©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Post-transcriptional Regulation of Na/Glucose Cotransporter (SGTL1) Gene Expression in LLC-PK Cells
INCREASED MESSAGE STABILITY AFTER CYCLIC AMP ELEVATION OR DIFFERENTIATION INDUCER TREATMENT (*)

(Received for publication, May 11, 1995; and in revised form, June 20, 1995)

Hua Peng Julia E. Lever (§)

From the Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have further investigated the molecular basis of increased differentiation-regulated expression of SGTL1, a Na/glucose cotransporter, in the renal epithelial cell line LLC-PK(1). Treatment of confluent monolayers either with the differentiation inducer hexamethylene bisacetamide (HMBA) or with cyclic AMP-elevating agents promoted increased levels of the SGLT1 mRNA, the immunodetectable 75-kDa cotransporter subunit, and the transport activity. Two molecular species of SGLT1 mRNA (2.2 and 3.9 kilobases (kb)) are transcribed from the same gene in LLC-PK(1) cells and differ only in the length of the 3`-untranslated region. The larger transcript is less stable (t = 2 h) than the smaller one (t = 10 h) in control, confluent monolayers. The 3.9-kb species was stabilized from degradation after either cyclic AMP elevation (t = 14 h) or HMBA addition (t = 8 h), with negligible effects on the stability of the 2.2-kb species (t = 11 h). Inhibition of translation by cycloheximide resulted in a 10-fold increase in the t of the 3.9-kb transcript and a 2-fold increase in that of the 2.2-kb species in control monolayers. Our results demonstrate that post-transcriptional regulation of message stability plays a major role in differentiation-dependent SGTL1 expression promoted by either HMBA or cyclic AMP.


INTRODUCTION

The SGTL1 gene family consists of a number of structurally related transporters (Hediger et al., 1987; Wright, 1993) which catalyze Na-coupled secondary active transport. SGTL1, a Na/glucose cotransporter, is restricted in expression to intestinal and renal proximal tubule apical membranes (Lever, 1992) where it plays an essential role in concentrative transepithelial transport of glucose. The inherited human disorder glucose/galactose malabsorption results from a single mutation in SGTL1 (Turk et al., 1991), which maps on human chromosome 22 (Hediger et al., 1989). Expression of this transporter is developmentally regulated in the LLC-PK(1) polarized epithelial cell line of renal proximal tubule origin (Mullin et al., 1980; Amsler and Cook, 1982). Whereas SGLT1 mRNA is undetectable in sparse, actively dividing cultures of LLC-PK(1) cells (Yet et al., 1994) the development of a confluent cell density is accompanied by tight junction formation, membrane polarization, and expression of SGTL1 together with other differentiated markers characteristic of renal proximal tubule (Mullin et al., 1980; Yoneyama and Lever, 1984). Levels of SGTL1 transport activity (Amsler and Cook, 1982, 1985; Peng and Lever, 1993), the 75-kDa transporter protein subunit (Wu and Lever, 1989; Peng and Lever, 1993), and SGLT1 mRNA (Yet et al., 1994) are significantly increased by independent and synergistic mechanisms after elevation of cAMP levels or treatment with the differentiation inducer hexamethylene bisacetamide (HMBA). (^1)An LLC-PK(1) cell mutant deficient in protein kinase A activity was also impaired in Na/glucose cotransport activity (Amsler et al., 1991). Shioda et al.(1994) have recently demonstrated that activation of protein kinase C results in the loss of SGLT1 mRNA.

Alternative cleavage and polyadenylation results in the accumulation of two SGTL1 transcripts in LLC-PK(1) cells (Ohta et al., 1990), a 3.9-kilobase (kb) form and a 2.2-kb form. In the present study, we demonstrate that the 3.9-kb SGTL1 transcript is relatively unstable in comparison with the 2.2-kb form. Induction of increased SGTL1 expression following exposure to either cyclic AMP-elevating agents or HMBA is accompanied by a pronounced stabilization of the 3.9-kb SGTL1 transcript with negligible effects on the 2.2-kb form. These results indicate that post-transcriptional mechanisms play an important role in regulating levels of SGTL1 following cell differentiation.


EXPERIMENTAL PROCEDURES

Materials

[alpha-P]UTP, 3000 Ci/mmol, was purchased from ICN (Costa Mesa, CA), [^14C]alpha-methylglucopyranoside (alpha-MGP) was from DuPont NEN, cycloheximide, IBMX, dibutyryl cyclic AMP, and actinomycin D were from Sigma, forskolin was from Calbiochem, and HMBA was from Aldrich. Fetal bovine serum was from Sterile Systems (Logan, UT). H-89 was purchased from Seikagaku America, Inc. Other chemicals were molecular biology grade.

Methods

Cell Culture

The porcine renal cell line LLC-PK(1) clone G8 was maintained in a 50:50 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (ICN/Flow Laboratories) supplemented with 10% fetal bovine serum, 5 mM glutamine, and 15 mM HEPES as described previously (Peng and Lever, 1993). Cells were plated at a density of 10^4 cells/cm^2 on plastic tissue culture dishes.

