Targeting TGF-beta1 increases hepatocyte growth factor (HGF/SF) levels in external auditory canal cholesteatoma (EACC) epithelial cell culture.
OBJECTIVE
The transforming growth factor (TGF)-beta 1 is known to have pro- and anti-angiogenic actions. Hepatocyte growth factor/scatter factor (HGF/SF) antagonizes TGF-beta1 by stabilizing SMAD transcriptional co-repressor TGIF. HGF/SF is a multifunctional polypeptide with morphogenic, motogenic, angiogenic, and proliferative capabilities. We assume HGF to be a pivotal factor during the pathogenesis of the external auditory canal cholesteatoma (EACC). In this study, we investigate the effect of antisense targeting TGF-beta1 on HGF/SF levels in the epithelial EACC-culture.


METHODS
For 48 h, epithelial EACC cell culture was incubated with 3 and 6 mumol antisense targeting TGF-beta1, respectively. Levels of HGF/SF were determined and normalized to cellular protein. Untreated EACC cell culture and scrambled TGF-beta1-antisense served as control. In the second experiment, EACC cells were incubated with rh TGF-beta1 (2 and 4 ng/ml) for 48 h and HGF/SF was determined.


RESULTS
After incubation with 3 mumol TGF-beta1-antisense, the average level of HGF/SF was measured at 43.68 pg/ml. Incubation with 6 micromol TGF-beta1-antisense showed 64.95 pg/ml. In untreated EACC (control), the average level of HGF/SF after 48 was 34.55 pg/ml. Incubation with scrambled TGF-beta1 oligonucleotide showed an average HGF/SF level of 34.41 (3 micromol) and 35.66 (6 micromol), respectively. The difference between the scrambled antisense and the targeting antisense TGF-beta1 was significant (p<0.05). After incubation with 2 ng/ml TGF-beta1, the HGF/SF levels were at 22.16 pg/ml. TGF-beta1, 4 ng/ml, resulted in 15.33 pg/ml of HGF/SF. The difference of the levels of HGF/SF after incubation with exogenous TGF-beta1 was significant (p<0.05).


CONCLUSIONS
In this study, levels of HGF/SF increased in the epithelial EACC cell culture after incubation with 3 and 6 mumol antisense TGF-beta1 oligonucleotides depending on the concentration of the antisense. In reverse, TGF-beta1 acted as inhibiting cytokine on HGF/SF levels. In conclusion, TGF-beta1 may be a useful therapeutic agent for managing EACC.
Naim
Ramin
R
Department of Otolaryngology, Head and Neck Surgery, University Hospital Mannheim, Germany.
Chang
Ray C
RC
Alfano
Suzanne S
SS
Riedel
Frank
F
Bayerl
Christiane
C
Sadick
Haneen
H
Bran
Gregor
G
Hormann
Karl
K
D015153:Blotting, Western; D002478:Cells, Cultured; D002781:Cholesteatoma / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D004424:Ear Canal / Q000378:metabolism / Q000473:pathology; D004847:Epithelial Cells / Q000166:cytology; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D007118:Immunoassay; D015603:Keratinocytes / Q000166:cytology; D008954:Models, Biological; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
D016207:Cytokines; D016376:Oligonucleotides, Antisense; D010455:Peptides; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017228:Hepatocyte Growth Factor