TRANSCRIPTIONAL REGULATION OF THE GENE ENCODING THE HUMAN C-TYPE LECTIN LEUKOCYTE RECEPTOR AIM CD69 AND FUNCTIONAL-CHARACTERIZATION OF ITS TUMOR NECROSIS FACTOR-ALPHA-RESPONSIVE ELEMENTS/

The human activation antigen CD69 is a member of the C-type animal lec tin superfamily that functions as a signal-transmitting receptor. Alth ough the expression of CD69 can be induced in vitro on cells of most h ematopoietic lineages with a wide variety of stimuli, in vivo it is ma inly expressed by T-lymphocytes located in the inflammatory infiltrate s of several human diseases. To elucidate the mechanisms that regulate the constitutive and inducible expression of CD69 by leukocytes, we i solated the promoter region of the CD69 gene and carried out its funct ional characterization. Sequence analysis of the 5'-flanking region of the CD69 gene revealed the presence of a potential TATA element 30 ba se pairs upstream of the major transcription initiation site and sever al putative binding sequences for inducible transcription factors (NF- kappa 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 s panning positions -78 to +16 contained the cis-acting sequences necess ary for basal and phorbol 12-myristate 13-acetate-inducible transcript ion of the CD69 gene. Removal of the upstream sequences located betwee n positions -78 and -38 resulted in decreased promoter strength and ab olished the response to phorbol 12-myristate 13-acetate. We also found that tumor necrosis factor-alpha (TNF-alpha) is capable of inducing t he surface expression of the CD69 molecule as well as the promoter act ivity of fusion plasmids that contain 5'-flanking sequences of the CD6 9 gene, suggesting that this cytokine may regulate in vivo the express ion of CD69. In addition, cotransfection experiments demonstrated that the CD69 gene promoter can be activated by the NF-kappa B/Rel family members c-Rel and RelA. The deletion of the sequence spanning position s -255 to -170 abolished both the response to TNF-alpha and the transa ctivation by NF-kappa B. These results indicate that the NF-kappa B-bi nding site located at position -223 is necessary for the TNF-alpha-ind uced expression of the CD69 gene. Mobility shift assays showed that th e two NF-kappa B motifs located in the proximal promoter region (posit ions -223 and -160) bind various NF-kappa B-related complexes, in elud ing 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-alpha has a key role in the expressi on of this molecule at sites of chronic inflammation.