Introduction

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The sodium and potassium-adenosine triphosphatase (Na⁺, K⁺-ATPase, E.C. 3.6.1.3), present in the plasma membrane of all eukaryotic cells, hydrolyzes ATP and uses the free energy to drive the transport of potassium into the cell and sodium out of the cell, against their electrochemical gradients (Glynn, 1985; Skou et al., 1988). This key enzyme determines directly Na⁺ and K⁺ transmembrane gradients and indirectly intracellular Ca²⁺ concentration, cell volume, and electrical membrane potential (Rayson, 1993; Vasilets and Schwarz, 1993). The importance of the activity of Na⁺, K⁺-ATPase for the processes of cell growth and cell proliferation has also been documented (Marakhova et al., 1998; Gentile et al., 1996; Peng et al., 1996). Cardiotonic steroids such as ouabain or digoxin bind specifically to the  subunit of the Na⁺, K⁺-ATPase, and this interaction results in the inhibition of the enzyme activity and Na⁺ and K⁺ transport (Hoffman and Bigger, 1990). Although the main pharmacological effect of these compounds is the increase in the force of contraction of heart muscle, they were also shown to be involved with the modification of cell proliferation as well as apoptosis (Hoffman and Bigger, 1990; Peng et al., 1996). The molecular mechanism by which the inhibitor influences cellular signaling has not yet been elucidated.

It has been suggested that the binding site for cardiotonic steroids is actually a receptor for unidentified endogenous digitalis-like compounds (for review, see Blaustein, 1996; Lichtstein et al., 1992). Recently, several laboratories have identified steroidal digitalis-like compounds in animal tissues. An ouabain isomer was identified in human plasma (Hamlyn et al., 1991) and bovine hypothalamus (Tymiak et al., 1993); ouabain has been identified in bovine adrenal glands (Schneider et al., 1998); digoxin was shown to be present in human urine and mammalian adrenal (Goto et al., 1990; Shaikh et al., 1991); 19-norbufalin and its peptide derivative were identified in cataractous human lenses (Lichtstein et al., 1993), and a substance from human placenta was tentatively identified as 3,14,20,21-bufadienolide (Hilton et al., 1996). In addition, marinobufogenin-like immunoreactivity (Bagrov et al., 1995) and proscillaridin A-like immunoreactivity (Li et al., 1998) have been demonstrated in human plasma and bovine adrenal and hypothalamus, respectively.

Na⁺, K⁺-ATPase is present in the lens, and its activity is essential for the maintenance of lens integrity and transparency (Moseley et al., 1996; Neville et al., 1978; Samuelov and Lichtstein, 1997). We have recently demonstrated that the inhibition of Na⁺, K⁺-ATPase activity by cardenolides and bufodienolides such as ouabain and bufalin, as well as by the endogenous bufodienolide 19-norbufalin derivative, causes protein leakage and structural changes in the lens (Lichtstein et al., 1998). The important role of Na⁺, K⁺-ATPase and the presence of endogenous Na⁺, K⁺-ATPase inhibitors in the lens prompted us to study the effects of these compounds on gene expression in this tissue to investigate the molecular mechanisms that occur following Na⁺ pump inhibition. In this study, we have used the technique of mRNA differential display (Liang and Pardee, 1992) to detect genes whose expression is altered following treatment with exogenous (ouabain, bufalin) as well as by the endogenously present (19-norbufalin derivative) Na⁺, K⁺-ATPase inhibitors. This study demonstrates that, following exposure of the lens for 24 h to 10 µM these compounds, the mRNA of several isoforms of the 14-3-3 proteins, highly conserved, ubiquitous, multifunctional regulatory proteins (Aitken, 1995), are down-regulated.

	Materials and Methods

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Lens Culture. Lenses from Sprague-Dawley rats (75-100 g) were dissected from the globe in accordance with the guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Each lens was incubated separately in 2 ml of modified TC-199 medium with or without Na⁺, K⁺-ATPase inhibitors at 37°C in 5% CO₂ atmosphere according to published methods (Zigler and Hess, 1985). Stock solutions of ouabain, bufalin, and 19-norbufalin derivative were prepared in ethanol and diluted into the wells so that the final alcohol concentration did not exceed 1%. Digitalis-like compounds used in this study was a synthetic 19-norbufalin derivative. The synthesis procedure will be published elsewhere (J. Deutsch and D. L., in preparation). A concentration of 10 µM each of the compounds was chosen because past work had shown that at this concentration all the compounds had an effect, but they did not kill the lens cells at the 24-h time point.

