Caspase-9 activation in hypoxic human corneal epithelial cells.

The purpose of this study was to determine the effect of hypoxia on caspase-8 and -9 gene and protein expression and activity in corneal epithelium. Non-transformed human corneal epithelial cells (HCEC) were cultured in 2% oxygen. A cDNA expression array coupled with densitometric analysis was used to compare relative mRNA expression levels of 96 apoptosis-related genes in hypoxic and normoxic HCEC. Caspase-8, caspase-9, FLIP, Fas, FasL, and TNFalpha protein expression was assessed further using Western blot analysis and ELISA. Caspase-8 and -9 activities were measured using a fluorometric activity assay. Hypoxia did not affect caspase-8 or -9 gene or protein expression in HCEC, however caspase-9 activity was significantly increased. Hypoxia significantly suppressed the activity of caspase-8. FLIP and Fas gene and protein expression were not significantly altered in hypoxic cells compared to normoxic controls. mRNA and protein levels of TNFalpha and TNFR-1 were significantly decreased, while FasL mRNA and proteins levels were significantly increased in hypoxic HCEC. In corneal epithelium stressed by hypoxia caspase-9 activity is upregulated, suggesting that apoptosis proceeds via the mitochondrial pathway. Caspase-8 activity may be suppressed because the loss of TNFalpha and TNFR-1 gene and protein expression inhibits the initial formation of a death signaling complex.

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.

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.

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.

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.