©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Development of Na-dependent Glucose Transport during Differentiation of an Intestinal Epithelial Cell Clone Is Regulated by Protein Kinase C (*)

Olivier Delézay (1), Stephen Baghdiguian (3), Jacques Fantini (2)(§)

From the (1) From INSERM U270 and (2) CNRS URA 1455, Faculté de Médecine Nord, Bd Piere Dramard, 13916 Marseille Cedex 20 and the (3) Laboratoire de Pathologie comparée CNRS-INRA URA 1186, Université Montpellier 2, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The sodium-dependent glucose transporter SGLT1 is expressed on the apical plasma membrane of fully differentiated enterocytes. Recently, we have found that the cotransport function appears gradually during the process of differentiation of the human intestinal epithelial cell clone HT-29-D4. However, the SGLT1 protein was detected in both undifferentiated and differentiated HT-29-D4 cells suggesting that sodium-glucose cotransport was dependent on post-translational events controlling the efficient targeting of the protein in the plasma membrane. In the present study, we have analyzed the molecular mechanisms controlling the functional expression of the SGLT1 protein during the course of HT-29-D4 differentiation. We show that the appearance of the cotransport function in the apical membrane is blocked by 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl (H-7), a potent inhibitor of protein kinase C activity. Moreover, H-7 treatment was associated with an unability of HT-29-D4 cells to organize into a polarized monolayer of differentiated cells. Reciprocally, short term treatment (15 min) of undifferentiated cells by 0.1 µM phorbol myristyl acetate resulted in the appearance of the cotransport function. In contrast, inhibition of cAMP and cGMP-dependent protein kinases by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl did not prevent the development of sodium-glucose cotransport during the differentiation of HT-29-D4 cells. In addition, stimulation of cAMP-dependent protein kinases by 8-Cl-cAMP did not induce the cotransport function in undifferentiated HT-29-D4 cells. By using immunogold labeling at the electron microscopy level, we demonstrated that phorbol myristyl acetate induced the redistribution of SGLT1 protein from intracellular sites to the plasma membrane. In conclusion, our data show that the appearance of a functional sodium-glucose cotransporter in HT-29-D4 cells is controlled, at least in part, by intracellular pathways regulated by the activity of protein kinase C.


INTRODUCTION

The study of transport mechanisms across the intestinal epithelium has been considerably facilitated by the availability of several cultured epithelial cell lines with differentiation characteristics mimicking enterocytic maturation (1, 2) . Among them, the HT-29-D4 clonal cell line possesses the advantage to undergo differentiation following a simple alteration of the culture medium, that is, the replacement of glucose by galactose (3) . This differentiation process occurs without cell loss nor progressive adaptation, so that the differentiated cells can be considered as the true counterpart of undifferentiated ones (4) . HT-29-D4 cells differentiated in galactose medium (HT-29-D4 gal) exhibit morphological, biochemical, and electrophysiological properties of fully differentiated intestinal absorptive cells (3, 4, 5, 6, 7) , including the presence of a functional sodium-glucose cotransporter (SGLT1) located in the apical membrane (8). Interestingly, undifferentiated HT-29-D4 cells (HT-29-D4 glu) are unable to absorb glucose using this pathway. This property is dependent on the efficient plasma membrane targeting of the cotransporter, since the SGLT1 protein was expressed in both HT-29-D4 gal and glu cells, but remained inside undifferentiated cells.()

In the present study, we have analyzed the role of protein kinases in the mechanims associated with the appearance of a functional sodium-glucose cotransporter in the plasma membrane. Our results demonstrate that protein kinase C is involved in the morphogenesis of the differentiated epithelial phenotype associated with a functional sodium-glucose cotransport. Moreover, we show that stimulation of this kinase by phorbol myristyl acetate (PMA)() induces the occurrence of SGLT1 function in undifferentiated HT-29-D4 cells. In contrast, stimulation of cAMP-dependent protein kinases had no effect under the same experimental conditions. We conclude that the efficient targeting of the sodium-glucose cotransporter is modulated by protein kinase C.


EXPERIMENTAL PROCEDURES

Materials

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine-HCl (H-7) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl (HA-1004) were purchased from France Biochem (Meudon, France). -Methyl-D-glucopyranoside (AMG) was purchased from Sigma. [U-C--Methyl]D-glucopyranoside was from Du Pont de Nemours (France). Phlorizin was obtained from Aldrich Chimie (France) and prepared as a 100 mM stock solution in MeSO.