Transport Assay

Na-dependent glucose uptake activity was assayed using cell monolayers on 35-mm dishes as described previously (Peng and Lever, 1993). The nonmetabolizable glucose analog [^14C]alpha-MGP, 200 µM, 1 mCi/mmol, was used as substrate. Results are shown after subtraction of Na-independent uptake and normalized to protein content.

Northern Blot Analysis

LLC-PK(1) monolayers were washed twice with ice-cold phosphate-buffered saline, and total RNA was extracted by the guanidine isothiocyanate procedure (Davis et al., 1986). Due to the low abundance of SGTL1 transcripts, isolation of poly(A) RNA was required for detection by Northern blot analysis. Poly(A) RNA was purified using oligo(dT)-cellulose columns (Davis et al., 1986) and then denatured and fractionated on a 1% agarose gel containing formaldehyde (Maniatis et al., 1989). The RNA was then transferred to a Duralon-UV membrane (Stratagene, La Jolla, CA) and UV-cross-linked using a Stratalinker 1800 (Stratagene). A 324-bp cDNA encoding amino acid residues 538-645 of the LLC-PK(1) Na/glucose symporter (SGTL1) amino acid sequence (Ohta et al., 1990) inserted into the pCR1000 vector (InVitrogen, San Diego, CA) at the HindIII/EcoRI site was constructed as described in Yet et al.(1994). The plasmid was linearized with EcoRI and a P-labeled antisense RNA probe was prepared by in vitro transcription using T7 RNA polymerase (Stratagene). A 1.27-kb XbaI fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA from plasmid pUC-GAPDH13, kindly provided by Dr. Shyu (University of Texas, Houston), was subcloned into pBluescript II SK(-) at the XbaI site. The resulting plasmid was named pBS-GAPDH13 and contains 1260 nucleotides of the rat GAPDH with 91 nucleotides of plasmid. A 864-nucleotide antisense transcript was synthesized from the NsiI-linearized pBS-GAPDH13, using T3 RNA polymerase (Stratagene) as described above, and used as an internal standard for recovery and integrity of RNA samples.

Filters were prehybridized for 3 h and then hybridized overnight at 55 °C in hybridization solution containing 50% formamide, 5 SSPE (20 SSPE = 3 M NaCl, 0.2 M NaH(2)PO(4), 0.02 M EDTA, pH 7.4), 1 PE (1 PE = 50 mM Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, and 5 mM EDTA) and 150 µg/ml denatured salmon sperm DNA. Labeled probes were added to the hybridization solution to give a final concentration of 10^6 cpm/ml. Blots were given two 15-min washes at room temperature in 0.1 SSC, 0.1% SDS, then washed for 15 min at 55 °C and were given a final 30 min-wash at 65 °C. Filters were exposed at -80 °C to X-Omat AR films with double intensifying screens. The hybridization signals on the autoradiograms were quantitated by computer-assisted image processing analysis (Bio Image® System, Millipore). For quantitative experiments two different exposures were analyzed.

For determination of mRNA half-life, poly(A) RNA was prepared from monolayers at various times following addition of 5 µg/ml actinomycin D, and SGLT1 mRNA levels were determined by quantitation of Northern blots. Half-life was estimated by the equation: t = 0.693/k, where k = 2.303 slope of the plot of log mRNA versus time).

Western Blot Analysis

Monolayers were solubilized in lysis buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1% Triton X-100 and harvested with a rubber policeman. The lysate was centrifuged at 12,000 g for 2 min at 4 °C. Supernatants (50 µg of protein) were resolved by SDS-polyacrylamide gel electrophoresis (10% acrylamide). Proteins and prestained molecular weight markers (Bio-Rad) were electrophoretically transferred to nitrocellulose (Schleicher & Schuell) at room temperature using an ABN model SD 1000 electrotransfer unit. Blots were incubated in a blocking solution (PBS containing 5% nonfat milk and 0.02% Tween-20) for 3 h at room temperature and then incubated with polyclonal antibody against the pig renal Na/glucose symporter (Peng and Lever, 1993) at 1:2500 dilution in blocking solution for 3 h at room temperature followed by four 15-min washes with 250 ml of PBS containing 0.02% Tween 20 (PBS-T). Subsequently, blots were incubated in blocking solution with horseradish peroxidase-conjugated goat-anti-rabbit IgG (Zymed) as secondary antibody at 1:2,000 dilution for 3 h at room temperature followed by four washes with 250 ml of PBS-T and a final wash with PBS. Detection was carried out using an enhanced chemiluminescence detection kit (Amersham Corp.) with an exposure time to autoradiography film (Hyperfilm-ECL, Amersham Corp.) of 1-10 s, followed by quantitation using densitometry.


RESULTS

HMBA and IBMX Synergistically Increase Na/Glucose Cotransport Activity and Levels of Its Protein Subunit

Previous work from this laboratory has demonstrated that LLC-PK(1) cells treated with either HMBA, an inducer of differentiation, or the cyclic AMP phosphodiesterase inhibitor IBMX exhibited increased Na-coupled glucose transport activity, accompanied by a corresponding increase in levels of the 75-kDa Na/glucose cotransporter subunit (Peng and Lever, 1993) and SGLT1 mRNA (Yet et al., 1994).

