Anchoring of protein kinase A-regulatory subunit IIalpha to subapically positioned centrosomes mediates apical bile canalicular lumen development in response to oncostatin M but not cAMP

Abstract

Oncostatin M and cAMP signaling stimulate apical surface-directed membrane trafficking and apical lumen development in hepatocytes, both in a protein kinase A (PKA)-dependent manner. Here, we show that oncostatin M, but not cAMP, promotes the A-kinase anchoring protein (AKAP)-dependent anchoring of the PKA regulatory subunit (R)IIalpha to subapical centrosomes and that this requires extracellular signal-regulated kinase 2 activation. Stable expression of the RII-displacing peptide AKAP-IS, but not a scrambled peptide, inhibits the association of RIIalpha with centrosomal AKAPs and results in the repositioning of the centrosome from a subapical to a perinuclear location. Concomitantly, common endosomes, but not apical recycling endosomes, are repositioned from a subapical to a perinuclear location, without significant effects on constitutive or oncostatin M-stimulated basolateral-to-apical transcytosis. Importantly, however, the expression of the AKAP-IS peptide completely blocks oncostatin M-, but not cAMP-stimulated apical lumen development. Together, the data suggest that centrosomal anchoring of RIIalpha and the interrelated subapical positioning of these centrosomes is required for oncostatin M-, but not cAMP-mediated, bile canalicular lumen development in a manner that is uncoupled from oncostatin M-stimulated apical lumen-directed membrane trafficking. The results also imply that multiple PKA-mediated signaling pathways control apical lumen development and that subapical centrosome positioning is important in some of these pathways.

Figure 1.

Localization of centrosomes in polarized HepG2 cells. Seventy-two hours after plating, HepG2 cells were stained with anti-γ-tubulin antibody to visualize centrosomes (A) and TRITC-phalloidin to stain apical surfaces (B). In polarized cells, most centrosomes (arrows) localize at the apical surface (D), some of which are positive for PKA-RIIα as shown in the bottom panel (A′, B′, and D′). Nuclei were stained with Hoechst (C and C′). (E–G) A close-up shows that PKA-RIIα (F) is localized at the pericentriolar mesh, surrounding and partially overlapping with the pericentrin-positive centrioles (E). Merged image in G. Bars, 20 μm (A–D′) and 0.5 μm (E–G).

Figure 2.

OSM promotes the association of PKA-RIIα with subapically positioned centrosomes. (A) Polarized HepG2 cells were treated at 37°C for 4 h with 10 ng/ml OSM or 1 mM db-cAMP. Cells were then fixed and immunolabeled for γ-tubulin and labeled with TRITC-phalloidin to determine the number of centrosomes positioned within a 2-μm radius from the apical surface. (B) OSM stimulates the recruitment of PKA-RIIα at the centrosomes in an ERK-, but not PKA-dependent manner. HepG2 cells were treated with db-cAMP or OSM at 37°C for 4 h. In other experiments, cells were treated with OSM in the presence of the MEK inhibitor PD98059 at 50 μM, or with the PKA inhibitors KT5720 at 10 μM or H89 at 10 μM. Cells were then fixed and immunolabeled for PKA-RIIα and γ-tubulin to determine the number of PKA-RIIα–positive centrosomes. (C) Cells were stimulated with OSM in the presence of absence of KT-5720, or H89 or db-cAMP, fixed, and (immuno)labeled for PKA-RIIα and TRITC-phalloidin to determine the number of PKA-RIIα–positive centrosomes within a 2-μm radius from the apical lumen. (D) PD98059 at 50 μM blocks the OSM-stimulated phosphorylation of ERK1/2, evidenced by Western blotting. (E) Western blot showing the expression level of AKAP350 and PKA-RIIα in untreated HepG2 cells and cells treated with 10 ng/ml OSM or 1 mM db-cAMP at 37°C for 4 h. (F) Quantification of the cellular expression of pericentrin in untreated cells and cells treated with OSM or db-cAMP. Quantification was based on the measured fluorescence intensity of immunolabeled coverslips (see Materials and Methods). All data are expressed as mean ± SD of at least three independent experiments. Statistical significance (p < 0.05; indicated with asterisks) was determined using a Student's t test.

