Tyrosine 51 residue of the syndecan-2 extracellular domain is involved in the interaction with and activation of pro-matrix metalloproteinase-7.

Although syndecan-2 is known to interact with the matrix metalloproteinase-7 (MMP-7), the details of their interaction were unknown. Our experiments with a series of syndecan-2 extracellular domain deletion mutants show that the interaction is mediated through an interaction of the extracellular domain of syndecan-2 (residues 41 to 60) with the α2 helix-loop-α3 helix in the pro-domain of MMP-7. NMR and molecular docking model show that Glu7 of the α1 helix, Glu32 of the α2 helix, and Gly48 and Ser52 of the α2 helix-loop-α3 helix of the MMP-7 pro-domain form the syndecan-2-binding pocket, which is occupied by the side chain of tyrosine residue 51 (Tyr51) of syndecan-2. Consistent with this notion, the expression of a syndecan-2 mutant in which Tyr51 was changed to Ala diminished the interaction between the syndecan-2 extracellular domain and the pro-domain of MMP-7. Furthermore, HT-29 colon adenocarcinoma cells expressing the interaction-defective mutant exhibited reductions in the cell-surface localization of MMP-7, the processing of pro-MMP-7 into active MMP-7, the MMP-7-mediated extracellular domain shedding of both syndecan-2 and E-cadherin, and syndecan-2-mediated anchorage-independent growth. Collectively, these data strongly suggest that Tyr51 of the syndecan-2 extracellular domain mediates its interaction with and activating processing of pro-MMP-7 and regulates MMP-7-dependent syndecan-2 functions.

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.

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.

Probing protein complexes inside living cells using a silicon nanowire-based pull-down assay.

Most proteins perform their functions as interacting complexes. Here we propose a novel method for capturing an intracellular protein and its interacting partner out of living cells by utilizing intracellular access of antibody modified vertical silicon nanowire arrays whose surface is covered with a polyethylene glycol layer to prevent strong cell adhesion. Such a feature facilitates the removal of cells by simple washing, enabling subsequent detection of a pulled-down protein and its interacting partner, and further assessment of a drug-induced change in the interacting complex. Our new SiNW-based tool is thus suitable for authentication of protein networks inside living cells.

Shed syndecan-2 enhances tumorigenic activities of colon cancer cells.

Because earlier studies showed the cell surface heparan sulfate proteoglycan, syndecan-2, sheds from colon cancer cells in culture, the functional roles of shed syndecan-2 were assessed. A non-cleavable mutant of syndecan-2 in which the Asn148-Leu149 residues were replaced with Asn148-Ile149, had decreased shedding, less cancer-associated activities of syndecan-2 in vitro, and less syndecan-2-mediated metastasis of mouse melanoma cells in vivo, suggesting the importance of shedding on syndecan-2-mediated pro-tumorigenic functions. Indeed, shed syndecan-2 from cancer-conditioned media and recombinant shed syndecan-2 enhanced cancer-associated activities, and depletion of shed syndecan-2 abolished these effects. Similarly, shed syndecan-2 was detected from sera of patients from advanced carcinoma (625.9 ng/ml) and promoted cancer-associated activities. Furthermore, a series of syndecan-2 deletion mutants showed that the tumorigenic activity of shed syndecan-2 resided in the C-terminus of the extracellular domain and a shed syndecan-2 synthetic peptide (16 residues) was sufficient to establish subcutaneous primary growth of HT29 colon cancer cells, pulmonary metastases (B16F10 cells), and primary intrasplenic tumor growth and liver metastases (4T1 cells). Taken together, these results demonstrate that shed syndecan-2 directly enhances colon cancer progression and may be a promising therapeutic target for controlling colon cancer development.

Growth factors in synaptic function.

Synapses are increasingly recognized as key structures that malfunction in disorders like schizophrenia, mental retardation, and neurodegenerative diseases. The importance and complexity of the synapse has fuelled research into the molecular mechanisms underlying synaptogenesis, synaptic transmission, and plasticity. In this regard, neurotrophic factors such as netrin, Wnt, transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), and others have gained prominence for their ability to regulate synaptic function. Several of these factors were first implicated in neuroprotection, neuronal growth, and axon guidance. However, their roles in synaptic development and function have become increasingly clear, and the downstream signaling pathways employed by these factors have begun to be elucidated. In this review, we will address the role of these factors and their downstream effectors in synaptic function in vivo and in cultured neurons.

Targeting syndecans: a promising strategy for the treatment of cancer.

