CXCR4 expression and biologic activity in acute myeloid leukemia are dependent on oxygen partial pressure.

The CXCR4/SDF-1 axis has been studied extensively because of its role in development and hematopoiesis. In acute myeloid leukemia (AML), elevated expression of CXCR4 has been shown to correlate with shortened survival. Hy-poxia increases CXCR4 in several tumor models, but the impact of reduced O(2) partial pressure (pO(2)) on expression and biologic function of CXCR4 in AML is unknown. We determined pO(2) in bone marrows of AML patients as 6.1% (+/-1.7%). At this pO(2), CXCR4 surface and total expression were up-regulated within 10 hours in leukemic cell lines and patient samples as shown by Western blotting, fluorescence-activated cell sorting, and microscopy. Interestingly, hypoxic cells failed to internalize CXCR4 in response to SDF-1, and upon reoxygenation at 21% O(2), surface and total expression of CXCR4 rapidly decreased independent of adenosine triphosphate or proteasome activity. Instead, increased pO(2) led to alteration of lipid rafts by cholesterol depletion and structural changes and was associated with increased shedding of CXCR4-positive microparticles, suggesting a novel mechanism of CXCR4 regulation. Given the importance of CXCR4 in cell signaling, survival, and adhesion in leukemia, the results suggest that pO(2) be considered a critical variable in conducting and interpreting studies of CXCR4 expression and regulation in leukemias.

Factor H binds to platelet integrin alphaIIbbeta3.

Factor H is a plasma protein that regulates activity of the alternative complement pathway in plasma and on cell surface. Binding of factor H to a cell surface protects that cell against complement-induced damage. Factor H binds to glycosoaminoglycans, surface-immobilized C3b, L selectin, and integrins such as alpha(M)beta(1) (a direct binding) or alpha(V)beta(3) (an indirect binding mediated through intermediary plasma proteins). We studied the binding of factor H to platelets and to integrin alpha(IIb)beta(3) (glycoprotein IIb-IIIa), the most abundant integrin on platelets. We measured binding of purified factor H to platelets or heterologous cells expressing recombinant alpha(IIb)beta(3) using flow cytometry. We also measured binding of factor H to alpha(IIb)beta(3) in cell free systems using either surface plasmon resonance or enzyme-linked immunosorbent assay. We found that factor H directly binds to alpha(IIb)beta(3) and this binding has a dissociation constant (Kd) of 131 +/- 60.9 nM and is not dependent on active conformation of alpha(IIb)beta(3) or on the presence of cations. Considering the high affinity of this interaction, the abundance of alpha(IIb)beta(3) integrin on platelets, and the high concentration of factor H in plasma, alpha(IIb)beta(3) provides a constitutive presence of factor H on platelets. Activation of platelets increases platelet-bound factor; however, this increase in binding of factor H cannot be explained by additional binding of factor H to alpha(IIb)beta(3) and perhaps involves other binding sites for factor H on platelets.

Epigenetic control of PRV-1 expression on neutrophils.

OBJECTIVE: Polycythemia rubra vera-1 (PRV-1) is a GPI-linked protein that is expressed on a subgroup of neutrophils. The number of PRV-1-expressing neutrophils increases in pregnancy and sepsis, or after administration of granulocyte colony-stimulating factor. Expression of the PRV-1 gene is also increased in patients with polycythemia vera (PV) and essential thrombocythemia (ET). We investigated whether DNA methylation of the PRV-1 gene has a role in regulation of transcription and expression of the PRV-1 protein. METHODS: We compared the level of methylation of the PRV-1 gene and expression of the PRV-1 mRNA in normal neutrophils expressing PRV-1 to those that are PRV-1-negative. We also studied PRV-1 methylation and mRNA expression in patients with Philadelphia chromosome-negative myeloproliferative disorders and in an in vitro model of DNA demethylation. RESULTS: We found that methylation of CpG dinucleotides close to initiation codon of the PRV-1 gene was inversely related to expression of PRV-1 in normal neutrophils. Furthermore, overexpression of the PRV-1 gene in PV and ET is associated with a decrease in methylation of this gene. Among patients with PV and ET, methylation of the PRV-1 gene is also inversely correlated with the presence of the JAK2(V617F) somatic mutation. In an in vitro model, exposure of KG1 and KG1a cells to a DNA demethylating agent caused a decrease in methylation of the PRV-1 gene and increased its mRNA level. CONCLUSION: DNA methylation regulates PRV-1 expression under physiologic and pathologic conditions.

The effect of the JAK2 V617F mutation on PRV-1 expression.

