Ischemic injury is invoked as a mechanism contributing to end-organ damage and other complications of sickle cell disease (SCD). However, the intrinsic sensitivity of tissues in SCD to ischemic insults has never been addressed. We examined the effect of renal ischemia in a transgenic mouse expressing human sickle hemoglobin. Twenty-four hours after bilateral, total renal artery occlusion for 15 minutes, transgenic sickle mice exhibited worse renal function and more marked histological injury. With bilateral renal ischemia of greater duration (22.5 minutes), and after 6 hours, transgenic sickle mice exhibited massive vascular congestion, sickling of red blood cells, more marked histological injury in the kidney, and more prominent congestion in the capillary beds in the lungs and heart. Additionally, serum amyloid P-component, the murine homologue of C-reactive protein, was markedly increased in transgenic sickle mice as compared to wild-type mice. Twenty-four hours after bilateral renal ischemia for 22.5 minutes, transgenic sickle mice exhibited 28% mortality, with no mortality observed in any other group. With bilateral renal ischemia of short or long duration, renal expression of caspase-3 was most prominent in transgenic sickle mice subjected to ischemia. Thus, renal ischemia in this murine model induces more severe renal injury and extrarenal complications. We conclude that tissues in SCD exhibit heightened vascular congestion and sensitivity to ischemia and that clinically apparent or silent episodes of ischemia may contribute to the complications of SCD. Vaso-occlusive disease contributes dominantly to the morbidity and mortality of sickle cell disease (SCD).

Early B cell factor (EBF) is a transcription factor essential for specification and commitment to the B cell fate. In this study, we show downregulation of a developmentally regulated cluster of hoxa genes, notably hoxa9, coincides with induction of EBF at the Pro-B cell stage of B cell differentiation. Analysis of the hematopoietic progenitor compartment in Hoxa9−/− mice revealed significantly reduced frequencies and expression levels of Flt3, a cytokine receptor important for lymphoid priming and the generation of B cell precursors (BCPs). We show that Hoxa9 directly regulates the flt3 gene. Chromatin immunoprecipitation analysis revealed binding of Hoxa9 to the flt3 promoter in a lymphoid progenitor cell line. Knockdown of Hoxa9 significantly reduced Flt3 transcription and expression. Conversely, forced expression of Hoxa9 increased Flt3 transcription and expression in a Pro-B cell line that expressed low levels of Flt3. Hoxa9 inversely correlated with ebf1 in ex vivo-isolated bone marrow progenitors and BCPs, suggesting that EBF might function to silence a Hoxa9 transcriptional program. Restoration of EBF function in an EBF−/− cell line induced B lineage gene expression but did not directly suppress hoxa9 transcription, revealing alternate mechanisms of Hoxa9 regulation in BCPs. These data provide new insight into Hoxa9 function and regulation during lymphoid and B cell development. Furthermore, they suggest that failure to upregulate Flt3 provides a molecular basis for the lymphoid/early B cell deficiencies in Hoxa9−/− mice.

The generation of B-cell precursors (BCP) from lymphohematopoietic progenitors (LHP) in bone marrow is dependent on signals provided by the receptor tyrosine kinase Flt3 and its ligand, . Mice deficient in FL exhibit striking reductions in LHP and BCP. Currently, the mechanism by which Flt3 regulates lymphoid lineage/B-cell development is unknown. Here, we show that haploinsufficiency of FL (FL 1/À ) reduced the numbers of LHP, common lymphoid progenitors, and pro-B cells, suggesting that FL levels set a threshold for B lymphopoiesis. Limiting dilution analysis confirmed reduced BCP frequency in FL 1/À mice. Real-time PCR of LHP from FL 1/À animals showed increased transcripts of the B lineage inhibitor id1. However, targeted deletion of id1 did not restore the lymphoid/B lineage deficiencies in FL À/À mice, supporting Id1-independent mechanisms. BrdU incorporation studies established that FL is not essential for the proliferation of Flt3 1 multipotential progenitors. Analysis of FL À/À progenitors expressing low levels of Flt3 revealed decreased levels of the pro-survival factor Mcl1. Consequently, the Flt3 1 LHP progeny of Flt3 low LSK 1 cells exhibited increased Annexin V staining. Together, these data suggest that Flt3 signaling initiates a cascade of events in Flt3 low precursors that promote the survival of LHP from which BCP are derived.Key words: Apoptosis . B-cell development . Cell differentiation . Flt3-ligand IntroductionLymphopoiesis is a stepwise process dependent on signals from the microenvironment. In bone marrow (BM), differentiating multipotential progenitors (MPP) integrate microenvironmental signals to generate lymphoid progenitors from which B-cell precursors (BCP) are derived. Fms-like tyrosine kinase (Flt3) and its ligand, , are critical regulators of lymphoid progenitors and their B lineage progeny. However, the mechanism by which Flt3 signaling regulates the generation of BCP from lymphohematopoietic progenitors (LHP) in vivo is not well understood.HSC and MPP can be identified in BM by a lack of lineage markers (Lin -), expression of stem cell antigen-1 (Sca-1), and high levels of the receptor tyrosine kinase, c-kit [1]. These cells are collectively termed LSK 1 . LHP are Flt3 1 MPP that are functionally distinct from Flt3 À/low MPP in having lost megakaryocyte/erythroid differentiation potential [2]. LHP can be distinguished within MPP by a variety of criteria including GFP knocked into the recombinase-activating gene one (RAG1) coding region [3,4]. LHP defined by these criteria lack surface expression of the IL-7R. Another way to distinguish LHP in vivo is differential expression of Flt3 and VCAM-1 [5]. The ability to distinguish LHP within MPP provides a means to identify and characterize cellular and molecular circuits that regulate lymphoid lineage development.Abundant experimental evidence suggests that the molecular circuitry initiating lymphoid lineage specification within MPP ResultsHaploinsufficiency of FL reduces LHP and BCP Exogenous administration of FL in vivo increases ...

