SCF Receptor Research Articles

Stem cell factor and erythropoietin-independent production of cultured reticulocytes.

Cultured reticulocytes can supplement transfusion needs and offer promise for drug delivery and immune tolerization. They can be produced from induced pluripotent stem cells (iPSCs), but the 45-day culture time and cytokine costs make large-scale production prohibitive. To overcome these limitations, we have generated IPSCs that express constitutive SCF receptor and jak2 adaptor alleles. We show that iPSC lines carrying these alleles can differentiate into self-renewing erythroblast (SRE) that can proliferate for up to 70 cell-doubling in a cost-effective, chemically-defined, albumin- and cytokine-free medium. These kitjak2 SREs retain the ability to enucleate at a high rate up to senescence. Kitjak2 derived cultured reticulocytes should be safe for transfusion because they can be irradiated to eliminate residual nucleated cells. The kitjak2 cells express blood group 0 and test negative for RhD and other clinically significant RBCs antigens and have sufficient proliferation capacity to meet global RBC needs.

Sex‐Specific Protein Expression Differences in Peri‐Lymphatic Adipose of Obese and Lean Zucker Rats.

Lymphatic abnormalities have been reported to accompany obesity, however little is understood about the mechanisms that lead to these changes. To gain novel insight, we performed a proteomic study of the adipose tissue surrounding mesenteric lymphatic vessels in obese and lean Zucker rats of both sexes in combination of a study of passive wall mechanics of isolated mesenteric collecting lymphatic vessels. At 12‐weeks of age, body weights were recorded and the rats were euthanized for collection of mesetneric lymphatic vessels for study plus blood via cardiac puncture and peri‐lymphatic adipose tissue (PLAT) from the mesentery for proteomic analysis. Mesenteric collecting lymphatic vessels were mounted on a pressure myograph and wall thickness and diameter were measured across a physiological pressure range (1–5 cm water). Serum and protein lysate from the adipose were were then analyzed through the RayBiotech Rat Cytokine Array Q67 testing service. The cohort for study included ten obese and ten lean Zucker rats, with equal numbers of males and females in each group. The mean body weights of this cohort were: lean males 275.8 ± 17.9 g; lean females 190.8 ± 6.3 g; obese males 458.4 ± 11.8 g; and obese females 440.0 ± 6.2 g. (P<0.0001). Across both sexes, the lymphatic vessels from obese rats had a significantly greater wall thickness compared to vessels from lean rats. In the proteomic study of PLAT, 5 targets were significantly elevated in obese rats of both sexes (CCL2, galectin‐1, galectin‐3, neuropilin‐1, and PPBP). Additionally, there were 11 targets were elevated in obese males, but not females (b‐NGF, CINC‐3, fractalkine, IL‐6, IL‐13, PDGF‐AA, prolactin receptor, RAGE, TIMP‐1, VEGF‐A, FGF‐BP), and 24 targets that were elevated in obese females, but not males (4‐1BB, adiponectin, B7‐1, CTACK, EphA5, Flt‐3L, Gas‐1, GFR alpha‐1, IL‐1ra, IL‐7, IL‐17F, IL‐22, JAM‐A, neuropilin‐2, NOPE, Notch‐1, Notch‐2, P‐cadherin, prolactin, RANTES, SCF, TIMP‐2, and TWEAK receptor). In serum from obese males, there were 4 targets that were increased (JAM‐A, GM‐CSF, ICAM‐1, neuropilin‐2) and 2 targets decreased (GP130, Ppbp) compared to lean males. In serum from obese females, 1 target was significantly increased (CCL2) and 4 targets were decreased (decorin, fractalkine, NOPE, prolactin) compared to lean females. There were also four targets that were different across the sexes regardless of lean/obese status (FLT‐3L, Gas‐1, GFR‐alpha‐1, TIMP‐1). Analysis of the targets elevated in the PLAT in both males and females with the STRING database revealed that the elevated proteins belong to clusters pertaining to VEGF signaling, positive regulation of cell adhesion, regulation of angiogenesis, and response to stress. Collectively, the results suggest that in addition to some common signaling proteins that are elevated in the PLAT of obese Zucker rats compared to lean counterparts, there are also several differential signals across the sexes. Future studies that may reveal differences in lymphatic function or dysfunction across the sexes are warranted.Support or Funding InformationSupported by NIH R01GM120774 and L40HL097863.

Expression of c-Kit discriminates between two functionally distinct subsets of human type 2 innate lymphoid cells.

Human type 2 innate lymphoid cells (ILC2) are the only ILC subset that shows heterogeneous expression of the SCF receptor c-Kit (CD117). Despite its use as surface marker to distinguish ILC populations, its influence on ILC2 biology has not been investigated. Here, we show that c-Kit expression of peripheral blood ILC distinguishes two functionally distinct ILC2 subsets (c-Kithi and c-Kitlo ). When examined for their potential for functional plasticity we found that c-Kitlo ILC2 displayed greater potential to produce type 2 cytokines, possibly representing fully mature and lineage committed ILC2. On the other hand, c-Kithi ILC2 coexpressed the ILC3-marker and chemokine receptor CCR6 and were able to mount a significant IL-17A response under ILC3-promoting conditions. In addition, c-Kithi ILC2 produced higher levels of IFN-γ than c-Kitlo ILC2 under ILC1-conditions. Although costimulation with SCF did not further influence ILC2 plasticity, it augmented type 2 cytokine production. We conclude that c-Kit marks distinct subpopulations of ILC2, which has therapeutic implications for conditions in which ILC2 are involved, such as allergy and asthma.

