Normalization of hyperosmotic-induced inositol uptake by renal and endothelial cells is regulated by NF-kappa B

Mark A. Yorek1, Joyce A. Dunlap1, Wenli Liu2, and William L. Lowe Jr.2

1 Department of Internal Medicine, Diabetes-Endocrinology Research Center and Veterans Affairs Medical Center, University of Iowa, Iowa City, Iowa 52246; and 2 Department of Medicine, Veterans Affairs Lakeside Medical Center, and Northwestern University Medical School, Chicago, Illinois 60611


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hyperosmolarity is a stress factor that has been shown to cause an increase in the transcription of the Na+-dependent myo-inositol cotransporter (SMIT). However, regulation of the reversion of SMIT mRNA levels and transporter activity following removal of hyperosmotic stress is less understood. Previously we have shown that postinduction normalization of SMIT mRNA levels and myo-inositol accumulation following removal of hyperosmotic stress is inhibited by actinomycin D and cycloheximide, suggesting that normalization requires RNA transcription and protein synthesis. We now demonstrate that removal of hyperosmotic stress causes an activation of the transcription factor NF-kappa B in renal and endothelial cells. Inhibiting NF-kappa B activation with pyrrolidine dithiocarbamate (PD) blocks the normalization of SMIT mRNA levels and myo-inositol accumulation on removal of the cells from hyperosmotic medium. These studies demonstrate that the downregulation of the myo-inositol transporter following reversal of hyperosmotic induction is regulated via the activation of NF-kappa B.

myo-inositol; hyperosmolarity; nuclear factor kappa B; sodium-dependent myo-inositol cotransporter


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MYO-INOSITOL HAS AT LEAST two important functions in mammalian cells. First, it plays an integral role in signal transduction pathways by virtue of its incorporation into phosphoinositides and subsequent release as second messengers on activation of a phosphoinositide specific phospholipase C or phosphatidylinositol 3-kinase (3, 12, 23). Second, myo-inositol is an important osmolyte, serving to protect cells exposed to hyperosmotic stress (4). This protective role is shared with other osmolytes such as sorbitol, betaine, taurine, and glycerophosphorylcholine; however, these osmolytes may differ in their role as osmotic regulators because of their specific tissue localization and mechanisms responsible for their accumulation/metabolism (17, 26).

In most mammalian cells the intracellular concentration of myo-inositol is maintained at levels many times higher than circulating concentrations (7, 14). This gradient is regulated and maintained by a Na+/myo-inositol cotransporter (SMIT) that is widely expressed in mammalian cells and by an efflux mechanism, which is poorly understood (8, 13). In mammalian cells, hyperosmolarity is the most potent means to increase the activity of the SMIT and, thus, myo-inositol accumulation (21, 25, 27). The hyperosmolarity-induced increase in myo-inositol transport is dependent on increased transcription of the SMIT gene (27). Although osmotic regulation of the organic osmolytes is physiologically important to renal cells, we and others have shown that hyperosmolarity also regulates SMIT activity and mRNA levels in endothelial, neural, and glial cells (15, 18, 21, 24, 25).

It has been shown that, following removal of the hyperosmotic stimulus, the level of intracellular myo-inositol and myo-inositol uptake by mammalian cells rapidly returns to normal (15, 18, 24, 29). In previous studies, we demonstrated that the reversion of myo-inositol accumulation and SMIT mRNA levels, once the hyperosmotic stimulus had been removed, was inhibited by actinomycin D and cycloheximide, suggesting that normalization of SMIT activity and mRNA levels following hyperosmotic induction requires RNA transcription and protein synthesis (29). In the present study, we show that removal of the hyperosmotic stimulus causes an activation of nuclear factor kappa B (NF-kappa B) and that activation of this transcription factor is associated with the postinduction normalization of SMIT mRNA levels and myo-inositol uptake.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Chemicals were from Sigma (St. Louis, MO) unless otherwise noted. Ethanol, chloroform, isoamyl alcohol, Corning 75-cm2 flasks, and Falcon six-well plates were from Fisher Scientific (Fair Lawn, NJ). Phenol was from Bethesda Research Laboratories (Gaithersburg, MD). Ethidium bromide and proteinase K were from Boehringer Mannheim (Indianapolis, IN). SDS was from BDH (Poole, England). Pyridine, trimethylchlorosilane, and hexamethyldisilazane were from Pierce (Rockford, IL). Acrylamide, bis-acrylamide, dextran sulfate, and N,N,N',N'-tetramethylethylenediamine were from Bio-Rad (Hercules, CA). Transcription buffer, dithiothreitol, RNasin, ATP, CTP, UTP, GTP, T7 and SP6 RNA polymerase, and deoxyribonuclease were from Promega (Madison, WI). Myo-[2-3H]inositol, [alpha -32P]dATP, and [32P]UTP were from Amersham (Arlington Heights, IL). Safety-Solve, cesium chloride, and scintillation vials were from RPI (Mount Prospect, IL). cDNA probes for the beta -actin gene were obtained from Ambion (Austin, TX). Media were obtained from the Diabetes Endocrinology Research Center, University of Iowa (Iowa City, IA).

