Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β) gene with differential expression patterns and regulatory functions

Background

Glycogen synthase kinase 3 (GSK3α and GSK3β) are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer’s disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment.

Methodology

Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms.

Conclusions

We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.     

Mutations in the C-terminal domain of topoisomerase II affect meiotic function and interaction with the casein kinase 2β subunit

Topoisomerase II is a major target of the protein kinase casein kinase 2 (PK CK2) in vivo. All major phosphorylation acceptor sites in the yeast enzyme are found in the C-terminal 350aa. The acceptor sites are generally clustered such that there is more than one modified Ser or Thr within a short peptide. Mutagenesis of the predicted acceptor sites have confirmed that five of the eight predicted sites are targeted in vitro and in vivo by PK CK2. Mutation to nonphosphorylatable, neutral residues provokes at most a 10% increase in mitotic doubling time. Truncation of the enzyme leaves the enzyme catalytically active, but slightly lengthens the doubling time during mitotic growth and impedes progress through meiosis. Since this could reflect the loss of interaction with an important ligand, we have examined whether the C-terminal domain of the yeast enzyme mediates interaction with the regulatory subunit of PK CK2, which was previously reported to bind topoisomerase II. We find that point mutation of the phospho-acceptor sites does not abrogate the interaction with a small region of PK CK2 , while truncation at aal276 or aal236 does. The site of interaction within PK CK2 does not coincide with the highly negatively charged spermine binding site.     

Quercetin inhibits macrophage polarization through the p-38α/β signalling pathway and regulates OPG/RANKL balance in a mouse skull model

Aseptic loosening caused by wear particles is a common complication after total hip arthroplasty. We investigated the effect of the quercetin on wear particle-mediated macrophage polarization, inflammatory response and osteolysis. In vitro, we verified that Ti particles promoted the differentiation of RAW264.7 cells into M1 macrophages through p-38α/β signalling pathway by using flow cytometry, immunofluorescence assay and small interfering p-38α/β RNA. We used enzyme-linked immunosorbent assays to confirm that the protein expression of M1 macrophages increased in the presence of Ti particles and that these pro-inflammatory factors further regulated the imbalance of OPG/RANKL and promoted the differentiation of osteoclasts. However, this could be suppressed, and the protein expression of M2 macrophages was increased by the presence of the quercetin. In vivo, we revealed similar results in the mouse skull by μ-CT, H&E staining, immunohistochemistry and immunofluorescence assay. We obtained samples from patients with osteolytic tissue. Immunofluorescence analysis indicated that most of the macrophages surrounding the wear particles were M1 macrophages and that pro-inflammatory factors were released. Titanium particle-mediated M1 macrophage polarization, which caused the release of pro-inflammatory factors through the p-38α/β signalling pathway, regulated OPG/RANKL balance. Macrophage polarization is expected to become a new clinical drug therapeutic target.     

Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain

Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe^II₄-MTα and Co^II₄-MTα species existing in water. Furthermore, the Fe^II₄-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe^II₄-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6 h at 25 °C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.     

Hypothalamic glycogen synthase kinase 3β has a central role in the regulation of food intake and glucose metabolism

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lepob/ob mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.     

Elevated CCN2 expression in scleroderma: a putative role for the TGFβ accessory receptors TGFβRIII and endoglin

The ability of TGFβ1 to act as a potent pro-fibrotic mediator is well established, potently inducing the expression of fibrogenic genes including type I collagen (COL1A2) and CCN2. Previously we have shown elevated expression of the TGFβ accessory receptor, endoglin on Systemic Sclerosis (SSc) dermal fibroblasts. Here we sought to assess the cell surface expression of the TGFβ receptor complex on SSc dermal fibroblasts (SDF), and investigate their role in maintaining the elevated expression of CCN2. SDF exhibited elevated expression of the TGFβ accessory receptors betaglycan/TGFβRIII and endoglin, but not type I or type II receptors. To determine the effect of altered receptor repertoire on TGFβ responses, we investigated the effect of exogenous TGFβ on expression of two pro-fibrotic genes. SDF exhibited higher basal expression of COL1A2 and CCN2 compared to healthy controls. TGFβ induced a marked increase in the expression of these genes in normal dermal fibroblasts, whereas SDF exhibited only a modest increase. We next sought to determine if higher basal expression in SDF was a result of autocrine expression of TGFβ. Surprisingly basal expression was not affected by a pan-neutralizing TGFβ antibody. To explore if altered accessory receptor expression alone could account for these changes, we determined their effects on CCN2 promoter activity. Endoglin inhibited CCN2 promoter activity in response to TGFβ. TGFβRIII alone or in combination with endoglin was sufficient to enhance basal CCN2 promoter activity. Thus TGFβ accessory receptors may play a significant role in the altered expression of fibrogenic genes in SDF.     

Differences in the force–interval relationship of isolated human myocardium with chronic coronary artery disease with and without type 2 diabetes mellitus and the role of sarcoplasmic reticulum Ca<Superscript>2+</Superscript>-ATPase

The dynamics of the force–interval relationship of the human myocardium in coronary artery disease (CAD) and CAD with concomitant diabetes mellitus was studied, and its dependence on the level of sarcoplasmic reticulum (SR) Ca²⁺-ATPase expression was evaluated. The study was performed on myocardial biopsy material obtained during coronary bypass operation using cardiopulmonary techniques. Patients with chronic CAD and patients with CAD associated with type II diabetes mellitus were enrolled in the study. It was found that CAD patients with and without diabetes mellitus with similar clinical parameters had either negative or positive dynamics of the force–interval relationship. The positive force–interval relationship was associated with a “high level” of Ca²⁺-ATPase, while the negative force–interval relationship was associated with a “low level” of this protein. In CAD associated with diabetes mellitus of short duration, the positive dynamics of the force–interval relationship is more pronounced and corresponds to a higher level of SR Ca²⁺-ATPase expression than in CAD alone.     