Phosphoproteomic Landscaping Unveils Constitutive cKIT Activation in Human Erythroblasts from Polycythemia Vera (PV) Patients

PV is characterized by the gain-of-function V617F mutation in JAK2, the gene encoding the first signaling element of the cytokine receptor superfamily. Progenitor cells from PV are more sensitive in vitro to Imatinib, which also inhibits the SCF receptor cKIT, than those from normal sources (Gaikwad, Exp Hemat 2007;35:931) and clinical trials with similar tyrosine kinase inhibitors have been reported to have some efficacy in PV (Nussenzveig, Int J Hematol 2009;90:58; Silver, Leuk Res 2012;36:156). These data led us to examine the mechanism by which this tyrosine kinase inhibitor affects erythropoiesis in PV.We observed that in cultures containing SCF PV progenitor cells generated similar numbers of erythroid cells (Ery) as those from adult (AB) and cord (CB) blood by day 10 [fold increase (FI) ~1-2] but by day 13 PV cells generated significantly greater numbers of Ery than AB and CB [PV FI=11±0.2, p=0.021 vs AB; CB FI=6.2±1.9, p=0.025 vs PV and 0.0055 vs AB; AB FI=2.6±0.5]. Since by day 10 progenitor cells were no longer detected, we hypothesized that increases in Ery at day 13 reflect intrinsically greater Ery proliferation potentials. To test this hypothesis, we compared the phosphoproteomic landscaping of day 10 Ery from 3 PV, 3 AB and 3 CB by Reverse Phase Protein Array (RPPA) using as target >160 signaling events (data are at http://capmm.gmu.edu/data). Overall, 40 proteins were statistically different between PV and AB and 30 proteins were statistically different between CB and AB. Pathway analyses of significant hits identified that PV and CB Ery differ from the AB ones in the activation states of 1-2 proteins involved in stemness and cell cycle control inferring that there is no major change in their cycling or differentiation state. By contrast, the 3 populations showed numerous differences in cKIT signaling. PV Ery differed from AB cells by expressing greater levels of cKITY719 and cKIT703, which were reduced to barely detectable levels by the pan-JAK inhibitor Ruxolitinib, and of elements of PI3K (eNOS/NosIII, PDK1 and PKCd) and MAPK (pMARCKS, MSK1, AMPKα1 and β1 and p38 MAPK) signaling downstream, respectively, to cKITY719 and cKIT703. PV Ery expressed also greater levels of JAK2Y1007/1008 and of its downstream target STAT3Y705. CB Ery showed lower levels of cKIT, cKITY703, cKITY719 and CD63, a member of tetraspanin superfamily that binds cKITY719 switching its intracellular fate from recycling to lysosome degradation, greater phosphorylation of proteins of MAPK (pMARCKS, MSK1, PTEN and Src) and PI3K (PKCd, mTOR, p70S6K and panPKC/βII) signaling than AB Ery. These results were stoichiometrically validated by WB and indicate that PV Ery express greater degrees of cKIT activation than AB Ery suggesting that greater response to SCF might account for their greater amplification in culture. This hypothesis was tested by RPPA analyses (and stoichiometric validation by WB) of Ery from PV, AB and CB growth factor deprived (GFD) for 4h and then stimulated with SCF for 15’ and 2h. GFD altered the activation state of 25 proteins (22 de-activated and 3 activated) in PV, of 12 proteins (10 de-activated and 2 activated) in AB and 8 proteins (4 de-activated and 4 activated) in CB. SCF altered the activation state of 36 proteins in PV (18 activated and 18 de-activated), 23 proteins in CB (all activations) and 6 proteins in AB (all activations). In PV and CB Ery, GFD decreased cKITY719 and cKITY703 and the activation state of their downstream targets JAK2Y1007/1008, MAPKs and mTOR while SCF increased the stoichiometric levels of cKITY719 and cKITY703 and the activation of mTOR. SCF also increased cKITY703 and cKITY719 but did not activate mTOR in Ery from AB. In agreement with the hypothesis that Ery from PV and CB respond more readily to SCF than those from AB, SCF induced greater cell-surface cKIT down-modulation (by flow cytometry) and lower intra-cytoplasmic cKIT/CD63 association (by confocal microscopy and WB) in PV and CB Ery than in AB Ery. Screening of 97 inhibitors against targets analysed by RPPA which are approved for clinical use by FDA revealed that growth of PV Ery was more sensitive than that of AB only to JAK and cKIT inhibitors. In addition, shRNA-CD63 reduced the growth of PV Ery (FI=0.9 vs 1.3 p=0.012) while increased by 2-fold (p=0.02) that of AB Ery. These results provide the first phosphoproteomic landscaping of cKIT signalling in Ery from PV and normal sources and confirm that cKIT is an important therapeutic target for PV. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Insecticidal Exposure may be the cause of Progression of Immune Disorders and Inflammatory Diseases Through Dys-Regulation of Cytokines

