Fionnuala B. Hickey

BCR-ABL Regulates Phosphatidylinositol 3-Kinase-p110γ Transcription and Activation and Is Required for Proliferation and Drug Resistance

The BCR-ABL oncogene is the hallmark of chronic myeloid leukemia, a clonal hematopoietic stem cell disorder. BCR-ABL displays constitutive tyrosine kinase activity, required for its transformation ability. Although the molecular mechanisms behind this malignancy are not fully understood, a role for phosphatidylinositol (PI) 3-kinase has been repeatedly described. Here we report the specific up-regulation of the class IB catalytic subunit of PI 3-kinase (p110γ) in response to BCR-ABL expression. We demonstrate that this upregulation is due to increased transcription and is dependent on both PI 3-kinase and MEK activity. We performed in vitro kinase activity assays and show that BCR-ABL also leads to increased p110γ activity and that this activation requires both G protein-coupled receptor and Ras signaling. In addition, by transfection of cells with dominant negative p110γ, we determined that this specific PI 3-kinase isoform is involved in both proliferation and the apoptosis resistance associated with chronic myeloid leukemia. The data presented here define for the first time the ability of BCR-ABL to alter the expression levels of PI 3-kinase isoforms and also demonstrate a previously unreported link between BCR-ABL and p110γ.

Adenylate cycalse toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL‐10‐secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL‐10 mRNA and protein expression in DC but significantly inhibited IL‐12p70 production. CyaA enhanced LPS‐induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF‐κB suppressed IL‐10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF‐κB did not reverse the inhibitory effect of CyaA on TLR agonist‐induced IL‐12 production. Furthermore, CyaA suppression of IL‐12 was independent of IL‐10. In contrast, CyaA suppressed LPS‐ and IFN‐γ‐induced IFN‐regulatory factor‐1 (IRF‐1) and IRF‐8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme‐inactive mutant of CyaA failed to modulate TLR‐induced signaling in DC, whereas the effects of the wild‐type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist‐induced IL‐10 and IL‐12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF‐1 and IRF‐8 expression and that this is mediated by elevation of intercellular cAMP concentrations.

Diabetic kidney disease and immune modulation

Immune modulation is now known to contribute to the development of glomerulosclerosis, tubulointerstitial fibrosis and end-stage renal disease in a large number of kidney diseases. Similarly, diabetic nephropathy is increasingly considered an inflammatory disease, with immune modulation being involved in both the development and progression of the disease. Infiltration of immune cells including macrophages, T cells, B cells and mast cells into the kidney has been reported. A number of pro-inflammatory cytokines and chemokines also play a major role in pathogenesis of diabetic nephropathy. Consequently, a variety of therapeutic strategies involving modulation of the immune response are currently being investigated in diabetic kidney disease.

IHG-1 Promotes Mitochondrial Biogenesis by Stabilizing PGC-1α

Increased expression of Induced-by-High-Glucose 1 (IHG-1) associates with tubulointerstitial fibrosis in diabetic nephropathy. IHG-1 amplifies TGF-β1 signaling, but the functions of this highly-conserved protein are not well understood. IHG-1 contains a putative mitochondrial-localization domain, and here we report that IHG-1 is specifically localized to mitochondria. IHG-1 overexpression increased mitochondrial mass and stabilized peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). Conversely, inhibition of IHG-1 expression decreased mitochondrial mass, downregulated mitochondrial proteins, and PGC-1α-regulated transcription factors, including nuclear respiratory factor 1 and mitochondrial transcription factor A (TFAM), and reduced activity of the TFAM promoter. In the unilateral ureteral obstruction model, we observed higher PGC-1α protein expression and IHG-1 levels with fibrosis. In a gene-expression database, we noted that renal biopsies of human diabetic nephropathy demonstrated higher expression of genes encoding key mitochondrial proteins, including cytochrome c and manganese superoxide dismutase, compared with control biopsies. In summary, these data suggest that IHG-1 increases mitochondrial biogenesis by promoting PGC-1α-dependent processes, potentially contributing to the pathogenesis of renal fibrosis.

Bcr-Abl regulates osteopontin transcription via Ras, PI-3K, aPKC, Raf-1, and MEK

Chronic myeloid leukemia (CML) is caused by the constitutively active Bcr‐Abl tyrosine kinase. This fusion protein is generated by the Philadelphia translocation t(9;22). CML is a progressive condition that invariably advances from a drug‐sensitive to a drug‐resistant, aggressive, acute leukemia. The mechanisms responsible for this progression are largely unknown; however, in many cases, progression is accompanied by an increase in Bcr‐Abl expression. Osteopontin (OPN) expression has been shown to be involved in the progression and increased aggression and invasiveness of many solid tumors. Here, we demonstrate that OPN expression is induced in a model of leukemia, and we describe the identification of specific signaling pathways required for the induction of OPN expression by p210 Bcr‐Abl. We have determined that high levels of Bcr‐Abl activate a signaling cascade involving the sequential activation of Ras, phosphatidylinositol‐3 kinase, atypical protein kinase C, Raf‐1, and mitogen‐activated protein kinase kinase, leading to the ultimate expression of OPN. Our results suggest that these molecules represent a single pathway and also that there is no redundancy in this pathway, as inhibition of any individual component results in a block in the induction of OPN. The data presented here define for the first time the ability of Bcr‐Abl to stimulate the expression of OPN and also identify the signaling pathway involved. This may not only prove important in understanding the mechanisms of progression of CML but also highlights a pathway that may prove significant in many other cases of oncogenesis, where OPN expression is implicated.

