The method of affinity coelectrophoresis was used to study the binding of nine representative glycosaminoglycan (GAG)-binding proteins, all thought to play roles in nervous system development, to GAGs and proteoglycans isolated from developing rat brain. Binding to heparin and non-neural heparan and chondroitin sulfates was also measured. All nine proteins-laminin-1, fibronectin, thrombospondin-1, NCAM, L1, protease nexin-1, urokinase plasminogen activator, thrombin, and fibroblast growth factor-2-bound brain heparan sulfate less strongly than heparin, but the degree of difference in affinity varied considerably. Protease nexin-1 bound brain heparan sulfate only 1.8-fold less tightly than heparin (Kdvalues of 35 vs. 20 nM, respectively), whereas NCAM and L1 bound heparin well (Kd approximately 140 nM) but failed to bind detectably to brain heparan sulfate (Kd>3 microM). Four proteins bound brain chondroitin sulfate, with affinities equal to or a few fold stronger than the same proteins displayed toward cartilage chondroitin sulfate. Overall, the highest affinities were observed with intact heparan sulfate proteoglycans: laminin-1's affinities for the proteoglycans cerebroglycan (glypican-2), glypican-1 and syndecan-3 were 300- to 1800-fold stronger than its affinity for brain heparan sulfate. In contrast, the affinities of fibroblast growth factor-2 for cerebroglycan and for brain heparan sulfate were similar. Interestingly, partial proteolysis of cerebroglycan resulted in a >400-fold loss of laminin affinity. These data support the views that (1) GAG-binding proteins can be differentially sensitive to variations in GAG structure, and (2) core proteins can have dramatic, ligand-specific influences on protein-proteoglycan interactions.

Cerebroglycan is a glycosylphosphatidylinositol-linked integral membrane heparan sulfate proteoglycan found exclusively in the developing nervous system. In the rodent, cerebroglycan mRNA first appears in regions containing newly generated neurons and typically disappears 1 to several days later (Stipp et al., 1994, J. Cell Biol. 124:149-160). To gain insight into the roles that cerebroglycan plays in the developing nervous system, monospecific antibodies were prepared and used to localize cerebroglycan protein. In the rat, cerebroglycan was prominantly expressed on axon tracts throughout the developing brain and spinal cord, where it was found at times when axons are actively growing, but generally not after axons have reached their targets. Cerebroglycan was also found on neuronal growth cones both in vivo and in vitro. Interestingly, cerebroglycan immunoreactivity was rarely seen in or around neuronal cell bodies. Indeed, by examining the hippocampus at a late stage in development-when most neurons no longer express cerebroglycan but newly generated granule neurons do-evidence was obtained that cerebroglycan is strongly polarized to the axonal, and excluded from the somatodendritic, compartment of neurons. The timing and pattern of cerebroglycan expression are consistent with a role for this cell-surface heparan sulfate proteoglycan in regulating the growth or guidance of axons.

The glypicans are a family of glycosylphosphatidylinositol (GPI)-anchored proteoglycans that, by virtue of their cell-surface localization and possession of heparan sulfate chains, may regulate the responses of cells to numerous heparin-binding growth factors, cell adhesion molecules, and extracellular matrix components. Mutations in one glypican cause a syndrome of human birth defects, suggesting important roles for these proteoglycans in development. Glypican-1, the first-discovered member of this family, was originally found in cultured fibroblasts, and later shown to be a major proteoglycan of the mature and developing brain. Here we examine the pattern of glypican-1 mRNA and protein expression more widely in the developing rodent, concentrating on late embryonic and early postnatal stages. High levels of glypican-1 expression were found throughout the brain and skeletal system. In the brain, glypican-1 mRNA was widely, and sometimes only transiently, expressed by zones of neurons and neuroepithelia. Glypican-1 protein localized strongly to axons and, in the adult, to synaptic terminal fields as well. In the developing skeletal system, glypican-1 was found in the periosteum and bony trabeculae in a pattern consistent with expression by osteoblasts, as well as in the bone marrow. Glypican-1 was also observed in skeletal and smooth muscle, epidermis, and in the developing tubules and glomeruli of the kidney. Little or no expression was observed in the developing heart, lung, liver, dermis, or vascular endothelium at the stages examined. The tissue-, cell type-, and in some cases stage-specific expression of glypican-1 revealed in this study are likely to provide insight into the functions of this proteoglycan in development.

During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.

During metastasis, cancer cells are exposed to potentially destructive hemodynamic forces including fluid shear stress (FSS) while en route to distant sites. However, prior work indicates that cancer cells are more resistant to brief pulses of high-level FSS in vitro relative to non-transformed epithelial cells. Herein, we identify a mechano-adaptive mechanism of FSS resistance in cancer cells. Our findings demonstrate that cancer cells activate RhoA in response to FSS, which protects them from FSS-induced plasma membrane damage. We show that cancer cells freshly isolated from mouse and human tumors are resistant to FSS, that formin and myosin II activity protects circulating tumor cells (CTCs) from destruction, and that short-term inhibition of myosin II delays metastasis in mouse models. Collectively, our data indicate that viable CTCs actively resist destruction by hemodynamic forces and are likely to be more mechanically robust than is commonly thought.