Industrial revolution has dangerously increased the load of environmental or environmentally induced diseases. Recently, the insecticides (a class of pesticides killing various insects) have become one of the most important environmental pollutants. This Review discusses the association of insecticides, cytokines dys-regulation and the development of immuno-regulatory disorders and chronic inflammatory conditions. In spite of abundant reviewing and reporting of various immuno-inflammatory diseases individually, efforts lack to understand the central pathological mechanism underlying these diseases collectively. The cytokines are extensively involved in inter-cellular communications; they are regarded attractive markers for risk stratification or patient prognosis. In this Review, an understanding has been generated about the role of various receptors, genes and signaling pathways etc. in the trianglular link of cytokines, immuno-inflammatory diseases and insecticides. Such role, in a number of instances, had been those of (1) a receptor tyrosine kinase designated as c-kit or SCF receptor and (2) NFκB, the most excellently described transcription factor. The conclusion of the Review is that insecticides are linked with increasing susceptibility to various diseases especially any type of chronic inflammation through cytokine dys-regulation. Moreover, insecticidal toxicity can cause deviations in certain parameters of diagnosis. So insecticide applicators must be stressed to ensure only recommended doses and formulations of insecticides if really necessary.

In vitro generation of allo-reactive human NK cells from umblillical cord blood CD34+ progenitor cells

Compare to autologous NK cells, allogeneic NK cells have more anti-tumor cytotoxicity without GVHD. Adoptive transfer of allo-reactive NK cell can be ideally used combined with other immunotherapy. However, in vivo persistency of transferred NK cells in the recipients are still an obstacle in NK cell-based immunotherapy. As most of NK cells purified from peripheral blood of donors are fully differentiated, their short lifespan might be a reason. In this study, we developed a new method of “young” human NK cell generation which can be used for immune cell therapy. CD34+ hematopoietic precursor/stem cells were purified from umbilical cord blood and differentiated into NK cells in vitro. Stem cell expansion and lineage commitment can be modulated by stromal cells and exogenous cytokines. Most of CD34+ cells express receptors for IL-3, Flt3-L, and SCF, while a small subset expressed IL-7Rα. Without feeder cells, CD34+ cells were initially expanded with exogenous cytokines (IL-3, Flt3-L, IL-15, SCF, and IL-7) but most of cells were differentiated into myeloid cells. When the precursor cells were co-cultured with feeder cells (murine embryonic liver stromal cell line EL08-1D2), CD56+ NK cells were successfully generated after 3 weeks (x20,000 fold expansion). Interestingly, as use of feeder cells was necessary for NK cell differentiation during early days (D0 to 5), feeder cell interaction may have a role on the proliferation of early lymphoid precursors or myeloid/lymphoid fate decision of hematopoietic stem cells. When we used EL08-1D2 feeder cells transduced with human Notch ligand DLL4, most of hematopoietic precursors were differentiated into lymphoid lineage cells. Some of generated CD56+ cells were non-cytotoxic group3 innate lymphoid cells (ILC3s), and ILC3s do not express most of NK cytotoxic receptors (NKG2D, NKp30, NKp46, 2B4, CD94, NKG2A, DNAM-1, and CD16). Cytotoxic NK cells can be discriminated from ILC3s based on CD11a/CD18 (LFA-1) expression, and this was confirmed using CD107a assay and IFN-γ staining. While ILC3s require SCF and IL-7 for their generation, NK cells can be successfully generated with IL-15 and Flt-3L only. As small populations of the generated NK cells express KIRs which are essential for allogeneic cytotoxicity, we epigenetically induced KIR expression using a demethylating agent 5-aza-2′-deoxycytidine during NK cell differentiation. When 5-aza-2′-deoxycytidine was treated on immature NK cells (D14 to 21), it efficiently induced KIR expression while it did not on fully differentiated NK cells (at D21). In conclusion, this new method can be used for novel NK cell based immunotherapy.

FILAMENTOUS FLOWER Is a Direct Target of JAZ3 and Modulates Responses to Jasmonate.