Both SGTL1 and SGLT2 cotransporter isoforms are expressed in LLC-PK(1) cells (Kong et al., 1993; Mackenzie et al., 1994). The Na-dependent glucose transport properties of this cell line, notably sensitivity to competitive inhibition by galactose (Moran et al., 1988) and a 2:1 Na:glucose stoichiometry (Moran et al., 1982) are characteristic of properties expressed by the cloned SGTL1 isoform (Ikeda et al., 1989), but not the SGLT2 isoform (Mackenzie et al., 1994), and suggest that SGTL1 is functionally the dominant isoform expressed in these cultures.

Addition of HMBA to newly confluent cultures at 3 days after seeding at a density of 1 10^4 cells/cm^2 induced an increase in Na-coupled glucose transport activity, measured by Na-coupled alpha-MGP uptake, as a function of exposure time and concentration (Fig. 1, A and B). HMBA effects on transport exhibited a 4-day lag period, followed by an increase in transport activity for another 8 days (Fig. 1A). Addition of 1 mM IBMX to either subconfluent cultures (0.6-0.8 10^5 cells/cm^2) (Fig. 1C) or early confluent cultures (10^5 cells/cm^2) (Fig. 1D) for 12 days caused a 4-5-fold increase in symport activity after a 4-day lag period. If IBMX was added to post-confluent cultures (2.0-2.2 10^5 cells/cm^2), the lag period was shortened to 2 days, reaching a peak of 5-fold induction beginning at day 4 (Fig. 1E). This result revealed that cell density does not appreciably change the magnitude of induction (4 6-fold), but the lag period was shortened at higher cell density. By contrast, in the case of HMBA, optimal induction was observed when early confluent cells were used, and a reduced induction of symport activity was noted the higher the cell density at the time of inducer addition (not shown). Since the cell density at the time of inducer addition significantly influences the magnitude of induction and exhibits a different optimum for each inducer, in all subsequent experiments unless otherwise stated, we added 5 mM HMBA to newly confluent cultures at a density of 10^5 cells/cm^2 (3 days after initial seeding), and 1 mM IBMX to post-confluent cultures at a density of 2 10^5 cells/cm^2 (4-5 days after initial seeding).


Figure 1: Induction of Na/glucose symport activity by HMBA and IBMX. A, time course of induction by HMBA; B, HMBA concentration dependence. (), untreated control; (black square), HMBA (2 mM); (bullet), HMBA (5 mM). C-E, effect of cell density on symporter induction by IBMX. IBMX was added to medium at (C) 2 days (subconfluent), (D) 3 days (early confluent), and (E) 4 days (late confluent) after plating LLC-PK(1) cells at a density of 10^4 cells/cm^2. (), control; (bullet), IBMX-induced. F, synergistic interaction between HMBA and IBMX. Early confluent cells were treated for 8 days with 1 mM IBMX (I) or HMBA (H) (2 mM or 5 mM as indicated), separately or in combination, for comparison with untreated control cultures. All values are means ± S.E. of triplicate determinations averaged from two experiments.



HMBA and IBMX have synergistic effects on induction of symporter expression. Addition of IBMX in the presence of either a suboptimal level of HMBA (2 mM), which by itself does not influence symport activity, or optimal levels of HMBA (5 mM), resulted in a significantly greater induction of transport activity than produced by either agent alone (Fig. 1F).

Western blot analysis using a polyclonal antibody specific for the 75-kDa pig kidney Na/glucose cotransporter subunit confirmed the separate and synergistic effects of these inducers (Fig. 2). Results shown in Fig. 2A indicated that addition of either IBMX or HMBA to newly confluent cultures for 8 days induced a 7- and 8-fold increase in levels of the 75-kDa symporter subunit, respectively (lanes 2 and 3). The protein kinase A inhibitor H-89 (50 µM) (Chijiwa et al., 1990) inhibited spontaneous expression of the 75-kDa subunit in control cells (lane 4) and abolished IBMX-mediated induction (lane 6) but had only a moderate effect in the presence of HMBA (20%) (lane 5). Addition of cAMP-elevating agents, either forskolin or IBMX, to post-confluent cultures for 4 days resulted in an increased expression of the 75-kDa symporter subunit, by 2.5- and 7.2-fold, respectively (Fig. 2B, lanes 3 and 4). Combination of IBMX and forskolin did not produce an additional effect compared to that of IBMX alone (Fig. 2B, lane 5). Treatment of cells with IBMX (1 mM) in the presence of 2 mM HMBA (a suboptimal dose) induced a higher expression of the transporter subunit (Fig. 2B, lane 6) than observed with either inducer alone, showing a synergistic effect (compare with lanes 2 and 4).


