blister and lysophosphatidic-acid

The enhanced migration found in tumor cells is often caused by external stimuli and the sequential participation of cytoskeleton-related signaling molecules. However, until now, the molecular connection between the lysophosphatidic acid (LPA) receptor and nonmuscle myosin II (NM II) has not been analyzed in detail for LPA-induced migration. Here, we demonstrate that LPA induces migration by activating the LPA(1) receptor which promotes phosphorylation of the 20 kDa NM II light chain through activation of Rho kinase (ROCK). We show that LPA-induced migration is insensitive to pertussis toxin (PTX) but does require the LPA(1) receptor as determined by siRNA and receptor antagonists. LPA activates ROCK and also increases GTP-bound RhoA activity, concomitant with the enhanced membrane recruitment of RhoA. LPA-induced migration and invasion are attenuated by specific inhibitors including C3 cell-permeable transferase and Y-27632. We demonstrate that NM II plays an important role in LPA-induced migration and invasion by inhibiting its cellular function with blebbistatin and shRNA lentivirus directed against NM II-A or II-B. Inhibition or loss of either NM II-A or NM II-B in 4T1 cells results in a decrease in migration and invasion. Restoration of the expression of NM II-A or NM II-B also rescued LPA-induced migration. Taken together, these results suggest defined pathways for signaling through the LPA(1) receptor to promote LPA-mediated NM II activation and subsequent cell migration in 4T1 breast cancer cells.

Topics: Amides; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Female; Heterocyclic Compounds, 4 or More Rings; Lysophospholipids; Mice; Myosin Light Chains; Pertussis Toxin; Phosphorylation; Pyridines; Receptors, Lysophosphatidic Acid; rho-Associated Kinases; rhoA GTP-Binding Protein; RNA, Small Interfering; Signal Transduction

Three-dimensional (3D) cell-matrix cultures provide a useful model to analyze and dissect the structural, functional, and mechanical aspects of cell-matrix interactions and motile behavior important for cell and tissue morphogenesis. In the current studies we tested the effects of serum and physiological growth factors on the morphogenetic behavior of human fibroblasts cultured on the surfaces of 3D collagen matrices. Fibroblasts in medium containing serum contracted into clusters, whereas cells in medium containing platelet-derived growth factor (PDGF) were observed to migrate as individuals. The clustering activity of serum appeared to depend on lysophosphatidic acid, required cell contraction based on inhibition by blocking Rho kinase or myosin II, and was reversed upon switching to PDGF. Oncogenic Ras transformed human fibroblasts did not exhibit serum-stimulated cell clustering. Our findings emphasize the importance of cell-specific promigratory and procontractile growth factor environments in the differential regulation of cell motile function and cell morphogenesis.

Topics: Actins; Amides; Cell Aggregation; Cell Culture Techniques; Cell Line, Transformed; Cell Movement; Cell Shape; Collagen; Enzyme Inhibitors; Fibroblasts; Heterocyclic Compounds, 4 or More Rings; Humans; Intercellular Signaling Peptides and Proteins; Lysophospholipids; Male; Myosin Type II; Oncogene Protein p21(ras); Platelet-Derived Growth Factor; Pyridines; rho-Associated Kinases; Serum; Sphingosine; Vinculin

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.

Topics: Actomyosin; Adherens Junctions; Amides; Animals; Cell Adhesion Molecules; Cell Line; Chlorocebus aethiops; COS Cells; Cytoskeletal Proteins; Dogs; Enzyme Inhibitors; Heterocyclic Compounds, 4 or More Rings; Lysophospholipids; Membrane Proteins; Mice; Microscopy, Electron, Transmission; Myosin Type II; Nonmuscle Myosin Type IIB; Occludin; Platelet-Derived Growth Factor; Pyridines; Rats; rho GTP-Binding Proteins; RNA, Small Interfering; Tight Junctions; Vinculin