Markku Salmivirta

Neurite growth-promoting protein (amphoterin, p30) binds syndecan

A new ligand for syndecan (a cell surface heparan sulfate-rich proteoglycan) has been discovered. In the solid-phase binding assay utilizing small nitrocellulose discs to immobilize matrix molecules, binding of syndecan to neurite growth-promoting protein, p30/amphoterin, was observed. This binding was strongly dependent on the concentration of amphoterin used to coat the discs, but was saturable with an excess amount of syndecan. The interaction was inhibitable with heparan sulfate and heparin but less effectively with chondroitin sulfate, indicating that heparan sulfate chains of syndecan were involved in the binding. Anti-amphoterin antibodies inhibited the binding partially. Mouse mammary epithelial cells were shown to bind amphoterin directly but not after trypsin treatment or in the presence of heparin and to produce amphoterin in the extracellular space. Both syndecan and amphoterin were found to localize on lateral surfaces of newly adhered mammary epithelial cells. Toward confluency amphoterin amounts decreased. Because amphoterin can be localized to the same sites with syndecan and because of their interaction, amphoterin is a new putative pericellular ligand for syndecan. These interactions may be involved in the regulation of cell behavior.

Syndecan-1 supports integrin α2β1-mediated adhesion to collagen

Several different receptor molecules act in concert to regulate cell adhesion. Among these are cell-surface proteoglycans and integrins, which collaborate extensively in mediating binding of cells to extracellular matrix molecules fibronectin and vitronectin. However, very little is known about possible functional synergism between proteoglycans and integrins during adhesion of cells to collagen, although collagen is the most abundant protein in the human body. Here we show that cell-surface heparan sulphate proteoglycans (HSPGs) support integrin alpha2beta1-mediated adhesion to collagen. Cells made devoid of HSPGs either by genetic means or by enzymatic digestions were unable to adhere to collagen via alpha2beta1 integrin. HSPG-deficient cells also displayed impaired spreading and actin organization on collagen. Among different HSPG molecules syndecan-1 was found to play an important role in supporting alpha2beta1 integrin-mediated adhesion. Using overexpression and knock-down experiments we demonstrated that syndecan-1, but not syndecan-2 or -4, enhanced binding of alpha2beta1 to collagen. Moreover, syndecan-1 co-localized with alpha2beta1 integrin and contributed to proper organization of cortical actin. Finally, crosstalk between syndecan-1 and alpha2beta1 integrin was found to enhance the transcription of matrix metalloproteinase-1 in response to collagen binding. Our findings thus suggest that a previously unknown link between integrin alpha2beta1 and syndecan-1 is important in regulating cell adhesion to collagen and in triggering integrin downstream signalling.

Protein kinase A balances the growth factor-induced Ras/ERK signaling

Protein kinase A (PKA) has been proposed to regulate the signal transduction through the Ras/extracellular‐regulated kinase (ERK) pathway. Here we demonstrate that when the PKA activity was inhibited prior to growth factor stimulus the signal flow through the Ras/ERK pathway was significantly increased. Furthermore, the data indicated that this PKA‐mediated regulation was simultaneously targeted to the upstream kinase Raf‐1 and to the ERK‐specific phosphatase mitogen‐activated protein kinase phosphatase‐1 (MKP‐1). Moreover, our data suggested that the level of PKA activity determined the transcription rate of mkp‐1 gene, whereas the Ras/ERK signal was required to protect the MKP‐1 protein against degradation. These results point to a tight regulatory relationship between PKA and the growth factor signaling, and further suggest an important role for basal PKA activity in such regulation. We propose that PKA adjusts the activity of the Ras/ERK pathway and maintains it within a physiologically appropriate level.

Altered expression of syndecan-1 in prostate cancer.

Syndecan‐1 is a cell surface heparan sulfate proteoglycan expressed by epithelial cells. It interacts with growth factors, matrix components, and other extracellular proteins, and is thought to be involved in processes such as cell growth, differentiation and adhesion. The expression of syndecan‐1 appears generally downregulated in human carcinomas and in experimental cancer models, whereas transfectional expression of syndecan‐1 in cultured cancer cells has been shown to inhibit their growth and other aspects of malignant behavior. These findings suggest that analysis of syndecan‐1 expression might be of prognostic value in cancer diagnosis, and studies on some carcinomas indeed point to an inverse correlation between syndecan‐1 expression and cancer prognosis. So far, little information has been available on the expression of syndecan‐1 in human prostate and prostate disease. We have generated and characterized novel antibodies against syndecan‐1 and applied them to immunohistochemical staining of specimens representing normal prostate as well as benign and malignant (n=23) prostate disease. The results indicate that syndecan‐1 expression is altered but not uniformly absent in prostate cancer, which is in contrast to the expression of high‐molecular‐weight cytokeratins. The data initially suggest an inverse correlation between syndecan‐1 expression and Gleason grade of the tumor, and warrant a larger study to assess the potential prognostic value of analysing syndecan‐1 expression in prostate carcinoma.

