Polo-like Kinase 2 Research Articles

Polo-like kinase 2 targeting as novel strategy to sensitize mutant p53-expressing tumor cells to anticancer treatments.

Polo-like kinase 2 (Plk2) belongs to a family of serine/threonine kinases, and it is involved in tumorigenesis of diverse kind of tissues. We previously reported that Plk2 gene was a transcriptional target of the mutant p53/NF-Y oncogenic complex. Plk2 protein can bind to and phosphorylate mutant p53 triggering an oncogenic autoregulatory feedback loop involved in cancer cell proliferation and chemoresistance. In this study, we aimed to assess whether the specific inhibition of Plk2 kinase activity by the selective TC-S 7005 inhibitor could decrease cell proliferation and migration inhibiting mutant p53 phosphorylation, thus disarming its oncogenic potential. We found that the Plk2 inhibitor treatment sensitized the cells to the irradiation and chemotherapy drugs, thereby overcoming the mutant p53-dependent chemoresistance. Taken together, we provided results that Plk2 could be considered a tractable pharmacological target for cancers expressing mutant p53 proteins. The combined treatment with conventional chemotherapeutic drugs and Plk2 inhibitors may represent a new candidate intervention approach, which may be considered for improving tumor cell sensitivity to DNA damaging drugs. KEY MESSAGES : Missense mutations are present in the TP53 gene in about half of all human cancers and correlate with poor patient outcome. Mutant p53 proteins exert gain of function (GOF) activities in tumor cells such as increased proliferation, genomic instability and resistance to therapies. Polo-like kinase 2 (PLK2) binds and phosphorylates mutant p53 protein strengthening its GOF activities. Pharmacologically targeting PLK2 weakens mutant p53 proteins and sensitizes tumor cells to therapeutic treatments.

Pharmacological inhibition of PLK2 kinase activity mitigates cognitive decline but aggravates APP pathology in a sex-dependent manner in APP/PS1 mouse model of Alzheimer's disease

Converging evidence from clinical and experimental studies suggest the potential significance of Polo-like kinase 2 (PLK2) in regulating the phosphorylation and toxicity of the Alzheimer's disease (AD)-related protein, amyloid precursor protein (APP). These findings have prompted various experimental trials aimed at inhibiting PLK2 kinase activity in different transgenic mouse models of AD. While positive impacts on cognitive decline were reported in these studies, the cellular effects remained controversial. In the present study, we sought to assess the cognitive and cellular consequences of chronic PLK2 inhibitor treatment in the APP/PS1 transgenic mouse model of AD. First, we confirmed that inhibiting PLK2 prevented cognitive decline in a sex-dependent manner, particularly by enhancing working memory in male APP/PS1 mice. Surprisingly, cellular analysis revealed that treatment with PLK2 inhibitor increased the load of amyloid plaques and elevated levels of soluble amyloid β (Aβ) 40 and Aβ42 in the cortex, as well as insoluble Aβ42 in the hippocampus of female mice, without affecting APP pathology in males. These results underscore the potential of PLK2 inhibition to mitigate cognitive symptoms in males. However, paradoxically, it intensifies amyloid pathology in females by enhancing APP amyloidogenic processing, creating a controversial aspect to its therapeutic impact. Overall, these data highlight the sex-dependent nature of the effects of PLK2 inhibition, which may also be influenced by the genetic background of the transgenic mouse model utilized.

#1996 Identification of the biological bases associated with the mTOR-I-related podocytopathy through high-resolution mass spectrometry

