Genetically modified dermal keratinocytes express high levels of transforming growth factor-beta1.
In an attempt to genetically modify cultured keratinocytes with transforming growth factor-beta1 (TGF-beta1), which has been proven to be one of the most important cytokines involved in wound healing, two constructs were made. One, designated pG3Z:K14-TGF-beta1, is a plasmid in which the expression of TGF-beta1 is driven by the keratin 14 promoter. The other, designated pLin-TGF-beta1, is a retroviral vector in which the retroviral 5' long-terminal repeat promoter drives expression. In both constructs, the deletion of a small fragment of the noncoding region of the TGF-beta1 gene was made to differentiate the transcript from that for endogenously expressed TGF-beta1. Different types of cells were transfected with the pG3Z:K14-TGF-beta1 construct using the calcium phosphate method. The pLin-TGF-beta1 construct was propagated in a retroviral packaging cell line and conditioned medium that contained high titers of the virus was used to transduce keratinocytes or other types of cells grown in standard culture. Northern analysis, used to evaluate the expression of TGF-beta1 mRNA in the pG3Z:K14-TGF-beta1 transfected keratinocyte C1-177 cell line, showed a smaller TGF-beta1 transcript compared with that endogenously expressed by dermal fibroblasts. The level of TGF-beta1 protein evaluated by enzyme-linked immunosorbent assay was significantly higher in medium conditioned by either the K14-TGF-beta1 transfected or the pLin-TGF-beta1 transduced keratinocytes, compared with that obtained from control cells; however, the level of TGF-beta1 protein was unchanged in cultures of pG3Z:K14-TGF-beta1 transfected nonkeratinocyte cells such as fetal and adult fibroblasts. Using the mink lung epithelial cell growth inhibition assay, we found an increase in TGF-beta1 activity in conditioned medium from the pG3Z:K14-TGF-beta1 transfected cells. To evaluate possible paracrine effects of the keratinocyte derived TGF-beta1, a coculture system was established with pLin-TGF-beta1 transduced keratinocytes grown in the upper chamber and dermal fibroblasts in the lower chamber. The results showed that TGF-beta1 released from keratinocytes diffused to the lower chamber where it stimulated collagen production by dermal fibroblasts. In summary, we demonstrate here that primary cultured keratinocytes can be genetically modified to express high levels of TGF-beta1 and suggest that this offers a potential approach for the therapy of dermal lesions such as nonhealing wounds.
Ghahary
A
A
Department of Surgery, Wound Healing Research Group, Edmonton, Alberta, Canada.
Tredget
E E
EE
Chang
L J
LJ
Scott
P G
PG
Shen
Q
Q
D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D053547:Keratin-14; D015603:Keratinocytes / Q000378:metabolism*; D007633:Keratins / Q000235:genetics; D005810:Multigene Family / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012867:Skin / Q000166:cytology*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
C507540:KRT14 protein, human; D053547:Keratin-14; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D007633:Keratins