Figure 2: Levels of the immunodetectable 75-kDa Na/glucose symporter subunit. A, effect of inducers and inhibitors. Cells were plated at 10^4 cell/cm^2, and treated for 8 days with the indicated additions at 3 days after seeding (early confluent). Lane 1, control; lane 2, 1 mM IBMX; lane 3, 5 mM HMBA; lane 4, H-89 (50 µM); lane 5, HMBA plus H-89; lane 6, IBMX plus H89. B, synergy between HMBA and IBMX on expression of the transporter protein. Cells were plated at 10^4 cells/cm^2 and treated for 4 days with the indicated additions at 4 days after seeding (late-confluent). Lane 1, control; lane 2, 2 mM HMBA; lane 3, forskolin (100 µM); lane 4, 1 mM IBMX; lane 5, IBMX plus forskolin; lane 6, IBMX plus 2 mM HMBA. Samples (50 µg/lane) of cell extracts were resolved on 12% SDS-polyacrylamide gel electrophoresis and subjected to Western blot analysis using a polyclonal antibody to the pig renal Na/glucose cotransporter. Densitometric values are shown below.



Effect of Inducers on Steady-state Levels of SGLT1 mRNA

Two SGTL1 transcripts, of 2.2 and 3.9 kb, respectively, are expressed in LLC-PK(1) cells as a result of alternative usage of two AATAAA polyadenylation sites (Ohta et al., 1990). The two transcripts contain an identical coding region and differ only in the length of 3`-untranslated region (Ohta et al., 1990). We have confirmed this point using Northern blot analysis by demonstrating that a 952-bp antisense RNA probe corresponding to nucleotide residues 2697-3649 of the 3`-untranslated region of LLC-PK(1)SGTL1 hybridized only with the 3.9-kb transcript (not shown).

Our earlier report demonstrating increased steady-state SGLT1 mRNA levels following inducer treatment utilized roller bottle-grown cells for mRNA isolation (Yet et al., 1994). Since the rate of differentiation is strongly influenced by growth conditions, we have reinvestigated several parameters of SGLT1 mRNA induction, using cells grown on dishes, for optimal comparison with results from transport assay and Western blot analysis also obtained using cell monolayers on dishes. A 324-bp antisense RNA probe specific for the SGTL1 isoform (Yet et al., 1994), which did not cross-hybridize with SGLT2, was used. Blots were stripped and rehybridized with a probe for GAPDH mRNA, as a control for recovery and integrity of the samples.

The steady-state levels of SGLT1 mRNAs increased as a function of exposure time to inducers (Fig. 3). HMBA stimulated a maximal induction (3-fold) peaking at day 8, with a 4-day lag period before increased message levels could be detected. Since GAPDH mRNA levels increased within the first 4 days (Fig. 3A, lower panel), it is possible that an increase in SGLT1 mRNA within this time period was masked by the normalization. In the case of HMBA, the peak in mRNA levels preceded the peak in transport activity by 4 days (compare Fig. 1A and 3A). In the case of IBMX, 1 mM, an induction of SGLT1 mRNA was observed after a 2-day lag and reached a peak (8-fold increase) at 4 days of exposure (Fig. 3B), coincident with the peak in transport activity of postconfluent cells (compare with Fig. 1E). Maximal induction was observed at 5 mM HMBA (Fig. 4A) and 1 mM IBMX (Fig. 4B), when measured at the peak induction periods of 8 and 4 days, respectively. The dose-response pattern observed for the increase in transport activity resembled that observed for the increase in message in the case of HMBA (Fig. 1A) and IBMX (not shown).


Figure 3: Time course of induction of SGLT1 mRNA. Cells were exposed to either (A) 5 mM HMBA or (B) 1 mM IBMX. Poly(A) RNA (1.5 µg) samples were subjected to Northern blot analysis using a P-labeled SGTL1-specific antisense RNA probe (upper panels) and reprobed with an antisense GAPDH RNA probe (lower panels). mRNA levels were quantitated by optical densitometry. Values for SGLT1 mRNA are normalized using GAPDH as internal standard and expressed as means ± S.E. of two independent experiments. Empty symbols, untreated controls; solid symbols, inducer-treated. (, bullet), 2.2-kb SGTL1 message; (, black square), 3.9-kb SGTL1 message; (, up triangle, filled), GAPDH mRNA.




Figure 4: Induction of SGLT1 mRNA as a function of inducer concentration. A, samples (3 µg) of poly(A) RNA from LLC-PK(1) cells treated for 8 days with the indicated concentrations of HMBA, or B, samples (1.5 µg) of poly(A) RNA from LLC-PK(1) cells treated for 4 days with indicated concentrations of IBMX, were analyzed by Northern blot using P-labeled SGTL1-specific and GAPDH antisense RNA probes.



To further examine whether the inducer-stimulated increase in steady-state SGLT1 mRNA levels is associated with elevation of cyclic AMP levels and activation of protein kinase A, cells were treated for 4 days with either the adenylyl cyclase activator forskolin, or the cyclic AMP analogs dibutyryl cyclic AMP or 8-bromo-cyclic AMP. All of these cyclic AMP-elevating agents resulted in increased steady-state SGLT1 mRNA levels (Fig. 5A). The protein kinase A inhibitor H-89 down-regulated SGLT1 mRNA in control cultures, and blocked the induction of SGLT1 mRNA by IBMX (Fig. 5B). These results strongly indicated that cyclic AMP elevation and resultant protein kinase A activation is required to induce SGLT1 mRNA levels.