Cell Culture

HT-29-D4 cells were routinely grown in 75-cm flasks (Falcon) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 25 mM glucose and supplemented with 10% fetal calf serum, penicillin (40 units/ml) and streptomycin (40 µg/ml). To induce differentiation, half-confluent HT-29-D4 cells were grown in glucose-free Dulbecco's modified Eagle's medium supplemented with 5 mM galactose and 10% dialyzed fetal calf serum. Cells cultured in galactose medium will be referred to as HT-29-D4 gal cells. Cells grown in glucose medium will be referred to as HT-29-D4 glu cells.

AMG Uptake Measurements

Confluent monolayers of HT-29-D4 cells were washed in modified Earle's solution (B medium: 137 mM NaCl, 5.36 mM KCl, 0.4 mM NaHPO, 0.8 mM MgCl, 1.8 mM CaCl, 20 HEPES adjusted to pH 7.4 with NaOH). Then, cells were washed twice in sodium-free B medium (137 mM choline chloride instead of NaCl and 0.4 mM KHPO instead of NaHPO adjusted to pH 7.4 with KOH). The cells were incubated at 37 °C, with 0.1 mMC-labeled AMG (0.15 µCi/ml) in B medium or in sodium-free B medium. At the end of the incubation, the medium was removed and the monolayer was washed three times with 1 ml of B medium or sodium-free B medium at 4 °C. Cells were disrupted in 0.5 ml of 0.1 N NaOH, 0.1% SDS, and the radioactivity was determined using a Packard counter. The results were expressed as nanomoles of AMG/milligram of proteins. The protein content was evaluated using a Pierce kit and bovine serum albumin as standard.

Membrane Preparations

HT-29-D4 cells grown in 75-cm flasks were washed in B medium containing 5.5 mM glucose and scraped in the same medium using a rubber policeman. Cells were pelleted for 7 min at 150 g and were homogenized in a glass Teflon Potter homogenizer (10 strokes), in a hypotonic solution (10 mM HEPES, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride) at 4 °C. The homogenate obtained was diluted in 1 M sucrose to obtain a final concentration of 0.25 M sucrose and was spun at 150 g for 10 min. The supernatant was centrifuged at 15,000 g for 30 min, and the resulting membrane pellet was resuspended in the centrifugation medium and stored at -80 °C.

Western Blot Analysis

A sequence-specific (Ser-Lys) antibody was prepared against a synthetic peptide (19 amino acids) corresponding to the known amino acid sequence of the sodium glucose transporter (9) . A cysteine was added to the NH-terminal end to link the peptide to keyhole limpet hemocyanin, and antibodies were raised in rabbit as described previously (8) . Specific IgG were isolated from serum using affinity chromatography. Electrotransfer of 10 µg of membrane proteins from 7.5% SDS-polyacrylamide gel electrophoresis to nitrocellulose membrane (Hybond-ECL, Amersham) was carried out for 2 h at 100 V according to Towbin et al.(10) . After transfer, the membrane was preincubated overnight at 4 °C in blocking buffer (5% non-fat milk and 0.05% Tween 20 in PBS, pH 7.4) to reduce nonspecific binding. The membrane was then probed with affinity-purified rabbit IgG in blocking buffer. After 1 h of incubation, the nitrocellulose paper was washed with 0.05% Tween 20 in PBS for 30 min, then incubated with anti-rabbit IgG peroxidase-coupled antibodies (1/5000, Amersham) for 1 h in blocking buffer. Peroxidase-loaded proteins were revealed using the ECL detection system (Amersham).

Transmission Electron Microscopy

HT-29-D4 cells were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 1 h, washed for 10 min in the same buffer containing 6.84% sucrose, post-fixed in 1% osmium tetroxide followed by uranyl acetate treatment, then dehydrated in ethanol, and embedded in Epon. Ultrathin sections were cut perpendicularly to the plane of the cell layer and observed with a transmission electron microscope (Jeol 1200X).

