Mobilization of hematopoietic stem cells (HSCs) from bone marrow (BM) to peripheral blood (PB) by cytokine granulocyte colony-stimulating factor (G-CSF) or the chemical antagonist of CXCR4, AMD3100, is important in the treatment of blood diseases. Due to clinical conditions of each application, there is a need for continued improvement of HSC mobilization regimens. Previous studies have shown that genetic ablation of the Rho GTPase Cdc42 in HSCs results in their mobilization without affecting survival. Here we rationally identified a Cdc42 activity-specific inhibitor (CASIN) that can bind to Cdc42 with submicromolar affinity and competitively interfere with guanine nucleotide exchange activity. CASIN inhibits intracellular Cdc42 activity specifically and transiently to induce murine hematopoietic stem/progenitor cell egress from the BM by suppressing actin polymerization, adhesion, and directional migration of stem/progenitor cells, conferring Cdc42 knockout phenotypes. We further show that, although, CASIN administration to mice mobilizes similar number of phenotypic HSCs as AMD3100, it produces HSCs with better long-term reconstitution potential than that by AMD3100. Our work validates a specific small molecule inhibitor for Cdc42, and demonstrates that signaling molecules downstream of cytokines and chemokines, such as Cdc42, constitute a useful target for long-term stem cell mobilization.

Whether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage.

Using an unbiased genetic screen in mice, Hartmut Geiger et al. found that the thrombomodulin–activated protein C pathway, which controls blood coagulation among other functions, also protects from radiation damage. Administration of a recombinant variant of thrombomodulin or of recombinant activated protein C protected mice from total body irradiation, pointing to potential therapeutic applications. Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy1,2. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)–activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

Aged hematopoietic stem cells (HSCs) are impaired in supporting hematopoiesis. The molecular and cellular mechanisms of stem cell aging are not well defined. HSCs interact with nonhematopoietic stroma cells in the bone marrow forming the niche. Interactions of hematopoietic cells with the stroma/microenvironment inside bone cavities are central to hematopoiesis as they regulate cell proliferation, self-renewal, and differentiation. We recently hypothesized that one underlying cause of altered hematopoiesis in aging might be due to altered interactions of aged stem cells with the microenvironment/niche. We developed time-lapse 2-photon microscopy and novel image analysis algorithms to quantify the dynamics of young and aged hematopoietic cells inside the marrow of long bones of mice in vivo. We report in this study that aged early hematopoietic progenitor cells (eHPCs) present with increased cell protrusion movement in vivo and localize more distantly to the endosteum compared with young eHPCs. This correlated with reduced adhesion to stroma cells as well as reduced cell polarity upon adhesion of aged eHPCs. These data support a role of altered eHPC dynamics and altered cell polarity, and thus altered niche biology in mechanisms of mammalian aging.

