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).
Conclusion: 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).