A novel negative regulatory element in the human collagenase-3 proximal promoter region

We have identified in the human collagenase-3 promoter a novel negative regulatory element, GAAAAGAAAAAG, designated AGRE (AG-Rich Element). The AGRE site functionality was characterized in human osteoarthritic (OA) chondrocytes as well as four cell lines. The cells were transfected with a plasmid consisting of the first 133 bp of the collagenase-3 promoter and its AGRE mutated or deleted derivatives. The absence of a functional AGRE site resulted in a statistically significant increase of the collagenase-3 basal transcription that was not affected by the collagenase-3 inducers IL-1beta and TGF-beta1. Two specific protein-AGRE binding complexes were detected by EMSA, and their presence depended on the physiological state of the cell. Indeed, normal chondrocytes and synovial fibroblasts and the four cell lines showed only a slower-migrating complex (complex 1). In OA chondrocytes, the type of complex discriminated two groups--the low-OA chondrocytes, showing low collagenase-3 basal levels and high inducibility of IL-1beta stimulation (complex 1), and the high-OA chondrocytes with high collagenase-3 basal levels and low IL-1beta inducibility (a faster-migrating complex, designated complex 2). UV cross-linking revealed the presence of 48 and 97 kDa proteins in complex 1 and 27, 35, and 73 kDa proteins in complex 2. These findings suggest that the AGRE site plays a rate-limiting role in human collagenase-3 production.