ABSTRACT
We and others have shown central roles of the Na(+)/H(+) exchanger NHE1 in cell motility. The aim of this study was to determine the roles of NHE1 and of the Na(+), HCO(3)(-) cotransporter NBCn1 in motility of serum-starved MCF-7 breast cancer cells expressing constitutively active ErbB2 (ΔNErbB2). ΔNErbB2 expression elicited NBCn1 upregulation, Ser(703)-phosphorylation of NHE1, and NHE1-inhibitor (EIPA)-sensitive pericellular acidification, in conjunction with increased expression of ß1 integrin and ERM proteins. Active ERM proteins and NHE1 colocalized strongly to invadopodial rosettes, the diameter of which was increased by ΔNErbB2. Adhesion and migration on collagen-I were augmented by ΔNErbB2, unaffected by the NBC inhibitor S0859, and further stimulated by EIPA in a manner potentiated by PI3K-Akt-inhibition. These findings demonstrate that NHE1 inhibition can enhance cancer cell motility, adding an important facet to the understanding of NHE1 in cancer.
Subject(s)
Cation Transport Proteins/metabolism , Cell Movement/physiology , Receptor, ErbB-2/metabolism , Sodium-Bicarbonate Symporters/metabolism , Sodium-Hydrogen Exchangers/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Benzamides/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cation Transport Proteins/genetics , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Chromones/pharmacology , Culture Media, Serum-Free/pharmacology , Cytoskeletal Proteins/metabolism , Enzyme Inhibitors/pharmacology , Female , Humans , Hydrogen-Ion Concentration , Immunoblotting , Integrin beta1/metabolism , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-2/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Sodium-Bicarbonate Symporters/antagonists & inhibitors , Sodium-Bicarbonate Symporters/genetics , Sodium-Hydrogen Exchanger 1 , Sodium-Hydrogen Exchangers/genetics , Sulfonamides/pharmacologyABSTRACT
Long-term osmotic stress results in altered gene transcription, however, with the exception of the TonE/TonEBP system, the underlying mechanisms are poorly understood. We previously showed that upon osmotic shrinkage of Ehrlich Lettré Ascites (ELA) fibroblasts, the MEK1-ERK1/2 pathway is transiently inhibited while p38 MAPK is activated, in turn impacting on cell survival (Pedersen et al., 2007, Cell Physiol Biochem 20: 735-750). Here, we show that downstream of these kinases, two transcription factors with major roles in control of cell proliferation and death, serum response factor (SRF) and cAMP response element-binding protein (CREB) are differentially regulated in ELA cells. SRF Ser(103) phosphorylation and SRF-dependent transcriptional activity were strongly augmented 5-30 min and 24 h, respectively, after hyperosmotic stress (50% increase in extracellular ionic strength), in a p38 MAPK-dependent manner. In contrast, CREB Ser(133) was transiently dephosphorylated upon osmotic shrinkage. The ERK1/2 effector ribosomal S kinase (RSK) and the ERK1/2- and p38 MAPK effector mitogen- stress-activated protein kinase 1 (MSK1) both phosphorylate CREB at Ser(133) . RSK and MSK1 were dephosphorylated within 5 min of shrinkage. MSK1 phosphorylation recovered within 30 min in a p38-MAPK-dependent manner. CREB was transiently dephosphorylated after shrinkage in a manner exacerbated by p38 MAPK inhibition or MSK1 knockdown, but unaffected by inhibition of RSK. In conclusion, in ELA cells, hyperosmotic stress activates SRF in a p38 MAPK-dependent manner and transiently inactivates CREB, likely due to MSK1 inactivation. We suggest that these events contribute to shrinkage-induced changes in gene transcription and death/survival balance.
