Correlation of syndecan gene amplification with metastatic potential and clinical outcomes in carcinomas.

Cell surface receptors play crucial roles in cellular responses to extracellular ligands, helping to modulate the functions of a cell based on information coming from outside the cell. Syndecan refers to a family of cell adhesion receptors that regulate both extracellular and cytosolic events. Alteration of syndecan expression disrupts regulatory mechanisms in a cell type-specific fashion, often leading to serious diseases, notably cancer. Given the multifaceted functions and distinct tissue distributions of syndecan, it will be important to unravel the gene-level intricacies of syndecan expression and thereby further understand its involvement in various carcinogenic processes. Although accumulating evidence indicates that the protein expression patterns of syndecan family members are significantly altered in cancer cells, the underlying gene-level mechanisms remain largely unknown. This review endeavors to explore syndecan gene expression levels across different cancer types by scrutinizing extensive cancer genome datasets using tools such as cBioPortal. Our analysis unveils that somatic mutations in SDC genes are rare occurrences, whereas copy number alterations are frequently observed across diverse cancers, particularly in SDC2 and SDC4. Notably, amplifications of SDC2 and SDC4 correlate with heightened metastatic potential and dismal prognosis. This underscores the recurrent nature of SDC2 and SDC4 amplifications during carcinogenesis and sheds light on their role in promoting cancer activity through augmented protein expression. The identification of these amplifications not only enriches our understanding of carcinogenic mechanisms but also hints at the potential therapeutic avenue of targeting SDC2 and SDC4 to curb cancer cell proliferation and metastasis.

Everolimus exerts anticancer effects through inhibiting the interaction of matrix metalloproteinase-7 with syndecan-2 in colon cancer cells.

Previous work showed that matrix metalloproteinase-7 (MMP-7) regulates colon cancer activities through an interaction with syndecan-2 (SDC-2) and SDC-2-derived peptide that disrupts this interaction and exhibits anticancer activity in colon cancer. Here, to identify potential anticancer agents, a library of 1,379 Food and Drug Administration (FDA)-approved drugs that interact with the MMP-7 prodomain were virtually screened by protein-ligand docking score analysis using the GalaxyDock3 program. Among five candidates selected based on their structures and total energy values for interacting with the MMP-7 prodomain, the known mechanistic target of rapamycin kinase (mTOR) inhibitor, everolimus, showed the highest binding affinity and the strongest ability to disrupt the interaction of the MMP-7 prodomain with the SDC-2 extracellular domain in vitro. Everolimus treatment of the HCT116 human colon cancer cell line did not affect the mRNA expression levels of MMP-7 and SDC-2 but reduced the adhesion of cells to MMP-7 prodomain-coated plates and the cell-surface localization of MMP-7. Thus, everolimus appears to inhibit the interaction between MMP-7 and SDC-2. Everolimus treatment of HCT116 cells also reduced their gelatin-degradation activity and anticancer activities, including colony formation. Interestingly, cells treated with sirolimus, another mTOR inhibitor, triggered less gelatin-degradation activity, suggesting that this inhibitory effect of everolimus was not due to inhibition of the mTOR pathway. Consistently, everolimus inhibited the colony-forming ability of mTOR-resistant HT29 cells. Together, these data suggest that, in addition to inhibiting mTOR signaling, everolimus exerts anticancer activity by interfering with the interaction of MMP-7 and SDC-2, and could be a useful therapeutic anticancer drug for colon cancer.NEW & NOTEWORTHY The utility of cancer therapeutics targeting the proteolytic activities of MMPs is limited because MMPs are widely distributed throughout the body and involved in many different aspects of cell functions. This work specifically targets the activation of MMP-7 through its interaction with syndecan-2. Notably, everolimus, a known mTOR inhibitor, blocked this interaction, demonstrating a novel role for everolimus in inhibiting mTOR signaling and impairing the interaction of MMP-7 with syndecan-2 in colon cancer.

Light-Dependent Circadian Rhythm Governs O-GlcNAc Cycling to Influence Cognitive Function in Adult Zebrafish.