Hematopoietic stem cell transplantation has become a standard of care for the treatment of many hematological diseases. Transplantation of mobilized peripheral blood stem cells has replaced bone marrow (BM) transplantation as the preferred method for hematopoietic recovery. To date, G-CSF mobilized hematopoietic stem/progenitor cell (HSPC) harvest is the main FDA-approved preparative regiment for transplantation protocols, but this application has several limitations in utilities including diverse individual variability and potential side effects in several patient populations. Although AMD3100, a chemical CXCR4-blocker, has been found effective for HSPC mobilization, the development of additional HSPC mobilization agents that work through well defined molecular mechanisms remains in need. Previously our laboratory has shown in a conditional knockout mouse model that deficiency of the Rho GTPase Cdc42 in the BM causes impaired adhesion, homing, lodging and retention of HSPCs, leading to massive egress of HSPCs from BM to the peripheral blood without compromising their proliferative potential. From an array of small molecule inhibitors of PIP2-induced actin-polymerization discovered in a high throughput screening, we identified CASIN, a novel Cdc42 Activity-Specific Inhibitor, that is effective in suppressing Cdc42 activity in a dose-dependent manner in murine fibroblasts and low density bone marrow (LDBM) cells and human CD34+ umbilical cord blood (HCB) cells in vitro, and in murine LDBM cells in vivo. The inhibitory effect by CASIN appears to be specific to Cdc42 and is reversible. We subsequently tested the hypothesis that pharmacological targeting Cdc42 by CASIN may transiently mimic the Cdc42 knockout phenotype leading to HSPC mobilization. In the dose range of 5–10 uM, CASIN does not show detectable toxicity in wild type or Cdc42 knockout HSPCs in cell survival and colony-forming unit activity assays. CASIN treatment of 32D murine myeloid progenitor cells or freshly isolated progenitor cells results in a reversible inhibition of F-actin polymerization induced by SDF-1α and blockade of α5β1 integrin mediated adhesion to fibronectin fragment CH296. Its effects on actin organization and adhesion are associated with an inhibition of directional migration of the colony-forming cells toward SDF-1α. In contrast, CASIN does not show a detectable effect on the adhesion and migration activities of Cdc42 knockout HSPCs, suggesting that it works specifically through Cdc42 to affect cell actin structure and adhesion. Upon injection into mice (5mg/Kg, intraperitoneally), CASIN is effective in stimulating mobilization of progenitor activity into the peripheral blood (~ 6-fold increase compared to control at 40 hrs post injection). Subsequent serial transplantation experiments show that the PB harvested from CASIN treated mice could reconstitute various lineages of blood cells in primary, secondary, and tertiary recipients, indicating that long-term hematopoietic stem cells were mobilized from the BM of CASIN-treated donor mice. Consistent with the mobilization phenotype, FACS analysis shows that intravenous injection of CASIN can cause transient reduction of long-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34−) and short-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34+) from BM. Similar to the effects on murine HSPCs, CASIN is active on CD34+ HCB cells in transiently suppressing F-actin assembly, adhesion to fibronectin, and SDF-1α induced migration without detectable toxicity in vitro. Whether CASIN is effective in mobilizing HCB-engrafted NOD/SCID mice is currently under investigation. Our studies suggest that the novel concept of pharmacological targeting of Cdc42, that transiently and reversibly mimics the effect of Cdc42 knockout, may be developed into a mobilization regiment with a well defined molecular and cellular mechanism.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into peripheral blood by the cytokine G-CSF has become the preferred source of HSPCs for clinical stem cell transplants. However, up to 10% of donors fail to mobilize sufficient numbers of stem cells impeding autologous transplants or significantly delaying transplant recovery time. Consequently, novel regimens are warranted to increase the number of stem cells in peripheral blood upon mobilization. Using a forward genetic approach in the mouse, we map the epidermal growth factor receptor (EGFR) to a genetic region on murine chromosome 11 modifying G-CSF-mediated HSPC mobilization. Expression levels of EGFR in HSPCs were inversely correlated with HSPC mobilization, implying a negative role for EGFR signaling in mobilization. Genetic reduction of EGFR activity (waved2 mice) or treatment with the EGFR inhibitor erlotinib increased stem cell mobilization up to 5-fold in combination with G-CSF. Increased mobilization due do alteration of EGFR activity correlated with reduced activity of Cdc42 and consequently, inhibition of Cdc42 activity in vivo by a specific Cdc42 inhibitor similarly enhanced mobilization. Our findings reveal a novel signaling pathway regulating stem cell mobilization and thus provide new rationale for targeted pharmacological approaches to further improve HSPC mobilization and thus transplantation outcomes.

