Abstract
We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.
Highlights
The mammalian intestine is covered by a single layer of epithelial cells that is renewed every 4 –5 days along the cryptvillus axis [1]
G protein-coupled receptor (GPCR) Agonists Induce Protein kinase D1 (PKD1) and PKD2 Activation with Different Kinetics in IEC-18 Cells—To determine the kinetics of PKD activation by agonists that stimulate endogenously expressed Gq-coupled receptors in intestinal epithelial IEC-18 cells, cultures of these cells were stimulated for various times (2.5–240 min) with either angiotensin II (ANGII) (Fig. 1A) or vasopressin (Fig. 1B)
Concluding Remarks—The sequential proliferation, lineagespecific differentiation, crypt-villus migration, and cell death of the epithelial cells of the intestinal mucosa is a highly regulated process involving a broad range of regulatory peptides, differentiation signals, and luminal stimuli
Summary
Stock cultures of IEC-18 and IEC-6 cells [38, 39] were maintained at 37 °C in DMEM supplemented with 5% fetal bovine serum in a humidified atmosphere containing 10% CO2 and 90% air. IEC-18 or IEC-6 cells were seeded in 35-mm dishes at a density of 2 ϫ 105 cells/dish the day before transfection. Our previous studies established that these cells express Gq-coupled receptors for angiotensin II (ANGII) and vasopressin (13, 40 – 44)
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