Inhibition of cell differentiation by Galpha q in the renal epithelial cell line LLC-PK1

Lihyun Sun1, Debora J. Weaver2, Kurt Amsler3, and Ellen R. Weiss1

1 Department of Cell Biology and Anatomy, The University of North Carolina at Chapel Hill, Chapel Hill 27599; 2 Department of Biological Sciences, Campbell University, Buies Creek, North Carolina 27506; and 3 Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

LLC-PK1, an epithelial cell line derived from the kidney proximal tubule, was used to study the ability of the G protein alpha -subunit, Galpha q, to regulate cell differentiation. A constitutively active mutant protein, alpha qQ209L, was expressed using the LacSwitch-inducible mammalian expression system. Induction of alpha qQ209L expression with isopropyl-beta -D-thiogalactopyranoside (IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold. Increasing concentrations of IPTG progressively inhibited the activity of two differentiation markers, Na+-dependent hexose transport and alkaline phosphatase activity. Induction of alpha qQ209L expression also caused a change from an epithelial to a spindle-shaped morphology. The effects of alpha qQ209L expression on cell differentiation were similar to those observed with 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. However, protein kinase C (PKC) levels were downregulated in TPA-treated cells but not in alpha qQ209L-expressing cells, suggesting that the regulation of PKC by Galpha q may be different from regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not inhibit the effect of IPTG on the development of Na+-dependent hexose transport in alpha qQ209L-expressing cells. These data implicate PKCdelta and PKCepsilon in the pathway used by Galpha q to block the development of Na+-dependent hexose transport in IPTG-treated cells.

phospholipase C; kidney; proximal tubule; protein kinase C; G protein

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE RENAL EPITHELIAL cell line LLC-PK1 has been used extensively as a model for investigating the regulation of kidney differentiation in the proximal tubule (2-4, 28, 32, 43). These cells display the characteristic morphology of a polarized renal epithelium on reaching confluence (8, 9). They progressively acquire several properties typical of the brush border of the kidney proximal tubule, such as expression of Na+-dependent hexose transport, gamma -glutamyltranspeptidase, and alkaline phosphatase activities (7, 20, 43). The development of Na+-dependent hexose transport activity is accelerated in response to compounds that raise cAMP levels and is significantly decreased in LLC-PK1 mutants deficient in cAMP-dependent protein kinase (3, 4). Phorbol esters, such as 12-O-tetradecanoylphorbol 13-acetate (TPA), which activate protein kinase C (PKC), can prevent or drastically delay the expression of several of these differentiation markers, including the Na+-dependent hexose transporter (3, 8, 37), gamma -glutamyltranspeptidase, and alkaline phosphatase (5, 7). The conversion from undifferentiated, actively growing cells to a culture expressing proximal tubule-specific traits and the ability to manipulate the expression of these differentiation markers make these cells particularly useful for studying the regulation of epithelial cell differentiation. The inhibitory influence of TPA on the expression of differentiation markers in this cell line has suggested the participation of PKC in their regulation. However, the effect of physiological activation of PKC through the stimulation of phospholipase C (PLC) has not been studied. Members of the Galpha q family of G proteins are known to mediate the activation of phosphoinositide (PI)-specific PLCbeta in response to stimulation of a variety of G protein-coupled receptors (31). PI-PLCbeta hydrolyzes phosphatidylinositol bisphosphate (PIP2), generating diacylglycerol (DAG), and inositol trisphosphate (IP3). Both of these metabolic products play critical roles in cell signaling; DAG is known to be a physiological activator of PKC, and IP3 stimulates the release of Ca2+ from intracellular stores (34). Therefore these pathways may be important in the regulation of cell differentiation.

The introduction of constitutively active mutants of G protein alpha -subunits into cells has been used by many laboratories to define the signaling pathways regulated by a particular G protein without the necessity of activating the appropriate G protein-coupled receptor (17). The mutations that generate constitutive activation are in the GTP binding domain of the alpha -subunit, resulting in loss of the ability to hydrolyze GTP. These mutants have been found to constitutively activate their downstream effectors. In fibroblasts, expression of constitutively active mutants of members of the Galpha q family has profound effects, inducing mitogenesis and transformation in NIH/3T3 cells (10, 11, 18) and inhibiting the proliferation of Swiss-3T3 cells (29). The present study represents the first investigation of the regulation of growth control and differentiation in epithelial cells by a member of the Galpha q family.