Syndecan and tenascin: Induction during early tooth morphogenesis and possible interactions

Cell-matrix interactions in morphogenesis The important role of the extraceUular matrix (ECM) in regulation of organ morphogenesis has been recognized for several decades (Grobstein, 1967; Bernfield et al., 1972; Lash and Vasan, 1978; Hay, 1981; Toole, 1981). When the main categories of matrix constituents, the collagens, other glycoproteins and proteoglycans with their different glycosaminoglycan side chains had been identified, their roles were studied in the development of different embryonic organs. Experiments where their accumulation was perturbed indicated developmental significance for molecules from all different categories, and metabolic labelling studies suggested that they were developmentally regulated (KaUman and Grobstein, 1966; Bernfield et al., 1972; Hetem et al., 1985). Subsequently, the localization of individual molecules by specific antibodies and immunohistology indicated that the distribution patterns of different matrix molecules correlated with morphogenetic changes, suggesting again developmental roles for the ECM

Syndecan — A Cell Surface Proteoglycan that Selectively Binds Extracellular Effector Molecules

Syndecan is a cell surface proteoglycan (Saunders et al., 1989), which was originally isolated from mouse mammary epithelial (NMuMG) cells as a hybrid proteoglycan containing both heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) chains (Rapraeger et al., 1985). The ratio and total amount of GAG bound to syndecan is known to vary in different tissues (Sanderson & Bernfield, 1988; Salmivirta et al., 1991) and cells (Rapraeger 1989). Syndecan binds several extracellular matrix molecules such as fibrillar collagens of interstitial matrix (Koda et al., 1985), fibronectin (Saunders & Bernfield, 1988) and thrombospondin (Sun et al., 1989) but shows no binding to some other heparin binding matrix molecules, like vitronectin and laminin (Koda et al., 1985; Elenius et al., 1990). Syndecan also binds growth factors, like bFGF (Kiefer et al., 1990), and could therefore play a major role in the regulation of cell adhesion, growth and differentiation (for a review see Jalkanen et al., 1991).

Removal of cell surface heparan sulfate increases TACE activity and cleavage of ErbB4 receptor

BackgroundNuclear localization of proteolytically formed intracellular fragment of ErbB4 receptor tyrosine kinase has been shown to promote cell survival, and nuclear localization of ErbB4 receptor has been described in human breast cancer. Tumor necrosis factor alpha converting enzyme (TACE) initiates the proteolytic cascade leading to ErbB4 intracellular domain formation. Interactions between matrix metalloproteases and heparan sulfate have been described, but the effect of cell surface heparan sulfate on TACE activity has not been previously described.ResultsAs indicated by immunodetection of increased ErbB4 intracellular domain formation and direct enzyme activity analysis, TACE activity was substantially amplified by enzymatic removal of cell surface heparan sulfate but not chondroitin sulfate.ConclusionIn this communication, we suggest a novel role for cell surface heparan sulfate. Removal of cell surface heparan sulfate led to increased formation of ErbB4 intracellular domain. As ErbB4 intracellular domain has previously been shown to promote cell survival this finding may indicate a novel mechanism how HS degradation active in tumor tissue may favor cell survival.

Syndecan from embryonic tooth mesenchyme binds tenascin

Syndecan is a cell surface heparan sulfate-rich proteoglycan found on various epithelial cells but also in some embryonic mesenchymal tissues. We have immunoisolated syndecan from embryonic tooth mesenchyme that appeared as a 250-300-kDa molecule (Kav = 0.3 in Sepharose 4B), containing only heparan sulfate side chains (Mr = 35,000). Northern analysis of whole tooth germs and tooth mesenchymes also revealed high expression of syndecan mRNAs (2.6 and 3.4 kilobases). In the binding assay utilizing nitrocellulose as a solid phase to immobilize matrix molecules, syndecan immunoisolated from tooth mesenchyme revealed binding to tenascin, and this interaction was shown to be mediated via heparan sulfate side chains. In contrast, syndecan from mouse mammary epithelial cells showed only weak interaction with tenascin. We propose that syndecan and tenascin may represent interactions of a cell surface receptor and a matrix ligand involved in mesenchymal cell condensation and differentiation during early organogenesis.