Treatment of RSMC with the antiCHMG1 antibody alone, or an unspecific antibody, also gave statistically significant results (0.05 0.01). 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 Gi/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. IP1 Thus, HMG1 has all the hallmarks of a molecule that can promote atherosclerosis and restenosis after Fruquintinib vascular damage. toxin and its mutant were provided by Dr. M.G. Pizza (I.R.I.S., Siena, Italy) (Fazioli et al. 1997). AntiCRAGE antibody was Fruquintinib a gift of Dr. A.M. Schmidt (Columbia University, New York, NY) (Taguchi et al. 2000). HMG1 purified from calf thymus was a gift of Jordi Bernus (C.S.I.C., Barcelona, Spain). Collagen I and fibronectin were purchased from Roche. The polyclonal rabbit antiCHMG1 was purchased from BD PharMingen. The monoclonal mouse antiphosphorylated MAP kinases (ERK 1 and 2) was from New England Biolabs, Inc. Fluorescein-conjugated F(ab)2 fragments of antiCrabbit immunoglobulins and fluorescein-conjugated F(ab)2 fragments of antiCmouse immunoglobulins were from Chemicon. Nonspecific rabbit polyclonal immunoglobulins, nonspecific monoclonal mouse IgG1 (MOPC-21), TRITC-conjugated phalloidin, and fMLP (formyl-methionine-leucine-proline) were from Sigma-Aldrich. Expression and Purification of HMG1 and Derivatives HMG1/M1-176 is usually a truncated form of HMG1 that lacks the COOH-terminal acidic domain name of the intact HMG1 molecule; for the sake of simplicity, it will be called Box A+B. The plasmids pRNHMG1/M1-V176, pT7HMG1bA, and pT7HMG1bB coding for Box A+B, Box A, and Box B, respectively, have been previously described, as well as the protocols for expression and purification of the single and double boxes (Bianchi et al. 1992). The plasmid pT7-7-rHMG1cm used for the expression of full-length HMG1 in bacteria was a kind gift of Prof. J.O. Thomas (Cambridge University, Cambridge, UK). Expression and purification of full-length HMG1 was performed in BL21(? ) strain following the protocol of Studier and Moffatt 1986, with slight modifications: transformed bacteria was grown in M9 medium supplemented with Cas-aminoacids 20 g/liter, glycerol 0.5%, yeast extract 5 g/liter, glucose 0.4%. Chloramphenicol 100 g/ml was used as selective agent. Temperature was shifted from 37 to 23C during the 16-h induction period. The procedure for the expression and purification of full-length HMG1 in yeast (test for pairwise comparisons of treatments, or an ANOVA model for the evaluation of treatments with increasing doses of a reagent. Results HMG1 Induces RMSC Migration The chemotactic effect of HMG1 was decided with a chemotaxis assay using modified Boyden chambers. We tested several preparations Fruquintinib of HMG1: HMG1 purified from calf thymus, recombinant HMG1 expressed from (Mistry et al. 1997). 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 (Fig. 1 D, and results not shown), and antiCHMG1 antibodies abolished the effect of recombinant HMG1 as well (not shown). Open in a separate window Physique 1 HMG1 has chemotactic activity on RSMC. Chemotaxis.