Immunogold Labeling

Undifferentiated HT-29-D4 cells were grown in Transwell cell culture chambers (catalog number 3426, Costar, Cambridge, MA) and treated or not with 0.1 µM PMA for 1 h at 37 °C. The cells were then fixed in Sorensen-Phosphate 0.1 M, pH 7.4, containing 1% glutaraldehyde and 0.2% picric acid for 3 h. After 70% ethanol treatment, the cells were embedded in LR White (Agar Scientific Ltd, Essex, United Kingdom). Ultrathin sections were deposited onto 200-mesh nickel grids, quenched in PBS containing 50 mM NHCl, saturated in PBS 10% normal goat serum, and subsequently incubated with primary antibodies (anti--tubulin monoclonal antibody was from Boehringer Mannhein and anti-CD26 monoclonal antibody was from Immunotech, Marseille, France). The labeling was detected using protein A-gold 10 nm (Sigma). The cells were contrasted with uranyl acetate and observed with a Jeol 1200X transmission electron microscope.

Immunofluorescence Analysis

HT-29-D4 cells grown on glass coverslips were washed with PBS containing 0.1 mM CaCl and 1 mM MgCl (PBS-CM). They were fixed in 3.7% paraformaldehyde in PBS-CM at 4 °C. All subsequent steps were performed at room temperature. Cells were incubated in PBS-CM containing 10% normal goat serum to block nonspecific binding sites. Monolayers were incubated for 45 min with the monoclonal antibody MAC 601 raised against CEA (Biosys, Compiègne, France). After several washings, the primary antibody was revealed with fluorescein-conjugated goat anti-mouse antibodies (Immunotech, Marseille, France). Immunofluorescence analysis was performed with a Zeiss epifluorescence microscope.


RESULTS

To investigate the role of protein kinases during the functional differentiation of HT-29-D4 cells, we first tested the influence of the protein kinase C inhibitor H-7 on the cotransport function. In these experiments, HT-29-D4 glu cells were switched to the galactose medium in either the absence or presence of 30 µM H-7 (i.e. 5-fold the value of the k for protein kinase C) and their capacity to absorb AMG was tested. This non-metabolizable analog of glucose is specific for the sodium-glucose pathway as already described (11) . AMG uptake experiments were performed with or without sodium or in the presence of 100 µM phlorizin (a specific inhibitor of the cotransporter) by time point analysis (10 min). As shown in Fig. 1a, a significant uptake of AMG was measured as soon as 5 days after switching HT-29-D4 glu cells to galactose medium. This uptake increased with time and was completely abolished in the absence of sodium or in the presence of 100 µM phlorizin. In contrast, no significant sodium-dependent, phlorizin-sensitive AMG uptake could be observed when the cells were cultured in the presence of the protein kinase C inhibitor H-7. The absence of the cotransport function in H-7-treated cells was not due to an effect on cellular proliferation as assessed by determination of cellular protein content (data not shown).


Figure 1: Effect of H-7 and HA-1004 treatments on AMG uptake during HT-29-D4 cell differentiation. HT-29-D4 glu cells were switched in galactose medium at day 0 with (filled symbols) or without (open symbols) 30 µM H-7 (a) or 11 µM HA-1004 (b). AMG uptake measurements were analyzed for 10 min in the presence of 140 mM sodium chloride (squares), choline chloride (circles), or sodium chloride and 100 µM phlorizin (triangles). Values reported are means ± S.D. of three replicate determinations.



These results indicate that the functional differentiation of HT-29-D4 cells is regulated by protein kinases. However, the type of the protein kinase(s) involved is uncertain since H-7, although known as a potent inhibitor of protein kinase C, may also alter the function of cGMP- and cAMP-dependent protein kinases (12) . To further document this point, we have tested the influence of an inhibitor of both cAMP- and cGMP-dependent protein kinases (HA-1004) on the appearance of sodium-glucose cotransport function. The results indicate that HT-29-D4 cells treated with HA-1004 (at 11 µM, a concentration that corresponds to 5-fold the K for protein kinase A and 10-fold the value for protein kinase G) express the cotransport function (Fig. 1b). However, one should note that this function is not detectable after 6 days of culture in galactose medium in contrast with untreated control cells. Therefore, HA-1004 does not inhibit the appearance of the cotransport function but moderately delays it in time. Indeed, the values of AMG uptake after 20 days are similar for both HA-1004-treated and control cells. Thus, it seems that the functional appearance of the sodium-glucose cotransport function is mainly dependent on protein kinase C activity.