The GTPase-deficient constitutively active mutant protein alpha qQ209L (containing a mutation of Gln-209 to Leu) was stably expressed in LLC-PK1 cells using the LacSwitch-inducible system, which allows for the stable integration of this gene into cells without disturbing their normal pattern of growth and differentiation. The use of an inducible promoter circumvents the problem of lethality (29) or possible compensation in cells due to constitutive expression of GTPase-deficient mutants (22). In the present study, we show that induction of alpha qQ209L expression enhances PLC activity and results in inhibition of cell differentiation in LLC-PK1 cells. Although alpha qQ209L expression exerts effects on cell differentiation similar to treatment with TPA, total PKC activity is downregulated in TPA-treated cells but not in induced alpha qQ209L-expressing cells. Therefore the activation of the PLC pathway by its physiological regulator, Galpha q, may have effects that are distinct from those observed with TPA.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. The LLC-PK1 cell line was received from the American Type Culture Collection. This cell line is maintained in alpha -MEM supplemented with 10% fetal bovine serum in 5% CO2-95% air at 37°C. Medium is replenished every 3-4 days. When cells reach confluence, routine passage is carried out by washing cell monolayers in Ca2+-, Mg2+-free PBS (in mM: 137 NaCl, 2.7 KCl, 4.3 Na2HPO4, and 1.4 KH2PO4, pH 7.4), followed by trypsin digestion [0.25% trypsin and 1 mM EDTA in Hanks' balanced salt solution (HBSS)].

Stable transfection of LLC-PK1 cells. The LacSwitch-inducible mammalian expression system (Stratagene) was used. The Lac repressor vector (p3'SS) was transfected into the LLC-PK1 cells by calcium phosphate precipitation (16). Cells (106/10-cm dish) were transfected with 10 µg of the plasmid DNA. Stable clones were selected for resistance to 1 mg/ml hygromycin (LC Laboratories) and examined for the expression of the Lac repressor by indirect immunofluorescence using a polyclonal antibody to the Lac repressor (Stratagene). A clone that showed the highest expression of the Lac repressor based on the brightness of nuclear staining was selected. This clone was stably transfected with the pOPRSVI operator vector alone or pOPRSVI containing the cDNA for mutant alpha qQ209L (a gift from Dr. Gary L. Johnson, National Jewish Medical and Research Center, Denver, CO) using calcium phosphate precipitation. For the second transfection, 2 × 106 cells were transfected with 1 µg of the specific DNA and 10 µg of pSP72 carrier DNA per 10-cm dish. G418 (GIBCO BRL) at 1 mg/ml was added into the media to select for clones containing the lac operator vector.

Screening of clones by RT-PCR. To measure the expression of alpha qQ209L in the presence of isopropyl-beta -D-thiogalactopyranoside (IPTG, Stratagene), hygromycin- and G418-resistant clones were screened by RT-PCR. For each clone, 5 mM IPTG was added to a 10-cm dish of cells for 12 h. Total RNA was isolated from each clone. After reverse transcription with Moloney murine leukemia virus RT (Boehringer Mannheim), cDNA was amplified by PCR with Taq DNA polymerase (Promega) using a 31-base 5'-primer corresponding to nucleotides 739-769 in the alpha q cDNA and a 31-base 3'-primer corresponding to the 5'-end of the thymidine kinase poly(A) consensus sequence in pOPRSVI. The predicted size of the PCR product is 351 bp.

PLC assay. Cells were plated at a density of 2 × 105/35-mm dish in hygromycin- and G418-free media and allowed to grow for the indicated times in culture. One day before measurement, cells were washed with PBS and labeled with myo-[2-3H]inositol (Amersham) at 1 µCi/ml for 16-24 h in serum-free alpha -MEM containing 0.1% BSA at 37°C. After labeling, cells were washed twice with serum-free alpha -MEM-0.1% BSA and once with serum-free alpha -MEM-0.1% BSA containing 20 mM LiCl. Cells were then incubated for 30 min in alpha -MEM-0.1% BSA containing 20 mM LiCl to prevent degradation of inositol phosphates (IPs). Different concentrations of IPTG were added as described in the text. At the end of the incubation, cells were fixed in 1 ml of ice-cold acidified methanol (methanol-HCl, 100:1), scraped, and transferred into a tube containing 2 ml of chloroform, 1 ml of H2O, and 1 ml of acidified methanol. After mixing and centrifugation, the aqueous phase containing IPs was passed through an AG1-X8 (200-400 mesh, formate form, Bio-Rad) anion-exchange column. Total IPs were eluted with 0.1 M formic acid-1.0 M ammonium formate (6). Aliquots were assayed for radioactivity by liquid scintillation spectroscopy. Total [3H]inositol incorporation into lipid was determined by measuring the radioactivity in the organic phase. The IPs produced were calculated as a percentage of the total labeled lipids.