Cell culture. Bovine aortic endothelial (BAE) cells originated from freshly slaughtered steers and were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 294 µg/ml glutamine as previously described (8, 13). Rat inner medullary collecting duct (IMCD) cells were kindly provided by Dr. John Stokes, University of Iowa, and were grown in DMEM/F-12 medium supplemented with 5% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 294 µg/ml glutamine as previously described (25, 29). All cells were propagated in Corning 75-cm2 flasks in an incubator maintained at 37°C with 5% CO2 in humidified air. Cells were passed weekly at a dilution ranging from 1:10 to 1:20 and fed three times per week by replacing the medium. For myo-inositol accumulation studies, the cells were seeded onto Falcon six-well cluster plates, and assays were conducted in triplicate when the cells reached confluence. For the SMIT mRNA studies and electrophoretic mobility shift assays, the cells were seeded in Corning 25 or 75 cm2 flasks.

Myo-inositol accumulation. For these studies IMCD and BAE cells were exposed to medium containing 150 mM raffinose (490 mosM) for 24 h and then washed with isotonic medium (~300 mosM) and resuspended in isotonic medium or this medium containing 100 µM PD for 16 h. Cells were preincubated with PD for 1 h before resuspension in isotonic medium. Myo-inositol accumulation was also determined in cells maintained in isotonic medium or exposed to hyperosmotic medium for 24 h. Myo-inositol accumulation was determined as previously described (25, 29).

Quantification of SMIT mRNA levels. To quantify SMIT mRNA levels, IMCD and BAE cells were incubated in medium containing 150 mM raffinose for 24 h and then washed with isotonic medium and resuspended in isotonic medium or this medium containing 100 µM PD for 6 h. Cells were preincubated with PD for 1 h before resuspension in isotonic medium. SMIT mRNA levels were also determined in cells that were maintained in isotonic medium or hyperosmotic medium for the 24-h period. SMIT mRNA levels were quantified using a solution hybridization-RNase protection assay as previously described (25, 29). Briefly, for the IMCD cells, 32P-labeled antisense SMIT mRNAs were transcribed using SP6 RNA polymerase and a rat SMIT cDNA construct in pGEM-3Zf(+) that had been linearized with EcoR I (26). Due to the high degree of homology of the SMIT gene, the 32P-labeled antisense murine SMIT mRNA probe was found to hybridize with bovine RNA to give a protected band similar in size to the protected RNA band derived using RNA prepared from murine cells. Thus, to determine SMIT mRNA levels in BAE cells, 32P-labeled antisense SMIT mRNAs were transcribed using T7 RNA polymerase and a murine SMIT cDNA construct in pGEM-3Zf(+) that had been linearized with Hind III. Details of the cloning of the murine and rat SMIT cDNAs have been described previously (25, 26). Antisense SMIT mRNA was incubated at 45°C in 75% formamide-0.4 M NaCl with 20 µg of total RNA. After 16 h incubation, the samples were digested with RNases A and T1. The protected double-stranded hybrids were collected by ethanol precipitation and electrophoresed through an 8% polyacrylamide-8 M urea denaturing gel. To confirm equal loading of the gel, beta -actin mRNA levels were determined simultaneously with the use of commercially available beta -actin antisense control templates. The antisense beta -actin RNA probes were generated per the manufacturer's instructions using T7 polymerase. A sufficient quantity of each of the antisense SMIT mRNA and beta -actin probes was added to each sample to insure the presence of an excess of labeled antisense RNA (25). SMIT mRNA was represented as a single band on the autoradiogram of the gel, with the intensity of the band being proportional to the SMIT mRNA level in the sample. SMIT mRNA levels were quantified by scanning densitometry of the autoradiogram using a GS 300 transmittance/reflectance scanning densitometer (Hoefer, San Francisco, CA) interfaced with a model HP 3396A integrator and standardized to the intensity of the beta -actin mRNA band.

