RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells.
In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.
Jiang
Xiaohua
X
Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine, CURE: Digestive Diseases Research Center and Molecular Biology Institute, University of California, Los Angeles, California 90095-178622, USA.
Jacamo
Rodrigo
R
Zhukova
Elena
E
Sinnett-Smith
James
J
Rozengurt
Enrique
E
D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects*; D003599:Cytoskeleton / Q000201:enzymology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007422:Intestines / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008246:Lysophospholipids / Q000494:pharmacology*; D011554:Pseudopodia / Q000201:enzymology; D034622:RNA Interference
D008246:Lysophospholipids; D051416:Focal Adhesion Protein-Tyrosine Kinases; C032881:lysophosphatidic acid