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

2004; Elsevier BV; Volume: 279; Issue: 26 Linguagem: Inglês

10.1074/jbc.m311838200

ISSN

1083-351X

Autores

Lilly Bourguignon, Patrick A. Singleton, Falko Diedrich, Robert Stern, Eli Gilad,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

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. 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. CD44 is a multifunctional transmembrane glycoprotein expressed in many cells and tissues including breast tumor cells and carcinoma tissues (1Bourguignon L.Y.W. J. Mammary Gland Biol. & Neoplasia. 2000; 6: 287-297Crossref Scopus (144) Google Scholar, 2Dall P. Heider K.-H. Sinn H.-P. 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The external portion of CD44 binds to extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; siRNAs, small interfering RNAs; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid; ROK, Rho kinase; EIPA, S-(N-ethyl-N-isopropyl) amiloride; NHE1, Na+-H+ exchanger; GST, glutathione S-transferase; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BCECF, 2′,7′-bis(carboxyethyl)-5-(and-6)-carboxyfluorescein; GPI, glycosylphosphatidylinositol; ELISA, enzyme-linked immunosorbent assay; Hyal-2, hyaluronidase-2; GTPγS, guanosine 5′-3-O-(thio)triphosphate; LYB, lysosensor yellow/blue.1The abbreviations used are: ECM, extracellular matrix; HA, hyaluronan; siRNAs, small interfering RNAs; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid; ROK, Rho kinase; EIPA, S-(N-ethyl-N-isopropyl) amiloride; NHE1, Na+-H+ exchanger; GST, glutathione S-transferase; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BCECF, 2′,7′-bis(carboxyethyl)-5-(and-6)-carboxyfluorescein; GPI, glycosylphosphatidylinositol; ELISA, enzyme-linked immunosorbent assay; Hyal-2, hyaluronidase-2; GTPγS, guanosine 5′-3-O-(thio)triphosphate; LYB, lysosensor yellow/blue. components, hyaluronan (HA) (8Turley E.A. 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A. 2001; 98: 4443-4448Crossref PubMed Scopus (294) Google Scholar). Clearly, Hyal-2 is closely associated with tumor progression. In this study we have described a novel CD44 interaction with NHE1 and Hyal-2 in lipid rafts, leading to intracellular/extracellular acidification, HA modification, cathepsin B activation, and breast tumor cell invasion. This new information can provide new insights toward a better understanding of HA-CD44 signaling-regulated malignancy of solid tumor cancers such as breast cancers. Cell Culture—The breast tumor cell line (MDA-MB-231 cells) was obtained from the American Type Culture Collection and grown in Eagle's minimum essential medium supplemented with Earle's salt solution, essential and nonessential amino acids, vitamins, and 10% fetal bovine serum. Antibodies and Reagents—Monoclonal rat anti-CD44 antibody (clone, 020; isotype, IgG2b; obtained from CMB-TECH, Inc., San Francisco) recognizes a common determinant of the HA binding region of CD44 isoforms including CD44s, CD44E, and CD44 variant species. This rat anti-CD44 was routinely used for HA-related blocking experiments. Polyclonal rabbit anti-CD44s antibody, which was raised against a unique N-/O-glycosylated moiety of 85-kDa CD44s, is mono-specific for most of the glycosylated CD44s expressed on the cell surface and displays no cross-reactivity to other CD44 isoforms (e.g. CD44E or CD44 variant species). This reagent was routinely used for immunoblotting experiments. Rabbit anti-Rho kinase (ROK) was prepared according to procedures described previously (13Bourguignon L.Y.W. Zhu H. Shao L. Zhu D. Chen Y.W. Cell Motil. Cytoskeleton. 1999; 43: 269-287Crossref PubMed Scopus (145) Google Scholar). For the preparation of polyclonal rabbit anti-Hyal-2 antibody, specific synthetic peptides (∼15–17 amino acids unique for the Hyal-2 sequence) were prepared by the Peptide Laboratories by using an Advanced ChemTech automatic synthesizer (model ACT350). All polyclonal antibodies were prepared by using conventional DEAE-cellulose chromatography and were tested to be monospecific (by immunoblot assays). Rabbit anti-phosphothreonine antibody and rabbit anti-phosphoserine antibody were obtained from Zymed Laboratories Inc.