Sphingolipid transport to the apical plasma membrane domain in human hepatoma cells is controlled by PKC and PKA activity: a correlation with cell polarity in HepG2 cells - PubMed (original) (raw)
Sphingolipid transport to the apical plasma membrane domain in human hepatoma cells is controlled by PKC and PKA activity: a correlation with cell polarity in HepG2 cells
M M Zegers et al. J Cell Biol. 1997.
Abstract
The regulation of sphingolipid transport to the bile canalicular apical membrane in the well differentiated HepG2 hepatoma cells was studied. By employing fluorescent lipid analogs, trafficking in a transcytosis-dependent pathway and a transcytosis-independent ('direct') route between the trans-Golgi network and the apical membrane were examined. The two lipid transport routes were shown to operate independently, and both were regulated by kinase activity. The kinase inhibitor staurosporine inhibited the direct lipid transport route but slightly stimulated the transcytosis-dependent route. The protein kinase C (PKC) activator phorbol-12 myristate-13 acetate (PMA) inhibited apical lipid transport via both transport routes, while a specific inhibitor of this kinase stimulated apical lipid transport. Activation of protein kinase A (PKA) had opposing effects, in that a stimulation of apical lipid transport via both transport routes was seen. Interestingly, the regulatory effects of either kinase activity in sphingolipid transport correlated with changes in cell polarity. Stimulation of PKC activity resulted in a disappearance of the bile canalicular structures, as evidenced by the redistribution of several apical markers upon PMA treatment, which was accompanied by an inhibition of apical sphingolipid transport. By contrast, activation of PKA resulted in an increase in the number and size of bile canaliculi and a concomitant enhancement of apical sphingolipid transport. Taken together, our data indicate that apical membrane-directed sphingolipid transport in HepG2 cells is regulated by kinases, which could play a role in the biogenesis of the apical plasma membrane domain.
Figures
Figure 1
Transfer of fluorescent lipid analog to bile canaliculi via the direct pathway and via transcytosis. HepG2 cells were labeled at 4°C with 3 μM C6-NBD–Cer to reveal the direct pathway and C6-NBD–SM to visualize the transcytosis-dependent pathway, as described in Materials and Methods. Cells labeled with C6-NBD–SM were incubated at 37°C for 15 min to allow endocytic uptake of the lipid. To remove C6-NBD–SM remaining in the basolateral plasma membrane, the cells were back exchanged after the 37°C incubation. Cells labeled with C6-NBD–Cer were incubated at 37°C for 60 min to allow synthesis and subsequent transport of C6-NBD–lipid products in the absence of back exchange medium. After the 37°C incubation, the cells were back exchanged. (a) Cells labeled with C6-NBD– SM display a bright labeling of the bile canaliculus (arrowhead) and an additional, vesicular staining pattern. (c) Cells that were labeled with C6-NBD–Cer show an accumulation of NBD fluorescence, typical for localization at the Golgi apparatus and in the bile canaliculus (arrowhead). Note the absence of a vesicular staining pattern in cells that were labeled with C6-NBD–SM (a). (b and d are phase contrast images corresponding to a and c, respectively.) Bars, 10 μm.
Figure 2
Effect of staurosporine on apical lipid transport. HepG2 cells were preincubated with 100 nM staurosporine in HBSS at 37°C for 30 min. Cells were then cooled and, in the presence of staurosporine, labeled and incubated with 3 μM C6-NBD–Cer for determining lipid transport in the direct pathway (A) and 3 μM C6-NBD–SM or -GlcCer for that in the transcytosis-dependent pathway (B). Cells that were labeled with C6-NBD–Cer were incubated at 37°C both in the absence and presence of back-exchange medium. Apical transport of lipids was determined by assessing the percentage of labeled bile canaliculi as described in Materials and Methods. In each experiment a minimum of 50 bile canalicular structures was counted. Each column represents the mean of three to five independent experiments ±SEM. A shows the effect of staurosporine (SSP) on the direct pathway in cells incubated for 60 min in the presence of back-exchange medium, or 30 and 60 min in the absence of back- exchange medium. B shows the effect of staurosporine on the transcytosis of C6-NBD–GlcCer (white bars) and -SM (black bars).
