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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 has chemotactic activity on RSMC. Chemotaxis assays were performed using modified Boyden chambers. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). The data represent the mean ± SD (n = 3). (A) Concentration-dependent migratory response of RSMC to HMG1 purified from calf thymus. The statistical significance of the result is P < 0.0001 in an ANOVA model. (B) Comparison of the chemotactic effect of HMG1 proteins, either purified from calf thymus or expressed in yeast, with those of the well-characterized chemoattractants fMLP and bFGF. All treatments increase the migratory response relative to the control (P < 0.0001 in Student's t test). (C) Effect of anti–HMG1 antibodies on fMLP- and HMG1-induced migration. *Treatments where the migratory response was statistically different from the control beyond the P = 0.0001 limit in Student's t test. Treatment of RSMC with the anti–HMG1 antibody alone, or an unspecific antibody, also gave statistically significant results (0.05 < P < 0.01). Treatment with HMG1 plus anti–HMG1 gave results that did not differ statistically from the untreated control. (D) Concentration-dependent migratory response of RSMC to HMG1 expressed in yeast (P. pastoris). The statistical significance of the result is P < 0.0001 in an ANOVA model.
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Figure 1: HMG1 has chemotactic activity on RSMC. Chemotaxis assays were performed using modified Boyden chambers. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). The data represent the mean ± SD (n = 3). (A) Concentration-dependent migratory response of RSMC to HMG1 purified from calf thymus. The statistical significance of the result is P < 0.0001 in an ANOVA model. (B) Comparison of the chemotactic effect of HMG1 proteins, either purified from calf thymus or expressed in yeast, with those of the well-characterized chemoattractants fMLP and bFGF. All treatments increase the migratory response relative to the control (P < 0.0001 in Student's t test). (C) Effect of anti–HMG1 antibodies on fMLP- and HMG1-induced migration. *Treatments where the migratory response was statistically different from the control beyond the P = 0.0001 limit in Student's t test. Treatment of RSMC with the anti–HMG1 antibody alone, or an unspecific antibody, also gave statistically significant results (0.05 < P < 0.01). Treatment with HMG1 plus anti–HMG1 gave results that did not differ statistically from the untreated control. (D) Concentration-dependent migratory response of RSMC to HMG1 expressed in yeast (P. pastoris). The statistical significance of the result is P < 0.0001 in an ANOVA model.

Mentions: HMG1 from calf thymus stimulated migration of RSMC in a concentration-dependent manner, starting at doses as low as 0.1 ng/ml and with a 2.5-fold maximal response at 100 ng/ml (Fig. 1 A). The effect of HMG1 was comparable in amplitude to the effects of the well-characterized attractants fMLP and bFGF (Baggiolini et al. 1994; van Leeuwen 1996; Degryse et al. 1999) (Fig. 1 B, and results not shown). Polyclonal antibodies against HMG1, but not nonspecific control antibodies, totally blocked the migratory response (Fig. 1 C), showing that this was specifically due to HMG1. These antibodies failed to alter the effect of the chemoattractant peptide fMLP used as positive control, and they affected cell migration only marginally. Similar results were obtained with recombinant HMG1 produced in yeast and E. coli (Fig. 1 D, and results not shown), and anti–HMG1 antibodies abolished the effect of recombinant HMG1 as well (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 has chemotactic activity on RSMC. Chemotaxis assays were performed using modified Boyden chambers. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). The data represent the mean ± SD (n = 3). (A) Concentration-dependent migratory response of RSMC to HMG1 purified from calf thymus. The statistical significance of the result is P < 0.0001 in an ANOVA model. (B) Comparison of the chemotactic effect of HMG1 proteins, either purified from calf thymus or expressed in yeast, with those of the well-characterized chemoattractants fMLP and bFGF. All treatments increase the migratory response relative to the control (P < 0.0001 in Student's t test). (C) Effect of anti–HMG1 antibodies on fMLP- and HMG1-induced migration. *Treatments where the migratory response was statistically different from the control beyond the P = 0.0001 limit in Student's t test. Treatment of RSMC with the anti–HMG1 antibody alone, or an unspecific antibody, also gave statistically significant results (0.05 < P < 0.01). Treatment with HMG1 plus anti–HMG1 gave results that did not differ statistically from the untreated control. (D) Concentration-dependent migratory response of RSMC to HMG1 expressed in yeast (P. pastoris). The statistical significance of the result is P < 0.0001 in an ANOVA model.
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Related In: Results  -  Collection

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Figure 1: HMG1 has chemotactic activity on RSMC. Chemotaxis assays were performed using modified Boyden chambers. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). The data represent the mean ± SD (n = 3). (A) Concentration-dependent migratory response of RSMC to HMG1 purified from calf thymus. The statistical significance of the result is P < 0.0001 in an ANOVA model. (B) Comparison of the chemotactic effect of HMG1 proteins, either purified from calf thymus or expressed in yeast, with those of the well-characterized chemoattractants fMLP and bFGF. All treatments increase the migratory response relative to the control (P < 0.0001 in Student's t test). (C) Effect of anti–HMG1 antibodies on fMLP- and HMG1-induced migration. *Treatments where the migratory response was statistically different from the control beyond the P = 0.0001 limit in Student's t test. Treatment of RSMC with the anti–HMG1 antibody alone, or an unspecific antibody, also gave statistically significant results (0.05 < P < 0.01). Treatment with HMG1 plus anti–HMG1 gave results that did not differ statistically from the untreated control. (D) Concentration-dependent migratory response of RSMC to HMG1 expressed in yeast (P. pastoris). The statistical significance of the result is P < 0.0001 in an ANOVA model.
Mentions: HMG1 from calf thymus stimulated migration of RSMC in a concentration-dependent manner, starting at doses as low as 0.1 ng/ml and with a 2.5-fold maximal response at 100 ng/ml (Fig. 1 A). The effect of HMG1 was comparable in amplitude to the effects of the well-characterized attractants fMLP and bFGF (Baggiolini et al. 1994; van Leeuwen 1996; Degryse et al. 1999) (Fig. 1 B, and results not shown). Polyclonal antibodies against HMG1, but not nonspecific control antibodies, totally blocked the migratory response (Fig. 1 C), showing that this was specifically due to HMG1. These antibodies failed to alter the effect of the chemoattractant peptide fMLP used as positive control, and they affected cell migration only marginally. Similar results were obtained with recombinant HMG1 produced in yeast and E. coli (Fig. 1 D, and results not shown), and anti–HMG1 antibodies abolished the effect of recombinant HMG1 as well (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