RNA Extraction. Total RNA was isolated from five rat lenses for each of the experimental conditions using the Stratagene kit and protocol (Stratagene, La Jolla, CA). Each lens was cultured individually, but to ensure adequate mRNA for experimentation, five lenses were combined for each condition. All experiments were done at least two times. Lenses were placed in a 1.5-ml Eppendorf tube and thoroughly homogenized in a Dounce homogenizer in 0.5 ml of guanidinium isothiocyanate (solution D) containing -mercaptoethanol. Following the addition of 0.05 ml of 2 M sodium acetate (pH 4), the tubes were mixed by inversion, and 0.5 ml of phenol and 0.1 ml of chloroform:isoamyl alcohol (24:1) were added. After shaking vigorously for 10 s, samples were incubated on ice for 15 min. The samples were then centrifuged at 10,000g for 20 min at 4°C. The aqueous layer was transferred to a clean Eppendorf tube and mixed with an equal volume of isopropanol. Samples were incubated at 20°C for at least 1 h followed by centrifugation at 10,000g for 20 min at 4°C. The supernatant was decanted and the pellet gently resuspended in 0.15 ml solution D, followed by the addition of an equal amount of isopropanol. The samples were mixed and incubated at 20°C overnight. Pellets were obtained by centrifugation as before, and the supernatant was decanted. The pellets were washed with 75% ethanol, centrifuged, and dried using a speed-vac evaporator (Speed-Vac Concentrator, Savant Instruments Inc., Farmingdale, NY) for 5 min. Residual DNA was removed by treatment with DNase I. Pellets were resuspended in 25 µl of diethylpyrocarbonate-treated dH₂O followed by the addition of 50 mM Tris, pH 7.5, 10 mM MgCl₂, 20 U of RNase-free DNase I (Boehringer Mannheim, Indianapolis, IN), and 20 U of RNasin (Promega Corp., Madison, WI) in a total volume of 50 µl. Samples were incubated at 37°C for 30 min followed by the addition of 200 µl of phenol/chloroform (1:1), inversion, and centrifugation at 10,000g for 2 min at 4°C. The aqueous phase was transferred to a clean Eppendorf tube, and the RNA was precipitated by the addition of 15 µl of 3 M sodium acetate, pH 5.2, and 1 ml of 95% ethanol, incubation at 20°C for 2 h, and centrifugation at 10,000g for 10 min at 4°C. The ethanol was decanted, and the pellets were then washed twice with 70% ethanol and once with 95% ethanol. The pellets were then dried using a Speed-Vac Concentrator for 5 min, followed by resuspension in 20 µl of diethylpyrocarbonate-treated dH₂O. RNA concentration and purity was determined by measuring optical density at 260 and 280 nm using a Shimadzu spectrophotometer (Shimadzu Corp., Columbia, MD). RNA integrity was monitored by electrophoresing RNA samples on 1% agarose gels in 1× TAE (FMC Bioproducts, Rockland, ME). The 28S and 18S RNA bands were visualized on the ethidium bromide-stained gel by exposure to UV light.

Differential Display (DD). Changes in gene expression were determined by DD (Liang and Pardee, 1992) using the Hieroglyph mRNA profile kit (Genomyx, Foster City, CA) according to the manufacturer's specifications. First-strand synthesis was performed for each of the four experimental conditions using 0.2 µg of RNA. Each set of RNA samples was reverse transcribed with SuperScript II RT (Life Technologies, Inc., Gaithersburg, MD) in the presence of one of each of the 12 3'-anchored primers in a Perkin-Elmer GeneAmp 9600 thermal cycler (Perkin-Elmer Corp., Foster City, CA). Samples were then stored at 70°C or used in the DD-polymerase chain reaction (PCR).