Figure 2
Effect of staurosporine on apical lipid transport. HepG2 cells were preincubated with 100 nM staurosporine in HBSS at 37°C for 30 min. Cells were then cooled and, in the presence of staurosporine, labeled and incubated with 3 μM C6-NBD–Cer for determining lipid transport in the direct pathway (A) and 3 μM C6-NBD–SM or -GlcCer for that in the transcytosis-dependent pathway (B). Cells that were labeled with C6-NBD–Cer were incubated at 37°C both in the absence and presence of back-exchange medium. Apical transport of lipids was determined by assessing the percentage of labeled bile canaliculi as described in Materials and Methods. In each experiment a minimum of 50 bile canalicular structures was counted. Each column represents the mean of three to five independent experiments ±SEM. A shows the effect of staurosporine (SSP) on the direct pathway in cells incubated for 60 min in the presence of back-exchange medium, or 30 and 60 min in the absence of back- exchange medium. B shows the effect of staurosporine on the transcytosis of C6-NBD–GlcCer (white bars) and -SM (black bars).
Figure 3
The effect of staurosporine on the total cellular uptake of C6-NBD–SM and -GlcCer. HepG2 cells were preincubated with 100 nM staurosporine in HBSS at 37°C for 30 min. Cells were then cooled and, in the presence of staurosporine, labeled and incubated with 3 μM C6-NBD–SM or -GlcCer at 4°C, as described in Materials and Methods. Cells were then washed and incubated at 37°C for the indicated time intervals. After the incubation, cells were washed with cold PBS, back exchanged, and scraped from the culture dish. Lipids from the cells were extracted and NBD lipids were quantified as described. The data are shown as the mean of three independent experiments ±SEM. Open symbols represent internalization of C6-NBD–GlcCer in control (○) and staurosporine-treated (▿) cells. Closed symbols reflect internalization of C6-NBD–SM in control (•) and staurosporine-treated (▾) cells.
Figure 4
PKC activity inhibits apical transport of sphingolipids. A shows the effect of activator (PMA) or inhibitor (BIM) of PKC activity on direct transport of sphingolipids. In the presence of PMA and/or BIM, cells were labeled with 3 μM C6-NBD– Cer at 4°C, as described in Materials and Methods. Cells were then incubated at 37°C for 60 min in back-exchange medium, while PMA and/or BIM remained in the solution. Subsequently, cells were washed and apical transport was determined as described in Materials and Methods. B shows the inhibition of transcytosis of C6-NBD–SM and -GlcCer by PKC activity. Cells were incubated with 3 μM C6-NBD–GlcCer (white bars) or -SM (black bars) at 4°C in the presence of PMA and/or BIM. After a 15-min incubation at 37°C in the presence of PMA and/or BIM, the cells were washed, cooled, and back exchanged. Apical transport was determined as described in Materials and Methods. Note that in all experiments, the inhibitor BIM (100 nM) was preincubated with the cells at 37°C for 30 min.
Figure 11
The effect of PMA, cAMP, and H89 on total cellular uptake of C6-NBD–SM and -GlcCer. HepG2 cells were preincubated with 20 nM PMA, 1 mM dBcAMP, or 10 μM H89 in HBSS at 37°C for 30 min. Cells were then cooled and, in the presence of the drugs, labeled and incubated with 3 μM C6-NBD–SM or -GlcCer at 4°C. Subsequent incubations were carried out at 37°C for the indicated time intervals. After the incubation, the cells were washed with cold PBS, back exchanged, and scraped from the culture dish. Lipids were extracted, and NBD lipids were quantified as described. Data points represent the mean of three independent experiments ±SEM. (A) Internalization of C6-NBD–GlcCer in control (○), PMA-treated (□), dBcAMP-treated (▵), and H89-treated (▿) cells. (B) Internalization of C6-NBD–SM in control (•), PMA-treated (▪), dBcAMP-treated (▴), and H89-treated (▾) cells.
Figure 5
Villin is specifically located in bile canaliculi in HepG2 cells. HepG2 cells were fixed and stained for villin using a monoclonal antibody. Note the specific location of villin in the bile canalicular microvilli. Bar, 10 μm.
Figure 6
PMA causes a reorganization of actin filaments and depolarization of HepG2 cells. Cells were washed and incubated with 0.1% DMSO (control; a), or 100 nM PMA in HBSS at 37°C for 60 min (b) and 4 h (c). Cells were then fixed and stained for actin using TRITC-labeled phalloidin, as described. Note that actin filaments, located around bile canaliculi in control cells (a), redistribute upon PMA treatment (b and c). Bars, 10 μm.
