Binding, internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts

Abstract

Binding, internalization, and degradation of 125I-labeled, antiproliferative, or nonantiproliferative heparan sulfate by human embryonic lung fibroblasts was investigated. Both L-iduronate-rich, antiproliferative heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan sulfate types were bound with approximately the same affinity to one high-affinity site (Kd approximately 10(-8) M) and to one low-affinity site (Kd approximately 10(-6) M), respectively. Results of Hill-plot analysis suggested that the two sites are independent. Competition experiments with unlabeled glycosaminoglycans indicated that the binding sites had a selective specificity for sulfated, L-iduronate-rich heparan sulfate. Dermatan sulfate, which is also antiproliferative, was weakly bound to the cells. The antiproliferative effects of heparan and dermatan sulfate appeared to be additive. Hence, the two glycosaminoglycans probably exert their effect through different mechanisms. At concentrations above 5 micrograms/ml (approximately 10(-7) M), heparan sulfate was taken up by human embryonic lung fibroblasts, suggesting that the low-affinity site represents an endocytosis receptor. The antiproliferative effect of L-iduronate-rich heparan sulfate species was also exerted at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran sulfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural analysis of the inactive and active heparan sulphate preparations indicated that although sulphated L-iduronate appears essential for antiproliferative activity, it is not absolutely required for binding to the cells. Degradation of internalized heparan sulfate was analyzed by polyacrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiproliferative one was only marginally affected.

Quantitative proteomic analysis of fibroblast nuclear proteins after stimulation with mitogen activated protein kinase inhibiting heparan sulfate

“…HS6 binds to one high affinity site and to one low affinity site on the cell surface of fibroblasts [14]. As shown previously DexS, is without significant effect on cell proliferation [13] and MAP kinase activation (Fig.…”

“…HS2, CS nor DexS, which do not inhibit cell proliferation or antiproliferative DS had any significant effect on ERK activation, whereas antiproliferative HS (HS6) and heparin had strong anti-ERK effects. Arroyo-Yanguas et al [14] showed previously that antiproliferative DS do not bind very well to the cell surface, whereas HS does, suggesting that the inhibitory effects of MAP kinase are mediated through interactions on the cell surface. Both HS6 and heparin have high contents of both sulfation and IdoA, whereas HS2, with lower amounts of sulfation and IdoA was unable to affect MAP kinase activity.…”

“…Final preparation step was separation on a Sepharose 6 column under dissociative conditions to remove cytoactive components bound to the HS. The procedure for the determination of total hexosamine, uronic acid and sulfate content has earlier been described [13,14,[26][27][28]. The DS was obtained and characterized by methods described previously.…”

“…HS poor in sulfate and IdoA have no effect on either the level of PDGF receptors or on proliferation indicating that the structural specificity is of importance for the above described effects [12,13]. To further explain the mechanism behind these results, a number of binding and endocytosis studies have been performed [14][15][16]. ArroyoYanguas et al found that HS with antiproliferative capacity bind to the cell surface with one low affinity site and a high affinity site.…”

Antiproliferative heparan sulfate inhibiting hyaluronan and transforming growth factor-β expression in human lung fibroblast cells

“…The final preparation step was separation on a Sepharose 6 column under dissociative conditions to remove cytoactive components bound to the HS. The procedure for the determination of total hexosamine, IdoUA, and sulfate content has earlier been described (18)(19)(20). Chemical data for the HS2 and HS6 used in the study are summarized in Table 1.…”