The cytoskeletal protein ezrin regulates EC proliferation and angiogenesis via TNF-α–induced transcriptional repression of cyclin A
Raj Kishore, Gangjian Qin, Corinne Luedemann, Evelyn Bord, Allison Hanley, Marcy Silver, Mary Gavin, David Goukassain, Douglas W. Losordo
Raj Kishore, Gangjian Qin, Corinne Luedemann, Evelyn Bord, Allison Hanley, Marcy Silver, Mary Gavin, David Goukassain, Douglas W. Losordo
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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

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Ezrin mediates TNF-α–induced inhibition of EC proliferation. (A–F) Overe...
Ezrin mediates TNF-α–induced inhibition of EC proliferation. (A–F) Overexpression of WT or DN ezrin does not affect actin cytoskeleton. ECs either untransfected (A) or transfected with WT or DN ezrin (C and E, respectively) and treated (B, D, and F) or not treated with 20 ng/ml of TNF-α were stained with rhodamine-phalloidin for the detection of actin filaments, using the protocol described elsewhere (59). (G) Quiescent BAECs transiently transfected with empty vector (EV), WT ezrin, or DN ezrin were cultured in serum (white bars) or serum containing 20 ng/ml of TNF-α (black bars) for 24 hours. Cells were labeled with BrdU (50 μg/ml) for the last 6 hours of culture. Cells were fixed and stained with anti-BrdU antibodies. BrdU-positive cells were counted in at least 6 different visual fields. Average data from 3 similar experiments are shown. *P < 0.01; **P < 0.001.