These data would suggest that the inhibition of protein kinase C activity affect either the expression of the SGLT1 protein or its cellular localization. Therefore, we have analyzed by Western blot the presence of the protein in cells treated or not by the H-7 inhibitor. Using affinity-purified antibodies directed against a nonadecapeptide (Ser-Lys) issued from the sequence of SGLT1 (9, 13) , we detected the specific 64-kDa protein in both cell extracts, showing that SGLT1 expression was not altered by H-7 treatment (Fig. 2). Thus, the absence of the cotransport function could not be related to an effect at the level of protein synthesis.


Figure 2: Immunoblotting of SGLT1 protein in membrane extracts form HT-29-D4 control and H-7-treated cells. 10 µg of membrane preparation from HT-29-D4 untreated cells (lane A) or H-7 treated cells (lane B) were solubilized in reduction buffer and resolved by 7.5% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to the nitrocellulose paper and probed with anti-SGLT1 affinity-purified antibodies. The arrow indicates the specific band of 64 ± 2 kDa.



Next, we studied the effect of H-7 treatment on the overall organization of HT-29-D4 cells by transmission electron microscopy. When HT-29-D4 cells are cultured for more than 20 days in galactose medium, the cells organize into a monolayer of highly polarized columnar cells resembling normal enterocytes (Fig. 3a). Treatment with the protein kinase C inhibitor H-7 (30 µM) resulted in a marked perturbation of the cellular organization (Fig. 3b). In this case, the cells formed a multilayer of non-polarized cells in which tight junctions were only occasionally observed. Several cells showed typical intracellular lumina lined with regularly arranged microvilli (Fig. 3d), structures which are representative of an abortive targeting of the apical plasma membrane (14, 15, 16) . Large intercellular lumina with rare microvilli were also noted (Fig. 3c). The H-7-induced disorganization observed at the ultrastructural level was further documented by an analysis of the localization of an apical marker, CEA, by immunofluorescence techniques. As shown in Fig. 4a, the anti-CEA mAb labeled the apical side of differentiated HT-29-D4 cells. In contrast, a fundamentally different pattern of CEA labeling was observed in H-7-treated cells (Fig. 4b). In the latter case, the periphery of the cells was strongly fluorescent, indicating that the antibody had access to the entire plasma membrane. This result is consistent with the electron microsopy study, confirming that H-7 inhibited the organization of HT-29-D4 cells into a polarized monolayer.


Figure 3: Ultrastructure of HT-29-D4 cells upon H-7 treatment. HT-29-D4 cells were cultured for 30 days in galactose medium in either the absence (a) or the presence (b-d) of 30 µM H-7. The cells were fixed, and ultrathin sections cut perpendicularly to the cell layer were analyzed by transmission electron microscopy. In absence of H-7, the cells are fully differentiated and formed a regular monolayer of columnar enterocyte-like cells (a). In the presence of 30 µM H-7, the cells grew as unorganized multilayers displaying large intercellular lumina (b and c). In some cells, the presence of intracellular lumina lined with regularly arranged microvilli was evidenced (b and d). In some areas, an apical striated border was observed locally in cells belonging the last cell layer (b). d, desmosomes; g, Golgi apparatus; il, intracellular lumen; is, intercellular space; itl, intercellular lumen; ly, lysosomes; m, mithochondria; mv, microvilli; n, nucleus; seb, striated border; tj, tight junction. Bars: a-c, 5 µm; d, 1 µm.




Figure 4: Immunofluorescence localization of the CEA antigen. HT-29-D4 grown in galactose medium in either the absence (A) or presence (B) of 30 µM H-7 were fixed as described under ``Experimental Procedures,'' labeled with anti-CEA monoclonal antibody, then revealed with fluorescene isothiocyanate-conjugated anti-mouse antibodies. Bar, 20 µm.



If the inhibition of protein kinases is associated with a defect of the appearance of differentiated parameters in HT-29-D4 gal cells, one would expect that, in HT-29-D4 glu cells, the activation of these kinases would entail the expression of such functions. To investigate this possibility, we tested the influence of PMA and 8-Cl-cAMP on the AMG uptake. As shown in Fig. 5a, HT-29-D4 glu cells treated for 15 min by 0.1 µM PMA express a functional sodium-glucose cotransporter. This expression increased as a function of time to reach a plateau after 1 h of PMA treatment. Under these conditions, the AMG uptake was phlorizin sensitive, which was not the case for untreated control cells. In contrast, the activation of cAMP-dependent protein kinase by 0.5 mM 8-Cl-cAMP did not induce the appearance of the cotransport function even after 2 h of treatment (Fig. 5b).