Na+-dependent hexose transport activity. To measure the activity of the Na+-dependent hexose transporter, uptake of the nonmetabolizable glucose analog, methyl-alpha -D-glucopyranoside (alpha -MeG) was performed as described by Amsler and Cook (3). Cells were plated at a density of 2 × 105/35-mm dish in hygromycin- and G418-free media. Different concentrations of IPTG were added as described in the figure legends. Where indicated, cells were also incubated with 2 µM GF-109203X (GFX), 50 µg/ml of oleyl-2-acetylglycerol (OAG), or 100 nM A-23187, a Ca2+ ionophore. At the desired time points, the medium was aspirated, and cells were incubated in HBSS without glucose (in mM: 137 NaCl, 5 KCl, 1.3 CaCl2, 0.4 MgSO4 · 7H2O, 0.5 MgCl2 · 6H2O, 0.3 Na2HPO4, 0.3 KH2PO4, and 20 mM HEPES, pH 7.2) at 37°C for 2 min. The uptake assay was initiated by adding 1 ml of HBSS containing 100 µM alpha -[14C]MeG (0.2 µCi/ml; alpha -[14C]MeG was from New England Nuclear; alpha -MeG was from Sigma), and dishes were incubated at 37°C for 60 min. Uptake was terminated by aspiration of the solution, and dishes were rinsed three times in ice-cold Tris-buffered saline [15 mM Tris · HCl (pH 7.4)-0.15 M NaCl]. Cell monolayers were solubilized in 0.2% SDS, and radioactivity was determined by liquid scintillation spectroscopy. Protein was measured using a modified Lowry assay (Bio-Rad) at 750 nm, with BSA as a standard.

Alkaline phosphatase activity. Alkaline phosphatase activity was measured using a modification of the method described by Amsler (2). Cells were plated at a density of 2 × 105/35-mm dish in hygromycin- and G418-free media. IPTG at different final concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following day and replaced every other day. At the desired time points, cells were rinsed three times with ice-cold HBSS and solubilized in the assay buffer (in mM: 150 NaCl, 5 KCl, 1 CaCl2, 1 MgSO4, and 10 Tris · HCl, pH 10) containing 1% Triton X-100. Approximately 200-300 µg of cell protein per sample were incubated in the assay buffer containing 10 mM p-nitrophenyl phosphate (Sigma) for 60 min at 37°C. The reaction was stopped by the addition of 0.2 N NaOH. Activity was determined by measurement of optical density at 405 nm using p-nitrophenol (Sigma) as a standard. Protein was determined from the same dishes using the Bradford assay.

Counting cells. Cells were plated at a density of 2 × 105/35-mm dish, and IPTG at a final concentration of 5 mM was added the following day. Cells were trypsinized on various days indicated in the legend to Fig. 2 and diluted into alpha -MEM. Trypan blue was added to a final concentration of 0.04%, and the number of viable cells was determined using a hemocytometer.

Microscopy. Cells were plated at the density of 5 × 104/35-mm dish, and IPTG at a final concentration of 5 mM was added the next day. Morphological changes induced by IPTG were viewed with a Zeiss IM35 phase-contrast light microscope containing a ×16 Zeiss lens (0.35 NA). Photographs were taken with an Olympus OM-2 camera.

PKC assay. Cells were plated at the density of 1.4 × 106/10-cm plate in hygromycin- and G418-free media. IPTG at different final concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following day (day 0) and replaced every 2 days throughout the experiments. The kinase assay was performed on day 7 using the PKC Assay System (GIBCO BRL) according to the manufacturer's protocols. Briefly, total cellular PKC was extracted and partially purified by DEAE-cellulose ion-exchange column chromatography. Kinase reactions were performed by measuring the phosphorylation of an acetylated synthetic peptide from myelin basic protein in the presence of 20 µM [gamma -32P]ATP (5 µCi/ml, Amersham) at 30°C for 5 min. TPA at a final concentration of 10 µM was included in the reaction to maximally activate PKC. The specificity of the reaction for PKC was confirmed using the PKC pseudosubstrate inhibitor peptide. Aliquots of the reaction mixtures were spotted onto phosphocellulose disks, washed twice in 1% phosphoric acid and twice in water. The radioactivity was determined by liquid scintillation spectroscopy. Proteins were precipitated with 10% TCA and dissolved in 0.5 M NaOH for quantification using the modified Lowry assay as described above.

