The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-α–induced transcriptional repression of cyclin A
Raj Kishore, … , David Goukassain, Douglas W. Losordo
Raj Kishore, … , David Goukassain, Douglas W. Losordo
Published July 1, 2005
Citation Information: J Clin Invest. 2005;115(7):1785-1796. https://doi.org/10.1172/JCI22849.
View: Text | PDF | Corrigendum | Erratum
Research Article Vascular biology

The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-α–induced transcriptional repression of cyclin A

Abstract

TNF-α modulates EC proliferation and thereby plays a central role in new blood vessel formation in physiologic and pathologic circumstances. TNF-α is known to downregulate cyclin A, a key cell cycle regulatory protein, but little else is known about how TNF-α modulates EC cell cycle and angiogenesis. Using primary ECs, we show that ezrin, previously considered to act primarily as a cytoskeletal protein and in cytoplasmic signaling, is a TNF-α–induced transcriptional repressor. TNF-α exposure leads to Rho kinase–mediated phosphorylation of ezrin, which translocates to the nucleus and binds to cell cycle homology region repressor elements within the cyclin A promoter. Overexpression of dominant-negative ezrin blocks TNF-α–induced modulation of ezrin function and rescues cyclin A expression and EC proliferation. In vivo, blockade of ezrin leads to enhanced transplanted EC proliferation and angiogenesis in a mouse hind limb ischemia model. These observations suggest that TNF-α regulates angiogenesis via Rho kinase induction of a transcriptional repressor function of the cytoskeletal protein ezrin and that ezrin may represent a suitable therapeutic target for processes dependent on EC proliferation.

Authors

Raj Kishore, Gangjian Qin, Corinne Luedemann, Evelyn Bord, Allison Hanley, Marcy Silver, Mary Gavin, David Goukassain, Douglas W. Losordo

×

Figure 1

Options: View larger image (or click on image) Download as PowerPoint
Ezrin is a TNF-α–inducible cyclin A CHR–binding protein. (A) Nuclear ext...
Ezrin is a TNF-α–inducible cyclin A CHR–binding protein. (A) Nuclear extracts from serum-stimulated (S) and serum-stimulated and TNF-α–treated (T) BAECs were incubated with biotinylated WT or mutant CHR oligonucleotides. Protein-DNA complexes were captured on avidin-agarose columns. Eluted proteins from columns were analyzed for ezrin protein expression in Western blots. (B) Nuclear extracts from serum-treated or TNF-α–treated cells were immunodepleted (ID) of ezrin by anti-ezrin antibodies. Depleted extracts alone or depleted extracts from TNF-α–treated cells reconstituted with recombinant-ezrin protein (GST-ezrin) or control DP1 protein (GST-DP1) were then analyzed for CHR-binding activity. ×100, 100-fold molar excess of competing unlabeled oligonucleotide. (C) GST-ezrin or GST-DP1 protein (1 μg) was incubated with radiolabeled oligonucleotides spanning CHR-binding elements in EMSA experiments. For competition experiments, the indicated molar concentration of unlabeled oligonucleotides was added to the reaction mix before the addition of radiolabeled probe. Autoradiographs shown are representative of at least 4 similar experiments.