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Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.

Fitzgerald ML, Wang Z, Park PW, Murphy G, Bernfield M - J. Cell Biol. (2000)

Bottom Line: Ledbetter, D.M.These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors.Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

View Article: PubMed Central - PubMed

Affiliation: Division of Newborn Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

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Syndecan ectodomains are shed by proteolytic cleavage at the cell surface. Live P3X63 mouse plasma cells were incubated for 30 min at 4°C with biotinylated mAb 281-2 against the syndecan-1 ectodomain before treatment with or without 0.5 μM PMA for 30 min at 37°C. Cells were fixed and labeled with FITC-avidin for immunofluorescent detection of cell surface syndecan-1 on untreated (A) or PMA-treated (B) cells.
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Figure 4: Syndecan ectodomains are shed by proteolytic cleavage at the cell surface. Live P3X63 mouse plasma cells were incubated for 30 min at 4°C with biotinylated mAb 281-2 against the syndecan-1 ectodomain before treatment with or without 0.5 μM PMA for 30 min at 37°C. Cells were fixed and labeled with FITC-avidin for immunofluorescent detection of cell surface syndecan-1 on untreated (A) or PMA-treated (B) cells.

Mentions: P3X63 cells, a mouse plasma cell line, express abundant cell surface syndecan-1, have barely detectable levels of intracellular syndecan-1, and shed syndecan-1 at a low constitutive rate. Immunoprecipitation and dot blot analysis of P3X63 cell lysates and conditioned media showed that PMA treatment causes cell surface syndecan-1 to be rapidly lost with the concomitant appearance of the shed syndecan-1 ectodomains in the conditioned media (Fitzgerald, M.L., J.-S. Chun, and M. Bernfield, American Society of Cell Biology. 1994. 1813 (Abstr.)). We have now labeled syndecan-1 at the surface of these cells with biotinylated mAb 281-2 at 4°C to avoid constitutive shedding. Bright fluorescence and occasional punctate stain was seen at the cell surface (Fig. 4 A). After PMA treatment of these cells, the syndecan-1 stain was reduced after 5 min and was completely lost after 30 min, except for some residual punctate stains (Fig. 4 B). To confirm that shedding is from the cell surface, we asked whether trypsinization of unlabeled P3X63 cells at 4°C, which removes cell surface syndecan-1 quantitatively (Jalkanen et al. 1987), prevents subsequent PMA-induced shedding. No syndecan-1 ectodomain was detected in the conditioned media after PMA treatment of trypsinized cells (data not shown). Moreover, pretreatment of cells with cytochalasin D (1 μg/ml) or colchicine (50 μM) does not inhibit PMA-accelerated shedding, and antibody-induced cross-linking of cell surface syndecan-1 actually prevents accelerated shedding (data not shown). Thus, accelerated syndecan-1 ectodomain shedding is a cell surface event.


Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase.

Fitzgerald ML, Wang Z, Park PW, Murphy G, Bernfield M - J. Cell Biol. (2000)

Syndecan ectodomains are shed by proteolytic cleavage at the cell surface. Live P3X63 mouse plasma cells were incubated for 30 min at 4°C with biotinylated mAb 281-2 against the syndecan-1 ectodomain before treatment with or without 0.5 μM PMA for 30 min at 37°C. Cells were fixed and labeled with FITC-avidin for immunofluorescent detection of cell surface syndecan-1 on untreated (A) or PMA-treated (B) cells.
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Related In: Results  -  Collection

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Figure 4: Syndecan ectodomains are shed by proteolytic cleavage at the cell surface. Live P3X63 mouse plasma cells were incubated for 30 min at 4°C with biotinylated mAb 281-2 against the syndecan-1 ectodomain before treatment with or without 0.5 μM PMA for 30 min at 37°C. Cells were fixed and labeled with FITC-avidin for immunofluorescent detection of cell surface syndecan-1 on untreated (A) or PMA-treated (B) cells.
Mentions: P3X63 cells, a mouse plasma cell line, express abundant cell surface syndecan-1, have barely detectable levels of intracellular syndecan-1, and shed syndecan-1 at a low constitutive rate. Immunoprecipitation and dot blot analysis of P3X63 cell lysates and conditioned media showed that PMA treatment causes cell surface syndecan-1 to be rapidly lost with the concomitant appearance of the shed syndecan-1 ectodomains in the conditioned media (Fitzgerald, M.L., J.-S. Chun, and M. Bernfield, American Society of Cell Biology. 1994. 1813 (Abstr.)). We have now labeled syndecan-1 at the surface of these cells with biotinylated mAb 281-2 at 4°C to avoid constitutive shedding. Bright fluorescence and occasional punctate stain was seen at the cell surface (Fig. 4 A). After PMA treatment of these cells, the syndecan-1 stain was reduced after 5 min and was completely lost after 30 min, except for some residual punctate stains (Fig. 4 B). To confirm that shedding is from the cell surface, we asked whether trypsinization of unlabeled P3X63 cells at 4°C, which removes cell surface syndecan-1 quantitatively (Jalkanen et al. 1987), prevents subsequent PMA-induced shedding. No syndecan-1 ectodomain was detected in the conditioned media after PMA treatment of trypsinized cells (data not shown). Moreover, pretreatment of cells with cytochalasin D (1 μg/ml) or colchicine (50 μM) does not inhibit PMA-accelerated shedding, and antibody-induced cross-linking of cell surface syndecan-1 actually prevents accelerated shedding (data not shown). Thus, accelerated syndecan-1 ectodomain shedding is a cell surface event.

Bottom Line: Ledbetter, D.M.These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors.Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

View Article: PubMed Central - PubMed

Affiliation: Division of Newborn Medicine, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated. To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.

Show MeSH
Related in: MedlinePlus