Immediate early gene X-1 (IEX-1) modulates apoptosis, cellular growth, mechanical strain-induced cardiac hypertrophy, and vascular intimal hyperplasia. To determine how IEX-1 alters apoptosis, we performed yeast two-hybrid studies using IEX-1 as the "bait" protein, and examined interactions between IEX-1 and proteins expressed by a human kidney cDNA expression library. We found that IEX-1 interacts with several proteins of which at least four are known to play a role in the regulation of apoptosis: (1) calcium-modulating cyclophilin ligand; (2) tumor necrosis factor-related apoptosisinducing ligand (tumor necrosis factor superfamily, member 10); (3) ML-1 myeloid cell leukemia gene encoded protein; and (4) BAT3, a gene present in the major histo-compatibility complex. Our data suggest that IEX-1 may regulate apoptosis by directly interacting with various proteins involved in the control of apoptotic pathways. KeywordsIEX-1; gly96; 1,25-Dihydroxyvitamin D; ApoptosisThe human immediate early gene X-1 (IEX-1, also known as Dif2, and murine orthologs gly96 and PRG1) plays a role in the control of apoptosis and cellular growth [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The gene (gly96) was initially identified in mouse fibroblasts as a serum-regulated, glycosylated, immediate early gene [1]. The messenger RNA of a similar gene (p22/PRG1) is induced in rat pancreatic cells by pituitary adenylate cyclase activating peptide (PACAP) [20]. The human ortholog of gly96 and PRG1, IEX-1, was identified as an X-radiation induced in fibroblasts by Kondratyev et al. [2], and as a UV-radiation and 1α, 25-dihydroxyvitamin D 3 -regulated gene in human keratinocytes [3,4].Increased expression of IEX-1 is associated with an increase in the growth rate of keratinocytes and HeLa cells, and disruption of IEX-1 gene expression in HeLa cells and 293 cells is associated with a decrease in cellular proliferation [3,7,8,21]. IEX-1 has been shown to be induced in cardiomyocytes and vascular smooth muscle cells by mechanical stretch, and overexpression of IEX-1 in vascular smooth muscle cells protects against stretch-induced hypertrophy [9,15] IEX-1 is regulated by a variety of factors. IEX-1 expression is increased by serum, X-, and UV-irradiation, growth factors such as epidermal growth factor, inflammatory stimuli such as lipopolysaccharide and ceramide, retinoids such as all-trans-retinoic acid or cis-retinoic acid, and by over-expression of Sp1 in cells [1][2][3]6,10,11,20,[24][25][26][27][28][29][30][31]. The gene is repressed by steroid hormones such as 1α, 25-dihydroxyvitamin D 3 and by over-expression of p53 [3,4,30,32]. 1α, 25-Dihydroxyvitamin D 3 also results in a redistribution of IEX-1 from the nucleus into the peri-nuclear and cytoplasmic space. The ratio of Sp1 to p53 within the cell appears to regulate the amount of IEX-1 expression present within the cell [30]. Slight modifications of IEX-1 transcription are observed by mutating putative elements for p300, Sox, NFκB, and AP4 within the prom...

gly96/IEX 1 is a growth- and apoptosis-regulating, immediate early gene that is widely expressed in epithelial and vascular tissues. In vascular tissues, expression of the gene is induced by mechanical stretch, and overexpression of the gene prevents injury-induced vascular smooth muscle hypertrophy and neointimal hyperplasia. We now show that deletion of the gly96/IEX-1 gene in mice is associated with development of elevated blood pressure, cardiac hypertrophy, and diminished fractional shortening of the left ventricle. Systolic blood pressure in conscious male gly96/IEX-1-/- mice is 20-25 mmHg higher than in gly96/IEX-1+/+ mice. Serum and/or urine concentrations of sodium, potassium, creatinine, angiotensin II, corticosterone, aldosterone, epinephrine, norepinephrine, prostaglandin E2, thromboxane B2, prostaglandin-6-keto-1alpha, nitrites and nitrates, cAMP, and cGMP are normal in gly96/IEX-1-/- mice. Alterations in dietary sodium intake do not alter blood pressure in gly96/IEX-1-/- mice. Aortic mRNAs for endothelial nitric oxide synthase, guanylate cyclase-alpha, and cGMP kinase-1 are increased in gly96/IEX-1-/- mice. Treatment with Nomega-nitro-L-arginine methyl ester or L-arginine does not alter blood pressure in gly96/IEX-1-/- mice. Gly96/IEX-1-/- mice respond to infused sodium nitroprusside with decrements in blood pressure similar to those seen in wild-type littermate mice. In contrast to gly96/IEX-1 transgenic mice that have abnormalities in immune function, gly96/IEX-1-/- mice have normal lymphoid tissue architecture and a normal complement of T and B cells in lymphoid tissues. Ablation of the gly96/IEX-1 gene results in hypertension and cardiac hypertrophy, suggesting a novel role for this gene in cardiovascular physiology.