Transforming growth factor(β)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly

James V. Jester, Jiying Huang, Patricia A. Barry-Lane, Winston W Y Kao, Walter M Petroll, Harrison D Cavanagh

Research output: Contribution to journalArticle

156 Citations (Scopus)

Abstract

Purpose. Recent studies indicate that transforming growth factor (TGF)(β) is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, α-actin (α-SMA). Although TGF(β) is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating α-SMA gene expression and ultimately myofibroblast development. Methods. Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGF(β) (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 μM), Gly-Arg-AL- Asp-Ser-Pro (GRADSP; 100 μM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for α-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit α-SMA. All experiments were repeated at least three times. Results. Keratocytes exposed to TGF(β) showed expression of α-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and α-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGF(β)-associated tyrosine phosphorylation of paxillin, pp125(tak), p130, PLCγ, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGF(β) showed a dose-dependent loss of α-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. Conclusions. The data suggest that TGF(β)-mediated α-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.

Original languageEnglish (US)
Pages (from-to)1959-1967
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume40
Issue number9
StatePublished - Aug 1999

Fingerprint

Myofibroblasts
Transforming Growth Factors
Fibronectins
Actins
Tyrosine
Phosphotyrosine
glycyl-arginyl-alanyl-aspartyl-seryl-proline
Phosphorylation
glycyl-arginyl-glycyl-aspartic acid
Proteins
seryl-proline
Smooth Muscle
Paxillin
Rabbits
Gene Expression
Messenger RNA
Focal Adhesions
Muscle Proteins
Extracellular Matrix Proteins
Serum-Free Culture Media

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Transforming growth factor(β)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly. / Jester, James V.; Huang, Jiying; Barry-Lane, Patricia A.; Kao, Winston W Y; Petroll, Walter M; Cavanagh, Harrison D.

In: Investigative Ophthalmology and Visual Science, Vol. 40, No. 9, 08.1999, p. 1959-1967.

Research output: Contribution to journalArticle

@article{d4e6f50c786c429cb1d8f618f6d19775,
title = "Transforming growth factor(β)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly",
abstract = "Purpose. Recent studies indicate that transforming growth factor (TGF)(β) is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, α-actin (α-SMA). Although TGF(β) is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating α-SMA gene expression and ultimately myofibroblast development. Methods. Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGF(β) (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 μM), Gly-Arg-AL- Asp-Ser-Pro (GRADSP; 100 μM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for α-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit α-SMA. All experiments were repeated at least three times. Results. Keratocytes exposed to TGF(β) showed expression of α-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and α-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGF(β)-associated tyrosine phosphorylation of paxillin, pp125(tak), p130, PLCγ, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGF(β) showed a dose-dependent loss of α-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. Conclusions. The data suggest that TGF(β)-mediated α-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.",
author = "Jester, {James V.} and Jiying Huang and Barry-Lane, {Patricia A.} and Kao, {Winston W Y} and Petroll, {Walter M} and Cavanagh, {Harrison D}",
year = "1999",
month = "8",
language = "English (US)",
volume = "40",
pages = "1959--1967",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "9",

}

TY - JOUR

T1 - Transforming growth factor(β)-mediated corneal myofibroblast differentiation requires actin and fibronectin assembly

AU - Jester, James V.

AU - Huang, Jiying

AU - Barry-Lane, Patricia A.

AU - Kao, Winston W Y

AU - Petroll, Walter M

AU - Cavanagh, Harrison D

PY - 1999/8

Y1 - 1999/8

N2 - Purpose. Recent studies indicate that transforming growth factor (TGF)(β) is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, α-actin (α-SMA). Although TGF(β) is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating α-SMA gene expression and ultimately myofibroblast development. Methods. Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGF(β) (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 μM), Gly-Arg-AL- Asp-Ser-Pro (GRADSP; 100 μM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for α-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit α-SMA. All experiments were repeated at least three times. Results. Keratocytes exposed to TGF(β) showed expression of α-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and α-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGF(β)-associated tyrosine phosphorylation of paxillin, pp125(tak), p130, PLCγ, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGF(β) showed a dose-dependent loss of α-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. Conclusions. The data suggest that TGF(β)-mediated α-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.

AB - Purpose. Recent studies indicate that transforming growth factor (TGF)(β) is a potent inducer of corneal myofibroblast differentiation and expression of smooth muscle-specific, α-actin (α-SMA). Although TGF(β) is known to enhance synthesis of extracellular matrix proteins and receptors, little is known about how it modulates the expression of smooth muscle proteins in nonmuscle cells. The purpose of this study was to identify the role of Arg-Gly-Asp (RGD)-dependent tyrosine phosphorylation in regulating α-SMA gene expression and ultimately myofibroblast development. Methods. Because cell culture in serum-containing media mimics myofibroblast transformation, all experiments were performed on freshly isolated rabbit keratocytes plated in defined, serum-free media. Cells were exposed to TGF(β) (1 ng/ml), Gly-Arg-Gly-Asp-D-Ser-Pro (GRGDdSP, 50 μM), Gly-Arg-AL- Asp-Ser-Pro (GRADSP; 100 μM), or herbimycin A (0.1-10 nM) at 24 hours (sparse) or 7 days (confluent). Cells were evaluated by immunocytochemistry and proteins and RNA collected for western and northern blot analyses using antibodies specific for α-SMA, fibronectin, focal adhesion proteins, and phosphotyrosine (clones 4G10 and PY20); and probes directed against rabbit α-SMA. All experiments were repeated at least three times. Results. Keratocytes exposed to TGF(β) showed expression of α-SMA that coincided with the intracellular reorganization of the actin cytoskeleton and the extracellular assembly of fibronectin fibrils. Addition of RGD containing but not control peptides blocked the organization of intracellular actin, extracellular fibronectin, and α-SMA protein and mRNA. Immunoprecipitation of cell proteins with 4G10 or PY20 identified the TGF(β)-associated tyrosine phosphorylation of paxillin, pp125(tak), p130, PLCγ, and tensin, which was blocked by addition of GRGDdSP. Addition of herbimycin A to keratocytes exposed to TGF(β) showed a dose-dependent loss of α-SMA protein and mRNA which correlated with loss of tyrosine phosphorylation, absence of actin reorganization, and fibronectin assembly. Conclusions. The data suggest that TGF(β)-mediated α-SMA gene expression leading to myofibroblast transformation may involve an RGD-dependent phosphotyrosine signal transduction pathway.

UR - http://www.scopus.com/inward/record.url?scp=0032842569&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032842569&partnerID=8YFLogxK

M3 - Article

C2 - 10440249

AN - SCOPUS:0032842569

VL - 40

SP - 1959

EP - 1967

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 9

ER -