Potential conflict of interest: Nothing to report.
Cyclic adenosine monophosphate (cAMP) stimulates hepatic bile acid uptake by translocating sodium-taurocholate (TC) cotransporting polypeptide (Ntcp) from an endosomal compartment to the plasma membrane. Rab4 is associated with early endosomes and involved in vesicular trafficking. This study was designed to determine the role of Rab4 in cAMP-induced TC uptake and Ntcp translocation. HuH-Ntcp cells transiently transfected with empty vector, guanosine triphosphate (GTP) locked dominant active Rab4 (Rab4(GTP)), or guanosine diphosphate (GDP) locked dominant inactive Rab4 (Rab4(GDP)) were used to study the role of Rab4. Neither Rab4(GTP) nor Rab4(GDP) affected either basal TC uptake or plasma membrane Ntcp level. However, cAMP-induced increases in TC uptake and Ntcp translocation were enhanced by Rab4(GTP) and inhibited by Rab4(GDP). In addition, cAMP increased GTP binding to endogenous Rab4 in a time-dependent, but phosphoinositide-3-kinase–independent manner. Conclusion: Taken together, these results suggest that cAMP-mediated phosphoinositide-3-kinase–independent activation of Rab4 facilitates Ntcp translocation in HuH-Ntcp cells. (HEPATOLOGY 2008.)
Cyclic adenosine monophosphate (cAMP) rapidly stimulates hepatic taurocholate (TC) uptake by translocating Na+-TC cotransporting polypeptide (Ntcp) from an endosomal pool to the sinusoidal membrane.1 Previous studies indicate that the effect of cAMP on Ntcp is mediated via the phosphoinositide-3-kinase (PI3K) signaling pathway and involves PI3K/protein kinase B (PKB) and PI3K/protein kinase Cζ (PKCζ) pathways.2 Similar signaling pathways have also been suggested for insulin-induced rapid translocation of GLUT4 to plasma membrane in myocytes and adipocytes.3, 4 It is now evident that rapid translocation of transporters involves recruitment of transporter containing vesicles to the plasma membrane. A number of proteins, including Rab proteins, have been suggested to play an important role in this process.
Rab proteins, which belong to the Ras guanosine triphosphatase (GTPase) superfamily and consist of more than 60 members,5 are involved in endocytosis, protein trafficking, and apically directed transcytosis in hepatocytes.6–8 Rab proteins cycle between guanosine diphosphate (GDP)-bound inactive form and a guanosine triphosphate (GTP)-bound active form. The general mechanism by which Rab proteins stimulate vesicular transport involves the binding of these proteins to the donor membrane (on the target vesicle), attachment of the Rab-associated vesicle to cytoskeletal motor proteins through various effectors molecules, and docking and fusion of the vesicle to the acceptor membrane (plasma membrane) through the soluble N-ethylmaleimide sensitive fusion protein attachment receptor complex.9, 10 This process can also occur in reverse, allowing transport of proteins from the plasma membrane to the intracellular compartment.
Rab proteins have been shown to regulate a number of transporters. Thus, insulin-mediated glucose transporter 4 translocation is facilitated by Rab proteins, specifically Rab4, Rab5, and Rab11.11, 12 Rab4 has been reported to regulate cystic fibrosis transmembrane conductance regulator (CFTR) function in HT-29 cells13 and amiloride-sensitive sodium channel in colonic epithelia.14 In hepatocytes, Rab11 has been proposed to be involved in the translocation of adenosine triphosphate (ATP)–binding cassette transporters to the apical membrane, and Rab4 plays a role in endocytosis and vesicle fusion in hepatocytes.15, 16 However, whether Rab proteins are involved in cAMP-mediated Ntcp translocation is not known.
Cyclic AMP is known to stimulate vesicular trafficking in hepatocytes.16, 17 Rab4 has been found to colocalize with Ntcp vesicles in rat hepatocytes.18 Because Rab4 is involved in endosomal trafficking and receptor recycling,19, 20 it is possible that cAMP-mediated translocation of Ntcp involves Rab4. The aim of the present study was to investigate whether Rab4 facilitates/mediates cAMP-mediated Ntcp translocation. Results of our studies using dominant active and dominant inactive Rab4 suggest that Rab4 facilitates cAMP-mediated Ntcp translocation.
ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CFP, cyan fluorescent protein; CPT-cAMP, 8-chlorophenylthio cAMP; GDP, guanosine diphosphate; GTP, guanosine triphosphate; GTPase, guanosine triphosphatase; PI3K, phosphoinositide-3-kinase; PKB, protein kinase B; PKC, protein kinase C; Rab4(GDP), GDP locked dominant inactive Rab4; Rab4(GTP), GTP locked dominant active Rab4; SEM, standard error of the mean; TC, taurocholate; Ntcp, Na+-taurocholate cotransporting polypeptide; wt-Rab4, wild-type Rab4.