Electrophoretic mobility shift assay. IMCD or BAE cells were incubated in isotonic medium or hyperosmotic medium for 24 h followed by incubation for 1 h in isotonic medium in the absence or presence of 100 µM PD. Before this experiment, cells induced by hyperosmolarity were resuspended in isotonic medium for 15-180 min to determine the time course for activation of NF-kappa B by reversal of hyperosmotic induction. Following these incubations, cells were washed and harvested using PBS at 4°C and low-speed centrifugation. The cells were then resuspended in 1.5 ml of buffer A (10.0 mM HEPES, pH 8.0, 1.5 mM MgCl2, 10.0 mM KCl, 0.5 mM dithiothreitol, 300 mM sucrose, 0.1% Nonidet P-40, 1 µg/ml of pepstatin, antipain, chymostatin, and aprotinin, 0.1 µg/ml leupeptin, and 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 5 min. The crude nuclear pellet was then collected by microcentrifugation for 2 min at 4°C. Afterward, the pellet was quickly washed with buffer A and resuspended in buffer B (20 mM HEPES, pH 8.0, 20% glycerol, 100 mM KCl, 100 mM NaCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, 1 µg/ml of pepstatin, antipain, chymostatin, and aprotinin, and 0.1 µg/ml leupeptin). The isolated nuclei were sonicated for 10 s at 4°C and clarified by microcentrifugation. Protein concentration of the extract was determined, and the extract was stored at -70°C until assayed. For gel mobility shift assays, annealed oligonucleotides containing the consensus sequence for NF-kappa B (5'-TTTCGCGGGGACTTTCCCGCGC-3'; 5'-TTTGCGCGGGAAAGTCCCCGCG-3') and the E-box of the adenovirus major late transcription factor promoter (5'-ATAGGTGTAGGCCACGTGACCGGGTGT-3'; 5'-ACACCCGGTCACGTG-3') were radiolabeled with [alpha -32P]dATP and unlabeled dGTP, dCTP, and dTTP using Klenow DNA polymerase and gel purified as previously described (30). Fifteen micrograms of nuclear extract protein were preincubated for 10 min at 25°C with 1-µg poly(dIdC) · poly(dIdC) under ionic conditions. Radiolabeled probe (5 × 104 cpm, ~2 ng) was added to each 20-µl reaction and incubated for 15 min at 37°C. For a nonspecific control, a 50-fold excess of unlabeled oligonucleotide was included in some incubations. For supershift analysis, nuclear extracts from IMCD and BAE cells incubated in control medium, hyperosmolarity medium, or hyperosmolarity medium followed by incubation in isotonic medium (reversal) were preincubated for 15 min at room temperature with 1 µg of NF-kappa B anti-p50 or anti-p65 rabbit polyclonal antibody (Santa Cruz, CA). Afterward, radiolabeled oligonucleotide was added and examined as described above. Samples were analyzed on a 5% nondenaturing polyacrylamide gel in 0.5× tris(hydroxymethyl)aminomethane-borate-EDTA [45 mM tris(hydroxymethyl)aminomethane-borate, 1 mM EDTA, pH 8.0] and electrophoresed at 115 V for 3 h at 25°C. Gels were then dried, and autoradiographs were exposed for the appropriate period at -70°C with intensifying screens.