The plant hormone jasmonate (JA) plays an important role in regulating growth, development, and immunity. Activation of the JA-signaling pathway is based on the hormone-triggered ubiquitination and removal of transcriptional repressors (JASMONATE-ZIM DOMAIN [JAZ] proteins) by an SCF receptor complex (SCF(COI1)/JAZ). This removal allows the rapid activation of transcription factors (TFs) triggering a multitude of downstream responses. Identification of TFs bound by the JAZ proteins is essential to better understand how the JA-signaling pathway modulates and integrates different responses. In this study, we found that the JAZ3 repressor physically interacts with the YABBY (YAB) family transcription factor FILAMENTOUS FLOWER (FIL)/YAB1. In Arabidopsis thaliana, FIL regulates developmental processes such as axial patterning and growth of lateral organs, shoot apical meristem activity, and inflorescence phyllotaxy. Phenotypic analysis of JA-regulated responses in loss- and gain-of-function FIL lines suggested that YABs function as transcriptional activators of JA-triggered responses. Moreover, we show that MYB75, a component of the WD-repeat/bHLH/MYB complex regulating anthocyanin production, is a direct transcriptional target of FIL. We propose that JAZ3 interacts with YABs to attenuate their transcriptional function. Upon perception of JA signal, degradation of JAZ3 by the SCF(COI1) complex releases YABs to activate a subset of JA-regulated genes in leaves leading to anthocyanin accumulation, chlorophyll loss, and reduced bacterial defense.

Peripheral blood CD56brightNK cells respond to stem cell factor and adhere to its membrane‐bound form after upregulation of c‐kit

CD56(bright) NK cells express receptors for IL-2, IL-7, IL-15, and SCF. We found that human peripheral blood CD56(bright) NK cells responded to IL-2, IL-7, IL-15 by phosphorylating STAT-5, ERK, and Akt but did not respond to SCF. However, CD56(bright) NK cells in culture upregulated c-kit transcription three to fourfold, which led to a steady increase in c-kit and a concomitant acquisition of responsiveness to SCF. After 44 h, CD56(bright) NK cells had upregulated c-kit approximately 20-fold and phosphorylated ERK and Akt in response to SCF concentrations well below levels present in plasma. CD56(bright) NK cells cultured in IL-15 maintained c-kit transcription/expression at ex vivo levels and did not become responsive to SCF. Furthermore, SCF-responsive, CD56(bright) c-kit(high) NK cells swiftly downregulated c-kit and stopped responding to SCF after IL-15 stimulation. However, commitment of CD56(bright) NK cells to a c-kit-negative, SCF-unresponsive state did not occur, as after 5 days of culture, withdrawal of IL-15 restored c-kit to maximal levels and reestablished SCF-responsiveness. CD56(bright) NK cells that had upregulated c-kit firmly adhered to COS cells transfected with the membrane form of SCF. Furthermore, SCF signaling significantly increased the capacity of CD56(bright) NK cells to degranulate. Collectively, our data suggest that c-kit on human CD56(bright) NK cells is a functional receptor that is downregulated in peripheral blood, possibly to render CD56(bright) NK cells unresponsive to the SCF therein.

Phenotyping Of Leukemic Stem Cells In Ph+ ALL and Ph- ALL Reveals Unique Profiles Of Markers and Targets In Distinct Disease Variants

Acute lymphoblastic leukemia (ALL) is a life-threatening hematopoietic neoplasm characterized by abnormal growth and accumulation of lymphatic blast cells in various hematopoietic tissues. In a substantial number of patients, the Philadelphia (Ph) chromosome and the related oncoprotein BCR/ABL, are detectable. Despite recent advances in the management and therapy of patients with ALL, including the use of BCR/ABL1 tyrosine kinase inhibitors (TKI), the prognosis is still poor. Therefore, several attempts have been made to improve targeted treatment approaches in ALL. One strategy is to identify markers and targets expressed on leukemic stem cells (LSC) in these patients and to apply targeted drugs in order to eliminate LSC. In patients with Ph+ ALL, the leukemia-initiating cell-population is considered to reside within a CD34+/CD38- fraction of the clone. In the present study, we examined the expression of various stem cell markers and target antigens in CD34+/CD38- stem cells and in more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL (n=12), Ph- ALL (n=13), Ph+ CML (n=20), and in control bone marrow (BM) samples (unexplained cytopenia, n=10). Surface expression of target antigens was analyzed by multicolor flow cytometry, and mRNA expression levels by qPCR. As assessed by flow cytometry, CD34+/CD38- cells were found to co-express CD19, the stem cell-homing receptor CD44, the Campath-1 antigen (CD52), AC133 (CD133), FLT3 (CD135), and CXCR4 (CD184) in all ALL patients examined. In a majority of the ALL patients tested (14/25), LSC also expressed Siglec-3 (CD33). In CML, LSC were found to express a similar profile of antigens, including CD33, CD44, CD52, CD133, CD135, and CXCR4, but these cells did not express CD19. In control BM samples, CD34+/CD38- cells expressed a similar phenotype, but the levels of CD33 and CD52 were lower compared to LSC in ALL and CML. The IL-1RAP was found to be expressed on LSC in patients with Ph+ CML and Ph+ ALL, but not on LSC in Ph- ALL or in normal BM stem cells. By contrast, the SCF receptor KIT (CD117) was found to be expressed on LSC in Ph+ CML but was hardly detectable on LSC in patients with Ph+ ALL or Ph- ALL. The IL-2RA (CD25) and the SDF-1-degrading surface enzyme dipeptidyl-peptidase IV (DPPIV=CD26) were expressed on LSC in patients with CML and in all patients with Ph+ ALL exhibiting BCR/ABL-p210, whereas in Ph+ ALL with BCR/ABL-p190, LSC variably expressed CD25, and did not express CD26. In patients with Ph- ALL and in the normal BM, CD34+/CD38- cells did not express CD25 or CD26. The target receptor CD20 was detectable on ALL LSC in 7/18 patients examined. All target receptors tested were also detectable on more mature CD34+/CD38+ progenitor cells in patients with Ph+ ALL and Ph- ALL. In consecutive studies, expression of target antigens was confirmed at the mRNA level by qPCR analyses of highly enriched ALL LSC. Finally, we were able to show that the CD52-targeting drug alemtuzumab induces rapid lysis of CD34+/CD38- ALL LSC in all patients examined (Figure). In summary, our data show that LSC in Ph+ ALL and Ph- ALL express a unique phenotype, including clinically relevant cytokine receptors and cell surface target antigens, including the Campath-1 antigen, CD52. In Ph+ ALL with BCR/ABL-p210, the phenotype of ALL LSC largely resembles the phenotype of LSC in Ph+ CML, confirming the close relationship and similar pathogenesis of these two types of leukemias. [Display omitted] Ficoll-isolated MNC of 4 patients with Ph+ ALL were incubated in control medium (Co) or in various concentrations of alemtuzumab (10-300 µg/ml) in RPMI 1640 medium in the presence of 30% human serum at 37°C for 1 hour. After washing, cells were stained with fluorochrome-conjugated mAb against CD34, CD38 and CD45 for 15 minutes. DAPI-staining was used to evaluate the percentage of viable cells. Cells were analysed using a FACSCanto II and FlowJo software. Results show the numbers of viable CD34+/CD38- cells and are expressed as percent of control (Co). Values represent the mean±S.D. of four independent experiments. Asterisk (*): p<0.05 compared to control. Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