INTRODUCTION: Syndecans are cell surface adhesion receptors that play important functional roles. Their deregulation has been linked to several pathologies, and their aberrant regulation via diverse mechanisms is critically involved in the pathogenesis of various cancers. Similar to other receptors, syndecan functions are altered by ligand binding, overexpression, and extracellular domain shedding. These regulatory mechanisms may suggest new strategies for specifically controlling cancer cells. AREAS COVERED: In this review, we describe the biological functions of syndecans in cancer, discuss their potential therapeutic relevance, and examine the current syndecan-targeting therapeutic options. EXPERT OPINION: Since syndecans have diverse, specific, and novel regulatory functions in various cancers, therapeutic control of syndecan function has become an attractive approach for potential anticancer strategies. Syndecan and its associated proteins may also be strong candidate markers for the diagnosis and management of cancer, potentially opening a new era in cancer treatment.

The matrix metalloproteinase-7 regulates the extracellular shedding of syndecan-2 from colon cancer cells.

The cell surface heparan sulfate proteoglycan syndecan-2 regulates the activation of matrix metalloproteinase-7 (MMP-7) as a docking receptor. Here, we demonstrate the role of MMP-7 on syndecan-2 shedding in colon cancer cells. Western blot analysis showed that shed syndecan-2 was found in the culture media from various colon cancer cells. Overexpression of MMP-7 enhanced syndecan-2 shedding, whereas the opposite was true when MMP-7 levels were knocked-down using small inhibitory RNAs. Consistently, HT29 cells treated with MMP-7, but neither MMP-2 nor MMP-9, showed increased shed syndecan-2 in a time- and concentration-dependent manner. Furthermore, MALDI-TOF MS analysis and N-terminal amino acid sequencing revealed that MMP-7 cleaved both recombinant syndecan-2 and an endogenously glycosylated syndecan-2 ectodomain in the N-terminus at Leu(149) residue in vitro. Taken together, the data suggest that MMP-7 directly mediates shedding of syndecan-2 from colon cancer cells.

Syndecan-2 cytoplasmic domain regulates colon cancer cell migration via interaction with syntenin-1.

The cell surface heparan sulfate proteoglycan, syndecan-2, is crucial for the tumorigenic activity of colon cancer cells. However, the role played by the cytoplasmic domain of the protein remains unclear. Using colon cancer cells transfected with various syndecan-2-encoding genes with deletions in the cytoplasmic domain, it was shown that syndecan-2-induced migration activity requires the EFYA sequence of the C-terminal region; deletion of these residues abolished the rise in cell migration seen when the wild-type gene was transfected and syndecan-2 interaction with syntenin-1, suggesting that syntenin-1 functioned as a cytosolic signal effector downstream from syndecan-2. Colon cancer cells transfected with the syntenin-1 gene showed increased migratory activity, whereas migration was decreased in cells in which syntenin-1 was knock-down using small inhibitory RNA. In addition, syntenin-1 expression potentiated colon cancer cell migration induced by syndecan-2, and syndecan-2-mediated cell migration was reduced when syntenin-1 expression diminished. However, syntenin-1-mediated migration enhancement was not noted in colon cancer cells transfected with a gene encoding a syndecan-2 mutant lacking the cytoplasmic domain. Furthermore, in line with the increase in cell migration, syntenin-1 mediated Rac activation stimulated by syndecan-2. Together, the data suggest that the cytoplasmic domain of syndecan-2 regulates colon cancer cell migration via interaction with syntenin-1.

Shedding; towards a new paradigm of syndecan function in cancer.

Syndecans, cell surface heparansulfate proteoglycans, have been proposed to act as cell surface receptors and/or coreceptors to play critical roles in multiple cellular functions. However, recent reports suggest that the function of syndecans can be further extended through shedding, a cleavage of extracellular domain. Shedding constitutes an additional level for controlling the function of syndecans, providing a means to attenuate and/or regulate amplitude and duration of syndecan signals by modulating the activity of syndecans as cell surface receptors. Whether these remaining cleavage products are still capable of functioning as cell surface receptors to efficiently transduce signals inside of cells is not clear. However, shedding transforms cell surface receptor syndecans into soluble forms, which, like growth factors, may act as novel ligands to induce cellular responses by association with other cell surface receptors. It is becoming interestingly evident that shed syndecans also contribute significantly to syndecan functions in cancer biology. This review presents current knowledge about syndecan shedding and its functional significance, particularly in the context of cancer.

Syndecan-2 functions as a docking receptor for pro-matrix metalloproteinase-7 in human colon cancer cells.

Although elevated syndecan-2 expression is known to be crucial for the tumorigenic activity in colon carcinoma cells, how syndecan-2 regulates colon cancer is unclear. In human colon adenocarcinoma tissue samples, we found that both mRNA and protein expression of syndecan-2 were increased, compared with the neighboring normal epithelium, suggesting that syndecan-2 plays functional roles in human colon cancer cells. Consistent with this notion, syndecan-2-overexpressing HT-29 colon adenocarcinoma cells showed enhanced migration/invasion, anchorage-independent growth, and primary tumor formation in nude mice, paralleling their morphological changes into highly tumorigenic cells. In addition, our experiments revealed that syndecan-2 enhanced both expression and secretion of matrix metalloproteinase-7 (MMP-7), directly interacted with pro-MMP-7, and potentiated the enzymatic activity of pro-MMP-7 by activating its processing into the active MMP-7. Collectively, these data strongly suggest that syndecan-2 functions as a docking receptor for pro-MMP-7 in colon cancer cells.