Figure 5: Regulation of SGLT1 mRNA levels by cAMP and protein kinase A. Samples (1.5 µg) of poly(A) RNA from untreated control cells or cells treated for 4 days with various compounds as indicated were analyzed by Northern blot using a P-labeled SGTL1 antisense RNA probe. A, lane 1, untreated control; lane 2, 1 mM IBMX; lane 3, 100 µM forskolin; lane 4, 100 µM dibutyryl cAMP (dbcAMP); lane 5, 250 µM 8-bromo-cAMP (8Br-cAMP). B, the protein kinase A inhibitor H-89 down-regulates SGLT1 mRNA levels in LLC-PK(1) cells. Lane 1, untreated control; lane 2, 1 mM IBMX; lane 3, 1 mM IBMX plus 50 µM H-89; lane 4, 50 µM H-89.



mRNA Stability Studies

We next explored the possibility that increased mRNA stability contributes to the increased levels of SGTL1 message observed after inducer treatment. The half-lives of SGTL1 transcripts were measured in cultures treated with the transcription inhibitor actinomycin D (5 µg/ml) in the presence or absence of inducers. As shown in Fig. 6and 7, the 2.2-kb SGTL1 transcript is quite stable in control cultures, with a half-life of 10 h for the 4- and 8-day controls (Table 1, Fig. 6and Fig. 7). The 3.9-kb transcript is less stable (t = 2-3 h). Addition of either IBMX (Fig. 6) or HMBA (Fig. 7) significantly increased the half-life of the 3.9-kb SGTL1 species, by 3-fold in the case of HMBA and 8-fold in the case of IBMX. By contrast, the stability of the 2.2-kb SGTL1 transcript was much less responsive to inducer treatment, exhibiting only a slight increase in half-life. These results indicate that HMBA and IBMX stabilize the 3.9-kb SGTL1 transcript with no significant effect on the half-life of the 2.2-kb transcript.


Figure 6: Stabilization of the 3.9-kb SGLT1 transcript after IBMX treatment. Cells were treated with 1 mM IBMX for 4 days before addition of 5 µg/ml actinomycin D (Act D). At the indicated time intervals after addition of ActD, poly(A) RNA was isolated. SGLT1 mRNA levels were analyzed by Northern blot analysis and quantitated by densitometry. A, autoradiogram; B, half-life was determined from densitometric data. Values are means ± S.E. of two to three experiments. Empty symbols, control; solid symbols, IBMX-treated cells. (, bullet), 3.9-kb SGLT1 mRNA; (, black square), 2.2-kb SGLT1 mRNA.






Figure 7: HMBA differentially stabilizes the SGLT1 3.9-kb message. Cells were treated with 5 mM HMBA for 8 days before addition of 5 µg/ml actinomycin D (Act D) and SGLT1 mRNA half-life determination as described in the legend to Fig. 6. A, autoradiogram from a typical experiment; B, scanning densitometry. Values are means ± S.E. of two or three experiments. Empty symbols, control; solid symbols, HMBA-treated. (, bullet), 3.9-kb SGLT1 mRNA; (, black square), 2.2-kb mRNA.



Due to the low abundance of the SGTL1 message and the limited sensitivity of the nuclear run-on assay, a measurable transcriptional activity under both control and inducer-treated conditions was not detectable, although positive controls demonstrated an unchanged rate of GAPDH transcription after inducer treatment (data not shown). Thus we were unable to evaluate the possible contribution of transcriptional activation to changes in steady-state SGTL1 message levels.

Requirement for New Protein Synthesis

The turnover rate of mRNAs can be modified by either altered production of specific ribonucleases and RNA-binding proteins (Sachs, 1993) or altered interactions between these protein factors and mRNA transcripts. The rate of turnover of many mRNAs is closely associated with translation (Maquat et al., 1981; Graves et al., 1987). To determine whether regulation of the stability of SGLT1 mRNA requires the synthesis of new protein, the protein synthesis inhibitor cycloheximide, 50 µg/ml, was added to cells for 2 h before the addition of 5 µg/ml actinomycin D. Poly(A) RNA was isolated and the half-lives of SGTL1 transcripts were measured (Fig. 8). Cycloheximide treatment resulted in a 10-fold increase in the half-life of the 3.9-kb transcript in control cells, but only a 1.5-fold increase in that of the 2.2-kb form. This result suggested that the relative instability of the larger transcript in control cells is closely associated with translation and may possibly require the action of one or more labile, destabilizing protein factor(s). These may be specific ribonucleases or other types of instability determinants.


