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Recently there have been reports that identify two transient receptor potential channels in cell–matrix junctions known as focal adhesions. These are the calcium channel TRP canonical 7 and the calcium-activated monovalent ion channel, TRP melastatin (TRPM) 4. Here, we report on the occurrence of TRPM4 in focal adhesions of fibroblasts. Of three commercial antibodies recognizing this channel, only one yielded focal adhesion staining, while the other two did not. The epitope recognized by the focal adhesion-localizing antibody was mapped to the extreme C-terminus of the TRPM4 protein. The other two antibodies bind to N-terminal regions of the TRPM4 proteins. Deletion of the TRPM4 gene by CRISPR/cas9 techniques confirmed that this channel is a bona fide focal adhesion component, while expression of full-length TRPM4 proteins suggested that processing may occur to yield a form that localizes to focal adhesions. Given the reports that this channel may influence migratory behavior of cells and is linked to cardiovascular disease, TRPM4 functions in adhesion should be explored in greater depth. (J Histochem Cytochem 71: 495–508, 2023).

Syndecans are transmembrane heparan sulfate proteoglycans present on most mammalian cell surfaces. They have a long evolutionary history, a single syndecan gene being expressed in bilaterian invertebrates. Syndecans have attracted interest because of their potential roles in development and disease, including vascular diseases, inflammation and various cancers. Recent structural data is providing important insights into their functions, which are complex, involving both intrinsic signaling through cytoplasmic binding partners and co-operative mechanisms where syndecans form a signaling nexus with other receptors such as integrins and tyrosine kinase growth factor receptors. While the cytoplasmic domain of syndecan-4 has a well-defined dimeric structure, the syndecan ectodomains are intrinsically disordered, which is linked to a capacity to interact with multiple partners. However, it remains to fully establish the impact of glycanation and partner proteins on syndecan core protein conformations. Genetic models indicate that a conserved property of syndecans links the cytoskeleton to calcium channels of the transient receptor potential class, compatible with roles as mechanosensors. In turn, syndecans influence actin cytoskeleton organization to impact motility, adhesion and the extracellular matrix environment. Syndecan clustering with other cell surface receptors into signaling microdomains has relevance to tissue differentiation in development, for example in stem cells, but also in disease where syndecan expression can be markedly up-regulated. Since syndecans have potential as diagnostic and prognostic markers as well as possible targets in some forms of cancer, it remains important to unravel structure/function relationships in the four mammalian syndecans.

Cell surface proteoglycans are known to be important regulators of many aspects of cell behavior. The principle family of transmembrane proteoglycans is the syndecans, of which there are four in mammals. Syndecan-1 is mostly restricted to epithelia, and bears heparan sulfate chains that are capable of interacting with a large array of polypeptides, including extracellular matrix components and potent mediators of proliferation, adhesion and migration. For this reason, it has been studied extensively with respect to carcinomas and tumor progression. Frequently, but not always, syndecan-1 levels decrease as tumor grade, stage and invasiveness and dedifferentiation increase. This parallels experiments that show depletion of syndecan-1 can be accompanied by loss of cadherin-mediated adhesion. However, in some tumors, levels of syndecan-1 increase, but the characterization of its distribution is relevant. There can be loss of membrane staining, but acquisition of cytoplasmic and/or nuclear staining that is abnormal. Moreover, the appearance of syndecan-1 in the tumor stroma, either associated with its cellular component or the collagenous matrix, is nearly always a sign of poor prognosis. Given its relevance to myeloma progression, syndecan-1-directed antibody—toxin conjugates are being tested in clinical and preclinical trials, and may have future relevance to some carcinomas.

