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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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Accelerated shedding is inhibited by peptide hydroxamates. (A and B) NMuMG cells were incubated with or without PMA (0.5 μM for 30 min) in the presence or absence of peptide hydroxamate BB-2116 or BB-1101 at the indicated concentration. (C and D) SVEC4-10 cells were incubated for 2 h with or without 10 ng/ml EGF or 100 μM TRAP in the presence or absence of 1 μM BB-1101. (E and F) NMuMG cells were incubated for 2 h with or without 100 μM ceramide in the presence or absence of 1 μM BB-1101. Syndecan-1 (A, C, and E) and syndecan-4 (B, D, and F) ectodomains in the conditioned media were assayed by dot blot analysis as in Fig. 1. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3). For each shedding agonist assayed, incubation with inhibitor alone (1 μM) reduced the level of shedding compared with the untreated control.
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Figure 7: Accelerated shedding is inhibited by peptide hydroxamates. (A and B) NMuMG cells were incubated with or without PMA (0.5 μM for 30 min) in the presence or absence of peptide hydroxamate BB-2116 or BB-1101 at the indicated concentration. (C and D) SVEC4-10 cells were incubated for 2 h with or without 10 ng/ml EGF or 100 μM TRAP in the presence or absence of 1 μM BB-1101. (E and F) NMuMG cells were incubated for 2 h with or without 100 μM ceramide in the presence or absence of 1 μM BB-1101. Syndecan-1 (A, C, and E) and syndecan-4 (B, D, and F) ectodomains in the conditioned media were assayed by dot blot analysis as in Fig. 1. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3). For each shedding agonist assayed, incubation with inhibitor alone (1 μM) reduced the level of shedding compared with the untreated control.

Mentions: Thus, we tested the effects of hydroxamic acid-based MP inhibitors, initially developed to inhibit shedding of pro-TNF-α (Odake et al. 1994; Sayama et al. 1995; Wojtowicz-Praga et al. 1997; Pratt et al. 1998) on syndecan-1 and -4 shedding accelerated by PMA, receptor activation, and cellular stress. The peptide hydroxamates BB-2116 and BB-1101 reduced the shedding of syndecan-1 (Fig. 7 A) and syndecan-4 (Fig. 7 B) ectodomains from PMA-treated NMuMG cells in a concentration-dependent manner without affecting cell morphology or viability; the IC50 was ∼0.1 μM. Similar results were observed when P3X63 and SVEC4-10 cells were used (data not shown). EGF-, TRAP-, and ceramide-accelerated shedding of syndecan-1 (Fig. 7C and Fig. E) and syndecan-4 (Fig. 7D and Fig. F) ectodomains were also inhibited by hydroxamate BB-1101. The control hydroxamate compound BB-3861, which lacks the zinc-binding domain, had no effect on syndecan shedding at concentrations up to 10 μM (data not shown). These results suggest that syndecan-1 and -4 shedding, accelerated by each of the agonists tested, depends on metalloproteinase activity.


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)

Accelerated shedding is inhibited by peptide hydroxamates. (A and B) NMuMG cells were incubated with or without PMA (0.5 μM for 30 min) in the presence or absence of peptide hydroxamate BB-2116 or BB-1101 at the indicated concentration. (C and D) SVEC4-10 cells were incubated for 2 h with or without 10 ng/ml EGF or 100 μM TRAP in the presence or absence of 1 μM BB-1101. (E and F) NMuMG cells were incubated for 2 h with or without 100 μM ceramide in the presence or absence of 1 μM BB-1101. Syndecan-1 (A, C, and E) and syndecan-4 (B, D, and F) ectodomains in the conditioned media were assayed by dot blot analysis as in Fig. 1. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3). For each shedding agonist assayed, incubation with inhibitor alone (1 μM) reduced the level of shedding compared with the untreated control.
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Related In: Results  -  Collection

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Figure 7: Accelerated shedding is inhibited by peptide hydroxamates. (A and B) NMuMG cells were incubated with or without PMA (0.5 μM for 30 min) in the presence or absence of peptide hydroxamate BB-2116 or BB-1101 at the indicated concentration. (C and D) SVEC4-10 cells were incubated for 2 h with or without 10 ng/ml EGF or 100 μM TRAP in the presence or absence of 1 μM BB-1101. (E and F) NMuMG cells were incubated for 2 h with or without 100 μM ceramide in the presence or absence of 1 μM BB-1101. Syndecan-1 (A, C, and E) and syndecan-4 (B, D, and F) ectodomains in the conditioned media were assayed by dot blot analysis as in Fig. 1. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3). For each shedding agonist assayed, incubation with inhibitor alone (1 μM) reduced the level of shedding compared with the untreated control.
Mentions: Thus, we tested the effects of hydroxamic acid-based MP inhibitors, initially developed to inhibit shedding of pro-TNF-α (Odake et al. 1994; Sayama et al. 1995; Wojtowicz-Praga et al. 1997; Pratt et al. 1998) on syndecan-1 and -4 shedding accelerated by PMA, receptor activation, and cellular stress. The peptide hydroxamates BB-2116 and BB-1101 reduced the shedding of syndecan-1 (Fig. 7 A) and syndecan-4 (Fig. 7 B) ectodomains from PMA-treated NMuMG cells in a concentration-dependent manner without affecting cell morphology or viability; the IC50 was ∼0.1 μM. Similar results were observed when P3X63 and SVEC4-10 cells were used (data not shown). EGF-, TRAP-, and ceramide-accelerated shedding of syndecan-1 (Fig. 7C and Fig. E) and syndecan-4 (Fig. 7D and Fig. F) ectodomains were also inhibited by hydroxamate BB-1101. The control hydroxamate compound BB-3861, which lacks the zinc-binding domain, had no effect on syndecan shedding at concentrations up to 10 μM (data not shown). These results suggest that syndecan-1 and -4 shedding, accelerated by each of the agonists tested, depends on metalloproteinase activity.

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