Daizo Murakami

Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway

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.

Cell–matrix interaction via CD44 is independently regulated by different metalloproteinases activated in response to extracellular Ca2+ influx and PKC activation

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.

Presenilin-dependent γ-secretase activity mediates the intramembranous cleavage of CD44

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 γ-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by γ-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/γ-secretase activity in the functional regulation of adhesion molecules.

Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins

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-RasVal-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 Ha-RasVal-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.

An asymptomatic intraorbital foreign body for 30 years.

Intraorbital foreign bodies (IOFBs) are usually accompanied by notable skin wounds and/or ocular wounds, and often there is a clear history of a penetrating object. We report herein on a patient who had an asymptomatic IOFB for 30 years. To the best of our knowledge, there is only one other case of a patient with an IOFB for a longer asymptomatic period after the injury. IOFBs may be overlooked because a small penetrating wound may be accompanied by no signs of inflammation for many decades as in this patients clinical course.

Radiotherapy for T3N0 glottic carcinoma without cord fixation: elective nodal irradiation or not?

Background Although the T3 category has been changed in the sixth edition of the TNM staging system proposed by the Union for International Cancer Control (UICC), the appropriate clinical target volume (CTV) of elective nodal irradiation (ENI) for T3N0 glottic carcinoma without cord fixation, which was formerly treated as a T1-2N0 disease, is not fully discussed. Materials and Methods We retrospectively analyzed 64 patients staged or restaged as T3N0 disease without cord fixation. All patients received irradiation to the primary lesion alone using opposed lateral fields. Surgery was performed in 10 patients without tumor regression after the delivery of 40 Gy. The other 54 patients received a median total dose of 66 Gy. Concurrent chemoradiotherapy (CRT) with low-dose cisplatin and UFT (low-dose CRT) and docetaxel, cisplatin, and 5-fluorouracil (TPF-CRT) were performed in 23 and 19 patients, respectively. Results Eighteen (28.1%) patients suffered treatment failure; all were recorded as local failure alone. The 5-year local control rates for RT alone, low-dose CRT, and TPF-CRT groups were 51.7%, 61.6%, and 93.8%, respectively (p = 0.027). The 5-year laryngeal preservation rates for RT alone, low-dose CRT, and TPF-CRT groups were 57.4%, 81.6%, and 89.5%, respectively (p = 0.048). Conclusions The rate of regional failure was zero when irradiating the primary lesion alone using opposed lateral fields. This treatment technique covers the most level III regions; hence, CTV for ENI should include level III alone.BACKGROUND Although the T3 category has been changed in the sixth edition of the TNM staging system proposed by the Union for International Cancer Control (UICC), the appropriate clinical target volume (CTV) of elective nodal irradiation (ENI) for T3N0 glottic carcinoma without cord fixation, which was formerly treated as a T1-2N0 disease, is not fully discussed. MATERIALS AND METHODS We retrospectively analyzed 64 patients staged or restaged as T3N0 disease without cord fixation. All patients received irradiation to the primary lesion alone using opposed lateral fields. Surgery was performed in 10 patients without tumor regression after the delivery of 40 Gy. The other 54 patients received a median total dose of 66 Gy. Concurrent chemoradiotherapy (CRT) with low-dose cisplatin and UFT (low-dose CRT) and docetaxel, cisplatin, and 5-fluorouracil (TPF-CRT) were performed in 23 and 19 patients, respectively. RESULTS Eighteen (28.1%) patients suffered treatment failure; all were recorded as local failure alone. The 5-year local control rates for RT alone, low-dose CRT, and TPF-CRT groups were 51.7%, 61.6%, and 93.8%, respectively (p = 0.027). The 5-year laryngeal preservation rates for RT alone, low-dose CRT, and TPF-CRT groups were 57.4%, 81.6%, and 89.5%, respectively (p = 0.048). CONCLUSIONS The rate of regional failure was zero when irradiating the primary lesion alone using opposed lateral fields. This treatment technique covers the most level III regions; hence, CTV for ENI should include level III alone.

Radiation therapy for nasopharyngeal carcinoma: the predictive value of interim survival assessment

Pretreatment characteristics are suggested as predictive and/or prognostic factors for nasopharyngeal carcinoma (NPC); however, individual tumor radiosensitivities have previously not been considered. As boost planning is recommended for NPC, we performed interim assessments of magnetic resonance (MR) images for boost planning and retrospectively evaluated their predictive value for the survival of NPC patients. Radiation therapy via elective nodal irradiation (median dose: 39.6 Gy) with/without chemotherapy was used to treat 63 NPC patients. Boost irradiation (median total dose: 70 Gy) was performed based on the interim assessment. The largest lymph node (LN) was measured on MR images acquired at the time of interim assessment. The site of first failure was local in 8 (12.7%), regional in 7 (11.1%), and distant in 12 patients (19.0%). All 7 patients with regional failure harbored LNs ≥15 mm at interim assessment. We divided the 63 patients into two groups based on LN size [large (≥15 mm), n = 10 and small (<15 mm), n = 53]. Univariate analysis showed that 5-year overall survival (OS) and cause-specific survival (CSS) rates for large LNs were significantly lower than for small LNs (OS: 12.5% vs 70.5%, P < 0.001 and CSS: 25.0% vs 80.0%, P < 0.001). Multivariate analysis showed that large LNs were a significantly unfavorable factor for both OS (hazard ratio = 4.543, P = 0.002) and CSS (hazard ratio = 6.020, P = 0.001). The results suggest that LN size at interim assessment could predict survival in NPC patients.