The syndecans are the major family of transmembrane proteoglycans, usually bearing multiple heparan sulfate chains. They are present on virtually all nucleated cells of vertebrates and are also present in invertebrates, indicative of a long evolutionary history. Genetic models in both vertebrates and invertebrates have shown that syndecans link to the actin cytoskeleton and can fine-tune cell adhesion, migration, junction formation, polarity and differentiation. Although often associated as co-receptors with other classes of receptors (e.g. integrins, growth factor and morphogen receptors), syndecans can nonetheless signal to the cytoplasm in discrete ways. Syndecan expression levels are upregulated in development, tissue repair and an array of human diseases, which has led to the increased appreciation that they may be important in pathogenesis not only as diagnostic or prognostic agents, but also as potential targets. Here, their functions in development and inflammatory diseases are summarized, including their potential roles as conduits for viral pathogen entry into cells.

Cell surface proteoglycans are known to be important regulators of many aspects of cell behavior. The principle family of transmembrane proteoglycans is the syndecans, of which there are four in mammals. Syndecan-1 is mostly restricted to epithelia, and bears heparan sulfate chains that are capable of interacting with a large array of polypeptides, including extracellular matrix components and potent mediators of proliferation, adhesion and migration. For this reason, it has been studied extensively with respect to carcinomas and tumor progression. Frequently, but not always, syndecan-1 levels decrease as tumor grade, stage and invasiveness and dedifferentiation increase. This parallels experiments that show depletion of syndecan-1 can be accompanied by loss of cadherin-mediated adhesion. However, in some tumors, levels of syndecan-1 increase, but the characterization of its distribution is relevant. There can be loss of membrane staining, but acquisition of cytoplasmic and/or nuclear staining that is abnormal. Moreover, the appearance of syndecan-1 in the tumor stroma, either associated with its cellular component or the collagenous matrix, is nearly always a sign of poor prognosis. Given its relevance to myeloma progression, syndecan-1-directed antibody—toxin conjugates are being tested in clinical and preclinical trials, and may have future relevance to some carcinomas.

The syndecans are the major family of transmembrane proteoglycans, usually bearing multiple heparan sulfate chains. They are present on virtually all nucleated cells of vertebrates and are also present in invertebrates, indicative of a long evolutionary history. Genetic models in both vertebrates and invertebrates have shown that syndecans link to the actin cytoskeleton and can fine-tune cell adhesion, migration, junction formation, polarity and differentiation. Although often associated as co-receptors with other classes of receptors (e.g. integrins, growth factor and morphogen receptors), syndecans can nonetheless signal to the cytoplasm in discrete ways. Syndecan expression levels are upregulated in development, tissue repair and an array of human diseases, which has led to the increased appreciation that they may be important in pathogenesis not only as diagnostic or prognostic agents, but also as potential targets. Here, their functions in development and inflammatory diseases are summarized, including their potential roles as conduits for viral pathogen entry into cells.

In multicellular organisms, the cells are surrounded by persistent, dynamic extracellular matrix (ECM), the largest calcium reservoir in animals. ECM regulates several aspects of cell behavior including cell migration and adhesion, survival, gene expression and differentiation, thus playing a significant role in health and disease. Calcium is reported to be important in the assembly of ECM, where it binds to many ECM proteins. While serving as a calcium reservoir, ECM macromolecules can directly interact with cell surface receptors resulting in calcium transport across the membrane. This chapter mainly focusses on the role of cell-ECM interactions in cellular calcium regulation and how calcium itself mediates these interactions.

In multicellular organisms, the cells are surrounded by persistent, dynamic extracellular matrix (ECM), the largest calcium reservoir in animals. ECM regulates several aspects of cell behavior including cell migration and adhesion, survival, gene expression and differentiation, thus playing a significant role in health and disease. Calcium is reported to be important in the assembly of ECM, where it binds to many ECM proteins. While serving as a calcium reservoir, ECM macromolecules can directly interact with cell surface receptors resulting in calcium transport across the membrane. This chapter mainly focusses on the role of cell-ECM interactions in cellular calcium regulation and how calcium itself mediates these interactions.

Transforming growth factor-β (TGF-β) is an important regulator of many cellular and immunological functions. It is often deposited in extracellular matrices in a latent form. This commentary is to draw attention to the likelihood that preparing cell-free matrices from tissue cultures by high pH buffers, such as ammonium hydroxide, can activate the TGF-β. Therefore, cells subsequently seeded onto such matrices may respond to the presence of active TGF-β in addition to interactions with macromolecular extracellular matrix components.

