TY - JOUR
T1 - TGF-β is not involved in early phase growth inhibition of keratinocytes by 1α,25(OH) 2vitamin D 3
AU - Shirakata, Yuji
AU - Ueno, Hikaru
AU - Hanakawa, Yasushi
AU - Kameda, Kenji
AU - Yamasaki, Kenshi
AU - Tokumaru, Sho
AU - Yahata, Yoko
AU - Tohyama, Mikiko
AU - Sayama, Koji
AU - Hashimoto, Koji
N1 - Funding Information:
We thank Ms. Eriko Tan, Ms. Teruko Tsuda, and Ms. Akiko Kon for their excellent technical assistance. We also thank Dr. Kohei Miyazono (Tokyo, Japan) for providing the plasmids containing the luciferase reporter gene. This work was partly supported by Health Sciences Research Grants for Research on Specific Diseases from the Ministry of Health, Labor, and Welfare of Japan (to K.H.), and a Grant-in-Aid of Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Y.S., K.H.).
PY - 2004/10
Y1 - 2004/10
N2 - It has been proposed that transforming growth factor-β (TGF-β) is involved in the growth inhibition of normal human epidermal keratinocytes (NHEK) by 1α,25-dihydoxyvitamin D 3 (1α,25(OH) 2D 3), although this is still controversial because of the difficulty in blocking TGF-β activity completely. To determine whether TGF-β is involved in early phase growth inhibition by 1α,25(OH) 2D 3. TGF-β mRNA was detected by ribonuclease protection assay (RPA), and biological active TGF-β was determined by a luciferase reporter assay. To block intrinsic TGF-β activity completely, we constructed an adenovirus vector expressing a truncated TGF-β type II receptor with a dominant negative effect (AdexTβTR) that blocks TGF-β signal transduction. 1α,25(OH) 2D 3 slightly upregulated TGF-β1 and TGF-β2 after 24 h according to an RPA and a luciferase reporter assay, however growth inhibition by 1α,25(OH) 2D 3 occurred at 6 h. The addition of 10 -6 M of 1α,25(OH) 2D 3 to NHEK infected with AdexTβTR 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 AdexTβTR or AdexLacZ-infected cells treated with 1α,25(OH) 2D 3. Since 1α,25(OH) 2D 3 rapidly inhibits NHEK growth regardless of the prevention of TGF-β signal transduction, TGF-β is not involved in early phase growth inhibition by 1α,25(OH) 2D 3.
AB - It has been proposed that transforming growth factor-β (TGF-β) is involved in the growth inhibition of normal human epidermal keratinocytes (NHEK) by 1α,25-dihydoxyvitamin D 3 (1α,25(OH) 2D 3), although this is still controversial because of the difficulty in blocking TGF-β activity completely. To determine whether TGF-β is involved in early phase growth inhibition by 1α,25(OH) 2D 3. TGF-β mRNA was detected by ribonuclease protection assay (RPA), and biological active TGF-β was determined by a luciferase reporter assay. To block intrinsic TGF-β activity completely, we constructed an adenovirus vector expressing a truncated TGF-β type II receptor with a dominant negative effect (AdexTβTR) that blocks TGF-β signal transduction. 1α,25(OH) 2D 3 slightly upregulated TGF-β1 and TGF-β2 after 24 h according to an RPA and a luciferase reporter assay, however growth inhibition by 1α,25(OH) 2D 3 occurred at 6 h. The addition of 10 -6 M of 1α,25(OH) 2D 3 to NHEK infected with AdexTβTR 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 AdexTβTR or AdexLacZ-infected cells treated with 1α,25(OH) 2D 3. Since 1α,25(OH) 2D 3 rapidly inhibits NHEK growth regardless of the prevention of TGF-β signal transduction, TGF-β is not involved in early phase growth inhibition by 1α,25(OH) 2D 3.
KW - 1α,25(OH) D
KW - Adenovirus vector
KW - Dominant negative TGF-β receptor
KW - Growth inhibition
KW - Keratinocyte
KW - Luciferase reporter assay
KW - RNase protection assay
KW - TGF-β
UR - http://www.scopus.com/inward/record.url?scp=5644244250&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=5644244250&partnerID=8YFLogxK
U2 - 10.1016/j.jdermsci.2004.07.002
DO - 10.1016/j.jdermsci.2004.07.002
M3 - Article
C2 - 15488704
AN - SCOPUS:5644244250
VL - 36
SP - 41
EP - 50
JO - Journal of Dermatological Science
JF - Journal of Dermatological Science
SN - 0923-1811
IS - 1
ER -