CD44 Shedding Research Articles

Abstract 5214: HYAL4 splice variant induces treatment-resistance in advanced bladder cancer

Abstract Introduction and Objective: Precision-based treatment approaches could improve the prognosis of patients with bladder cancer (BC). We recently discovered a first-in-class human Chondroitinase (Chase) that degrades chondroitin sulfate. This Chase is a splice variant of HYAL4 (V1) and promotes a malignant phenotype and chemoresistance in BC cells. We evaluated V1/Chase expression and functions in BC and inhibitors to overcome V1/Chase-mediated Gemcitabine (Gem) resistance. Methods: Cohort 1: 653 voided urine specimens (BC, 160; non-BC, 493); cohort 2: 40 cystectomy specimens from MIBC patients who received adjuvant Gemcitabine plus cisplatin (G+C) treatment. Gene expression was measured by q-PCR and urine was assayed by the Chase test. V1 was expressed in BC cell lines or silenced and the transfectants were analyzed for sensitivity to Gem and Cisplatin. The mechanism of Gem resistance was evaluated in preclinical in vitro and in vivo models. Results: In cohort 1, Chase levels were 7-10-fold elevated in patients with BC compared to patients with benign genitourinary conditions, history of BC, or other cancers. Chase test detected BC with 94.4% and 87.4% specificity. Chase test had 84% accuracy in correctly diagnosing the cases with atypical cytology inference. Stem cell marker CD44 was the major substrate of Chase in BC cells. V1’s Chase activity induced CD44 cleavage and shedding. Intracellularly, CD44 cleavage induced JAK2/Stat3 pathway and subsequently upregulation of cytidine deaminase. CDA induces Gem metabolism and efflux of dFdU, an inactive Gem metabolite. HPLC analysis showed, V1 transfectants have increased levels of dFdU and higher efflux. STAT3 and CDA inhibitors synergistically re-sensitized V1-expressing cells to Gem. In cohort 2, V1, p-JAK2 and CDA expression predicted failure to G+C treatment (P<0.0001). JAK2, STAT3 and CDA inhibitors synergistically re-sensitized V1-expressing cells to Gem. V1-induced muscle invasive tumors that were metastatic. While V1-expressing tumors were resistant, combination of Gem with a CDA or JAK-2 inhibitor abrogated V1 tumor growth with minimal toxicity. Conclusion: V1/Chase is a potential diagnostic and prognostic biomarker for bladder cancer and drives muscle-invasion, metastasis, and Gem resistance in BC. Inhibitors of the Chase signaling pathway overcome V1-induced Gem resistance, suggesting a precision-based treatment approach to improve treatment response. Support: 1R01CA227277-05A1; 1R01CA283699-01 Citation Format: Karina Aguilar, Sunita Panda, Joshua Van Der Eerden, Luis Lopez, Martha Terris, Pablo Santamaria, Bal Lokeshwar, Vinata B. Lokeshwar. HYAL4 splice variant induces treatment-resistance in advanced bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5214.

Curcumin Modulates the Membrane Raft Integrity via Phase Separation and Induces CD44 Shedding in Tumor Cells.

CD44 is a transmembrane cell adhesion molecule that is cleaved by the membrane proteinase, a disintegrin and metalloproteinase 10 (ADAM10), on the cell surface via ectodomain shedding after cholesterol depletion. Lipid raft-mediated CD44 shedding is essential for cancer cell invasion. As cell-cell and cell-matrix adhesions are critical for cancer progression, lipid raft-targeting agents may be effective for cancer therapy. Here, we found that curcumin and its derivatives induced the ADAM10-mediated shedding of CD44 in tumor cells. We also found that curcumin and the derivatives are membrane-active compounds whose effect depends on its planar backbone and the spatial arrangement of methoxy groups substituted on the two aromatic rings using giant unilamellar and plasma membrane vesicles. Curcumin and its derivatives with rigid backbones and hydroxy groups exerted membrane-domain-modulating activity, which may account for their pleiotropic effects via multiple signaling pathways involving membrane receptors. This study provides a basis for the use of membrane-active compounds, such as curcuminoids, to elucidate the roles of lipid rafts in cellular signaling, regulation of membrane-bound ADAM metalloproteinases, and the development of novel membrane lipid-based therapies.