This study explores the 24-h rhythmic cycle of protein O-GlcNAcylation within the brain and highlights its crucial role in regulating the circadian cycle and neuronal function based on zebrafish as an animal model. In our experiments, disruption of the circadian rhythm, achieved through inversion of the light-dark cycle or daytime melatonin treatment, not only impaired the rhythmic changes of O-GlcNAcylation along with altering expression patterns of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in zebrafish brain but also significantly impeded learning and memory function. In particular, circadian disruption affected rhythmic expression of protein O-GlcNAcylation and OGT in the nuclear fraction. Notably, the circadian cycle induces rhythmic alterations in O-GlcNAcylation of H2B histone protein that correspond to changes in H3 trimethylation. Disruption of the cycle interfered with these periodic histone code alterations. Pharmacological inhibition of OGT with OSMI-1 disrupted the wake-sleep patterns of zebrafish without affecting expression of circadian rhythm-regulating genes. OSMI-1 inhibited the expression of c-fos, bdnf, and calm1, key genes associated with brain function and synaptic plasticity, and decreased the binding of O-GlcNAcylated H2B and OGT to promoter regions of these genes. The collective findings support the potential involvement of circadian cycling of the O-GlcNAc histone code in regulating synaptic plasticity and brain function. Overall, data from this study provide evidence that protein O-GlcNAcylation serves as a pivotal posttranslational mechanism integrating circadian signals and neuronal function to regulate rhythmic physiology.

Emerging role of syndecans in maintaining homeostasis of colon epithelium during inflammation.

The syndecans are a family of transmembrane proteoglycans that are widespread in mammalian tissues. Located at the cell surface membrane, they contribute to modulating the composition of the extracellular matrix via glycosaminoglycan chains (GAGs) attached to their extracellular domains. Syndecans can interact with a variety of extracellular ligands through their core proteins and GAGs, and may also transmit signals through their transmembrane domain to regulate intracellular functions. These properties enable syndecan to modulate glycocalyx formation, epithelial cell-to-cell connections for cell barrier formation, and epithelial cell-lamina propria interactions in the colon epithelium, all of which are crucial for the homeostasis of this tissue. Inflammation induces structural alterations of the colon epithelium, and accumulating evidence suggests that syndecan expression might play important regulatory functions during inflammation. This review summarizes the possible roles of syndecans in maintaining tissue homeostasis in the colon epithelium, especially under inflammation.

Substituted Syndecan-2-Derived Mimetic Peptides Show Improved Antitumor Activity over the Parent Syndecan-2-Derived Peptide.

We previously showed that a synthetic peptide (S2-P) corresponding to a portion of the human syndecan-2 (SDC2) sequence can bind to the pro-domain of matrix metalloproteinase-7 (MMP-7) to inhibit colon cancer activities. Since S2-P had a relatively weak binding affinity for the MMP-7 pro-domain, we herein modified the amino acid sequence of S2-P to improve the anticancer potential. On the basis of the interaction structure of S2-P and MMP-7, four peptides were generated by replacing amino acids near Tyr 51, which is critical for the interaction. The SDC2-mimetic peptides harboring an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-D) or with an Ala-to-Phe substitution at the N-terminal side of Tyr 51 and an Ala-to-Asp substitution at the C-terminal side of Tyr 51 (S2-FE) showed improved interaction affinities for the MMP-7 pro-domain. Compared to S2-P, S2-FE was better able to inhibit the SDC2-MMP-7 interaction, the cell surface localization of MMP-7, the gelatin degradation activity of MMP-7, and the cancer activities (cell migration, invasion, and colony-forming activity) of human HCT116 colon cancer cells in vitro. In vivo, S2-FE inhibited the primary tumor growth and lung metastasis of CT26 mouse colon cancer cells in a xenograft mouse model. Together, these data suggest that S2-FE could be useful therapeutic anticancer peptides for colon cancer.

Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain.

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

We previously reported that syndecan-2 expression is increased on the colonic epithelium during chronic inflammation. Here, we report that syndecan-2 exhibits a different pattern of site-specific colonic expression during acute inflammation. Syndecan-2 expression was up-regulated predominantly in the proximal colon of dextran sulfate sodium-induced colitis mice. The colitis-associated up-regulation of syndecan-2 was barely detected in Rag-1-/- (recombination activating gene 1 knockout) mice under colitis-inducing conditions. Increased syndecan-2 expression correlated with increased levels of infiltrated CD4+ IL-17A+ T cells in the proximal colon. Serum levels of IL-17A were increased during the acute inflammatory response in normal mice but not Rag-1-/- mice. IL-17A directly induced IL-17 receptor (IL-17RA) and syndecan-2 expression in ex vivo-cultured proximal colon tissues and adenoma cell lines from proximal colon. IL-17RA knockdown reduced the IL-17A-mediated syndecan-2 expression in SNU1235 cells. No elevation of syndecan-2 or IL-17RA was observed in colonic tissues from IL-17A-/- mice during colitis induction. Finally, increased expression of syndecan-2 and IL-17RA was observed in the proximal colons of cecal ligation and puncture-induced sepsis mice and infectious pan colitis patients. Together, these data suggest that acute inflammation induces syndecan-2 expression predominantly in the proximal colon via IL-17A-IL-17RA signaling during the early stage of the inflammatory response and that proximal colonic syndecan-2 might be a biomarker for acute inflammation.-Hong, H., Song, H.-K., Hwang, E. S., Lee, A. R., Han, D. S., Kim, S.-E., Oh, E.-S. Up-regulation of syndecan-2 in proximal colon correlates with acute inflammation.