Figure 5: Stimulation of protein kinases in HT-29-D4 undifferentiated cells. HT-29-D4 undifferentiated cells grown in glucose medium were incubated with 0.1 µM PMA (a) or 0.5 mM 8-Cl-cAMP (b) and tested for their capacity to absorb AMG by time point analysis (10 min). AMG measurments were performed in either the presence (filled squares) or absence (open squares) of 100 µM phlorizin. Values reported are means ± S.D. of three replicate determinations.



These data would suggest that protein kinase C is involved in the translocation of the SGLT1 protein from intracellular sites to the plasma membrane. However, one cannot rule out the alternative possibility that protein kinase C may directly phosphorylate the cotransporter already localized to the plasma membrane. Two experiments were thus conducted in order to further clarify this particular point. First, we tested the influence of PMA and H-7 treatments on the cotransport activity in fully differentiated HT-29-D4 cells. As shown in Fig. 6, the sodium-dependent phlorizin-sensitive AMG uptake was not altered in the presence of either 0.1 µM PMA or 30 µM H-7, suggesting that the cotransport activity is not directly influenced by protein kinase C phosphorylation. Moreover, immunocytochemical studies were conducted to confirm that the PMA-induced appearance of the cotransport function in undifferentiated HT-29-D4 cells (Fig. 5a) was associated with SGLT1 protein plasma membrane targeting. In these experiments, anti--tubulin antibodies were used as a control for intracellular labeling (Fig. 7b), while anti-CD26 (dipeptidylpeptidase IV) antibodies served as a reference for plasma membrane proteins (Fig. 7c). When affinity-purified antibodies directed against SGLT1 protein were used, the labeling was exclusively detected in restricted areas corresponding to intracellular membranes (Fig. 7d). Upon PMA treatment, the SGLT1 labeling was found in vesicles close to the plasma membrane (Fig. 7e) and, in some cells, unambiguously at the level of the plasma membrane (Fig. 7, f-h). This process could not be evidenced in all observed cells, in agreement with the level of cotransport activity induced by PMA in undifferentiated HT-29-D4 cells (Fig. 5a). However, the redistribution of the SGLT1 protein appeared to be highly specific since the localization of -tubulin and CD26 was not affected by PMA (not shown). Taken together, these data strongly support the concept that protein kinase C activation results in the SGLT1 protein translocation from intracellular compartments to the plasma membrane.


Figure 6: Effect of PMA and H-7 treatment on sodium-glucose cotransport. Fully differentiated HT-29-D4 cells were either treated or not treated with 0.1 µM PMA or 30 µM H-7 for 4 h. AMG uptake experiments were performed as described under ``Experimental Procedures'' with (filled bars) or without (open bars) 140 mM sodium or in the presence of 100 µM phlorizin (hatched bars). Values reported are means ± S.D. of three replicate determinations.




Figure 7: Immunolocalization of SGLT1 protein. Undifferentiated HT-29-D4 cells were either treated (e-h) or not treated (a-d) with 0.1 µM PMA for 1 h and then embedded in LR White for immunogold labeling. a, negative control with the primary antibody omitted; only two gold particles were found in 100 observed cells. b, immunolocalization of -tubulin in a cytoplasm area with numerous cytoskeletal fibrillar elements. c, CD26 labeling exclusively localized on the plasma membrane. d, typical SGLT1 labeling associated with intracellular membranes in a HT-29-D4-untreated cell. e, localization of SGLT1 in a vesicle close to the plasma membrane in a PMA-treated cell. f-h, localization of SGLT1 protein on the plasma membrane of PMA-treated HT-29-D4 cells. Abbreviations:as, apical space; is, intercellular space; m, mitochondria; n, nucleus; in f, the gold particles are indicated with arrowheads. Bars: 100 nm (a-d, g, and h); 30 nm (e); 1 µm (f).




