Changes in PtdIns(4,5)P2 Induced by Etoposide Treatment Modulates Small Intestinal P-Glycoprotein via Radixin

Abstract

Previously, we reported that repeated oral administration of etoposide (ETP) increases P-glycoprotein (P-gp) expression in association with activation of ezrin/radixin/moesin (ERM) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in the small intestine. However, the detailed mechanisms of this pathway have yet to be fully elucidated. Recently, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], one of the most abundant phosphoinositides in the plasma membrane, has attracted attention regarding its involvement in the plasma membrane localization of various membrane proteins. PtdIns(4,5)P2 is an essential factor in the dissociation and subsequent membrane translocation (activation) of ERM, and its synthetic pathway is known to be highly regulated by RhoA/ROCK signaling. Here, we examined the involvement of PtdIns(4,5)P2 in the mechanism by which ETP treatment increases small intestinal P-gp levels, and we determined which protein within ERM contributes to this phenomenon. Repeated oral treatment with ETP (10 mg/kg/d) over 5 d significantly increased PtdIns(4,5)P2 expression in the ileal membrane as measured by dot blot. Furthermore, this increase was suppressed by co-administration of a RhoA inhibitor, rosuvastatin (5 mg/kg/d, per os (p.o.)), or a ROCK inhibitor, fasudil (5 mg/kg/d, p.o.). In immunoprecipitation assays, radixin (but not ezrin or moesin) binding to PtdIns(4,5)P2 was observed to increase in association with the up-regulation of P-gp in the same fraction, and immunofluorescence studies indicated that radixin co-localized with PtdIns(4,5)P2 in the ileal tissue. In conclusion, ETP treatment appears to up-regulate PtdIns(4,5)P2 expression via RhoA/ROCK signaling, leading to the activation of ERM, presumably through the physical interaction of radixin with PtdIns(4,5)P2. This in turn increases the expression of ileal P-gp.

We previously reported that repeated oral treatment with etoposide (ETP), an anticancer drug that is a substrate for P-glycoprotein (P-gp),¹⁾ activates ezrin/radixin/moesin (ERM), which are scaffold proteins for P-gp in the small intestine.^2,3) Activated ERM may in turn increase P-gp expression in the plasma membrane of the small intestine under ETP treatment.^2,3) ERM activation is regulated by Ras homolog gene family member A (RhoA) and Rho-associated coiled-coil containing protein kinase (ROCK) signaling, as demonstrated in our previous studies^2,3) and others.^4,5) In our reports we proposed the possibility that of the ERM proteins, radixin may contribute to the above phenomena more than ezrin or moesin⁶⁾; although the detailed mechanism remained unclear.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is one of the most abundant phosphoinositides in the plasma membrane.⁷⁾ PtdIns(4,5)P2 regulates many cellular events such as cytoskeleton remodeling and vesicle trafficking.⁷⁾ Recently, a novel role of PtdIns(4,5)P2 has been proposed in the regulation of the plasma membrane localization of various ion transporters and channels.^8–14) Interestingly, type 1 phosphatidylinositol 4-phosphate 5-kinase (PI4P5K), a major enzyme in the production of intracellular PtdIns(4,5)P2,^15,16) is one of the downstream effectors of RhoA.^17,18) Furthermore, since ROCK also modulates PI4P5K kinase activity,^16,19) the RhoA/ROCK pathway plays a significant role in PtdIns(4,5)P2 production.^16,17,19,20) Of particular note is that PtdIns(4,5)P2 is an essential factor in the dissociation and subsequent membrane translocation and activation of ERM, thereby allowing ERM to exert their linker protein activities.^21–26) These previous observations raise the possibility that PtdIns(4,5)P2 has potential as a therapeutic target aimed at controlling P-gp expression, via ERM.

Here, we examined the involvement of PtdIns(4,5)P2 in the mechanism by which repeated oral treatment with ETP increases the levels of small intestinal P-gp, and we determined which protein within ERM contributes to this phenomena.