The melanocortin-1 receptor reversely regulates the melanin synthesis and migration of melanoma cells via dimerization-induced conformational changes.

We previously reported that the melanocortin-1 receptor (MC1R), a key regulator of melanogenesis, regulates cell migration; however, the detailed mechanism remained unknown. Since the homo-dimerization of MC1R by four inter-subunit disulfide bonds is known to be functionally important for melanogenesis, we investigated the importance of MC1R dimerization for cell migration. Unlike the wild-type MC1R, the dimerization-defective mutant MC1R in which four critical Cys residues were replaced with Ala residues (Cys35-267-273-275Ala) significantly inhibited melanin synthesis but enhanced cell migration in human MNT-1 and A375 melanoma cells. This suggests that there may be a reverse correlation between melanin synthesis and cell migration. Interestingly, melanoma cells expressing the dimerization-defective mutant exhibited enhanced expression of the cell surface heparan sulfate proteoglycan, syndecan-2, and knockdown of syndecan-2 expression decreased the mutant-mediated cell migration. Consistently, ASIP, an antagonist of MC1R, enhanced syndecan-2 expression and cell migration and reversed the α-melanocyte-stimulating hormone (α-MSH)-mediated inhibition of syndecan-2 expression. Furthermore, α-MSH reduced the cell migration of MNT1 cells expressing wild-type MC1R but not its dimerization-defective mutant. Together, these data strongly suggest that MC1R reversely regulates melanin synthesis and migration via the conformational changes induced by dimerization.

Syndecan-2 cytoplasmic domain up-regulates matrix metalloproteinase-7 expression via the protein kinase Cγ-mediated FAK/ERK signaling pathway in colon cancer.

The syndecan family of heparan sulfate proteoglycans contributes to cell adhesion and communication by serving as co-receptors for cell signaling and extracellular matrix molecules. Syndecan-2 is located at the cell surface, and we previously reported that it induces matrix metalloproteinase-7 (MMP-7) expression in colon cancer cells. However, the underlying regulatory mechanisms are unknown. Here, we report that overexpression of syndecan-2 in HT-29 colon cancer cells increases the phosphorylation of focal adhesion kinase (FAK) and ERK in parallel with up-regulated MMP-7 expression, but a syndecan-2 mutant lacking the cytoplasmic domain showed significant reductions in these effects. Consistent with this observation, FAK inhibition via FAK-related non-kinase expression or inhibition of ERK with the ERK1/2 inhibitor SCH772984 diminished the syndecan-2-mediated up-regulation of MMP-7. Activation of PKC enhanced syndecan-2-mediated MMP-7 expression, whereas inhibition of PKC had the opposite effect. Of note, the exogenous expression of syndecan-2 triggered localization of PKCγ to the membrane. Expression of syndecan-2 harboring a phosphomimetic (S198E) mutation of the variable region of the cytoplasmic domain enhanced MMP-7 expression and FAK phosphorylation. Finally, experimental suppression of shedding of the syndecan-2 extracellular domain did not significantly affect the syndecan-2-mediated up-regulation of MMP-7 in the early period after syndecan-2 overexpression. Taken together, these findings suggest that syndecan-2's cytoplasmic domain up-regulates MMP-7 expression in colon cancer cells via PKCγ-mediated activation of FAK/ERK signaling.

Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells.