Data analysis. Data for myo-inositol accumulation are reported as nanomoles per milligram cell protein. Significance of differences was determined by ANOVA and Student's t-test. For analysis of SMIT mRNA levels, statistical comparisons for significance were performed using the one-tailed multiple-comparison procedure of Dunnett.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of NF-kappa B following postinduction normalization of hyperosmolarity. Previously we had shown that normalization of SMIT mRNA levels and increased accumulation of myo-inositol by mammalian cells exposed to hyperosmotic conditions were prevented by actinomycin D and cycloheximide (29). This suggests that turnover of SMIT mRNA levels and activity following hyperosmotic induction requires RNA transcription and protein synthesis. Moreover, we have also shown that tumor necrosis factor-alpha (TNF-alpha ) activates NF-kappa B and causes a decrease in SMIT mRNA levels and myo-inositol accumulation by cultured endothelial cells (30). The effect of TNF-alpha on SMIT mRNA levels and transport activity was prevented by various inhibitors of NF-kappa B activation such as pyrrolidine dithiocarbamate, genistein, and Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK) (30). To further examine the regulation of the normalization of SMIT mRNA levels and myo-inositol transporter activity following removal of hyperosmotic induction, we investigated whether the transcription factor NF-kappa B regulates the postinduction normalization of SMIT mRNA levels and activity. For the present studies, we chose to examine the effect of reversal of hyperosmotic induction on NF-kappa B activation and SMIT mRNA levels and transporter activity using a cultured renal cell line (rat IMCD cells) and BAE cells. Less extensive studies were also conducted with murine cerebral microvessel endothelial cells and murine cortical collecting duct cells, and results similar to those observed with IMCD and BAE cells were obtained.

Data in Fig. 1 demonstrate that exposing IMCD and BAE cells to hyperosmolarity for 24 h causes a small decrease in NF-kappa B activity compared with the basal activity of NF-kappa B observed in cells maintained in isotonic medium. Transferring cells from the hyperosmotic medium to isotonic medium causes a time-dependent activation of NF-kappa B compared with basal activity. Within 30-60 min after reversal of the hyperosmotic conditions, NF-kappa B is maximally activated. After 60 min, NF-kappa B activity begins to decline and approaches basal levels of activity after 180 min (data not shown). Data in Fig. 2 demonstrate that DNA protein binding for an unrelated probe containing the consensus sequence for the E-box of the adenovirus major late transcription factor promoter was not affected by hyperosmolarity or reversal of the hyperosmotic condition (30). Data in Fig. 3 show that a 50-fold excess of unlabeled NF-kappa B oligonucleotide competed for binding of the NF-kappa B-labeled probe in nuclear extracts prepared from IMCD and BAE cells incubated in control medium, hyperosmotic medium, or hyperosmotic medium followed by isotonic medium for 1 h. Data in Fig. 4 show that, in nuclear extracts prepared from IMCD and BAE cells incubated in control medium, hyperosmotic medium, or hyperosmotic medium followed by isotonic medium for 1 h, the NF-kappa B p65 antibody caused a gel retardation (supershift) in the binding complex (see arrow). In contrast, the NF-kappa B p50 antibody had no effect. This suggests that p65 is the major NF-kappa B isoform activated by hyperosmotic reversion. This was the same NF-kappa B isoform activated by TNF-alpha in cultured BAE cells (30). In a separate study we found that the activity of the transcription factor AP-1 was not affected by removing the cells from hyperosmolarity (data not shown).


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Fig. 1.   Effect of reversal of hyperosmolarity on the activation of NF-kappa B in inner medullary collecting duct (IMCD) and bovine aortic endothelial (BAE) cells. To induce hyperosmolarity, cells were incubated for 24 h in medium containing 150 mM raffinose. Afterward, the cells were resuspended in isotonic medium for 15-60 min. Control and hyperosmolarity-conditioned cells were incubated in isotonic medium or medium containing 150 mM raffinose, respectively, for the entire 24-h period. After these incubations, nuclear extracts were prepared, and gel mobility shift assays performed as described in MATERIALS AND METHODS. Representative autoradiograph from a single experiment that was repeated at least 4 times with similar results.