MATERIALS AND METHODS

Ethics Approval of the Study Protocol

All procedures were in accordance with the Guiding Principles for the Care and Use of Laboratory Animals as adopted by the Japanese Pharmacological Society. The study protocol was approved by the Animal Ethics Committee of Kobe Gakuin University, Kobe, Japan (approval number: A090130-1).

Animals

Male ddY mice (4–5 weeks old; Japan SLC, Inc., Shizuoka, Japan) were housed in an animal room maintained at 24°C and 55±5% humidity on a 12 h light–dark cycle (light phase, 08:00–20:00). Mice were provided with food and water ad libitum.

Reagents for Immunoblotting

The reagents used in the immunoprecipitation and Western blot analyses were skimmed milk (Wako Pure Chemical Industries, Ltd., Osaka, Japan); bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, U.S.A.); the primary antibodies for ezrin (rabbit polyclonal antibody ab41672, 1 : 8000 dilution; Abcam plc., Cambridge, U.K.), radixin (rabbit monoclonal antibody clone EP1862Y, 1 : 5000 dilution; Abcam plc.), moesin (rabbit monoclonal antibody clone EPR2428 (2), 1 : 8000 dilution; Abcam plc.), P-gp (mouse monoclonal antibody C219, 1 : 200 dilution; Merk Millipore KGaA, San Diego, CA, U.S.A.), PtdIns(4,5)P2 (mouse monoclonal antibody 2C11, 1 : 20000 dilution; Abcam) or actin (goat polyclonal antibody clone SC-1616, 1 : 2000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.); horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) secondary antibody (Kirkegaard and Perry Laboratories, Guildford, U.K.); anti-goat IgG secondary antibody (Kirkegaard and Perry Laboratories) or anti-rabbit IgG secondary antibody (Kirkegaard and Perry Laboratories).

Drug Administration

The drugs used in this study were etoposide phosphate (ETP; Sequoia Research Products, Pangbourne, U.K.), rosuvastatin calcium (rosuvastatin; Toronto Research Chemicals Inc., North York, ON, Canada) and fasudil hydrochloride (fasudil; Tocris Bioscience, Bristol, U.K.). All drugs were dissolved in water. Each group was compared against a vehicle (water; 0.1 mL/10 g body weight) only group. Mice were treated with ETP (10 mg/kg, per os (p.o.)) or the vehicle once a day for 5 d. Rosuvastatin (5 mg/kg, p.o.), fasudil (5 mg/kg, p.o.) or the vehicle only were each co-administered with ETP. The drug doses and the experimental schedule used here were chosen based on our previous publications.^1–3,27)

Preparation of Membrane Fractions from the Ileal Mucosa

These experiments were conducted as described previously.^27–29) The protein concentrations of the obtained fractions were measured using the Lowry method (DC Protein Assay kit; Bio-Rad, Hercules, CA, U.S.A.). The membrane fractions were used for subsequent immunoprecipitation, dot blot or Western blot analyses.

Dot Blot Analysis

This experiment was performed as described previously^30–32) with some modifications. Briefly, using a narrow-mouth pipette tip, the ileal membrane fractions suspended in chilled Tris buffer containing 20 mM Tris–HCl (pH 7.4), 2 mM ethylenediamine tetraacetic acid and 0.5 mM ethylene glycol tetraacetic acid (0.10 µg total protein dose in 2 µL) were spotted slowly onto the nitrocellulose membrane at the center of each grid drawn by pencil. The membranes were then blocked with blocking buffer containing 5% skimmed milk (Wako Pure Chemical Industries, Ltd.) in TBS for 60 min at room temperature. Next, membranes were incubated with primary antibodies for PtdIns(4,5)P2 in blocking buffer overnight at 4°C with gentle shaking. Membranes were then washed ten times for 3 min each with TBS-T. Membranes were subsequently incubated with HRP-conjugated anti-mouse secondary antibody (1 : 10000 dilution) for 60 min at room temperature with gentle shaking and then washed ten times for 3 min each with TBS-T. The immunoreactive dots were visualized using the Light Capture system (ATTO, Tokyo, Japan) together with the Pierce Western blotting substrate (Thermo Scientific, Rockford, IL, U.S.A.). The densitometric intensity of the dot blot bands were calculated using image analysis software (Image J; NIH, Bethesda, MD, U.S.A.).

