CD44 interaction with Na+-H+ exchanger (NHE1) creates acidic microenvironments leading to hyaluronidase-2 and cathepsin B activation and breast tumor cell invasion.
We have explored CD44 (a hyaluronan (HA) receptor) interaction with a Na(+)-H(+) exchanger (NHE1) and hyaluronidase-2 (Hyal-2) during HA-induced cellular signaling in human breast tumor cells (MDA-MB-231 cell line). Immunological analyses demonstrate that CD44s (standard form) and two signaling molecules (NHE1 and Hyal-2) are closely associated in a complex in MDA-MB-231 cells. These three proteins are also significantly enriched in cholesterol and ganglioside-containing lipid rafts, characterized as caveolin and flotillin-rich plasma membrane microdomains. The binding of HA to CD44 activates Na(+)-H(+) exchange activity which, in turn, promotes intracellular acidification and creates an acidic extracellular matrix environment. This leads to Hyal-2-mediated HA catabolism, HA modification, and cysteine proteinase (cathepsin B) activation resulting in breast tumor cell invasion. In addition, we have observed the following: (i) HA/CD44-activated Rho kinase (ROK) mediates NHE1 phosphorylation and activity, and (ii) inhibition of ROK or NHE1 activity (by treating cells with a ROK inhibitor, Y27632, or NHE1 blocker, S-(N-ethyl-N-isopropyl) amiloride, respectively) blocks NHE1 phosphorylation/Na(+)-H(+) exchange activity, reduces intracellular acidification, eliminates the acidic environment in the extracellular matrix, and suppresses breast tumor-specific behaviors (e.g. Hyal-2-mediated HA modification, cathepsin B activation, and tumor cell invasion). Finally, down-regulation of CD44 or Hyal-2 expression (by treating cells with CD44 or Hyal-2-specific small interfering RNAs) not only inhibits HA-mediated CD44 signaling (e.g. ROK-mediated Na(+)-H(+) exchanger reaction and cellular pH changes) but also impairs oncogenic events (e.g. Hyal-2 activity, hyaluronan modification, cathepsin B activation, and tumor cell invasion). Taken together, our results suggest that CD44 interaction with a ROK-activated NHE1 (a Na(+)-H(+) exchanger) in cholesterol/ganglioside-containing lipid rafts plays a pivotal role in promoting intracellular/extracellular acidification required for Hyal-2 and cysteine proteinase-mediated matrix degradation and breast cancer progression.
Bourguignon
Lilly Y W
LY
Department of Medicine, University of California, Veterans Affairs Medical Center, San Francisco 94121, USA. lillyb@itsa.ucsf.edu
Singleton
Patrick A
PA
Diedrich
Falko
F
Stern
Robert
R
Gilad
Eli
E
D000577:Amides / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology / Q000378:metabolism*; D002401:Cathepsin B / Q000378:metabolism*; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D045744:Cell Line, Tumor; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009361:Neoplasm Invasiveness; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054460:rho-Associated Kinases
D000577:Amides; C495540:CAV1 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D004791:Enzyme Inhibitors; D018960:Hyaluronan Receptors; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011725:Pyridines; D034741:RNA, Small Interfering; D017923:Sodium-Hydrogen Exchangers; C106354:flotillins; C069139:growth factor-activatable Na-H exchanger NHE-1; C108830:Y 27632; D000584:Amiloride; D006820:Hyaluronic Acid; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D006821:Hyaluronoglucosaminidase; D002401:Cathepsin B; C039614:ethylisopropylamiloride