Background information. The c-Met-dependent, β-actin-rich, blebbed pseudopodia of MSV-MDCK-INV (invasive Moloney-sarcoma-virus-transformed Madin-Darby canine kidney) cells are induced by Rho/ROCK (Rho kinase) activation, and are morphologically distinct from flat extended lamellipodia.Results. Microtubules were shown to extend to these actin-rich pseudopodial domains, and microtubule depolymerization by nocodazole treatment resulted in progressive cellular blebbing, initiating in the pseudopodial domains and resulting in transient cellular rounding and blebbing after 30 min. The blebbing response was dependent on autocrine HGF (hepatocyte growth factor) activation of c-Met and prevented by inhibition of RhoA, ROCK and p38 MAPK (p38 mitogen-activated protein kinase), but not ERK (extracellular-signal-regulated kinase) or PI3K (phosphoinositide 3-kinase). Phospho-p38 MAPK was present in pseudopodia, localizing activation of this signalling pathway to this protrusive membrane structure. In serum-starved cells, LPA (lysophosphatidic acid) activation of RhoA induced p38 MAPK-dependent pseudopodial protrusions, and inhibition of p38 MAPK prevented pseudopodial protrusion and displacement of MSV-MDCK-INV cells. MSV-MDCK-INV cells exhibited intermittent blebbing and rounding, which may represent an integral part of their motile behaviour.Conclusions. The localized activation of an autocrine HGF/c-Met loop regulates Rho/ROCK activation of p38 MAPK signalling to stimulate both membrane blebbing and pseudopod formation.

A NHE1 variant that exhibits very high resistance to (3-methyl sulfonyl-4-piperidinobenzoyl) guanidine methane sulfonate (HOE694), a potent inhibitor of Na(+)-H(+) exchangers, was selected and characterized. Sequencing of the coding region corresponding to the N-terminal domain of this variant revealed the presence of only one mutation located within membrane-spanning segment 9 (M9). This base pair change replaces a glutamate (Glu) with an aspartate (Asp). We reproduced this amino acid change in wild-type NHE1 and found that this mutation alone is responsible for the huge decrease in sensitivity to the HOE694 compound and to ethylisopropylamiloride (EIPA). We found that the NHE1-Glu(346)Asp mutant was more than 2000-fold more resistant to HOE694 and up to 300-fold more resistant to EIPA than wild-type NHE1, with the size, rather than the charge, of the amino acid in position 346 having the greatest effect. Interestingly, its affinity for Na(+) was at least 4-fold lower than that of wild-type NHE1. Mutation of amino acids in the vicinity of Glu(346) did not change the sensitivity of mutated NHE1 proteins to inhibitors. We suggest there is a direct interaction of Glu(346) or involvement of Glu(346) in a coordination site with NHE inhibitors and with Na(+).

Cell motility is required for physiological processes of wound repair and organogenesis as well as for the pathologic process of tumor invasion; a critical element of cell motility and invasion is the de novo polarized protrusion of pseudopodia via localized actin polymerization (1-5). Pseudopodia formation is associated with cell motility in vitro and tumor cell motility in vivo, and pseudopodial protrusion of surrounding extracellular matrix has been well characterized as an essential element of tumor cell invasion (6 -15).Hepatocyte growth factor (HGF) 1 /scatter factor, secreted by cells of mesodermal and mesenchymal origin, was originally identified for its ability to disrupt epithelial cell-cell interactions and to trigger invasive growth (16 -23). HGF exhibits powerful mitogenic, motogenic, and morphogenic activities on epithelial and endothelial cells expressing the HGF receptor (HGF-R), also known as the met proto-oncogene (17, 24), whose two end results are the modification of the actin cytoskeleton and the disruption of epithelial cell-cell adhesions (16). HGF-R activation promotes receptor auto-phosphorylation on tyrosine residues and activation of downstream signaling events including the ras (25), phosphatidylinositol 3-kinase (26, 27) phospholipase C-␥ (28), and mitogen-activated protein kinase (29) related pathways. Deregulated control of the invasive-growth phenotype by oncogenically activated Met confers invasive and metastatic properties to cancer cells (18,22). Breast (30), ovarian (31), prostate (32), gastric (33), thyroid (34), hepatocellular (35), and renal (36) carcinomas as well as osteosarcoma (37, 38) and myeloma (39) are indeed associated with HGF-R overexpression or increased HGF-R tyrosine phosphorylation. Notably, protein overexpression was found to be associated with amplification of the met gene in only a few primary carcinomas, but in a significant proportion of the metastases examined (18,33,38). Targeting of a constitutively active Tpr-Met to the plasma membrane via a c-Src myristoylation signal induces enhanced cellular transformation and the formation of cellular protrusions in MDCK cells (40). However, whether the acquisition of a motile and metastatic phenotype by tumor cells due to HGF-R activation is indirectly due to disruption of epithelial cell-cell contacts and induction of an epithelial-mesenchymal transformation or whether autocrine HGF-R activation specifically induces cell motility and, more particularly, pseudopodial protrusion, has yet to be directly demonstrated.To determine the molecular basis for the acquisition of motile and invasive properties following transformation of polarized epithelial cells, we have established a model system based on