Materials and Methods
The Lipofectamine reagent kit was purchased from Life Technologies/GIBCO (Carlsbad, CA). 8-Chlorophenylthio-cAMP (CPT-cAMP), taurocholate (TC) (Na-salt), LY294002, wortmannin, aprotinin, leupeptin, okadaic acid, and collagenase were obtained from Sigma Chemical Company (St. Louis, MO). [3H]TC (56 mCi/mmol), and [α-32P]GTP were purchased from Perkin-Elmer (Boston, MA). Antifusion protein antibodies to the cloned Ntcp were prepared as described.21 Rab4 antibody was obtained from BD Transduction Laboratories. Wild-type Rab4 (wt-Rab4) and mutant Rab4s were cloned in Clontech C1-cyan fluorescent protein (CFP) vector; Rab4(GTP) and Rab4(GDP) represent dominant active, GTP locked, GTPase deficient, Rab4Q67L and dominant inactive, GDP locked, Rab4S22N, respectively.19 HuH-Ntcp cells are HuH-7 cells, a human hepatocellular carcinoma cell line, stably transfected with rat Ntcp22 and were generously provided by Dr. Gores (Rochester, MN). Male Wistar rats (200-300 g) obtained from Charles River Laboratories (Wilmington, MA) served as liver donors.
Cell Culture and Transfections.
HuH-Ntcp cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 100,000 units/L penicillin, 100 mg/L streptomycin, 1.2 g/L G418, and 25 μg/mL amphotericin B at 37°C in a 5% CO2/95% O2 air incubator. The cells were transfected with CFP-tagged empty vector, wt-Rab4, Rab4(GTP), and Rab4(GDP) using Lipofectamine as described.23 The transfection efficiency based on cells exhibiting CFP immunofluorescence ranged from 25%-48%. Immunoblot analysis for CFP was performed with anti-green fluorescent protein antibody to verify protein expression of the Rab4 mutants.
TC Uptake and Ntcp Translocation.
TC uptake in HuH-Ntcp cells was determined as described.23 Briefly, following pretreatment with 100 μM CPT-cAMP for 15 minutes, cells were incubated with 20 μM TC containing 3H-TC (100 decays per minute/pmol) for 2 minutes. Cells were then washed twice with 0.5 mL ice-cold [4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid] buffer and lysed in 0.25 mL of a lysis buffer. Aliquots of cell lysate were counted for radioactivity and TC uptake rate was expressed in pmol/minute/mg protein. A cell surface protein biotinylation method as described23, 24 was used to quantitate Ntcp translocation in HuH-Ntcp cells. Briefly, following various treatments cell surface proteins were biotinylated by exposing HuH-Ntcp cells to sulfo-NHS-LC-Biotin (0.5 mg/mL; Pierce) followed by preparation of cell lysate used to determine biotinylated and total Ntcp mass. Biotinylated proteins were isolated using streptavidin-agarose beads and then subjected to immunoblot analysis to determine plasma membrane Ntcp using rat Ntcp antibody (1:2,000 dilution).
Rab4 GTP Overlay Assay.
The GTP binding ability of endogenous and mutant Rab4 was determined using a blot overlay assay for GTP binding proteins, as described,13, 19, 25, 26 with the exception that Rab4 was first immunoprecipitated. Briefly, Rab4s immunoprecipitated from cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. For GTP binding assays, gels were first soaked in 50 mM Tris pH 7.5%-20% glycerol for 1 hour, followed by transfer to nitrocellulose membranes. The membranes were then rinsed twice in wash buffer (50 mM Tris pH 7.6, 10 μM MgCl2, and 0.3% Tween-20) for 10 minutes at room temperature followed by incubation with an ATP overlay buffer (wash buffer + 100 mM dithiothreitol and 100 μM ATP) for 10 minutes at room temperature. Finally, the ATP overlay buffer was spiked with 1 μCi/mL of [α-32P]GTP and the membrane was incubated for 1 hour at room temperature. The membrane was washed, air-dried for 4 hours, and then autoradiographed to determine GTP-bound Rab4. The GTP blot was stripped and subjected to immunoblot analysis with Rab4 antibody to detect immunoprecipitated Rab4. Each assay was run in triplicate. To minimize variations due to differences in immunoprecipitated Rab4, GTP-bound Rab4 was expressed as a fraction of total immunoprecipitated Rab4.