We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions. Glucose itself dose dependently increased LPS-induced iNOS expression. LPS-induced MAPK and IκB-α phosphorylation did not significantly differ at normal and high glucose conditions. The activity of LPS-induced nuclear factor-κB (NF-κB) and DNA binding of c-Rel to the iNOS promoter were inhibited under high glucose conditions in comparison with no significant changes under normal glucose conditions. In addition, we found that the LPS-induced increase in O-GlcNAcylation as well as DNA binding of c-Rel to the iNOS promoter were further increased by GlcN under normal glucose conditions. However, both O-GlcNAcylation and DNA binding of c-Rel decreased under high glucose conditions. The NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited LPS-induced iNOS expression under high glucose conditions but it did not influence iNOS induction under normal glucose conditions. In addition, pyrrolidine dithiocarbamate inhibited NF-κB DNA binding and c-Rel O-GlcNAcylation only under high glucose conditions. By blocking transcription with actinomycin D, we found that stability of LPS-induced iNOS mRNA was increased by GlcN under normal glucose conditions. These results suggest that GlcN regulates inflammation by sensing energy states of normal and fuel excess.

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation.

Inflammatory hypoxia induces syndecan-2 expression through IL-1ß-mediated FOXO3a activation in colonic epithelia.

Chronic inflammation is known to be a key causative factor in tumor progression, but we do not yet fully understand the molecular mechanism through which inflammation leads to cancer. Here, we report that the dextran sulfate sodium (DSS)-induced mouse model of chronic colitis is associated with increases in the serum level of IL-1ß and the colonic epithelial expression of the cell-surface heparan sulfate proteoglycan, syndecan-2. We further show that IL-1ß stimulated the transcription of syndecan-2 via NF-κB-dependent FOXO3a activation in CCD841CoN normal colonic epithelial cells and early-stage HT29 colon cancer cells. Inflammatory hypoxia was observed in the colonic epithelia of mice with chronic colitis, suggesting that hypoxic stress is involved in the regulation of syndecan-2 expression. Consistently, experimental inflammatory hypoxia induced hypoxia inducible factor-1α-dependent FOXO3a expression and the p38 MAPK-mediated nuclear localization of FOXO3a. FOXO3a directly mediated syndecan-2 expression in both cell lines and the colonic epithelia of mice with DSS-induced colitis. Moreover, syndecan-2 expression was detected in azoxymethane/DSS-induced colon tumors. Together, these data demonstrate that inflammatory hypoxia up-regulates syndecan-2 via the IL-1ß-NF-κB-FOXO3a pathway. These findings provide new mechanistic insights into inflammatory hypoxia-mediated syndecan-2 expression to connect chronic inflammation and the development of colon cancer.-Choi, S., Chung, H., Hong, H., Kim, S. Y., Kim, S.-E., Seoh, J.-Y., Moon, C. M., Yang, E. G., Oh, E.-S. Inflammatory hypoxia induces syndecan-2 expression through IL-1ß-mediated FOXO3a activation in colonic epithelia.

Processing of syndecan-2 by matrix metalloproteinase-14 and effect of its cleavage on VEGF-induced tube formation of HUVECs.

Syndecans (SDCs) are transmembrane proteoglycans that are involved in cell adhesion and cell communication. Specifically, SDC2 plays a key role in tumorigenesis, metastasis, and angiogenesis. Previously, we found that rat SDC2 is shed by matrix metalloproteinase-7 (MMP-7) in colon cancer cells. Here, we analyzed the susceptibility of rat SDC2 to various MMPs. We found that the rat SDC2 ectodomain (ECD) fused to the C-terminal Fc region, which was expressed in mammalian cells, was cleaved more efficiently by MMP-14 than MMP-7. Likewise, when anchored on the surface of HeLa cells, rat SDC2 was cleaved more efficiently by the treatment of MMP-14 than MMP-7 and was shed more readily by membrane-anchored MMP-14 than soluble MMP-14. Furthermore, MMP-14 cleaved recombinant SDC2-ECD expressed in Escherichia coli into multiple fragments. Using N-terminal amino acid sequencing and the top-down proteomics approach, we determined that the major cleavage sites were S88↓L89, T98↓M99, T100↓L101, D132↓P133, and N148↓L149 for rat SDC2-ECD and S55↓G56, S65↓P66, P75↓K76, N92↓I93 D122↓P123, and S138↓L139 for human SDC2-ECD. Finally, the rat and human SDC2-ECD lost the ability to suppress vascular endothelial growth factor-induced formation of capillary-like tubes by human umbilical vein endothelial cells following cleavage by MMP-14, but its major cleavage-site mutant of rat SDC2-ECD did not. These results suggest that MMP-14 is a novel enzyme responsible for degrading SDC2 and impairing its physiological roles including angiogenesis.

The Conserved Phenylalanine in the Transmembrane Domain Enhances Heteromeric Interactions of Syndecans.