Statistical analysis. Statistics were calculated using the computer programs Statview and SuperANOVA from Abacus Concepts.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression of constitutively active alpha qQ209L increases PLC activity. To investigate the effect of Galpha q expression on cell differentiation, clones stably expressing the GTPase-deficient mutant protein, alpha qQ209L, were established. Although the expression of mRNA for alpha qQ209L could be detected by RT-PCR in cells treated with IPTG, protein could not be detected in whole cell homogenates (data not shown). This may be due to the inability of the antibody to detect small increases in protein expression against a background of endogenous Galpha q in these cells. Because alpha qQ209L is constitutively active, high levels of expression may not be necessary to cause a significant increase in Galpha q activity. PLC activity was assayed to determine the induction of alpha qQ209L expression. A time course (Fig. 1A) demonstrated a maximal increase in IP accumulation after a 12-h incubation in the presence of IPTG in one of the clones, alpha qQ2. No IP accumulation was observed in cultures incubated in the absence of IPTG. In contrast, a clone transfected with the vector alone (v1) demonstrated no increase in IP accumulation in the presence or absence of IPTG. PLC activity measured after a 12-h incubation with IPTG was selected to screen for IPTG inducibility in all clones found to express alpha qQ209L mRNA by RT-PCR. Among different alpha qQ209L-expressing clones, functional inducibility by IPTG varied from no induction to 6- to 7.5-fold induction (data not shown). Two clones, alpha qQ1 and alpha qQ2, demonstrated approximately two- and sixfold increases in IP accumulation, respectively (Fig. 1B). These clones were selected for further study because, in the absence of IPTG, both of them demonstrated PLC activity comparable to that of the vector control clone (v1), suggesting that no expression of alpha qQ209L occurred in uninduced cells. In addition, they represent two different levels of alpha qQ209L activity.


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Fig. 1.   Phospholipase C (PLC) activity in pOPRSVI and pOPRSVI-alpha qQ209L transfected LLC-PK1 cells. A: time course for isopropyl-beta -D-thiogalactopyranoside (IPTG) induction. Cells of v1, a control clone transfected with vector pOPRSVI (black-square, square ), and alpha qQ2, a pOPRSVI-alpha qQ209L-transfected clone (black-triangle, triangle ), were plated at 2 × 105 cells/35-mm dish and allowed to grow for 2 days before being labeled overnight with myo-[2-3H]inositol as described in MATERIALS AND METHODS. IPTG at a final concentration of 5 mM was added to some of dishes for 0, 6, 12, and 24 h during labeling with myo-[2-3H]inositol. PLC activity assayed in absence (open symbols) or presence (filled symbols) of IPTG was measured at each time point as accumulation of total inositol phosphates (IPs) for 30 min as described in MATERIALS AND METHODS. IPs produced were calculated as a percentage of total labeled lipids. Data are expressed as means ± SD of triplicate measurements. B: PLC activity in v1 cells and alpha qQ209L-expressing alpha qQ1 and alpha qQ2 cells. Procedures for cell plating and labeling were the same as those described for A. Cells were uninduced (-IPTG, filled bars) or induced (+IPTG, hatched bars) with 5 mM IPTG for 12 h in presence of myo-[2-3H]inositol. IP formation over a 30-min incubation period was measured. Total IPs produced were calculated as a percentage of total labeled lipids. Bars represent means ± SD of triplicate measurements.

alpha qQ209L expression inhibits Na+-dependent hexose transport activity. Na+-dependent hexose transport occurs at the apical membrane of proximal tubule cells and is a biochemical marker for the differentiation of LLC-PK1 cells (8, 9, 21). To study the effect of alpha qQ209L expression on differentiation in this cell line, the development of Na+-dependent hexose transport activity in the absence or presence of IPTG was compared over a 14-day culture period (Fig. 2A). In the absence of IPTG, both v1 and the alpha qQ209L-expressing clone, alpha qQ2, acquired transport activity progressively over time. Incubation with IPTG blocked the development of this activity in alpha qQ2, but not in v1. Transport activity was also plotted as a function of protein amount, as an indicator of cell density (Fig. 2B). In clones v1 (in either absence or presence of IPTG) and alpha qQ2 (in the absence of IPTG), the ability to transport alpha -MeG was apparent when the cells reached 400-500 µg of protein/dish. In contrast, IPTG-treated alpha qQ2 cells exhibited almost no acquisition of transport activity even at cell densities far above those of untreated alpha qQ2 and v1 cells. Furthermore, the alpha qQ2 clone was shown to reach higher cell densities in the presence of IPTG compared with uninduced cells (Fig. 2C). Na+-dependent hexose transport activity was also examined in alpha qQ1, the Galpha q-expressing clone that showed lower inducibility of IP accumulation by IPTG (Fig. 2D). After a 7-day exposure to IPTG, Na+-dependent hexose transport activity was reduced to a lesser extent than observed in the alpha qQ2 clone. Because TPA has been shown to drastically delay the development of Na+-dependent hexose transport activity in wild-type LLC-PK1 cells (3), its effect was also examined in the alpha qQ1 cells. As shown in Fig. 2D, 0.1 µM TPA caused a greater inhibition of the development of transport activity in alpha qQ1 compared with IPTG induction, ruling out the possibility that transport activity is less sensitive to PKC activation in this clone. These results are consistent with the lower level of IP accumulation when this clone is treated with IPTG (Fig. 1B). The data suggest that alpha qQ209L expression inhibits the development of Na+-dependent hexose transport activity and that different levels of expression induced by IPTG, demonstrated by the differences in IP accumulation in the two Galpha q-expressing clones, result in different extents of inhibition of Na+-dependent hexose transport activity.


