Bourguignon Lilly Y W, Singleton Patrick A, Diedrich Falko, Stern Robert, Gilad Eli
Department of Medicine, University of California, Veterans Affairs Medical Center, San Francisco 94121, USA.
J Biol Chem. 2004 Jun 25;279(26):26991-7007. doi: 10.1074/jbc.M311838200. Epub 2004 Apr 16.
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.
我们研究了在人乳腺肿瘤细胞（MDA-MB-231细胞系）中透明质酸（HA）诱导的细胞信号传导过程中，CD44（一种透明质酸受体）与钠氢交换体（NHE1）及透明质酸酶-2（Hyal-2）之间的相互作用。免疫分析表明，在MDA-MB-231细胞中，标准形式的CD44（CD44s）与两种信号分子（NHE1和Hyal-2）紧密结合形成复合物。这三种蛋白质在富含胆固醇和神经节苷脂的脂筏中也显著富集，脂筏是富含小窝蛋白和膜联蛋白的质膜微结构域。HA与CD44的结合激活了钠氢交换活性，进而促进细胞内酸化并创造出酸性细胞外基质环境。这导致Hyal-2介导的HA分解代谢、HA修饰以及半胱氨酸蛋白酶（组织蛋白酶B）激活，从而引发乳腺肿瘤细胞侵袭。此外，我们还观察到：（i）HA/CD44激活的Rho激酶（ROK）介导NHE1的磷酸化及活性，（ii）抑制ROK或NHE1活性（分别用ROK抑制剂Y27632或NHE1阻滞剂S-（N-乙基-N-异丙基）阿米洛利处理细胞）可阻断NHE1磷酸化/钠氢交换活性，降低细胞内酸化程度，消除细胞外基质中的酸性环境，并抑制乳腺肿瘤特异性行为（如Hyal-2介导的HA修饰、组织蛋白酶B激活及肿瘤细胞侵袭）。最后，下调CD44或Hyal-2的表达（用CD44或Hyal-2特异性小干扰RNA处理细胞）不仅抑制HA介导的CD44信号传导（如ROK介导的钠氢交换体反应及细胞pH变化），还会损害致癌事件（如Hyal-2活性、透明质酸修饰、组织蛋白酶B激活及肿瘤细胞侵袭）。综上所述，我们的结果表明，在富含胆固醇/神经节苷脂的脂筏中，CD44与ROK激活的NHE1（一种钠氢交换体）之间的相互作用在促进Hyal-2和半胱氨酸蛋白酶介导的基质降解及乳腺癌进展所需的细胞内/细胞外酸化过程中起关键作用。