Abstract Background and Aims mTOR inhibitors (mTOR-I) often cause podocytopathy with the development of proteinuria (sometimes in the nephrotic range) in kidney transplant patients, but the biological mechanisms underlying this effect have been only partially defined. Method Immortalized human podocytes were treated in vitro with different everolimus (EVE) doses (10, 100, 200, 500 nM) for 24 h. Total cell lysates and supernatants were subjected to high-resolution mass spectrometry (Proteomics analysis). Innovative tests were used for bioinformatics analysis, including the Support Vector machine (SVM) and partial least squares discriminant analysis (PLS-DA). The results were validated in vitro and in human urine samples from 30 patients treated with EVE-based immunosuppressive treatment, using various molecular biology techniques (western blot, ELISA). Results Proteomic evaluation identified more than 7000 differentially expressed proteins. Among these, after a conservative statistical analysis, 78 showed an expression level significantly and directly correlated with EVE dosage. Functional analysis of the selected proteins showed a biological interconnection that involved several pathways, including kinases, cell cycle regulation, epithelial–mesenchymal transition, and protein synthesis. Osteopontin (SPP1) and Polo Like Kinase 2 (PLK2) were two of the most significantly correlated proteins (p < 0.00001, FDR<0.5%). The data were then confirmed through biomolecular experiments in vitro and in vivo in urine samples from kidney transplant recipients who received immunosuppressive treatment with EVE. Conclusion This study, for the first time, analyzed the biological impact of mTOR-I on podocytes, helping to understand not only the molecular basis of its therapeutic effect but also the biological reasons underlying their adverse events (particularly proteinuria). Validation of our data on larger cohorts of patients treated with this category of drugs will help identify new therapeutic targets to minimize adverse effects.

Emerging role of immunogenic cell death in cancer immunotherapy.

Cancer immunotherapy, such as immune checkpoint blockade (ICB), has emerged as a groundbreaking approach for effective cancer treatment. Despite its considerable potential, clinical studies have indicated that the current response rate to cancer immunotherapy is suboptimal, primarily attributed to low immunogenicity in certain types of malignant tumors. Immunogenic cell death (ICD) represents a form of regulated cell death (RCD) capable of enhancing tumor immunogenicity and activating tumor-specific innate and adaptive immune responses in immunocompetent hosts. Therefore, gaining a deeper understanding of ICD and its evolution is crucial for developing more effective cancer therapeutic strategies. This review focuses exclusively on both historical and recent discoveries related to ICD modes and their mechanistic insights, particularly within the context of cancer immunotherapy. Our recent findings are also highlighted, revealing a mode of ICD induction facilitated by atypical interferon (IFN)-stimulated genes (ISGs), including polo-like kinase 2 (PLK2), during hyperactive type I IFN signaling. The review concludes by discussing the therapeutic potential of ICD, with special attention to its relevance in both preclinical and clinical settings within the field of cancer immunotherapy.

Icariin Inhibits Overexpression and Aggregation of α-Synuclein in A53T α-Synuclein Transgenic Mice by Regulating Parkin and PLK2.

Synucleinopathies, which are major pathological features of Parkinson's disease (PD), are characterized by misfolded aggregates of α-synuclein in the peripheral and central nervous system. Icariin (ICA) is the main active component of Epimedium flavonoids. Our previous study found that ICA decreases α-synuclein expression in APPV717I transgenic mice. The aim of the present study was to examine the potential applications and mechanisms of ICA in PD using A53T α-synuclein transgenic (A53T Tg) mice. After 3 months of intragastric ICA administration, rotarod and pole tests were used to assess behavioral changes in A53T Tg mice at 8 and 13 months of age. SH-SY5Y cells over-expressing wild-type α-synuclein were used to further examine the pharmacological effect and underlying mechanism of ICA. Western blotting and immunocytochemistry were used to detect the expression levels of α-synuclein and its related proteins. ICA significantly improved the impaired motor function and coordination in A53T Tg mice. It also decreased the expression, Ser129 phosphorylation, and aggregation of α-synuclein in SH-SY5Y cells transfected with α-synuclein and the striatum of A53T Tg mice. Moreover, ICA increased the expression of parkin, which is associated with the ubiquitin-proteasome system (UPS), and decreased the level of polo-like kinase 2 (PLK2), an enzyme that phosphorylates α-synuclein. ICA alleviated motor impairments in A53T mice, an effect which may be associated with the decreased phosphorylation and aggregation of α-synuclein through PLK2 and parkin regulation.