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Fig. 2.   Effect of reversal of hyperosmolarity on the electrophoretic mobility shift assay for the adenovirus major late transcription factor promoter (E-box) in IMCD and BAE cells. Cells were incubated as described in Fig. 1. Afterward, nuclear extracts were prepared and gel mobility shift assays performed as described in MATERIALS AND METHODS. Representative autoradiograph from a single experiment that was repeated at least 4 times with similar results.



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Fig. 3.   Competitive electrophoretic mobility shift assay of NF-kappa B. Cells were incubated as described in Fig. 1. The time point for the reversal period (isotonic) was selected as 1 h. Nuclear extracts prepared from these cells were incubated with or without a 50-fold excess of unlabeled NF-kappa B oligonucleotide. Afterward, the gel mobility shift assay was performed as described in the MATERIALS AND METHODS.



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Fig. 4.   Electrophoretic mobility supershift assay of NF-kappa B. Cells were incubated as described in Fig. 1. The time point for the reversal period (isotonic) was selected as 1 h. Nuclear extracts prepared from these cells were preincubated with or without 1 µg of the NF-kappa B antibody p50 or p65 for 15 min at room temperature. Afterward, radiolabeled NF-kappa B oligonucleotide probe was added to the incubation mixture and the gel mobility shift assay performed as described in MATERIALS AND METHODS. Arrow, the NF-kappa B p65 antibody caused a gel retardation (supershift) in the binding complex.

The activation of NF-kappa B following removal of IMCD and BAE cells from hyperosmotic medium to isotonic medium was completely prevented by PD (100 µM) and to a lesser extent by TLCK (50 µM; Fig. 5). In contrast, genistein (50 µM) and sulfasalazine (100 µM) were not effective in preventing the activation of NF-kappa B when hyperosmotic-conditioned cells were transferred to isotonic medium.


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Fig. 5.   Effect of reversal of hyperosmolarity in the absence or presence of genistein (Gen), pyrrolidine dithiocarbamate (PD), Nalpha -p-tosyl-L-lysine chloromethyl ketone (TLCK), or sulfasalazine (Sul) on the activation of NF-kappa B in IMCD and BAE cells. Cells were incubated in medium as described in Fig. 1, except that, for 1 h before the transfer of cells treated with hyperosmotic medium to isotonic medium, some cells were pretreated with 50 µM genistein, 100 µM PD, 50 µM TLCK, or 100 µM sulfasalazine. After this 1-h preincubation, cells pretreated with inhibitors were resuspended in isotonic medium containing the same concentration of each inhibitor and the incubation continued for 1 h. Other hyperosmolarity-conditioned cells were resuspended in isotonic medium alone for 1 h. Control and hyperosmolarity-conditioned cells were incubated in isotonic medium or medium containing 150 mM raffinose, respectively, for the entire 24-h period. After these incubations, nuclear extracts were prepared and gel mobility shift assays performed as described in MATERIALS AND METHODS. Representative autoradiograph from a single experiment that was repeated at least 4 times with similar results.

Effect of NF-kappa B activation on SMIT mRNA levels and myo-inositol transport activity following hyperosmotic postinduction normalization. Incubating IMCD and BAE cells for 24 h in medium containing 150 mM raffinose (hyperosmolarity) caused a significant increase in SMIT mRNA levels (Figs. 6 and 7). Returning IMCD and BAE cells that were exposed for 24 h to hyperosmotic medium to normal medium for 6 h (isotonic) resulted in normalization of SMIT mRNA levels. The normalization of SMIT mRNA levels following hyperosmotic induction and resuspension in isotonic medium was prevented in part by the addition of 100 µM PD to the isotonic medium. Exposing control IMCD or BAE cells for 6 h to isotonic medium containing 100 µM PD had no effect on SMIT mRNA levels. After 6 h incubation in isotonic medium containing 100 µM PD, SMIT mRNA levels in IMCD and BAE cells were 88 ± 17 and 117 ± 11% of control, respectively. For these studies we focused on the effects of PD, because studies presented in Fig. 5 demonstrated that it was most effective in preventing the activation of NF-kappa B when hyperosmotic-conditioned cells were transferred to isotonic medium. Levels of beta -actin mRNA were unchanged by these incubation conditions (Fig. 6).