JAK2(V617F)an acquired mutation of JAK2, is present in a majority of patients with polycythemia vera and to a lesser extent among patients with the other myeloproliferative disorders. We analyzed the effect of JAK2(V617F) on the expression of polycythemia rubra vera 1(PRV-1), using an in vitro model. Compared to wild-type JAK2, the presence of JAK2(V617F) increased both PRV-1 protein and mRNA levels in murine myeloid cells. A JAK2 inhibitor eliminated the V617F-induced increase in PRV-1 expression.

Paradoxical upregulation of tumor suppressor protein p53 in serum-stimulated vascular smooth muscle cells: a novel negative-feedback regulatory mechanism.

BACKGROUND: The proliferative response of vascular smooth muscle cells (VSMCs) to various growth stimuli is critical for atherosclerosis and postangioplasty restenosis. Although tumor suppressor protein p53 plays a critical role in the elimination of cancerous cells, recent genetic studies have indicated that it also protects against atherosclerosis and restenosis. METHODS AND RESULTS: We examined the levels of p53 protein in normal VSMCs before and after serum stimulation. The p53 protein levels increased robustly on stimulation. Upregulated p53 protein was capable of binding to the p53 consensus sequence, as shown by electrophoretic mobility shift assay. In addition, p53 upregulation was associated with increases in the transcript and protein levels of p21WAF1/CIP1 and Bax, as shown by real-time reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Furthermore, the upregulation of p21WAF1/CIP1 and Bax was followed by cell-cycle arrest and apoptosis induction, as shown by 5-bromo-2'-dUTP incorporation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining, respectively. Finally, double-staining analyses showed that the majority of p53-expressing cells also expressed p21WAF1/CIP1 and Bax proteins. CONCLUSIONS: p53 protein expression in quiescent VSMCs is paradoxically increased by application of a growth stimulus. Through the mediation of p21WAF1/CIP1 and Bax, the induced p53 protein negatively regulates the growth of dividing VSMCs, thereby minimizing the inappropriate accumulation of VSMCs. Therefore, p53 may be a negative regulator of VSMC growth.

Adenoviral gene transfer of fortilin attenuates neointima formation through suppression of vascular smooth muscle cell proliferation and migration.

BACKGROUND: Fortilin, a recently characterized nuclear antiapoptotic factor structurally distinct from inhibitor of apoptosis proteins (IAPs) and Bcl-2 family member proteins, has been suggested to be involved in cell survival and regulation of apoptosis within the cardiovascular system. In this continued investigation, we characterized the influence of adenovirus-mediated fortilin (Ad-fortilin) gene delivery on vascular remodeling after experimental angioplasty. METHODS AND RESULTS: Vessel wall expression of Ad-fortilin or adenoviral luciferase (Ad-luc) was demonstrated 72 hours and 14 days after rat carotid artery (CA) balloon angioplasty. Morphometric analyses 14 days after injury revealed significantly diminished neointima development in the Ad-fortilin-treated CAs compared with Ad-luc or PBS controls, with no changes in medial wall morphometry observed between the 3 groups. The Ad-fortilin-treated CAs demonstrated a 50% reduction in medial wall proliferating cell nuclear antigen (PCNA) labeling after 72 hours, with significantly reduced neointimal and medial wall PCNA labeling and cell counts after 14 days. Terminal dUTP nick-end labeling results and morphological changes characteristic of programmed cell death suggest a trend toward reduced apoptosis in the fortilin-transfected balloon-injured vessels compared with Ad-luc injured controls. Temporal analysis of human aorta smooth muscle cell (SMC) proliferation demonstrated a marked time-dependent inhibition in Ad-fortilin treated SMCs without the influence of elevated apoptosis. Thymidine incorporation was significantly inhibited in the Ad-fortilin-treated cells compared with Ad-luc controls. Ad-fortilin transfected SMCs also demonstrated significantly decreased migration compared with Ad-luc controls. CONCLUSIONS: These cumulative results suggest that the novel antiapoptotic protein fortilin may play important redundant pathophysiological roles in modulating the vascular response to experimental angioplasty through suppression of SMC proliferation and migration concomitant with reduction of vessel wall apoptosis.

Expression of polycythemia rubra vera-1 decreases the dependency of cells on growth factors for proliferation.

An increase in the level of polycythemia rubra vera -1 (PRV-1) mRNA has been reported in some myeloproliferative disorders. We have studied the effects of PRV-1 on cell proliferation and cell survival. In cell growth assays, the number of heterologous cells expressing PRV-1 increased faster than sham-transfected cells, a difference that was more pronounced in serum-free media. Even after 5 days of exposure to serum-free media, cells expressing PRV-1 continued to proliferate, whereas the control cells ceased to proliferate. We conclude that PRV-1 is a pro-proliferation molecule, and hypothesize that its overexpression may have a role in the pathogenesis of myeloproliferative disorders.

The critical role of the intrinsic VSMC proliferation and death programs in injury-induced neointimal hyperplasia.