There is significant individual variation in humans in their ability to mobilize hematopoietic stem and progenitor cells (HSPC) from bone marrow (BM) to peripheral blood upon G-CSF stimulation. Understanding the molecular mechanism underlying this variation may provide clinically significant targets to improve HSPC mobilization. Using forward genetics, we have previously demonstrated that a locus on chromosome 11 is responsible for murine inter-strain variation in G-CSF mediated HSPC mobilization (Geiger et. al., Exp. Hematol., 2004). Novel subcongenic animals were generated that further confined the locus to a 5 Mbp interval. Of the 12 genes in this interval, epidermal growth factor receptor (EGFR) was identified as a candidate gene for regulating inter-strain differences in mobilization. Using real-time PCR, we demonstrated that EGFR is expressed in both murine and human HSPC and that the level of EGFR expression is inversely correlated with GCSF-induced mobilization proficiency of murine HSPC. To further demonstrate a role of EGFR signaling in mobilization, C57BL/6 mice were treated with G-CSF and increasing concentrations of murine recombinant epidermal growth factor (EGF). Our results demonstrated that a single dose of EGF (0.8 ug/g) significantly inhibited mobilization efficiency (approx. 4-fold) (46.7±13 CFC/37.5ul PB with G-CSF treated mice vs. 12.67±1.5 CFC/37.5ul PB with G-CSF + EGF treated mice). To determine whether the reduction in mobilization efficiency is dependent upon EGFR activity, EGFR mutant mice (waved-2+/−) were treated with G-CSF and EGF. In contrast to control mice, EGF had no significant inhibitory effect on G-CSF-mediated mobilization in waved-2+/− mice indicating that EGFR signaling is necessary for inhibition of mobilization by EGF (46.7±13.0 vs. 12.67±1.5 CFC/37.5ul PB, G-CSF vs G-CSF+EGF treated control mice compared to 14.2±3.7 vs. 12.1±3.6 CFC/37.5ul PB with waved-2+/− mice). To determine if EGFR-signaling regulated mobilization is HSC intrinsic, we performed “competitive mobilization experiments” in which equal numbers of BM cells (4x106) from either control or waved-2−/− mice and competitor Ly5.1 cells were transplanted into lethally irradiated BoyJ mice and subsequently treated with G-CSF and EGF. Animals transplanted with control cells demonstrated a significant reduction in mobilization efficiency in response to EGF compared to animals transplanted with waved-2−/− cells (20.2±7.2 vs. 7.89±1.2 CFC/37.5ul PB, G-CSF vs G-CSF+EGF treated control transplanted mice compared to 20.3±4.5 vs. 18.67±1.2 CFC/37.5ul PB with waved-2−/− transplanted mice) indicating that EGFR activity in hematopoietic cells is necessary to confer inhibition of mobilization efficiency by EGF. Taken together, these findings identify EGFR signaling as a negative regulator and a genetic modifier of G-CSF induced mobilization proficiency and a potential novel target to enhance HSPC mobilization efficiency.

Surfactant protein SP-B is absolutely required for the surface activity of pulmonary surfactant and postnatal lung function. The results of a previous study indicated that the N-terminal segment of SP-B, comprising residues 1–9, is specifically required for surface activity, and suggested that prolines 2, 4, and 6 as well as tryptophan 9, may constitute essential structural motifs for protein function. In this work, we assessed the role of these two motifs in promoting the formation and maintenance of surface-active films. Three synthetic peptides were synthesized including a peptide corresponding to the N-terminal 37 amino acids of native SP-B and two variants in which prolines 2, 4, 6, or tryptophan 9 were substituted by alanines. All three synthetic peptides were surface-active, as expected from their amphipathic structure. The peptides were also able to insert into dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol (7:3 w/w ratio) monolayers preformed at pressures >30 mN/m, indicating that they perturb and insert into membranes. Substitution of alanine for tryptophan at position 9 significantly decreased both the rate of adsorption/insertion of the peptide into the interface and reinsertion of surface-active material excluded from the film during successive compression-expansion cycles. Substitution of alanines for prolines at positions 2, 4, and 6 did not produce substantial changes in the rate of adsorption/insertion; however, reinsertion of surface-active material into the expanding interface film was not as effective as in the presence of the nativelike peptide. These results suggest that W9 is critical for optimal interface affinity, whereas prolines may promote a conformation that facilitates rapid insertion of the peptide into phospholipid monolayers compressed to the highest pressures during compression-expansion cycling.

Hematopoietic stem and progenitor cells (HSPCs) are located in the bone marrow in close association with a highly organized 3-dimensional structure formed by stroma cells, referred to as the niche. Mobilization of HSPCs from bone marrow to peripheral blood in response to granulocyte colony-stimulating factor (G-CSF) requires de-adhesion of HSPCs from the niche. The influence of aging of HSPCs on cell-stroma interactions has not been determined in detail. Using a mouse model of G-CSF-induced mobilization, we demonstrated that the ability to mobilize hematopoietic stem cells is approximately 5-fold greater in aged mice. Competitive mobilization experiments confirmed that enhanced mobilization ability was intrinsic to the stem cell. Enhanced mobilization efficiency of primitive hematopoietic cells from aged mice correlated with reduced adhesion of hematopoietic progenitor cells to stroma and with elevated levels of GTP-bound Cdc42. These results might indicate that stroma-stem cell interactions are dynamic over a lifetime and result in physiologically relevant changes in the biology of primitive hematopoietic cells with age.

