It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar protontranslocating ATPase (V-ATPases) at the cell surface, whereas the poorly invasive MCF7 cell line does not. Bafilomycin, a specific V-ATPase inhibitor, reduces the in vitro invasion of MB231 cells but not MCF7 cells. Targeting of V-ATPases to different cellular membranes is controlled by isoforms of subunit a. mRNA levels for a subunit isoforms were measured in MB231 and MCF7 cells using quantitative reverse transcription-PCR. The results show that although all four isoforms are detectable in both cell types, levels of a3 and a4 are much higher in MB231 than in MCF7 cells. Isoform-specific small interfering RNAs (siRNA) were employed to selectively reduce mRNA levels for each isoform in MB231 cells. V-ATPase function was assessed using the fluorescent indicators SNARF-1 and pyranine to monitor the pH of the cytosol and endosomal/lysosomal compartments, respectively. Cytosolic pH was decreased only on knockdown of a3, whereas endosome/lysosome pH was increased on knockdown of a1, a2, and a3. Treatment of cells with siRNA to a4 did not affect either cytosolic or endosome/lysosome pH. Measurement of invasion using an in vitro transwell assay revealed that siRNAs to both a3 and a4 significantly inhibited invasion of MB231 cells. Immunofluorescence staining of MB231 cells for V-ATPase distribution revealed extensive intracellular staining, with plasma membrane staining observed in ϳ18% of cells. Knockdown of a4 had the greatest effect on plasma membrane staining, leading to a 32% reduction. These results suggest that the a4 isoform may be responsible for targeting V-ATPases to the plasma membrane of MB231 cells and that cell surface V-ATPases play a significant role in invasion. However, other V-ATPases affecting the pH of the cytosol and intracellular compartments, particularly those containing a3, are also involved in invasion.The leading cause of mortality from cancer is metastasis, making inhibition of metastasis an important strategy in controlling cancer progression. The metastatic cascade involves a series of steps that include escape of cells from the site of the primary tumor into the circulation or lymphatic system and the extravasation of cells from the circulation or lymphatic system into secondary sites (1, 2). Both of these processes require the tumor cells to display an invasive phenotype in which they degrade extracellular matrix of the surrounding tissue. The vacuolar H ϩ -ATPases (or V-ATPases) 2 are a family of ATP-dependent proton pumps that have been implicated in tumor cell invasion (3). V-ATPases are up-regulated in tissue samples from highly invasive pancreatic carcinomas (4), and treatment of a human cancer cell line with antisense oligonucleotides to the V-ATPase c subunit decreased invasion in vitro (5). Inhibition of V-ATPase expression in hepatocellular carcinoma cells using siRNAs reduces invasiveness of these cells in vitro and metastasis in vivo (6). In addition, treatment of hig...

Eukaryotic cells have evolved a family of ATP-dependent proton pumps known as the vacuolar (H(+))-ATPases (or V-ATPases) to regulate the pH of intracellular compartments, the extracellular space, and the cytoplasm. V-ATPases present within intracellular compartments are important for such normal cellular processes as receptor-mediated endocytosis and intracellular membrane traffic, protein processing and degradation and coupled transport of small molecules and ions. They also facilitate the entry of a number of envelope viruses and bacterial toxins, including influenza virus and anthrax toxin. V-ATPases present in the plasma membranes of cells are also important in normal physiology. They facilitate bone resorption by osteoclasts, acid secretion by intercalated cells of the kidney, pH homeostasis in macrophages and neutrophils, angiogenesis by endothelial cells, and sperm maturation and storage in the male reproductive tract. In the insect midgut, they establish a membrane potential used to drive K(+) secretion. Plasma membrane V-ATPases are especially important in human disease, with genetic defects in V-ATPases expressed in osteoclasts and intercalated cells leading to the diseases osteopetrosis and renal tubule acidosis, respectively. Plasma membrane V-ATPases have also been implicated in tumor cell invasion. V-ATPases are thus emerging as potential targets in the treatment of diseases such as osteoporosis and cancer.

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for both acidification of intracellular compartments and, for certain cell types, proton transport across the plasma membrane. Intracellular V-ATPases function in both endocytic and intracellular membrane traffic, processing and degradation of macromolecules in secretory and digestive compartments, coupled transport of small molecules such as neurotransmitters and ATP and in the entry of pathogenic agents, including envelope viruses and bacterial toxins. V-ATPases are present in the plasma membrane of renal cells, osteoclasts, macrophages, epididymal cells and certain tumor cells where they are important for urinary acidification, bone resorption, pH homeostasis, sperm maturation and tumor cell invasion, respectively. The V-ATPases are composed of a peripheral domain (V(1)) that carries out ATP hydrolysis and an integral domain (V(0)) responsible for proton transport. V(1) contains eight subunits (A-H) while V(0) contains six subunits (a, c, c', c'', d and e). V-ATPases operate by a rotary mechanism in which ATP hydrolysis within V(1) drives rotation of a central rotary domain, that includes a ring of proteolipid subunits (c, c' and c''), relative to the remainder of the complex. Rotation of the proteolipid ring relative to subunit a within V(0) drives active transport of protons across the membrane. Two important mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V(1) and V(0) domains and changes in coupling efficiency of proton transport and ATP hydrolysis. This review focuses on recent advances in our lab in understanding the structure and regulation of the V-ATPases.

The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation, renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and an integral V(0) domain that carries out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking studies, unique cysteine residues were introduced into structurally defined sites within the B and C subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact surfaces between subunits within the integral V(0) domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A subunit play in controlling reversible dissociation in vivo.

Studies indicate secreted cathepsins are involved in metastasis. V-ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V-ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA-MB231 cells. We investigated whether V-ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre-pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V-ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V-ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsin B in conditioned media and cathepsins B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V-ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V-ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.