Expansion of interferon inducible gene pool via USP18 inhibition promotes cancer cell pyroptosis

While immunotherapy has emerged as a breakthrough cancer therapy, it is only effective in some patients, indicating the need of alternative therapeutic strategies. Induction of cancer immunogenic cell death (ICD) is one promising way to elicit potent adaptive immune responses against tumor-associated antigens. Type I interferon (IFN) is well known to play important roles in different aspects of immune responses, including modulating ICD in anti-tumor action. However, how to expand IFN effect in promoting ICD responses has not been addressed. Here we show that depletion of ubiquitin specific protease 18 (USP18), a negative regulator of IFN signaling, selectively induces cancer cell ICD. Lower USP18 expression correlates with better survival across human selected cancer types and delays cancer progression in mouse models. Mechanistically, nuclear USP18 controls the enhancer landscape of cancer cells and diminishes STAT2-mediated transcription complex binding to IFN-responsive elements. Consequently, USP18 suppression not only enhances expression of canonical IFN-stimulated genes (ISGs), but also activates the expression of a set of atypical ISGs and NF-κB target genes, including genes such as Polo like kinase 2 (PLK2), that induce cancer pyroptosis. These findings may support the use of targeting USP18 as a potential cancer immunotherapy.

Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5.

Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress.

Inhibition of PLK2 activity affects APP and tau pathology and improves synaptic content in a sex-dependent manner in a 3xTg mouse model of Alzheimer's disease

Converging lines of evidence suggest that abnormal accumulation of the kinase Polo-like kinase 2 (PLK2) might play a role in the pathogenesis of Alzheimer's disease (AD), possibly through its role in regulating the amyloid β (Aβ) cascade. In the present study, we investigated the effect of inhibiting PLK2 kinase activity in in vitro and in vivo models of AD neuropathology. First, we confirmed that PLK2 overexpression modulated APP and Tau protein levels and phosphorylation in cell culture, in a kinase activity dependent manner. Furthermore, a transient treatment of triple transgenic mouse model of AD (3xTg-AD) with a potent and specific PLK2 pharmacological inhibitor (PLK2i #37) reduced some neuropathological aspects in a sex-dependent manner. In 3xTg-AD males, treatment with PLK2i #37 led to lower Tau burden, higher synaptic protein content, and prevented learning and memory deficits. In contrast, treated females showed an exacerbation of Tau pathology, associated with a reduction in amyloid plaque accumulation. Overall, our findings suggest that PLK2 inhibition alters key components of AD neuropathology in a sex-dependent manner and might display a therapeutic potential for the treatment for AD and related dementia.

PLK2 targets GSK3β to protect against cisplatin-induced acute kidney injury

Cisplatin-induced acute kidney injury (AKI), which is accompanied by a rapid decline in renal function and a high risk of death, is a complex critical illness with no effective or specific treatment. Polo-like kinase 2 (PLK2), a serine/threonine kinase, is involved in the progression of multiple diseases, including cancers, cardiac fibrosis, diabetic nephropathy, etc. Here, by integrating two Gene Expression Omnibus (GEO) datasets of cisplatin-induced AKI animal models, we identified PLK2 as a significantly up-regulated gene in AKI renal tissues, which was then verified in different AKI animal models and cell models. Suppressing PLK2 using siRNAs or inhibitors could enhance cisplatin-induced AKI by inducing severe apoptosis and oxidative stress damage, while enforced PLK2 expression could prevent renal dysfunction induced by cisplatin. We further discovered that PLK2 might phosphorylate glycogen synthase kinase 3β (GSK3β) in the pathogenesis of AKI. In conclusion, our results show that PLK2 play a protective role in cisplatin-induced AKI and may be a new protective target of cisplatin nephrotoxicity.

A Major Diplotaxis harra-Derived Bioflavonoid Glycoside as a Protective Agent against Chemically Induced Neurotoxicity and Parkinson's Models; In Silico Target Prediction; and Biphasic HPTLC-Based Quantification.