Immunoprecipitation Assays

These experiments were performed as described previously^3,33) with some modifications. Anti-PtdIns(4,5)P2 antibody (1 : 100 dilution) were applied to the supernatant obtained after purifying the ileal plasma membrane fractions (2000 µg of crude protein) by mixing with Protein A Sepharose 4 Fast Flow (GE Healthcare, Ltd., Little Chalfont, U.K.). Then, Protein A Sepharose 4 Fast Flow was added to the resulting fractions to react with the complex of PtdIns(4,5)P2 antibody and proteins co-immunoprecipitated with PtdIns(4,5)P2. After washing, the supernatant was used for Western blot analysis to detect the molecular interactions between PtdIns(4,5)P2 and P-gp with actin as well as each of the ERM proteins. The fractions, including proteins co-immunoprecipitated with PtdIns(4,5)P2, were heated for 7 min at 97°C and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5% w/v). Subsequent procedures were carried out under the same experimental conditions as described in our previous publication.⁶⁾

Immunofluorescence Analysis

Mice ileal tissue isolation and fixation followed by embedding and freezing of the tissue, preparation for frozen section, post-fixation and blocking were all performed as described previously.^6,34) The sections were then incubated in specific antibodies against PtdIns(4,5)P2 (1 : 100 dilution), ezrin (1 : 2000 dilution), radixin (1 : 200 dilution) or moesin (1 : 200 dilution) which were diluted in reaction buffer containing 0.01% Tween-20 and 1% BSA in phosphate buffered saline (PBS) overnight at 4°C. The next day, sections were washed with PBS-T and incubated in secondary antibody conjugated with Alexa Fluor 647 (goat polyclonal anti-mouse IgM, 1 : 200; Life Technologies, Inc.) for PtdIns(4,5)P2 or Alexa Fluor 488 (goat polyclonal anti-rabbit IgG, 1 : 200; Life Technologies, Inc., Carlsbad, CA, U.S.A.) for ezrin, radixin or moesin, all of which were diluted in reaction buffer, and incubated at room temperature for 2 h. Finally, sections were washed with PBS-T, then cover-slipped with Fluoromount Plus (Thermo Shandon Inc., Pittsburgh, PA, U.S.A.). Immunoreactive signals were detected with a confocal fluorescence microscope (FV1000; Olympus Corporation, Tokyo, Japan).

Statistical Analysis

Data are expressed as the mean±S.E.M. Statistical significance was assessed using the unpaired Student’s t-test or one-way ANOVA, followed by Scheffé’s test for single or multiple comparisons, respectively. A value of p<0.05 was considered significant.

RESULTS

Effect of Rosuvastatin or Fasudil on Increased PtdIns(4,5)P2 Expression in the Ileal Membrane after Repeated Oral Treatment with ETP

At 24 h after completing repeated oral treatment with ETP or vehicle only for 5 d, the expression levels of PtdIns(4,5)P2 in the ileal membrane were significantly higher than that of mice receiving vehicle-only treatment. The increased expression of PtdIns(4,5)P2 induced by ETP treatment was however suppressed upon co-administration of rosuvastatin (Fig. 1A) or fasudil (Fig. 1B) and these expression levels remained at those in vehicle-treated mice.

Fig. 1. Effect of Rosuvastatin or Fasudil on the Increased PtdIns(4,5)P2 Expressions in the Ileal Membrane after Repeated Oral Treatment with ETP

On 24 h after completing repeated oral treatment with ETP with rosuvastatin (A) or fasudil (B) for 5 d, the expression levels of PtdIns(4,5)P2 in the ileal membrane were analyzed by dot blot. Lower panels represent typical dot blot images of PtdIns(4,5)P2. Relative levels of PtdIns(4,5)P2 expressions were determined by using vehicle as a reference. Each column represents the mean±S.E.M. (A) vehicle (water) group n=6; vehicle (rosuvastatin) n=6; ETP (water) n=6; ETP (rosuvastatin) n=6, (B) vehicle (water) group n=6; vehicle (fasudil) n=6; ETP (water) n=6; ETP (fasudil) n=6, ** p<0.01, * p<0.05 vs. vehicle (water), ^##p<0.01, ^#p<0.05 vs. ETP (water), one-way ANOVA and Scheffé’s test.

