Analysis of expression patterns of protein phosphatase-1 and phosphatase-2A in rat and bovine lenses.

PURPOSE: The reversible phosphorylation and dephosphorylation at the serine and threonine residues on proteins play distinct roles in regulating multiple cellular activities. Whereas the protein serine-threonine kinases have been well studied in the lens system, very little is known about the expression and function of the serine-threonine phosphatases. The present article reports the expression patterns of protein phosphatase (PP)-1 and -2A in adult rat and bovine lenses. METHODS: Total RNAs and proteins were extracted from the epithelial and fiber cells of rat and bovine lenses. RT-PCR and Northern blot analysis were used to detect the mRNA expression levels in the epithelial cells and different fractions of fiber cells of these two types of lenses. Western blot was used to examine the protein expression levels in these different samples. An enzymatic assay was used to detect the activity distribution of PP-1 and -2A in these samples. RESULTS: The mRNAs for the PP-1 catalytic subunit (PP-1cs) and PP-2A catalytic subunit (PP-2Acs) were expressed in both epithelial and fiber cells of rat and bovine lenses. A detailed examination of the expression patterns of the two mRNAs in different fractions of fiber cells revealed that the cortical fiber cells (F1) contain the highest level of PP-1cs and -2Acs mRNAs (similar to those in the epithelial cells) among different fractions of fiber cells. The levels of the two mRNAs were sequentially decreased in the next layers of fiber cells (F2 and F3) and became barely detectable in the inner layers of fiber cells (F4 and N). In contrast to the mRNA expression patterns, the PP-1cs protein was mainly found in the epithelial cells. Among different layers of fiber cells, only cortical (F1) fiber cells contained detectable level of PP-1cs protein (bovine lenses contained a relatively higher level of PP-1cs than rat lenses in this region). In the remaining fiber cells, the PP-1cs protein was hardly detectable in rat lenses and slightly detectable in bovine lenses. The PP-2Acs protein was detectable only in the lens epithelial cells. Enzymatic assays revealed that the distribution patterns of PP-1 and -2A activities were similar to those of PP-1cs and -2Acs proteins. Furthermore, PP-1 activity was approximately four to five times higher than PP-2A activity in the lens epithelial cells. CONCLUSIONS: This study demonstrates that active PP-1 and -2A are mainly distributed in the lens epithelial cells, with PP-1 as a major phosphatase. The mRNAs and proteins for PP-1cs and -2Acs are differentially expressed in the epithelial and fiber cells of rat and bovine lenses.

Caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.

Phosphorylation and dephosphorylation are important cellular events regulating major metabolic activities such as signal transduction, gene expression, cell cycle progression, and apoptosis. It is well documented that okadaic acid, a potent inhibitor of protein phosphatase-1 (PP-1) and -2A (PP-2A), can induce apoptosis in a variety of cell lines. Our recent studies have revealed that in the immortal rabbit lens epithelial cell line, N/N1003A, inhibition of PP-1, but not PP-2A, leads to rapid apoptosis of the lens epithelial cells. This induction of cell death is associated with up-regulated expression of a set of genes, including the tumor-suppressor gene, p53, and the proapoptotic gene, bax. In the present study, we demonstrate that inhibition of PP-1 by okadaic acid in the primary cultures of rat lens epithelial cells also leads to apoptotic death. Moreover, we show that the cysteine protease, caspase-3, is important in the execution of okadaic acid-induced apoptosis. Treatment of the primary cultures of rat lens epithelial cells with 100 nM okadaic acid up-regulates expression of caspase-3 at the mRNA, protein, and enzyme activity levels. Inhibition of the caspase-3 activity with a chemically synthesized inhibitor prevents okadaic acid-induced apoptosis in rat lens epithelial cells. Similar results are also observed in the immortal cell line N/N1003A. Furthermore, stable expression of the mouse gene encoding lens alphaB crystallin inhibits okadaic acid-induced apoptosis, and this inhibition is associated with repression of the okadaic acid-induced up-regulation of caspase-3 activity. Taken together, these results demonstrate that caspase-3 is actively involved in okadaic acid-induced lens epithelial cell apoptosis.

Differential vulnerability of primary cultured cholinergic neurons to nitric oxide excess.

Many neuronal nitric oxide synthase (nNOS)-expressing brain neurons, including some cholinergic populations, are resistant to disease or to certain forms of excitotoxicity. Vulnerability to NO excess of forebrain (medial septal/diagonal band; MS-ACh) and brainstem (pedunculopontine/laterodorsal tegmental nuclei; BS-ACh) cholinergic neurons was compared in E16-E18 primary rat brain cultures. MS-ACh cells were approximately 300-fold more sensitive to the NO donor S-nitro-N-acetyl-D,L-penicillamine (SNAP) than were BS-ACh cells. Most (69%) MS-ACh cells contained nuclear DNA fragments by 2 h after addition of SNAP, while only 21% BS-ACh cells were TUNEL-positive after NO excess. Depletion of glutathione content did not potentiate the effect of SNAP on MS-ACh cells, but sensitized BS-ACh cells to the NO donor. Caffeic acid, a putative NF-kappa B inhibitor, enhanced the toxicity of SNAP to cholinergic neurons in both preparations. Our experiments show that cholinergic neurons in mixed primary cultures from different brain regions possess biochemical differences with respect to their vulnerability to NO excess.

