Growth hormone and the growth-promoting peptide, insulin growth factor (IGF-1), decline in blood following puberty and are the only two substances known to increase thymic cellularity in adult and aged animals. Growth hormone injections increase the number of bone marrow-derived multipotential hematopoietic progenitors by 10- fold in young mice, and this probably occurs by inducing the synthesis of IGF- 1. It has been discovered that stem cell factor (SCF; c-kit ligand, steel factor) and IL-3, both of which stimulate multipotential progenitors in bone marrow, induce abundant expression of IGF-1 mRNA and protein. SCF permits survival and self-renewal of the earliest known progenitors and has recently been shown to support development of progenitor T lymphocytes. IGF-1 prevents programmed cell death in IL-3-deprived bone marrow cells. Collectively, these findings strongly support the hypothesis that hematopoietic growth factors prevent apoptosis by stimulating IGF-1 synthesis in very primitive progenitors of all lineages and that an important mechanism for the impressive effect of growth hormone in augmenting thymopoiesis is linked to its induction of IGF-1 synthesis. The proposed experiments consist of four specific objectives designed to determine the expression, function and regulation of IGF-1 in hematopoietic progenitors from aged animals. Early multipotential hematopoietic progenitors from marrow will be derived by culturing in either SCF or IL-3+GM-CSF, whereas M-CSF will be used to generate lineage-specific cells.
Specific Aim 1 proposes to determine whether expression of transcripts and protein for IGF-1 and its receptor decline in hematopoietic progenitors derived from marrow of aged rats. This objective utilizes RNase protection assays followed by quantitation using phosphorimaging, Western blotting, flow cytometry and nuclear run- on experiments.
Specific Aim 2 proposes to concentrate on the hypothesis that IGF-1 and growth hormone act as survival factors by preventing apoptosis of primitive hematopoietic progenitors. The application predicts that early progenitors and thymocytes from aged rats will display a greater incidence of apoptosis which may be accompanied by an increase in expression of Fas antigen and reduced synthesis of the proto- oncogene, bcl-2. Growth hormone and IGF-1 are likely to reduce Fas antigen expression and increase synthesis of bcl-2. IGF-1 expressing progenitor cells will also be treated with antibodies to growth hormone or IGF-1 or anti-sense IGF-1 and IGF-1 receptor nuclease-resistant phosphorothioate oligonucleotides. The biological activities of IGF-1 binding proteins regulate the expansion of hematopoietic progenitors and that these proteins are primarily synthesized by macrophages. Affinity cross-linking with 125I-IGF-1, as well as cloning and sequencing, will be used to identify and quantitate these IGF binding proteins.
Specific Aim 3 proposes to determine if aged rats synthesize any IGFBP and whether IGFBP-3 affects proliferation, colony formation or apoptosis in early BM-derived progenitors. Also, studies will determine if all 6 known IGF binding proteins are synthesized by hematopoietic cells and do the transcripts of these binding proteins decline with age.
Specific Aim 4 proposes to determine whether growth hormone treatment in vivo and in vitro increases expression of IGF-1 in hematopoietic progenitors and in the involuted thymus glands of aged rats using a novel, competitive RT-PCR technique that we have developed. IFN-gamma is now known to increase expression of Fas antigen, and our preliminary data which shows that IFN-gamma also reduces expression of IGF-1 will be expanded to hematopoietic cells of aged rats. Collectively, these experiments will be the first to characterize the expression of IGF-1 and its receptors in hematopoietic progenitors, including those for T lymphocytes, in aged rats. The biology of hematopoiesis will be significantly advanced by exploring the novel idea that this peptide functions as a survival factor for SCF-derived hematopoietic progenitors by inhibiting programmed cell death.
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