Cholesterol depletion decreases adhesion of non-small cell lung cancer cells to E-selectin.

Lipid microdomains, ordered membrane phases containing cholesterol and glycosphingolipids, play an essential role in cancer cell adhesion and ultimately metastasis. Notably, elevated levels of cholesterol-rich lipid microdomains are found in cancer cells relative to their normal counterparts. Therefore, alterations of lipid microdomains through cholesterol modulation could be used as a strategy to prevent cancer metastasis. In this study, methyl-beta-cyclodextrin (MβCD), sphingomyelinase (SMase), and simvastatin (Simva) were used to investigate the effects of cholesterol on the adhesive behaviors of four non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A549) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule that initiates circulating tumor cell recruitment at metastatic sites. Under hemodynamic flow conditions, the number of adherent NSCLC cells on E-selectin significantly decreased by MβCD and Simva treatments, whereas SMase treatment did not show a significant effect. Significant increases in rolling velocities were detected only for H1299 and H23 cells after MβCD treatment. In contrast, cholesterol depletion did not affect SCLC cell attachment and rolling velocities. Moreover, cholesterol depletion by MβCD and Simva induced CD44 shedding and resulted in an enhanced membrane fluidity in the NSCLC cells, whereas it did not affect the membrane fluidity of the SCLC cells which lacked detectable expression of CD44. Our finding suggests that cholesterol regulates the E-selectin-mediated adhesion of NSCLC cells by redistributing the CD44 glycoprotein and thus modulating the membrane fluidity.NEW & NOTEWORTHY This study investigates the effects of cholesterol on the adhesive behaviors of lung cancer cells in recruitment at metastatic sites. Using cholesterol-modulating compounds, we found that reducing cholesterol decreases the adhesion of non-small cell lung cancer (NSCLC) cells while having no significant effect on small cell lung cancer (SCLC) cells. The study suggests that cholesterol regulates NSCLC cell metastasis by redistributing the adhesion proteins on the cells and modulating cells' membrane fluidity.