Subcloning and Sequencing. The reamplified DD-PCR products were subcloned using the pCRII-TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Before sequencing, candidate clones were selected based on the presence of a subcloned insert identified by PCR analysis. The PCR reaction mixture consisted of 300 to 500 ng of template, 10 µM each dNTP, 0.2 µM each vector-specific primer (5'-GCCAGTGTGCTGGAATTCGC-3' and 5'-TGATGGATATCTGCAGAATTCGCC-3'), 1× VENT buffer, 1× BSA solution, and 2 U of Vent DNA Polymerase (New England Biolabs, Beverly, MA) in a total reaction volume of 50 µl. The thermal cycling program consisted of one cycle at 94°C for 2 min followed by 25 cycles at 94°C for 1 min, 50°C for 20 s, and 72°C for 2 min, and terminated with a final extension at 72°C for 10 min. The presence of insert was analyzed by 1% agarose gel electrophoresis in 1× TBE.

Reverse Transcription (RT)-PCR and Northern Blot Analysis. RT-PCR was carried out using the SuperScript One-Step RT-PCR System (Life Technologies, Inc.) and the Titan One-Tube RT-PCR System (Boehringer Mannheim) according to the manufacturer's protocol with a few modifications. Each reaction contained 100 ng of one of the following primer sets: rat 14-3-3  (5'-CAGCAGCAAATGAAGAATGCATAAGG-3' and 5'-GCTTGATACACTGAATG AGACTCC-3'), rat 14-3-3  (5'-TGGAGGGTCATCAGCAGCAT-3' and 5'-ATAGTCCCCTTTCATCTTCA-3'), rat 14-3-3 zeta (5'-CACAGCAAGCATACAAGAA-3' and 5'-AGAATGAGGCAGACAAAGGT-3'), rat 14-3-3  (5'-CTTAGCCAAACAAGCCTTCG-3' and 5'-ATCTGAATAGCTGTGCTGCC-3') in combination with rat  crystallin (5'-CTAGAGGAGAAAAGTAGAGTCTCAAAATGCC-3' and 5'-CGAAAGAGATGACTTCAGAGGACAAATGTC-3'), 1 × reaction mix, 1 µl of SuperScript II RT/Taq mix, or Titan master mix. RNA (1-5 µl, 100 ng/µl) and diethylpyrocarbonate-treated dH₂O up to a final volume of 50 µl per sample were added. In the SuperScript system, the thermal cycling program consisted of 1 cycle at 42°C for 30 min and 94°C for 2 min followed by 25 cycles at 94°C for 30 s, 57°C for 1 min, and 72°C for 1.5 min, and terminating with an extension at 72°C for 10 min. In the Titan system, the cycling program consisted of 1 cycle at 42°C for 30 min and 95°C for 5 min followed by 25 cycles at 95°C for 1 min, 50.7°C for 1 min, and 72°C for 1 min. Fifteen microliters of each sample was analyzed on a 2% or 1.5% agarose gel in 1× TBE. Bands were visualized on the ethidium bromide-stained gel upon exposure to UV light, and the gel was photographed and analyzed using a Kodak Digital Science camera and software. PCR products were purified by agarose gel electrophoresis followed by band excision and DNA purification utilizing the Jetsorb kit (Genomed Inc., Research Triangle Park, NC). Products were sequenced by fluorescent dye terminator cycle sequencing to confirm their identity. For the Northern blot analysis, the  isomer DNA fragment from the DD was radiolabeled with [-³²P]dCTP according to the Random Primers DNA Labeling System (Life Technologies, Inc.) and purified with Microspin G-50 Columns (Pharmacia Biotech Inc., Piscataway, NJ).

