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The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

Degryse B, Bonaldi T, Scaffidi P, Müller S, Resnati M, Sanvito F, Arrigoni G, Bianchi ME - J. Cell Biol. (2001)

Bottom Line: HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays.HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells.These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Milan, 20133 Milan, Italy. degryse@scripps.edu

ABSTRACT
HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.

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HMG1 is released by necrotic and damaged cells. (A) Western-blot analysis showing the release of HMG1 by necrotic or permeabilized HeLa cells and HUVEC. Cells were induced into necrosis by adding to the medium 5 μM ionomycin plus 20 μM CCCP (CCCP), or 6 mM deoxyglucose plus 10 mM sodium azide (deoxygluc), for 16 h. Alternatively, 0.1% NP-40 was added for 10 min. The presence of the protein in supernatants (S) and cell pellets (P) was detected by Western blot using an anti–HMG1 antibody. The presence of HMG1 in the left two P lanes is due to the fact that only ∼50% of the treated cells underwent necrosis. (B) Immunofluorescence performed on living and necrotic HeLa cells. The cells (untreated or treated with ionomycin + CCCP) were fixed and stained for HMG1 and DNA.
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Figure 8: HMG1 is released by necrotic and damaged cells. (A) Western-blot analysis showing the release of HMG1 by necrotic or permeabilized HeLa cells and HUVEC. Cells were induced into necrosis by adding to the medium 5 μM ionomycin plus 20 μM CCCP (CCCP), or 6 mM deoxyglucose plus 10 mM sodium azide (deoxygluc), for 16 h. Alternatively, 0.1% NP-40 was added for 10 min. The presence of the protein in supernatants (S) and cell pellets (P) was detected by Western blot using an anti–HMG1 antibody. The presence of HMG1 in the left two P lanes is due to the fact that only ∼50% of the treated cells underwent necrosis. (B) Immunofluorescence performed on living and necrotic HeLa cells. The cells (untreated or treated with ionomycin + CCCP) were fixed and stained for HMG1 and DNA.

Mentions: We had previously shown that HMG1 is loosely bound to chromatin (Falciola et al. 1997). Therefore, we tested whether damaged cells, or cells undergoing necrosis, could release HMG1 in the medium. HeLa cells or HUVEC, which contain a significant amount of HMG1, were permeabilized by the addition of the nonionic detergent NP-40 (Falciola et al. 1997). Alternatively, cells were forced into necrosis by addition of ionomycin and the membrane uncoupler CCCP, or by treatment with deoxyglucose and azide. The number of cells undergoing necrosis was scored morphologically, and when it approached 50% the supernatant was collected. HMG1 was recovered in the supernatant of both necrotic cells and damaged cells (Fig. 8 A). The amount of HMG1 found in the supernatant of HUVEC, even when permeabilized with detergent, was always a fraction of the total HMG1 originally present in the cells, because HMG1 binds to the extracellular matrix secreted by HUVEC (not shown).


The high mobility group (HMG) boxes of the nuclear protein HMG1 induce chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

Degryse B, Bonaldi T, Scaffidi P, Müller S, Resnati M, Sanvito F, Arrigoni G, Bianchi ME - J. Cell Biol. (2001)

HMG1 is released by necrotic and damaged cells. (A) Western-blot analysis showing the release of HMG1 by necrotic or permeabilized HeLa cells and HUVEC. Cells were induced into necrosis by adding to the medium 5 μM ionomycin plus 20 μM CCCP (CCCP), or 6 mM deoxyglucose plus 10 mM sodium azide (deoxygluc), for 16 h. Alternatively, 0.1% NP-40 was added for 10 min. The presence of the protein in supernatants (S) and cell pellets (P) was detected by Western blot using an anti–HMG1 antibody. The presence of HMG1 in the left two P lanes is due to the fact that only ∼50% of the treated cells underwent necrosis. (B) Immunofluorescence performed on living and necrotic HeLa cells. The cells (untreated or treated with ionomycin + CCCP) were fixed and stained for HMG1 and DNA.
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Related In: Results  -  Collection

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Figure 8: HMG1 is released by necrotic and damaged cells. (A) Western-blot analysis showing the release of HMG1 by necrotic or permeabilized HeLa cells and HUVEC. Cells were induced into necrosis by adding to the medium 5 μM ionomycin plus 20 μM CCCP (CCCP), or 6 mM deoxyglucose plus 10 mM sodium azide (deoxygluc), for 16 h. Alternatively, 0.1% NP-40 was added for 10 min. The presence of the protein in supernatants (S) and cell pellets (P) was detected by Western blot using an anti–HMG1 antibody. The presence of HMG1 in the left two P lanes is due to the fact that only ∼50% of the treated cells underwent necrosis. (B) Immunofluorescence performed on living and necrotic HeLa cells. The cells (untreated or treated with ionomycin + CCCP) were fixed and stained for HMG1 and DNA.
Mentions: We had previously shown that HMG1 is loosely bound to chromatin (Falciola et al. 1997). Therefore, we tested whether damaged cells, or cells undergoing necrosis, could release HMG1 in the medium. HeLa cells or HUVEC, which contain a significant amount of HMG1, were permeabilized by the addition of the nonionic detergent NP-40 (Falciola et al. 1997). Alternatively, cells were forced into necrosis by addition of ionomycin and the membrane uncoupler CCCP, or by treatment with deoxyglucose and azide. The number of cells undergoing necrosis was scored morphologically, and when it approached 50% the supernatant was collected. HMG1 was recovered in the supernatant of both necrotic cells and damaged cells (Fig. 8 A). The amount of HMG1 found in the supernatant of HUVEC, even when permeabilized with detergent, was always a fraction of the total HMG1 originally present in the cells, because HMG1 binds to the extracellular matrix secreted by HUVEC (not shown).

Bottom Line: HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays.HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells.These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, University of Milan, 20133 Milan, Italy. degryse@scripps.edu

ABSTRACT
HMG1 (high mobility group 1) is a ubiquitous and abundant chromatin component. However, HMG1 can be secreted by activated macrophages and monocytes, and can act as a mediator of inflammation and endotoxic lethality. Here we document a role of extracellular HMG1 in cell migration. HMG1 (and its individual DNA-binding domains) stimulated migration of rat smooth muscle cells in chemotaxis, chemokinesis, and wound healing assays. HMG1 induced rapid and transient changes of cell shape, and actin cytoskeleton reorganization leading to an elongated polarized morphology typical of motile cells. These effects were inhibited by antibodies directed against the receptor of advanced glycation endproducts, indicating that the receptor of advanced glycation endproducts is the receptor mediating the HMG1-dependent migratory responses. Pertussis toxin and the mitogen-activated protein kinase kinase inhibitor PD98059 also blocked HMG1-induced rat smooth muscle cell migration, suggesting that a G(i/o) protein and mitogen-activated protein kinases are required for the HMG1 signaling pathway. We also show that HMG1 can be released by damage or necrosis of a variety of cell types, including endothelial cells. Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after vascular damage.

Show MeSH
Related in: MedlinePlus