Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH 2 -terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.The thrombospondin (TSP) 1 family of proteins includes at least five related extracellular matrix glycoproteins encoded by separate genes (1). Each thrombospondin contains NH 2 -and COOH-terminal globular domains flanking different structural modules ( Ref. 2; Fig. 1A). The modules common to all TSP proteins include epidermal growth factor-like type 2 and Ca 2ϩ -binding type 3 repeats. In addition to these common features, TSP1 and TSP2 share two additional modules: a procollagen homology region and three properdin-like type 1 repeats (TSR).The NH 2 and COOH termini contain regions that are low in cysteine content, do not possess internal repeating motifs, and do not show homology with other proteins (3). The aminoterminal domain is largely responsible for the heparin binding properties of thrombospondins (4, 5). The procollagen region contains a region homologous to a cysteine-rich domain in the NH 2 -terminal propeptide of the ␣ 1 -chain of type 1 procollagen (6). The type 1 repeats are homologous to sequences in a variety of proteins found in protozoans (7-9), invertebrates (10), and mammals (11-16).Thrombospondin 1 has been the most extensively studied TSP protein to date. TSP1 contains a growing number of sites that have been implicated in interactions with more than 30 cell surface and matrix proteins, including structural proteins (e.g. collagen and fibronectin), cell surface receptors (e.g. integrins, syndecans, and CD36), enzymes (e.g. elastase and plasmin), and cytokines (e.g. transforming growth factor-1) (17). Because of its ability to interact with such a wide variety of proteins, TSP1 has been implicat...
Acrolein is a respiratory irritant that can be generated during cooking and is in environmental tobacco smoke. More plentiful in cigarette smoke than polycyclic aromatic hydrocarbons (PAH), acrolein can adduct tumor suppressor p53 (TP53) DNA and may contribute to TP53-mutations in lung cancer. Acrolein is also generated endogenously at sites of injury, and excessive breath levels (sufficient to activate metalloproteinases and increase mucin transcripts) have been detected in asthma and chronic obstructive pulmonary disease (COPD). Because of its reactivity with respiratory-lining fluid or cellular macromolecules, acrolein alters gene regulation, inflammation, mucociliary transport, and alveolar-capillary barrier integrity. In laboratory animals, acute exposures have lead to acute lung injury and pulmonary edema similar to that produced by smoke inhalation whereas lower concentrations have produced bronchial hyperreactivity, excessive mucus production, and alveolar enlargement. Susceptibility to acrolein exposure is associated with differential regulation of cell surface receptor, transcription factor, and ubiquitin-proteasome genes. Consequent to its pathophysiological impact, acrolein contributes to the morbidly and mortality associated with acute lung injury and COPD, and possibly asthma and lung cancer.
We have previously shown that reductions in c-Myb-dependent transcription inhibit cell cycle progression and decrease intracellular Ca2+ concentrations in vascular smooth muscle cells (VSMC). We now report that these effects are largely mediated by a 4- to 10-fold increased rate of La(3+)-sensitive 45Ca extrusion, which is associated with 2- to 4-fold increased levels of plasma membrane Ca(2+)-ATPase 1 (PMCA1) mRNA and protein. PMCA4 mRNA, present at much lower concentrations, undergoes similar changes during suppression of c-Myb activity. We also report that PMCA1 expression is regulated during VSMC cell cycle progression, such that levels of PMCA1 are 40% lower at the G1/S interface than at G0. Moreover, transient overexpression of PMCA1a in VSMC elevates the 45Ca efflux rate by approximately 2-fold, decreases resting and peak thapsigargin-releasable Ca2+ concentrations at G1/S by 43% (68 nM) and 52% (160 nM), respectively, and reduces the rate of cell proliferation by over 2.5-fold. These data define a mechanism for c-Myb-dependent Ca2+ homeostasis and support a critical role for PMCA in the regulation of VSMC growth.
Considerable controversy surrounds the role of the c-myb proto-oncogene in vascular smooth muscle cells (VSMCs). Previous investigations using antisense approaches have suggested a relationship between c-myb expression, cell cycle progression, and cytoplasmic Ca2+ concentration ([Ca2+]cyt). However, the ability of certain antisense oligonucleotides to bind and inactivate growth factors allows alternative explanations. To define more specifically the role of c-Myb in cultured VSMCs (SVE and A10 cell lines), we have generated stable cell clones expressing a dominant-negative c-Myb lacking critical elements of the DNA binding domain (delta5-SVE) and transiently transfected cell populations (GRE-MEn-SVE and GRE-MEn-A10) expressing a glucocorticoid-inducible chimeric protein that targets the Drosophila Engrailed repressor domain to c-Myb-responsive promoters. The delta5-SVE clones and GRE-MEn cell populations exhibit a 60% reduction in mean intracellular c-Myb activity, as measured by cotransfection assays with a c-Myb-responsive reporter, a 42% decrease in the mean S phase entry of growth-arrested (G[0]) cells after serum stimulation, and a 36% inhibition of mean cell proliferation over 4 days. These cells also display 28% (34-nmol/L) and 30% (42-nmol/L) reductions in mean [Ca2+]cyt at G(0) and at the G1/S interface, respectively, as well as significant reductions in the peak [Ca2+]cyt responses to thapsigargin (5 micromol/L) and caffeine (10 mmol/L). These latter reductions in operationally defined Ca2+ pools were observed both at different stages of the cell cycle and after transient induction of the dominant-interfering construct, suggesting that c-Myb regulates these releasable Ca2+ stores independent of its effects on cell cycle progression.
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