Oxidative stress and chronic inflammation have a role in developing neurodegenerative diseases such as Parkinson’s disease (PD) and inflammatory movement disorders such as rheumatoid arthritis that affect millions of populations. In searching for antioxidant and anti-inflammatory molecules from natural sources that can counteract neurodegenerative diseases and arthritis, the flavonoid-rich extract of Diplotaxis harra (DHE) was selected based on its in vitro antioxidant and anti-inflammatory activities. DHE could inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages from 100% to the level of 28.51 ± 18.67 and 30.19 ± 5.00% at 20 μg/mL, respectively. A TLC bioautography of DHE fractions using 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) led to the isolation of a major antioxidant compound which was identified by X-ray diffraction analysis as isorhamnetin-3-O-β-D-glucoside (IR3G). IR3G also exhibited a potent anti-inflammatory activity, particularly by suppressing the upregulation of iNOS expression, similar to that of dexamethasone (DEX) at 10 μM to the level of 35.96 ± 7.80 and 29.34 ± 6.34%, respectively. Moreover, IR3G displayed a strong neuroprotectivity (>60% at 1.0−4–1.0−3 μM) against 6-hydroxydopamine (6-OHDA)-challenged SHSY5Y neuroblastoma, an in vitro model of dopaminergic neurons for Parkinson’s disease (PD) research. Accordingly, the in vivo anti-Parkinson potentiality was evaluated, where it was found that IR3G successfully reversed the 6-OHDA-induced locomotor deficit in a zebrafish model. A study of molecular docking and molecular dynamic (MD) simulation of IR3G and its aglycone isorhamnetin (IR) against human acetylcholine esterase (AChE), monoamine oxidase B (MAO-B), and Polo-like kinase-2 (PLK2) was performed and further outlined a putative mechanism in modulating neurodegenerative diseases such as PD. The free radical scavenging, anti-inflammatory through anti-iNOS and anti-COX-2 expression, and neuroprotective activities assessed in this study would present partial evidence for the potentiality of D. harra-derived IR3G as a promising natural therapeutic agent against neurodegenerative diseases and inflammatory arthritis. Finally, a biphasic HPTLC method was developed to estimate the biomarker IR3G in D. harra quantitatively.

Deficient immunoproteasome assembly drives gain of α-synuclein pathology in Parkinson's disease

Aberrant α-synuclein (α-Syn) accumulation resulting from proteasome dysfunction is considered as a prominent factor to initiate and aggravate the neurodegeneration in Parkinson's disease (PD). Although the involvement of 26S proteasome in proteostasis imbalance has been widely accepted, our knowledge about the regulation of immunoproteasome function and its potential role in α-Syn pathology remains limited. Immunoproteasome abundance and proteolytic activities depend on the finely tuned assembly process, especially β-ring formation mediated by the only well-known chaperone proteasome maturation protein (POMP). Here, we identified that α-Syn overexpression was associated with a reduction in immunoproteasome function, which in turn limited the degradation of polo-like kinase 2 (PLK2), exacerbated α-Syn Ser129 phosphorylation and aggregation, ultimately leading to the neurodegeneration. These effects could be dramatically attenuated by β5i overexpression. Mechanistically, α-Syn suppressed the transcriptional regulation of POMP by nuclear factor erythroid 2-related factor 2 (NRF2), thereby preventing the assembly of immunoproteasome β subunits. Dopaminergic neurons-specific overexpression of NRF2-POMP axis effectively rescued the aggregation of α-Syn and PD-like phenotypes. These findings characterized abnormal immunoproteasome assembly as a key contributor governing α-Syn accumulation and neurodegeneration, which might open up a new perspective for the implication of immunoproteasome in PD and provide approaches of manipulating immunoproteasome assembly for therapeutic purposes.

Polo-like kinase 2 inhibition reduces serine-129 phosphorylation of physiological nuclear alpha-synuclein but not of the aggregated alpha-synuclein.

Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson’s disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology.

Diminished PLK2 Induces Cardiac Fibrosis and Promotes Atrial Fibrillation.

[Figure: see text].

Systemic mesalazine treatment prevents spontaneous skin fibrosis in PLK2-deficient mice