Figure 6
PMA causes a reorganization of actin filaments and depolarization of HepG2 cells. Cells were washed and incubated with 0.1% DMSO (control; a), or 100 nM PMA in HBSS at 37°C for 60 min (b) and 4 h (c). Cells were then fixed and stained for actin using TRITC-labeled phalloidin, as described. Note that actin filaments, located around bile canaliculi in control cells (a), redistribute upon PMA treatment (b and c). Bars, 10 μm.
Figure 7
PKC activity causes the redistribution of a bile canalicular antigen. Cells were preincubated with 500 nM of the PKC inhibitor BIM in HBSS at 37°C for 30 min (c and d), followed by an incubation for 60 min in HBSS, containing 0.1% DMSO (control; a), 100 nM PMA (b), 500 nM BIM (c), or both (d). Finally, the cells were fixed and stained with MAB442 as described in Materials and Methods. Note that the bile canalicular antigen to which MAB442 is directed, redistributes upon PMA treatment, which is counter-acted by BIM. Bars, 10 μm.
Figure 8
Opening of tight junctions and depolarization of HepG2 cells depends on the concentration of PMA. Cells were labeled with 3 μM C6-NBD–SM at 4°C, as described in Materials and Methods. The cells were then washed and allowed to accumulate the fluorescent lipid in the apical domain, as accomplished by an incubation in HBSS at 37°C for 30 min. Cells were then treated with increasing concentrations of PMA in HBSS at 37°C for 30 min. As a positive control, tight junctions were opened by treatment with 25 mM EDTA in HBSS without Ca2+, at 37°C for 15 min. After the incubations, the cells were washed, cooled, and back exchanged. Cells that were treated with EDTA were back exchanged in back-exchange medium without Ca2+. The percentage of NBD-positive bile canaliculi was determined (crosshatched bars). In a parallel experiment, the total number of bile canaliculi was determined by staining the cells with the _P_-glycoprotein–specific antibody C219. The total number of bile canaliculi in PMA-treated cells, and cells that were treated with EDTA, was expressed as a percentage of control values (grey bars). Data represent the mean ±SEM of three independent experiments.
Figure 9
PMA treatment does not change levels of villin in depolarized cells. Cells were washed and incubated with 0.1% DMSO (control; lane 1), 100 nM PMA (lane 2), 500 nM BIM (lane 3), or both (lane 4) at 37°C in HBSS for 4 h. Gel electrophoresis and Western blotting using a monoclonal antibody against villin were performed as described in Materials and Methods. Note that both PKC modulators do not affect the levels of villin. Bars show quantitation of the Western blot by laser densitometric scanning and represent the mean of two independent experiments.
Figure 10
PKA activity stimulates apical transport of sphingolipids. HepG2 cells were preincubated with various PKA modulators in HBSS at 37°C for 30 min. The compounds were kept present during further incubations. A shows the effect of PKA activity on direct transport. Cells were labeled with 3 μM C6-NBD–Cer at 4°C, as described in Materials and Methods. After an incubation at 37°C for 60 min in back-exchange medium, the cells were washed and apical transport was determined as described. Data represent the mean ±SEM of three to five independent experiments of cells treated with 0.1% DMSO (control), 1 μM forskolin (1 μM FSK), 50 μM forskolin combined with 1 mM IBMX (50 μM FSK/IBMX), 1 mM dBcAMP (dBcAMP), or 10 μM H89 (H 89). B shows the stimulation of transcytosis of exogeneously inserted C6-NBD–SM and -GlcCer by dBcAMP. Cells were labeled with 3 μM C6-NBD–GlcCer (white bars) or -SM (black bars) at 4°C. After warming at 37°C for 15 min in HBSS, the cells were subsequently washed, cooled, and back exchanged. Apical transport was determined as described in Materials and Methods. Data represent the mean of three independent experiments ±SEM.
Figure 12
cAMP induces a PKA-dependent increase in number of and enlargement of bile canaliculi. HepG2 cells were incubated for 4 h at 37°C in HBSS (a) supplemented with either 1 mM dBcAMP (b), 50 μM forskolin and 1 mM IBMX (c), or 1 mM dBcAMP and 10 μM H89 (d). In the latter case cells had been preincubated with 10 μM H89 in HBSS at 37°C for 30 min before the addition of dBcAMP. After the incubation the cells were washed, fixed in ethanol, and bile canaliculi stained, using the actin stain TRITC–phalloidin, as described in Materials and Methods. Note the enlargement of bile canaliculi, when compared to control cells (a), in cells that have been treated with agents that induce an increase in intracellular cAMP concentration (b and c) but not in cells that were treated with both dBcAMP and the PKA inhibitor H89 (d). Bars, 10 μm.
References
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