Hyperglycemia-induced effects on glycocalyx components in the retina

PurposeDiabetic retinopathy is a vision-threatening complication of diabetes characterized by endothelial injury and vascular dysfunction. The loss of the endothelial glycocalyx, a dynamic layer lining all endothelial cells, contributes to several microvascular pathologies, including an increase in vascular permeability, leukocyte plugging, and capillary occlusion, and may drive the progression of retinopathy. Previously, a significant decrease in glycocalyx thickness has been observed in diabetic retinas. However, the effects of diabetes on specific components of the retinal glycocalyx have not yet been studied. Therefore, the aim of our study was to investigate changes in synthesis, expression, and shedding of retinal glycocalyx components induced by hyperglycemia, which could provide a novel therapeutic target for diabetic retinopathy. MethodsPrimary rat retinal microvascular endothelial cells (RRMECs) were grown under normal glucose (5 mM) or high-glucose (25 mM) conditions for 6 days. The mRNA and protein levels of the glycocalyx components were examined using qRT-PCR and Western blot analysis, respectively. Further, mass spectrometry was used to analyze protein intensities of core proteins. In addition, the streptozotocin-induced Type 1 diabetic rat model was used to study changes in the expression of the retinal glycocalyx in vivo. The shedding of the glycocalyx was studied in both culture medium and in plasma using Western blot analysis. ResultsA significant increase in the shedding of syndecan-1 and CD44 was observed both in vitro and in vivo under high-glucose conditions. The mRNA levels of syndecan-3 were significantly lower in the RRMECs grown under high glucose conditions, whereas those of syndecan-1, syndecan-2, syndecan-4, glypican-1, glypican-3, and CD44 were significantly higher. The protein expression of syndecan-3 and glypican-1 in RRMECs was reduced considerably following exposure to high glucose, whereas that of syndecan-1 and CD44 increased significantly. In addition, mass spectrometry data also suggests a significant increase in syndecan-4 and a significant decrease in glypican-3 protein levels with high glucose stimulation. In vivo, our data also suggest a significant decrease in the mRNA transcripts of syndecan-3 and an increase in mRNA levels of glypican-1 and CD44 in the retinas of diabetic rats. The diabetic rats exhibited a significant reduction in the retinal expression of syndecan-3 and CD44. However, the expression of syndecan-1 and glypican-1 increased significantly in the diabetic retina. ConclusionsOne of the main findings of our study was the considerable diversity of glucose-induced changes in expression and shedding of various components of endothelial glycocalyx, for example, increased endothelial and retinal syndecan-1, but decreased endothelial and retinal syndecan-3. This indicates that the reported decrease in the retinal glycocalyx in diabetes in not a result of a non-specific shedding mechanism. Moreover, mRNA measurements indicated a similar diversity, with increases in endothelial and/or retinal levels of syndecan-1, glypican-1, and CD44, but a decrease for syndecan-3, with these increases in mRNA potentially a compensatory reaction to the overall loss of glycocalyx.

Simvastatin promotes restoration of chondrocyte morphology and phenotype

In this study we examined whether the action of simvastatin affects re-differentiation of passaged chondrocytes and if so, whether this was mediated via changes in cholesterol or cholesterol intermediates. Bovine articular chondrocytes, of varying passage number, human knee chondrocytes and rat chondrosarcoma chondrocytes were treated with simvastatin and examined for changes in mRNA and protein expression of markers of the chondrocyte phenotype as well as changes in cell shape, proliferation and proteoglycan production. In all three models, while still in monolayer culture, simvastatin treatment alone promoted changes in phenotype and morphology indicative of re-differentiation most prominent being an increase in SOX9 mRNA and protein expression. In passaged bovine chondrocytes, simvastatin stimulated the expression of SOX9, ACAN, BMP2 and inhibited the expression of COL1 and α-smooth muscle actin. Co-treatment of chondrocytes with simvastatin plus exogenous cholesterol—conditions that had previously reversed the inhibition on CD44 shedding, did not alter the effects of simvastatin on re-differentiation. However, the co-treatment of chondrocytes with simvastatin together with other pathway intermediates, mevalonate, geranylgeranylpyrophosphate and to a lesser extent, farnesylpyrophosphate, blocked the pro-differentiation effects of simvastatin. Treatment with simvastatin stimulated expression of SOX9 and COL2a and enhanced SOX9 protein in human OA chondrocytes. The co-treatment of OA chondrocytes with mevalonate or geranylgeranylpyrophosphate, but not cholesterol, blocked the simvastatin effects. These results lead us to conclude that the blocking of critical protein prenylation events is required for the positive effects of simvastatin on the re-differentiation of chondrocytes.

A disintegrin and metalloproteinase 15-mediated glycocalyx shedding contributes to vascular leakage during inflammation.