PKB (Akt) activity was determined from the ratio of phosphorylated PKB (active form) to total PKB as described.27 The Lowry method was used to determine cell protein.28 The blots were scanned in grayscale using Adobe PhotoShop (Adobe Systems Incorporated, San Jose, CA) and the relative band densities were quantitated using Sigmal Gel (Jandel Scientific Software, San Rafael, CA). All values are expressed as mean ± standard error of the mean (SEM). Analysis of variance followed by Fisher's least significant difference test was used to statistically analyze data, with P < 0.05 considered significant.
Results and Discussion
Role of Rab4 in cAMP-Stimulated TC Uptake and Ntcp Translocation.
We studied the effect of Rab4(GTP) and Rab4(GDP) to determine the role of Rab4, an approach used by others to define regulation of other transporters by Rab4.13, 14, 29 If Rab4 is involved, Rab4(GTP) should facilitate while Rab(GDP) should inhibit cAMP-stimulated TC uptake and Ntcp translocation. To test this, HuH-Ntcp cells were transiently transfected with CFP-tagged Rab4(GTP) or Rab4(GDP), followed by determination of TC uptake and Ntcp translocation in the presence and absence of cAMP.
The expression of Rab4 mutants was confirmed by immunoblot analysis with green fluorescent protein antibody, which detects CFP. The result showed expression of CFP-tagged Rab4 in Rab4(GTP) transfected cells and Rab4(GDP) transfected cells, while only CFP was detected in cells transfected with the empty vector (Fig. 1A). Immunoblot analysis with Rab4 antibody detected both endogenous and expressed CFP-tagged Rab4 in cells transfected with Rab4(GTP) and Rab4(GDP), while only endogenous Rab4 was detected in cells transfected with the empty vector (Fig. 1B). The CFP-tagged Rab4(GTP) and Rab4(GDP) represented 40 ± 4.5% and 35 ± 5.2% (mean ± SEM, n = 4), respectively, of total Rab4 (endogenous + expressed). Thus, both Rab4 mutants were adequately expressed.
Next, we determined the effect of Rab4 mutants on TC uptake and Ntcp translocation in HuH-Ntcp cells. As we reported,23 cAMP increased TC uptake (Fig. 2A) and Ntcp translocation (Fig. 2B) by 65% and 69%, respectively, in cells expressing the empty vector. Expression of neither Rab4(GTP) nor Rab4(GDP) affected basal level of TC uptake and plasma membrane Ntcp compared to cells expressing the empty vector. On the other hand, expression of Rab4(GTP) further augmented cAMP-induced stimulation of TC uptake and Ntcp translocation from 65% to 152% and from 69 to 162%, respectively (Fig. 2A,B). In contrast, cAMP failed to stimulate TC uptake and Ntcp translocation in cells expressing Rab4(GDP). These findings are similar to a study showing that expression of a dominant negative Rab4 does not affect basal, but inhibits insulin stimulated glucose transporter 4 translocation in 3T3-L1 cells.29 Since Rab4(GTP) and Rab4(GDP) do not affect basal TC uptake and Ntcp translocation, but augment and inhibit, respectively, the effect of cAMP, these results would suggest that Rab4 activation is necessary for cAMP to stimulate TC uptake and Ntcp translocation.
We then verified the dominant active and inactive properties of Rab4(GTP) and Rab4(GDP) by determining GTP binding using GTP overlay assay. For this study, Rab4 immunoprecipitated from cells transfected with CFP-tagged wt-Rab4, Rab4(GTP), and Rab4(GDP) was subjected to GTP overlay assay and immunoblotting with anti-Rab4 antibody (Fig. 3). While wt-Rab4 and Rab4(GTP) efficiently bound GTP, Rab4(GDP) failed to bind GTP. The Rab4 immunoblot revealed expression of all CFP-tagged Rab4 constructs. As expected, cells expressing the empty vector did not show GTP binding or expression of CFP-Rab4. These results confirmed the active and inactive status of Rab4(GTP) and Rab4(GDP), respectively, as reported.13, 14, 19 Analysis of the extent of GTP binding (GTP binding/CFP-Rab4 expression) showed that wt-Rab4 bound 50 ± 8.4% (mean ± SEM, n = 3) less GTP than Rab4(GTP). This is expected since Rab4(GTP) can only bind GTP, while wt-Rab4 will be in configurations allowing binding of GTP or GDP. This result suggests that about 50% of Rab4 is in the active form in HuH-Ntcp cells and that GTP overlay assay can be used to determine whether a stimulant can activate Rab4.