Figure 8: Effect of cycloheximide on the stability of SGLT1 mRNA. Cultures were treated with either A, HMBA, 5 mM, for 8 days or B, IBMX, 1 mM for 4 days. Cycloheximide (CHX), 50 µg/ml, was added to medium 2 h before addition of 5 µg/ml actinomycin D (Act D) and additions were present thereafter. Half-lives of the SGTL1 transcripts were determined as described in the legend to Fig. 6. Open symbols, control; solid symbols, inducer-treated. Dashed lines, with CHX; solid lines, without CHX. (, bullet), 3.9-kb SGLT1 mRNA; (, black square), 2.2-kb SGLT1 mRNA.



In the case of cultures pretreated with inducers for the optimal time period before addition of these inhibitors, a different response was observed. Inhibition of protein synthesis did not prevent the stabilization of the 3.9-kb SGTL1 transcript observed in either IBMX- or HMBA-treated cells, suggesting that preexisting proteins may mediate the stabilization. In fact, an additional 2-fold stabilization was observed after inhibition of protein synthesis, perhaps due to inhibition of the synthesis of destabilizing factors.


DISCUSSION

The considerable variation in stability of eukaryotic mRNA is an important aspect of the regulation of the expression of genetic information. Linkage of mRNA turnover rates to activation of signal transduction pathways provides a means to rapidly fine-tune rates of protein expression in response to changes in the cell environment.

By use of two alternate polyadenylation sites, transcription of the Na/glucose cotransporter gene SGTL1 in the renal kidney cell line LLC-PK(1) gives rise to two mRNA transcripts of 3.9 and 2.2 kb that differ in the length of their 3`-untranslated regions (Ohta et al., 1990). In the present study, we provide evidence that the stability of SGLT1 mRNA is differentially regulated by cAMP and the differentiation inducer HMBA. In untreated differentiated cells, the 2.2-kb transcript is quite stable, with a measured half-life of 10-12 h. By comparison, the 3.9-kb transcript is 5-6-fold less stable with a half-life of about 2 h. Both HMBA and IBMX significantly increase the half-life of the larger transcript (3-fold for HMBA and 9-fold for IBMX) but have only a slight effect on that of the smaller form. Our present results provide the first demonstration that these agents act, at least in part, at the post-transcriptional level to stabilize the larger SGTL1 transcript.

It is interesting to note that the magnitude of the cAMP-stimulated increase in the 3.9-kb mRNA half-life is comparable with the observed increase in the steady-state level of the 3.9-kb transcript and also the observed increase in the steady-state level of the 75-kDa transporter subunit (Table 2). There is increasing evidence that the presence of the 3`-untranslated region controls the efficiency of translation (reviewed in Jackson and Standart (1990)). Our observation that the increase in the 3.9-kb transcript alone (not the sum of the 2.2- plus 3.9-kb transcripts) correlates with the increase in the protein (Table 2) suggests the possibility that the 3.9-kb SGTL1 transcript may be preferentially translated. In the case of HMBA, the increase in the transporter subunit is about 2-fold higher than predicted from the levels of the 3.9-kb message, suggesting the possibility that translational or post-translational effects may also be involved in HMBA action in addition to effects on message stability. Although HMBA and cyclic AMP exert synergistic effects on the transport activity and the levels of the transporter subunit ( Fig. 1and Fig. 2), they clearly act on different targets in the cell. HMBA effects on transporter expression appear, at least in part, to involve effects on polyamines and protein kinase C (Peng and Lever, 1993) with only minor involvement of protein kinase A (Fig. 2A); the mechanism of action of HMBA remains poorly understood.



Our results, taken together with recent findings by Shioda et al.(1994) that protein kinase C activation destabilizes the SGTL1 message, suggest that two separate and opposing signaling pathways intersect to regulate post-transcriptional processing and turnover of the SGTL1 message (Fig. 9). Thus, activation of protein kinase A in confluent cultures would up-regulate SGTL1 expression by message stabilization whereas activation of protein kinase C would down-regulate expression by message destabilization. Protein kinase A activation may also explain in part the message stabilization by HMBA since cyclic AMP levels in LLC-PK(1) cells are also elevated after HMBA treatment (Lever, 1992). As discussed above, HMBA effects cannot be explained solely by changes in cyclic AMP levels.


Figure 9: Scheme summarizing the post-transcriptional regulation of SGLT1 mRNA in LLC-PK(1) cells. PKC and PKA, protein kinases C and A.



Both stabilizing and destabilizing effects of cAMP elevation on mRNA turnover have been reported in various systems (reviewed in Williams et al.(1993)). In examples of cell differentiation where the precise signaling pathways are not well understood, changes in mRNA stability accompany developmental transitions. For example, erythroleukemic differentiation is associated with stabilization of globin message and destabilization of non-globin messages (Volloch and Housman, 1981). Activation of protein kinase C has been associated with regulation of mRNA turnover in a number of different systems, including the stabilization of cytokine and lymphokine mRNA's accompanying cell differentiation (reviewed in Williams et al.(1993)). Phorbol 12-O-tetradecanoate 13-acetate activation of protein kinase C has been previously implicated in the down-regulation of Na/glucose cotransport activity in LLC-PK(1) cells (Amsler and Cook, 1982) and induced a rapid loss of SGLT1 mRNA due to message destabilization (Shioda et al., 1994). In LLC-PK(1) cells, protein kinase C, assayed either with histone (Dawson and Cook, 1987) or a synthetic peptide (Shioda et al., 1994) as substrate, is mostly in the particulate activated form in subconfluent, undifferentiated cells and in the inactive, cytosolic form in confluent, differentiated cultures. Our observations taken together with those of Shioda et al. (1994) suggest that, under various conditions which induce message destabilization and de-differentiation, the SGTL1 message has a half-life of 1.5-2 h, whereas under conditions which induce message stabilization (differentiation), the SGTL1 message has a half-life of 12 h. The differentiated cultures used by Shioda et al.(1994), 6 10^5 cells/cm^2, maintained at confluence for 10 days, are probably at a stage of differentiation comparable to our HMBA- or IBMX-treated cultures.

