The FXYD proteins are a family of small membrane proteins that share an invariant four amino acid signature motif F-X-Y-D and act as tissue-specific regulatory subunits of the Na,KATPase. FXYD5 (also termed dysadherin or RIC) is a structurally and functionally unique member of the FXYD family. As other FXYD proteins, FXYD5 specifically interacts with the Na,K-ATPase and alters its kinetics by increasing V max . However, unlike other family members FXYD5 appears to have additional functions, which cannot be readily explained by modulation of transport kinetics. Knockdown of FXYD5 in MDA-MB-231 breast cancer cells largely decreases expression and secretion of the chemokine CCL2 (MCP-1). A related effect has also been observed in renal cell carcinoma cells. The current study aims to further characterize the relationship between the expression of FXYD5 and CCL2 secretion. We demonstrate that transfection of M1 epithelial cell line with FXYD5 largely increases lipopolysaccharide (LPS) stimulated CCL2 mRNA and secretion of the translated protein. We have completed a detailed analysis of the molecular events leading to the above response. Our key findings indicate that FXYD5 generates a late response by increasing the surface expression of the TNF␣ receptor, without affecting its total protein level, or mRNA transcription. LPS administration to mice demonstrates induced secretion of CCL2 and TNF␣ in FXYD5-expressing lung peripheral tissue, which suggests a possible role for FXYD5 in normal epithelia during inflammation.FXYD is a family of type I plasma membrane proteins characterized by the extracellular motif Phe-Xxx-Tyr-Asp. All seven mammalian members of this family specifically interact with the Na,K-ATPase and they have been described to act as tissue specific regulators or auxiliary subunits of the Na,KATPase whose role is to adjust the pump's activity to the cell environment (1). FXYD5 (also termed dysadherin or RIC) is a structurally and functionally unique member of the FXYD family. As other FXYD proteins, FXYD5 specifically interacts with the Na,K-ATPase and alters its kinetics by increasing V max (2, 3). However, in addition to regulating Na,K-ATPase kinetics, FXYD5 appears to have other functions. FXYD5 has been identified as a cancer-associated protein whose expression inhibits E-cadherin and promotes metastasis (4). Clinical studies have demonstrated correlations between the abundance of FXYD5 and the progression of malignancies and survival chances of patients with various human cancers (for review see Ref. 5). Silencing FXYD5 decreases individual and collective cell motility, while its overexpression has the opposite effect (6 -8). In addition, the expression of FXYD5 has been associated with changes in cytoskeletal organization, altered cell shape and impairment of tight and adherence junctions (6 -10). The above observations are consistent with the fact that the extracellular domain of FXYD5 is much longer than that of other FXYD proteins and presumably undergoes excessive O-glycosylation (4, 11).Prev...

FXYD5 is a Na-K-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5-specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5-related morphological changes are mediated through its interaction with Na-K-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression may downregulate the β1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5-expressing cells may correlate with matrix metalloproteinase 9 secretion and adhesion changes in 4T1 wild-type cells.