Vasopressin-mediated mitogenic signaling in intestinal epithelial cells

Abstract

Here, we demonstrate that arginine vasopressin (AVP) induces multiple intracellular signal transduction pathways in rat intestinal epithelial IEC-18 cells via a V 1A receptor. Addition of AVP to these cells induces a rapid and transient increase in cytosolic Ca 2ϩ concentration and promotes protein kinase D (PKD) activation through a protein kinase C (PKC)-dependent pathway, as revealed by in vitro kinase assays and immunoblotting with an antibody that recognizes autophosphorylated PKD at Ser 916 . AVP also stimulates the tyrosine phosphorylation of the nonreceptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2) and promotes Src family kinase phosphorylation at Tyr 418 , indicative of Src activation. AVP induces extracellular signal-related kinase (ERK)-1 (p44 mapk ) and ERK-2 (p42 mapk ) activation, a response prevented by treatment with mitogen-activated protein kinase kinase (MEK) inhibitors (PD-98059 and U-0126), specific PKC inhibitors (GF-I and Ro-31-8220), depletion of Ca 2ϩ (EGTA and thapsigargin), selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (tyrphostin AG-1478, compound 56), or the selective Src family kinase inhibitor PP-2. Furthermore, AVP acts as a potent growth factor for IEC-18 cells, inducing DNA synthesis and cell proliferation through ERK-, Ca 2ϩ -, PKC-, EGFR tyrosine kinase-, and Src-dependent pathways. arginine vasopressin; protein kinase D; protein kinase C; Src; proline-rich tyrosine kinase 2; intestinal epithelial proliferation THE SEQUENTIAL PROLIFERATION, lineage-specific differentiation, migration, and cell death of epithelial cells of the intestinal mucosa is a tightly regulated process that is modulated by a broad spectrum of regulatory peptides (8,36,70). The nontransformed IEC-6 and IEC-18 cells, derived from rat small intestinal crypt (56), have provided an in vitro model to examine intestinal epithelial cell migration, differentiation, and proliferation (16,27,58,70). Previous studies demonstrated that the proliferation and migration of these intestinal epithelial cells is regulated by a variety of polypeptide growth factors, including epidermal growth factor (EGF), insulin-like growth factor I, and hepatocyte growth factor, which act via single-pass transmembrane tyrosine kinase receptors (3,17,51). Neuropeptides and vasoactive peptides that signal through G protein-coupled receptors (GPCRs), characterized by seven-transmembrane helices, also act as potent cellular growth factors for a variety of cell types (63,64,66,67). However, the role of GPCRs and their ligands in intestinal epithelial cell signaling and proliferation remains poorly understood.The neurohypophysial nonapeptide arginine vasopressin (AVP), also known as antidiuretic hormone, is traditionally recognized for its role as a vasoconstrictor hormone acting on vascular smooth muscle cells and its antidiuretic effect via the renal collecting system. In addition to its function in the regulation of body fluid osmolality, vascular tone, and blood pressure, AVP acts as a growth-promo...

Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells

“…1C, closed bars), reflecting YAP translocation from the nucleus to the cytoplasm in most cells of the population. Rapid nuclear-cytoplasmic shuttling of YAP was also provoked by stimulation of IEC-18 cells with the GPCR agonist vasopressin (39) or by serum growth factors (Fig. 1D).…”

“…These cells endogenously express G␣ q/11 -coupled receptors for angiotensin II (ANG II) and vasopressin (23,39,40,(43)(44)(45)(46) and have been extensively used as a model system to examine signal transduction pathways in response to GPCR activation (23,36,38,39,(43)(44)(45)47). ANG II and vasopressin act as potent growth factors for IEC-18 and IEC-6 cells (23,39,(43)(44)(45)47). Because the levels of YAP/TAZ influence their association with transcriptional cofactors especially when expressed at elevated levels, we studied the localization, phosphorylation, and activity of the endogenous YAP protein.…”

ANG II and LPA induce Pyk2 tyrosine phosphorylation in intestinal epithelial cells: role of Ca2+, PKC, and Rho kinase

“…The IEC-18 and IEC-6 small intestinal cell lines have provided a useful model for studying these functions in culture (7,9,33,40,55). Although growth factor-induced proliferation and motility have been described in these cells, the associated intracellular signaling mechanisms are not well characterized.…”

“…The nontransformed IEC-18 and IEC-6 intestinal epithelial cell lines, derived from rat small intestinal crypt epithelium (40), have been widely used as a model of epithelial cell proliferation, migration, and differentiation (7,9,33,55). In the "artificial wound" model of epithelial restitution, IEC-6 cell migration is promoted by transforming growth factors-␣ and -␤ (TGF-␣ and -␤), epidermal growth factor (EGF), and lysophosphatidic acid (LPA).…”

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

“…Importantly, AVP acts as a growth factor inducing hypertrophy and cell growth in a variety of cell types [8][9][10][11][12][13]. AVP-stimulated cellular responses are mediated by three AVP receptors subtypes (V 1 , V 2 and V 3 ), which belong to the superfamily of G-protein-coupled…”