The Na(+)/H(+) exchanger NHE1 is involved in intracellular pH homeostasis and cell volume regulation and accumulates with actin in the lamellipodia of fibroblasts. In order to determine the role of NHE1 following epithelial transformation and the acquisition of motile and invasive properties, we studied NHE1 expression in polarized MDCK cells, Moloney Sarcoma virus (MSV) transformed MDCK cells and an invasive MSV-MDCK cell variant (MSV-MDCK-INV). Expression of NHE1 was significantly increased in MSV-MDCK-INV cells relative to MSV-MDCK and MDCK cells. NHE1 was localized with b-actin to the tips of MSV-MDCK-INV cell pseudopodia by immunofluorescence. Sensitivity of NHE1-mediated (22)Na uptake to ethylisopropylamiloride, a specific inhibitor of NHE1, was increased in MSV-MDCK cells relative to MDCK cells. Changes in intracellular pH induced upon EIPA treatment were also of higher magnitude in MSV-MDCK and MSV-MDCK-INV cells compared to wild-type MDCK cells, especially in Hepes-buffered DMEM medium. Inhibition of NHE1 by 50 microM ethylisopropylamiloride induced the disassembly of actin stress fibers and redistribution of the actin cytoskeleton in all cell types. However, in MSV-MDCK-INV cells, the effect of ethylisopropylamiloride treatment was more pronounced and associated with the increased reversible detachment of the cells from the substrate. Videomicroscopy of MSV-MDCK-INV cells revealed that within 20 minutes of addition, ethylisopropylamiloride induced pseudopodial retraction and inhibited cell motility. The ability of ethylisopropylamiloride to prevent nocodazole-induced formation of actin stress fibers in MSV-MDCK cells was more pronounced in Hepes medium relative to NaHCO(3) medium, showing that NHE1 can regulate actin stress fiber assembly in transformed MSV-MDCK cells via its intracellular pH regulatory effect. These results implicate NHE1 in the regulation of the actin cytoskeleton dynamics necessary for the adhesion and pseudopodial protrusion of motile, invasive tumor cells.

Liver cell pH and volume regulation are perturbed by prolonged cold storage in University of Wisconsin solution and subsequent rewarming, but the molecular basis of this effect remains unknown. We prepared membranes from hepatocytes subjected to variable periods of cold preservation with or without subsequent rewarming and probed them by Western blotting with specific antibodies against the Na+ -H+ exchanger isoform NHE-1 and the Na+ -K+ ATPase alpha subunit. Results were compared with the content of GLUT-2, an abundant basolateral protein. NHE-1 decreased significantly as cold preservation times exceeded 10 h. Subsequent rewarming by short-term culture at 37 degrees C did not further reduce this parameter. On the other hand, expression of Na+ -K+ ATPase remained stable during cold storage times lasting up to 48 h, whereas rewarming resulted in a dramatic reduction in cells cold preserved beyond 10 h. In contrast, the membrane content of GLUT-2 was unaffected by cold preservation with or without subsequent rewarming. The results indicate that cold storage and rewarming respectively and selectively modulate the expression of specific hepatocellular membrane transport proteins.