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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 of syndecan-1 ectodomains results from cleavage at a juxtamembrane site in the core protein. (A) Schematic depiction of wild-type (WT) syndecan-1 and the CD4-juxtamembrane (JM) mutant syndecan-1. The JM region of the ectodomain is shown both with the WT amino acid sequence and with the CD4-JM domain mutant sequence (bold face). (B) COS-7 cells were transiently transfected with either the WT syndecan-1 or the CD4-JM mutant. Cells were incubated with or without PMA (0.5 μM for 15 min) in the absence or presence of peptide hydroxamate BB-1101 (1 μM). Syndecan-1 ectodomains in the conditioned media were assayed by dot blot analysis and quantitation was done as in Fig. 1. Each point represents the mean ± SD (n = 3).
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Figure 6: PMA-accelerated shedding of syndecan-1 ectodomains results from cleavage at a juxtamembrane site in the core protein. (A) Schematic depiction of wild-type (WT) syndecan-1 and the CD4-juxtamembrane (JM) mutant syndecan-1. The JM region of the ectodomain is shown both with the WT amino acid sequence and with the CD4-JM domain mutant sequence (bold face). (B) COS-7 cells were transiently transfected with either the WT syndecan-1 or the CD4-JM mutant. Cells were incubated with or without PMA (0.5 μM for 15 min) in the absence or presence of peptide hydroxamate BB-1101 (1 μM). Syndecan-1 ectodomains in the conditioned media were assayed by dot blot analysis and quantitation was done as in Fig. 1. Each point represents the mean ± SD (n = 3).

Mentions: We constructed a putative uncleavable syndecan-1 mutant by replacing 15 amino acids of the wild-type syndecan-1 juxtamembrane domain with the corresponding amino acid sequence of the human CD4 T cell transmembrane antigen (Fig. 6 A), and tested whether the mutant was resistant to PMA-accelerated shedding. CD4 does not undergo PMA-accelerated shedding (Wang et al. 1987; Shin et al. 1991), suggesting that CD4 is not cleaved by the proteolytic activity that sheds syndecan-1. Whereas PMA treatment of COS-7 cells transfected with wild-type syndecan-1 (syn1-WTJM) released the soluble ectodomain, an activity inhibited by a peptide hydroxamate (cf. below), PMA did not accelerate release of syndecan-1 from cells transfected with the syn1-CD4 juxtamembrane mutant (Fig. 6 B). Similar results were observed using CHO cells and HT-1080 human fibrosarcoma cells (data not shown). Thus, the core protein is cleaved in the juxtamembrane region within 15 amino acids from the cell surface.


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 of syndecan-1 ectodomains results from cleavage at a juxtamembrane site in the core protein. (A) Schematic depiction of wild-type (WT) syndecan-1 and the CD4-juxtamembrane (JM) mutant syndecan-1. The JM region of the ectodomain is shown both with the WT amino acid sequence and with the CD4-JM domain mutant sequence (bold face). (B) COS-7 cells were transiently transfected with either the WT syndecan-1 or the CD4-JM mutant. Cells were incubated with or without PMA (0.5 μM for 15 min) in the absence or presence of peptide hydroxamate BB-1101 (1 μM). Syndecan-1 ectodomains in the conditioned media were assayed by dot blot analysis and quantitation was done as in Fig. 1. Each point represents the mean ± SD (n = 3).
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Figure 6: PMA-accelerated shedding of syndecan-1 ectodomains results from cleavage at a juxtamembrane site in the core protein. (A) Schematic depiction of wild-type (WT) syndecan-1 and the CD4-juxtamembrane (JM) mutant syndecan-1. The JM region of the ectodomain is shown both with the WT amino acid sequence and with the CD4-JM domain mutant sequence (bold face). (B) COS-7 cells were transiently transfected with either the WT syndecan-1 or the CD4-JM mutant. Cells were incubated with or without PMA (0.5 μM for 15 min) in the absence or presence of peptide hydroxamate BB-1101 (1 μM). Syndecan-1 ectodomains in the conditioned media were assayed by dot blot analysis and quantitation was done as in Fig. 1. Each point represents the mean ± SD (n = 3).
Mentions: We constructed a putative uncleavable syndecan-1 mutant by replacing 15 amino acids of the wild-type syndecan-1 juxtamembrane domain with the corresponding amino acid sequence of the human CD4 T cell transmembrane antigen (Fig. 6 A), and tested whether the mutant was resistant to PMA-accelerated shedding. CD4 does not undergo PMA-accelerated shedding (Wang et al. 1987; Shin et al. 1991), suggesting that CD4 is not cleaved by the proteolytic activity that sheds syndecan-1. Whereas PMA treatment of COS-7 cells transfected with wild-type syndecan-1 (syn1-WTJM) released the soluble ectodomain, an activity inhibited by a peptide hydroxamate (cf. below), PMA did not accelerate release of syndecan-1 from cells transfected with the syn1-CD4 juxtamembrane mutant (Fig. 6 B). Similar results were observed using CHO cells and HT-1080 human fibrosarcoma cells (data not shown). Thus, the core protein is cleaved in the juxtamembrane region within 15 amino acids from the cell surface.

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