Signal Transducer and Activator of Transcription (STAT)-3 Induces Granulocyte/Macrophage Colony-Stimulating Factor (GM-CSF) Receptor-α Expression in Chronic Lymphocytic Leukemia (CLL) Cells,

Abstract 3879 GM-CSF stimulates proliferation of granulocytes, macrophages, and hematopoietic progenitors. Upon binding to its cellular receptor (R), GM-CSF induces dimerization of the GM-CSFR α and β subunits, phosphorylation of Janus kinase (JAK)-2, and activation of downstream signaling pathways. Because GM-CSF improved the response to Rituximab monotherapy in patients with CLL (Ferrajoli A. Leuk Lymphoma 50:514, 2009) and was found to upregulate CD20 cell surface antigen expression (Vanugopal P. et al. Leuk Res 24:411, 2000), we investigated the effect of GM-CSF on CLL cells. Incubation of peripheral blood (PB) CLL cells with increasing concentrations of GM-CSF (0.05 to 1.0 μM) did not induce the phosphorylation of STAT3, STAT5, AKT, or ERK as assessed by western immunoblot, or phosphorylation of JAK2 as assessed by immunoprecipitation. Therefore, we investigated whether CLL cells express GM-CSFR. Western immunoblot studies revealed that, like normal B lymphocytes, CLL cells do not express GM-CSFRβ but, unlike normal B cells, CLL cells express high levels of GM-CSFRα. Flow cytometry analysis of PB cells from 8 patients with CLL showed that 13 to 59% of CLL cells (CD19+/CD5+) co-expressed CD116 (GM-CSFRα) but not CD131 (GM-CSFRβ). Thus, we wondered what induces GM-CSFRα expression in CLL cells. Because STAT3 is constitutively activated in CLL and sequence analysis revealed that the GM-CSFRα promoter harbors γ-interferon activation sequence (GAS)-like elements typically activated by STAT3, we sought to determine whether STAT3 activates GM-CSFRα. In MM1 cells, interleukin (IL)-6 induced STAT3 phosphorylation and up-regulated GM-CSFRα whereas STAT3-siRNA down-regulated both STAT3 and GM-CSFRα protein levels, suggesting that STAT3 activates transcription of GM-CSFRα. To clarify these findings, we cloned the human GM-CSFRα promoter, generated a series of truncated promoter constructs and assessed their activity using a luciferase assay. We found that IL-6 augmented luciferase activity of GM-CSFRα promoter −4012 – +23, −3018 – +23, −2517 – +23, and −496 – +23, suggesting that IL-6 enhanced GM-CSFRα expression by activating STAT3. Furthermore, we established that regions, located between bp −3581 TTGTTGAAAA −3572, −2984 TTTTCTTAA −2976 and −77 TTTCCCAA −70, harbor GAS-like elements that activated the GM-CSFRα promoter upon exposure to IL-6. Binding of STAT3 to those regions in IL-6-stimulated MM1 cells was confirmed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). To test whether STAT3 induced transcription of GM-CSFRα in CLL, we obtained fresh PB CLL cells and, by using the same GAS-like element-containing probes, performed EMSA. CLL cell nuclear protein bound these probes and anti-STAT3 and -phosphoserine STAT3 antibodies attenuated the binding. CLL cell ChIP confirmed that STAT3 binds to the promoter of GM-CSFRα as well as the promoters of the STAT3-regulated genes STAT3, c-Myc and P21, but not to that of the control gene RPL30. Finally, using qRT-PCR and western blot analysis we determined that STAT3-shRNA down-regulated GM-CSFRα, STAT3 and STAT3-regulated gene mRNAs, and STAT3 and GM-CSFRα protein levels. Taken together, these data suggest that constitutively activated STAT3 binds to the GM-CSFRα promoter, activates its transcription, and induces production of GM-CSFRα protein in CLL cells. Disclosures: No relevant conflicts of interest to declare.

