Development, characterization, and wound healing of the keratin 14 promoted transforming growth factor-beta1 transgenic mouse.
Transforming growth factor-beta1 is a fibrogenic cytokine that is important in the development of fibroproliferative disorders of the skin after injury. To investigate the role of transforming growth factor-beta1 produced by keratinocytes during wound healing, a plasmid with the human transforming growth factor-beta1 gene coupled with the keratin 14 promoter (pG3Z: K14-TGF-beta1) was constructed. The construct was tested successfully in vitro before being used to generate transgenic animals, which were subsequently bred into homozygous and heterozygous lines. Genotype screening of founders and progeny was performed by Southern blotting and targeting of the transgene to the epidermis by the keratin 14 promoter was shown by reverse transcription polymerase chain reaction. The major phenotypic change observed in the transgenic animals was "scruffiness" of the fur attributed to transgene expression in the skin, seen primarily in the homozygous line. A significant reduction in the rate of reepithelialization of full-thickness excisional wounds of dorsal skin was seen in homozygous animals compared with normal litter-mate controls at day 7 (p < 0.05, Fisher's Exact test) and day 9 (p < 0.01) postwounding. Wounds in heterozygous animals also healed more slowly at day 9 (p < 0.01). Northern analysis of mRNA extracted from the wounds showed increased human transforming growth factor-beta1 message levels in homozygous and heterozygous animals, maximal at day 5. Significant increases in transforming growth factor-beta1 activity in healing wounds measured using the plasminogen activator inhibitor-1/luciferase assay were found in the transgenic strains at day 9 postinjury as compared with the normal litter-mate control mice (p < 0.001, ANOVA). Type I procollagen mRNA expression was higher in the homozygous and heterozygous animals, with the highest levels reached at day 9. By day 5 postwounding, biopsies of both homozygous and heterozygous tissues were significantly higher in collagen as compared with wounds in control animals (p < 0.05, ANOVA). Based on these data, the K14-TGF-beta1 transgenic mouse shows that excessive latent transforming growth factor-beta1 produced in the epidermal layer of the skin delays reepithelialization in excisional wounds but subsequently the cells of the epidermis stimulate dermal fibroblasts leading to fibrosis through a paracrine mechanism.
Chan
Teddy
T
Wound Healing Research Group, University of Alberta, Alberta, Canada.
Ghahary
Aziz
A
Demare
Jack
J
Yang
Liju
L
Iwashina
Takashi
T
Scott
Paul G
PG
Tredget
Edward E
EE
D000818:Animals; D003094:Collagen / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004817:Epidermis / Q000502:physiology; D007150:Immunohistochemistry; D053547:Keratin-14; D015603:Keratinocytes / Q000502:physiology*; D007633:Keratins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D023421:Models, Animal; D010957:Plasmids; D011347:Procollagen / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1; D014945:Wound Healing / Q000502:physiology*
C507540:KRT14 protein, human; D053547:Keratin-14; C507446:Krt14 protein, mouse; D011347:Procollagen; D012333:RNA, Messenger; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D007633:Keratins; D003094:Collagen