Myofibroblast differentiation of normal human keratocytes and hTERT, extended-life human corneal fibroblasts.
OBJECTIVE
The purpose of this study was to determine whether TGFbeta induces myofibroblast differentiation in cultured human keratocytes and in telomerase (hTERT)-immortalized human corneal fibroblast cell lines.


METHODS
Normal human corneal keratocytes were isolated from donor corneas of various ages and grown under serum-free (cultured keratocytes) or serum-added (corneal fibroblasts) conditions. Corneal fibroblasts were infected with the MPSV-hTERT retroviral vector, and selected clones were isolated and characterized by chromosomal karyotyping. The responses of normal cultured keratocytes and serum-starved corneal fibroblasts to TGFbeta in the presence or absence of Arg-Gly-Asp (RGD)-containing peptides and neutralizing antibodies to platelet-derived growth factor (PDGF) were characterized by immunocytochemistry, Western blot analysis, and real-time PCR, to identify assembly of actin filaments, formation of focal adhesions, and expression of alpha-smooth muscle actin (alpha-SMA).


RESULTS
Treatment of cultured keratocytes with TGFbeta (1 ng/mL) induced cell spreading, assembly of actin filaments, formation of focal adhesions, and expression of alpha-SMA, which was blocked by the addition of RGD-containing peptides (100 microM). A similar response was identified in hTERT-expressing human corneal fibroblast cell lines, showing a 69-fold increase in alpha-SMA message. Furthermore, treatment of hTERT corneal fibroblasts with RGD or anti-PDGF inhibited myofibroblast differentiation. Karyotype analysis of hTERT corneal fibroblasts identified age-dependent chromosomal aberrations in cells of older donors but not in those of a 10-year-old donor.


CONCLUSIONS
Induction of myofibroblast differentiation by TGFbeta in cultured human keratocytes and hTERT corneal fibroblasts occurs through a similar signal transduction pathway to that previously identified in the rabbit, which involves an autocrine PDGF feedback loop.
Jester
James V
JV
Departments of Ophthalmology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA. james.jester@utsouthwestern.edu
Huang
Jiying
J
Fisher
Stephen
S
Spiekerman
Jennifer
J
Chang
Jin Ho
JH
Wright
Woodring E
WE
Shay
Jerry W
JW
D000199:Actins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002648:Child; D003319:Corneal Stroma / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D008875:Middle Aged; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D019098:Telomerase / Q000502:physiology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000199:Actins; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D009842:Oligopeptides; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; C047981:arginyl-glycyl-aspartic acid; D019098:Telomerase