	Results

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To identify genes whose expressions were altered in response to Na⁺, K⁺-ATPase inhibitors in rat lens, we compared the repertoires of genes expressed by control lenses to those treated with ouabain, bufalin, and 19-norbufalin derivative. This was achieved utilizing a differential display technique (Liang and Pardee, 1992). Rat lenses were incubated in the presence of 10 µM ouabain, bufalin, or 19-norbufalin derivative. After 24 h, total RNA was extracted and subjected to differential display using twelve 3' anchor primers in combination with four 5' arbitrary primers. Thirty-three percent of the resulting cDNA products have been analyzed, revealing 10 genes that appeared either up- or down-regulated by the Na⁺ pump inhibitors. An example of the separation is shown in Fig. 1. The enlargement demonstrates three cDNA fragments that were modified by the treatment. Fragments A and B were up-regulated, whereas fragment C was down-regulated by the three inhibitors.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Autoradiograph of a polyacrylamide gel showing differential gene expression in rat lenses exposed to Na+, K+-ATPase inhibitors. mRNA differential display reactions were performed as described in Materials and Methods. 33P-Labeled PCR products were visualized by autoradiography. The cDNA fragments obtained from mRNA extracted from control and ouabain, bufalin, and 19-norbufalin derivative-treated lenses are demonstrated.

To identify the gene denoted as the differential display product C, the candidate DNA fragment was eluted from the gel, reamplified, subcloned, and sequenced. The DNA sequence obtained from product C was used to search GenBank for homologous sequences. A 98% identity was found between the sequences of the down-regulated gene (product C) and the known 14-3-3  isoform.

Northern blot analysis and RT-PCR confirmed the down-regulation of 14-3-3  isoform transcripts by Na⁺, K⁺-ATPase inhibitors. A Northern blot of control and treated lens samples is shown in Fig. 2. The results demonstrate down-regulation of the 14-3-3  mRNA transcripts by ouabain, bufalin, and 19-norbufalin derivative. The quantification of the results is based on the ethidium bromide stain of the 18S RNA from each sample. The mRNA for the  isoform in each of the treated groups was less than 20% of the control.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Northern blot and histogram of relative band intensities of 14-3-3  isoform expression in control and Na+, K+-ATPase inhibitor-treated lenses. A nylon membrane containing treated and control lens total RNA was hybridized with a 1.2-kb  32P-labeled probe. Ethidium bromide staining of 18S RNA was used to normalize for RNA loading. Quantitation was by Eagle Eye II (Stratagene) and by Kodak Biomax ID (Image Analysis Software).

RT-PCR of RNA extracted from control and treated lenses, using 14-3-3  and -crystallin-specific primers, also demonstrated (Fig. 3) that the three Na⁺, K⁺-ATPase inhibitors induce down-regulation of the 14-3-3  isoform. The mRNA for -crystallin was chosen as a reference as this mRNA did not vary in the 24-h time period of this experiment. The quantification showed that the ouabain, bufalin, and 19-norbufalin derivative reduced 14-3-3 mRNA by more than 80%.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   RT-PCR products of the 14-3-3  isoform and -crystallin using RNA from control and ouabain-, bufalin-, and 19-norbufalin-treated lenses. The products were quantified (Kodak Biomax ID, Image Analysis Software) and normalized to -crystallin, which did not change under the different experimental conditions.

To examine whether other 14-3-3 isoforms are altered in the same way, RT-PCR analysis, using different 14-3-3 isoform primers, was conducted. The RT-PCR products were sequenced to verify their identity (data not shown) and quantified following gel electrophoresis. As shown in Fig. 4, in comparison to the -crystallin bands, 14-3-3  and [] isoforms were also down-regulated by Na⁺ pump inhibition, whereas the  isoform was down-regulated by bufalin and slightly by 19-norbufalin derivative but up-regulated by ouabain. Aside from the  isoform, the influence of the Na⁺, K⁺-ATPase inhibitors was considerably less dramatic than with the  isoform, with 14-3-3 mRNA levels being about 60 to 70% of the control.

View larger version (53K): [in this window] [in a new window]   Fig. 4.   RT-PCR products of 14-3-3 , , and  isoforms and -crystallin using RNA from control and ouabain, bufalin, and 19-norbufalin derivative-treated lenses. Table indicates ratio of net intensity of 14-3-3 mRNA levels divided by net intensity of -crystallin mRNA levels, quantified using Kodak Biomax ID (Image Analysis Software).