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Fig. 6.   Effect of reversal of hyperosmolarity in the absence or presence of PD on Na+-dependent myo-inositol cotransporter (SMIT) mRNA levels in IMCD and BAE cells. Cells were incubated in medium as described in Fig. 1, except that, for 1 h before the transfer of hyperosmolarity-conditioned cells to isotonic medium, one set of cells were pretreated with 100 µM PD. After this 1-h preincubation, cells pretreated with PD were resuspended in isotonic medium containing 100 µM PD, and the incubation continued for another 6 h. Other hyperosmolarity-conditioned cells were resuspended in isotonic medium alone for 6 h. Control and hyperosmolarity-conditioned cells were incubated in isotonic medium or medium containing 150 mM raffinose, respectively, for the entire 30-h period. After these incubations, RNA was isolated and SMIT and beta -actin mRNA levels determined as described in MATERIALS AND METHODS. Representative autoradiograph from a single experiment that was repeated 6 times.



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Fig. 7.   Effect of reversal of hyperosmolarity in the absence or presence of PD on SMIT mRNA levels in IMCD and BAE cells. The experiment described in Fig. 6 was repeated 6 times and the data presented as a percentage of control with the level of SMIT mRNA in control cells assigned a value of 100%. SMIT mRNA levels were standardized using beta -actin mRNA. Each value is mean ± SE from 6 separate experiments. * P < 0.05, compared with control. + P < 0.05, compared with isotonic treated cells. Hyper, hypertonic; Iso, isotonic.

Consistent with the effect of hyperosmotic medium on SMIT mRNA levels, treatment of IMCD and BAE cells for 24 h with medium containing 150 mM raffinose (hyperosmolarity) caused a significant increase in myo-inositol accumulation (Table 1). Returning cells incubated in hyperosmotic medium for 24 h to isotonic medium for 24 h resulted in a normalization of myo-inositol accumulation that was prevented by the addition of 100 µM PD to the isotonic medium. Exposing control IMCD and BAE cells for 24 h to isotonic medium containing 100 µM PD had no effect on myo-inositol accumulation.

                              
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Table 1.   Effect of pyrrolidine dithiocarbamate on reversibility of hyperosmotic-induced myo-inositol accumulation by IMCD and BAE cells


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells react to increased osmolarity with numerous changes in gene expression. The specific genes affected differ between species, but the known osmoprotective effects of the gene products are remarkably similar, particularly with regard to cellular accumulation of compatible organic osmolytes (5). The transcription of the gene encoding the Na+-dependent myo-inositol cotransporter (SMIT) is increased by hyperosmolarity, and, following exposure to hyperosmotic conditions, many cells have been shown to increase myo-inositol accumulation, suggesting that myo-inositol is an important organic osmolyte (6, 10, 15, 18, 21, 24, 25, 27). After removal of the hyperosmotic stress, SMIT mRNA levels and, subsequently, cellular myo-inositol content and transport rapidly return to normal (15, 18, 24, 25, 29). However, the process regulating the normalization of SMIT mRNA levels and myo-inositol transport following removal of hyperosmotic stress in not well understood. Returning cells from a hyperosmotic medium to isotonic medium has been shown to activate a myo-inositol efflux pathway that is Na+ independent and inhibited by quinidine, quinine, anion transport blockers, and cis-unsaturated fatty acids (2, 11, 22). In our studies, we have examined the effect of postinduction normalization of hyperosmolarity on SMIT mRNA levels and myo-inositol transporter activity. Previously we have shown that postinduction normalization of SMIT mRNA levels and myo-inositol accumulation requires RNA and protein synthesis (29).