Skin fibrosis is a complex biological remodeling process occurring in disease like systemic sclerosis, morphea, or eosinophilic fasciitis. Since the knowledge about the underlying pathomechanisms is still incomplete, there is currently no therapy, which prevents or reverses skin fibrosis sufficiently. The present study investigates the role of polo-like kinase 2 (PLK2) and the pro-fibrotic cytokine osteopontin (OPN) in the pathogenesis of cutaneous fibrosis and demonstrates the antifibrotic effects of systemic mesalazine treatment in vivo. Isolated primary dermal fibroblasts of PLK2 wild-type (WT) and knockout (KO) mice were characterized invitro. Skin thickness and histoarchitecture were studied in paraffin-embedded skin sections. The effects of mesalazine treatment were examined in isolated fibroblasts and PLK2 KO mice, which were fed 100 µg/g mesalazine for 6 months via the drinking water. Compared to WT, PLK2 KO fibroblasts displayed higher spontaneous myofibroblast differentiation, reduced proliferation rates, and overexpression of the fibrotic cytokine OPN. Invitro, 72 h of treatment with 10 mmol/L mesalazine induced phenotype conversion in PLK2 KO fibroblasts and attenuated OPN expression by inhibiting ERK1/2. In vivo, dermal myofibroblast differentiation, collagen accumulation, and skin thickening were prevented by mesalazine in PLK2 KO. Plasma creatinine levels indicated good tolerability of systemic long-term mesalazine treatment. The current study reveals a spontaneous fibrotic skin phenotype and ERK1/2-dependent OPN overexpression in PLK2 KO mice. We provide experimental evidence for the antifibrotic effectiveness of systemic mesalazine treatment to prevent fibrosis of the skin, suggesting further investigation in experimental and clinical settings.

Sustained Reduction of Intracellular Calcium Is Important to Maximise GA101-Mediated Direct Cell Death in B-Cell Lymphomas

For over a decade, anti-CD20 monoclonal antibodies (mAbs) have successfully been used in the therapy of B-cell malignancies. Obinutuzumab (GA101), a type-II glycoengineered anti-CD20 mAb approved for treatment of Chronic Lymphocytic Leukaemia (CLL) and Follicular Lymphoma, was designed to have enhanced antibody-dependant cellular cytotoxicity compared with Rituximab. Pre-clinical data showed that in addition, GA101 also exhibited significantly enhanced direct cell death. The complete mechanism of increased direct cell death is still unknown.The biological role of CD20 remains unclear but it has been shown to act as part of an ion channel complex, a component of the store operated calcium (Ca2+) entry system. Numerous studies have investigated the immediate impact of anti-CD20 mAbs on intracellular Ca2+. Yet to date, none have assessed intracellular Ca2+ over a longer period.To investigate the effect of anti-CD20 mAbs on intracellular Ca2+, we initially used 4 B-cell lymphoma cell lines to assess Ca2+ flux immediately after treatment with either Rituximab or GA101. Cells were first stained with a fluorescent calcium indicator and baseline measurements recorded by flow cytometry. Cells were then treated with each antibody and fluorescence measurements were recorded for a further 5-10 minutes. Next, we conducted a time course experiment to visualise the kinetics of intracellular Ca2+ after 1, 2, 4, 8, and 24 hour treatments with either Rituximab or GA101; Trastuzumab was used as an isotype control. After treatment, samples were stained with a labelled calcium indicator before quantifying intracellular Ca2+ using imaging flow cytometry. We validated our findings in primary CLL samples using the same method. We conducted liquid chromatography tandem mass spectrometry based label-free phosphoproteomics on SUDHL4 cells in biological triplicates after 1 and 24 hour treatments with GA101 and Rituximab. Expression levels of >8500 phosphorylation sites were measured across treatments to establish changes in intracellular signalling and kinase activity.In line with published data, we observed an influx of Ca2+ when cells were treated with Rituximab but intracellular Ca2+ was not significantly altered for any of the extended treatments. In contrast, GA101 treatment did not induce an immediate influx of Ca2+,and a reduction in intracellular Ca2+ was observed after 1 hour treatment which was sustained across all extended treatment periods. This difference in kinetics was validated in primary CLL samples. Global phosphoproteomic analysis found that the activity of Polo-Like Kinase 2 (PLK2) was significantly increased following treatment with GA101. The increase in the activity of this kinase was specific to GA101 and was unchanged following treatment with Rituximab. Simultaneously there was a significant increase in the phosphorylation of Calumenin at Ser44, a known binding site of PLK2. Samples treated with GA101 for 24 hours exhibited a larger downregulation of pathways associated with calcium signalling compared to Rituximab-treated and control samples.Calumenin, a member of the CREC family, is able to bind 7 Ca2+ ions. It can be localised in the cytosol, in compartments of the secretory pathway, on the plasma membrane or in the extracellular space. We hypothesise that once phosphorylated, this protein bound with Ca2+ is secreted extracellularly, resulting in reduced intracellular Ca2+.In summary we report for the first time that intracellular Ca2+ is significantly decreased as a result of GA101 treatment in cell lines and primary samples. Using a global unbiased approach we have identified a significant increase in PLK2 activity and we observed a large increase in phosphorylation of Calumenin. Our data suggests that these proteins may contribute to the increased direct cell death observed as a result of GA101 treatment. We believe that expression and activity of these proteins are crucial in order to maximise GA101-mediated direct cell death. Ongoing studies are examining the functional roles of PLK2 and Calumenin in the reduction of intracellular Ca2+, comparing Ca2+ levels in response to antibody treatment in primary samples from different CLL subtypes, and assessing and comparing levels of apoptosis following antibody treatment to see whether there is a correlation with reduced intracellular Ca2+. DisclosuresGribben:Celgene: Honoraria; TG Therapeutics: Honoraria; Genentech/Roche: Honoraria; Karyopharm: Honoraria; Kite: Honoraria; Abbvie: Honoraria; Pharmacyclics: Honoraria; Janssen: Honoraria; Acerta: Honoraria.

