Three distinct stages of lens opacification in transgenic mice expressing the HIV-1 protease.

Scanning electron microscopy of the lenses from transgenic mice (TG(72)) containing the HIV-1 protease linked to the lens alphaA-crystallin promoter showed structural changes around postnatal day 16. Frank opacification of the lens was observed at day 24. To relate the biochemical and biophysical changes that occur during the process of cataract development, high-resolution two-dimensional gel electrophoresis (2D), quantitative image analysis and ion measurements were carried out on lenses from postnatal day 10 and on days 15-24. The phase separation temperature (Tc), a measure of molecular interactions between proteins, was also determined for normal and transgenic lenses. A comparison of the transgenic and normal lenses on day 10 revealed no significant differences in any of the measured parameters. However, starting around day 16 or the first stage of observed structural changes, the TG(72)crystallin profiles of the alphaA- alphaB-, betaA3-, betaA4-, betaB3 and one gamma-crystallin began to deviate from the normal. By postnatal day 20, a second stage was initiated with an influx of calcium and sodium ions that was accompanied by modifications of betaB1- and betaB2-crystallin. In the third and final stage of the cataract process, a large increase in the proteolysis of crystallins was accompanied by the appearance of the frank cataract on day 24. The Tc initially increased in all of the mouse lenses until just prior to eyelid opening. After that time, the Tc decreased in all lenses. Although the Tc continued to decrease in the normal lenses with age, for the homozygous transgenic mice it exhibited a dramatic increase that began on day 20. Thus, in the TG(72)transgenic mouse, cataract formation occurs in a three-stage process. Tc and other biophysical parameters previously measured appeared to be insensitive to the modifications that occur during stage 1. However, during the second stage of cataract formation, there was a correspondence between abnormal Tc and the abnormal interactions between cellular constituents apparently resulting from lens hydration, the loss of ion homeostasis and continued proteolysis. The last stage of cataract formation results in a total loss of lens transparency and leakage of lens proteins.

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.

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.

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.

Phosphorylation of small molecular weight polypeptides in the iris-ciliary complex, aqueous humor and vitreous humor.

The polypeptides of vitreous humor, aqueous humor and iris-ciliary complex cells of eyes were phosphorylated with [gamma-32P]ATP without exogenous protein kinase. Phosphorylated polypeptides were analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The phosphorylated polypeptides of rabbit vitreous humor showed many high molecular weight prominent bands, but no detectable phosphoproteins were found in the 12 kDa or lower range. Bovine vitreous humor has predominantly acidic polypeptides and some of them are below 20 kDa. Rabbit and bovine iris-ciliary complex and rabbit aqueous humor showed a prominent common 4 kDa phosphopolypeptide which could also be synthesized by cloned populations of cells from the bovine iris and the rabbit iris-ciliary body. It is possible that the 4 kDa phosphopolypeptide of the aqueous humor is synthesized by the iris-ciliary complex cells.

Studies on the possible role of vitreous humor on protein synthesis and morphology of organ cultured adult rabbit lens. II. Epithelial cells.

The protein synthetic activities of epithelial cells of lenses organ-cultured without adhered vitreous humor manifest significant increase compared to the epithelial cells of lenses incubated with attached vitreous humor. This effect is not due to trauma of vitreous removal, as the addition of freeze-dried vitreous humor to the culture medium of lenses without attached vitreous humor could inhibit protein synthesis. However, the protein synthesis inhibitor in the vitreous humor has no visible effect on the lens morphology. It was also found that the factor from vitreous humor has no effect on mRNA production or cell-free protein synthesis. Thus, it seems that the effect on protein synthesis must be mediated via some other pathway.