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Fig. 2.   Na+-dependent hexose transport activity in v1 and alpha qQ209L-expressing LLC-PK1 cells. A: acquisition of methyl-alpha -D-glucopyranoside (alpha -MeG) uptake capacity in v1 cells (black-square, square ) and alpha qQ209L-expressing alpha qQ2 cells (black-triangle, triangle ). Cells were plated at 2 × 105/35-mm dish 1 day before IPTG was added and maintained in absence (open symbols) or presence (filled symbols) of 5 mM IPTG from day 0 through day 13. Medium was replenished every other day. alpha -MeG uptake was measured at 37°C in HBSS containing 100 µM alpha -[14C]MeG (0.2 µCi/ml) for 60 min. Level of uptake was normalized to the amount of protein determined from the same dishes. Data are expressed as means ± SD of triplicate determinations in a single experiment and are representative of 2 independent experiments. B: analysis of alpha -MeG uptake capacity in v1 (black-square, square ) and alpha qQ2 (black-triangle, triangle ) cells as a function of protein amount/dish. Data from the time course shown in A were replotted as a function of protein level in cultures. C: comparison of number of cells per culture for uninduced alpha qQ2 cells (triangle ) and alpha qQ2 cells induced with IPTG (black-triangle). Cells were cultured as described in MATERIALS AND METHODS. IPTG was added on day 0. Cells were trypsinized on days 3, 7, and 13 and counted as described in MATERIALS AND METHODS. Error bars represent SD of 6 determinations from a single experiment. D: alpha -MeG uptake capacity in alpha qQ1 cells. Cells were plated as described above. On day 0, cells were either untreated (control) or treated with IPTG (5 mM; +IPTG) or 12-O-tetradecanoylphorbol 13-acetate (0.1 µM; +TPA). Medium containing these reagents was replenished every other day up to day 7 when alpha -MeG uptake was measured as described in MATERIALS AND METHODS. Bars represent means ± SD of triplicate measurements.

On the basis of the results shown in Fig. 2, we tested the hypothesis that varying IPTG concentrations would induce different levels of alpha qQ209L expression, resulting in modulation of the development of Na+-dependent hexose transport activity. As shown in Fig. 3A, there is a progressive increase in IP accumulation with increasing concentrations of IPTG. IP accumulation reached a maximum of approximately fivefold over basal activity at 2 mM IPTG. The half-maximal increase was at 0.03 mM IPTG. A corresponding decrease in Na+-dependent hexose transport activity to ~50% of maximum at an IPTG concentration of 0.01 mM was observed (Fig. 3B). At 0.05 mM IPTG, transport activity declined to ~13% of maximal levels and reached its lowest level at 2 mM IPTG. These results suggest that cell differentiation is modulated in an IPTG concentration-dependent manner, presumably by regulating the level of alpha qQ209L expression in a manner directly proportional to the induction of PLC activity.


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Fig. 3.   IPTG-dependent changes in PLC and Na+-dependent hexose transport activities in alpha qQ2. A: alpha qQ2 cells were plated at a density of 2 × 105/35-mm dish. IPTG was added the following day (day 0) at 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 mM and replaced every other day throughout experiment. Cells were labeled with myo-[2-3H]inositol on day 6, and PLC activity was measured on day 7 as described in MATERIALS AND METHODS. Total IPs accumulated over a 30-min period were calculated as a percentage of total labeled lipids. Symbols represent means ± SD of triplicate determinations. B: alpha qQ2 cells were plated at a density of 2 × 105/35-mm dish. IPTG at different final concentrations of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, and 5 mM was added the following day (day 0) and replaced every other day. alpha -MeG uptake was measured on day 7 over a 60-min incubation period at 37°C and normalized to protein amount determined from the same dishes, as described in MATERIALS AND METHODS. Symbols represent means ± SD of triplicate determinations.

Expression of alpha qQ209L inhibits alkaline phosphatase activity. To examine whether IPTG-dependent expression of alpha qQ209L can inhibit other differentiation markers, the activity of alkaline phosphatase in alpha qQ2-expressing cells was assayed. Alkaline phosphatase is another differentiation marker in LLC-PK1 cells that demonstrates increased expression at the apical surface over time in culture (20, 30, 43). As shown in Fig. 4, alkaline phosphatase activity was higher on day 7 than on day 1 in both the control cell line v1 and alpha qQ2 cells in the absence of IPTG. TPA treatment reduced acquisition of this differentiated function in both v1 and alpha qQ2 cells on day 7. Similarly, increasing concentrations of IPTG reduced alkaline phosphatase activity in alpha qQ2 cells. There was also a gradual decrease in alkaline phosphatase activity with increasing concentrations of IPTG. Therefore regulated expression of alpha qQ209L also inhibited alkaline phosphatase activity.


