CREB is a critical regulator of normal hematopoiesis and leukemogenesis

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation

Influence of increased metabolic rate on [^(13)C]bicarbonate washout kinetics

The effect of changes in metabolic rate on the dynamics of CO_2 exchange among its various compartments in the human body is not well understood. We examined CO_2 dynamics in six healthy male subjects using an intravenous bolus of [^(13)C]bicarbonate. Subjects were studied while resting, during light exercise [50% of the lactate threshold (LT), 3-4 times resting O_2 uptake (V_(O_2))], and during moderate exercise (95% of the LT, 6 times resting V_(O_2)). The sum of three exponential terms well described the washout of ^(13)CO_2 in exhaled breath both at rest and during each exercise level despite substantial increases in metabolic rate accompanying the exercise studies. Average recovery of ^(13)C label rose from 67% during rest to 80% during light and moderate exercise (P less than 0.01). The estimate of CO_2 elimination (V_(CO_2)) calculated from the washout parameters and corrected for recovery was in very good agreement with the V_(CO_2) directly measured simultaneously breath by breath (r = 0.993, SE for V_(CO_2) = 0.079 l/min). By use of a three-compartment mammillary model, the quantity of CO_2 in the central pool (Q_1) doubled from rest to light exercise (233 +/- 60 to 479 +/- 76 mmol, P less than 0.01) but did not change further with moderate exercise (458 +/- 74 mmol). Rate constants for exchange between pools and for irreversible loss from the system tended to increase with metabolic rate, but there was large variation in the responses. We conclude that the compartmental dynamics of CO_2 transport and storage are very sensitive to changes in metabolic rate induced by exercise

Oral [^(13)C]bicarbonate measurement of CO_2 stores and dynamics in children and adults

During exercise, less additional CO_2 is stored per kilogram body weight in children than in adults, suggesting that children have a smaller capacity to store metabolically produced CO_2. To examine this, tracer doses of [^(13)C]bicarbonate were administered orally to 10 children (8-12 yr) and 12 adults (25-40 yr) at rest. Washout of ^(13)CO_2 in breath was analyzed to estimate recovery of tracer, mean residence time (MRT), and size of CO_2 stores. CO_2 production (VCO_2) was also measured breath by breath using gas exchange techniques. Recovery did not differ significantly between children [73 ± 13% (SD)] and adults (71 ± 9%). MRT was shorter in children (42 ± 7 min) compared with adults (66 ± 15 min, P < 0.001). VCO_2 per kilogram was higher in the children (5.4 ± 0.9 ml.min^(-1).kg^(-1)) compared with adults (3.1 ± 0.5, P less than 0.0001). Tracer estimate of CO_2 production was correlated to VCO_2 (r = 0.86, P < 0.0001) and when corrected for mean recovery accurately predicted the VCO_2 to within 3 ± 14%. There was no difference in the estimate of resting CO_2 stores between children (222 ± 52 ml CO_2/kg) and adults (203 ± 42 ml CO_2/kg). We conclude that orally administered [^(13)C]bicarbonate can be used to assess CO_2 transport dynamics. The data do not support the hypothesis of lower CO_2 stores under resting conditions in children

Increased abscess formation and defective chemokine regulation in CREB transgenic mice.

Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression in vivo, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNFα protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1β (Interleukin)-1β was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNFα, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFκB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation in vivo and aberrant cytokine and chemokine response, and neutrophil function in vitro