MiR-101-3p Contributes to α-Synuclein Aggregation in Neural Cells through the miR-101-3p/SKP1/PLK2 Pathway.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive neuronal loss in different brain regions, including the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc). The aggregation of α-synuclein (α-Syn) plays an essential role in the progression of PD-related neuron toxicity. In this study, bioinformatic analysis was used to confirm differentially expressed genes between patients with PD and healthy donors. Immunofluorescence was used to study the aggregation of α-Syn. Flow cytometry was used to confirm the apoptosis of neurons. Western blot was used to investigate the underlying mechanism. Coimmunoprecipitation (co-IP) was used to verify the interaction between proteins. Luciferase activity assay was used to confirm the target gene of miRNA. In vitro protein ubiquitination assay was used to ascertain the role of S-phase kinase-associated protein 1 (SKP1) on the ubiquitination processes of polo-like kinase 2 (PLK2). The result indicated that miR-101-3p was overexpressed in the substantia nigra of the postmortem brains of patients with PD. The underlying role was investigated in the SH-SY5Y cell line. The overexpression of α-Syn did not result in toxicity or aggregation. However, the co-overexpression of miR-101-3p and α-Syn promoted aggregation and neuron toxicity. Luciferase activity assay indicated that SKP1 is a target gene of miR-101-3p. The co-IP experiment confirmed that SKP1 could directly interact with PLK2. In vitro protein ubiquitination assay confirmed that SKP1 could promote the ubiquitination and subsequent protein degradation of PLK2. We also observed that the cotransfection of short hairpin RNA that targets PLK2 and α-Syn overexpression plasmid results in the endoplasmic reticulum stress of neurons. Our results collectively provide evidence that miR-101-3p contributes to α-Syn aggregation in neurons through the miR-101-3p/SKP1/PLK2 pathway.

PLK2 protects retinal ganglion cells from oxidative stress by potentiating Nrf2 signaling via GSK-3β.

Oxidative stress of retinal ganglion cells (RGCs) has been established as a main contributor to retinal degeneration in the pathogenesis of glaucoma. Polo-like kinase 2 (PLK2) has recently been reported to be a potent antioxidant protein that enhances cell survival in response to oxidative stress. To date, the involvement of PLK2 in RGC-associated oxidative stress is undermined. In the present work, we evaluated whether PLK2 regulates oxidative stress evoked by hydrogen peroxide (H2 O2 ) in RGCs. PLK2 expression was induced by H2 O2 stimulation in RGCs. Upregulation of PLK2 had a profoundly cytoprotective effect on H2 O2 -stimulated RGCs by attenuating cellular apoptosis and reactive oxygen species (ROS) level. Further data revealed that upregulation of PLK2 strikingly enhanced the activation of Nrf2 signaling. Moreover, PLK2 overexpression promoted glycogen synthase kinase (GSK)-3β phosphorylation, whereas PLK2 knockdown reduced the levels of GSK-3β phosphorylation. Notably, GSK-3β inhibition using a chemical inhibitor markedly abrogated the suppressive effects of PLK2 knockdown on Nrf2 activation. Repression of Nrf2 blocked the PLK2 overexpression-induced protective effects in H2 O2 -stimulated RGCs. Overall, this study elucidates that upregulation of PLK2 protects RGCs against H2 O2 -induced oxidative stress injury by upregulating Nrf2 activation via modulation of GSK-3β phosphorylation. These findings underline the pivotal role of PLK2 in mediating oxidative stress-evoked retinal degeneration in the pathogenesis of glaucoma.

