TNF ALPHA AND IMPAIRED WOUND HEALING OF CACHEXIA

University Of North Carolina Chapel Hill, Chapel Hill NC

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Abstract

Wound healing is a dynamic process characterized by the integrated actions of different cells, cytokines, and the extracellular matrix. Patients with cachexia, who frequently require surgery for the underlying illness, have impaired wound healing. The underlying hypothesis of this proposal is that this impaired wound-healing results from effects of excessive tumor necrosis factor alpha (TNFalpha) on extracellular matrix proteins. TNFalpha inhibits fibrillar collagen gene expression and stimulates cytokines and interstitial collagenase (MMP1 and MMP13) gene expression. TNFalpha is a potent activator of the intracellular signaling molecules cJun N-terminal kinase (JNK or SAPK), p38 MAPK, and NF-KB. To gain new insights into the molecular pathogenesis of wound healing, this proposal will focus on specific aspects of the effects of TNFalpha on intracellular signaling which in turn regulate expression of the type I collagen alpha1(I), interstitial collagenase, and cytokines. The specific aims of this proposal are: 1. To identify the regulatory elements in the collagen alpha1(I) gene by which TNFalpha inhibits its transcription. 2. To characterize the role of transforming growth factor beta (TGF-beta) in the inhibition of collagen alpha1(I) by TNFalpha. 3. To investigate the effect of TNFalpha on the stability of mRNAs for collagen alpha1(I), interstitial collagenase, and IL-6. 4. To characterize the signal transduction pathway by which TNFalpha inhibits collagen alpha1(I) gene expression. 5. To study the effect of IL-6 on interstitial collagenase. These studies will use primary cultures of two collagen producing cell lines; fibroblasts and hepatic stellate cells. Our laboratory has developed unique reagents required for in-depth study of the molecular mechanisms of TNFalpha's effects. We are developing novel transgenic mouse lines to study the cis- acting elements in the alpha1(I) collagen gene. We are developing recombinant adenoviruses that will deliver activating or dominant-negative forms of signaling molecules, which will modulate the genetic expression of entire populations of cultured primary cells. Finally, we are combining the above reagents with well validated assays of signaling kinases in order to use the genetics of knockout mice to further assess the role of specific signaling pathways in TNFalpha's down-stream effects.