C‐Kit SCF receptor (CD117) expression and KIT gene mutation in conjunctival pigmented lesions

AbstractPurpose to investigate the presence of KIT gene mutations and immunoreactivity in 85 conjunctival melanocytic tumors and clarify the role of KIT as a potential therapeutic target in this group of patients.Methods 85 conjunctival pigmented tumors (27 melanomas, 12 PAMs and 46 nevi) were immunostained for KIT. Intensity and pattern of expression were evaluated. Molecular analysis to identify KIT mutations was performed in 15 selected cases (tumor‐rich areas &gt;50%). KIT immunostaining score and pattern were statistically related to patients’ age, sex, diagnostic category, presence of relapse, disease‐free survival, presence of metastases, metastasis‐free survival, limbal versus non‐limbal tumor location and thickness of melanomas.Results KIT stains was documented in 48% of melanomas, 50% of PAMs and 24% of nevi. The mean score of KIT staining in the melanomas/PAMs group was significantly different from nevi (p=0.0076). No statistically significant differences were detected between either c‐kit immunostaining score or pattern and each of the other clinico‐pathologic parameters considered. No KIT gene mutations were detected in melanomas and nevi. A silent mutation/polymorphism in KIT exon 13 was found in one PAM.Conclusion Despite the high level of KIT immunostains in conjunctival PAM and melanoma, this parameter seems not to be a good predictor for the presence of molecular mutations. KIT‐activating mutations should be considered an uncommon event in these tumors.

C-Kit SCF receptor (CD117) expression andKITgene mutation in conjunctival pigmented lesions

To investigate the presence of KIT gene mutations and immunoreactivity in 85 conjunctival melanocytic tumours and to clarify the role of KIT as a potential therapeutic target in this group of patients. Eighty-five conjunctival pigmented tumours [27 melanomas, 12 primary acquired melanosis (PAMs) and 46 nevi] were immunostained for KIT. Intensity and pattern of expression were evaluated. Molecular analysis to identify KIT mutations was performed in 15 selected cases (tumour-rich areas >50%). KIT immunostaining score and pattern were statistically related to patients' age, sex, diagnostic category, presence of relapse, disease-free survival, presence of metastases, metastasis-free survival, limbal versus nonlimbal tumour location and thickness of melanomas. KIT stains were documented in 48% of melanomas, 50% of PAMs and 24% of nevi. The mean score of KIT staining in the melanomas/PAMs group was significantly different from nevi (p = 0.0076). No statistically significant differences were detected between either c-kit immunostaining score or pattern and each of the other clinico-pathologic parameters considered. No KIT gene mutations were detected in melanomas and nevi. A silent mutation/polymorphism in KIT exon 13 was found in one PAM. Despite the high level of KIT immunostains in PAMs and melanomas, this parameter seems not to be a good predictor of the presence of molecular mutations. KIT-activating mutations should be considered an uncommon event in this tumour.