The possible effect of protein translation on the stability of SGLT1 mRNA was examined in the present study using the protein synthesis inhibitor cycloheximide. Cycloheximide treatment stabilized both transcripts in uninduced cells, resulting in a >10-fold increase in the half-life of the larger transcript and 1.5-2-fold increase in the 2.2-kb species. In both HMBA- and IBMX-treated cells, cycloheximide did not prevent the inducer-augmented stabilization of the 3.9-kb transcript, indicating that preexisting protein factor(s) may be involved. However, cycloheximide treatment caused an approximately 2-fold increase in half-lives of both transcripts in either HMBA- or IBMX-treated cells compared with inducer-treated cells in the absence of cycloheximide. Shioda et al.(1994) observed that inhibition of transcription abrogated the protein kinase C-induced SGTL1 destabilization, suggesting a requirement for the regulated expression of phorbol 12-O-tetradecanoate 13-acetate-induced genes that inhibit SGTL1 gene transcription and/or enhance SGLT1 mRNA degradation. Since the protein kinase C-mediated SGTL1 message destabilization observed by Shioda et al.(1994) involved effects on both the 2.2- and 3.9-kb transcripts, whereas our observed protein kinase A-mediated SGTL1 message stabilization involved only the 3.9-kb transcript, these kinases clearly influence SGTL1 message stability by different mechanisms and stability determinants.

Our results suggest that certain labile protein factor(s) may be required for degradation of both the 2.2- and 3.9-kb transcripts in control, differentiated cultures, and the process of decay is translation-associated. The labile factor(s) may be a free cytosolic endonuclase or a destabilizing mRNA binding protein which either directly binds to target mRNA to degrade the mRNA molecule or tags the mRNA molecule for nucleolytic attack. It has also been suggested that initial cleavages of some mRNAs may be triggered by a ribosome-bound nuclease, which would become active when the ribosome reaches a certain site in the coding region (Graves et al., 1987; Yen et al., 1988). Thus the requirement for translation could be explained by either the need for continuing protein synthesis to provide a labile factor, or to unmask a target site for binding of a destabilizing factor or ultimately to activate a ribosome-bound nuclease by unfolding of the mRNA during ribosome movement. Our results suggest that the sensitivity of the SGTL1 message to degradation can be modulated by a cAMP-mediated mechanism, most likely by protein kinase A-mediated protein phosphorylation. In a few instances, specific cis-acting sequences in either the 3`-untranslated region or other portions of the transcript have been shown responsible for hormonal or developmental changes in message stability (reviewed in Jackson(1993) and Williams et al.(1993)). The interaction of regulatory cytoplasmic proteins with these sequences and the modulation of this interaction by various kinase systems would provide a mechanism for linkage to cellular signaling mechanisms. Evidence for such a mechanism has been obtained in the case of GLUT1 mRNA, which encodes a facilitative glucose transporter with no amino acid sequence or functional similarity to SGTL1 (Stephens et al., 1992).

Since the 2.2- and 3.9-kb SGTL1 transcripts differ only in the length of the 3`-untranslated region, it is reasonable to propose that sequences in this region may regulate SGTL1 turnover. Degradation by specific endo- and exonucleases would be modified by altered interaction of cellular RNA binding proteins with these sequences in response to cAMP fluctuation.


FOOTNOTES

*
This work was supported by U.S. Public Health Service Grant DK 27400 (to J. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225. Tel.: 713-792-5600; Fax: 713-794-4150; jlever{at}utmmg.med.uth.tmc.edu.

(^1)
The abbreviations used are: HMBA, N,N`-hexamethylene bisacetamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IBMX, 3-isobutyl-1-methylxanthine; H-89, N-[1-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; alpha-MGP, alpha-D-methylglucopyranoside; kb, kilobase(s); PBS, phosphate-buffered saline.


ACKNOWLEDGEMENTS

We thank Dr. Ann-Bin Shyu for helpful comments and Beto Zuniga for preparation of the figures.


