Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts

Among its plethora of activities as an inflammatory mediator, TNF-alpha has potent regulatory control on extracellular matrix production and degradati on. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characteriz ation of the transcription factors involved has been elusive. In the presen t study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) repor ter gene constructs, we have characterized the TNF-alpha response element o f the COL1A2 promoter. The TNF-alpha response element was attributed to a s pecific region that comprises noncanonical activator protein-1 (AP-1) (CGAG TCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was elimin ated bp a 2-bp substitution mutation in the NF-kappa B1 binding half site o f the NF-kappa B cia element, Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B 1 and ReLA homodimers, but not AP-1, were capable of binding this element, Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-alpha stimulation , which reached a plateau by 1 h and was not affected by preincubation of c ells with cycloheximide. Gel supershift assays identified the complex as th e NF-kappa B (p50/p65) heterodimer, whereas ribs to nuclear factor of activ ated T cells (NF-AT) and Jun family members failed to recognize the complex , These data suggest that in fibroblasts TNF-alpha activates and initiates the nuclear translocation of NF-kappa B that binds a divergent NF-kappa B e lement and plays a critical role in the observed inhibition of alpha 2(I) c ollagen gene transcription.