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J. Biol. Chem. 279 (26): 26991-27007

© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

CD44 Interaction with Na+-H+ Exchanger (NHE1) Creates Acidic Microenvironments Leading to Hyaluronidase-2 and Cathepsin B Activation and Breast Tumor Cell Invasion*

Lilly Y. W. Bourguignon{ddagger}§, Patrick A. Singleton{ddagger}, Falko Diedrich{ddagger}, Robert Stern¶, , and Eli Gilad{ddagger}

{ddagger}Department of Medicine, University of California, and Endocrine Unit, Veterans Affairs Medical Center and the Department of Pathology, University of California, San Francisco, California 94121

Abstract: 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.

Received for publication October 29, 2003. Revision received April 15, 2004.

* This work was supported by United States Public Health Grants R01 CA66163, R01 CA 78633, and P01 AR39448 from the National Institutes of Health and a Veterans Affairs Merit Review grant. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence and reprint requests should be addressed: Endocrine Unit (111N), Dept. of Medicine, University of California, San Francisco, and Veterans Affairs Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-221-4810 (Ext. 3321); Fax: 415-383-1638; E-mail: lillyb{at}itsa.ucsf.edu.

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