Proteoglycans can be difficult molecules to isolate and analyze due to large mass, charge, and tendency to aggregate or form macromolecular complexes. This unit describes detailed methods for purification of matrix, cell surface, and cytoskeleton-linked proteoglycans. Methods for analysis of glycoaminoglycan size and type and of core protein species are described.

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.

IGF-IR is highly associated with the behaviour of breast cancer cells. In ERα-positive breast cancer, IGF-IR is present at high levels. In clinical practice, prolonged treatment with anti-estrogen agents results in resistance to the therapy with activation of alternative signaling pathways. Receptor Tyrosine Kinases, and especially IGF-IR, have crucial roles in these processes. Here, we report a nodal role of IGF-IR in the regulation of ERα-positive breast cancer cell aggressiveness and the regulation of expression levels of several extracellular matrix molecules. In particular, activation of IGF-IR, but not EGFR, in MCF-7 breast cancer cells results in the reduction of specific matrix metalloproteinases and their inhibitors. In contrast, IGF-IR inhibition leads to the depletion by endocytosis of syndecan-4. Global important changes in cell adhesion receptors, which include integrins and syndecan-4 triggered by IGF-IR inhibition, regulate adhesion and invasion. Cell function assays that were performed in MCF-7 cells as well as their ERα-suppressed counterparts indicate that ER status is a major determinant of IGF-IR regulatory role on cell adhesion and invasion. The strong inhibitory role of IGF-IR on breast cancer cells aggressiveness for which E2-ERα signaling pathway seems to be essential, highlights IGF-IR as a major molecular target for novel therapeutic strategies.

Syndecans are a small family of four transmembrane proteoglycans in mammals. They have similar structural organization, consisting of an N-terminal ectodomain, single transmembrane domain and C-terminal cytoplasmic domain. Over the years, the association between syndecans and the actin cytoskeleton has been established, which has consequences for the regulation of cell adhesion and migration. Specifically, ecto- and cytoplasmic domains are responsible for the interaction with extracellular matrix molecules and intracellular kinases, respectively. These interactions indicate syndecans as key molecules during cancer initiation and progression. Particularly syndecans interact with other cell surface receptors, such as growth factor receptors and integrins, which lead to activation of downstream signaling pathways, which are critical for the cellular behavior. Moreover, this review describes the key role of syndecans in intracellular calcium regulation and homeostasis. The syndecan-mediated regulation of calcium metabolism is highly correlated with cells’ adhesion phenotype through the actin cytoskeleton and formation of junctions, with implications during differentiation and disease progression.

Cell surface proteoglycans comprise a transmembrane or membrane-associated core protein to which one or more glycosaminoglycan chains are covalently attached.They are ubiquitous receptors on nearly all animal cell surfaces.In mammals, the cell surface proteoglycans include the six glypicans, CD44, NG2 (CSPG4), neuropilin-1 and four syndecans.A single syndecan is present in invertebrates such as nematodes and insects.Uniquely, syndecans are receptors for many classes of proteins that can bind to the heparan sulphate chains present on syndecan core proteins.These range from cytokines, chemokines, growth factors and morphogens to enzymes and extracellular matrix (ECM) glycoproteins and collagens.Extracellular interactions with other receptors, such as some integrins, are mediated by the core protein.This places syndecans at the nexus of many cellular responses to extracellular cues in development, maintenance, repair and disease.The cytoplasmic domains of syndecans, while having no intrinsic kinase activity, can nevertheless signal through binding proteins.All syndecans appear to be connected to the actin cytoskeleton and can therefore contribute to cell adhesion, notably to the ECM and migration.Recent data now suggest that syndecans can regulate stretchactivated ion channels.The structure and function of the syndecans and the ion channels are reviewed here, along with an analysis of ion channel functions in cell-matrix adhesion.This area sheds new light on the syndecans, not least since evidence suggests that this is an evolutionarily conserved relationship that is also potentially important in the progression of some common diseases where syndecans are implicated.