ELEVATION OF MATRIX METALLOPROTEINASES 3 AND 9 IN CEREBROSPINAL FLUID AND BLOOD IN PATIENTS WITH SEVERE TRAUMATIC BRAIN INJURY

OBJECTIVE Traumatic brain injury (TBI) causes an increase in matrix metalloproteinases (MMPs), which are associated with neuroinflammation, blood-brain barrier disruption, hemorrhage, and cell death. We hypothesized that patients with TBI have an increase in MMPs in ventricular cerebrospinal fluid (CSF) and plasma. METHODS Patients with TBI and a ventricular catheter were entered into the study. Samples of CSF and plasma were collected at the time of catheter placement and at 24 and 72 hours after admission. Seven TBI patients were entered into the study, with 6 having complete data for analysis. Only patients who had a known time of insult that fell within a 6-hour window from initial insult to ventriculostomy were accepted into the study. Control CSF came from ventricular fluid in patients undergoing shunt placement for normal pressure hydrocephalus. Both MMP-2 and MMP-9 were measured with gelatin zymography and MMP-3 with Western immunoblot. RESULTS We found a significant elevation in the levels of the latent form of MMP-9 (92-kD) in the CSF obtained at the time of arrival (P < 0.05). Elevated levels of MMP-2 were detected in plasma at 72 hours, but not in the CSF. Using albumin from both CSF and blood, we calculated the MMP-9 index, which was significantly increased in the CSF, indicating endogenous MMP production. Western immunoblot showed elevated levels of MMP-3 in CSF at all times measured, whereas MMP-3 was not detected in the CSF of normal pressure hydrocephalus. CONCLUSION We show that MMPs are increased in the CSF of TBI patients. Although the number of patients was small, the results were robust and clearly demonstrated increases in MMP-3 and MMP-9 in ventricular CSF in TBI patients compared with controls. Although these preliminary results will need to be replicated, we propose that MMPs may be important in blood-brain barrier opening and hemorrhage secondary to brain injury in patients.

Zinc and DHA have opposing effects on the expression levels of histones H3 and H4 in human neuronal cells

Zn and DHA have putative neuroprotective effects and these two essential nutrients are known to interact biochemically. We aimed to identify novel protein candidates that are differentially expressed in human neuronal cell line M17 in response to Zn and DHA that would explain the molecular basis of this interaction. Two-dimensional gel electrophoresis and MS were applied to identify major protein expression changes in the protein lysates of human Ml7 neuronal cells that had been grown in the presence and absence of Zn and DHA. Proteomic findings were further investigated using Western immunoblot and real-time PCR analyses. Four protein spots, which had significant differential expression, were identified and selected for in-gel trypsin digestion followed by matrix-assisted laser desorption ionisation MS analysis. The resultant peptide mass fingerprint for each spot allowed their respective identities to be deduced. Two human histone variants H3 and H4 were identified. Both H3 and H4 were downregulated by Zn in the absence of DHA (Zn effect) and upregulated by DHA (DHA effect) in the presence of Zn (physiological condition). These proteomic findings were further supported by Western immunoblot and real-time PCR analyses using H3- and H4-specific monoclonal antibodies and oligonucleotide primers, respectively. We propose that dietary Zn and DHA cause a global effect on gene expression, which is mediated by histones. Such novel information provides possible clues to the molecular basis of neuroprotection by Zn and DHA that may contribute to the future treatment, prevention and management of neurodegenerative diseases such as Alzheimer's disease.

Apoprotein(A) Isoforms and Plasma LP(A) Concentration in Members of Four Families

Apoprotein(A) Isoforms and Plasma LP(A) Concentration in Members of Four FamiliesApoprotein(a) is a multikringle protein which shows a genetically inherited size polymorphism. The APO(a) gene is located at the telomeric region of chromosome 6q2.6-q 2.7. Apo(a) size polymorphism is a major determinant of Lp(a) levels. The aim of this study is to describe the influence of apo(a) size polymorphism on the plasma Lp(a) levels in the members of four families. K3EDTA plasma was obtained from every subject after over-night fast. Apo(a) isoforms were determined by 3-15% SDS-PAGE followed by Western immunoblot technique. Plasma Lp(a) level was de - termined with immunonephelometric method. Every child inherited one isoform from its mother and the other from its father. The children from the first family had Lp(a) levels similar to those measured in their parents. The daughters from the second and fourth family inherited the dominant S3 apo(a) isoform from their mothers and also mother's high Lp(a) levels (0.365 g/L - daughter from the second, and 0.465 g/L and 0.446 g/L - daughter from the fourth family respectively). The elder daughter from the third family, carrier of double banded S4S1 apo(a) isoform, had the highest Lp(a) level among the children from all four families. We found out a generation decrease of the Lp(a) level in two families. On the basis of our findings we concluded that the inheritance of the apo(a) isoforms in the members of all four families is in accordance with the simple Mendelian's model and that the apo(a) size polymorphism influences the Lp(a) level in the blood of the examined subjects.