Diabetes mellitus and apoptosis: inflammatory cells.

Since the early observation that similarities between thyroiditis and insulitis existed, the important role played by inflammation in the development of diabetes has been appreciated. More recently, experiments have shown that inflammation also plays a prominent role in the development of target organ damage arising as complications, with both elements of the innate and the adaptive immune system being involved, and that cytokines contributing to local tissue damage may arise from both infiltrating and resident cells. This review will discuss the experimental evidence that shows that inflammatory cell-mediated apoptosis contributes to target organ damage, from beta cell destruction to both micro- and macro-vascular disease complications, and also how alterations in leukocyte turnover affects immune function.

Insulin receptor substrate 2 and FoxO3a signalling are involved in E‐cadherin expression and transforming growth factor‐β1‐induced repression in kidney epithelial cells

Insulin receptor substrate (IRS) proteins comprise a family of adaptor molecules that integrate extracellular signals from insulin and other ligands to intracellular effectors such as phosphoinositide 3‐kinase and mitogen‐activated protein kinase. The predominant forms of IRS protein in humans, IRS1 and IRS2, are widely expressed. Despite structural similarities, IRS1 and IRS2 display distinct signalling modalities, and mice lacking these proteins present with distinct phenotypes. Transforming growth factor (TGF)‐β1 is the primary cytokine shown to induce epithelial–mesenchymal transition. Recent data have demonstrated a role for IRS1 in TGF‐β1‐induced epithelial–mesenchymal transition in lung epithelial cells. In the present study, we report data showing that TGF‐β1 signals via IRS2 in kidney epithelial cells. Small interfering RNA (siRNA)‐mediated targeting of IRS2 increased E‐cadherin expression, although it did not alter TGF‐β1‐mediated E‐cadherin repression. Phosphorylation of the downstream target of IRS2/Akt signalling, FoxO3a, was induced on Ser253 and, to a lesser extent, on Thr32. Transfection of FoxO3aThr32Ala mutant for 24 h greatly reduced FoxO3a phosphorylation on Ser253 but over‐expression of FoxO3a Ser253Ala did not effect Thr32 phosphorylation, suggesting that a distinct order of phosphorylation of FoxO3a is required for physiological function in cells. Transfection of FoxO3a Ser253Ala mutant partially inhibited TGF‐β1‐mediated E‐cadherin repression at 24 h. Taken together, these data highlight novel roles for IRS2 and FoxO3a in the regulation of kidney epithelial cells by E‐cadherin.

Identification of transcriptional targets associated with the expression of p210 Bcr-Abl.

Abstract:  Objectives: Chronic myeloid leukaemia is caused by the expression of the p210 Bcr‐Abl fusion protein which results from the Philadelphia translocation, t(9;22). This oncogene has been the focus of extensive research. However, the molecular mechanisms responsible for the haematological malignancy are not fully understood. The main objective of the current study was to identify novel transcriptional targets of Bcr‐Abl. Methods: In order to achieve this, microarrays were employed in order to conduct a genome‐wide expression analysis comparing 32D cells with a transfected clone expressing high levels of p210 Bcr‐Abl. Quantitative RT‐PCR was employed in order to confirm the observed increase/decrease in expression for a number of the deregulated genes. Results and conclusions: This comparison identified 138 genes of known function showing altered expression in response to Bcr‐Abl‐mediated signalling. Among the genes found to be upregulated in response to p210 Bcr‐Abl were aldolase 1A and phosphofructokinase, both of which encode key enzymes in the glycolytic pathway. As a consequence of this, we demonstrate that the rate of glycolysis is significantly increased in Bcr‐Abl expressing cells in a PI3K‐dependent manner. Our results also indicate altered expression of genes involved in cell proliferation, cell adhesion and cell signalling.

Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies

ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

IHG-1 Increases Mitochondrial Fusion and Bioenergetic Function

Induced in high glucose-1 (IHG-1) is a conserved mitochondrial protein associated with diabetic nephropathy (DN) that amplifies profibrotic transforming growth factor (TGF)-β1 signaling and increases mitochondrial biogenesis. Here we report that inhibition of endogenous IHG-1 expression results in reduced mitochondrial respiratory capacity, ATP production, and mitochondrial fusion. Conversely, overexpression of IHG-1 leads to increased mitochondrial fusion and also protects cells from reactive oxygen species–induced apoptosis. IHG-1 forms complexes with known mediators of mitochondrial fusion—mitofusins (Mfns) 1 and 2—and enhances the GTP-binding capacity of Mfn2, suggesting that IHG-1 acts as a guanine nucleotide exchange factor. IHG-1 must be localized to mitochondria to interact with Mfn1 and Mfn2, and this interaction is necessary for increased IHG-1–mediated mitochondrial fusion. Together, these findings indicate that IHG-1 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following oxidant stress. We propose that in diabetic kidney disease increased IHG-1 expression protects cell viability and enhances the actions of TGF-β, leading to renal proximal tubule dedifferentiation, an important event in the pathogenesis of this devastating condition.