The transmembrane domain (TMD) of the syndecans, a family of transmembrane heparin sulfate proteoglycans, is involved in forming homo- and heterodimers and oligomers that transmit signaling events. Recently, we reported that the unique phenylalanine in TMD positively regulates intramolecular interactions of syndecan-2. Besides the unique phenylalanine, syndecan-2 contains a conserved phenylalanine (SDC2-Phe-169) that is present in all syndecan TMDs, but its function has not been determined. We therefore investigated the structural role of SDC2-Phe-169 in syndecan TMDs. Replacement of SDC2-Phe-169 by tyrosine (S2F169Y) did not affect SDS-resistant homodimer formation but significantly reduced SDS-resistant heterodimer formation between syndecan-2 and -4, suggesting that SDC2-Phe-169 is involved in the heterodimerization/oligomerization of syndecans. Similarly, in an in vitro binding assay, a syndecan-2 mutant (S2(F169Y)) showed a significantly reduced interaction with syndecan-4. FRET assays showed that heteromolecular interactions between syndecan-2 and -4 were reduced in HEK293T cells transfected with S2(F169Y) compared with syndecan-2. Moreover, S2(F169Y) reduced downstream reactions mediated by the heterodimerization of syndecan-2 and -4, including Rac activity, cell migration, membrane localization of PKCα, and focal adhesion formation. The conserved phenylalanine in syndecan-1 and -3 also showed heterodimeric interaction with syndecan-2 and -4. Taken together, these findings suggest that the conserved phenylalanine in the TMD of syndecans is crucial in regulating heteromeric interactions of syndecans.

Screening of breast cancer stem cell inhibitors using a protein kinase inhibitor library.

BACKGROUND: Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)'s Integrated Colony Enumerator (NICE) with a screening of protein kinase library. METHODS: Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated. RESULTS: Automated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IκB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-ß1-induced migration, colony formation and EMT of MCF-7 cells. CONCLUSIONS: We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.

Trans-regulation of Syndecan Functions by Hetero-oligomerization.

Syndecans, a family of transmembrane heparansulfate proteoglycans, are known to interact through their transmembrane domains to form non-covalently linked homodimers, a process essential for their individual functions. Because all syndecan transmembrane domains are highly conserved and thus might mediate interactions between different members of the syndecan family, we investigated syndecan interactions in detail. All recombinant syndecan-2 and -4 protein variants containing the transmembrane domain formed not only sodium dodecyl sulfate (SDS)-resistant homodimers but also SDS-resistant heterodimers. Biochemical and structural data revealed that recombinant syndecan-2 and -4 formed intermolecular interactions in vitro, and the GXXXG motif in transmembrane domain mediated this interaction. When exogenously expressed in rat embryonic fibroblasts, syndecan-2 interacted with syndecan-4 and vice versa. Furthermore, bimolecular fluorescence complementation-based assay demonstrated specific hetero-molecular interactions between syndecan-2 and -4, supporting hetero-oligomer formation of syndecans in vivo. Interestingly, hetero-oligomerization significantly reduced syndecan-4-mediated cellular processes such as protein kinase Cα activation and protein kinase Cα-mediated cell adhesion as well as syndecan-2-mediated tumorigenic activities in colon cancer cells such as migration and anchorage-independent growth. Taken together, these data provide evidence that hetero-oligomerization produces distinct syndecan functions and offer insights into the underlying signaling mechanisms of syndecans.

A unique phenylalanine in the transmembrane domain strengthens homodimerization of the syndecan-2 transmembrane domain and functionally regulates syndecan-2.

The syndecans are a type of cell surface adhesion receptor that initiates intracellular signaling events through receptor clustering mediated by their highly conserved transmembrane domains (TMDs). However, the exact function of the syndecan TMD is not yet fully understood. Here, we investigated the specific regulatory role of the syndecan-2 TMD. We found that syndecan-2 mutants in which the TMD had been replaced with that of syndecan-4 were defective in syndecan-2-mediated functions, suggesting that the TMD of syndecan-2 plays one or more specific roles. Interestingly, syndecan-2 has a stronger tendency to form sodium dodecyl sulfate (SDS)-resistant homodimers than syndecan-4. Our structural studies showed that a unique phenylalanine residue (Phe(167)) enables an additional molecular interaction between the TMDs of the syndecan-2 homodimer. The presence of Phe(167) was correlated with a higher tendency toward oligomerization, and its replacement with isoleucine significantly reduced the SDS-resistant dimer formation and cellular functions of syndecan-2 (e.g. cell migration). Conversely, replacement of isoleucine with phenylalanine at this position in the syndecan-4 TMD rescued the defects observed in a mutant syndecan-2 harboring the syndecan-4 TMD. Taken together, these data suggest that Phe(167) in the TMD of syndecan-2 endows the protein with specific functions. Our work offers new insights into the signaling mediated by the TMD of syndecan family members.

