Alternative pre-mRNA splicing switches modulate gene expression in late erythropoiesis.

Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.

A proposed vestigial translation initiation motif in VP1 of hepatitis A virus.

The internal ribosome entry site (IRES) of picornaviruses has a 3' polypyrimidine tract (PPT) 16-24 bases upstream of an AUG triplet (PPT/AUG motif). This motif is critical in determining the efficiency of cap-independent translation. HAV has a conserved PPT/AUG motif consisting of a nine base sequence (AGGUUUUUC) 23 bases upstream of the preferred AUG start codon. This HAV-specific PPT/AUG motif is repeated and conserved in VP1 of HAV, but not of other picornaviruses. We proposed that the PPT/AUG motif in the open reading frame initiated translation and/or had an impact on the life cycle of the virus. In vitro translation of mutant bicistronic mRNAs and growth in cell culture of mutant viruses provided no evidence that the VP1 PPT/AUG motif had any impact on either translation or growth. HAV differs from other picornaviruses in its inefficient growth in cell culture. Since the HAV-specific PPT/AUG motif is found in only 1 in 300,000 reported viral sequences outside the hepatovirus genus, this motif may be a vestigial translation initiation element and may have played a role in determining the unusual phenotype of HAV.

Loss of specific chaperones involved in membrane glycoprotein biosynthesis during the maturation of human erythroid progenitor cells.

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34(+) erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61alpha), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.

Growth control of multiple myeloma cells through inhibition of glycogen synthase kinase-3.

Anti-apoptotic pathways play a central role in the survival of multiple myeloma cells. The contribution of PI3-kinase and Akt kinase in mediating myeloma cell survival is well established although the role of glycogen synthase kinase-3 (GSK3) is less defined. In this study we determined the contribution of GSK3 in growth regulation of myeloma cells. We treated six different multiple myeloma cell lines with a Thiadiazolidinone (TDZD), a non-competitive inhibitor of GSK3 and determined its effects on proliferation and apoptosis. In addition we determined the activation of forkhead transcription factors (FOXO) in response to TDZD. TDZD inhibited proliferation and induced apoptosis of all myeloma cell lines. TDZD was also effective in inducing apoptosis of primary myeloma cells whereas CD34 positive normal hematopoietic cells were protected from apoptosis. Furthermore, TDZD-mediated inhibition of GSK3 resulted in dephosphorylation and activation of FOXO3a. In primary myeloma cells FOXO transcription factors were highly phosphorylated where as the levels of GSK3 phosphorylation was quite low. The levels of the pro-apoptotic proteins Fas ligand (FasL) and IkappaBalpha increased after treatment with TDZD in myeloma cell lines. These studies provide the basis for further testing of GSK3 inhibitors in the clinical setting.

Osteopontin regulates actin cytoskeleton and contributes to cell proliferation in primary erythroblasts.

Erythropoietin and stem cell factor are the key cytokines that regulate early stages of erythroid differentiation. However, it remains undetermined whether additional cytokines also play a role in the differentiation program. Here, we report that osteopontin (OPN) is highly expressed and secreted by erythroblasts during differentiation. We also demonstrate that OPN-deficient human and mouse erythroblasts exhibit defects in F-actin filaments, and addition of exogenous OPN to OPN-deficient erythroblasts restored the F-actin filaments in these cells. Furthermore, our studies demonstrate that OPN contributes to erythroblast proliferation. OPN knock-out male mice exhibit lower hematocrit and hemoglobin levels compared with their wild-type counterparts. We also show that OPN mediates phosphorylation or activation of multiple proteins including Rac-1 GTPase and the actin-binding protein, adducin, in human erythroblasts. In addition, we show that the OPN effects include regulation of intracellular calcium in human erythroblasts. Finally, we demonstrate that human erythroblasts express CD44 and integrins beta1 and alpha4, three known receptors for OPN, and that the integrin beta1 receptor is involved in transmitting the proliferative signal. Together these results provide evidence for signal transduction by OPN and contribution to multiple functions during the erythroid differentiation program in human and mouse.

The immunodominant CD8+ T cell epitope region of Theiler's virus in resistant C57BL/6 mice is critical for anti-viral immune responses, viral persistence, and binding to the host cells.

Theiler's virus infection induces an immune-mediated demyelinating disease, providing a relevant animal model of human multiple sclerosis. VP2(121-130)-specific CD8+ T cells in resistant H-2b mice account for the majority of CNS-infiltrating CD8+ T cells. To further study the role of the CD8(+) T cells, we generated a panel of mutant viruses substituted with L, G, or T at the anchor residue (M130) of the VP2(121-130) epitope. M130L virus (M130L-V) with a substitution of M with L displayed similar properties as wild-type virus (WT-V). However, M130G-V and M130T-V could not establish a persistent infection in the CNS. The level of both virus-specific CD8+ and CD4+ T cell responses is significantly reduced in mice infected with these variant viruses. While all mutant and wild-type viruses replicate comparably in BHK cells, replication of M130G-V and M130T-V in macrophages was significantly lower compared to those infected with WT-V and M130L-V. Interestingly, these mutant viruses deficient in replication in primary mouse cells showed drastically reduced binding ability to the cells. These results suggest that the anchor residue of the predominant CD8+ T cell epitope of TMEV in resistant mice is critical for the virus to infect target cells and this deficiency may result in poor viral persistence leading to correspondingly low T cell responses in the periphery and CNS. Thus, selection of the cellular binding region of the virus as the predominant epitope for CD8+ T cells in resistant mice may provide a distinct advantage in controlling viral persistence by preventing escape mutations.

Friend of GATA-1-independent transcriptional repression: a novel mode of GATA-1 function.

The GATA-1-interacting protein Friend Of GATA-1 (FOG-1) is essential for the proper transcriptional activation and repression of numerous GATA-1 target genes. Although FOG-1-independent activation by GATA-1 has been described, all known examples of GATA-1-mediated repression are FOG-1 dependent. In the GATA-1-null G1E cell line, estrogen receptor ligand binding domain (ER) chimeras of either wild-type GATA-1 or a FOG-1-binding defective mutant of GATA-1 repressed several genes similarly upon activation with beta-estradiol. Repression also occurred in a FOG-1-null cell line expressing ER-GATA-1 and during ex vivo erythropoiesis. At the Lyl1 and Rgs18 loci, we found highly restricted occupancy by GATA-1 and GATA-2, indicating that these genes are direct targets of GATA factor regulation. The identification of genes repressed by GATA-1 independent of FOG-1 defines a novel mode of GATA-1-mediated transcriptional regulation.

Rhinovirus 16 3C protease induces interleukin-8 and granulocyte-macrophage colony-stimulating factor expression in human bronchial epithelial cells.

Rhinovirus (RV), a member of the Picornaviridae family, accounts for many virus-induced asthma exacerbations. RV induces airway cell chemokine expression both in vivo and in vitro. Because of the known interactions of proteases with cellular functions, we hypothesized that RV 3C protease is sufficient for cytokine up-regulation. A cDNA encoding RV16 3C protease was constructed by PCR amplification and transfected into 16HBE14o- human bronchial epithelial cells. 3C protease induced expression of both IL-8 and GM-CSF, as well as transcription from both the IL-8 and GM-CSF promoters. 3C expression also induced activator protein 1 and NF-kappaB transcriptional activation. Finally, mutation of IL-8 promoter AP-1 and NF-kappaB promoter sequences significantly reduced 3C-induced responses. Together, these data suggest expression of RV16 3C protease is sufficient to induce chemokine expression in human bronchial epithelial cells, and does so in an AP-1- and NF-kappaB-dependent manner.