The human activation antigen CD69 is a member of the C-type animal lectin superfamily that functions as a signal-transmitting receptor. Although the expression of CD69 can be induced in vitro on cells of most hematopoietic lineages with a wide variety of stimuli, in vivo it is mainly expressed by T-lymphocytes located in the inflammatory infiltrates of several human diseases. To elucidate the mechanisms that regulate the constitutive and inducible expression of CD69 by leukocytes, we isolated the promoter region of the CD69 gene and carried out its functional characterization. Sequence analysis of the 5`-flanking region of the CD69 gene revealed the presence of a potential TATA element 30 base pairs upstream of the major transcription initiation site and several putative binding sequences for inducible transcription factors (NF-B, Egr-1, AP-1), which might mediate the inducible expression of this gene. Transient expression of CD69 promoter-based reporter gene constructs in K562 cells indicated that the proximal promoter region spanning positions -78 to +16 contained the cis-acting sequences necessary for basal and phorbol 12-myristate 13-acetate-inducible transcription of the CD69 gene. Removal of the upstream sequences located between positions -78 and -38 resulted in decreased promoter strength and abolished the response to phorbol 12-myristate 13-acetate. We also found that tumor necrosis factor- (TNF-) is capable of inducing the surface expression of the CD69 molecule as well as the promoter activity of fusion plasmids that contain 5`-flanking sequences of the CD69 gene, suggesting that this cytokine may regulate in vivo the expression of CD69. In addition, cotransfection experiments demonstrated that the CD69 gene promoter can be activated by the NF-B/Rel family members c-Rel and RelA. The deletion of the sequence spanning positions -255 to -170 abolished both the response to TNF- and the transactivation by NF-B. These results indicate that the NF-B-binding site located at position -223 is necessary for the TNF--induced expression of the CD69 gene. Mobility shift assays showed that the two NF-B motifs located in the proximal promoter region (positions -223 and -160) bind various NF-B-related complexes, including the heterodimers p50/RelA and p50/c-Rel and homodimers of p50 (KBF-1) and RelA. Our findings help to explain the regulated synthesis of CD69 in vivo and suggest that TNF- has a key role in the expression of this molecule at sites of chronic inflammation.

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