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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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A membrane-associated proteolytic activity is responsible for accelerated shedding. P3X63 cells were coincubated for 15 min with human ARK cells that had been pretreated with or without PMA for 15 min and washed twice with serum-free media. Cells were (A) mixed together or (B) separated from each other by a Transwell membrane. A constant number of P3X63 cells (5 × 106) and a varying number of ARK cells were used. Conditioned media were analyzed by dot blot using mAb 281-2 to detect syndecan-1 shed from the surface of P3X63 cells. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3).
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Figure 5: A membrane-associated proteolytic activity is responsible for accelerated shedding. P3X63 cells were coincubated for 15 min with human ARK cells that had been pretreated with or without PMA for 15 min and washed twice with serum-free media. Cells were (A) mixed together or (B) separated from each other by a Transwell membrane. A constant number of P3X63 cells (5 × 106) and a varying number of ARK cells were used. Conditioned media were analyzed by dot blot using mAb 281-2 to detect syndecan-1 shed from the surface of P3X63 cells. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3).

Mentions: We next evaluated whether the proteolytic activity responsible for cleavage of the core protein and shedding of the ectodomain is soluble or membrane-associated. For these assays, PMA-treated human ARK cells and untreated mouse P3X63 cells were either mixed together or separated from each other by a Transwell membrane. In both assays, a constant number of P3X63 cells and a varying number of ARK cells were used. Human and mouse syndecan-1 ectodomains were distinguished using mAb DL-101 and 281-2, respectively. When cells were mixed to allow cell–cell contact, the PMA-treated ARK cells accelerated shedding of the syndecan-1 ectodomain from the untreated P3X63 cells (Fig. 5 A). However, when a Transwell membrane separated the cells, the PMA-treated ARK cells did not accelerate shedding from the untreated P3X63 cells (Fig. 5 B). Similar results were observed when the same experiments were performed using PMA-treated P3X63 cells and untreated ARK cells (data not shown). These results indicate that the proteolytic activity responsible for shedding is cell-associated rather than soluble. Further, because this proteolytic activity can act on adjacent cells, it likely acts at 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)

A membrane-associated proteolytic activity is responsible for accelerated shedding. P3X63 cells were coincubated for 15 min with human ARK cells that had been pretreated with or without PMA for 15 min and washed twice with serum-free media. Cells were (A) mixed together or (B) separated from each other by a Transwell membrane. A constant number of P3X63 cells (5 × 106) and a varying number of ARK cells were used. Conditioned media were analyzed by dot blot using mAb 281-2 to detect syndecan-1 shed from the surface of P3X63 cells. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169376&req=5

Figure 5: A membrane-associated proteolytic activity is responsible for accelerated shedding. P3X63 cells were coincubated for 15 min with human ARK cells that had been pretreated with or without PMA for 15 min and washed twice with serum-free media. Cells were (A) mixed together or (B) separated from each other by a Transwell membrane. A constant number of P3X63 cells (5 × 106) and a varying number of ARK cells were used. Conditioned media were analyzed by dot blot using mAb 281-2 to detect syndecan-1 shed from the surface of P3X63 cells. Quantitation was done as in Fig. 1 and each point represents the mean ± SD (n = 3).
Mentions: We next evaluated whether the proteolytic activity responsible for cleavage of the core protein and shedding of the ectodomain is soluble or membrane-associated. For these assays, PMA-treated human ARK cells and untreated mouse P3X63 cells were either mixed together or separated from each other by a Transwell membrane. In both assays, a constant number of P3X63 cells and a varying number of ARK cells were used. Human and mouse syndecan-1 ectodomains were distinguished using mAb DL-101 and 281-2, respectively. When cells were mixed to allow cell–cell contact, the PMA-treated ARK cells accelerated shedding of the syndecan-1 ectodomain from the untreated P3X63 cells (Fig. 5 A). However, when a Transwell membrane separated the cells, the PMA-treated ARK cells did not accelerate shedding from the untreated P3X63 cells (Fig. 5 B). Similar results were observed when the same experiments were performed using PMA-treated P3X63 cells and untreated ARK cells (data not shown). These results indicate that the proteolytic activity responsible for shedding is cell-associated rather than soluble. Further, because this proteolytic activity can act on adjacent cells, it likely acts at 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