Expression of phosphorylated STAT5 in bone marrow hematopoietic stem cells of patients with paroxysmal nocturnal hemoglobinuria before and after in vitro G-CSF or SCF stimulation

To study the expressions of phosphorylated STAT5 (P-STAT5) in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH) before and after in vitro G-CSF or SCF stimulation, then evaluate the functions of G-CSF and SCF receptors in PNH clone cells. Bone marrow mononuclear cells (BMMNC) of 26 PNH patients and 14 normal controls were stimulated in vitro with G-CSF (100 ng/ml) or SCF (100 ng/ml) for 10 min. Before and after these stimulations, the mean fluorescence intensity (MFI) of P-STAT5 in CD34(+)CD59(+) BMMNC and CD34(+)CD59(-) BMMNC were measured by flow cytometry. (1) The P-STAT5 MFI was (24 ± 18) in unstimulated CD34(+)CD59(-) cells. And it was significantly lower than that in unstimulated CD34(+)CD59(+) cells of PNH patients (64 ± 49) and normal controls (61 ± 33) (both P < 0.01). No statistic difference existed between the latter two. (2) The P-STAT5 MFI was (36 ± 35) in G-CSF stimulated CD34(+)CD59(-) cells of PNH patients. And it was significantly lower than that in G-CSF stimulated CD34(+)CD59(+) cells of PNH patients (124 ± 84) and normal controls (116 ± 59) (both P < 0.01). There was no statistic difference between the latter two. (3) The P-STAT5 MFI was (34 ± 27) in SCF stimulated CD34(+)CD59(-) cells of PNH patients. And it was significantly lower than that in SCF stimulated CD34(+)CD59(+) cells of PNH patients (124 ± 97) and normal controls (128 ± 62) (both P < 0.01). And no statistic difference existed between the latter two. (4) The increased P-STAT5 MFI in G-CSF stimulated CD34(+)CD59(-) cells of PNH patients was significantly lower than that in G-CSF stimulated CD34(+)CD59(+) cells of PNH patients and normal controls (both P < 0.01). No statistic difference existed between the latter two. The increase of P-STAT5 MFI in SCF stimulated CD34(+)CD59(-) cells of PNH patients was significantly lower than that in SCF stimulated CD34(+)CD59(+) cells of PNH patients and normal controls (both P < 0.01). No statistic difference existed between the latter two. There is a lower expression of P-STAT5 expressed in G-CSF or SCF stimulated PNH clone cells compared to that in normal clone cells.

2094 ADENOVIRUS-MEDIATED DAX1GENE TRANSFER ENHANCES THE FUNCTION OF SERTOLI CELLS VIA THE SCF/C-KIT PATHWAY

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20122094 ADENOVIRUS-MEDIATED DAX1GENE TRANSFER ENHANCES THE FUNCTION OF SERTOLI CELLS VIA THE SCF/C-KIT PATHWAY Satoshi Kurokawa, Yoshiyuki Kojima, Kentaro Mizuno, Akihiro Nakane, Takahiro Yasui, Noriyasu Kawai, Keiichi Tozawa, Tetsuji Maruyama, Yutaro Hayashi, and Kenjiro Kohri Satoshi KurokawaSatoshi Kurokawa Nagoya, Japan More articles by this author , Yoshiyuki KojimaYoshiyuki Kojima Nagoya, Japan More articles by this author , Kentaro MizunoKentaro Mizuno Nagoya, Japan More articles by this author , Akihiro NakaneAkihiro Nakane Nagoya, Japan More articles by this author , Takahiro YasuiTakahiro Yasui Nagoya, Japan More articles by this author , Noriyasu KawaiNoriyasu Kawai Nagoya, Japan More articles by this author , Keiichi TozawaKeiichi Tozawa Nagoya, Japan More articles by this author , Tetsuji MaruyamaTetsuji Maruyama Nagoya, Japan More articles by this author , Yutaro HayashiYutaro Hayashi Nagoya, Japan More articles by this author , and Kenjiro KohriKenjiro Kohri Nagoya, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2261AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES DAX1, an essential transcription factor in sex differentiation, is expressed in the cells of the hypothalamic-pituitary-testicular axis. DAX1 plays important roles in both steroidogenesis in Leydig cells and spermatogenesis in Sertoli cells in the testes. DAX1 expression is synchronized with the spermatogenic cycle, especially in Sertoli cells. However, DAX1 function in Sertoli cells is unclear. We therefore analyzed the function of DAX1 in Sertoli cells using the adenoviral gene transfer technique. METHODS 1) In vitro study: An adenovirus vector containing full-length mouse Dax1 cDNA was constructed from the human adenovirus serotype 5. This vector was tested on the mouse Sertoli cell line, TM4, to assess the efficiency of gene transfer at various multiplicities of infections (MOIs): 1, 10, and 100. TM4 cells were harvested and mRNA was extracted 48 hours after gene transfer. The expression level of 16 Sertoli cell-specific genes was examined by quantitative RT-PCR. 2) In vivo study: Sl/Sld and W/Wv mice with male infertility were used. Ten-week-old male mice were sacrificed and their testes were harvested. Dax1 expression in the testes was analyzed by quantitative RT-PCR and western blotting. RESULTS 1) More than 70% of TM4 cells showed gene transfer with the adenovirus vector at a MOI of 100. Among the Sertoli cell-specific genes, Scf, Sox9, and Star were expressed at significantly higher levels after Dax1 gene transfer. Scf showed the largest expression level increase (3.1 fold). 2) Dax1 mRNA expression was significantly higher in the testes of Sl/Sld and W/Wv mice than in normal mice. Strong DAX1 staining was detected in the nuclei of Sertoli cells. CONCLUSIONS Strong Scf expression was observed after Dax1 gene transfer, indicating that the nuclear receptor DAX1 controls SCF expression in Sertoli cells. The Sl/Sld mouse has mutant SCF and the W/Wv mouse has mutant C-KIT, which is a SCF receptor. DAX1 expression was accelerated in the testes of these mice, which may be caused by a feedback system based on the functional loss of SCF. SCF, which is produced in the Sertoli cells of the testis, binds to C-KIT on the spermatogonia and plays an important role in the differentiation and proliferation of germ cells. DAX1 may activate Sertoli cells to stimulate spermatogenesis via a SCF/C-KIT signaling pathway. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e844 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Satoshi Kurokawa Nagoya, Japan More articles by this author Yoshiyuki Kojima Nagoya, Japan More articles by this author Kentaro Mizuno Nagoya, Japan More articles by this author Akihiro Nakane Nagoya, Japan More articles by this author Takahiro Yasui Nagoya, Japan More articles by this author Noriyasu Kawai Nagoya, Japan More articles by this author Keiichi Tozawa Nagoya, Japan More articles by this author Tetsuji Maruyama Nagoya, Japan More articles by this author Yutaro Hayashi Nagoya, Japan More articles by this author Kenjiro Kohri Nagoya, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Amine‐surface‐modified superparamagnetic iron oxide nanoparticles interfere with differentiation of human mesenchymal stem cells

Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used for stem cell labeling and tracking. Surface modification has been known to improve biocompatibility, biodistribution, and labeling efficiency of SPIO nanoparticles. However, the effects of amine (NH 3+)-surface-modified SPIO nanoparticles on proliferation and differentiation of human mesenchymal stem cells (hMSCs) remain unclear. The purpose of this study is to investigate how amine-surface-modified SPIO nanoparticles affected hMSCs. In this study, intracellular uptake and the contiguous presence of amine-surface-modified SPIO nanoparticles in hMSCs were demonstrated by Prussian blue staining, transmission electron microscopy and magnetic resonance imaging. Moreover, accelerated cell proliferation was found to be associated with cellular internalization of amine-surface-modified SPIO nanoparticles. The osteogenic and chondrogenic differentiation potentials of hMSCs were impaired after treating with SPIO, while adipogenic potential was relatively unaffected. Altered cytokine production profile in hMSCs caused by amine-surface-modified SPIO nanoparticles may account for the increased proliferation and impaired differentiation potentials; concentrations of the growth factors in the SPIO-labeled condition medium including amphiregulin, glial cell-derived neurotrophic factor, heparin-binding EGF-like growth factor and vascular endothelial growth factor, as well as soluble form of macrophage colony-stimulating factor receptor and SCF receptor, were higher than in the unlabeled-condition medium. In summary, although amine-surface-modified SPIO labeling is effective for cell tracking, properties of hMSCs may alter as a consequence and this needs to be taken into account when evaluating therapeutic efficacies of SPIO-labeled stem cells in vivo.

Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

BackgroundNerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.ResultsTo study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, tropomyosin-receptor-kinase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis.ConclusionNGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.

Antitumour activity of oral E7080, a novel inhibitor of multiple tyrosine kinases, in human sarcoma xenografts

E7080 is an inhibitor of multiple tyrosine kinases, several of which have pro-angiogenic properties, including receptors for VEGF, FGF, SCF and PDGF. We undertook our study to evaluate the preclinical activity of E7080 in human sarcomas. The antitumour activity of orally administered E7080 was tested in ten human tumour xenografts representing different sarcoma histotypes. Concomitant changes in microvessel density were assayed by immunohistochemistry to CD31. Immunohistochemistry was also used to assess the expression of kinases that E7080 is known to inhibit. The MTS assay was applied to determine effects on tumour cell viability in vitro. At the Q1D5 × 2 schedule, E7080 (30 mg/kg) was active (T/C<40%) in 7/10 xenografts. The effects were accompanied by marked decrease in microvessel densities. Given at the Q1D5 × 4 schedule, E7080 (30, 10, 3 mg/kg) showed antitumour activity in a dose dependent manner in two different xenografts. E7080 growth inhibition did not correlate with the expression of VEGFR1-3, PDGFRA, PDGFRB, FGFR1 or KIT on tumour cells but was significantly correlated with expression of VEGFR2 on tumour microvessels. In vitro E7080 did not show potent effects on tumour cell viability in four different sarcoma cell lines, with IC50 values ≥ 10 μM. In conclusion, E7080 showed broad in vivo antitumour activity in sarcoma, mainly attributable to angiogenesis inhibition. E7080 was also active in xenografts resistant to one or more clinically relevant reference drugs given at MTD (doxorubicin, cisplatin or ifosfamide). The present results encourage further investigation of a potential role of E7080 in sarcoma therapy in the clinic.

