Inhibition of Protein Kinase C Epsilon Causes Ciliated Bovine Bronchial Cell Detachment

Abstract

This study defines the in vitro phenomenon of ciliated bovine bronchial epithelial cell (BBEC) detachment from the basal epithelium and regulation of cilia motility mediated through protein kinase C epsilon (PKCε). The authors determined the time course of activation and downregulation of PKCε by the known PKC activator phorbol 12-myristate 13-acetate (PMA) and demonstrate that chemical inhibition of PKC by calphostin C or the novel PKC isoform inhibitor Ro 31-8220 induced striking detachment of ciliated BBECs from the basal cell monolayer within 1 hour, independent of apoptosis or necrotic cell death. The results of this study support a possible novel PKCε-mediated signaling pathway through which ciliated cell attachment is maintained. Keywords airway epithelium; bronchial epithelial cell; cell detachment; cilia; kinase; protein kinase C epsilonThe protein kinase C (PKC) family of serine/threonine kinases transmits cellular signals via phosphorylation in response to diverse stimuli, regulating biological processes such as proliferation and differentiation. PKCε has been shown in multiple cell types to be activated by second messengers such as diacylglyercerol (DAG), fatty acids, and phosphatidylinositol 3,4,5,-triphosphate (PIP3). The activated kinase translocates from the cytoplasm to the membrane or cytoskeleton in response to DAG or PIP3, whereas binding of certain fatty acids causes translocation to the Golgi network [1]. Specific subcellular localization of PKCε is also enhanced by association with the selective adaptor protein beta'-COP (also called RACK2) within the Golgi apparatus [2], whereas binding to the scaffolding protein receptor of activated C kinase 1 (RACK1) at the cellular membrane can enhance focal adhesion assembly and induce migration and spreading of certain mammalian cell types [3,4]. Phosphorylation by PKCε has been implicated in the regulation of several diverse mammalian systems such as (1) modulation of stress signals via heat-shock proteins [5]; (2) induction of neurite outgrowth [6] MATERIALS AND METHODS Cell Culture and TreatmentsPrimary bovine bronchial epithelial cells (BBECs) were prepared from bovine lung as described previously [18]. Briefly, bronchi were dissected from the lung and digested overnight at 4°C in 0.1% bacterial protease type IV in minimum essential medium (M199 with Earl's salts; Gibco, Carlsbad, CA). The following day, the bronchi were repeatedly rinsed in M199 containing 10% fetal bovine serum (FBS; Gibco) to collect a mixture >95% viable ciliated and basal epithelial cells lining the lumen. These cells were then washed in M199 medium, counted with a hemacytometer, and 3×10 6 cells were plated on 60-mm tissue culture dishes coated with 1% type I collagen (Vitrogen; Cohesion, Palo Alto, CA). BBECs were grown in M199 complete medium containing 10% FBS, 50 U/mL penicillin and streptomycin (Gibco), and 2 μg/mL fungizone (Gibco) in a humidified 95% air/5% CO 2 incubator at 37°C. Confluent monolayers of primary BBECs (a mixed cell population of clumped, ci...

RACK1, a PKC Targeting Protein, Is Exclusively Localized to Basal Airway Epithelial Cells

“…In our localization studies of RACK1 in airway epithelial cells, we anticipated that a targeting protein for PKC might be localized to the apical region of both ciliated cells and basal airway cells, because PKCe is an important regulator of cilia motility and ciliated cell attachment (Slager et al 2006). Contrary to this hypothesis, our IHC results showed that RACK1 is localized exclusively on non-ciliated basal mammalian airway cells.…”

“…In the latter phenomenon, inhibition of PKCe by a novel PKC inhibitor, Ro 31-8220, mimics the effects seen in viral infections or cigarette smoke exposure (Sisson et al 1994), producing widespread detachment of ciliated cells and cilia loss (Slager et al 2006). We were interested in determining how this mechanism of ciliated cell detachment is regulated and hypothesized that RACK1 would be localized to the cilia or apical region of ciliated cells as a Figure 4 RACK1 is not localized to human airway goblet cells.…”

“…We have previously shown that PKCe is an important regulator of several important airway functions, including cytokine release after environmental insult from organic dust (Wyatt et al in press) and ciliated cell detachment from the basal cell layer (Slager et al 2006) after epithelial injury. In the latter phenomenon, inhibition of PKCe by a novel PKC inhibitor, Ro 31-8220, mimics the effects seen in viral infections or cigarette smoke exposure (Sisson et al 1994), producing widespread detachment of ciliated cells and cilia loss (Slager et al 2006).…”

“…For example, we have shown that inhibition of PKCe causes preferential ciliated cell detachment from the basal monolayer in cultured bovine bronchial epithelial cells (BBECs); however, the PKCe-dependent signaling mechanism controlling these events is not well understood (Slager et al 2006). Additionally, we have recently shown that PKCe signaling mediates cytokine release from bronchial epithelial cells in response to organic dust exposure (Wyatt et al in press).…”

Aging causes a slowing in ciliary beat frequency, mediated by PKCε

“…In addition to CBF, SAVA was used to determine the number of motile cilia as previously described (23). Briefly, the number of motile points from each digital video image was determined using a software algorithm in SAVA.…”