Role of Intestinal Epithelial Cell Responses to Transforming Growth Factor (TGF)-β in Trichinella Spiralis Infection

Abstract

Background: Protein kinase D1 (PKD1) is the founding member of a new protein kinase family within the CAMK group and separate from the previously identified PKCs. In most unstimulated cells, PKD1 is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing cysteine-rich zinc finger-like motifs and a pleckstrin homology domain. PKD1 can be activated within intact cells by multiple stimuli via phosphorylation of Ser744 and Ser748 in the PKD1 activation loop. Recent studies demonstrated rapid PKC-dependent and sustained PKC-independent PKD1 activation in response to G protein-coupled receptor (GPCR) agonists. Here, we tested the hypothesis that sustained PKD1 activation plays a key role in intestinal epithelial cell proliferation. Results: In order to determine the kinetics of PKD family activation by agonists that stimulate endogenous Gq-coupled receptors in intestinal epithelial cells (IEC-18 and IEC-6), these cells were stimulated for various times with either angiotensin II (ANG II) or vasopressin. The kinetics of autophosphorylation of PKD1 and PKD2 (indicative of their activation) were strikingly different. The autophosphorylation of PKD2 declined rapidly toward base-line levels whereas that of PKD1 remained elevated for up to 24h, indicating that PKD1 activation is sustained whereas that of PKD2 is transient in the same intracellular environment. Assays of catalytic activity, multi-site phosphorylation and sub-cellular localization demonstrated that GPCR agonists stimulated sustained PKD1 activation via a sequential mechanism consisting of an early PKC-dependent phase and a late PKC-independent phase in both IEC-18 and IEC-6 cells. The sustained activation of PKD1 coincided with its nuclear localization, raising the possibility that this PKD isoform was preferentially associated to long-term cellular responses, including stimulation of DNA synthesis and cell proliferation. To determine the role of endogenous PKD1 in GPCR-induced mitogenesis in intestinal epithelial cells, we depleted its expression using siRNAs that target specifically this PKD isoform. PKD1 knockdown (90%) prevented c-Fos accumulation, increase in DNA synthesis and cell number induced by the GPCR agonists. Conclusion: Our results support the hypothesis that PKD1 plays a key role in mediating GPCR-induced cell proliferation in cultured intestinal epithelial cells. In an accompanying Abstract, we show that PKD1 also plays a role in promoting crypt cell proliferation In Vivo.