© 1995 American Heart Association, Inc.
Articles |
Porcine Aortic Smooth Muscle Cells Secrete a Serine Protease for Insulin-like Growth Factor Binding Protein-2
From the Department of Medicine, University of North Carolina School of Medicine, Chapel Hill.
Correspondence to David R. Clemmons, MD, Department of Medicine CB #7170, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7170.
Abstract Porcine aortic smooth muscle cells secrete two forms
of insulin-like growth factor (IGF) binding proteins (IGFBP-2 and -4),
and both forms have been shown to modulate IGF-I actions in this cell
type. Recently, we showed that IGFBP-4 inhibited IGF-I action and that
the cells produced a protease that cleaved IGFBP-4 into non-IGF binding
fragments. After the cleavage of IGFBP-4, the cellular DNA synthesis
response to IGF-I was enhanced. This study reports that these cells
also secrete a protease for IGFBP-2. Like the IGFBP-4 protease, this
protease is also secreted constitutively, but unlike the IGFBP-4
protease, its secretion is enhanced if the cells are serum-deprived for
24 hours before the collection of conditioned medium. The protease
cleaved IGFBP-2 into 25- and 16-kD fragments, which had reduced IGF-I
binding activity. Protease activity was enhanced by coincubation with
IGF-I or IGF-II, and IGF-II was more potent than IGF-I. The protease is
a serine protease, since its activity can be inhibited by
3,4-dichloroisocoumarin and aprotinin. It is also inhibited by EDTA,
and its activity can be restored with calcium but not zinc. The
heparin-binding serpins, specifically, heparin cofactor II and
antithrombin III, are inhibitory. Heparin alone also had
activity, and the combination of antithrombin III plus heparin
caused complete inhibition. The conditioned medium also contained
proteolytic activities for IGFBP-4 and -5 but it did not cleave IGFBP-1
and -3. Chromatography of the conditioned medium on heparin-sepharose
indicated that the IGFBP-2 protease bound very weakly to heparin, since
it was eluted with 0.2 mol/L NaCl. This contrasts with the IGFBP-5
protease activity, which required 2.0 mol/L NaCl to elute most of the
activity. 
1-Antichymotrypsin inhibited the IGFBP-2
protease, but it had no effect on the IGFBP-5 protease. Exposure to
other growth factors such as transforming growth factor-ß, fibroblast
growth factor, or platelet-derived growth factor did not alter protease
activity. In summary, porcine aortic smooth muscle cells secrete a
serine protease for IGFBP-2. This protease cleaves IGFBP-2 into two
fragments that have reduced IGF binding, and its activity is enhanced
by prior serum deprivation of the cells. The protease has the potential
to significantly modify IGF actions in this cell type.
Key Words: insulin-like growth factor I • atherosclerosis • proteolysis • somatomedin
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