Granulocyte colony-stimulating factor (G-CSF), by dimerizing its receptor (G-CSFR), effects all stages of myelopoiesis. Two functionally distinct regions within the cytoplasmic domain exist. proliferative signaling is through the membrane-proximal 55 amino acids, but the carboxy-terminal 98 amino acids are essential to drive neutrophilic maturation. The four known alternatively spliced G-CSFR mRNA isoforms (class I-IV) differ in their structure of these signaling regions. The class I isoform contains both signaling regions, but the class IV isoform, through alternative splicing, deletes much of the carboxy-terminus, making it differentiation defective. Experimental evidence suggests that differentiation defective receptor isoforms may exert a dominant-negative effect on the maturational signal by heterodimerizing with the class I isoform. Preliminary results from our laboratory indicate the leukemic cell lines HL60, NB4, and EM3 express increased levels of the differentiation defective class IV isoform than do mature neutrophils. From these results we propose two hypotheses. First, over-expression of the class IV isoform is an aberrant process in leukemic cell, producing to a maturational block and contributing to leukemogenesis. To test this we will determine the relative G-CSFR isoform expression in fresh AML cells using a quantitative reverse transcription/polymerase chain reaction method (Q-RT/PCR). Results will be correlated with proliferative and maturational response to G-CSF. The second hypothesis, that the increased levels of G-CSFR class IV isoform in leukemic cells reflects their physiologic stage of maturational arrest. This will be examined in three human models of myelopoiesis, using Q-RT/PCR methods to measure relative G-CSFR isoform expression as cells terminally differentiate. While defining the role of G-CSFR class IV in leukemogenesis and granulopoiesis, we will use three murine models of G-CSFR class I driven neutrophilic differentiation to determine if the class IV isoform can exert a dominant-negative effect on the maturation signal in these models. To better understand how myeloid cells regulate the relative expression of the G-CSFR class I and class IV isoforms, we will devise PCR generated mutants of G-CSFR cytoplasmic domain genomic sequence to study the mRNA sequences relevant to mechanisms of alternative splicing. The results of these studies will define the physiologic mechanisms and relevance of G-CSFR isoform expression in granulocytic development from mRNA transcription to protein expression. A better understanding of relative G-CSFR isoform expression will provide insight into a variety of disorders of myelopoiesis, such myelogenous leukemia or myelodysplastic syndromes.
|White, S M; Alarcon, M H; Tweardy, D J (2000) Inhibition of granulocyte colony-stimulating factor-mediated myeloid maturation by low level expression of the differentiation-defective class IV granulocyte colony-stimulating factor receptor isoform. Blood 95:3335-40|