Syndecan-2 regulates the migratory potential of melanoma cells.

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, alpha-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by alpha-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells.

Syndecan-4 promotes the retention of phosphatidylinositol 4,5-bisphosphate in the plasma membrane.

Although phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates syndecan-4 function, the potential influence of syndecan-4 on PIP(2) remains unknown. GFP containing PIP(2)-binding-PH domain of phospholipase Cdelta (GFP-PHdelta) was used to monitor PIP(2). Syndecan-4 overexpression in COS-7 cells enhanced membrane translocation of GFP-PHdelta, while the opposite was observed when syndecan-4 was knocked-down. PIP(2) levels were higher in total phospholipids extracted from rat embryo fibroblasts expressing syndecan-4. Syndecan-4-induced membrane targeting of GFP-PHdelta was further enhanced by phosphoinositide-3-kinase inhibitor, but not by phospholipase C (PLC) inhibitor. Besides, both ionomycin and epidermal growth factor caused dissociation of GFP-PHdelta from plasma membrane, an effect that was significantly delayed by syndecan-4 over-expression. Collectively, these data suggest that syndecan-4 promotes plasma membrane retention of PIP(2) by negatively regulating PLC-dependent PIP(2) degradation.

Syndecan-2 overexpression regulates adhesion and migration through cooperation with integrin alpha2.

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is known to serve as an adhesion receptor, but details of the regulatory mechanism governing syndecan-2 cell adhesion and migration remain unclear. Here, we examined this regulatory mechanism, showing that overexpression of syndecan-2 enhanced collagen adhesion, cell migration and invasion of normal rat intestinal epithelial cells (RIE1), and increased integrin alpha2 expression levels. Interestingly, RIE1 cells transfected with either syndecan-2 or integrin alpha2 showed similar adhesion and migration patterns, and a function-blocking anti-integrin alpha2 antibody abolished syndecan-2-mediated adhesion and migration. Consistent with these findings, transfection of integrin alpha2 siRNA diminished syndecan-2-induced cell migration in HCT116 human colon cancer cells. Taken together, these results demonstrate a novel cooperation between syndecan-2 and integrin alpha2beta1 in adhesion-mediated cell migration and invasion. This interactive dynamic might be a possible mechanism underlying the tumorigenic activities of colon cancer cells.

Phosphorylation of focal adhesion kinase at Tyrosine 407 negatively regulates Ras transformation of fibroblasts.

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs, via tyrosine phosphorylation at specific residues. We recently reported that FAK Tyr-407 phosphorylation negatively regulates the enzymatic and biological activities of FAK, unlike phosphorylation of other tyrosine residues. In this study, we further investigated the effect of FAK Tyr-407 phosphorylation on cell transformation. We found that FAK Tyr-407 phosphorylation was lower in H-Ras transformed NIH3T3 and K-Ras transformed rat-2 fibroblasts than in the respective untransformed control cells. Consistently, FAK Tyr-407 phosphorylation was decreased in parallel with cell transformation in H-Ras-inducible NIH3T3 cells and increased during trichostatin A-induced detransformation of both K-Ras transformed rat-2 fibroblasts and H-Ras transformed NIH3T3 cells. In addition, overexpression of a phosphorylation-mimicking FAK Tyr-407 mutant inhibited morphological transformation of H-Ras-inducible NIH3T3 cells and inhibited invasion activity and anchorage-independent growth of H-Ras-transformed NIH3T3 cells. Taken together, these data strongly suggest that FAK Tyr-407 phosphorylation negatively regulates transformation of fibroblasts.

Protein kinase Calpha can undergo membrane localization via an alternative phosphatidylinositol 4,5-bisphosphate-dependent pathway.

Protein kinase Calpha (PKCalpha) activation is known to be dependent on the metabolic product of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC). Here we report that fibroblasts may have an additional PIP2-dependent mechanism for membrane localization of PKCalpha. We observed PKCalpha membrane localization in both wild type and PLCgamma1 -/- mouse embryonic fibroblasts. Treatment of cells with a specific PLC inhibitor U73122 resulted in increased PIP2 levels and enhanced membrane localization of PKCalpha. PKCalpha levels in the membrane fraction decreased following incubation with PLCgamma, but increased following treatment with U73122 or addition of exogenous PIP2 in vitro. In addition, PKCalpha interacted with PIP2-conjugate bead and mixed micelles containing PIP2. Finally, we found that PIP2 is involved in syndecan-4-mediated membrane localization of PKCalpha. Taken together, these data suggest that PIP2 might contribute to directly regulating the membrane localization of PKCalpha.