Richard C. Stahl

Syndecan-1 Expression Is Down-regulated during Myoblast Terminal Differentiation MODULATION BY GROWTH FACTORS AND RETINOIC ACID

Syndecan-1 is an integral membrane proteoglycan involved in the interaction of cells with extracellular matrix proteins and growth factors. It is transiently expressed in several condensing mesenchymal tissues after epithelial induction. In this study we evaluated the expression of syndecan-1 during skeletal muscle differentiation. The expression of syndecan-1 as determined by Northern blot analyses and immunofluorescence microscopy is down-regulated during differentiation. The transcriptional activity of a syndecan-1 promoter construct is also down-regulated in differentiating muscle cells. The decrease in syndecan-1 gene expression is not dependent on the presence of E-boxes, binding sites for the MyoD family of transcription factors in the promoter region, or myogenin expression. Deletion of the region containing the E-boxes or treatment of differentiating cells with sodium butyrate, an inhibitor of myogenin expression, had no effect on syndecan-1 expression. Basic fibroblast growth factor and transforming growth factor type β, which are inhibitors of myogenesis, had little effect on syndecan-1 expression. When added together, however, they induced syndecan-1 expression. Retinoic acid, an inducer of myogenesis, inhibited syndecan-1 expression and abolished the effect of the growth factors. These results indicate that syndecan-1 expression is down-regulated during myogenesis and that growth factors and retinoic acid modulate syndecan-1 expression by a mechanism that is independent of myogenin.

cDNA Cloning, Genomic Organization, and in Vivo Expression of Rat N-syndecan

The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120,000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3′-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.

Glypican-1 and α4(V) Collagen Are Required for Schwann Cell Myelination

Schwann cell myelination requires interactions with the extracellular matrix (ECM) mediated by cell surface receptors. Previously, we identified a type V collagen family member, α4(V) collagen, which is expressed by Schwann cells during peripheral nerve differentiation. This collagen binds with high affinity to heparan sulfate through a unique binding motif in the noncollagenous N-terminal domain (NTD). The principal α4(V) collagen-binding protein on the Schwann cell surface is the heparan sulfate proteoglycan glypican-1. We investigated the role of α4(V) collagen and glypican-1 in Schwann cell terminal differentiation in cultures of Schwann cells and dorsal root ganglion neurons. Small interfering RNA-mediated suppression of glypican-1 expression decreased binding of α4(V)-NTD to Schwann cells, adhesion and spreading of Schwann cells on α4(V)-NTD, and incorporation of α4(V) collagen into Schwann cell ECM. In cocultures, α4(V) collagen coassembles with laminin on the surface of polarized Schwann cells to form tube-like ECM structures that are sites of myelination. Suppression of glypican-1 or α4(V) collagen expression significantly inhibited myelination. These results demonstrate an important role for these proteins in peripheral nerve terminal differentiation.

Matrix metalloproteinase-dependent shedding of syndecan-3, a transmembrane heparan sulfate proteoglycan, in Schwann cells

Schwann cells transiently express the transmembrane heparan sulfate proteoglycan syndecan‐3 during the late embryonic and early postnatal periods of peripheral nerve development. Neonatal rat Schwann cells released soluble syndecan‐3 into the culture medium by a process that was blocked by inhibition of endogenous matrix metalloproteinase activity. When Schwann cells were plated on a substratum that binds syndecan‐3, the released proteoglycan bound to the substratum adjacent to the cell border. Membrane‐anchored syndecan‐3 was concentrated in actin‐containing filopodia that projected from the lateral edges of the Schwann cell membrane. Membrane shedding was specific for syndecan‐3 and was not observed for the related proteoglycan syndecan‐1. Analysis of Schwann cells transfected with wild‐type and chimeric syndecan‐1 and syndecan‐3 cDNAs revealed that membrane shedding was a property of the syndecan‐3 ectodomain. Inhibition of syndecan‐3 release significantly enhanced Schwann cell adhesion and process extension on dishes coated with the non‐collagenous N‐terminal domain of α4(V) collagen, which binds syndecan‐3 and mediates heparan sulfate‐dependent Schwann cell adhesion. Matrix metalloproteinase‐dependent syndecan‐3 shedding was also observed in newborn rat peripheral nerve tissue. Syndecan‐3 shedding in peripheral nerve tissue was age specific, and was not observed during later stages of postnatal nerve development. These results demonstrate that Schwann cell syndecan‐3 is subject to matrix metalloproteinase‐dependent membrane processing, which modulates the biological function of this proteoglycan.

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger.

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

Schwann Cells Secrete a Novel Collagen-like Adhesive Protein That Binds N-Syndecan

A heparin-binding glycoprotein was purified from conditioned medium of cultured rat Schwann cells. The protein, p200, which has an apparent molecular mass of approximately 200 kDa, was identified by its ability to bind the cell surface heparan sulfate proteoglycan N-syndecan (syndecan-3) in a membrane overlay assay. Soluble heparin but not chondroitin sulfate inhibited the binding, suggesting the involvement of heparan sulfate chains of proteoglycan in the interaction. Purified p200 promoted the attachment and spreading of Schwann cells. Adhesion to p200 was blocked by heparin, suggesting that heparan sulfate proteoglycans are cell surface receptors for p200. The tissue distribution of p200 was determined by immunoblot analysis with anti-p200 antibodies. Among neonatal rat tissues examined p200 was detected only in sciatic nerve and, at lower levels, in skeletal muscle. p200 expression in sciatic nerve was detectable only during the first 2-3 weeks of postnatal development and was not detected in adult rats. Immunofluorescent staining of rat sciatic nerve showed that p200 was localized in the extracellular matrix surrounding individual Schwann cells-axon units. Two tryptic peptides from p200 were purified and sequenced. These contained multiple GXX collagen-like repeats. Bacterial collagenase digestion of p200 produced a product with an apparent molecular mass of approximately 90 kDa. These data suggest that Schwann cells secrete an apparently novel collagen-like adhesive protein that interacts with cells through cell surface heparan sulfate proteoglycans.