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Fig. 4.   Alkaline phosphatase activity in v1 and alpha qQ2 cells. Cells were plated at a density of 2 × 105 in 35-mm dishes and allowed to grow for 1 day. IPTG at different final concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following day (day 0). Alkaline phosphatase activities were measured as described in MATERIALS AND METHODS. On day 1 (filled bars), when cells were still actively growing and subconfluent, alkaline phosphatase activity was determined from cells treated without or with 5 mM IPTG or with 0.1 µM TPA. On day 7 (hatched bars), when cells reached confluence and were differentiated, alkaline phosphatase activity was determined in cells incubated in 0.005, 0.05, 0.5, and 5 mM IPTG. Amounts of p-nitrophenol produced from ~300 µg of protein in day 1 cultures and from 200 µg protein in day 7 cultures were measured for 60 min at 37°C. Results were normalized to amount of protein determined from the same dishes. Data are expressed as means ± SD of triplicate measurements and are representative of 2 independent experiments. Ability of IPTG to reduce the level of alkaline phosphatase activity in alpha qQ2 cells treated for 7 days was analyzed using a Dunnett ANOVA test. * Concentrations of IPTG caused a statistically significant reduction (P < 0.01) in the level of alkaline phosphatase activity compared with cells without IPTG.

Morphological changes in alpha qQ209L-expressing LLC-PK1 cells. The morphology of uninduced and induced alpha qQ2 cells was examined at the light-microscopic level (Fig. 5). Uninduced subconfluent cells exhibit the islandlike clusters of closely apposed cells typical of cultured epithelia, which are indistinguishable from the control v1 clone. In the presence of IPTG, these cells acquire a spindle-shaped morphology, extending long, spiny processes with ruffled edges, and appear more loosely associated. In larger islands, uninduced cells form a monolayer of rounded cells, whereas IPTG-treated cells are more disorganized and appear to overgrow each other in multicellular aggregates. Interestingly, Mullin et al. (27) observed that PKCalpha is expressed preferentially in LLC-PK1 cells in areas that are multilayered. Untreated confluent monolayers of these cells were able to form domes, whereas IPTG-treated cells did not. Similar morphological changes were observed in five different clones that demonstrated at least a twofold induction in IP accumulation by IPTG. These morphological changes could be observed within 12 h of IPTG incubation (data not shown).


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Fig. 5.   Morphological changes are induced by IPTG in subconfluent alpha qQ209L-expressing LLC-PK1 cells. The alpha qQ209L-expressing clone, alpha qQ2, was plated at a density of 5 × 104 cells/35-mm dish and 5 mM IPTG was added the following day. After treatment with (+IPTG) or without (-IPTG) IPTG for 1 day, the morphology of these cells at lower (L) and higher (H) densities was recorded as described in MATERIALS AND METHODS.

alpha qQ209L expression does not downregulate PKC activity. The activation of Galpha q stimulates PLC-mediated hydrolysis of PIP2, leading to the release of DAG and IP3. Because DAG stimulates PKC, the effect of IPTG-induced expression of alpha qQ209L on the levels of PKC was examined. Prolonged treatment with phorbol esters has been shown to downregulate PKC, a process attributed to the proteolytic degradation of the activated enzymes (39). We were therefore interested in the levels of total PKC activity in cells incubated with IPTG compared with cells chronically exposed to TPA. As illustrated in Fig. 6, chronic TPA treatment caused downregulation of PKC activity in both control (v1) and alpha qQ209L-expressing cells (alpha qQ2), as reported previously for LLC-PK1 cells (26). However, alpha qQ209L expression induced by IPTG did not downregulate PKC. The total PKC activity in alpha qQ2 cells treated with IPTG was comparable to that of untreated alpha qQ2 cells and of untreated and treated v1 cells.


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Fig. 6.   Protein kinase C (PKC) activity in v1 and alpha qQ2 cells. Cells were plated at a density of 1.4 × 106 in 10-cm plates. IPTG at different final concentrations (0.005, 0.05, 0.5, and 5 mM) or TPA (0.1 µM) was added the following day (day 0). PKC assays were performed on day 7 as described in MATERIALS AND METHODS. PKC activities were calculated as picomoles of phosphate per minute per milligram of protein determined from the same dishes and normalized as a percentage to the level of activity in v1 cells in absence of IPTG in the same experiment, which was regarded as 100%. Data represent average of 2 independent experiments performed in triplicate. Error bars represent means ± SE of 6 measurements.