AimsEndothelial hyperpermeability exacerbates multiple organ damage during inflammation or infection. The endothelial glycocalyx, a protective matrix covering the luminal surface of endothelial cells (ECs), undergoes enzymatic shedding during inflammation, contributing to barrier hyperpermeability. A disintegrin and metalloproteinase 15 (ADAM15) is a sheddase capable of cleaving the ectodomains of membrane-bound molecules. Herein, we tested whether and how ADAM15 is involved in glycocalyx shedding and vascular leakage during sepsis.Methods and resultsDextran-150kD exclusion assay revealed lipopolysaccharide (LPS) significantly reduced glycocalyx thickness in mouse cremaster microvessels. Consistently, shedding products of glycocalyx constituents, including CD44 ectodomain, were detected with an increased plasma level after cecal ligation and puncture (CLP)-induced sepsis. The direct effects of CD44 ectodomain on endothelial barrier function were evaluated, which revealed CD44 ectodomain dose-dependently reduced transendothelial electrical resistance (TER) and caused cell–cell adherens junction disorganization. Furthermore, we examined the role of ADAM15 in CD44 cleavage and glycocalyx shedding. An in vitro cleavage assay coupled with liquid chromatography-tandem mass spectrometry confirmed ADAM15 cleaved CD44 at His235-Thr236 bond. In ECs with ADAM15 knockdown, LPS-induced CD44 cleavage and TER reduction were greatly attenuated, whereas, ADAM15 overexpression exacerbated CD44 cleavage and TER response to LPS. Consistently, ADAM15 knockout in mice attenuated CLP-induced increase in plasma CD44. Intravital and electron microscopic images revealed ADAM15 deficiency prevented LPS-induced glycocalyx injury in cremaster and pulmonary microvasculatures. Functionally, ADAM15−/− mice with better-preserved glycocalyx exhibited resistance to LPS-induced vascular leakage, as evidenced by reduced albumin extravasation in pulmonary and mesenteric vessels. Importantly, in intact, functionally vital human lungs, perfusion of LPS induced a significant up-regulation of ADAM15, accompanied by elevated CD44 in the effluent and increased vascular permeability to albumin.ConclusionTogether, our data support the critical role of ADAM15 in mediating vascular barrier dysfunction during inflammation. Its mechanisms of action involve CD44 shedding and endothelial glycocalyx injury.

Expression and shedding of CD44 in the endometrium of women with endometriosis and modulating effects of vitamin D: A randomized exploratory trial

Endometriosis is an estrogen-dependent disease. The impaired estrogen and progesterone signaling over-activates the Wnt/β-catenin pathway in endometriosis patients, which can explain the increased invasion potency of endometrial cells derived from the endometrium of women with endometriosis. The regulatory effects of vitamin D on Wnt/β-catenin pathway were demonstrated by previous studies. According to gene prioritization method, among Wnt target genes, CD44 was in high ranking in relation to endometriosis. The aim of this study is to investigate the expression of CD44 in the endometrium of women with endometriosis and to study the effects of vitamin D on its expression. This prospective study was performed, during a 12 months period from December 2015 to November 2016, on healthy women as the control group (n = 14) and endometriosis patients (n = 34). The endometriosis patients randomly divided into two groups: One group treated according to the routine protocol and the other group, alongside the routine protocol, took 50,000 IU vitamin D weekly for 12–14 weeks. Blood, endometrial fluid, and endometrial tissue samples were obtained from the control group and endometriosis groups before and after the intervention. We used in silico gene prioritization to study the relevance of CD44. The expression of CD44 was evaluated using the techniques of Western blot, real-time polymerase chain reaction, and ELISA. The eutopic endometrium of women with endometriosis in mid-secretory phase expressed significantly higher levels of CD44s, CD44V, and CD44v6. The concentration of soluble CD44 in the serum and endometrial fluid of endometriosis patients was higher than of healthy women. The expression level of CD44s, CD44V, and CD44v6 in the eutopic endometrium as well as the concentration of soluble CD44 in the endometrial fluid was decreased after modification of the circulating levels of 25(OH)D. It seems that the increased expression and extensive shedding of CD44 in eutopic endometrium play a role in the pathogenesis of endometriosis. Vitamin D can control and modify this process at least in part. We suggest more in vivo investigations on the therapeutic potency of vitamin D in endometriosis.

Disruption of TACE-filamin interaction can inhibit TACE-mediated ectodomain shedding

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells.

