REGULATION OF NEUROKININ RECEPTORS IN BONE MARROW STROMA

DESCRIPTION (Adapted from applicant's abstract): Neurohematopoietic modulation is beginning to be unravelled. This understanding has lead to the addition of neuropeptides, such as the mammalian tachykinins to the list of hematopoietic regulators. The mechanisms by which neuropeptides regulate hematopoiesis will add to the general understanding of the pathophysiology of diseases such as anemias and leukemias. Substance P (SP) and neurokinin A (NK-A), members of the tachykinins, modulate hematopoiesis partly through cytokine induction in bone marrow (BM) stroma. Studies with specific antagonists indicate that SP/NK-A interact with two (NK-1 and NK-2) of the three cloned neurokinin receptors (NK-R) in stromal cells. IL-1 and c-kit ligand modulate the numbers and affinities of SP receptors (SP-R) in stroma. Amplification with specific NK-1 primers indicated that the size of fragments induced by these cytokines are different than predicted. The change in binding affinities as well as difference in fragment size suggest that at least for NK-1, BM stroma may have different subtypes. Transcriptional regulation of the NK-Rs will help to elucidate the mechanisms by which the tachykinins regulate hematopoiesis. However, this will only be possible if the NK-R cDNA in BM stroma is cloned. This application will focus on cloning the NK-1 cDNA in human BM stroma and elucidate its/their transcriptional regulation by specific cytokines and neurotrophic factors. The working hypothesis is based on the abilities of cytokines and neurotrophic factors found within the BM to induce NK-1 in BM stroma. Since unstimulated stroma failed to amplify visible NK-1 specific DNA fragments, in aim 1, the investigators will use a cDNA library prepared from IL-1 stimulated BM stroma to clone the NK-1 cDNA. Specific probes, designed from the cloned cDNA(s) will be used to identify the inducers (cytokines/neurotrophic factors) of NK-1 subtype(s). The regions within the NK-1 gene that are sensitive to these 'inducers' will be identified in aim 2. To address this question, the sequence of the NK-1 promoter is required and this will be obtained after cloning of the human NK-1 gene from a commercially available genomic library. Different upstream sequences of the NK-1 gene will be used in aim 3 to prepare heterologous reporter constructs which will be transfected in immune and non- immune cell lines, BM stroma, and specific stromal cells. Transfected cells will be stimulated with cytokines/neurotrophic factors and reporter activities used to determine activation. The answers obtained in this application for a FIRST award will lead to the long term goal of this project which will include studies of intracellular signalling pathway by NK-Rs in specific BM cell populations and identification of transcriptional factors pertinent to the NK-R gene.