Recent data indicate that the heat shock response inhibits nuclear translocation of the proinflammatory transcription factor NF-κB. Under basal conditions NF-κB is retained in the cytoplasm by an inhibitory protein called I-κB which exists as two major isoforms: I-κBα and I-κBβ. Induction of the heat shock response in BEAS-2B cells, a human cell line representative of bronchial epithelium, increased expression of I-κBα mRNA in a time-dependent manner. Coincubation with actinomycin-D inhibited heat shock-mediated expression of I-κBα mRNA. Transient transfection assays with a plasmid containing the reporter gene firefly luciferase, under the control of the human I-κBα promoter, demonstrated that heat shock activated the I-κBα promoter. Heat shock-mediated induction of I-κBα was associated with inhibition of NF-κB activation. We conclude that heat shock increases I-κBα mRNA expression in BEAS-2B cells by activating the I-κBα promoter, and propose that heat shock-mediated up-regulation of I-κBα is a potential mechanism by which the heat shock response inhibits proinflammatory responses in lung cells.

I-kappaBalpha is an intracellular protein that functions as a primary inhibitor of the proinflammatory transcription factor NF-kappaB. Induction of the stress response with heat shock was previously demonstrated to induce I-kappaBalpha gene expression. Because the stress response can also be induced by nonthermal stimuli, we determined whether induction of the stress response with prostaglandin A1 (PGA1) would induce I-kappaBalpha gene expression. Treatment of human bronchial epithelium (BEAS-2B cells) with PGA1 induced nuclear translocation of heat shock factor 1, thus confirming that PGA1 induces the stress response in BEAS-2B cells. Induction of the stress response with PGA1 increased I-kappaBalpha mRNA expression in a time-dependent manner and increased I-kappaBalpha peptide expression. Transient transfection assays involving a human I-kappaBalpha promoter-luciferase reporter construct demonstrated that induction of the stress response with PGA1 activated the I-kappaBalpha promoter. Induction of the stress response with PGA1 and concomitant induction of I-kappaBalpha were associated with inhibition of TNF-alpha-mediated secretion of interleukin 8 and with inhibition of TNF-alpha-mediated nuclear translocation and activation of NF-kappaB. These data demonstrate that induction of the stress response, by a nonthermal stimulus, increases I-kappaBalpha gene expression by a mechanism involving activation of the I-kappaBalpha promoter. Coupled with previous data demonstrating heat shock-mediated induction of I-kappaBalpha gene expression, these data suggest that I-kappaBalpha may be considered to be one of the stress proteins. The functional consequences of stress response-mediated I-kappaBalpha gene expression may involve attenuation of cellular proinflammatory responses.

Pneumocystis carinii is a eukaryotic organism that causes pneumonia in immunocompromised hosts. The cell biology and life cycle of the organism are poorly understood primarily because of the lack of a continuous in vitro cultivation system. These limitations have prevented investigation of the organism's infectious cycle and hindered the rational development of new antimicrobial therapies and implementation of measures to prevent exposure to the organism or transmission. The interaction of P. carinii with its host and its environment may be critical determinants of pathogenicity and life cycle. Signal transduction pathways are likely to be critical in regulating these processes. G proteins are highly conserved members of the pathways important in many cellular events, including cell proliferation and environmental sensing. To characterize signal transduction pathways in P. carinii, we cloned a G-protein alpha subunit (G-alpha) of P. carinii carinii and P. carinii ratti by PCR amplification and hybridization screening. The gene encoding the G-alpha was present in single copy on a 450-kb chromosome of P.c. ratti. The 1,062-bp G-alpha open reading frame is interrupted by nine introns. The predicted polypeptide showed 29 to 53% identity with known fungal G-alpha proteins with greatest homology to Neurospora crassa Gna-2. Northern (RNA) blot analysis and immunoprecipitation demonstrated expression of the G-alpha mRNA and protein P. carinii isolated from heavily infected animals. Some alteration in the level of transcription was noted in short-term maintenance in starvation or rich medium. Characterization of signal transduction in P. carinii will permit a better understanding of the reproductive capacity and other cellular processes in this family or organisms that cannot be cultured continuously.

.......................................................................................................................................3 ACKNOWLEDGEMENTS ..................................................................................................................5 ABBREVIATIONS ...............................................................................................................................9 CHAPTER