Function of a Subunit Isoforms of the V-ATPase in pH Homeostasis and in Vitro Invasion of MDA-MB231 Human Breast Cancer Cells

Ayana Hinton,Souad R Sennoune,Sarah Bond,Min Fang,Moshe Reuveni,G Gary Sahagian,Daniel Jay,Raul Martinez-Zaguilan,Michael Forgac

doi:10.1074/jbc.m901201200

Abstract

It has previously been shown that highly invasive MDA-MB231 human breast cancer cells express vacuolar proton-translocating 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 approximately 18% 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.

Highlights

How might V-ATPases function in tumor cell invasion? Because V-ATPases are present in both intracellular compartments and the plasma membrane, there are a number of possibilities
MRNA Levels of a Subunit Isoforms in MDA-MB231 and MCF7 Cells as Measured Using Quantitative RT-PCR—Because of the role V-ATPases have been reported to play in invasiveness of breast cancer cells, it was of interest to compare the mRNA expression profiles for a subunit isoforms in the highly invasive breast cancer cell line MDA-MB231 with those of the poorly invasive cell line MCF7
Selective Knockdown of Each Subunit a Isoform mRNA Using small interfering RNAs (siRNA)—To evaluate the role of a subunit isoforms in V-ATPase function and invasiveness, we employed isoform-specific siRNAs to reduce the level of each a subunit isoform mRNA in MB231 cells

Summary

Introduction

How might V-ATPases function in tumor cell invasion? Because V-ATPases are present in both intracellular compartments and the plasma membrane, there are a number of possibilities. Intracellular V-ATPases may function in invasion by either aiding in the proteolytic activation of cathepsins or matrix metalloproteases within lysosomes or secretory vesicles or by. The a3 isoform is responsible for targeting the V-ATPase to the plasma membrane of osteoclasts, where it plays an essential role in bone resorption [18, 19, 23, 27], a3 has been localized to lysosomes and insulincontaining secretory vesicles in pre-osteoclasts [18] and pancreatic islet cells [17], respectively. The results suggest distinct functions of a subunit isoforms in control of cytosolic and endosome/lysosome pH and a role for both intracellular and plasma membrane V-ATPases in tumor cell invasion

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Conclusion