Alternative Splicing in Angiogenesis Bowler, Elizabeth; Oltean, Sebastian
International journal of molecular sciences,
04/2019, Volume:
20, Issue:
9
Journal Article
Peer reviewed
Open access
Alternative splicing of pre-mRNA allows the generation of multiple splice isoforms from a given gene, which can have distinct functions. In fact, splice isoforms can have opposing functions and there ...are many instances whereby a splice isoform acts as an inhibitor of canonical isoform function, thereby adding an additional layer of regulation to important processes. Angiogenesis is an important process that is governed by alternative splicing mechanisms. This review focuses on the alternative spliced isoforms of key genes that are involved in the angiogenesis process;
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative ...splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined.
PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003.
In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b.
Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mitochondria are critical to cardiac injury during reperfusion as a result of damage sustained during ischemia, including the loss of bcl-2. We asked if bcl-2 depletion not only leads to selective ...permeation of the outer mitochondrial membrane (MOMP) favoring cytochrome c release and programmed cell death, but also favors opening of the mitochondrial permeability transition pore (MPTP). An increase in MPTP susceptibility would support a role for bcl-2 depletion mediated cell death in the calcium overload setting of early reperfusion via MPTP as well as later in reperfusion via MOMP as myocardial calcium content normalizes.
Calcium retention capacity (CRC) was used to reflect the sensitivity of the MPTP opening in isolated cardiac mitochondria. To study the relationship between bcl-2 inhibition and MPTP opening, mitochondria were incubated with a bcl-2 inhibitor (HA14-1) and CRC measured. The contribution of preserved bcl-2 content to MPTP opening following ischemia-reperfusion was explored using transgenic bcl-2 overexpressed mice.
CRC was decreased in mitochondria following reperfusion compared to ischemia alone, indicating that reperfusion further sensitizes to MPTP opening. Incubation of ischemia-damaged mitochondria with increasing HA14-1concentrations increased calcium-stimulated MPTP opening, supporting that functional inhibition of bcl-2 during simulated reperfusion favors MPTP opening. Moreover, HA14-1 sensitivity was increased by ischemia compared to non-ischemic controls. Overexpression of bcl-2 attenuated MPTP opening in following ischemia-reperfusion. HA14-1 inhibition also increased the permeability of the outer membrane in the absence of exogenous calcium, indicating that bcl-2 inhibition favors MOMP when calcium is low.
The depletion and functional inhibition of bcl-2 contributes to cardiac injury by increasing susceptibility to MPTP opening in high calcium environments and MOMP in the absence of calcium overload. Thus, ischemia-damaged mitochondria with decreased bcl-2 content are susceptible to MPTP opening in early reperfusion and MOMP later in reperfusion when cytosolic calcium has normalized.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Local hypoxia occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to ...hypoxia. However, most research has focused on hypoxia‐inducible genes as opposed to those that are decreased in hypoxia. We demonstrate that chromatin accessibility is decreased in hypoxia, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing, and the R‐loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts, and in patient samples with hypoxic tumors. Most interestingly, we found that when DDX5 is rescued in hypoxia, replication stress and R‐loop levels accumulate further, demonstrating that hypoxia‐mediated repression of DDX5 restricts R‐loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R‐loop processing factors; however, as shown for DDX5, their role is specific and distinct.
Regions of hypoxia occur in solid tumors and are associated with cancer progression and therapy resistance. The repression of specific pathways in response to hypoxia like DNA repair and splicing factors was described previously. Here, we find that genes encoding proteins with roles in the R‐loop interactome are repressed in hypoxia and link this to decreased chromatin accessibility. DDX5 expression was decreased under hypoxia in cell lines (cancer and normal), tumor xenografts and hypoxic tumors from cancer patients leading to reduced R‐loop accumulation and replication stress.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Inhibition of the ATR kinase has emerged as a therapeutically attractive means to target cancer since the development of potent inhibitors, which are now in clinical testing. We investigated a ...potential link between ATR inhibition and the autophagy process in esophageal cancer cells using four ATR inhibitors including two in clinical testing. The response to pharmacological ATR inhibitors was compared with genetic systems to investigate the ATR dependence of the effects observed. The ATR inhibitor, VX-970, was found to lead to an accumulation of p62 and LC3-II indicative of a blocked autophagy. This increase in p62 occurred post-transcriptionally and in all the cell lines tested. However, our data indicate that the accumulation of p62 occurred in an ATR-independent manner and was instead an off-target response to the ATR inhibitor. This study has important implications for the clinical response to pharmacological ATR inhibition, which in some cases includes the blockage of autophagy.
