The effect of growth hormone administration on the regulation of mitochondrial apoptosis in-vivo

The purpose of this study was to determine whether recombinant human growth hormone (rhGH) would show any significant effects on the expression of apoptosis regulating proteins in peripheral blood mononuclear cells (PBMCs). Additionally, the potential for post-transcriptional regulation of gene expression by miRNA was assessed in two cellular compartments, the cytosol and the mitochondria. Ten male subjects were subcutaneously injected with either rhGH (1 mg) or saline (0.9%) for seven consecutive days in a double-blinded fashion. Blood sampling was undertaken prior to treatment administration and over a period of three weeks following treatment cessation. Bcl-2 and Bak gene and protein expression levels were measured in PBMCs, while attention was also directed to the expression of miR-181a and miR-125b, known translational inhibitors of Bcl-2 and Bak respectively. Results showed that rhGH significantly decreased Bak protein concentrations compared to placebo samples for up to 8 days post treatment. While cytosolic miRNA expression was not found to be significantly affected by rhGH, measurement of the expression of miR-125b in mitochondrial fractions showed a significant down-regulation eight days post-rhGH administration. These findings suggest that rhGH induces short-term anti-apoptotic effects which may be partially mediated through a novel pathway that alters the concentration of mitochondrially-associated miRNAs

Chemokines (CCL3, CCL4, CCL5) inhibit ATP-induced release of IL-1beta by monocytic cells

ATP and chemokines are among the first inflammatory mediators that can enter the circulation via damaged blood vessels at the site of injury, leading to an activation of the host’s immune response. The main function of chemokines is leukocyte mobilization, guiding immune cells towards the injured tissue along a chemotactic concentration gradient. In monocytes, ATP typically triggers inflammasome assembly, a multiprotein complex necessary for the maturation and secretion of IL-1beta. IL-1beta is a potent inflammatory cytokine of innate immunity, essential for pathogen defense. However, excessive IL-1beta may cause life-threatening systemic inflammation. Here, we hypothesize that chemokines control ATP-dependent secretion of monocytic IL-1beta, by engaging a cholinergic signaling pathway. LPS-primed human monocytic U937 cells were treated with chemokines in the presence or absence of nAChR antagonists or iPLA2beta inhibitors and concomitantly stimulated with the P2X7 agonist BzATP. IL-1beta concentration was determined in the cell culture supernatants. Silencing of the chemokine receptor and iPLA2b gene expression was achieved by transfecting cells with the appropriate siRNA. CCL3, CCL4, and CCL5 dose-dependently inhibited BzATP-stimulated release of IL-1beta, whereas CXCL16 was ineffective. The effect of CCL3 was confirmed for primary mononuclear leukocytes. The inhibitory effect of CCL3 was blunted after silencing CCR1 or iPLA2beta gene expression by siRNA and was sensitive to antagonists of nAChRs containing subunits alpha7 and alpha9/alpha10. U937 cells secreted small factors in response to CCL3 that mediated the inhibition of IL-1beta release. We suggest that CCL chemokines inhibit ATP-induced release of IL-1beta from U937 cells by a triple-membrane-passing mechanism involving CCR, iPLA2, release of small mediators, and nAChR subunits alpha7 and alpha9/alpha10. We speculate that whenever chemokines and ATP enter the circulation concomitantly, systemic release of IL-1beta is minimized

G-CSF receptor truncations found in SCN/AML relieve SOCS3-controlled inhibition of STAT5 but leave suppression of STAT3 intact

Truncated granulocyte colony-stimulating factor receptors (G-CSF-Rs) are implicated in severe congenital neutropenia (SCN) and the consecutive development of acute myeloid leukemia (AML). Mice expressing G-CSF-R truncation mutants (gcsfr-d715) show defective receptor internalization, an increased signal transducer and activator of transcription 5 (STAT5)/STAT3 activation ratio, and hyperproliferative responses to G-CSF treatment. We determined whether a lack of negative feedback by suppressor of cytokine signaling (SOCS) proteins contributes to the signaling abnormalities of G-CSF-R-d715. Expression of SOCS3 transcripts in bone marrow cells from G-CSF-treated gcsfr-d715 mice was approximately 60% lower than in wild-type (WT) littermates. SOCS3 efficiently suppressed STAT3 and STAT5 activation by WT G-CSF-R in luciferase reporter assays. In contrast, while SOCS3 still inhibited STAT3 activation by G-CSF-R-d715, STAT5 activation was no longer affected. This was due mainly to loss of the SOCS3 recruitment site Tyr729, with an additional contribution of the internalization defects of G-CSF-R-d715. Because Tyr729 is also a docking site for the Src homology 2-containing protein tyrosine phosphatase-2 (SHP-2), which binds to and inactivates STAT5, we suggest a model in which reduced SOCS3 expression, combined with the loss of recruitment of both SOCS3 and SHP-2 to the activated receptor complex, determine the increased STAT5/STAT3 activation ratio and the resulting signaling abnormalities projected by truncated G-CSF-R mutants

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues

Characterisation of the zinc fingers of Erythroid Kruppel-Like Factor

Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis

Characterization of an antisense oligonucleotide targeted at Phospholipase C gamma 1 in the mouse T-cell line EL4.1L-2

Master'sMASTER OF SCIENC

Characterizing the endoribonuclease activity of APE1.