CD44 is a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. Its interaction with appropriate extracellular matrix ligands promotes the migration and invasion processes involved in metastases. It was originally identified as a receptor for hyaluronan or hyaluronic acid and later to several other ligands including, osteopontin (OPN), collagens, and matrix metalloproteinases. CD44 has also been identified as a marker for stem cells of several types. Beside standard CD44 (sCD44), variant (vCD44) isoforms of CD44 have been shown to be created by alternate splicing of the mRNA in several cancer. Addition of new exons into the extracellular domain near the transmembrane of sCD44 increases the tendency for expressing larger size vCD44 isoforms. Expression of certain vCD44 isoforms was linked with progression and metastasis of cancer cells as well as patient prognosis. The expression of CD44 isoforms can be correlated with tumor subtypes and be a marker of cancer stem cells. CD44 cleavage, shedding, and elevated levels of soluble CD44 in the serum of patients is a marker of tumor burden and metastasis in several cancers including colon and gastric cancer. Recent observations have shown that CD44 intracellular domain (CD44-ICD) is related to the metastatic potential of breast cancer cells. However, the underlying mechanisms need further elucidation.

Increased expression of surface CD44 in hypoxia-DCs skews helper T cells toward a Th2 polarization.

A low partial oxygen pressure (hypoxia) occurs in many pathological environments, such as solid tumors and inflammatory lesions. Understanding the cellular response to hypoxic stress has broad implications for human diseases. As we previously reported, hypoxia significantly altered dendritic cells (DCs) to a DC2 phenotype and promoted a Th2 polarization of naïve T cells with increased IL-4 production. However, the underlying mechanisms still remain largely unknown. In this study, we found the over-expression of surface CD44 in DCs was involved in this process via ligand binding. Further investigation showed hypoxia could reduce the surface expression of membrane type 1 metalloprotease (MT1-MMP) via down-regulating the kinesin-like protein KIF2A, which subsequently alleviated the shedding of CD44 from DCs. Moreover, KIF2A expression was found negatively regulated by HIF-1α in hypoxic microenvironment. These results suggest a previously uncharacterized mechanism by which hypoxia regulates the function of DCs via KIF2A/MT1-MMP/CD44 axis, providing critical information to understand the immune response under hypoxia.

MT1-MMP recognition by ERM proteins and its implication in CD44 shedding.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a key enzyme involved in tumor cell invasion by shedding their cell-surface receptor CD44 anchored with F-actin through ezrin/radixin/moesin (ERM) proteins. We found the cytoplasmic tail of MT1-MMP directly binds the FERM domain of radixin, suggesting F-actin-based recruitment of MT1-MMP to CD44 for invasion. Our crystal structure shows that the central region of the MT1-MMP cytoplasmic tail binds subdomain A of the FERM domain, and makes an antiparallel β-β interaction with β2A-strand. This binding mode is distinct from the previously determined binding mode of CD44 to subdomain C. We showed that radixin simultaneously binds both MT1-MMP and CD44, indicating ERM protein-mediated colocalization of MT1-MMP and its substrate CD44 and anchoring to F-actin. Our study implies that ERM proteins contribute toward accelerated CD44 shedding by MT1-MMP through ERM protein-mediated interactions between their cytoplasmic tails.

Molecular mechanism for the action of the anti-CD44 monoclonal antibody MEM-85.