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•Inhibition of ATR using VX-970 leads to an accumulation of p62•VX-970-mediated accumulation of p62 occurs independently of ATR•VX-970-mediated accumulation of p62 is consistent with blocked autophagy
Biological Sciences; Biochemistry; Biochemical Mechanism; Cancer
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Alternative splicing is a key process required for the regulation of gene expression in normal development and physiology. It is regulated by splice factors whose activities are in turn regulated by ...splice factor kinases and phosphatases. The CDC-like protein kinases are a widespread family of splice factor kinases involved in normal physiology and in several diseases including cancer. In humans they include the CLK1, CLK2, CLK3 and CLK4 genes. The expression of CLK1 is regulated through alternative splicing producing both full-length catalytically active and truncated catalytically inactive isoforms, CLKT1 (arising from exon 4 skipping) and CLKT2 (arising from intron 4 retention). We examined CLK1 alternative splicing in a range of cancer cell lines, and report widespread and highly variable rates of exon 4 skipping and intron 4 retention. We also examined the effect of severe environmental stress including heat shock, osmotic shock, and exposure to the alkaloid drug harmine on CLK1 alternative splicing in DU145 prostate cancer cells. All treatments rapidly reduced exon 4 skipping and intron 4 retention, shifting the balance towards full-length CLK1 expression. We also found that the inhibition of CLK1 with the benzothiazole TG003 reduced exon 4 skipping and intron 4 retention suggesting an autoregulatory mechanism. CLK1 inhibition with TG003 also resulted in modified alternative splicing of five cancer-associated genes.
•The CDC2-like splice factor kinase CLK1 regulates the intracellular activities of key splice factors.•Both exon 4 skipping and intron 4 retention result in the expression of truncated CLK1 that lacks the catalytic domain.•We observe highly variable alternative splicing of CLK1 in a range of human cancer cell lines.•CLK1 intron 4 retention and exon 4 skipping rapidly diminish in conditions of severe cellular stress.•CLK1 inhibition with the benzothiazole TG003 results in an upregulation of CLK1, suggesting a feedback loop.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Working with Hypoxia Bowler, Elizabeth; Ladomery, Michael R
Methods in molecular biology (Clifton, N.J.),
01/2019, Volume:
1990
Journal Article
A hypoxic environment can be defined as a region of the body or the whole body that is deprived of oxygen. Hypoxia is a feature of many diseases, such as cardiovascular disease, tissue trauma, ...stroke, and solid cancers. A loss of oxygen supply usually results in cell death; however, when cells gradually become hypoxic, they may survive and continue to thrive as described for conditions that promote metastatic growth. The role of hypoxia in these pathogenic pathways is therefore of great interest, and understanding the effect of hypoxia in regulating these mechanisms is fundamentally important. This chapter gives an extensive overview of these mechanisms. Moreover, given the challenges posed by tumor hypoxia we describe the current methods to simulate and detect hypoxic conditions followed by a discussion on current and experimental therapies that target hypoxic cells.
Myositis-specific and associated autoantibodies are important biomarkers in routine clinical use. We assessed local testing performance for myositis autoantibodies by comparing line immunoassay (LIA) ...to protein radio-immunoprecipitation and identifying clinical characteristics associated with each myositis autoantibody in the MyoCite cohort.
Serum samples from patients within the MyoCite cohort, a well-characterised retro-prospective dataset of adult and juvenile idiopathic inflammatory myopathy (IIM) patients in Lucknow, India (2017-2020), underwent LIA at Sanjay Gandhi Postgraduate Institute of Medical Science (SGPGIMS), Lucknow. Immunoprecipitation of 147 IIM patient serum samples (125 adult-onset, 22 juvenile-onset) was conducted at the University of Bath, with researchers blind to LIA results. LIA performance was assessed against Immunoprecipitation as the reference standard, measuring sensitivity, specificity, and inter-rater agreement. Univariate and multivariate logistic regression determined clinical associations for specific MSA.
Immunoprecipitation identified myositis autoantibodies in 56.5% (n = 83) of patient samples, with anti-Jo1 (n = 16; 10.9%) as the most common, followed by anti-MDA5 (n = 14, 9.5%). While LIA showed good agreement for anti-Jo1, anti-PL7 and anti-PL12 (Cohen's κ 0.79, 0.83, and 1, respectively), poor agreement was observed in other subgroups, notably anti-TIF1γ (Cohen's κ 0.21). Strongly positive samples, especially in myositis-specific autoantibodies, correlated more with immunoprecipitation results. Overall, 59 (40.1%) samples exhibited non-congruence on LIA and Immunoprecipitation, and κ values for LIA's for anti-TIF1γ, anti-Ku, anti-PmScl, anti-Mi2, and anti-SAE ranged between 0.21-0.60.