Effect of PKC activators and inhibitors on Na+-dependent hexose transport activity. The activation of PLC by Galpha q results in the generation of DAG and IP3. Both DAG and IP3 can stimulate downstream signaling pathways; DAG activates members of the PKC family of kinases and IP3 induces the release of Ca2+ from intracellular stores (34). To determine the ability of these pathways acting separately or together to influence the development of Na+-dependent hexose transport, alpha qQ2 cells were incubated with the DAG analog OAG and the Ca2+ ionophore A-23187 (Fig. 7). After a 7-day incubation, cells treated with OAG showed ~50% decrease in the development of Na+-dependent hexose transport, whereas cells treated with A-23187 were not affected, suggesting that PKC activation, but not increased cytosolic Ca2+, is important in the regulation of Na+-dependent hexose transport. The addition of A-23187 together with OAG had no greater effect than OAG alone. The role of PKC was further investigated using the inhibitor GFX. At 2 µM, this inhibitor blocks the morphological changes observed in these cells (Sun and Weiss, unpublished observations). However, GFX had no effect on the ability of cells to develop Na+-dependent hexose transport activity in the presence or absence of IPTG in either v1 or alpha qQ2 cells (Fig. 8).


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Fig. 7.   Effect of oleyl-2-acetylglycerol (OAG) and the Ca2+ ionophore A-23187 on development of Na+-dependent hexose transport. alpha qQ2 cells were plated at a density of 2 × 105/35-mm dish on day 0 and incubated as described in MATERIALS AND METHODS. Cells were refed every day with medium containing 50 µg/ml OAG and every other day with 100 nM A-23187 and IPTG where indicated. Cultures were assayed on day 5 for Na+-dependent hexose transport activity as described in MATERIALS AND METHODS and in legend to Fig. 2. Error bars represent means ± SE of a single experiment representative of 2 independent experiments.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have described for the first time the inducible expression of a constitutively active mutant of Galpha q in a renal epithelial cell to investigate its participation in the regulation of proximal tubule-specific markers. Our results demonstrate that the expression of alpha qQ209L inhibits the acquisition of Na+-dependent hexose transport and alkaline phosphatase activities, both markers for the kidney proximal tubule. The inhibition of differentiation was regulated in a concentration-dependent manner by IPTG, suggesting that the level of active Galpha q directly affects the extent of inhibition. In addition, expression of constitutively active Galpha q induced a conversion from an epithelial to a spindle-shaped morphology. The cells became more disorganized and reached a higher cell density than those not treated with IPTG. The morphological changes observed in alpha qQ209L-expressing cells are similar to those reported previously on exposure to TPA (3, 26). OAG was also found to inhibit the development of Na+-dependent hexose transport activity, further implicating PKC in this regulatory pathway. Surprisingly, the PKC inhibitor GFX, which blocks the morphological changes observed in our cells and is reported to inhibit the TPA-induced disruption of tight junctions (23), had no effect on the inhibition of Na+-dependent hexose transport by IPTG. It has been shown that LLC-PK1 cells possess the PKC isoforms alpha , beta , delta , epsilon , and zeta  (5, 25). PKCalpha , PKCbeta , and PKCgamma are most sensitive to GFX, with IC50 values ranging from 0.01 to 0.02 µM. In contrast, the IC50 values are an order of magnitude higher (0.21 and 0.13 µM, respectively) for PKCdelta and PKCepsilon and 5.8 µM for PKCzeta (24, 36). Therefore GFX is unlikely to be an effective inhibitor of delta , epsilon , and zeta  in our experiments. Because PKCzeta is not regulated by DAG (12), PKCdelta and PKCepsilon are the most likely candidates for the regulation of Na+-dependent hexose transport activity by alpha qQ209L in LLC-PK1 cells.


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Fig. 8.   Development of Na+-dependent hexose transport activity in cultures treated with GF-109203X (GFX). v1 and alpha qQ2 cells were plated as described in legend to Fig. 2, and a time course was performed in presence or absence of 5 mM IPTG and 2 µM GFX. Cells were refed every other day. Na+-dependent hexose transport activity was assayed as described in MATERIALS AND METHODS. open circle , bullet , -IPTG; triangle , black-triangle, +IPTG; open symbols, -GFX; filled symbols, +GFX. Error bars represent means ± SE of a single experiment representative of at least 2 independent experiments.