Retinoic acid-mediated enhancement of the cholinergic/neuronal nitric oxide synthase phenotype of the medial septal SN56 clone: establishment of a nitric oxide-sensitive proapoptotic state.

It is unclear what mechanisms lead to the degeneration of basal forebrain cholinergic neurons in Alzheimer's or other human brain diseases. Some brain cholinergic neurons express neuronal nitric oxide (NO) synthase (nNOS), which produces a free radical that has been implicated in some forms of neurodegeneration. We investigated nNOS expression and NO toxicity in SN56 cells, a clonal cholinergic model derived from the medial septum of the mouse basal forebrain. We show here that, in addition to expressing choline acetyltransferase (ChAT), SN56 cells express nNOS. Treatment of SN56 cells with retinoic acid (RA; 1 microM) for 48 h increased ChAT mRNA (+126%), protein (+88%), and activity (+215%) and increased nNOS mRNA (+98%), protein (+400%), and activity (+15%). After RA treatment, SN56 cells became vulnerable to NO excess generated with S-nitro-N-acetyl-DL-penicillamine (SNAP) and exhibited increased nuclear DNA fragmentation that was blocked with a caspase-3 inhibitor. Treatment with dexamethasone, which largely blocked the RA-mediated increase in nNOS expression, or inhibition of nNOS activity with methylthiocitrulline strongly potentiated the apoptotic response to SNAP in RA-treated SN56 cells. Caspase-3 activity was reduced when SNAP was incubated with cells or cell lysates, suggesting that NO can directly inhibit the protease. Thus, whereas RA treatment converts SN56 cells to a proapoptotic state sensitive to NO excess, endogenously produced NO appears to be anti-apoptotic, possibly by tonically inhibiting caspase-3.

Okadaic acid-induced lens epithelial cell apoptosis requires inhibition of phosphatase-1 and is associated with induction of gene expression including p53 and bax.

It is well established that phosphorylation and dephosphorylation are key cellular events which regulate important metabolic activities such as gene expression, cell cycle progression, and apoptosis. The polyether fatty acid, okadaic acid has been shown previously to activate apoptosis in a variety of cell lines. Although this marine sponge toxin is known to inhibit protein phosphatase (PP)-2A and PP-1, it is not certain in most cases whether inhibition of PP-1 or PP-2A is necessary to activate apoptosis. Furthermore, it is not clear how inhibition of these phosphatases leads to apoptosis. Here we present evidence that inhibition of PP-2A by okadaic acid does not activate apoptosis in the lens system. However, when PP-1 is inhibited by okadaic acid, rabbit lens epithelial cells undergo rapid apoptosis. Associated with this process is the several-fold up-regulation of the tumor suppressor gene p53 and the pro-apoptotic gene bax at both mRNA and protein levels. Analyses of the temporal pattern of expression of the two genes reveal that the up-regulation is maximized in a few hours after treatment with okadaic acid, when the majority of the treated cells become committed to apoptosis. A brief treatment of the cells with a protein synthesis inhibitor can abolish okadaic acid-induced up-regulation of both P53 and Bax proteins. Concomitant with this inhibition, okadaic acid-induced apoptosis is also temporarily blocked. These results suggest that okadaic acid-induced expression of p53, bax, and other genes are necessary for the activation of the apoptotic programs in lens systems.

Low concentrations of cis-linoleic acid induce cell damage in epithelial cells from bovine lenses.

As low as 5 micromol/l of cis-linoleic acid proves to be cytotoxic for bovine lens epithelial cells in culture. Albumin eliminates the linoleic acid cytotoxicity completely, presumably by binding the fatty acid. However, the damaging effect appears again when the molar ratio of linoleic acid to albumin exceeds 1:1. The assumption that the linoleic acid-caused cell damage would be mediated by peroxidation products could not be confirmed. The results obtained rather favor the idea that linoleic acid molecules themselves injure lens epithelial cells. Obviously, cell damage even occurs in the presence of albumin if one molecule of albumin binds more than one molecule of linoleic acid. Micromolar concentrations of linoleic acid produce reversible bleb formation as well as cell retraction within 30 min. In primary culture 10 micromol/l linoleic acid damage lens epithelial cells irreversibly within some hours. Trans-linoleic acid, linolenic acid and oleic acid are also harmful to lens cells but to a lesser degree, while saturated fatty acids are without any effect. Bleb formation as a leading early sign hints at the plasma membrane as the primary target for linoleic acid induced cytotoxicity.