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. 2001 Mar 19;152(6):1197-206.
doi: 10.1083/jcb.152.6.1197.

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

B Degryse  1 , T Bonaldi, P Scaffidi, S Müller, M Resnati, F Sanvito, G Arrigoni, M E Bianchi
Affiliations

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

B Degryse et al. J Cell Biol. .

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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Figures

Figure 1
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.
Figure 2
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.
Figure 4
Figure 4
Effects of HMG1 and its HMG boxes on RSMC migration into a wound. Monolayers were wounded and allowed to recover for 48 h in the presence of the indicated molecules. Then, RSMC that had migrated into the wound were counted as described in Materials and Methods. 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 = 5). Statistical significance was 0.05 < P < 0.01 for treatment with bFGF and full-length E. coli–made HMG1, 0.01 < P < 0.001 for treatment with Box A and Box B, and 0.001 < P < 0.0001 for treatment with calf thymus HMG1.
Figure 3
Figure 3
Chemotactic response of RSMC to the HMG box domains of HMG1. (A) Concentration-dependent response to Box A and Box B, both expressed in E. coli. Random cell migration is referred to as 100% migration. The data represent the mean ± SD (n = 3). The statistical significance of the result is P < 0.0001 in an ANOVA model, for both Box A and Box B. (B) Effects of full-length HMG1 expressed in E. coli (100 ng/ml), Box A+B (100 ng/ml), Box A (10 ng/ml), or Box B (10 ng/ml) on actin cytoskeleton organization. Cells were incubated with the indicated molecule for 30 min. Actin filaments were visualized using TRITC-phalloidin.
Figure 5
Figure 5
HMG1 binds to the surface of RSMC and stimulates cell motility through RAGE. (A) Large amounts of HMG1 bind to the surface of RSMC. 1 million cells were incubated at 4°C with 800 ng Box A+B and 5 μg BSA. P shows the proteins associated with the cells, after washing, and S shows 20 μl of the medium containing unbound protein. (B) RSMC express RAGE. 1 million cells were lysed on the plate in SDS-PAGE sample buffer, heated for 5 min at 100°C, and then loaded on a 12% tricine gel. RAGE was detected by Western blot with an anti–RAGE antibody. (C) Anti–RAGE antibody inhibits HMG1-induced RSMC migration. 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). Statistical significance was 0.001 < P < 0.0001 for treatment with HMG1 and HMG1 + unspecific antibody. Treatments with HMG1 + anti–RAGE antibody, and HMG1 + anti–HMG1 antibody, had no statistically significant difference from the control. Treatment with anti–HMG1 and anti–RAGE antibodies alone also did not differ from the control.
Figure 6
Figure 6
Pertussis toxin (PT) inhibits HMG1-induced RSMC migration and actin cytoskeleton reorganization. (A) Chemotaxis assays. The data represent the mean ± SD. The value of 100% corresponds to the number of cells migrating in the absence of any stimulator (random cell migration). (B) Cytoskeleton reorganization. RSMC were pretreated for 6 h either with PT or mPT (both at 50 ng/ml), and were then stimulated (at time 0) with either 100 ng/ml HMG1, 10 ng/ml Box A, or 10 ng/ml Box B. The stimulation with HMG1 was carried on for 30 min. Actin filaments were visualized using TRITC-phalloidin.
Figure 7
Figure 7
The MAP kinase pathway is involved in HMG1 signaling. RSMC, pretreated or not for 1 h with 50 μM PD98059, were stimulated for 30 min with 100 ng/ml calf thymus HMG1. Cells were then stained with antibodies against phosphorylated ERK1/2 and DAPI. A separate sample of cells were stained with TRITC-phalloidin to visualize the reorganization of the cytoskeleton.
Figure 8
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.
Figure 9
Figure 9
HMG1 is present in the nuclei of endothelial cells, but not in those of vascular SMC. (A and B). HMG1 is present in the nuclei of endothelial cells, but is not detectable in the nuclei of vascular SMC. Shown is a section of a human pancreatic artery, stained with anti–HMG1 antibody and counterstained with hematoxylin, at low (A) and high (B) magnification. The red frame indicates the location of the area shown in B, and the arrows point to the nuclei of SMC. (C) Western blot analysis showing the expression level of HMG1 in RSMC in comparison to HeLa cells. The indicated number of cells were lysed directly in SDS-PAGE sample buffer and loaded onto the gel. After the detection of HMG1 (which is identical in rat and human), the blot was stripped and subsequently stained with antihistone antibodies to check for loading.
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