Inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-beta receptors

J Biol Chem. 1995 Sep 8;270(36):21144-50. doi: 10.1074/jbc.270.36.21144.

Abstract

Transforming growth factor-beta 1 (TGF-beta 1) induces angiogenesis in vivo and capillary morphogenesis in vitro. Two receptor serine/threonine kinases (types I and II) have been identified as signal transducing TGF-beta receptors. We explored the possibility of inhibiting TGF-beta-mediated events in glomerular capillary endothelial cells using a TGF-beta type II receptor (T beta R-II) transdominant negative mutant. A mutant TGF-beta type II receptor (T beta R-IIM), lacking the cytoplasmic serine/threonine kinase domain, was produced by polymerase chain reaction using rat T beta R-II cDNA as template. Since T beta R-II and TGF-beta type I receptor (T beta R-I) heterodimerize for signal transduction, the mutant receptor competes for binding to wild-type T beta R-I, hence acting in a dominant negative fashion. Glomerular capillary endothelial cells were stably transfected with T beta R-IIM, and four independent clones were expanded. That the T beta R-IIM mRNA was expressed was shown by reverse transcriptase-polymerase chain reaction, RNase protection assay, and Northern analysis. Presence of cell surface T beta R-IIM protein was shown by affinity cross-linking with 125I-TGF-beta 1. In wild-type endothelial cells, TGF-beta 1 (2 ng/ml) significantly inhibited [3H]thymidine incorporation to 63 +/- 10% of control (n = 4). In transfected endothelial cells carrying T beta R-IIM, TGF-beta 1 stimulated [3H]thymidine incorporation to 131 +/- 9% of control (n = 4, p < 0.005). Also, in wild-type endothelial cells, endogenous and exogenous TGF-beta 1 induced apoptosis and associated capillary formation. Both apoptosis and capillary formation were uniformly and entirely absent in transfected endothelial cells carrying T beta R-IIM. This represents the first demonstration that capillary morphogenesis in vitro is associated with apoptosis, and that interference with T beta R-II signaling inhibits this process in glomerular capillary endothelial cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies / pharmacology
  • Apoptosis* / drug effects
  • Base Sequence
  • Blood
  • Capillaries / cytology
  • Capillaries / growth & development*
  • Capillaries / metabolism
  • Cattle
  • Cell Membrane / metabolism
  • Cells, Cultured
  • DNA Primers
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / growth & development*
  • Endothelium, Vascular / metabolism
  • Genes, Dominant
  • Molecular Sequence Data
  • Morphogenesis
  • RNA, Messenger / genetics
  • Receptors, Transforming Growth Factor beta / genetics
  • Receptors, Transforming Growth Factor beta / physiology*
  • Thymidine / metabolism
  • Transforming Growth Factor beta / immunology
  • Transforming Growth Factor beta / pharmacology

Substances

  • Antibodies
  • DNA Primers
  • RNA, Messenger
  • Receptors, Transforming Growth Factor beta
  • Transforming Growth Factor beta
  • Thymidine