	Discussion

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Na⁺, K⁺-ATPase inhibitors have been shown to be endogenous constituents of mammalian tissues, and recent studies have demonstrated that these compounds are synthesized by the adrenal and released to the circulation in response to an increase in extracellular fluid volume (Laredo et al., 1994, 1995; Lichtstein et al., 1998b). The physiological role of these compounds has not been elucidated yet, although it is suggested that these compounds may be involved in the development of hypertension (Blaustein, 1996) and cataract (Lichtstein et al., 1993) in humans. The Na⁺, K⁺-ATPase inhibitors influence gene expression. In this study, the mRNA differential display method identified several isoforms of 14-3-3 proteins that are down-regulated by these compounds.

The 14-3-3 proteins are a family of acidic proteins named after their migration position on two-dimensional DEAE-cellulose chromatography and starch-gel electrophoresis (Aitken, 1995). The 14-3-3 proteins are highly conserved, ubiquitous, multifunctional regulatory proteins that have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several signaling proteins, such as protein kinase C, Raf-1 kinases, and phosphatidylinositol 3 kinase (Li et al., 1995; Robinson et al., 1994; Wheeler-Jones et al., 1996). These proteins have been shown to bind to the insulin receptor substrate-1 (Ogihara et al., 1997) as well as affect vesicular transport and Ras signaling in cells (Gelperin et al., 1995; Kometiani et al., 1998). They were shown to stimulate catecholamine secretion from adrenal chromaffin cells by reorganization of the cortical actin network (Roth and Burgoyne, 1995) and are thought to participate in the initiation of programmed cell death (Hsu et al., 1997). In view of this wide array of biological effects, it is evident that the down-regulation induced by the Na⁺, K⁺-ATPase inhibitors will have numerous implications in cell function. It is not known how the members of the 14-3-3 family interact with one another to bring about alterations in cellular signaling. It would seem from our results that the , , and  isoforms are influenced by the inhibition of the Na⁺ pump depending on the specific inhibitor. Interestingly, in contrast to all other effects observed, ouabain up-regulated the 14-3-3 zeta isoform level. The fact that different Na⁺, K⁺-ATPase inhibitors induce distinct gene expression is not surprising. Such a distinct pattern was shown in other systems. It has been demonstrated that bufalin, but not ouabain, induces apoptosis and influences the expression of apoptosis-related genes in human leukemia cells (Masuda et al., 1995). We have previously demonstrated that the inhibition of Na⁺, K⁺-ATPase by digitalis compounds causes numerous alterations in the lens structure and protein composition (Lichtstein et al., 1998a). These compounds cause not only swelling, which might be expected by inhibition of the pump, but also mitosis near the equatorial area and concomitant cell death in the anterior region of the lens. The relative abundance of the different isoforms of 14-3-3 may dictate the cellular response to the inhibitors and may initiate the cellular metabolic changes via the signal transduction pathways.

Alterations in gene expression as a result of pump inhibition were demonstrated to occur in other systems. It was shown, for example, that ouabain caused the induction of early response genes as well as the genes of skeletal -actin, atrial natriuretic peptide, and myosin light chain 2 in cardiac myocytes (Huang et al., 1997). Furthermore, it was shown recently that an increase in intracellular Na⁺ in A10 embryonic aortic smooth muscle cells causes up-regulation of the Na⁺, K⁺-ATPase RNA levels as well as other genes (Ruiz-Opazo et al., 1977). In the lens it was shown (Shinohara and Piatigorsky, 1977) that the intracellular ratio of Na⁺ and K⁺ in cultured embryonic chick lenses has an important role in the regulation of protein synthesis during cataractogenesis. Investigators from the same laboratory have also demonstrated that ouabain induced marked changes in synthesis, degradation, and leakage of protein in the rat lens (Piatigorsky et al., 1978), showing that ouabain treatment of the lens is a useful experimental system for studying cataract formation. Because the intensity of Na⁺, K⁺-ATPase inhibition is different depending on the inhibitor concentration and affinity, the consequent changes in intracellular Na⁺ and K⁺ could have discrete effects on second messenger pathways. At this point, the exact mechanism by which ouabain is able to influence gene expression is not clear. In view of the important roles for 14-3-3 in the regulation of intracellular signaling pathways, it is possible that the down-regulation by digitalis compounds, elucidated in this study, is a key event for the different effects of digitalis and the endogenous Na⁺, K⁺-ATPase inhibitors.