Recent evidence shows that mRNA stability and turn-over is an integral control point in the regulation of gene expression. The stability of various mRNAs within a eukaryotic cell can differ and this results in a magnitude of difference in mRNA abundance. An enzyme known as APE1, apurinic/apyrimidinic DNA endonuclease 1, has recently been discovered to possess an endoribonuclease activity against c-myc messenger RNA (mRNA) in vitro. The identification of APE1 as an endoribonuclease warranted this research to further characterize this novel activity both in vitro and in vivo. Previous studies have discovered the residues constituting an active site for apurinic/aprymidinic [sic] DNA (AP-DNA) incision activity of APE1. Whether these residues are shared in the RNA-cleaving activity of APE1 was unknown. The first objective of this thesis was to assess the role of these amino acid residues in contributing to the endoribonuclease activity of APE1. Our results revealed that APE1 indeed shared these residues to cleave both RNA and AP-DNA. However, we also discovered certain differences in the activities of one mutant (D283N) in carrying out AP-DNA and RNA incisions. This suggested that the roles of active site residues in each reaction are not entirely identical. In addition, we have assessed the RNA-cleaving activities of APE1 variants identified in the human population. For a few variants, RNA-cleaving activities were severely reduced while its AP-DNA incision activities were functional. These results suggested a possible unrecognized link between the reductions in the RNA-cleaving activity of the variants and their reported association in certain diseases. The second objective of this thesis was to establish the RNA secondary structures and sequences that are preferentially cleaved by APE1. Our results revealed that APE1 has preference for cleaving the single stranded regions or weakly base paired regions of the RNA. Also, preferred sequences of cleavage were determined to be UA, UG, and CA dinucleotides. Prevalent AThe original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b162505

RNA sequences and structures cleaved by a novel mammalian endoribonuclease in vitro.

No abstract available.The original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b130187

SHARPIN Is Essential for Cytokine Production, NF-κB Signaling, and Induction of Th1 Differentiation by Dendritic Cells

Spontaneous mutations of the Sharpin (SHANK-associated RH domain-interacting protein, other aliases: Rbckl1, Sipl1) gene in mice result in systemic inflammation that is characterized by chronic proliferative dermatitis and dysregulated secretion of T helper1 (Th1) and Th2 cytokines. The cellular and molecular mechanisms underlying this inflammatory phenotype remain elusive. Dendritic cells may contribute to the initiation and progression of the phenotype of SHARPIN-deficient mice because of their pivotal role in innate and adaptive immunity. Here we show by flow cytometry that SHARPIN- deficiency did not alter the distribution of different DC subtypes in the spleen. In response to TOLL-like receptor (TLR) agonists LPS and poly I:C, cultured bone marrow-derived dendritic cells (BMDC) from WT and mutant mice exhibited similar increases in expression of co-stimulatory molecules CD40, CD80, and CD86. However, stimulated SHARPIN-deficient BMDC had reduced transcription and secretion of pro-inflammatory mediators IL6, IL12P70, GMCSF, and nitric oxide. Mutant BMDC had defective activation of NF-κB signaling, whereas the MAPK1/3 (ERK1/2) and MAPK11/12/13/14 (p38 MAP kinase isoforms) and TBK1 signaling pathways were intact. A mixed lymphocyte reaction showed that mutant BMDC only induced a weak Th1 immune response but stimulated increased Th2 cytokine production from allogeneic naïve CD4+ T cells. In conclusion, loss of Sharpin in mice significantly affects the immune function of DC and this may partially account for the systemic inflammation and Th2-biased immune response

Purification, identification and characterization of mammalian endoribonucleases that degrade c-myc mRNA in vitro.

There is increasing evidence that mammalian endoribonucleases play a significant role in the degradation of messenger RNA (mRNA) and are key players in the regulation of gene expression particularly under conditions of cellular stress. One of the major challenges, however, is identifying these enzymes and assessing their significance within the context of normal/basal levels of gene expression. To this end, our understanding of this diverse set of enzymes, the mechanisms by which they operate, and their target substrates, remains somewhat mysterious. Previous studies in this laboratory (Bergstrom et al. 2006) have uncovered a mammalian hepatic-derived endoribonuclease with the ability to degrade c-myc CRD RNA in vitro. However, the identity of the enzyme(s) and the remaining co-purified proteins was not determined. The c-myc transcript is a regulator of cell proliferation, differentiation and apoptosis. Deregulated expression of c-myc prevents differentiation of many cell types, induces apoptosis, induces genomic instability, and is associated with several tumor phenotypes. In addition, there is widespread evidence that mRNA stability plays a critical role in the regulation of c-myc gene expression. The main goal of this thesis was to re-purify and conclusively identify the mammalian hepatic-derived endoribonuclease(s) and the proteins that co-purified with endonucleolytic activity against c-myc CRD RNA in vitro. The first aim of this investigation was to purify and identify enzyme(s) responsible for endoribonucleolytic activity. This portion of the study demonstrates that distinct mammalian proteins with molecular weights corresponding to 17 kDa and 35 kDa, respectively, exhibit endoribonuclease activity against c-myc CRD RNA. The second aim of this study was to further characterize the endoribonuclease(s) and to confirm the identity of the enzyme(s) by immunodepleting native endoribonuclease activity. This study revealed that the 17 kDa endoribonuclease activity was contributed by rat pancreatic ribonucleaseThe original print copy of this thesis may be available here: http://wizard.unbc.ca/record=b135329