Coagulation factor IX (F9) is an essential molecule for coagulation. Mutations of F9 cause hemophilia B,
which leads to abnormal bleeding. In the process of coagulation, F9 is activated by cleavages between
the light and heavy chains that release an activating peptide. The light and heavy chains are essential for
coagulation. The mechanism of action of this activating peptide is not well known.
Materials and Methods:
431 cells derived from human squamous cell carcinoma were treated with recombinant F9 or its mutants.
Migration of the cells was evaluated using a wound healing assay. For morphological analysis, cells
transfected with vinculin-GFP and actin-RFP were observed using time-lapse recording. The cell
adhesion force to the matrix was evaluated by counting the number of detached cells after shaking the
Activated F9 increased cell migration speed by 73% in a wound healing assay; in contrast, native F9
decreased migration speed by 20%. Experiments with F9 deletion mutants revealed that both an EGF
domain in the light chain (EGF-F9) and an activating peptide were responsible for the increases and
decreases in migration speed, respectively. Evaluation of the adhesion force revealed that recombinant
EGF-F9 weakened cell adhesion to the matrix, whereas recombinant activation peptide suppressed the
action of recombinant EGF-F9. Time-lapse recording showed that cells treated with recombinant EGF-F9
migrated leaving adhesion components back, suggesting that EGF-F9 could not elicit cooperation of the
adhesion apparatus. Enhanced de-adhesion at the tailing end of the migrating cell was observed in the
presence of EGF-F9, suggesting that increased migration speed arises from a weakened adhesion force.
To prove this hypothesis, a recombinant EGF-F9 was injected into one side of the cells. This injection
resulted in de-adhesion of the tailing end of cells and directional cell migration in the opposite direction.
Activated F9 accelerated the detachment at the cell tail and increased migration speed, whereas F9
fragments of the activation peptide suppressed migration speed. Our data suggest both that active F9
encourages cell migration in wounds and that native F9 regulates cell migration under normal conditions.
Nicotine induces invadosome formation and cell invasion in A7r5 and primary
human vascular smooth muscle cells
Molecular Pharmacology, Physiology & Biotechnology, Brown University, Providence, RI
Vascular smooth muscle cell invasion from media to intima participates in plaque formation in
atherosclerosis. Cigarette smoking is known to increase the risk of developing atherosclerosis, but the
mechanistic connection between nicotine exposure and vascular smooth muscle cell invasion is not clear.
Invadosomes—invadopodia, podosomes, podosome rosettes—are adhesive/invasive organelles that
enable cell invasion through extracellular matrix. We tested the hypothesis that nicotine induces
invadosome formation and cell invasion in A7r5 and primary human vascular smooth muscle cells. We
found that prolonged (6 hr) nicotine treatment of A7r5 vascular smooth muscle cells enabled the cells to
form podosome rosettes in response to PKC activation, accompanied by global extracellular matrix
degradation and internalization. In contrast, PKC activation without nicotine treatment induced the
formation of podosomes in A7r5 cells, accompanied by focal extracellular matrix degradation. Nicotinic
acetylcholine receptors colocalized with other podosome markers (vinculin, PKC-alpha, and
metalloproteinase-2) at podosomes and podosome rosettes in A7r5 cells. Matrigel-coated transwell
experiments indicated that nicotine treatment and PKC activation synergistically enhanced invasiveness
of A7r5 vascular smooth muscle cells. Inclusion of alpha-bungarotoxin (nicotinic acetylcholine receptor
antagonist) or cycloheximide (protein synthesis inhibitor) during nicotine treatment abolished nicotine-
induced podosome rosette formation in A7r5 vascular smooth muscle cells. Nicotine-treated primary
human aortic vascular smooth muscle cells also formed podosome rosettes in response to PKC
activation. Furthermore, nicotine treatment and PKC activation synergistically enhance invasiveness of
primary human aortic vascular smooth muscle cells. Altogether, data acquired from A7r5 and primary
human vascular smooth muscle cells are consistent in suggesting that nicotine enhances vascular
smooth muscle cell invasion by activating synergistic mechanisms between nicotinic acetylcholine
receptor and PKC signaling that result in invadosome formation and new protein synthesis. A potential
clinical implication of these findings is that replacing cigarette smoking by nicotine administration may