DISCUSSION

In this study, we demonstrate the influence of protein kinase C on the establishment of a functional state of differentiation in the human epithelial intestinal cell clone HT-29-D4. One of the main characteristic of this differentiation process is the organization of the cells into a polarized epithelial monolayer which absorbs glucose using the sodium-glucose cotransport pathway. This cotransport function is specifically expressed by differentiated HT-29-D4 cells, and it gradually appears when undifferentiated cells are switched in a differentiating medium. The establishment of this differentiation-associated function does not seem to be regulated at the level of gene or protein level since the SGLT1 protein was detected in both HT-29-D4 glu and HT-29-D4 gal cells. Thus, the functional appearance of the cotransport function in HT-29-D4 cells seems to be regulated by protein targeting in the apical plasma membrane, which may also be the case for normal intestinal epithelial cells (17, 18) .

The main result of the present study is that an inhibitor of protein kinase C (H-7) is able to disturb the organization of HT-29-D4 cells into a differentiated epithelial monolayer and to prevent the appearance of the cotransport function. Moreover, short term treatments of undifferentiated HT-29-D4 cells by PMA, a potent protein kinase C activator, was sufficient to induce the expression of the cotransport function. These data reinforce the hypothesis of the involvement of protein kinase C in the establishment of a functional state of differentiation in HT-29-D4 cells. This is consistent with a previous report showing that the expression of SGLT1 cotransporter in the LLCPK-1 cell line was inhibited by H-7 (19, 20) . In our study, the possible implication of protein kinase A in the regulation of the cotransport function has been evaluated by two ways. On the one hand, treatment of HT-29-D4 cells by HA-1004 (an inhibitor of protein kinase A) did not prevent the appearance of the function. The only effect of this inhibitor was a delay in the detection of the sodium-dependent AMG uptake during the course of HT-29-D4 differentiation. On the other hand, the cotransport function could not be detected following short term treatments of HT-29-D4 glu cells by 8-Cl-cAMP. These data suggest that protein kinase A is not involved in the early events leading to the establishment of a functional state of differentiation. In contrast, protein kinase C was found to play a critical role throughout the process of epithelial differentiation of HT-29-D4 cells. Therefore, the HT-29-D4 cell line clearly differs from LLCPK-1 since in the later, an increase in cAMP was associated with the development of the differentiated transport mechanisms (21) . Moreover, in Madin-Darby canine kidney cells, the exocytosis of vacuolar apical compartment, which consists of large vacuoles containing apical membrane proteins, was efficiently stimulated by 8-Br-cAMP, while PMA induced only a modest exocytic response (22) .

At the morphological level, the inhibition of protein kinase C activity was associated with an unability of the cells to organize into a polarized epithelium. Obviously, the expression of differentiated functions is highly dependent on the supracellular organization of epithelial cells. For this reason, it is difficult to separate the effect of protein kinase C on morphological versus functional parameters of HT-29-D4 differentiation. The fact that the cotransporter protein was expressed in both untreated and H-7-treated cells is consistent with the involvement of protein kinase C in the post-translational events leading to the correct targeting of the SGLT1 protein in the apical plasma membrane. According to these results, the activation of protein kinase C by short term treatments with PMA elicited the appearance of the cotransport function in HT-29-D4 glu cells. This function was detectable after 15 min of PMA treatment, which strongly suggests that the effect was not dependent on de novo protein synthesis. By using immunocytochemical techniques at the electron microscopy level (immunogold labeling), we demonstrated that PMA induced the translocation of the SGLT1 protein from intracellular sites to the plasma membrane in HT-29-D4 undifferentiated cells. Interestingly, similar data were recently reported for the -aminobutyric acid transporter whose plasma membrane targeting is dependent on protein kinase C activation (23) . In agreement with our results on the SGLT1 protein, the activity of this transporter is regulated by protein kinase C-mediated subcellular redistribution rather than direct phosphorylation of the protein.

In conclusion, our data show that protein kinase C regulates protein plasma membrane targeting and thus plays an important role in the morphogenesis and the functional differentiation of intestinal epithelial cells in vitro.


FOOTNOTES

*
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: CNRS URA 1455, Faculté de Médecine Nord, Bd Piere Dramard, 13916 Marseille Cedex 20, France. Tel.: 33-91698849; Fax: 33-91657595.

The abbreviations used are: PMA, phorbol myristyl acetate; H-7, 1-5-isoquinolinesulfonyl)-2-methylpiperazine-HCl; HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide-HCl; AMG, -methyl-D-glucopyranoside; PBS, phosphate-buffered saline.

O. Delézay, S. Baghdiguian, and J. Fantini, manuscript submitted.


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