PI3K-Independent Activation of Rab4 by cAMP.
The above studies suggest a regulatory role for Rab4 in cAMP-stimulated TC uptake and Ntcp translocation. However, it is not known whether cAMP activates Rab4 (i.e., increases GTP binding of Rab4) and thereby stimulates Ntcp translocation. Alternatively, cAMP may not activate Rab4, but the presence of active Rab4 may be required to facilitate the effect of cAMP. It may be noted that insulin activates Rab4 in rat adipocytes.29, 30 We have used the GTP overlay assay to determine whether cAMP stimulates GTP binding of Rab4 in HuH-Ntcp cells. Treatment of cells with cAMP resulted in a time-dependent increase in Rab4 GTP binding (Fig. 4); GTP-binding of Rab4 was increased significantly by cAMP as early as 5 minutes after treatment and remained increased at 10 minutes (Fig. 4). We determined the effect of cAMP on Rab4 GTP binding only up to 10 minutes to ascertain that the Rab4 activation was within the time frame (15 minutes) when cAMP stimulates TC uptake. Whether this effect of cAMP is sustained beyond 10 minutes is not known. This result would suggest that cAMP activates Rab4 by stimulating guanine nucleotide exchange on Rab4 and that the effect of cAMP on Ntcp translocation may be mediated via Rab4.
In a previous study, we reported that cAMP-stimulated Ntcp translocation is phosphoinositide-3-kinase (PI3K)-dependent.27 Thus, it is possible that the activation of Rab4 by cAMP may also be PI3K-dependent. PI3K-dependent activation of Rab4 by insulin has been reported in adipocytes.30 However, inhibition of PI3K by LY 294002 did not affect cAMP-induced Rab4 GTP-binding (Fig. 5). LY294002 did inhibit cAMP-induced PI3K-dependent activation of PKB27 in these cells (Fig. 5), indicating that PI3K was inhibited. Thus, cAMP-induced activation of Rab4 is not mediated via PI3K.
The mechanism by which cAMP activates Rab4 in hepatocytes is not known and is currently under investigation. One possibility may be that cAMP activates guanine nucleotide exchange factor involved in GTP/GDP exchange on Rab4 or inhibits Rab4-GTPase activating protein. The present finding of PI3K-independent activation of Rab4 may suggest that cAMP-induced TC uptake and Ntcp translocation may require activation of two independent pathways, as discussed below.
The translocation of Ntcp by cAMP is mediated via the PI3K signaling pathway; the downstream mediators being PKB, protein kinase C (PKC)ζ, and PKCδ.31, 32 A recent study showed that PKCζ is present in Ntcp-containing vesicles and is required for microtubule-based movement of these vesicles.18 However, the steps between cAMP→PI3K→PKB/PKCζ/PKCδ and membrane translocation are not known. One possibility may be that Rab4 is downstream of the cAMP→PI3K→PKB/PKCζ/PKCδ pathway. However, this seems unlikely since activation of Rab4 by cAMP is PI3K-independent (Fig. 5). This may also explain why Rab4(GTP) did not stimulate TC uptake or Ntcp translocation in the absence of cAMP (Fig. 2A,B); such an effect would have been expected if Rab4 was the downstream mediator of the above mentioned signaling pathway. Since cAMP-induced Ntcp translocation is enhanced by Rab4(GTP) and inhibited by Rab4(GDP), activation of Rab4 is necessary, but not sufficient for cAMP-induced Ntcp translocation. Thus, we propose that cAMP activates two parallel signaling pathways, namely cAMP→→Rab4 and cAMP→PI3K→PKB/PKCζ/PKCδ, and the activation of both pathways is necessary for cAMP-induced Ntcp translocation.
The mechanism by which Rab4-GTP facilitates cAMP-induced increases in cell surface Ntcp is not known. Rab4 could stimulate translocation to or inhibit translocation from the plasma membrane. In as much as Rab4 has been suggested to be involved in vesicle trafficking from early endosome to plasma membrane in nonpolarized cells,9, 33 it is likely that Rab4 stimulates translocation of Ntcp to the plasma membrane. However, the alternate possibility that Rab4-GTP decreases endocytosis can not be ruled out. Since Rab5 and Rab11 also colocalize with Ntcp vesicles,18 it is possible that Rab5 and Rab11 are also involved in cAMP-induced Ntcp translocation. Further studies similar to the present one will be needed to ascertain that possibility.
In summary, the present study showed for the first time that cAMP activates Rab4 and activated Rab4 facilitates cAMP-induced Ntcp translocation.
We thank Holly Jameson for excellent technical assistance.