Limits...
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.

Show MeSH

Related in: MedlinePlus

Accelerated shedding of syndecan-1 and -4 depends on protein tyrosine kinase activity. (A and B) NMuMG epithelial cells were incubated with 4αPDD (0.5 μM), an inactive PMA analogue or PMA for 30 min and in the presence or absence of tyrphostin A25 (5 μg/ml) or methyl 2,5, dihydroxycinnamate (5 μg/ml). NMuMG cells were incubated with or without the following: (C and D) 100 μM TRAP, or (E and F) 700 mOsm NaCl, or (G and H) 100 μM ceramide for 2 h in the presence or absence of tyrphostin A25 (5 μg/ml). Conditioned media were harvested and applied to cationic Immobilon-N membranes for dot blot analysis using ECL detection. Syndecan-1 (A, C, E, and G) and syndecan-4 (B, D, F, and H) ectodomains were detected by ECL using mAb 281-2 and MSE-4 antiserum, respectively. Results are expressed as the amount of syndecan ectodomain shed in AU quantified by densitometric scanning and analyzed with NIH image software. Each point represents the mean ± SD of triplicate determinations. For each shedding agonist assayed, incubation with inhibitor alone had no effect on the level of shedding compared with the untreated control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169376&req=5

Figure 1: Accelerated shedding of syndecan-1 and -4 depends on protein tyrosine kinase activity. (A and B) NMuMG epithelial cells were incubated with 4αPDD (0.5 μM), an inactive PMA analogue or PMA for 30 min and in the presence or absence of tyrphostin A25 (5 μg/ml) or methyl 2,5, dihydroxycinnamate (5 μg/ml). NMuMG cells were incubated with or without the following: (C and D) 100 μM TRAP, or (E and F) 700 mOsm NaCl, or (G and H) 100 μM ceramide for 2 h in the presence or absence of tyrphostin A25 (5 μg/ml). Conditioned media were harvested and applied to cationic Immobilon-N membranes for dot blot analysis using ECL detection. Syndecan-1 (A, C, E, and G) and syndecan-4 (B, D, F, and H) ectodomains were detected by ECL using mAb 281-2 and MSE-4 antiserum, respectively. Results are expressed as the amount of syndecan ectodomain shed in AU quantified by densitometric scanning and analyzed with NIH image software. Each point represents the mean ± SD of triplicate determinations. For each shedding agonist assayed, incubation with inhibitor alone had no effect on the level of shedding compared with the untreated control.

Mentions: Phorbol esters, PKC activators known to enhance ectodomain shedding of a diverse group of cell surface proteins (Hooper et al. 1997), rapidly (5 min) accelerate shedding of syndecan-1 and -4 ectodomains from a variety of cultured cells (Fitzgerald, M.L., J.-S. Chun, and M. Bernfield, American Society of Cell Biology. 1994. 1813 (Abstr.); Subramanian et al., 1997). Both tyrphostin A25 and methyl 2,5 dihydroxycinnamate, protein tyrosine kinase inhibitors, prevented the PMA-accelerated shedding of syndecan-1 (Fig. 1 A) and syndecan-4 (Fig. 1 B) ectodomains.


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 of syndecan-1 and -4 depends on protein tyrosine kinase activity. (A and B) NMuMG epithelial cells were incubated with 4αPDD (0.5 μM), an inactive PMA analogue or PMA for 30 min and in the presence or absence of tyrphostin A25 (5 μg/ml) or methyl 2,5, dihydroxycinnamate (5 μg/ml). NMuMG cells were incubated with or without the following: (C and D) 100 μM TRAP, or (E and F) 700 mOsm NaCl, or (G and H) 100 μM ceramide for 2 h in the presence or absence of tyrphostin A25 (5 μg/ml). Conditioned media were harvested and applied to cationic Immobilon-N membranes for dot blot analysis using ECL detection. Syndecan-1 (A, C, E, and G) and syndecan-4 (B, D, F, and H) ectodomains were detected by ECL using mAb 281-2 and MSE-4 antiserum, respectively. Results are expressed as the amount of syndecan ectodomain shed in AU quantified by densitometric scanning and analyzed with NIH image software. Each point represents the mean ± SD of triplicate determinations. For each shedding agonist assayed, incubation with inhibitor alone had no effect on the level of shedding compared with the untreated control.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Accelerated shedding of syndecan-1 and -4 depends on protein tyrosine kinase activity. (A and B) NMuMG epithelial cells were incubated with 4αPDD (0.5 μM), an inactive PMA analogue or PMA for 30 min and in the presence or absence of tyrphostin A25 (5 μg/ml) or methyl 2,5, dihydroxycinnamate (5 μg/ml). NMuMG cells were incubated with or without the following: (C and D) 100 μM TRAP, or (E and F) 700 mOsm NaCl, or (G and H) 100 μM ceramide for 2 h in the presence or absence of tyrphostin A25 (5 μg/ml). Conditioned media were harvested and applied to cationic Immobilon-N membranes for dot blot analysis using ECL detection. Syndecan-1 (A, C, E, and G) and syndecan-4 (B, D, F, and H) ectodomains were detected by ECL using mAb 281-2 and MSE-4 antiserum, respectively. Results are expressed as the amount of syndecan ectodomain shed in AU quantified by densitometric scanning and analyzed with NIH image software. Each point represents the mean ± SD of triplicate determinations. For each shedding agonist assayed, incubation with inhibitor alone had no effect on the level of shedding compared with the untreated control.
Mentions: Phorbol esters, PKC activators known to enhance ectodomain shedding of a diverse group of cell surface proteins (Hooper et al. 1997), rapidly (5 min) accelerate shedding of syndecan-1 and -4 ectodomains from a variety of cultured cells (Fitzgerald, M.L., J.-S. Chun, and M. Bernfield, American Society of Cell Biology. 1994. 1813 (Abstr.); Subramanian et al., 1997). Both tyrphostin A25 and methyl 2,5 dihydroxycinnamate, protein tyrosine kinase inhibitors, prevented the PMA-accelerated shedding of syndecan-1 (Fig. 1 A) and syndecan-4 (Fig. 1 B) ectodomains.

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