Stimulation of protein kinase C-dependent and -independent signaling pathways by bistratene A in intestinal epithelial cells

Abstract

The marine toxin bistratene A (BisA) potently induces cytostasis and differentiation in a variety of systems. Evidence that BisA is a selective activator of protein kinase C (PKC) delta implicates PKC delta signaling in the negative growth-regulatory effects of this agent. The current study further investigates the signaling pathways activated by BisA by comparing its effects with those of the PKC agonist phorbol 12-myristate 13-acetate (PMA) in the IEC-18 intestinal crypt cell line. Both BisA and PMA induced cell cycle arrest in these cells, albeit with different kinetics. While BisA produced sustained cell cycle arrest in G(0)/G(1) and G(2)/M, the effects of PMA were transient and involved mainly a G(0)/G(1) blockade. BisA also produced apoptosis in a proportion of the population, an effect not seen with PMA. Both agents induced membrane translocation/activation of PKC, with BisA translocating only PKC delta and PMA translocating PKC alpha, delta, and epsilon in these cells. Notably, while depletion of PKC alpha, delta, and epsilon abrogated the cell cycle-specific effects of PMA in IEC-18 cells, the absence of these PKC isozymes failed to inhibit BisA-induced G(0)/G(1) and G(2)/M arrest or apoptosis. The cell cycle inhibitory and apoptotic effects of BisA, therefore, appear to be PKC-independent in IEC-18 cells. On the other hand, BisA and PMA both promoted PKC-dependent activation of Erk 1 and 2 in this system. Thus, intestinal epithelial cells respond to BisA through activation of at least two signaling pathways: a PKC delta-dependent pathway, which leads to activation of mitogen-activated protein kinase and possibly cytostasis in the appropriate context, and a PKC-independent pathway, which induces both cell cycle arrest in G(0)/G(1) and G(2)/M and apoptosis through as yet unknown mechanisms.

Fig. 1

IEC-18 cell cycle arrest following exposure to PMA or BisA. Asynchronously growing IEC-18 cells were treated with either 100 nM PMA or 100 nM BisA for the indicated times (C, vehicle control). Cell cycle distribution was determined by flow cytometric analysis. Data shown are representative of three independent experiments.

Fig. 2

Selective translocation of PKC δ to the membrane fraction of intestinal epithelial cells by BisA. Asynchronously growing IEC-18 cell populations were treated with PMA or BisA for the indicated times (C, control). Cytosolic/soluble (S) and membrane (M) fractions were prepared and subjected to western blot analysis, using antibodies specific for individual PKC isozymes. The presence of more than one immunoreactive band in some of the samples likely reflects differential phosphorylation of these molecules [19, 29]. Data shown are representative of three independent experiments.

Fig. 3

Induction of PKC-independent intestinal epithelial cell cycle arrest by BisA. IEC-18 cells were pretreated with 1 μM PDBu for 16 hr to down-regulate PKC α, δ, and ε. (a) Whole cell lysates of untreated (U) or PDBu-pretreated (D) IEC-18 cells were examined by western blot analysis for PKC isozyme expression. (b) Untreated (U) or PKC-depleted (D) cells were exposed to 100 nM PMA or 100 nM BisA for 12 hr, and cell cycle distribution was determined by flow cytometric analysis. C, control. Data shown are representative of three independent experiments.

Fig. 4

Activation of Erk 1 and 2 by PMA and BisA in intestinal epithelial cells. Proliferating IEC-18 cell populations were treated with 100 nM PMA, 100 nM BisA, or vehicle (C) for the indicated times; PMA, BisA, or vehicle was added directly to the plating medium. Whole cell lysates were prepared and subjected to western blot analysis, using an antibody specific for the dually phosphorylated, active forms of Erk 1 and 2. A single representative vehicle control is shown, since changes in Erk phosphorylation were not observed in control cells over the 4-hr experimental period. Data shown are representative of three independent experiments.

Fig. 5

PKC-dependent activation of MAPK by PMA and BisA in IEC-18 cells. Untreated IEC-18 cells or cells pretreated with 1 μM PDBu to deplete PKC α, δ, and ε were exposed to 100 nM PMA or 100 nM BisA for 30 min. Whole cell lysates were prepared and subjected to western blot analysis using an antibody specific for the dually phosphorylated, active forms of Erk 1 and 2. U, untreated; U+, no depletion, treated with PMA or BisA for 30 min; D, PKC-depleted; D+, PKC-depleted, retreated with PMA or BisA for 30 min. Data shown are representative of three independent experiments.