The effects of GR on mRNA translation have mostly been studied in myocytes where its activation induces REDD1 that, by suppressing mTOR, reduces 4E-BP1(S65) and S6KA1(S235/236) inhibiting initiation of mRNA translation and protein biosynthesis (Wang JBC 2006;281:39128). It is then counterintuitive that GR agonists (glucocorticoids, GC) are effective in 60% of patients with DBA, a inherited form of pure RBC aplasia induced by mutations that impair protein biosynthesis by reducing the initial steps of mRNA translation (mRNA binding to the ribosomal subunit 40S and ribosme assembly). To address this paradox, we analyzed the phosphoproteomic profiling of erythroid cells generated in vitro either with or without the GR agonist dexamethasone by adult blood (AB) and cord blood (CB) (http://capmm.gmu.edu/data) for proteins affecting mRNA translation. By contrast with muscle cells, Dex significantly (p=0.049) increased S6KA1(S235/236) (fold change (FC)=4), eIF4G(S1108)(FC=4.16) and mTOR(S2448)(FC=1.75) in erythroid cells from CB. However, Dex significantly decreased RSK3(T356/S360)(FC=0.45) and MSK1(S360)(FC=0.52) and increased DEPTOR(FC=1.51) in AB. These results indicate that GR exerts, respectively, positive and negative regulatory functions on mRNA translation in fetal and adult erythroid cells.
We previously reported that the single nucleotide polymorphism (SNP) rs6198 in NR3C1, the gene encoding GR, is present at frequency greater than normal in patients with DBA (Varricchio Blood 2011;118:473). This observation and the results described above led us to investigate whether additional clinically relevant NR3C1 SNPs are associated with disease manifestation in DBA. By polymerase chain reaction single strand conformation polymorphism analyses, we determined the frequency of 8 SNPs in 101 DBA patients, 67 from the Italian Registry and 34 from other European registries. Healthy volunteers (n=34) were analyzed as control. The SNPs investigated are: rs10482605 that decreases GRα and predicts GC resistance in major depression; rs10482616, rs7701443, rs6189/rs6190, rs860457, rs6198, that have unknown biological effects but predict GC resistance in Crohn's Disease; rs6196, that enhances expression of the dominant negative GR-β isoform predisposing to autoimmune diseases, myeloproliferative neoplasms and DBA, while reducing risks of diabetes in Cushing's Syndrome and rs33388/rs33389 that increase expression of GRγ which binds GC 10-times less efficiently than GRα and has not been associated with human diseases as yet. Cases versus control analyses suggests that the frequency of rs860457 (Table 1) and rs33388 (p=0.0689 by categorical analyses, not shown) is increased in this cohort of DBA patients. The reduced significance level of this observation may be due to the low numbers (34) of healthy controls used as comparison.
Demographic, causal mutations and disease manifestation (GR responsiveness and age of anemia onset) are known for patients from the Italian Registry only. The patients were 55.6% females and 44.4% males and their average age at diagnosis was 9.14±24.56 months. The causal mutations were RPL5 (n=12), RPL11 (n=8); RPS26 (n=12), RPS19 (n=28), RPS17 (n=1) and unknown (n=6). Treatment outcome is known for 58 of the 67 patients: 14 responded, 41 did not respond while three patients underwent spontaneous remission and were excluded from the analyses. Age of disease manifestation is known for 57 patients: 27 (29.82) displayed the disease before and 40 (70.18%) after 2 months. Gender and causal mutations were neither associated with response nor with age of disease manifestation. None of the SNPs met the threshold in the response vs non-responsive groups (Table 1). However, two SNPs (rs6196, p=0.0509 and rs33386, p=0.1249) met the threshold criteria in the age of disease manifestation analyses (Table 1). Of note rs6196 predicts GC resistance in Crohn's disease and rs33386 favors expression of GRγ. No significant association was observed when SNPs that met the threshold were analyzed in a multivariable model.
These results indicate that GR promotes protein biosynthesis in fetal but not in adult erythroid cells. The frequency of rs860457 which impairs GR functions is increased in DBA patients and rs6196 and rs33388 may be correlated with age to disease manifestation. These observations require further analyzes with larger cohorts of controls and/or patients.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.