Changes in the Protein–Protein Complex of PtdIns(4,5)P2 and P-gp with Ezrin, Radixin or Moesin in the Ileal Membrane after Repeated Oral Treatment with ETP

In the vehicle or ETP-treated groups, P-gp and actin with ezrin (Fig. 2A), radixin (Fig. 2B) or moesin (Fig. 2C) were all detected in the immunoprecipitates from the ileal membrane fractions pulled down using anti-PtdIns(4,5)P2 antibody. The amount of P-gp co-immunoprecipitated with PtdIns(4,5)P2 in the ileal membrane of mice exposed to repeated oral treatment with ETP for 5 d was significantly increased compared with that of vehicle-treated mice (Figs. 2D, E or F corresponding to A, B or C, respectively). Furthermore, the radixin levels under the same experimental conditions were also dramatically increased in mice treated with ETP compared with those of vehicle-treated mice (Fig. 2H). In contrast, those of ezrin (Fig. 2G) or moesin (Fig. 2I) were similar between the vehicle- and ETP-treated groups.

Fig. 2. Changes in the Protein–Protein Complex of PtdIns(4,5)P2, P-gp with Ezrin, Radixin or Moesin in the Ileal Membrane after Repeated Oral Treatment with ETP

On 24 h after final administration of repeated oral treatment with ETP or water for 5 d, the protein expressions of P-gp in addition to that of ezrin, radixin or moesin co-immunoprecipitated with anti-PtdIns(4,5)P2 antibody in the ileal membrane fractions were analyzed by using immunoprecipitation analysis followed by Western blot. Upper panel represents typical Western blot images of P-gp (180 kDa) and actin (42 kDa) in addition to ezrin (80 kDa) (A), radixin (80 kDa) (B) or moesin (75 kDa) (C), respectively. Each column represents the mean±S.E.M. Relative levels of P-gp (A-C) and ezrin (A), radixin (B) or moesin (C) protein expressions were analyzed by the ratio of P-gp/actin (D, E or F corresponding to A, B or C, respectively), ezrin/actin (G), radixin/actin (H) or moesin/actin (I), respectively. (A, D, G) vehicle n=4; ETP n=4 (B, E, H) vehicle n=7; ETP n=7 (C, F, I) vehicle n=6; ETP n=6, ** p<0.01, * p<0.05 vs. vehicle, unpaired Student’s t-test.

Immunohistological Co-localization of PtdIns(4,5)P2 with Radixin in the Ileal Tissue

Using antibodies for the detection of PtdIns(4,5)P2 (Fig. 3A) or radixin (Fig. 3B), a significant amount of signal was detected for both proteins in the ileal tissues including the villous surface and sub-epithelial stromal regions. Furthermore, these fluorescence signals were highly co-localized in both regions of the ileal tissue (Fig. 3C).

Fig. 3. Immunohistological Co-localization of PtdIns(4,5)P2 with Radixin in the Ileal Tissue

Immunohistological localization of PtdIns(4,5)P2 with radixin in the ileal tissue of normal mice was evaluated by single or double immunofluorescence staining. (A) magenta; PtdIns(4,5)P2 (1 : 100 dilution), (B) green; radixin (1 : 200 dilution) or (C) merged picture, respectively. Scale bar; 25 µm. (Color images were converted into gray scale.)

