ABSTRACT
Vascular smooth muscle cell (VSMC) migration is an important process in the development of vascular occlusive disease. To investigate mitogen regulation of VSMC migration, a cell-layer-scrape assay was used to measure migration 20 h after stimulation of VSMC with platelet-derived growth factor-BB (PDGF-BB), insulin-like growth factor I (IGF-I), or phorbol 12-myristate 13-acetate (PMA). The contributions of cell proliferation were eliminated by treatment of VSMC with hydroxyurea, which suppressed DNA synthesis.PDGF-BB stimulated VSMC migration 2.5-fold, while PMA and IGF-I stimulated migration 1.7- and 1.5-fold, respectively. The importance of protein kinase C (PKC), ERK, and phosphoinositide-3' kinase (PI3 kinase) in mitogen-stimulated migration was investigated, using specific inhibitors of these signaling molecules. PDGF-BB-stimulated migration was inhibited by the general PKC inhibitor RO 31-8220 (40%), the MEK inhibitor PD98059 (31%), and the PI3 kinase inhibitor wortmannin (22%) but not by PMA-induced downregulation of conventional and novel PKC isoforms. IGF-I-stimulated migration was inhibited by RO 31-8220 (34%) and wortmannin (37%) but was much less affected by PD98059 (19%) or PKC downregulation (10%). PMA-stimulated migration was inhibited by RO 31-8220 (53%), PD98059 (50%), wortmannin (45%), and PKC downregulation (47%). Western analysis confirmed that ERK was strongly activated by PDGF-BB and PMA but not by IGF-I. To examine potential in vivo negative regulators of VSMC migration, we analyzed the ability of heparin, an analogue of heparan sulfate, and TGFbeta to attenuate mitogen-stimulated migration. Heparin but not TGFbeta inhibited VSMC migration stimulated by all three mitogens. Delayed-addition experiments showed that RO 31-8220 retained substantial inhibitory activity even if added 3 h after PMA or IGF-I stimulation and 5 h after PDGF-BB addition, suggesting that sustained PKC activation is important for migration. The MEK inhibitor retained some effectiveness for 5 h after PDGF-BB stimulation but only 1 h after PMA addition. Western analysis showed ERK activation was transient after PMA treatment but sustained for 6 h after PDGF-BB treatment. Heparin strongly inhibited migration even if added 5-7 h after mitogen stimulation, suggesting that heparin may inhibit both short- and long-term signals necessary for migration. The present studies indicate that PMA and IGF-I activate a limited number of second messengers resulting in moderate stimulation of migration; in contrast PDGF-BB stimulates multiple signaling pathways resulting in strong stimulation of migration and lessened sensitivity to inhibitory signals.
Subject(s)
Anticoagulants/pharmacology , Cell Movement/drug effects , Insulin-Like Growth Factor I/pharmacology , Mitogen-Activated Protein Kinase Kinases , Muscle, Smooth, Vascular/cytology , Platelet-Derived Growth Factor/pharmacology , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Animals , Becaplermin , Cell Movement/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Growth Substances/pharmacology , Heparin/pharmacology , MAP Kinase Kinase 1 , Muscle, Smooth, Vascular/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-sis , Rats , Signal Transduction/physiology , Time Factors , Transforming Growth Factor beta/pharmacologyABSTRACT
The cloning and analysis of a cDNA clone encoding the soybean metalloproteinase obtained by polymerase chain reaction (PCR) and the rapid amplification of cDNA ends (RACE) reaction are described. The cDNA was constructed from poly(A)+ RNA isolated from 15-17 day old leaves. The deduced amino acid sequence of the cDNA reveals that the plant metalloproteinase is synthesized as a preproenzyme and the proenzyme form shares a structural motif, responsible for maintenance of inactive zymogen, with the matrix metalloproteinase (e.g. collagenase) family of enzymes from vertebrate origin. Northern and Western blot analysis demonstrated that the metalloproteinase transcript and protein are under a strict developmental program in that both are expressed only in leaf tissue and in a temporal fashion. The physiological function of the metalloproteinase still remains unclear although the data suggest that the enzyme is extracellular and a portion of the mature form of the enzyme is tightly bound to the cell wall.
Subject(s)
Glycine max/enzymology , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/chemistry , Amino Acid Sequence , Base Sequence , DNA, Complementary , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Humans , Metalloendopeptidases/isolation & purification , Molecular Sequence Data , Plant Leaves , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Random Amplified Polymorphic DNA Technique , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
A polypeptide present in intercellular wash fluids of young leaves of Glycine max has been purified to electrophoretic homogeneity. The protein has been identified as gamma-glutamyl hydrolase (GGH) based on the shared homology with a recently cloned cDNA from rat. The enzyme is present within the extracellular space of young leaves and a portion is bound to the cell wall. Northern and Western analysis confirm that this polypeptide is expressed only in young (1-15 d old) leaf, stem and root tissue and is therefore expressed under a strict developmental program. The primary sequence of gamma-glutamyl hydrolase shares amino acid identity with a cDNA clone from rat and two partially sequenced cDNAs from Arabidopsis. Although the complete in vivo function of gamma-glutamyl hydrolase in plants is unclear, it is known that the protein plays a critical role in folate metabolism and therefore likely in meeting the physiological demands of growing plant tissues.
Subject(s)
Glycine max/enzymology , Plant Leaves/enzymology , gamma-Glutamyl Hydrolase/isolation & purification , Blotting, Western , Cell Wall/enzymology , Cross Reactions , DNA, Complementary/genetics , Gene Expression , Metalloendopeptidases/immunology , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/genetics , RNA, Plant/metabolism , gamma-Glutamyl Hydrolase/chemistry , gamma-Glutamyl Hydrolase/genetics , gamma-Glutamyl Hydrolase/metabolismABSTRACT
A proteinase present in intercellular wash fluids from leaves of Glycine max has been purified 600-fold to electrophoretic homogeneity. The native protein is monomeric with a molecular mass of 60 kD, as estimated by denaturing gel electrophoresis, and has an isoelectric point of 7.7. The enzyme has a pH optimum of 9.5 when assayed with Azocoll as a substrate. The proteolytic activity is inhibited by p-chloromercuribenzoic acid and mercuric chloride and requires the presence of reducing agents. The enzyme activity is refractory to other classical sulfhydryl proteinases. The soybean leaf endoproteinase is present within the extracellular space of young leaves, and a portion is bound to the cell wall. Western blot analysis and activity measurements show that the enzyme is present only during the first 15 d postemergence of the leaf and is therefore under strict developmental control. We suggest that the enzyme may play a critical role in the extracellular milieu during rapid cell growth and leaf expansion.
