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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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PMA-accelerated shedding is specifically inhibited by TIMP-3. (A and B) NMuMG cells were treated with PMA (0.5 μM for 30 min) in the presence or absence of 20 μg/ml TIMP-1, -2, or -3. (C and D) NMuMG cells were treated with or without PMA (0.5 μM for 30 min) in the presence or absence of TIMP-3 at the indicated concentration. Syndecan-1 (A and C) and syndecan-4 (B and D) 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, treatment with TIMP-1, -2, or -3 alone (20 μM) had no effect on the level of shedding compared with the untreated control.
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Figure 8: PMA-accelerated shedding is specifically inhibited by TIMP-3. (A and B) NMuMG cells were treated with PMA (0.5 μM for 30 min) in the presence or absence of 20 μg/ml TIMP-1, -2, or -3. (C and D) NMuMG cells were treated with or without PMA (0.5 μM for 30 min) in the presence or absence of TIMP-3 at the indicated concentration. Syndecan-1 (A and C) and syndecan-4 (B and D) 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, treatment with TIMP-1, -2, or -3 alone (20 μM) had no effect on the level of shedding compared with the untreated control.

Mentions: Because the peptide hydroxamate MP inhibitors prevented syndecan-1 and -4 ectodomain shedding, we tested members of the tissue inhibitor of metalloproteinase (TIMP) family of secreted MMP inhibitors (Murphy and Willenbrock 1995; Anand-Apte et al. 1996; Gomez et al. 1997). TIMP family members have distinct expression patterns (Leco et al. 1994), form complexes with different progelatinases (Murphy and Willenbrock 1995; Gomez et al. 1997; Butler et al. 1999b; Murphy et al. 1999), and differ in their ability to bind heparin (Butler et al. 1999b). PMA-accelerated shedding of both syndecan-1 and -4 ectodomains from NMuMG cells (Fig. 8, A and B) is specifically inhibited by TIMP-3; TIMP-1 and TIMP-2 had no effect on shedding at concentrations up to 20 μg/ml. Similar results were observed when P3X63 and SVEC4-10 cells were used (data not shown). TIMP-3 inhibition was concentration-dependent; the IC50 was ∼5 μg/ml, and PMA-accelerated shedding was completely prevented in the presence of 20 μg/ml (∼0.8 μM) TIMP-3 (Fig. 8C and Fig. D). To determine whether shedding accelerated by physiological agonists is also inhibited by TIMP-3, we tested the effect of TIMP-3 on EGF-, TRAP-, and ceramide-accelerated shedding of syndecan-1 and -4 ectodomains. TIMP-3 reduced EGF and TRAP receptor–activated shedding from SVEC4-10 cells (Fig. 9A and Fig. B), and ceramide-induced shedding of these syndecans from NMuMG cells (Fig. 10C and Fig. D). Thus, shedding accelerated by PMA and a variety of physiological agonists requires a TIMP-3–sensitive MP 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)

PMA-accelerated shedding is specifically inhibited by TIMP-3. (A and B) NMuMG cells were treated with PMA (0.5 μM for 30 min) in the presence or absence of 20 μg/ml TIMP-1, -2, or -3. (C and D) NMuMG cells were treated with or without PMA (0.5 μM for 30 min) in the presence or absence of TIMP-3 at the indicated concentration. Syndecan-1 (A and C) and syndecan-4 (B and D) 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, treatment with TIMP-1, -2, or -3 alone (20 μM) had no effect on the level of shedding compared with the untreated control.
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Related In: Results  -  Collection

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Figure 8: PMA-accelerated shedding is specifically inhibited by TIMP-3. (A and B) NMuMG cells were treated with PMA (0.5 μM for 30 min) in the presence or absence of 20 μg/ml TIMP-1, -2, or -3. (C and D) NMuMG cells were treated with or without PMA (0.5 μM for 30 min) in the presence or absence of TIMP-3 at the indicated concentration. Syndecan-1 (A and C) and syndecan-4 (B and D) 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, treatment with TIMP-1, -2, or -3 alone (20 μM) had no effect on the level of shedding compared with the untreated control.
Mentions: Because the peptide hydroxamate MP inhibitors prevented syndecan-1 and -4 ectodomain shedding, we tested members of the tissue inhibitor of metalloproteinase (TIMP) family of secreted MMP inhibitors (Murphy and Willenbrock 1995; Anand-Apte et al. 1996; Gomez et al. 1997). TIMP family members have distinct expression patterns (Leco et al. 1994), form complexes with different progelatinases (Murphy and Willenbrock 1995; Gomez et al. 1997; Butler et al. 1999b; Murphy et al. 1999), and differ in their ability to bind heparin (Butler et al. 1999b). PMA-accelerated shedding of both syndecan-1 and -4 ectodomains from NMuMG cells (Fig. 8, A and B) is specifically inhibited by TIMP-3; TIMP-1 and TIMP-2 had no effect on shedding at concentrations up to 20 μg/ml. Similar results were observed when P3X63 and SVEC4-10 cells were used (data not shown). TIMP-3 inhibition was concentration-dependent; the IC50 was ∼5 μg/ml, and PMA-accelerated shedding was completely prevented in the presence of 20 μg/ml (∼0.8 μM) TIMP-3 (Fig. 8C and Fig. D). To determine whether shedding accelerated by physiological agonists is also inhibited by TIMP-3, we tested the effect of TIMP-3 on EGF-, TRAP-, and ceramide-accelerated shedding of syndecan-1 and -4 ectodomains. TIMP-3 reduced EGF and TRAP receptor–activated shedding from SVEC4-10 cells (Fig. 9A and Fig. B), and ceramide-induced shedding of these syndecans from NMuMG cells (Fig. 10C and Fig. D). Thus, shedding accelerated by PMA and a variety of physiological agonists requires a TIMP-3–sensitive MP 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