DISCUSSION

In this study, we observed that after repeated oral treatment with ETP for 5 d, the expression level of PtdIns(4,5)P2 was markedly increased in the ileal membrane fraction, as was similarly observed for P-gp in our previous publication.²⁾ Several studies have demonstrated that RhoA/ROCK pathway promotes the production of PtdIns(4,5)P2 by activating PI4P5K, a key enzyme in the regulation of PtdIns(4,5)P2 synthesis.^{17–19,35–37)}

To reveal whether this pathway is involved in PtdIns(4,5)P2 production, we performed additional experiments using rosuvastatin (an inhibitor of RhoA activity^27,38)) or fasudil (an inhibitor of ROCK activity^39–41)). The increase in PtdIns(4,5)P2 expression in the ileal membrane of mice exposed to ETP was suppressed by co-administration with either rosuvastatin or fasudil to the levels observed in the vehicle-only groups. We recently confirmed that activation of PI4P5K in the ileum induced by ETP treatment is also suppressed by inhibitors of RhoA or ROCK activity.⁴²⁾ Thus, our present results further support the idea that ETP treatment increases PtdIns(4,5)P2 levels through PI4P5K activation by modulating RhoA/ROCK signaling.⁴²⁾

PtdIns(4,5)P2 is a plasma membrane lipid known to play pivotal roles in a large number of signaling pathways, and acts as a substrate for the generation of potent intracellular second messengers.⁴³⁾ In contrast, some researchers have recently proposed a novel role of PtdIns(4,5)P2 as a potent regulator for the plasma membrane localization of various ion transporters or channels such as Na⁺/Ca²⁺ exchangers,^8,9) epithelial Na⁺ channels^10,11) and so forth.^12–14) In fact, Pochynyuk et al. made the remarkable discovery that activation of PI4P5K and ROCK via RhoA increased PtdIns(4,5)P2 levels, leading to the up-regulation of epithelial Na⁺ channel expression in the renal plasma membrane.¹⁰⁾ These observations raise the possibility that changes in PtdIns(4,5)P2 via RhoA/ROCK signaling may also be involved in the plasma membrane localization of drug transporters such as P-gp.

We therefore performed immunoprecipitation analysis using an anti-PtdIns(4,5)P2 antibody to analyze the molecular interactions between PtdIns(4,5)P2, P-gp and the actin cytoskeleton as well as with each of the ERM proteins. Immunoreactive bands of P-gp and actin with ezrin, radixin or moesin were all detected in these immunoprecipitates. Several studies have clearly demonstrated that PtdIns(4,5)P2 is a critical factor for promoting the dissociation of ERM,^22,25,26) which enables ERM translocation to the plasma membrane where the ERM proteins can interact with various membrane proteins.^44–49) Moreover, we recently demonstrated the existence of molecular interactions between P-gp and each of the ERM proteins in the small intestinal membrane by immunoprecipitation analysis using an anti-P-gp antibody.⁶⁾ Accordingly, PtdIns(4,5)P2 may physically interact with P-gp and the actin cytoskeleton possibly through the ERM proteins in the ileal membrane.

Of particular note is that repeated oral treatment with ETP for 5 d dramatically increased the amounts of radixin and P-gp, both of which were co-immunoprecipitated with PtdIns(4,5)P2, without affecting those of ezrin or moeisn. Since all of ERM proteins has a similar structural and functional characteristics, in particular, a common binding site for PtdIns(4,5)P2,^25,26) at this moment, it seemed to be difficult to clearly explain why ETP treatment specifically increased the number of molecular complex consists of PtdIns(4,5)P2, radixin and P-gp. However, in recent years, our laboratory and others have proposed the potential role of radixin to regulate the plasma membrane localization of P-gp and other membrane transporters not only in the canalicular membrane of the hepatocytes,^50,51) but also in the small intestine.^6,45,52) For example, knockdown of radixin dramatically reduced the plasma membrane localization or functional activity of P-gp compared with that of ezrin or moesin.⁵⁰⁾ Our present and previous study demonstrated that repeated oral treatment with ETP markedly increases the protein levels of P-gp and radixin via RhoA/ROCK signaling (Sup. Figs. 1A, B) as well as amounts of radixin co-immunoprecipitated with P-gp with no influence on ezrin or moesin in the ileal membrane fraction of mice.⁶⁾ Yano et al. recently reported that the plasma membrane localization and functional activity of P-gp in the small intestine were dramatically reduced in mice lacking radixin.⁵²⁾ On the other hands, all of ERM proteins almost equally contribute to the overexpression of P-gp in multi-drug resistance cancer cell lines.⁴⁴⁾ Accordingly, whether certain membrane proteins decide which to choose out of the ERM as each partner for their plasma membrane localization may be dependent on the kinds of tissues and/or membrane protein itself.^{6,45,48–50,52)} It also remains to be determined what increases in the amount of both radixin and P-gp bound to PI(4,5)P2 means under our experimental condition. Because of increases in the both protein expressions of the input as shown in our previously study,⁶⁾ it may be merely due to that contact probability of P-gp and radixin could be simply elevated, which may in turn magnifies the number of molecular complex at the small intestinal membrane. As Nakano et al. proposed in more recent study,⁴⁹⁾ another possibility is that ERM proteins (radixin at least in this case) are likely to have a potential to affect the membrane trafficking of P-gp from the intracellular pool to the plasma membrane^53–55) that is not merely caused by increment of their expressions, leading to an increase in the molecular complex production. In order to solute this unsettled question, further research is needed to analyze the mechanism by which ERM proteins increase the plasma membrane localization of P-gp not restricted in the small intestine but also in other tissues.