The most significant difference between TPA treatment and expression of constitutively active alpha q was on the levels of cellular PKC. Chronic TPA treatment downregulated PKC levels in both v1 and alpha qQ2 cells, consistent with previous reports in both LLC-PK1 and other cell types (13, 26). In contrast, long-term treatment of alpha qQ2 cells with IPTG did not significantly alter PKC levels, suggesting that physiological activation of PLC by alpha qQ209L expression does not result in downregulation of PKC. It should be noted that our experiments do not measure the activity of PKC in TPA-treated or Galpha q-expressing cells. Designing an assay that preserves and measures actual PKC activity in a cell under a given set of experimental conditions is difficult, since breaking open the cells changes compartmentalization, Ca2+ levels, and probably the state of activation of PKC. The reason for the difference in PKC levels observed for TPA treatment and IPTG induction is not clear but may be due to differences in the metabolism of DAG and TPA. DAG, the endogenous activator, is rapidly metabolized and appears to cause only transient activation of PKC. On the other hand, TPA is degraded very slowly in cells, causing persistent activation of PKC (39) and demonstrating greater potency than DAG in inducing the differentiation of macrophages (1, 40). Incubation with TPA has generally been shown to cause the complete loss of PKC, whereas this has been reported not to occur with DAG in the monoblastoid cell line U937 (19). In previous studies, phorbol ester and DAG displayed differences in phosphorylation substrates, the activation of specific enhancer elements, and the stimulation of cell differentiation (35, 38, 40). These data suggest that the downregulation of PKC observed in TPA-treated cells may not be a critical step in preventing the expression of proximal tubule-specific traits. Interestingly, TPA was previously shown to reduce the expression of PKCalpha but enhanced the expression of PKCdelta and had no effect on PKCepsilon (5). Therefore the levels of PKCs whose activities are known to be regulated by TPA in vitro may be differentially regulated in vivo. This may be due in part to compartmentalization of PKC isoforms within the cell as well as compartmentalization of the various factors that influence the levels of expression of these proteins.

The mechanism for the progressive, concentration-dependent inhibition of differentiation by Galpha q is unknown. The expression of Na+-dependent hexose transport activity that begins at confluence has been correlated with an increase in the synthesis of its mRNA and protein (41, 42). A 1-h treatment with TPA induces a rapid loss of transporter mRNA in postconfluent, differentiated LLC-PK1 cells, a phenomenon attributed to posttranscriptional degradation of the mRNA (33). These data are consistent with the observation that activation of PKC by phorbol esters or DAG analogs inhibits Na+-dependent hexose transport activity in renal proximal tubular primary cell culture through a decrease in maximal enzyme reaction rate (Vmax) (14), suggesting a change in the level rather than the activity of this protein. Phorbol esters can also block the expression of tissue-specific markers in the intestinal epithelial cell line, HT-29 (15). Therefore PKC is implicated in regulating the formation of the differentiated phenotype in both kidney and intestinal epithelia.

The expression of alpha qQ209L has been found to cause transformation in fibroblasts in a cell type-specific manner (10, 18). The expression of constitutively active mutants of the Galpha q family was either growth stimulatory or inhibitory in different fibroblasts (11, 29). With the inducible expression of alpha qQ209L, we were able to show a growth-stimulatory effect, indicated by an increase in the amount of protein per dish and in the number of cells, in these epithelial cells. Taken together with the data for cell differentiation, inducible expression of constitutively active alpha qQ209L resulted in stimulation of growth and inhibition of differentiation in LLC-PK1 epithelial cells. Future studies will include transformation assays to test whether these cells are transformed by the expression of alpha qQ209L. The mechanism whereby Galpha q-mediated pathways affect cell growth and differentiation remains to be elucidated. PKC phosphorylates a number of cellular substrates that could lead to changes in expression of proximal tubule markers in the LLC-PK1 cell line. Investigating the downstream events regulated by PKC in this cell line is in progress in the laboratory.

In summary, we have successfully expressed alpha qQ209L under the control of an inducible expression system in LLC-PK1 kidney epithelial cells. In an IPTG concentration-dependent manner, the expression of constitutively active Galpha q inhibited differentiation of these cells, as indicated by measurement of both Na+-dependent hexose transport and alkaline phosphatase activity, and caused changes in the epithelial morphology of these cells. From morphological changes occurring in the alpha qQ209L-expressing cells, cell-cell contacts seem to be altered in cells treated with IPTG (Sun and Weiss, unpublished observations). The inhibition of differentiation resulting from alpha qQ209L expression could be due to impaired cell-cell contacts induced by IPTG. Changes in cell-cell contacts, such as the adherens junctions, between these epithelial cells are currently under investigation.

    ACKNOWLEDGEMENTS

We thank Dr. Gary L. Johnson for the gift of the alpha qQ209L cDNA, Dr. Benjamin Peng for assistance with microscopy, and Dr. Shoji Osawa for review of the manuscript.

    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-43582 and a grant from the University Research Council, The University of North Carolina at Chapel Hill, and was performed during the tenure of E. R. Weiss as an Established Investigator of the American Heart Association.

Present address of L. Sun: Dept. of Neurology, San Francisco General Hospital, University of California, San Francisco, San Francisco, CA 94110.

Address for reprint requests: E. R. Weiss, Dept. of Cell Biology and Anatomy, CB7090, 108 Taylor Hall, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090.

Received 28 October 1996; accepted in final form 22 November 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(4):C1030-C1039
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