The proposed studies will analyze the expression of activin A and its regulatory control in hematopoietic tissues. We will also probe the possibility of using activin A to enhance the erythropoiesis in the human long-term marrow cultures in vitro, and analyze the distribution of activin A receptors in hematopoietic cells.
The specific aims are as follows: A. To identify and characterize activin A producing cells in marrow stromal cells and/or cloned stromal cell lines. We will use immunological techniques to detect the production of dimeric activin A, in situ hybridization for mRNA expression and bioassay/ELISA for bioactive and dimeric activin A among marrow stromal cells. In addition to marrow samples, we will utilize cloned stromal cell lines for the proposed studies. B. To analyze the 5' flanking region of activin A gene. Part of the 5' flanking region has been sequenced and analyzed. Several structural features are intriguing and have prompted us to analyze: (l) the transcription initiation site(s) and possible additional introns and exons in this flanking region; (2) the capacity of interacting with GATA binding proteins in a region where seven GATA motifs are present; and (3) promoter activities in this 5' flanking region, which may control the expression of activin A. C. To investigate the distribution of activin A receptors among hematopoietic cells. The reverse transcription PCR, using a pair of degenerate oligonucleotide primers, will make it possible to amplify the type II and various forms of type IIB receptors. These studies will be confirmed with immunocytochemical analysis of activin A receptors among specific cell lineages after fluorescence-activated cell sorting of bone marrow or peripheral blood mononuclear cells using lineage specific antibodies. In another project, we are developing specific antibodies against activin A receptor as GST fusion protein, which will be used in these studies when they are available. D. To analyze the potentiating effects of activin A on erythropoiesis in LTMC. We will investigate the dosage effect and lineage specificity of exogenously added activin A and various combinations of activin A /Epo and other recombinant hematopoietic factors, and the kinetics and proliferative states of various hematopoietic lineages in these cultures. We will also insert activin A cDNA into stromal cell populations and investigate whether the locally produced activin A in microenvironment sustains erythropoiesis longer than controls in LTMC. These studies will help to determine whether activin A or its combination with other factors have the potential as novel agent for red cell component therapy in the future.
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