Differential Expression of Procollagen Genes Between Mid- and Late-Gestational Fetal Fibroblasts

Background. Previously, we have shown that cutaneous wounds in mid-gestational (E15) mice heal in a scarless manner with decreased procollagen 1 and increased procollagen 3 production compared with wounds in late-gestational (EI8) mice, which heal with scars. The aim of the current work was to determine whether E 15 and E 18 fibroblasts respond to stimulation in culture with differential procollagen expression, suggesting they may preserve their phenotype in vitro. Further, we wanted to determine if fetal fibroblast gene expression patterns persisted in tissue culture. We measured expression of procollagen types 1 alpha 1 and 3 in response to TGF-beta 1 stimulation. We theorized that E15 fibroblasts would respond with a pattern of procollagen that would contribute to a more easily remodeled collagen. Methods. Mid- and late-gestational fetal fibroblasts were obtained from dorsal skin harvested from fetuses of time-dated CD-1 mice. Cells were grown to confluence in culture plates overnight. Cell monolayers were treated with 0.01% bovine serum albumin (BSA) plus 10 ng/well of TGF-beta 1. Cells were harvested at 6 and 24 h following treatment. Additional groups were treated with BSA alone (vehicle controls) and collected at 6 and 24 h. Another group without treatment was harvested after reaching confluence (0 time point). In a separate experiment to determine if gene expression patterns persisted, cells were treated with 0.01% BSA plus 10 ng/well of TGF-beta 1 for 24 h, then harvested. A second group of cells were treated again at 24 h and harvested at 48 h. Additional cells were treated with BSA alone for 24 and 48 h, and another group without treatment was harvested after reaching confluence (0 time point). Cells were processed to obtain mRNA, cDNA was made, and then samples analyzed by QPCR. Results were analyzed by ANOVA and Holm-Sidak method. Results. Procollagen 1 alpha 1 gene expression was decreased in E15 cells at 6 and 24 h following TGF-beta 1 treatment, P < 0.05. In contrast, procollagen 1 alpha 1 was increased in E18 cells, P < 0.05. Procollagen 3 gene expression was decreased in E18 cells at 6 and 24 h following treatment with TGF-beta 1, P < 0.05, whereas levels in E15 cells were unchanged at 6 h, and only trended lower at 24 h. We evaluated whether this differential expression of procollagen 3 persisted at 24 and 48 h. At 24 and 48 h, E15 control groups had increased procollagen 3 expression compared with E18 groups, P < 0.05. E15 and E18 cells in TGF-beta 1-treated groups had decreased procollagen 3 at 48h compared with their respective BSA control groups, P < 0.05. However, the degree of difference appeared to be greater in the E 15 group than the E 18 group. Conclusions. Our results from this in vitro work demonstrate a differential pattern of gene expression for procollagen 1 alpha 1 and 3 in E15 and E18 fibroblasts in response to TGF-beta 1. E15 cells showed decreased expression of procollagen 1 alpha 1, while E18 cells showed increased procollagen 1 alpha 1 and decreased procollagen 3 expression. These patterns of expression in E15 cells are suggestive of increased type 3 to 1 collagen ratio seen in scarless fetal wounds. Interestingly, treatment of either E15 or E18 cells with TGF-beta 1 significantly decreased procollagen 3 expression by 48 h, yet this was more profound in E15 groups. This suggests that after 24 h, E15 cells may transition towards an E18 phenotype and corresponding signaling. (C) 2009 Elsevier Inc. All rights reserved.