(South San Francisco, CA). Monoclonal mouse anti-caveolin, mouse anti-flotillin-1, and mouse anti-florillin-2 were purchased from BD Biosciences. Other immunoreagents such as rabbit anti-NHE1 were purchased from Alpha Diagnostic International (San Antonio, TX). High molecular weight HA polymers (∼500,000–1,000,000-dalton polymers) were purified by gel filtration column chromatography using Sephacryl S1000 columns. The purity of high molecular weight HA polymers used in our experiments was further verified by anion exchange high performance liquid chromatography. No small HA fragments were detected in these preparations. Y27632 and CA-074-Me were purchased from Calbiochem. EIPA was obtained from Sigma. Escherichia coli-derived GST-tagged RhoA was a gift from Dr. Martin Schwartz (Scripps Research Institute, La Jolla, CA). Both nigericin and PH 20 hyaluronidase were obtained from Sigma. In the Hiprep™ Sephacryl™ 400 HR (S-400HR, from Amersham Biosciences) gel filtration analyses, three protein markers such as catalase (232,000 daltons), ferritin (440,000 daltons), thyroglobulin (669,000 daltons) (Amersham Biosciences) plus two polysaccharide size standards including Healon HA (500,000 daltons) (prepared according to the procedures described by McKee et al. (18McKee C.M. Penno M.B. Cowman M. Burdick M.D. Strieter R.M. Bao C. Nobel P.W. J. Clin. Investig. 1996; 98: 2403-2413Crossref PubMed Scopus (691) Google Scholar)) and purified HA fragment (280,000 daltons) (ICN Biomedicals) were used. For agarose gel analyses, both Select-HA™ LoLadder (in the range of ∼27,000 to ∼500,000 daltons) and Select-HA™ HiLadder (in the range of ∼500,000 to 1,500,000 daltons) obtained from Hyalose (Oklahoma City, OK) were used as HA standards. Reverse Transcriptase (RT)-PCR—For the detection of Hyal-2 transcript expression, ∼3 μg of total RNA isolated from MDA-MB-231 cells was used for RT-PCR analysis using Superscript™ One-step RT-PCR with PlatinumR Taq system (Invitrogen). Two Hyal-2-specific primers (5′-GAATATTACCATCTTCTACCGCGA-3′ and 5′-CCAGGACACATTGACCACGTA-3′) were designed for this study. For loading controls, two GAPDH primers (5′-CATCCATGACAACTTTGGTATCGTG-3′ and 5′-GAGCTTGACAAAGTGGTCGTTGA-3′) were also used. The reactions were cycled after an initial 2 min of denaturation at 94 °C followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and polymerization at 72 °C for 2 min. The PCR products (∼859 bp for Hyal-2, and ∼442 bp for GAPDH) were analyzed using a 2.0% agarose gel electrophoresis and visualized by ethidium bromide staining. As controls, RT-PCR was carried out in the absence of Superscript II reverse transcriptase or in the absence of template in the PCR mixture. No amplification products were detected in these control samples. Preparations of CD44siRNA and Hyal-2si RNA—The siRNA sequence targeting human CD44 or Hyal-2 (from mRNA sequence, Gen-Bank™ accession number AJ251595) corresponds to the coding region relative to the first nucleotide of the start codon. Target sequences were selected using the software developed by Ambion Inc., UK. As recommended by Ambion, CD44 or Hyal-2-specific targeted regions were selected beginning 50–100 nucleotides downstream from the start codon. Sequences close to 50% G/C content were chosen. Specifically, CD44 target sequence 1 (5′-AAAAATGGTCGCTACAGCATC-3′), CD44 target sequence 2 (5′-AATAGCACCTTGCCCACAATG-3′), and scrambled sequences (5′-AAAAACGGTAGATGCATCAGC-3′) were used. Hyal-2 target sequence 1 (5′-AAGATGCTGCAGAAACGTGTG-3′), Hyal-2 target sequence 2 (5′-AAACGTGTGGAGCACTACATT-3′), and scrambled sequences (5′-AAAGTGCGTGAAGCAATTCGG-3′) were used. CD44 or Hyal-2-specific target sequences were then aligned to the human genome data base in a BLAST search to eliminate sequences with sig

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