Syndecan-2 enhances E-cadherin shedding and fibroblast-like morphological changes by inducing MMP-7 expression in colon cancer cells.

E-cadherin plays a mechanical role in mediating cell-cell interactions and maintaining epithelial tissue integrity, and the loss of E-cadherin function has been implicated in cancer progression and metastasis. Syndecan-2, a cell-surface heparan sulfate proteoglycan, is upregulated during the development of colon cancer. Here, we assessed the functional relationship between E-cadherin and syndecan-2. We found that stable overexpression of syndecan-2 in a human colorectal adenocarcinoma cell line (HT29) enhanced the proteolytic shedding of E-cadherin to conditioned-media. Either knockdown of matrix metalloproteinase 7 (MMP-7) or inhibition of MMP-7 activity using GM6001 significantly reduced the extracellular shedding of E-cadherin, suggesting that syndecan-2 mediates E-cadherin shedding via MMP-7. Consistent with this notion, enhancement of MMP-7 expression by interleukin-1α treatment increased the shedding of E-cadherin. Conversely, the specific reduction of either syndecan-2 or MMP-7 reduced the shedding of E-cadherin. HT29 cells overexpressing syndecan-2 showed significantly lower cell-surface expression of E-cadherin, decreased cell-cell contact, a more fibroblastic cell morphology, and increased expression levels of ZEB-1. Taken together, these data suggest that syndecan-2 induces extracellular shedding of E-cadherin and supports the acquisition of a fibroblast-like morphology by regulating MMP-7 expression in a colon cancer cell line.

Cytokeratin 18 is necessary for initiation of TGF-ß1-induced epithelial-mesenchymal transition in breast epithelial cells.

During epithelial-mesenchymal transition (EMT), epithelial cells lose key phenotypic markers (e.g., E-cadherin and cytokeratin 18) and acquire mesenchymal markers (e.g., N-cadherin and vimentin). Although the loss of cytokeratin 18 is a hallmark of EMT, the regulatory role of cytokeratin 18 in EMT is not yet fully understood. Here, we report that cytokeratin 18 is involved in the regulation of transforming growth factor-beta1 (TGF-ß1)-induced EMT in breast epithelial cells. When MCF10A cells were treated with TGF-ß1 for 24 h, considerable morphological changes, indicative of the early stages of EMT (e.g., loss of cell-cell contact), were observed and cytokeratin 18 was downregulated. However, E-cadherin levels were not altered until a later time point. This suggests that cytokeratin 18 may play an active role during the earlier stages of EMT. Consistent with this notion, siRNA-mediated knockdown of cytokeratin 18 delayed TGF-ß1-mediated EMT, and the associated downregulation of E-cadherin reduced the phosphorylation/nuclear localization of smad 2/3 and decreased the expression levels of snail and slug (which inhibit E-cadherin expression in epithelial cells as an early response to TGF-ß1). Taken together, these results suggest that cytokeratin 18 critically contributes to initiating TGF-ß1-induced EMT via the smad 2/3-mediated regulation of snail and slug expression in breast epithelial cells.

Inhibitory effects of N,N,N-trimethyl phytosphingosine-iodide on melanogenesis via ERK activation-mediated MITF degradation.

N,N,N-trimethyl phytosphingosine-iodide (TMP) was recently developed as an antitumor agent. We examined the effects of TMP on melanogenesis and its related signaling pathways in normal human melanocytes. Our results showed that melanin is significantly reduced in a dose-dependent manner in both cells following liposomal TMP treatment. We also investigated changes in the phosphorylation of extracellular signal-regulated kinase (ERK), which is related to the degradation of microphthalmia-associated transcription factor (MITF). Our results indicated that liposomal TMP treatment leads to the phosphorylation of ERK, which reduces both MITF and tyrosinase protein levels. Treatment with PD98059, an ERK pathway-specific inhibitor, restored liposomal TMP-induced reductions in melanin, abrogated reductions in tyrosinase activity, and downregulated MITF and tyrosinase protein. In conclusion, these results suggest that the inhibitory effects of TMP on melanogenesis are due to MITF and tyrosinase downregulation via ERK activation.