p200, a Collagen Secreted by Schwann Cells, Is Expressed in Developing Nerves and in Adult Nerves Following Axotomy

Previously we reported that cultured rat Schwann cells secrete p200, a collagen‐like heparin‐binding adhesive glycoprotein with a restricted pattern of expression. Here we report that p200 is secreted as a stable trimer, but only after treatment of Schwann cells with ascorbic acid, and was deposited in the fibrillar extracellular matrix. Heparin and heparitinase treatment inhibited incorporation of p200 into extracellular matrix, suggesting the involvement of Schwann cell heparan sulfate proteoglycans in this process. Pepsin digestion revealed that p200 secreted by ascorbate‐treated cells contains a collagenous domain of approximately 140 kDa. Immunofluorescent staining of rat embryos at different ages showed that p200 first appeared between embryonic days 15 and 18, and was confined to peripheral nerves. Staining of adult peripheral nerve was negative, but p200 expression was induced in adult sciatic nerve following nerve transection. These data suggest that p200 carries out unique functions during peripheral nerve development and regeneration and that its expression by Schwann cells is regulated by axon‐Schwann cell interaction. J. Neurosci. Res. 56:284–294, 1999.

Molecular cloning and characterization of an isoprenylated 67 kDa protein

The cDNA coding for a 67 kDa protein (p67) was isolated from a rat Schwann cell library. A recombinant form of p67 expressed in bacteria was used to produce polyclonal anti-p67 antibodies. By immunoblot analysis p67 was found to be expressed in most tissues and cell lines examined. Inspection of the deduced amino acid sequence revealed a COOH-terminal consensus sequence for isoprenylation. Consistent with this finding, p67 was a substrate for isoprenylation in vitro by geranylgeranylpyrophosphate. p67 was associated predominantly with the particulate fraction of rat smooth muscle cells. The rat p67 sequence was highly homologous to a family of recently described human and mouse gamma-interferon inducible, guanine nucleotide binding proteins.

Constitutive Release of α4 Type V Collagen N-terminal Domain by Schwann Cells and Binding to Cell Surface and Extracellular Matrix Heparan Sulfate Proteoglycans

During peripheral nerve development, Schwann cells synthesize collagen type V molecules that contain α4(V) chains. This collagen subunit possesses an N-terminal domain (NTD) that contains a unique high affinity heparin binding site. The α4(V)-NTD is adhesive for Schwann cells and sensory neurons and is an excellent substrate for Schwann cell and axonal migration. Here we show that the α4(V)-NTD is released constitutively by Schwann cells both in culture and in vivo. In cultures of neonatal rat Schwann cells, α4(V)-NTD release is increased significantly by ascorbate treatment, which facilitates collagen post-translational modification and collagen trimer assembly. In peripheral nerve tissue, the α4(V)-NTD is localized to the region of the outer Schwann cell membrane and associated extracellular matrix. The released α4(V)-NTD binds to the cell surface and extracellular matrix heparan sulfate proteoglycans of Schwann cells. Pull-down assays and immunofluorescent staining showed that the major α4(V)-NTD-binding proteins are glypican-1 and perlecan. α4(V)-NTD binding occurs via a mechanism that requires the high affinity heparin binding site and that is blocked by soluble heparin, demonstrating that binding to proteoglycans is mediated by their heparan sulfate chains.

α7β1 integrin is a receptor for laminin-2 on Schwann cells

The Schwann cell basal lamina acts as an organizer of peripheral nerve tissue and influences many aspects of cell behavior during development and regeneration. A principal component of the Schwann cell basal lamina is laminin‐2. This study was undertaken to identify Schwann cell receptors for laminin‐2. We found that among several Schwann cell integrins that can potentially interact with laminin‐2, only α7β1 bound to laminin‐2‐Sepharose. Dystroglycan, a non‐integrin Schwann cell receptor for laminin‐2 identified previously, was also found to bind to laminin‐2‐Sepharose. Antibody to the α7 integrin subunit partially inhibited Schwann cell adhesion to laminin‐2. Small interfering RNA‐mediated suppression of either α7 integrin or dystroglycan expression decreased adhesion and spreading of Schwann cells on laminin‐2, whereas knocking down both proteins together inhibited adhesion and spreading on laminin‐2 almost completely. α7 integrin and dystroglycan both colocalized with laminin‐2 containing basal lamina tubes in differentiating neuron–Schwann cell cocultures. The α7β1 integrin also coprecipitates with focal adhesion kinase in differentiating cocultures. These findings strongly suggest that α7β1 integrin is a Schwann cell receptor for laminin‐2 that provides transmembrane linkage between the Schwann cell basal lamina and cytoskeleton.