TGF-beta is not involved in early phase growth inhibition of keratinocytes by 1alpha,25(OH)2vitamin D3.
BACKGROUND
It has been proposed that transforming growth factor-beta (TGF-beta) is involved in the growth inhibition of normal human epidermal keratinocytes (NHEK) by 1alpha,25-dihydoxyvitamin D(3) (1alpha,25(OH)(2)D(3)), although this is still controversial because of the difficulty in blocking TGF-beta activity completely.


OBJECTIVE
To determine whether TGF-beta is involved in early phase growth inhibition by 1alpha,25(OH)(2)D(3).


METHODS
TGF-beta mRNA was detected by ribonuclease protection assay (RPA), and biological active TGF-beta was determined by a luciferase reporter assay. To block intrinsic TGF-beta activity completely, we constructed an adenovirus vector expressing a truncated TGF-beta type II receptor with a dominant negative effect (AdexTbetaTR) that blocks TGF-beta signal transduction.


RESULTS
1alpha,25(OH)(2)D(3) slightly upregulated TGF-beta1 and TGF-beta2 after 24 h according to an RPA and a luciferase reporter assay, however growth inhibition by 1alpha,25(OH)(2)D(3) occurred at 6 h. The addition of 10(-6) M of 1alpha,25(OH)(2)D(3) to NHEK infected with AdexTbetaTR or AdexLacZ (control vector) reduced DNA synthesis to 59.3 and 62.2% at 6 h, respectively. There was no significant difference in cell number after a 3-day incubation with AdexTbetaTR or AdexLacZ-infected cells treated with 1alpha,25(OH)(2)D(3).


CONCLUSIONS
Since 1alpha,25(OH)(2)D(3) rapidly inhibits NHEK growth regardless of the prevention of TGF-beta signal transduction, TGF-beta is not involved in early phase growth inhibition by 1alpha,25(OH)(2)D(3).
Shirakata
Yuji
Y
Department of Dermatology, Ehime University School of Medicine, Onsengun, Ehime 791-0295, Japan. shirakat@m.ehime-u.ac.jp
Ueno
Hikaru
H
Hanakawa
Yasushi
Y
Kameda
Kenji
K
Yamasaki
Kenshi
K
Tokumaru
Sho
S
Yahata
Yoko
Y
Tohyama
Mikiko
M
Sayama
Koji
K
Hashimoto
Koji
K
D000256:Adenoviridae / Q000235:genetics; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology*; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D005799:Genes, Dominant; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger / Q000378:metabolism; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry; D012260:Ribonucleases / Q000378:metabolism; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D015854:Up-Regulation
D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; C508960:TGFB1 protein, human; C509041:TGFB2 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D012260:Ribonucleases; D002117:Calcitriol; D001973:Bromodeoxyuridine