cells.

Isoproterenol treatment causes cytoskeletal reorganization in chicken lens fiber cells.

Cytoskeletal organization in organ cultured embryonic chicken lens was affected by isoproterenol treatment. Immunofluorescent localization of a 49 kD fiber cell-specific cytoskeletal protein showed a cytoplasmic and membrane localization in control lenses. Following isoproterenol treatment, fluorescence was predominantly membrane-associated. Changes in 49 kD distribution as determined by immunofluorescence occurred within minutes of isoproterenol application and were completely blocked by propranolol pretreatment. These results suggest that beta-adrenergic stimulation influences the apparent localization of a fiber cell-specific cytoskeletal protein.

Germ cell stimulation of Sertoli cell protein phosphorylation.

Cultures of Sertoli cells were treated with freshly isolated, intact germ cells to determine if germ cells were capable of influencing Sertoli cell function. By using two-dimensional polyacrylamide gel electrophoresis and autoradiography, germ cells were found to increase several-fold the incorporation of [32P]orthophosphate into two phosphoproteins (which we term germ cell-dependent phosphoproteins 1 and 2 or GC1 and GC2) of Sertoli cells. Increased phosphorylation of GC1 and GC2 was rapid, germ cell dose dependent, and calcium dependent. The increased phosphorylation of GC1 appears to involve calmodulin-dependent protein kinase, while phosphorylation of GC2 appears to involve the activation of calcium/phospholipid-dependent protein kinase (protein kinase C). Medium conditioned by germ cells was capable of eliciting the same response in Sertoli cells. Germ cells had no effect on the phosphorylation of proteins in Chinese hamster ovarian cells, but did result in increased phosphorylation of a protein in TR-ST cells, which migrated similarly to GC1 of Sertoli cells. Neither Chinese hamster ovarian nor TR-ST cells had any effect on Sertoli cell protein phosphorylation. These results indicate that germ cells may be directly involved in the local regulation of Sertoli cell function within the seminiferous epithelium. The results further suggest that the mechanism of germ cell-Sertoli cell interaction involves the mobilization of intracellular calcium, activation of Ca2+/calmodulin-dependent protein kinase, and protein kinase C. We infer from these results that germ cell-Sertoli cell interaction may operate via hydrolysis of Sertoli cell membrane phophatidylinositols.