Postangioplasty and in-stent restenosis remain ominous problems in percutaneous coronary intervention where good animal models of restenosis proneness and resistance are needed. We accidentally discovered that the carotid arteries (CAs) of the Harlan and Sasco substrains of Sprague-Dawley rats display drastically different restenosis phenotypes following balloon-induced endothelial denudation. When subjected to balloon injury, Sasco CAs exhibited significantly larger neointimal mass than did Harlan CAs at both days 14 and 32, as evidenced by a higher intima-to-media ratio and a greater number of intimal cells in Sasco CAs. This was due to a greater cell proliferation and to a less vigorous apoptosis of Sasco neointima, as assessed by 5-bromo-2'-deoxyuridine and terminal deoxynucleotidyl transferase-deoxyuridine nick-end labeling staining, respectively. At a cellular level, whereas vascular smooth muscle cells (VSMCs) isolated from Sasco and Harlan CAs were identical in morphology and in propensity to migrate, Sasco VSMCs proliferated more robustly and died far less, suggesting that under the exact same microenvironment, Sasco and Harlan VSMCs respond to growth and noxious stimuli in a drastically different fashion and that Sasco's significantly more robust neointimal proliferation after vascular injury in vivo can be accounted for by these intrinsic differences in VSMCs of these substrains in vitro. Sasco and Harlan Sprague-Dawley rats as well as VSMCs from these rats will prove to be powerful tools to study genes involved in the pathogenesis of restenosis.

Profound negative regulatory effects by resveratrol on vascular smooth muscle cells: a role of p53-p21(WAF1/CIP1) pathway.

We investigated the role of resveratrol, a polyphenol rich in red wine, in cell cycle progression and apoptosis of vascular smooth muscle cells (VSMCs). Resveratrol inhibited the growth of human aortic VSMCs at concentrations as low as 1 microM. This was due to the profound dose-dependent inhibition of DNA synthesis by resveratrol. DNA synthesis was more effectively inhibited when cells were pretreated with resveratrol. Resveratrol caused a dose-dependent increase in intracellular p53 and p21(WAF1/CIP1) levels. At lower concentrations (6.25-12.5 microM), resveratrol effectively blocked cell cycle progression of serum-stimulated VSMCs without inducing apoptosis, while the higher concentration of resveratrol (25 microM) selectively induced apoptosis in the same VSMCs. Intriguingly, however, the same high concentration of resveratrol could not induce apoptosis in quiescent VSMCs. These differential biological effects of resveratrol on quiescent and proliferating VSMCs suggest that resveratrol may be capable of selectively eliminating abnormally proliferating VSMCs of the arterial walls in vivo.

Physical and functional interaction between myeloid cell leukemia 1 protein (MCL1) and Fortilin. The potential role of MCL1 as a fortilin chaperone.

Myeloid cell leukemia 1 protein (MCL1) is an anti-apoptotic protein that is structurally related to Bcl-2. Unlike other Bcl-2 family proteins that are constitutively expressed, MCL1 is inducibly expressed in cells that are recently exposed to growth and differentiation stimuli. Here, we report the identification of fortilin as a novel MCL1-interacting protein by screening of a yeast two-hybrid library with MCL1 as bait. Fortilin specifically interacted with MCL1 both in vitro and in vivo. The intracellular localization of fortilin was predominantly nuclear and identical to that of MCL1, as shown by immunostaining and confocal microscopy analysis. Fortilin, like MCL1, was rapidly inducible in serum-stimulated human aortic vascular smooth muscle cells. Although the depletion of intracellular fortilin by small interfering RNA (siRNA) against fortilin (siRNA-fortilin) did not affect intracellular MCL1 level, the depletion of intracellular MCL1 by siRNA-MCL1 was associated with the significant reduction of the fortilin protein level, without affecting the fortilin transcript numbers. In addition, a pulse-chase experiment showed that the depletion of MCL1 by siRNA-MCL1 was associated with the rapid degradation of fortilin protein, which was found quite stable in the presence of MCL1. Furthermore, the half-life of fortilin(R21A), a point mutant of fortilin lacking the binding to MCL1, was significantly shorter than that of wild-type fortilin as shown by a pulse-chase experiment. These data suggest that MCL1, in addition to being an anti-apoptotic molecule, serves as a chaperone of fortilin, binding and stabilizing fortilin in vivo. Taken together with our previous observation that fortilin overexpression prevents cells from undergoing apoptosis (Li, F., Zhang, D., and Fujise, K. (2001) J. Biol. Chem. 276, 47542-47549), it is likely that MCL1, an anti-apoptotic protein inducible by growth and differentiation stimuli, stabilizes another anti-apoptotic protein fortilin maximizing the prosurvival environment in cells.