REFERENCES

  1. Amsler, K., and Cook, J. S. (1982) Am. J. Physiol. 242,C94-C101
  2. Amsler, K., and Cook, J. S. (1985) J. Cell. Physiol. 122,254-258 [Medline] [Order article via Infotrieve]
  3. Amsler, K., Ghatani, S., and Hemmings, B. A. (1991) Am. J. Physiol. 260,C1290-C1299
  4. Chijiwa, T., Mishima, A., Hagiwara, M., Sano, M., Hayashi, K., Inoue, T., Naito, K., Toshioka, T., and Hidaka, H. (1990) J. Biol. Chem. 265,5267-5272 [Abstract/Free Full Text]
  5. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology , Elsevier Science Publishing Co., New York
  6. Dawson, W. D., and Cook, J. S. (1987) J. Cell. Physiol. 132,104-110 [Medline] [Order article via Infotrieve]
  7. Graves, R. A., Pandey, N. B., Chodchoy, N., and Marzluff, W. F. (1987) Cell 48,615-626 [Medline] [Order article via Infotrieve]
  8. Hediger, M. A., Coady, M. J., Ikeda, T. S., and Wright, E. M. (1987) Nature 330,379-381 [CrossRef][Medline] [Order article via Infotrieve]
  9. Hediger, M. A., Budarf, M. L., Emanuel, B. S., Mohandas, T. K., and Wright, E. M. (1989) Genomics 4,297-300 [Medline] [Order article via Infotrieve]
  10. Ikeda, T. S., Hwang, H., Coady, M. J., Hirayama, B. A., Hediger, M. A., and Wright, E. M. (1989) J. Membr. Biol. 110,87-95 [Medline] [Order article via Infotrieve]
  11. Jackson, R. J. (1993) Cell 74,9-14 [Medline] [Order article via Infotrieve]
  12. Jackson, R. J., and Standart, N. (1990) Cell 62,15-24 [Medline] [Order article via Infotrieve]
  13. Kong, C.-T., Yet, S.-F., and Lever, J. E. (1993) J. Biol. Chem. 268,1509-1512 [Abstract/Free Full Text]
  14. Lever, J. E. (1992) in Membrane Transport in Biology (Shafer, J. A., Ussing, H. H., Kristensen, P., and Giebish, G. H., eds) Vol. 5, pp. 56-72, Springer-Verlag, Berlin
  15. Mackenzie, B., Panayotobva-Heiermann, M., Loo, D. D. F., Lever, J. E., and Wright, E. M. (1994) J. Biol. Chem. 269,22488-22491 [Abstract/Free Full Text]
  16. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  17. Maquat, L. E., Kinniburgh, A. J., Rachmeliwitz, E. A., and Ross, J. (1981) Cell 27,543-553 [Medline] [Order article via Infotrieve]
  18. Moran, A., Handler, J. S., and Turner, R. J. (1982) Am. J. Physiol. 243,C293-C298
  19. Moran, A., Davis, L. J., and Turner, R. J. (1988) J. Biol. Chem. 263,187-192 [Abstract/Free Full Text]
  20. Mullin, J. M., Weibel, J., Diamond, L., and Kleinzeller, A. (1980) J. Cell. Physiol. 104,375-389 [Medline] [Order article via Infotrieve]
  21. Ohta, T., Isselbacher, K. J., and Rhoads, D. B. (1990) Mol. Cell. Biol. 10,6491-6499 [Medline] [Order article via Infotrieve]
  22. Peng, H., and Lever, J. E. (1993) J. Cell. Physiol. 154,238-247 [Medline] [Order article via Infotrieve]
  23. Sachs, A. B. (1993) Cell 74,413-421 [Medline] [Order article via Infotrieve]
  24. Shioda, T., Ohta, T., Isselbacher, K. J., and Rhoads, D. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,11919-11923 [Abstract/Free Full Text]
  25. Stephens, J. M., Carter, B. Z., Pekala, P. H., and Malter, J. S. (1992) J. Biol. Chem. 267,8336-8341 [Abstract/Free Full Text]
  26. Turk, E., Zabel, B., Mundlos, S., Dyer, J., and Wright, E. M. (1991) Nature 350,354-356 [CrossRef][Medline] [Order article via Infotrieve]
  27. Volloch, V., and Housman, D. (1981) in Organization and Expression of Globin Genes (Stamatooyannopoulos, G., and Nienhuis, A. W., eds) pp. 251-257, Alan R. Liss, New York
  28. Williams, D. L., Sensel, M., McTigue, M., and Binder, R. (1993) in Control of Messenger RNA Stability (Belasco, J. G., and Brawerman, G., eds) pp. 161-197, Academic Press, New York
  29. Wright, E. M. (1993) Annu. Rev. Physiol. 55,575-589 [CrossRef][Medline] [Order article via Infotrieve]
  30. Wu, J.-S. R., and Lever, J. E. (1989) J. Cell. Biochem. 40,83-89 [Medline] [Order article via Infotrieve]
  31. Yen, T. J., Machlin, P. S., and Cleveland, D. W. (1988) Nature 334,580-585 [CrossRef][Medline] [Order article via Infotrieve]
  32. Yet, S.-F., Kong, C.-T., Peng, H., and Lever, J. E. (1994) J. Cell. Physiol. 158,506-512 [Medline] [Order article via Infotrieve]
  33. Yoneyama, Y., and Lever, J. E. (1984) J. Cell. Physiol. 121,64-73 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.