PLATELET GLYCOPROTEIN IIB GENE-EXPRESSION AS A MODEL OF MEGAKARYOCYTE-SPECIFIC EXPRESSION

Glycoprotein (GP)IIb/IIIa is an integrin grin complex normally restric ted in its expression to platelets and the megakaryocytes from which t hey are derived. This complex functions as a receptor for fibrinogen a nd other ligands and is involved in platelet aggregation. The receptor complex is expressed at high levels during final megakaryocyte differ entiation. Further, while GPIIIa is expressed in other tissues as part of the vitronectin receptor, GPIIb is only expressed on maturing mega karyocytes and the platelets derived from them. Thus studies of the GP IIb gene may serve as a model of gene regulation during this process. Over the past several gears, the genes for both GPIIb and IIIa have be en cloned and analyzed. The GPIIb gene contains 30 exons over 18 kilob ases (kb). The transcriptional start site has been determined and ther e does not appear to be a TATA-box immediately upstream of this site. Studies have been done to define regulatory elements upstream of the t ranscriptional start site. Most of these studies focused on the human promoter and on studies using megakaryocytic cell lines. These studies have defined several important tissue-specific promoter elements incl uding a GATA(454) site (454 basepairs upstream of the transcriptional start site that involves a GATA-binding consensus sequence), a GATA(54 ) site and an Ets(35) Site (that involves an Ets-binding consensus seq uence). Expression studies with megakaryocytic cell lines suggest that each of these sites effects expression approximately threefold. Furth er, an Ets(510) site was also described that had a similar effect. Whi le these studies were underway, we pursued studies of the rat 5'-flank ing region using a rat primary marrow expression system. Qualitatively , our data support the human data; however, quantitatively, we found s ignificant differences from the human studies done in cell lines. We f ound that the major tissue-specific promoter element was the GATA(454) site. Mutations altering this site result in an approximately fiftyfo ld drop in expression. In comparison, eliminating the Ets(510) site by truncation or point mutation had only a twofold effect on expression. Mutations at the Ets(35) site did effect expression at a high level, decreasing expression approximately fifteenfold, while mutations at th e GATA(54) site effected expression by approximately ninefold. In addi tion, using 50 hp deletions, we have preliminarily defined two domains from -450 to -351 bp and -150 to -101 bp upstream of the transcriptio nal start site that effected expression. The former appears to contain a positive regulatory element, while the latter appears to be a silen cer element. In expression studies with the rat GPIIb promoter in huma n megakaryocytic cell lines, we have obtained results similar to those seen with the human GPIIb promoter region in the same human cell line . Thus, we believe that the differences seen are not due to species di fferences in GPIIb gene regulation, but rather due to differences in s tudies involving terminal differentiation of megakaryoblasts into norm al megakaryocytes versus studies involving multilineage tumor cell lin es. The focus of future studies will be to fully delineate the promote r elements in the 5'-flanking region of the GPIIb gene, and how they d etermine tissue-specific expression.