Thyroid Hormone Receptor-interacting Protein 1 Modulates Cytokine and Nuclear Hormone Signaling in Erythroid Cells

Abstract

Erythropoietin (Epo) and thyroid hormone (T 3 ) are key molecules in the development of red blood cells. We have shown previously that the tyrosine kinase Lyn is involved in differentiation signals emanating from an activated erythropoietin receptor. Here we demonstrate that Lyn interacts with thyroid hormone receptor-interacting protein 1 (Trip-1), a transcriptional regulator associated with the T 3 receptor, providing a link between the Epo and T 3 signaling pathways. Trip-1 co-localized with Lyn and the T 3 receptor ␣ in the cytoplasm/plasma membrane of erythroid cells but translocated to discrete nuclear foci shortly after Epo-induced differentiation. Our data reveal that T 3 stimulated the proliferation of immature erythroid cells, and inhibited maturation promoted by erythropoietin. Removal of T 3 reduced cell division and enhanced terminal differentiation. This was accompanied by large increases in the cell cycle inhibitor p27Kip1 and by increasing expression of erythroid transcription factors GATA-1, EKLF, and NF-E2. Strikingly, a truncated Trip-1 inhibited both erythropoietin-induced maturation and T 3 -initiated cell division. This mutant Trip-1 acted in a dominant negative fashion by eliminating endogenous Lyn, elevating p27 Kip1 , and blocking T 3 response elements. These data demonstrate that Trip-1 can simultaneously modulate responses involving both cytokine and nuclear receptors.

Csk-binding Protein Mediates Sequential Enzymatic Down-regulation and Degradation of Lyn in Erythropoietin-stimulated Cells

“…The construction of the murine Lyn expression plasmids pCA-Lynwt, pCA-LynY397F, pCA-LynY508F (21), FLAGtagged murine SOCS1 expression vector pEFBos-SOCS1 (15), murine Lyn full-length and Lyn domain LexA fusion plasmids pBTM116-Lyn, pBTM116-Un, pBTM116-SH3, pBTM116-SH2, pBTM116-kinase (35,36), murine ARL-6 LexA fusion vector pBTM116-ARL-6 (39), murine Lyn SH2 domain VP16 fusion plasmid pVP16-SH2 (35,36), and murine GBP3 VP16 fusion plasmid pVP16-GBP3 have been described in detail previously (39).…”

“…Cell Culture, Inductions, Flow Cytometry, and Colony Assays-J2E (37), J397 (21), and R11 (21) cells were maintained in Dulbecco's modified Eagles medium (DMEM) with 5% fetal calf serum (FCS), while COS-7, HEK293, PA317, and Y2 cells were cultured in DMEM/10% fetal calf serum as described elsewhere (36). Cells were stimulated with Epo (5 units/ml) in media containing T 3 depleted FCS (36), and hemoglobin production detected by benzidine staining (21).…”

“…Cells were stimulated with Epo (5 units/ml) in media containing T 3 depleted FCS (36), and hemoglobin production detected by benzidine staining (21). Splenic erythroblasts were isolated from phenylhydrazinetreated Lyn ϩ/ϩ and Lyn Ϫ/Ϫ mice as described previously (26).…”

“…Mutation of HS1 and Trip1 in primary erythroid progenitors, or the Epo-responsive J2E cell line (37), impedes differentiation (35,36). At the time the yeast two-hybrid screen was conducted, one of the Lyn-binding proteins isolated was a novel molecule.…”

“…To biochemically define the Lyn signaling pathway, we have employed the yeast two-hybrid system and demonstrated that hemopoietic-specific protein 1 (HS1) and thyroid hormone receptor-interacting protein 1 (Trip1) are part of the Lyn pathway for red cell maturation (35,36). Mutation of HS1 and Trip1 in primary erythroid progenitors, or the Epo-responsive J2E cell line (37), impedes differentiation (35,36).…”