GENETICALLY-MODIFIED DERMAL KERATINOCYTES EXPRESS HIGH-LEVELS OF TRANSFORMING GROWTH-FACTOR-BETA-1

In an attempt to genetically modify cultured keratinocytes with transf orming growth factor-beta 1 (TGF-beta 1), which has been proven to be one of the most important cytokines involved in wound healing, two con structs were made. One, designated pG3Z:K14-TGF-beta 1, is a plasmid i n which the expression of TGF-beta 1 is driven by the keratin 14 promo ter. The other, designated pLin-TGF-beta 1, is a retroviral vector in which the retroviral 5' long-terminal repeat promoter drives expressio n. In both constructs, the deletion of a small fragment of the noncodi ng region of the TGF-beta 1 gene was made to differentiate the transcr ipt from that for endogenously expressed TGF-beta 1., Different types of cells were transfected with the pG3Z:K14-TGF-beta 1 construct using the calcium phosphate method. The pLin-TGF-beta 1 construct was propa gated 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-beta 1 mRNA in the pG3Z:K14-TG F-beta 1 transfected keratinocyte Cl-177 cell line, showed a smaller T GF-beta 1 transcript compared with that endogenously expressed by derm al fibroblasts, The level of TGF-beta 1 protein evaluated by enzyme-li nked immunosorbent assay was significantly higher in medium conditione d by either the K14-TGF-beta 1 transfected or the pLin-TGF-beta 1 tran sduced keratinocytes, compared with that obtained from control cells; however, the level of TGF-beta 1 protein was unchanged in cultures of pG3Z:K14-TGF-beta 1 transfected nonkeratinocyte cells such as fetal an d adult fibroblasts, Using the mink lung epithelial cell growth inhibi tion assay, we found an increase in TGF-beta 1 activity in conditioned medium from the pG3Z:K14-TGF-beta 1 transfected cells. To evaluate po ssible paracrine effects of the keratinocyte derived TGF-beta 1, a coc ulture system was established with pLin-TGF-beta 1 transduced keratino cytes grown in the upper chamber and dermal fibroblasts in the lower c hamber. The results showed that TGF-beta 1 released from keratinocytes diffused to the lower chamber where it stimulated collagen production by dermal fibroblasts, In summary, we demonstrate here that primary c ultured keratinocytes can be genetically modified to express high leve ls of TGF-beta 1 and suggest that this offers a potential approach for the therapy of dermal lesions such as nonhealing wounds.