Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

Zhenan Liu, Audrey N. Chang, Frederick Grinnell, Kathleen M. Trybus, Dianna M. Milewicz, James T. Stull, Kristine E. Kamm, Edward D. Korn

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2 Citations (Scopus)

Abstract

The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.

Original languageEnglish (US)
Pages (from-to)E5569-E5578
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number28
DOIs
StatePublished - Jul 11 2017

Fingerprint

Vascular Diseases
Smooth Muscle
Actins
Fibroblasts
Mutation
Thoracic Aortic Aneurysm
Dissection
Moyamoya Disease
Adventitia
Stress Fibers
Skin
Focal Adhesions
Telomerase
Missense Mutation
Vascular Smooth Muscle
Adenoviridae
Smooth Muscle Myocytes
Cell Movement
Transcription Factors
Western Blotting

Keywords

  • ACTA2 mutation
  • Cell migration
  • Human fibroblasts
  • Thoracic aortic aneurysms
  • Vascular disease

ASJC Scopus subject areas

  • General

Cite this

@article{f7f5986bc4904430a67f47c3b7c4e320,
title = "Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts",
abstract = "The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.",
keywords = "ACTA2 mutation, Cell migration, Human fibroblasts, Thoracic aortic aneurysms, Vascular disease",
author = "Zhenan Liu and Chang, {Audrey N.} and Frederick Grinnell and Trybus, {Kathleen M.} and Milewicz, {Dianna M.} and Stull, {James T.} and Kamm, {Kristine E.} and Korn, {Edward D.}",
year = "2017",
month = "7",
day = "11",
doi = "10.1073/pnas.1703506114",
language = "English (US)",
volume = "114",
pages = "E5569--E5578",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
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TY - JOUR

T1 - Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts

AU - Liu, Zhenan

AU - Chang, Audrey N.

AU - Grinnell, Frederick

AU - Trybus, Kathleen M.

AU - Milewicz, Dianna M.

AU - Stull, James T.

AU - Kamm, Kristine E.

AU - Korn, Edward D.

PY - 2017/7/11

Y1 - 2017/7/11

N2 - The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.

AB - The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2. We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2. Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.

KW - ACTA2 mutation

KW - Cell migration

KW - Human fibroblasts

KW - Thoracic aortic aneurysms

KW - Vascular disease

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U2 - 10.1073/pnas.1703506114

DO - 10.1073/pnas.1703506114

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JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

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