Cell-extracellular matrix (ECM) and cell-cell junctions that employ microfilaments are sites of tension. They are important for tissue repair, morphogenetic movements and can be emblematic of matrix contraction in fibrotic disease and the stroma of solid tumors. One cell surface receptor, syndecan-4, has been shown to regulate focal adhesions, junctions that form at the ends of microfilament bundles in response to matrix components such as fibronectin. Recently it has been shown that signaling emanating from this proteoglycan receptor includes regulation of Rho family GTPases and cytosolic calcium. While it is known that cell-ECM and cell-cell junctions may be linked, possible roles for syndecans in this process are not understood. Here we show that wild type primary fibroblasts and those lacking syndecan-4 utilize different cadherins in their adherens junctions and that tension is a major factor in this differential response. This corresponds to the reduced ability of fibroblasts lacking syndecan-4 to exert tension on the ECM and we now show that this may extend to reduced tension in cell-cell adhesion.

Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions.

Virtually all animal cells express heparan sulfate proteoglycans on the cell surface and in the extracellular matrix. Syndecans are a major group of transmembrane proteoglycans functioning as receptors that mediate signal transmission from the extracellular microenvironment to the cell. Their heparan sulfate chains, due to their vast structural diversity, interact with a wide array of ligands including potent regulators of adhesion, migration, growth and survival. Frequently, ligands interact with cell surface heparan sulfate in conjunction with high affinity receptors. The consequent signaling can therefore be complex, but it is now known that syndecans are capable of independent signaling. This review is divided in two sections, and will first discuss how the assembly of heparan sulfate, the anabolic process, encodes information related to ligand binding and signaling. Second, we discuss how, in partial catabolic processes, new roles for HSPGs emerge that affect cell behavior. Examples from tumor studies are emphasized, since HSPGs may be altered in composition and distribution and may also represent targets for the development of new therapeutics.

The PDZ domain-containing scaffold protein, syntenin-1, binds to the transmembrane proteoglycan, syndecan-4, but the molecular mechanism/function of this interaction are unknown. Crystal structure analysis of syntenin-1/syndecan-4 cytoplasmic domains revealed that syntenin-1 forms a symmetrical pair of dimers anchored by a syndecan-4 dimer. The syndecan-4 cytoplasmic domain is a compact intertwined dimer with a symmetrical clamp shape and two antiparallel strands forming a cavity within the dimeric twist. The PDZ2 domain of syntenin-1 forms a direct antiparallel interaction with the syndecan-4 cytoplasmic domain, inhibiting the functions of syndecan-4 such as focal adhesion formation. Moreover, C-terminal region of syntenin-1 reveals an essential role for enhancing the molecular homodimerization. Mutation of key syntenin-1 residues involved in the syndecan-4 interaction or homodimer formation abolishes the inhibitory function of syntenin-1, as does deletion of the homodimerization-related syntenin-1 C-terminal domain. Syntenin-1, but not dimer-formation-incompetent mutants, rescued the syndecan-4-mediated inhibition of migration and pulmonary metastasis by B16F10 cells. Therefore, we conclude that syntenin-1 negatively regulates syndecan-4 function via oligomerization and/or syndecan-4 interaction, impacting cytoskeletal organization and cell migration.

A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton.

Syndecans are transmembrane heparan sulfate proteoglycans, with roles in development, tumorigenesis and inflammation, and growing evidence for involvement in tissue regeneration. This is a fast developing field with the prospect of utilizing tissue engineering and biomaterials in novel therapies. Syndecan receptors are not only ubiquitous in mammalian tissues, regulating cell adhesion, migration, proliferation, and differentiation through independent signaling but also working alongside other receptors. Their importance is highlighted by an ability to interact with a diverse array of ligands, including extracellular matrix glycoproteins, growth factors, morphogens, and cytokines that are important regulators of regeneration. We also discuss the potential for syndecans to regulate stem cell properties, and suggest that understanding these proteoglycans is relevant to exploiting cell, tissue, and materials technologies.

