A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.
PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.
Department of Internal Medicine, Ulm University Medical Center, Ulm, Germany. johannes.waltenberger@medizin.uni-ulm.de
J
J
Waltenberger
A
A
Uecker
J
J
Kroll
H
H
Frank
U
U
Mayr
J D
JD
Bjorge
D
D
Fujita
A
A
Gazit
V
V
Hombach
A
A
Levitzki
F D
FD
Böhmer
D016475:3T3 Cells; D000818:Animals; D000077214:Becaplermin; D001808:Blood Vessels / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D016228:Endothelial Growth Factors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology; D051379:Mice; D008938:Mitosis / Q000187:drug effects*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects*; D010982:Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D020574:Proto-Oncogene Proteins c-sis; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor / Q000037:antagonists & inhibitors* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D019061:src-Family Kinases / Q000037:antagonists & inhibitors*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D016228:Endothelial Growth Factors; D004791:Enzyme Inhibitors; D008222:Lymphokines; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011720:Pyrazoles; D011743:Pyrimidines; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000077214:Becaplermin; D020794:Receptor Protein-Tyrosine Kinases; D020797:Receptor, Platelet-Derived Growth Factor beta; D017479:Receptors, Platelet-Derived Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases