Differential regulation of SOCS genes in normal and transformed erythroid cells

Abstract

The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells - erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1-/- mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.

Figure 1

Expression of SOCS-1, SOCS-3 and CIS mRNA in normal erythroid progenitors, (a) Cytocentrifuge preparations of splenic erythroid cells isolated from phenylhydrazine-treated mice stimulated in vitro for 0, 24 or 48 h with Epo (5 U/ml). (b) mRNA was isolated from splenocytes and subjected to RT-PCR analysis for 25, 30 or 35 cycles using primers specific for SOCS-1, SOCS-3, CIS and β-actin. (c) PCR products for SOCS-1, SOCS-3 and CIS were quantitated using NIH Image 1.6 and plotted relative to β-actin levels

Figure 2

Expression of SOCS-1, SOCS-3 and CIS mRNA in erythroleukemic cell lines, (a) Flow cytometry was used to characterize cell surface expression of c-Kit, Epo receptor (Epo-R), transferrin receptor (Tf-R) and Ter119 on erythroleukemic cell lines, (b) [3H]thymidine incorporation of unstimulated (white histogram) or Epo-stimulated (5U/ml; black histogram) cells is expressed as a per cent relative to unstimulated cells, (c), Northern analysis of poly A⁺ RNA from cells stimulated with Epo (5U/ml) for 0–6h. Membranes were probed with SOCS-1, followed by CIS and then GAPDH. Independent membranes were probed with SOCS-3 and GAPDH

Figure 3

Introduction of Lyn into immature erythroleukemic cell lines promotes maturation, (a) R11, 11RLyn, 11MLyn, J2E-NR and NMLyn cells were unstimulated (white histogram) or stimulated with Epo (5U/ml; black histogram) for 48 h, then hemoglobin synthesis measured by the benzidine staining, (b) [3H]thymidine incorporation of unstimulated (white histogram) or Epo-stimulated (5 U/ml; black histogram) cells is expressed as a per cent relative to unstimulated cells, (c) Cytocentrifuge preparations of R11, 11RLyn and 11MLyn cells in the absence of Epo. Note the enucleating erythroblasts and reticulocytes, indicated by arrows. The bar represents 10 μm. (d) Expression of c-Kit and Ter119 on the surface of R11, 11RLyn and 11MLyn cells was determined by flow cytometry. The gray area represents the background fluorescence, while the black area represents antibody binding, (e) Epo receptor numbers were determined by Scatchard analysis, (f) Cell lysates were immunoblotted with antibodies to NF-E2, GATA-1, EKLF, globin and v-Raf (loading control)

Figure 4

Lyn suppresses constitutive SOCS-1 and SOCS-3 mRNA expression, (a) Northern analysis of poly A⁺ RNA from immature erythroleukemic cell lines R11 and J2E-NR expressing exogenous Lyn (11RLyn, NMLyn) stimulated with Epo (5U/ml) for 0–6h. Membranes were probed with SOCS-1, followed by CIS and then GAPDH. Independent membranes were probed with SOCS-3 and GAPDH. (b) Northern analysis of total mRNA from unstimulated cells, probed with EKLF and GAPDH

Figure 5

Altered JAK/STAT signalling affects SOCS gene profile, (a) J2E cells and those bearing the mutant Δ321 Epo receptor (J-Δ321) and W282R Epo receptor (J-W282R) were unstimulated (white histogram) or stimulated with Epo (0.01 U/ml, striped histogram; 5 U/ml, black histogram) for 48 h and hemoglobin synthesis measured by the benzidine staining, (b) [3H]thymidine incorporation of unstimulated (white histogram) or Epo-stimulated (5 U/ml; black histogram) cells is expressed as a per cent relative to unstimulated cells, (c, d) Cells were stimulated with Epo (5 U/ml) for 0–60min and proteins (5 mg) were immunoprecipitated with anti-JAK2 (c) or anti-STAT5 (d) antibodies, then immunoblotted with antiphosphotyrosine antibodies followed by anti-JAK2 (c) or anti-STAT5 (d) antibodies, (e) Northern analysis of poly A⁺ RNA from cells stimulated with Epo (5 U/ml) for 0–6 h. Membranes were probed with SOCS-1, followed by CIS and then GAPDH. Independent membranes were probed with SOCS-3 and GAPDH

Figure 6

SOCS-1 promoter activity is not Epo-inducible. Schematic representation of the SOCS-1 promoter, putative STAT binding sites (TTCCNNNAA; triangles) and the various promoter constructs. pGL3E-SOCS-1 reporter constructs (10 μg) and 1 μg of pRL-SV40 were electroporated into J2E and J2E-NR cells, then harvested after 48 h of culture, with (black bar) or without (white bar) Epo (5 U/ml). Dual luciferase reporter assays were performed and levels of SOCS-1 promoter activity were expressed relative to the pGL3 control set at 100%

Figure 7

Erythroid progenitors lacking SOCS-1 are hypersensitive to Epo. Fetal liver cells isolated from SOCS-1+/+ (open circle), +/− (triangle) and −/− (closed circle) mice were plated in methylcellulose with varying concentrations of Epo. CFU-E numbers were determined 2 days later. The results shown are expressed as the percentage of the maximal colony numbers for the 1 U/ml Epo plates (+/− standard error; n = 6). Astericks indicate statistically significant (Student’s t-test) differences between −/−and +/− or +/+ cells with P-values displayed