A small group of cell surface receptors are proteoglycans, possessing a core protein with one or more covalently attached glycosaminoglycan chains. They are virtually ubiquitous and their chains are major sites at which protein ligands of many types interact. These proteoglycans can signal and regulate important cell processes, such as adhesion, migration, proliferation, and differentiation. Since many protein ligands, such as growth factors, morphogens, and cytokines, are also implicated in tumour progression, it is increasingly apparent that cell surface proteoglycans impact tumour cell behaviour. Here, we review some recent advances, emphasising that many tumour-related functions of proteoglycans are revealed only after their modification in processes subsequent to synthesis and export to the cell surface. These include enzymes that modify heparan sulphate structure, recycling of whole or fragmented proteoglycans into exosomes that can be paracrine effectors or biomarkers, and lateral interactions between some proteoglycans and calcium channels that impact the actin cytoskeleton.

Transmembrane heparan sulfate proteoglycans regulate multiple aspects of cell behavior, but the molecular basis of their signaling is unresolved. The major family of transmembrane proteoglycans is the syndecans, present in virtually all nucleated cells, but with mostly unknown functions. Here, we show that syndecans regulate transient receptor potential canonical (TRPCs) channels to control cytosolic calcium equilibria and consequent cell behavior. In fibroblasts, ligand interactions with heparan sulfate of syndecan-4 recruit cytoplasmic protein kinase C to target serine714 of TRPC7 with subsequent control of the cytoskeleton and the myofibroblast phenotype. In epidermal keratinocytes a syndecan-TRPC4 complex controls adhesion, adherens junction composition, and early differentiation in vivo and in vitro. In Caenorhabditis elegans, the TRPC orthologues TRP-1 and -2 genetically complement the loss of syndecan by suppressing neuronal guidance and locomotory defects related to increases in neuronal calcium levels. The widespread and conserved syndecan-TRPC axis therefore fine tunes cytoskeletal organization and cell behavior.

Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer.

Background: Cell surface proteoglycans interact with numerous regulators of cell behavior through their glycosaminoglycan chains. The syndecan family of transmembrane proteoglycans are virtually ubiquitous cell surface receptors that are implicated in the progression of some tumors, including breast carcinoma. This may derive from their regulation of cell adhesion, but roles for specific syndecans are unresolved.Methods: The MDA-MB231 human breast carcinoma cell line was exposed to exogenous glycosaminoglycans and changes in cell behavior monitored by western blotting, immunocytochemistry, invasion and collagen degradation assays. Selected receptors including PAR-1 and syndecans were depleted by siRNA treatments to assess cell morphology and behavior. Immunohistochemistry for syndecan-2 and its interacting partner, caveolin-2 was performed on human breast tumor tissue arrays. Two-tailed paired t-test and one-way ANOVA with Tukey¿s post-hoc test were used in the analysis of data.Results: MDA-MB231 cells were shown to be highly sensitive to exogenous heparan sulfate or heparin, promoting increased spreading, focal adhesion and adherens junction formation with concomitantly reduced invasion and matrix degradation. The molecular basis for this effect was revealed to have two components. First, thrombin inhibition contributed to enhanced cell adhesion and reduced invasion. Second, a specific loss of cell surface syndecan-2 was noted. The ensuing junction formation was dependent on syndecan-4, whose role in promoting actin cytoskeletal organization is known. Syndecan-2 interacts with, and may regulate, caveolin-2. Depletion of either molecule had the same adhesion-promoting influence, along with reduced invasion, confirming a role for this complex in maintaining the invasive phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover

In the 25 years, as the first of the syndecan family was cloned, interest in these transmembrane proteoglycans has steadily increased. While four distinct members are present in mammals, one is present in invertebrates, including C. elegans that is such a powerful genetic model. The syndecans, therefore, have a long evolutionary history, indicative of important roles. However, these roles have been elusive. The knockout in the worm has a developmental neuronal phenotype, while knockouts of the syndecans in the mouse are mild and mostly limited to post-natal rather than developmental effects. Moreover, their association with high-affinity receptors, such as integrins, growth factor receptors, frizzled and slit/robo, have led to the notion that syndecans are coreceptors, with minor roles. Given that their heparan sulphate chains can gather many different protein ligands, this gave credence to views that the importance of syndecans lay with their ability to concentrate ligands and that only the extracellular polysaccharide was of significance. Syndecans are increasingly identified with roles in the pathogenesis of many diseases, including tumour progression, vascular disease, arthritis and inflammation. This has provided impetus to understanding syndecan roles in more detail. It emerges that while the cytoplasmic domains of syndecans are small, they have clear interactive capabilities, most notably with the actin cytoskeleton. Moreover, through the binding and activation of signalling molecules, it is likely that syndecans are important receptors in their own right. Here, an overview of syndecan structure and function is provided, with some prospects for the future.

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

An appropriate balance in glycosylation of proteoglycans is crucial for their ability to regulate animal development. Here, we report that the Caenorhabditis elegans microRNA mir-79, an ortholog of mammalian miR-9, controls sugar-chain homeostasis by targeting two proteins in the proteoglycan biosynthetic pathway: a chondroitin synthase (SQV-5; squashed vulva-5) and a uridine 5'-diphosphate-sugar transporter (SQV-7). Loss of mir-79 causes neurodevelopmental defects through SQV-5 and SQV-7 dysregulation in the epidermis. This results in a partial shutdown of heparan sulfate biosynthesis that impinges on a LON-2/glypican pathway and disrupts neuronal migration. Our results identify a regulatory axis controlled by a conserved microRNA that maintains proteoglycan homeostasis in cells.

An appropriate balance in glycosylation of proteoglycans is crucial for their ability to regulate animal development. Here, we report that the Caenorhabditis elegans microRNA mir-79, an ortholog of mammalian miR-9, controls sugar-chain homeostasis by targeting two proteins in the proteoglycan biosynthetic pathway: a chondroitin synthase (SQV-5; squashed vulva-5) and a uridine 5'-diphosphate-sugar transporter (SQV-7). Loss of mir-79 causes neurodevelopmental defects through SQV-5 and SQV-7 dysregulation in the epidermis. This results in a partial shutdown of heparan sulfate biosynthesis that impinges on a LON-2/glypican pathway and disrupts neuronal migration. Our results identify a regulatory axis controlled by a conserved microRNA that maintains proteoglycan homeostasis in cells.

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine or autocrine effectors, or as competitive inhibitors of the intact proteoglycan. Tumour-associated MMPs are shown here to cleave the ectodomains of human syndecan-1 and syndecan-4. Two membrane proximal regions of both syndecan-1 and syndecan-4 are favoured MMP cleavage sites, six and 15 residues from the transmembrane domain. Other sites are 35-40 residues C-terminal from the heparan sulfate chain substitution sites in both syndecans. The MT1-MMP cleavage sites in syndecan-1 and syndecan-4 were confirmed by site-directed mutagenesis. These findings provide insights into the characteristics of syndecan shedding.

Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors, which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine or autocrine effectors, or as competitive inhibitors of the intact proteoglycan. Tumour-associated MMPs are shown here to cleave the ectodomains of human syndecan-1 and syndecan-4. Two membrane proximal regions of both syndecan-1 and syndecan-4 are favoured MMP cleavage sites, six and 15 residues from the transmembrane domain. Other sites are 35-40 residues C-terminal from the heparan sulfate chain substitution sites in both syndecans. The MT1-MMP cleavage sites in syndecan-1 and syndecan-4 were confirmed by site-directed mutagenesis. These findings provide insights into the characteristics of syndecan shedding.

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes., De två första författarna delar förstaförfattarskapet.De två sista författarna delar sistaförfattarskapet.