While LIA reliably detected anti-Jo1, anti-PL7, anti-PL12, anti-MDA5, and anti-NXP-2, it also displayed false positives and negatives. Its effectiveness in detecting other autoantibodies, such as anti-TIF1γ, was poor.
Abstract
Background/Aims
Idiopathic inflammatory myopathies (IIM) are multisystem diseases characterised by muscle inflammation. Over 60% of patients with IIM have a myositis-related autoantibody. ...Our laboratory specialises in autoantibody testing. We offer an extended spectrum autoantibody testing diagnostic service and have also screened over 3000 myositis patients enrolled in research studies for the presence of autoantibodies. We noticed a recurring pattern following K562 cell radio-immunoprecipitation that was present in several myositis patient sera and in a handful of samples that came to us via the diagnostic service. We set out to determine the antigenic target of this novel autoantibody.
Methods
We have previously screened 1319 serum/plasma samples from IIM patients enrolled in the UKMyoNet and 380 from patients with juvenile onset IIM enrolled in the JDCBS for autoantibodies by immunoprecipitation. Additional cohorts similarly investigated include >150 healthy control sera and >400 SLE patient sera. Patients with the novel autoantibody of interest were identified by a distinctive 120kDa band associated with a ‘smear’ on autoradiography following K562 cell immunoprecipitation and separation of autoantigens by SDS PAGE. ESI-QTOF mass spectrometry was used to identify the antigenic target in an example serum. Using a commercial anti-PARP1 antibody as a control, western blotting of K562 and PARP1 overexpressed cell lysate was used to confirm the antigenic target in remaining samples of interest. Indirect immunofluorescence was performed on HEp-2 cells according to manufacturers’ instructions.
Results
11 patient samples were identified as having the 120 complex ‘smear’ pattern of interest: six had been received via the diagnostic service and five were enrolled in UKMyoNet. Prevalence in the UKMyoNet cohort was 0.4%. Mass spectrometry identified PARP1 as the antigenic target. This was confirmed in all remaining samples by western blot. Table 1 shows available clinical data.
Conclusion
PARP1 is an autoantigen target in myositis patients in addition to those with other rheumatic diseases. Where data was available, patients with anti-PARP1 had features suggesting an anti-synthetase syndrome phenotype, but this may primarily relate to the other autoantibodies present. Further work is needed to determine the prevalence of anti-PARP1 in other cohorts and its clinical associations.
Disclosure
V. Riddell: Grants/research support; Bath Institute of Rheumatic Disease. Z. Betteridge: None. E. Bowler: None. H. Chinoy: None. P. Gordon: None. L. Wedderburn: None. S. Bagby: None. N. Mchugh: None. S. Tansley: None.
The reverse electron flow-induced ROS generation (RFIR) is decreased in ischemia-damaged mitochondria. Cardiac ischemia leads to decreased complex I activity and depolarized inner mitochondrial ...membrane potential (ΔΨ) that are two key factors to affect the RFIR in isolated mitochondria. We asked if a partial inhibition of complex I activity without alteration of the ΔΨ is able to decrease the RFIR.
Cardiac mitochondria were isolated from mouse heart (C57BL/6) with and without ischemia. The rate of H2O2 production from mitochondria was determined using amplex red coupled with horseradish peroxidase. Mitochondria were isolated from the mitochondrial-targeted STAT3 overexpressing mouse (MLS-STAT3E) to clarify the role of partial complex I inhibition in RFIR production.
The RFIR was decreased in ischemia-damaged mouse heart mitochondria with decreased complex I activity and depolarized ΔΨ. However, the RFIR was not altered in the MLS-STAT3E heart mitochondria with complex I defect but without depolarization of the ΔΨ. A slight depolarization of the ΔΨ in wild type mitochondria completely eliminated the RFIR.
The mild uncoupling but not the partially decreased complex I activity contributes to the observed decrease in RFIR in ischemia-damaged mitochondria.
The RFIR is less likely to be a key source of cardiac injury during reperfusion.
•Ischemia decreases the reverse electron flow-induced ROS generation (RFIR).•Ischemia inhibits complex I and depolarizes inner membrane potential (ΔΨ).•A genomic model of complex I deficiency without alteration to ΔΨ does not alter RFIR.•The decreased RFIR in ischemia-damaged mitochondria is due to depolarized ΔΨ.•RFIR is not a critical source of cardiac injury during reperfusion.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
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