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 degradation. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-alpha response element of the COL1A2 promoter. The TNF-alpha response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was eliminated by a 2-bp substitution mutation in the NF-kappa B1 binding half site of the NF-kappa B cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B1 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 cells with cycloheximide. Gel supershift assays identified the complex as the NF-kappa B (p50/p65) heterodimer, whereas Abs to nuclear factor of activated 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 element and plays a critical role in the observed inhibition of alpha 2(I) collagen gene transcription.
Kouba
D J
DJ
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Chung
K Y
KY
Nishiyama
T
T
Vindevoghel
L
L
Kon
A
A
Klement
J F
JF
Uitto
J
J
Mauviel
A
A
D002352:Carrier Proteins / Q000302:isolation & purification; D002478:Cells, Cultured; D003094:Collagen / Q000037:antagonists & inhibitors* / Q000235:genetics*; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D009154:Mutation / Q000276:immunology; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000276:immunology; D011485:Protein Binding / Q000276:immunology; D020218:Response Elements / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000502:physiology*
D002352:Carrier Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D003094:Collagen