Two Transient Receptor Potential Channels at Focal Adhesions.

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).

Calcium in Cell-Extracellular Matrix 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.

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.

Proteoglycans, ion channels and cell-matrix adhesion.

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 stretch-activated 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 and cell-cell adhesion are linked by syndecan-4.

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.

Syndecans - key regulators of cell signaling and biological functions.

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.

Minireview: Syndecans and their crucial roles during tissue regeneration.

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.

Extracellular matrix component signaling in cancer.

Cell responses to the extracellular matrix depend on specific signaling events. These are important from early development, through differentiation and tissue homeostasis, immune surveillance, and disease pathogenesis. Signaling not only regulates cell adhesion cytoskeletal organization and motility but also provides survival and proliferation cues. The major classes of cell surface receptors for matrix macromolecules are the integrins, discoidin domain receptors, and transmembrane proteoglycans such as syndecans and CD44. Cells respond not only to specific ligands, such as collagen, fibronectin, or basement membrane glycoproteins, but also in terms of matrix rigidity. This can regulate the release and subsequent biological activity of matrix-bound growth factors, for example, transforming growth factor-ß. In the environment of tumors, there may be changes in cell populations and their receptor profiles as well as matrix constitution and protein cross-linking. Here we summarize roles of the three major matrix receptor types, with emphasis on how they function in tumor progression.

Transmembrane proteoglycans control stretch-activated channels to set cytosolic calcium levels.

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.

Insights into the key roles of proteoglycans in breast cancer biology and translational medicine.

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.

Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells.

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 their expression levels were correlated in triple negative breast cancers. CONCLUSION: Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.

Fell-Muir Lecture: Syndecans: from peripheral coreceptors to mainstream regulators of cell behaviour.

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.

Protein kinase C, focal adhesions and the regulation of cell migration.

Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles and potential substrates.

An epidermal microRNA regulates neuronal migration through control of the cellular glycosylation state.

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.

Mapping of matrix metalloproteinase cleavage sites on syndecan-1 and syndecan-4 ectodomains.

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.

Heparan sulfate biosynthesis: methods for investigation of the heparanosome.

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.

Breast and ovarian cancers: a survey and possible roles for the cell surface heparan sulfate proteoglycans.

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.

Syndecan-1 and syndecan-4 are independent indicators in breast carcinoma.

Syndecan proteoglycans may be key regulators of tumor invasion and metastasis because this four-member family of transmembrane receptors regulates cell adhesion, proliferation, and differentiation. Their expression can also serve as prognostic markers. In breast carcinomas, syndecan-1 overexpression correlates with poor prognosis and aggressive phenotype. Syndecan-4 is expressed in most breast carcinoma cell lines, but its role in malignancy is unclear. A possible relationship between syndecan-1 and syndecan-4 expression and established prognostic factors in breast carcinomas was examined. Duplicate samples of 114 benign and malignant breast disease cases were stained for the two syndecans. Clinicopathological information was available for all cases. Syndecan-1 was detected in 72.8% of cases, with significant association between its expression and histological tumor type (p<0.05) and high grade tumors (p<0.05). Syndecan-4 was expressed in 66.7% of cases; expression correlated significantly with positive estrogen (p<0.01) and progesterone (p<0.01) receptor status. Independent expression of the two syndecans was noted from an analysis of single and double positive cases. There was a statistical relationship between syndecan-1 presence in high-grade tumors and absence of syndecan-4, whereas syndecan-4 presence in cases positive for estrogen and progesterone receptor associated with syndecan-1 absence. These syndecans may, therefore, have distinct roles in regulating breast carcinoma cell behavior.

Serine 34 phosphorylation of rho guanine dissociation inhibitor (RhoGDIalpha) links signaling from conventional protein kinase C to RhoGTPase in cell adhesion.

Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKCalpha can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDIalpha that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDIalpha on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDIalpha phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKCalpha in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA.

Heparan sulfate chain valency controls syndecan-4 function in cell adhesion.

Fibroblasts null for the transmembrane proteoglycan, syndecan-4, have an altered actin cytoskeleton, compared with matching wild-type cells. They do not organize alpha-smooth muscle actin into bundles, but will do so when full-length syndecan-4 is re-expressed. This requires the central V region of the core protein cytoplasmic domain, though not interactions with PDZ proteins. A second key requirement is multiple heparan sulfate chains. Mutant syndecan-4 with no chains, or only one chain, failed to restore the wild-type phenotype, whereas those expressing two or three were competent. However, clustering of one-chain syndecan-4 forms with antibodies overcame the block, indicating that valency of interactions with ligands is a key component of syndecan-4 function. Measurements of focal contact/adhesion size and focal adhesion kinase phosphorylation correlated with syndecan-4 status and alpha-smooth muscle actin organization, being reduced where syndecan-4 function was compromised by a lack of multiple heparan sulfate chains.