Other question is why ETP treatment increased protein expression of radixin in the ileal membrane. At this time, with the exception of a bit of article,⁵⁶⁾ there is a very little clear evidence about the transcriptional factor for radixin as well as ezrin and moesin which regulates their mRNA synthesis. Therefore, we cannot make a definite conclusion with respect to the influence of ETP treatment on the alteration of radixin mRNA level, however, we have a strong desire for future research identifying the critical transcriptional factors for ERM proteins. Taken together, these previous findings support our present conclusion that an increase in the amount of the molecular complex consists of PtdIns(4,5)P2 and radixin may, at least in part, involved in the regulation of P-gp levels in the ileal membrane following ETP treatment.

Several recent studies using histological analyses have suggested that in the small intestine, ezrin or moesin are predominantly localized on the epithelial cells of villi surfaces or the endothelial cells of the sub-epithelial capillaries, respectively.^48,57,58) By contrast, radixin appears to be widely distributed in the epithelial and/or endothelial cells of the small intestine as shown by immunofluorescence studies.^45,57,59) In fact, by means of immunofluorescence analyses, we confirmed that radixin positive signals were broadly distributed around the ileal tissue, while ezrin or moesin positive signals were limited on the epithelial regions of the villous surface or the endothelial cells located on the sub-epithelial regions, respectively (Sup. Figs. 2A, B). More critically, PtdIns(4,5)P2 and radixin were co-localized not only in the the villous surface, but also in the stromal regions of the ileum where we have already confirmed that radixin also co-localizes with P-gp. These results further support our hypothesis that repeated oral treatment with ETP increases the molecular complex including PtdIns(4,5)P2 and radixin, which may in turn contributes to the up-regulation of P-gp in the ileal membrane. Although there is a little evidence, however, regulating the expression and functional activity of drug transporters like P-gp via specific scaffold proteins such as radixin might have a therapeutic benefit to prevent the drug–drug interaction through drug transporters.

In conclusion, this study proposes a novel role of PtdIns(4,5)P2 in the mechanism by which ETP treatment increases P-gp expression in the small intestinal membrane, primarily by increasing the number of protein–protein complex between PtdIns(4,5)P2 especially with radixin in all of ERM (Fig. 4).

Fig. 4. Schematic Drawing of the Increased Expression of P-gp in the Ileal Membrane after Repeated Oral Treatment with ETP

Repeated oral treatment with ETP activates PI4P5K via RhoA/ROCK, leading to up-regulation of PtdIns(4,5)P2 production in the ileal membrane. Then, increased expression of PtdIns(4,5)P2 may recruit radixin especially among ERM, presumably contributing to an increase in the protein expression of P-gp in the ileal membrane.

Acknowledgment

This study was supported, in part, by Grants-in-Aid and Special Coordination Funds from Kobe Gakuin University Joint Research (C).

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