We have previously shown that the activation of NF-kappa B by the cytokine TNF-alpha may regulate the expression and transport activity of the SMIT (28, 30). In studies with cultured BAE cells and 3T3-L1 adipocytes, we demonstrated that TNF-alpha activates NF-kappa B and causes a decrease in SMIT mRNA levels and myo-inositol accumulation. Blocking the activation of NF-kappa B prevented the decrease in SMIT mRNA levels and myo-inositol accumulation mediated by TNF-alpha . Therefore, there is a precedent for mediation by NF-kappa B of SMIT mRNA levels and myo-inositol accumulation in cultured cells. This is further supported by the present studies that showed that removal of hyperosmotic stress was followed by a transient increase in NF-kappa B activity and a decrease in SMIT mRNA levels and myo-inositol accumulation by cultured renal and endothelial cells. However, one difference between the effect of TNF-alpha and reversal of hyperosmotic induction on NF-kappa B activity is that TNF-alpha causes a prolonged activation of NF-kappa B, whereas the activation of NF-kappa B by reversal of hyperosmolarity lasts for only hours. In these studies, we found that PD was most effective in preventing the activation of NF-kappa B following removal of hyperosmotic stress. In these studies, TLCK, a protease inhibitor, was also effective in blocking the activation of NF-kappa B following removal of hyperosmotic stress. The addition of PD also significantly inhibited the decrease in SMIT mRNA levels and myo-inositol accumulation following postinduction normalization. Other compounds that have been described to inhibit NF-kappa B activation, including genistein, a tyrosine kinase inhibitor, and sulfasalazine, were not effective in preventing the activation of NF-kappa B and subsequent decrease in SMIT mRNA levels and myo-inositol accumulation following postinduction normalization (data not shown). PD has been generally classified as an antioxidant (20). However, the mechanism for the inhibitory effect of PD on NF-kappa B activation has been reported to involve inhibition of binding of the transcription factor to DNA rather than an effect on the activation process (16). This mode of inhibition may explain why PD was the more potent inhibitor of NF-kappa B activation compared with the other compounds we tested. This result would also suggest that the activation of NF-kappa B by TNF-alpha and subsequent downregulation of SMIT mRNA levels and activity and the activation of NF-kappa B following removal of hyperosmolarity might be mediated by independent mechanisms.

NF-kappa B is primarily an activator of gene transcription. Therefore, the most likely explanation for the downregulation by NF-kappa B of SMIT mRNA levels and transporter activity is the regulation by NF-kappa B of the expression of proteins that regulate SMIT mRNA stability and/or protein turnover. Further studies are necessary to determine whether the 3'-untranslated region of the SMIT gene contains AU-enriched sequences that have been described to be recognition signals for trans-acting factors (regulatory proteins) that may affect the half-life of SMIT mRNA (1, 20). Another possible explanation for the decrease in myo-inositol uptake due to the reversal of hyperosmotic stress is the activation of either protein kinase A or protein kinase C. Preston et al. (19) in studies with MDCK cells demonstrated that activators of protein kinase A or protein kinase C reduce myo-inositol uptake in cells exposed to either isotonic or hypertonic conditions. However, there are conflicting reports regarding the possible role of protein kinase C in the posttranslational regulation of SMIT activity. Guzman and Crews (9) have reported that hyperglycemia increases myo-inositol transport in cultured mesangial cells by a mechanism mediated by the activation of protein kinase C. Furthermore, we have shown that phorbol myristate acetate, an activator of protein kinase C, did not affect myo-inositol uptake by 3T3-L1 adipocytes (28). It has not been reported whether hyperosmolarity or hypotonicity alters protein kinase A or protein kinase C activity. Thus it seems unlikely that increased protein kinase A or protein kinase C activity is responsible for mediating the effects of reversal of hyperosmolarity on SMIT mRNA levels and transporter activity. Nonetheless, at this time we can only state that the downregulation of SMIT mRNA levels and transporter activity by reversal of hyperosmotic induction parallels the activation of NF-kappa B and that inhibition of NF-kappa B activity prevents the decrease.

In summary, these studies demonstrate that postinduction normalization of hyperosmotic-induced SMIT mRNA levels and transporter activity is associated with the activation of NF-kappa B in cultured renal and endothelial cells.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45453, a Diabetes Center grant from the Medical Research Service of the Department of Veterans Affairs and Juvenile Diabetes Foundation, and a Merit Review grant from the Department of Veterans Affairs.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. A. Yorek, 3 E 17 Veterans Affairs Medical Center, Iowa City, IA 52246 (E-mail: myorek{at}icva.gov).

Received 5 August 1999; accepted in final form 6 December 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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