The hyaluronate receptor CD44 plays role in cell adhesion and migration and is involved in tumor metastasis. The extracellular domain of CD44 comprises the hyaluronate-binding domain (HABD) and the membrane-proximal stem region; the short intracellular portion interacts with adaptor proteins and triggers signaling pathways. Binding of hyaluronate to CD44 HABD induces an allosteric conformational change, which results in CD44 shedding. A poorly characterized epitope in human CD44 HABD is recognized by the murine monoclonal antibody MEM-85, which cross-blocks hyaluronate binding to CD44 and also induces CD44 shedding. MEM-85 is of therapeutic interest, as it inhibits growth of lung cancer cells in murine models. In this work, we employed a combination of biophysical methods to determine the MEM-85 binding epitope in CD44 HABD and to provide detailed insight into the mechanism of MEM-85 action. In particular, we constructed a single-chain variable fragment (scFv) of MEM-85 as a tool for detailed characterization of the CD44 HABD-antibody complex and identified residues within CD44 HABD involved in the interaction with scFv MEM-85 by NMR spectroscopy and mutational analysis. In addition, we built a rigid body model of the CD44 HABD-scFv MEM-85 complex using a low-resolution structure obtained by small-angle X-ray scattering. The MEM-85 epitope is situated in the C-terminal part of CD44 HABD, rather than the hyaluronate-binding groove, and the binding of MEM-85 induces a structural reorganization similar to that induced by hyaluronate. Therefore, the mechanism of MEM-85 cross-blocking of hyaluronate binding is likely of an allosteric, relay-like nature.

CD44 knock-down in bovine and human chondrocytes results in release of bound HYAL2

CD44 shedding occurs in osteoarthritic chondrocytes. Previous work of others has suggested that the hyaluronidase isoform HYAL2 has the capacity to bind to CD44, a binding that may itself induce CD44 cleavage. Experiments were developed to elucidate whether chondrocyte HYAL2: (1) was exposed on the extracellular plasma membrane of chondrocytes, (2) bound to CD44, (3) underwent shedding together with CD44 and lastly, (4) exhibited hyaluronidase activity within a near-neutral pH range. Enhancing CD44 shedding by IL-1β resulted in a proportional increase in HYAL2 released from human and bovine chondrocytes into the medium. CD44 knockdown by siRNA also resulted in increased accumulation of HYAL2 in the media of chondrocytes. By hyaluronan zymography only activity at pH3.7 was observed and this activity was reduced by pre-treatment of chondrocytes with trypsin. CD44 and HYAL2 were found to co-immunoprecipitate, and to co-localize within intracellular vesicles and at the plasma membrane. Degradation of hyaluronan was visualized by agarose gel electrophoresis. With this approach, hyaluronidase activity could be observed at pH4.8 under assay conditions in which CD44 and HYAL2 binding remained intact; additionally, weak hyaluronidase activity could be observed at pH6.8 under these conditions. This study suggests that CD44 and HYAL2 are bound at the surface of chondrocytes. The release of HYAL2 when CD44 is shed could provide a mechanism for weak hyaluronidase activity to occur within the more distant extracellular matrix of cartilage.

Targeting the T-cell membrane type-1 matrix metalloproteinase-CD44 axis in a transferred type 1 diabetes model in NOD mice.

This study tested the hypothesis that membrane-tethered type-1 matrix metalloproteinase (MT1-MMP)-induced proteolysis of T cell CD44 is important for defining the migration and function of autoreactive T cells, including diabetogenic, insulin-specific and Kd-restricted IS-CD8+ cells. To confirm the importance of MT1-MMP proteolysis of CD44 in type 1 diabetes (T1D), the anti-diabetic effects of three MMP inhibitors (3(S)-2,2-dimethyl-4[4-pyridin-4-yloxy-benzenesulfonyl]-thiomorpholine-3-carboxylic acid hydroxamate [AG3340], 2-(4-phenoxyphenylsulfonylmethyl) thiirane [SB-3CT] and epigallocatechin-3-gallate [EGCG]) were compared using an adoptive diabetes transfer model in non-obese diabetic (NOD) mice. Only AG3340 was capable of inhibiting both the activity of MT1-MMP and the shedding of CD44 in T cells; and the transendothelial migration and homing of IS-CD8+ T cells into the pancreatic islets. SB-3CT and EGCG were incapable of inhibiting T cell MT1-MMP efficiently. As a result, AG3340 alone, but not SB-3CT or EGCG, delayed the onset of transferred diabetes in NOD mice. In summary, the results of the present study emphasize that the MT1-MMP-CD44 axis has a unique involvement in T1D development. Accordingly, we suggest that a potent small-molecule MT1-MMP antagonist is required for the design of novel therapies for T1D.