Transcriptomic insights into the zinc homeostasis of MCF-7 breast cancer cells via next-generation RNA sequencing.

A significant gap in the knowledge of zinc homeostasis exists for breast cancer cells. In this study, we investigated the transcriptomic response of the luminal breast cancer cells (MCF-7) to the exposure of extracellular zinc using next-generation RNA sequencing. The dataset was collected for three time points (T0, T30, and T120) in the time course of zinc treatment, which revealed the dramatic increase, up to 869-fold, of the gene expression for metallothioneins (MT1B, MT1F, MT1X, and MT2A) and the zinc exporter ZnT1 (SLC30A1) at T30, continuingly through to T120. The similar dynamic expression pattern was found for the autophagy-related gene (VMP1) and numerous genes for zinc finger proteins (e.g. RNF165, ZNF365, ZBTB2, SNAI1, ZNF442, ZNF547, ZNF563, and ZNF296). These findings point to the all-hands-on-deck strategy adopted by the cancer cells for maintaining zinc homeostasis. The stress responsive genes encoding heat shock proteins (HSPA1A, HSPA1B, HSPA1L, HSPA4L, HSPA6, HSPA8, HSPH1, HSP90AA1, and HSP90AB1) and the MTF-1 biomarker genes (AKR1C2, CLU, ATF3, GDF15, HMOX1, MAP1A, MAFG, SESN2, and UBC) were also differentially up-regulated at T120, suggesting a role of heat shock proteins and the MTF-1 related stress proteins in dealing with zinc exposure. It is for the first time that the gene encoding Polo-like kinase 2 (PLK2) was found to be involved in zinc-related response. The top differentially expressed genes were validated by qRT-PCR and further extended to the basal type breast cancer cells (MDA-MB-231). It was found that the expression level of SLC30A1 in MDA-MB-231 was higher than MCF-7 in response to zinc exposure. Taken together, the findings contribute to our knowledge and understanding of zinc homeostasis in breast cancer cells.

Genetic Deletion of Polo-Like Kinase 2 Induces a Pro-Fibrotic Pulmonary Phenotype.

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.

Copper Dependent Modulation of α-Synuclein Phosphorylation in Differentiated SHSY5Y Neuroblastoma Cells.

Copper (Cu) dyshomeostasis plays a pivotal role in several neuropathologies, such as Parkinson’s disease (PD). Metal accumulation in the central nervous system (CNS) could result in loss-of-function of proteins involved in Cu metabolism and redox cycling, generating reactive oxygen species (ROS). Moreover, neurodegenerative disorders imply the presence of an excess of misfolded proteins known to lead to neuronal damage. In PD, Cu accumulates in the brain, binds α-synuclein, and initiates its aggregation. We assessed the correlation between neuronal differentiation, Cu homeostasis regulation, and α-synuclein phosphorylation. At this purpose, we used differentiated SHSY5Y neuroblastoma cells to reproduce some of the characteristics of the dopaminergic neurons. Here, we reported that differentiated cells expressed a significantly higher amount of a copper transporter protein 1 (CTR1), increasing the copper uptake. Cells also showed a significantly more phosphorylated form of α-synuclein, further increased by copper treatment, without modifications in α-synuclein levels. This effect depended on the upregulation of the polo-like kinase 2 (PLK2), whereas the levels of the relative protein phosphatase 2A (PP2A) remained unvaried. No changes in the oxidative state of the cells were identified. The Cu dependent alteration of α-synuclein phosphorylation pattern might potentially offer new opportunities for clinical intervention.