CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate–responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

CD44 is an adhesion molecule that interacts with hyaluronic acid (HA) and undergoes sequential proteolytic cleavages in its ectodomain and intramembranous domain. The ectodomain cleavage is triggered by extracellular Ca2+ influx or the activation of protein kinase C. Here we show that CD44-mediated cell–matrix adhesion is terminated by two independent ADAM family metalloproteinases, ADAM10 and ADAM17, differentially regulated in response to those stimuli. Ca2+ influx activates ADAM10 by regulating the association between calmodulin and ADAM10, leading to CD44 ectodomain cleavage. Depletion of ADAM10 strongly inhibits the Ca2+ influx-induced cell detachment from matrix. On the other hand, phorbol ester stimulation activates ADAM17 through the activation of PKC and small GTPase Rac, inducing proteolysis of CD44. Furthermore, depletion of ADAM10 or ADAM17 markedly suppressed CD44-dependent cancer cell migration on HA, but not on fibronectin. The spatio-temporal regulation of two independent signaling pathways for CD44 cleavage plays a crucial role in cell–matrix interaction and cell migration.

CD44 is the major adhesion molecule for the extracellular matrix components and is implicated in a wide variety of physiological and pathological processes including the regulation of tumor cell growth and metastasis. Our previous studies have shown that CD44 undergoes sequential proteolytic cleavages in the extracellular and transmembrane domains and the cleavage product derived from CD44 intramembranous cleavage acts as a signal transduction molecule. However, the underlying mechanism of the intramembranous cleavage of CD44 remains to be elucidated. In the present study, we report for the first time that CD44 is a substrate of the presenilin (PS)-dependent c-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-Otetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by c-secretase inhibitors in U251MG cells and that this cleavage is also inhibited in PS-deficient mouse embryonic fibroblasts. Furthermore, we showed that PS1 is redistributed to ruffling areas of the plasma membrane similarly to CD44 after TPA treatment, supporting our biochemical observation that PS1 is involved in the intramembranous cleavage of CD44. Our present findings suggest important implications for understanding CD44-dependent signal transduction and a potential role of PS/c-secretase activity in the functional regulation of adhesion molecules.

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CD44 is a cell surface adhesion molecule for several extracellular matrix components. We previously showed that CD44 expressed in cancer cells is proteolytically cleaved at the ectodomain through membrane-anchored metalloproteases and that CD44 cleavage plays a critical role in cancer cell migration. Therefore, cellular signals that promote the migration and metastatic activity of cancer cells may regulate the CD44 ectodomain cleavage. Here, we demonstrate that the expression of the dominant active mutant of Ha-Ras (Ha-RasVal-12 ) induces redistribution of CD44 to the newly generated membrane ruffling area and CD44 ectodomain cleavage. The migration assay revealed that the CD44 cleavage contributes to the Ha-RasVal-12 -induced migration of NIH3T3 cells on hyaluronate substrate. Treatment with LY294002, an inhibitor for phosphoinositide 3-OH kinase (PI3K), significantly inhibits Ha-Ras Val-12 -induced CD44 cleavage, whereas that with PD98059, an inhibitor for MEK, does not. The active mutant p110 subunit of PI3K has also been shown to enhance the CD44 cleavage, suggesting that PI3K mediates the Ras-induced CD44 cleavage. Moreover, the expression of dominant negative mutants of Cdc42 and Rac1 inhibits the HaRas Val-12 -induced CD44 cleavage. These results suggest that Ras > PI3K > Cdc42/Rac1 pathway plays an important role in CD44 cleavage and may provide a novel molecular basis to explain how the activated Ras facilitates cancer cell migration. CD44 is a transmembrane receptor for the extracellular matrix (ECM)1 components including hyaluronic acid (HA) (1) and is implicated in a wide variety of adhesion-dependent cellular processes including lymphocyte homing (2), cell migration (3, 4), and tumor cell metastasis and invasion (5-7). Our previous studies showed that CD44 expressed in tumor cells is proteolytically cleaved at the extracellular domain (ectodomain) through a membrane-associated metalloprotease and that this ectodomain cleavage generates a membrane-bound COOH-terminal cleavage product and a soluble NH 2 -terminal fragment released into the culture supernatant (8, 41). The cleavage was found to play a crucial role in an efficient cell detachment from a hyaluronate substrate during the cell migration and to promote the CD44-mediated cell migration of cancer cells (8). These results led us to speculate that cellular signals activated in the cancer cells having high motility and migration activity may contribute to the regulation of CD44 cleavage.Stimulations by various growth factors and ECM proteins are known to activate locomotion of tumor cells, which contributes to invasion and metastasis of the cells (9, 10). Ras small GTPases (Ha-Ras, Ki-Ras, and N-Ras) are indispensable for such cellular signaling. Signals from the growth factor-dependent activation of receptor tyrosine kinase or the integrin-dependent cell adhesion to ECM induce the activation and/or membrane recruitment of guanine nucleotide exchange factors, which convert inactive GDP-bound Ras into active GTP-bound Ras (11). The active form of...