Extracellular ATP induces CD44 shedding from macrophage‐like P388D1 cells via the P2X7 receptor

The P2X7 receptor (P2X7R) is a nucleotide receptor expressed predominantly on hemopoietic, bone, and epithelial cells. The P2X7R can be activated by extracellular ATP and induces the influx of calcium, releases cytokines, and participates in cell proliferation and apoptosis. CD44 is an adhesion molecule. The effects of CD44 include cell-cell and cell-matrix adhesion interactions, lymphocyte activation, and cell migration. Many studies have shown that P2X7R and CD44 play important roles in hematological malignancies, but no study exists regarding the relationship between P2X7R and CD44. In the present study, we characterized P388D1 cells for the surface expression of CD44 and analyzed ATP-induced shedding. The data showed that P388D1 cells express CD44. Incubation of P388D1 cells with ATP induced a rapid loss of CD44 from the P388D1 cell surface. In addition, using a receptor inhibitor and P2X7R short hairpin RNA, we showed that the loss of CD44 is mediated via the P2X7R. Finally, we demonstrated that activation of P2X7R by ATP induces CD44 shedding.

Low Cholesterol Triggers Membrane Microdomain-dependent CD44 Shedding and Suppresses Tumor Cell Migration

CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-β-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.

KIF5B and KIF3A/KIF3B kinesins drive MT1-MMP surface exposure, CD44 shedding, and extracellular matrix degradation in primary macrophages

The matrix metalloproteinase (MMP) MT1-MMP plays pivotal roles in leukocyte physiology such as monocyte diapedesis, dendritic cell migration, and T-cell homing. MT1-MMP is a surface-anchored "master switch" proteinase that cleaves a variety of substrates including extracellular matrix components, matrix receptors, and also other MMPs. However, little is known about the mechanisms enabling intracellular trafficking and exposure of MT1-MMP on the cell surface. We now show that, in primary human macrophages, MT1-MMP-positive vesicles travel bidirectionally along microtubules, in a process regulated by KIF5B and KIF3A/KIF3B kinesins. SiRNA-induced knockdown revealed that transport by KIF5B and KIF3A/KIF3B is crucial for delivery of MT1-MMP to the cell surface and also for surface-associated functions of MT1-MMP, such as shedding of the matrix receptors CD44 and syndecan-1 or degradation of extracellular matrix at podosomes. These data show that kinesin-mediated intracellular transport of MT1-MMP is a pivotal process that allows macrophages to dynamically modify their pericellular environment. These data also identify specific kinesins as potential targets for the early manipulation of MT1-MMP activity in tissues.

Two Novel Functions of Hyaluronidase-2 (Hyal2) Are Formation of the Glycocalyx and Control of CD44-ERM Interactions

It has long been predicted that the members of the hyaluronidase enzyme family have important non-enzymatic functions. However, their nature remains a mystery. The metabolism of hyaluronan (HA), their major enzymatic substrate, is also enigmatic. To examine the function of Hyal2, a glycosylphosphatidylinositol-anchored hyaluronidase with intrinsically weak enzymatic activity, we have compared stably transfected rat fibroblastic BB16 cell lines with various levels of expression of Hyal2. These cell lines continue to express exclusively the standard form (CD44s) of the main HA receptor, CD44. Hyal2, CD44, and one of its main intracellular partners, ezrin-radixin-moesin (ERM), were found to co-immunoprecipitate. Functionally, Hyal2 overexpression was linked to loss of the glycocalyx, the HA-rich pericellular coat. This effect could be mimicked by exposure of BB16 cells either to Streptomyces hyaluronidase, to HA synthesis inhibitors, or to HA oligosaccharides. This led to shedding of CD44, separation of CD44 from ERM, reduction in baseline level of ERM activation, and markedly decreased cell motility (50% reduction in a wound healing assay). The effects of Hyal2 on the pericellular coat and on CD44-ERM interactions were inhibited by treatment with the Na(+)/H(+) exchanger-1 inhibitor ethyl-N-isopropylamiloride. We surmise that Hyal2, through direct interactions with CD44 and possibly some pericellular hyaluronidase activity requiring acidic foci, suppresses the formation or the stability of the glycocalyx, modulates ERM-related cytoskeletal interactions, and diminishes cell motility. These effects may be relevant to the purported in vivo tumor-suppressive activity of Hyal2.