862 Association of polymorphisms in BRCA1 gene with breast cancer risk in Bulgarian familial cases

The erythroleukemia developed by spi-1/PU.1 transgenic mice is a multistage process characterized by an early arrest of the proerythroblast differentiation followed later on by malignant transformation. Herein, we report the presence of acquired mutations in the SCF receptor gene (Kit) in 86% of tumors isolated during the late stage of the disease. Kit mutations affect codon 814 or 818. Ectopic expression of Kit mutants in nonmalignant proerythroblasts confers erythropoietin independence and tumorigenicity to cells. Using PP1, PP2, and imatinib mesylate, we show that Kit mutants are responsible for the autonomous expansion of malignant cells via Erk1/2 and PI3K/Akt activations. These findings represent a proof of principle for oncogenic cooperativity between one proliferative and one differentiation blocking event for the development of an overt leukemia.

KGF Promotes Paracrine Activation of the SCF/c-KIT Axis from Human Keratinocytes to Melanoma Cells

The paracrine networks of the human melanoma microenvironment are able to influence tumor growth and progression. Among the paracrine growth factors involved in skin homeostasis, the KGF/FGF7 secreted by dermal fibroblasts promotes the epidermal proliferation and differentiation as well as the release from keratinocytes of other paracrine mediators. To evaluate the possible role played by KGF in affecting the behavior of different subtypes of melanoma carrying activating mutations or overexpression of the SCF receptor c-KIT, we used human melanoma cell lines, characterized by different expression levels of c-KIT and opposing responsivity to SCF, and HaCaT keratinocytes. Quantitative real-time reverse transcription-polymerase chain reaction assay and ELISA test on KGF-treated keratinocytes showed enhanced expression and secretion of SCF in response to KGF and dependent on functional KGF receptor. Immunofluorescence microscopy and biochemical analysis showed, in one of the selected melanoma cell models, SCF-dependent c-KIT activation induced by stimulation with the culture supernatants collected from KGF-treated keratinocytes. In keratinocyte-melanoma cocultures stained for the Ki67 proliferation marker, incubation with KGF induced enhanced growth not only of the keratinocytes but also of the melanoma cells, which could be blocked by the c-KIT inhibitor imatinib, demonstrating the establishment of a KGF-induced paracrine signaling network owing to the coexpression of biologically active SCF released from keratinocytes and functional c-KIT on melanoma cells.

Expression of stem cell factor receptor c-kit in human nontumoral and tumoral hepatic cells

The aim of this study was to examine and compare the stem cell factor receptor c-kit expression in hepatocellular carcinoma (HCC) with the corresponding peritumoral tissue. To confirm the immunohistochemical results in the investigated HCC tissues, HCC cell lines were analyzed for c-kit expression. Expression of c-kit (SCF receptor) has been evaluated in 72 HCC and in the corresponding surrounding nontumorous tissue. Additionally, immunohistochemical analysis reverse transcription-polymerase chain reaction was also used to examine the mRNA expression of c-kit protooncogene in tumor homogenates. Furthermore, three HCC cell lines (HUH-7, HepG2, and SK-Hep1) were used for gene-expression analysis of c-kit mRNA. C-kit expression was detected immunohistochemically in 70% of HCC with different degrees of intensity. Moreover, c-kit expression could also be found in about 90% of the corresponding peritumoral noncirrhotic as well as in cirrhotic liver tissues. C-kit mRNA was detectable in 83% of HCC and in 75% of the corresponding peritumoral noncirrhotic as well as in 100% of corresponding peritumoral cirrhotic samples. In addition, two of the three HCC cell lines (HUH-7 and SK-Hep1) showed a well detectable PCR-product for c-kit. Hepatocytes express the c-kit receptor at different grade of intensity under normal and altered pathological conditions. The presence of c-kit on HCC cell lines supports the assumption that SCF might play a role in the regulation of proliferative activity of tumorous and nontumorous hepatic cells.

IL-33 synergizes with IgE-dependent and IgE-independent agents to promote mast cell and basophil activation

Mast cell and basophil activation contributes to inflammation, bronchoconstriction, and airway hyperresponsiveness in asthma. Because IL-33 expression is inflammation inducible, we investigated IL-33-mediated effects in concert with both IgE-mediated and IgE-independent stimulation. Because the HMC-1 mast cell line can be activated by GPCR and RTK signaling, we studied the effects of IL-33 on these pathways. The IL-33- and SCF-stimulated HMC-1 cells were co-cultured with human lung fibroblasts and airway smooth muscle cells in a collagen gel contraction assay. IL-33 effects on IgE-mediated activation were studied in primary mast cells and basophils. IL-33 synergized with adenosine, C5a, SCF, and NGF receptor activation. IL-33-stimulated and SCF-stimulated HMC-1 cells demonstrated enhanced collagen gel contraction when cultured with fibroblasts or smooth muscle cells. IL-33 also synergized with IgE receptor activation of primary human mast cells and basophils. IL-33 amplifies inflammation in both IgE-independent and IgE-dependent responses.