Oncostatin M-stimulated Apical Plasma Membrane Biogenesis Requires p27^Kip1-Regulated Cell Cycle Dynamics

Abstract

Oncostatin M regulates membrane traffic and stimulates apicalization of the cell surface in hepatoma cells in a protein kinase A-dependent manner. Here, we show that oncostatin M enhances the expression of the cyclin-dependent kinase (cdk)2 inhibitor p27 Kip1 , which inhibits G 1 -S phase progression. Forced G 1 -S-phase transition effectively renders presynchronized cells insensitive to the apicalization-stimulating effect of oncostatin M. G 1 -S-phase transition prevents oncostatin M-mediated recruitment of protein kinase A to the centrosomal region and precludes the oncostatin M-mediated activation of a protein kinase A-dependent transport route to the apical surface, which exits the subapical compartment (SAC). This transport route has previously been shown to be crucial for apical plasma membrane biogenesis. Together, our data indicate that oncostatin M-stimulated apicalization of the cell surface is critically dependent on the ability of oncostatin M to control p27 Kip1 /cdk2-mediated G 1 -S-phase progression and suggest that the regulation of apical plasma membrane-directed traffic from SAC is coupled to centrosome-associated signaling pathways. INTRODUCTIONThe establishment and maintenance of apical and basolateral plasma membrane (PM) domains in epithelia require a careful orchestration of extra-and intracellular signals, triggering crucial cytoskeleton-, organelle-, and membrane traffic-linked machineries, as partly elucidated in systems as diverse as yeast, Drosophila, and mammalian cells (Lecuit and Pilot, 2003;Mostov et al., 2003;Baas et al., 2004). Impairment of these events may cause a loss of apical-basolateral asymmetry and may lead to deranged cell growth (Bilder et al., 2000;Vermeer et al., 2003;Baas et al., 2004) or spindle misorientation . Recent studies have reported an evolutionary conserved genetic program that may link the process of polarity development and proliferation (Bilder et al., 2000;Humbert et al., 2003;Rolls et al., 2003;Klezovitch et al., 2004). However, many aspects of such a link remain unclear, e.g., the extent to which cell proliferation can directly affect epithelial cell organization or vice versa. Indeed, epithelial cell proliferation and apical-basolateral polarity may be controlled by different signaling pathways (Liu et al., 2004).Signaling cascades stimulated by interleukin-6 family cytokines, including oncostatin M (OSM), elicit multiple responses in the cell. Originally, as its name indicates, oncostatin M was primarily recognized to inhibit the proliferation of tumor cells (Tanaka and Miyajima, 2003), presumably by modulating cell cycle regulatory proteins, including the cyclin-dependent kinase (cdk)2-inhibitor p27 Kip1 (Kortylewski et al., 1999;Klausen et al., 2000;Li et al., 2001). Disruption of the regulatory system that controls G 1 -phase progression is a common event in human hepatocarcinogenesis (Hui et al., 1998), and p27 Kip1 has been associated with differentiation, invasiveness, and metastasis of hepatocellular carcinoma tumors (Zhou et al., 2003). ...

Anchoring of Protein Kinase A-Regulatory Subunit IIα to Subapically Positioned Centrosomes Mediates Apical Bile Canalicular Lumen Development in Response to Oncostatin M but Not cAMP

“…In both AKAP-ISexpressing and control HepG2 cells the ratio BC/100 cells was ϳ15 (Figure 5), which, when taken into account that at least two cells participate in a single BC, means that at least 30% of the cells in the culture were polarized. Treatment of parental HepG2 cells or HepG2 cells expressing the scrambled peptide with OSM stimulated the number of apical lumens with ϳ30% (p Ͻ 0.05) (Figure 5), consistent with previous results (van der Wouden et al, 2002;van IJzendoorn et al, 2004a). Also, db-cAMP stimulated apical lumen development, evidenced by an increase in their numbers and circumference ( Figure 5).…”

“…Because the expression of the AKAP-IS peptide prevented the OSM-stimulated association of PKA-RII␣ with centrosomes ( Figure 3A), we next examined whether expression of the AKAP-IS peptide also prevented the previously reported (van der Wouden et al, 2002;van IJzendoorn et al, 2004a) stimulation of apical bile canalicular lumen development, i.e., cell polarity, by OSM. For this, AKAP-IS-expressing and control HepG2 cells (i.e., parental cells or HepG2 cells expressing the scrambled peptide) were treated with OSM or buffer (control) at 37°C for 4 h, fixed, and labeled with TRITC-conjugated phalloidin to identify BC and the nuclear stain Hoechst.…”

“…To confirm that the OSM-stimulated association of PKA-RII␣ with centrosomes occurs via an AKAP, and to investigate whether the OSM-stimulated association of PKA-RII␣ with centrosomes plays a role in OSM-stimulated bile canalicular lumen development (van der Wouden et al, 2002;van IJzendoorn et al, 2004a), HepG2 cells were stably transfected with epitope-tagged AKAP-IS, a peptide that specifically binds to PKA-RII with high affinity and displaces it from its natural anchoring sites, or, as a control, stably transfected with an epitope-tagged nonfunctional scrambled peptide (details with regard to these peptides are described in Alto et al, 2003). Stable transfectants were selected, and they were shown to express AKAP-IS or the scrambled peptide and displace PKA-RII␣ from natural anchoring sites or not, respectively (cf.…”

Formation of E-Cadherin/β-Catenin–based Adherens Junctions in Hepatocytes Requires Serine-10 in p27(Kip1)

“…In hepatocytes, the interleukin 6-family cytokine oncostatin M promotes p27 stability, inhibits cell proliferation, and stimulates cell-cell adhesion by promoting the mobilization of two main components of adherens junctions, E-cadherin and ␤-catenin, to the lateral cell surface (Klausen et al, 2000;Matsui et al, 2002;van IJzendoorn et al, 2004). To better correlate the different cellular effects, we exposed human hepatic HepG2 cells for a relatively short-term (4 h) to on-costatin M (10 ng/ml).…”

“…This effect of OSM on adherens junction formation is believed to be part of the complex mechanism by which OSM signaling controls liver development (Kamiya et al, 1999;Kinoshita and Miyajima, 2002), which also includes the establishment of apical-basolateral polarity (van der Wouden et al, 2002;Wojtal et al, 2007;van IJzendoorn et al, 2004). OSM, via the signal transducer protein gp130, typically stimulates two major signaling pathways that involve Ras/mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT; Heinrich et al, 1998).…”