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

Proteoglycans comprise a core protein to which one or more glycosaminoglycan chains are covalently attached. Although a small number of proteins have the capacity to be glycanated and become proteoglycans, it is now realized that these macromolecules have a range of functions, dependent on type and in vivo location, and have important roles in invertebrate and vertebrate development, maintenance, and tissue repair. Many biologically potent small proteins can bind glycosaminoglycan chains as a key part of their function in the extracellular matrix, at the cell surface, and also in some intracellular locations. Therefore, the participation of proteoglycans in disease is receiving increased attention. In this short review, proteoglycan structure, function, and localizations are summarized, with reference to accompanying reviews in this issue as well as other recent literature. Included are some remarks on proteoglycan and glycosaminoglycan localization techniques, with reference to the special physicochemical properties of these complex molecules.

Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has not been carried out in a detailed way using high-resolution microscopy. We have begun this process, using well-known markers for the various Golgi compartments, coupled with the use of characterized antibodies and cDNA expression. Laser scanning confocal microscopy coupled with line scanning provides high-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all the enzymes are clustered in a multimolecular complex or are distributed through the various compartments of the Golgi apparatus.

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.

Tumor markers are widely used in pathology not only for diagnostic purposes but also to assess the prognosis and to predict the treatment of the tumor. Because tumor marker levels may change over time, it is important to get a better understanding of the molecular changes during tumor progression. Occurrence of breast and ovarian cancer is high in older women. Common known risk factors of developing these cancers in addition to age are not having children or having children at a later age, the use of hormone replacement therapy, and mutations in certain genes. In addition, women with a history of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression.

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.

Heparan sulfate is perhaps the most complex polysaccharide known from animals. The basic repeating disaccharide is extensively modified by sulfation and uronic acid epimerization. Despite this, the fine structure of heparan sulfate is remarkably consistent with a particular cell type. This suggests that the synthesis of heparan sulfate is tightly controlled. Although genomics has identified the enzymes involved in glycosaminoglycan synthesis in a number of vertebrates and invertebrates, the regulation of the process is not understood. Moreover, the localization of the various enzymes in the Golgi apparatus has not been carried out in a detailed way using high-resolution microscopy. We have begun this process, using well-known markers for the various Golgi compartments, coupled with the use of characterized antibodies and cDNA expression. Laser scanning confocal microscopy coupled with line scanning provides high-quality resolution of the distribution of enzymes. The EXT2 protein, which when combined as heterodimers with EXT1 comprises the major polymerase in heparan sulfate synthesis, has been studied in depth. All the data are consistent with a cis-Golgi distribution and provide a starting point to establish whether all the enzymes are clustered in a multimolecular complex or are distributed through the various compartments of the Golgi apparatus.

Proteoglycans comprise a core protein to which one or more glycosaminoglycan chains are covalently attached. Although a small number of proteins have the capacity to be glycanated and become proteoglycans, it is now realized that these macromolecules have a range of functions, dependent on type and in vivo location, and have important roles in invertebrate and vertebrate development, maintenance, and tissue repair. Many biologically potent small proteins can bind glycosaminoglycan chains as a key part of their function in the extracellular matrix, at the cell surface, and also in some intracellular locations. Therefore, the participation of proteoglycans in disease is receiving increased attention. In this short review, proteoglycan structure, function, and localizations are summarized, with reference to accompanying reviews in this issue as well as other recent literature. Included are some remarks on proteoglycan and glycosaminoglycan localization techniques, with reference to the special physicochemical properties of these complex molecules.

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

Tumor markers are widely used in pathology not only for diagnostic purposes but also to assess the prognosis and to predict the treatment of the tumor. Because tumor marker levels may change over time, it is important to get a better understanding of the molecular changes during tumor progression. Occurrence of breast and ovarian cancer is high in older women. Common known risk factors of developing these cancers in addition to age are not having children or having children at a later age, the use of hormone replacement therapy, and mutations in certain genes. In addition, women with a history of breast cancer may also develop ovarian cancer. Here, the authors review the different tumor markers of breast and ovarian carcinoma and discuss the expression, mutations, and possible roles of cell surface heparan sulfate proteoglycans during tumorigenesis of these carcinomas. The focus is on two groups of proteoglycans, the transmembrane syndecans and the lipid-anchored glypicans. Both families of proteoglycans have been implicated in cellular responses to growth factors and morphogens, including many now associated with tumor progression.