Figure 3.

In HepG2 cells stably expressing AKAP-IS peptide centrosomes are positioned away from apical plasma membranes. (A). Parental HepG2 cells or HepG2 cells expressing the AKAP-IS or scrambled (control) peptide were treated with OSM or buffer (control) at 37°C for 4 h and immunolabeled for γ-tubulin and PKA-RIIα to determine the number of PKA-RIIα–positive centrosomes. Note that in cells expressing the AKAP-IS peptide, the association of PKA-RIIα with centrosomes is decreased and OSM is not able to stimulate the anchoring of PKA RIIα to centrosomes. Data are expressed as mean ± SD from three independent experiments. (B) Western blot showing phosphorylated ERK1/2 and total ERK1/2 in HepG2 cells and HepG2 cells expressing the AKAP-IS peptide treated for the indicated time intervals with OSM. (C) HepG2 cells (1 and 3) or HepG2 cells expressing the AKAP-IS peptide (2, 4, and 5) were immunolabeled for MRP2 and β-tubulin (3–5) or MRP2 and pericentrin (1 and 2). Note that in polarized HepG2 cells stably expressing AKAP-IS peptide, centrosomes (arrows) and the microtubule network are perinuclearly oriented. Image 5 is an enlargement of the boxed region in image 4. (E) HepG2 cells or HepG2 cells expressing the AKAP-IS or scrambled peptide were (immuno)labeled for γ-tubulin and TRITC-phalloidin to determine the number of centrosomes within a 2-μm radius from the apical surface. (F) HepG2 cells or HepG2 cells expressing the AKAP-IS or scrambled peptide were (immuno)labeled for PKA-RIIα and TRITC-phalloidin to determine the number of RIIα-positive centrosomes within a 2-μm radius of the apical surface. Data are expressed as mean ± SD from three independent experiments. Statistical significance was determined using a Student's t test. Bar, 10 μm.

Figure 4.

Subcellular distribution of the CE/SAC and ARE in HepG2 cells expressing the AKAP-IS peptide. The CE/SAC (A–D, open arrows; fluorescently labeled as described in Materials and Methods) are located close to the apical lumen (marked with closed arrow) in HepG2 cells (B; corresponding phase contrast in A, respectively). In cells expressing the AKAP-IS peptide, the CE/SAC (D, open arrows) is oriented away from the apical surface (corresponding phase contrast in C). In HepG2 cells, the rab11a-positive ARE (G) remains close to the apical surface (F and H). In HepG2 cells expressing the AKAP-IS peptide, rab11a (K) is also located close to the apical surface (J and L), whereas some immunoreactivity is observed at perinuclear structures reminiscent of centrosomes (K and L, arrows). Nuclei in E and I are stained with 4,6-diamidino-2-phenylindole. Bar, 10 μm.

Figure 5.

Displacement of PKA RIIα form its natural anchoring sites inhibits OSM-stimulated polarity development. Stimulation of parental HepG2 cells or HepG2 cells expressing the scrambled control peptide with either 10 ng/ml OSM, 1 mM db-AMP, or OSM + db-cAMP for 4 h stimulates the development of apical lumens. Expression of AKAP-IS peptide abrogates OSM-, but not db-cAMP-mediated stimulation of polarity. Data are expressed as mean ± SD from three independent experiments. Statistical significance was determined using a Student's t test.

Figure 6.

Basolateral-to-apical transcytosis of C₆-NBD-SM is stimulated in OSM-treated HepG2 cells that express or do not express the AKAP-IS peptide. HepG2 cells expressing the AKAP-IS peptide or not were labeled with 4 μM C₆-NBD-SM for 30 min at 4°C. Subsequently, the cells were washed and incubated with or without OSM for another 30 min at 4°C. Transport was triggered in the presence or absence of OSM by incubating the cells at 37°C. A–D show that the lipid analogue readily reaches the apical surface of (OSM-treated) cells expressing the AKAP-IS peptide, very similar to control HepG2 cells (cf. van der Wouden et al., 2002). After different time intervals, cells were cooled to block transport, and the number of NBD-positive BCs was determined (E). Data are expressed as mean ± SD from three independent experiments. Statistical significance was determined using a Student's t test.