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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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Effect of HMG1 on RSMC morphology and actin cytoskeleton organization. (A) Subconfluent (50–70%) cultures of RSMC were challenged for the indicated times with HMG1 (100 ng/ml), either purified from calf thymus or expressed in yeast or E. coli as indicated. Actin filaments were visualized using TRITC-phalloidin. (B) Anti–HMG1 rabbit antibodies, but not unspecific rabbit antibodies, inhibit HMG1-stimulated cytoskeleton reorganization. RSMC were pretreated overnight with either anti–HMG1 (2 μg/ml) or unspecific control antibodies (2 μg/ml), and then 100 ng/ml HMG1 (from calf thymus) was added. (C) RSMC were stimulated with 100 ng/ml HMG1. Quantification of the actin cytoskeleton reorganization was performed by taking low-magnification photographs and counting the cells in each state of cytoskeleton organization. Resting cells (state 1) exhibit numerous stress fibers. Nonresting cells (state 2) show a reorganization of actin cytoskeleton: a decrease of stress fibers content, membrane ruffling, actin semi-ring with an elongated polarized morphology characteristic of motile RSMC.
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Figure 2: Effect of HMG1 on RSMC morphology and actin cytoskeleton organization. (A) Subconfluent (50–70%) cultures of RSMC were challenged for the indicated times with HMG1 (100 ng/ml), either purified from calf thymus or expressed in yeast or E. coli as indicated. Actin filaments were visualized using TRITC-phalloidin. (B) Anti–HMG1 rabbit antibodies, but not unspecific rabbit antibodies, inhibit HMG1-stimulated cytoskeleton reorganization. RSMC were pretreated overnight with either anti–HMG1 (2 μg/ml) or unspecific control antibodies (2 μg/ml), and then 100 ng/ml HMG1 (from calf thymus) was added. (C) RSMC were stimulated with 100 ng/ml HMG1. Quantification of the actin cytoskeleton reorganization was performed by taking low-magnification photographs and counting the cells in each state of cytoskeleton organization. Resting cells (state 1) exhibit numerous stress fibers. Nonresting cells (state 2) show a reorganization of actin cytoskeleton: a decrease of stress fibers content, membrane ruffling, actin semi-ring with an elongated polarized morphology characteristic of motile RSMC.

Mentions: Chemoattractant-induced cell motility requires cytoskeleton reorganization and cell shape changes (Ridley 1994). Using RSMC, we have previously functionally connected induction of cell migration and cytoskeleton reorganization (Degryse et al. 1999). Subconfluent cultures of serum-starved RSMC were stimulated with 100 ng/ml HMG1 from calf thymus for increasing times from 5 to 120 min, and actin filaments were labeled with rhodamine-conjugated phalloidin. Low magnification pictures showed that stress fibers content, cell shape and size, and cytoskeleton organization changed within 30 min, but reversed after 120 min (Fig. 2 A). Higher magnification pictures showed that in control conditions most RSMC exhibited numerous stress fibers and a nonpolarized cell shape (Fig. 2 B). After 5 min, HMG1 induced a clear decrease in stress fiber content and stimulated membrane ruffling (data not shown). Within 15–30 min, a complete change of morphology and cytoskeleton organization had occurred: RSMC had an elongated, polarized morphology that reflected the spatial rearrangement of the actin cytoskeleton. Semi-ring structures of actin and membrane ruffling were observed at the leading part of the cell. Filaments of actin were also present flanking the nucleus and in the dragging trail. These effects of HMG1 slowly decreased. After 1–2 h, the stress fiber content increased back to a level similar to that of unstimulated cells, and cell morphology returned similar to that of unstimulated control cells.


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)

Effect of HMG1 on RSMC morphology and actin cytoskeleton organization. (A) Subconfluent (50–70%) cultures of RSMC were challenged for the indicated times with HMG1 (100 ng/ml), either purified from calf thymus or expressed in yeast or E. coli as indicated. Actin filaments were visualized using TRITC-phalloidin. (B) Anti–HMG1 rabbit antibodies, but not unspecific rabbit antibodies, inhibit HMG1-stimulated cytoskeleton reorganization. RSMC were pretreated overnight with either anti–HMG1 (2 μg/ml) or unspecific control antibodies (2 μg/ml), and then 100 ng/ml HMG1 (from calf thymus) was added. (C) RSMC were stimulated with 100 ng/ml HMG1. Quantification of the actin cytoskeleton reorganization was performed by taking low-magnification photographs and counting the cells in each state of cytoskeleton organization. Resting cells (state 1) exhibit numerous stress fibers. Nonresting cells (state 2) show a reorganization of actin cytoskeleton: a decrease of stress fibers content, membrane ruffling, actin semi-ring with an elongated polarized morphology characteristic of motile RSMC.
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Related In: Results  -  Collection

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Figure 2: Effect of HMG1 on RSMC morphology and actin cytoskeleton organization. (A) Subconfluent (50–70%) cultures of RSMC were challenged for the indicated times with HMG1 (100 ng/ml), either purified from calf thymus or expressed in yeast or E. coli as indicated. Actin filaments were visualized using TRITC-phalloidin. (B) Anti–HMG1 rabbit antibodies, but not unspecific rabbit antibodies, inhibit HMG1-stimulated cytoskeleton reorganization. RSMC were pretreated overnight with either anti–HMG1 (2 μg/ml) or unspecific control antibodies (2 μg/ml), and then 100 ng/ml HMG1 (from calf thymus) was added. (C) RSMC were stimulated with 100 ng/ml HMG1. Quantification of the actin cytoskeleton reorganization was performed by taking low-magnification photographs and counting the cells in each state of cytoskeleton organization. Resting cells (state 1) exhibit numerous stress fibers. Nonresting cells (state 2) show a reorganization of actin cytoskeleton: a decrease of stress fibers content, membrane ruffling, actin semi-ring with an elongated polarized morphology characteristic of motile RSMC.
Mentions: Chemoattractant-induced cell motility requires cytoskeleton reorganization and cell shape changes (Ridley 1994). Using RSMC, we have previously functionally connected induction of cell migration and cytoskeleton reorganization (Degryse et al. 1999). Subconfluent cultures of serum-starved RSMC were stimulated with 100 ng/ml HMG1 from calf thymus for increasing times from 5 to 120 min, and actin filaments were labeled with rhodamine-conjugated phalloidin. Low magnification pictures showed that stress fibers content, cell shape and size, and cytoskeleton organization changed within 30 min, but reversed after 120 min (Fig. 2 A). Higher magnification pictures showed that in control conditions most RSMC exhibited numerous stress fibers and a nonpolarized cell shape (Fig. 2 B). After 5 min, HMG1 induced a clear decrease in stress fiber content and stimulated membrane ruffling (data not shown). Within 15–30 min, a complete change of morphology and cytoskeleton organization had occurred: RSMC had an elongated, polarized morphology that reflected the spatial rearrangement of the actin cytoskeleton. Semi-ring structures of actin and membrane ruffling were observed at the leading part of the cell. Filaments of actin were also present flanking the nucleus and in the dragging trail. These effects of HMG1 slowly decreased. After 1–2 h, the stress fiber content increased back to a level similar to that of unstimulated cells, and cell morphology returned similar to that of unstimulated control cells.

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