The activity of a designer tissue inhibitor of metalloproteinases (TIMP)-1 against native membrane type 1 matrix metalloproteinase (MT1-MMP) in a cell-based environment

The surface-anchored membrane type 1 matrix metalloproteinase (MT1-MMP) degrades a wide range of extracellular matrix components that includes collagens, laminins, fibronectin and the structural proteoglycan aggrecan. The enzyme modulates cell motility and plays an important role in tumour invasion and proliferation. We have previously designed a variant of tissue inhibitor of metalloproteinase (TIMP)-1 bearing a triple mutation (V4A + P6V + T98L, or N-TIMP-1 mt1) that forms tight binary complex with the soluble catalytic domain of MT1-MMP [M.H. Lee, M. Rapti, G. Murphy, J. Biol. Chem. 278 (2003) 40224–40230]. Here, we report our latest findings on the cellular potency of this mutant against native MT1-MMP in cell-based environment. We show that N-TIMP-1 mt1 is a highly potent inhibitor against the ectodomain form of MT1-MMP ( K i 9.53 nM) with potential for further development as a therapeutic agent. The mutant is devoid of pro-MMP-2-activating capability but is highly effective in blocking MT1-MMP-mediated FITC-labelled collagen and gelatin film degradation in HTC75 fibrosarcoma and MCF7 breast cancer models. Most encouragingly, N-TIMP-1 mt1 is also effective against CD44 shedding in HTC75 cells and able to prevent tubule formation in human umbilical vascular endothelial cells (HUVEC) in a 3D fibrin gel model. We are interested in the development of the TIMPs as therapeutic agents against MT1-MMP related disorders such as cancers. Our findings here indicate the potential for the design of selective TIMPs with refined specificity and possibility for future therapeutic application.

ADAM10 Is the Constitutive Functional Sheddase of CD44 in Human Melanoma Cells

CD44 proteins are cell surface receptors for hyaluronic acid (HA), a component of the extracellular matrix that has multiple effects on cell behavior. CD44 can be shed from the cell surface by proteolytic cleavage. The resulting soluble form can interfere with the interaction between HA and membrane-bound CD44. Soluble CD44 can abolish the cell proliferation-promoting effect of HA on melanoma cell lines, suggesting that a better understanding of the shedding process might identify ways of blocking tumor cell proliferation. ADAM10, ADAM17, and MMP14 have previously been implicated in the shedding of CD44 from various tumor cells. Using immunohistochemistry we demonstrate that ADAM10 and ADAM17 but not MMP14 are significantly expressed on melanoma cells in histological sections. In human melanoma cell lines expression of these proteases could be blocked by transfection with appropriate siRNAs. However, only blocking of ADAM10 expression led to decreased shedding of CD44. In parallel, cell proliferation was promoted. Confocal microscopy demonstrated that ADAM10 and CD44 colocalize on the cell surface. We conclude that ADAM10 is the predominant protease involved in the constitutive shedding of endogenous CD44 from melanoma cells, and that enhancement of ADAM10 activity could be an approach to decrease the proliferation of melanoma cells.