Arabidopsis thaliana histone deacetylase 14 (HDA14) is an α-tubulin deacetylase that associates with PP2A and enriches in the microtubule fraction with the putative histone acetyltransferase ELP3

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.     

Interplay between α-, β-, and γ-Secretases Determines Biphasic Amyloid-β Protein Level in the Presence of a γ-Secretase Inhibitor

Amyloid-β (Aβ) is produced by the consecutive cleavage of amyloid precursor protein (APP) first by β-secretase, generating C99, and then by γ-secretase. APP is also cleaved by α-secretase. It is hypothesized that reducing the production of Aβ in the brain may slow the progression of Alzheimer disease. Therefore, different γ-secretase inhibitors have been developed to reduce Aβ production. Paradoxically, it has been shown that low to moderate inhibitor concentrations cause a rise in Aβ production in different cell lines, in different animal models, and also in humans. A mechanistic understanding of the Aβ rise remains elusive. Here, a minimal mathematical model has been developed that quantitatively describes the Aβ dynamics in cell lines that exhibit the rise as well as in cell lines that do not. The model includes steps of APP processing through both the so-called amyloidogenic pathway and the so-called non-amyloidogenic pathway. It is shown that the cross-talk between these two pathways accounts for the increase in Aβ production in response to inhibitor, i.e. an increase in C99 will inhibit the non-amyloidogenic pathway, redirecting APP to be cleaved by β-secretase, leading to an additional increase in C99 that overcomes the loss in γ-secretase activity. With a minor extension, the model also describes plasma Aβ profiles observed in humans upon dosing with a γ-secretase inhibitor. In conclusion, this mechanistic model rationalizes a series of experimental results that spans from in vitro to in vivo and to humans. This has important implications for the development of drugs targeting Aβ production in Alzheimer disease.     

MyD88 Drives the IFN-β Response to Lactobacillus acidophilus in Dendritic Cells through a Mechanism Involving IRF1, IRF3, and IRF7

Type I IFNs are induced by pathogens to protect the host from infection and boost the immune response. We have recently demonstrated that this IFN response is not restricted to pathogens, as the Gram-positive bacterium Lactobacillus acidophilus, a natural inhabitant of the intestine, induces high levels of IFN-β in dendritic cells. In the current study, we investigate the intracellular pathways involved in IFN-β upon stimulation of dendritic cells with L. acidophilus and reveal that this IFN-β induction requires phagosomal uptake and processing but bypasses the endosomal receptors TLR7 and TLR9. The IFN-β production is fully dependent on the TIR adapter molecule MyD88, partly dependent on IFN regulatory factor (IRF)1, but independent of the TIR domain-containing adapter inducing IFN-β MyD88 adapter-like, IRF and IRF7. However, our results suggest that IRF3 and IRF7 have complementary roles in IFN-β signaling. The IFN-β production is strongly impaired by inhibitors of spleen tyrosine kinase (Syk) and PI3K. Our results indicate that L. acidophilus induces IFN-β independently of the receptors typically used by bacteria, as it requires MyD88, Syk, and PI3K signaling and phagosomal processing to activate IRF1 and IRF3/IRF7 and thereby the release of IFN-β.     

A2a adenosine receptor agonist improves endoplasmic reticulum stress in MIN6 cell line through protein kinase A/ protein kinase B/ Cyclic adenosine monophosphate response element-binding protein/ and Growth Arrest And DNA-Damage-Inducible 34/ eukaryotic Initiation Factor 2α pathways

Endoplasmic reticulum (ER) stress is one of the main molecular events underlying pancreatic beta cell (PBC) failure, apoptosis, and a decrease in insulin secretion. Recent studies have highlighted the fundamental role of A2a adenosine receptor (A2aR) in potentiation of insulin secretion and proliferation of PBCs. However, possible protective effects of A2aR signaling against ER stress have not been elucidated yet. Thus, in the present study, we aimed to investigate the effects of A2aR activation in MIN6 beta cells undergoing tunicamycin (TM)-mediated ER stress. A2aR expression and activity were evaluated using real-time polymerase chain reaction and measurement of the cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), phospho-protein kinase B or Akt (p-Akt)/Akt, and phospho-Cyclic adenosine monophosphate response element-binding protein/CREB levels in response to a specific agonist (CGS 21680). Survival and proliferation in TM and CGS 21680 cotreated cells were evaluated using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining, colony formation, and 5-bromo-2′-deoxyuridine (Brdu) assays. In addition, the effects of A2aR stimulation on insulin secretion were evaluated using the enzyme-linked immunosorbent assay. B-cell lymphoma 2 (Bcl-2), phospho-eukaryotic Initiation Factor 2α (p-eIF2α)/eIF2α, growth arrest and DNA-damage-inducible 34 (GADD34), X-box binding protein 1 (XBP-1), spliced X-box binding protein 1 (XBP-1s), immunoglobulin heavy-chain-binding protein (BIP), and CCAAT-enhancer-binding protein homologous protein (CHOP) levels were evaluated using western blotting. Our results showed a decrease in A2aR expression and p-Akt/Akt and p-CREB/CREB levels in TM-pretreated cells. We also mentioned that CGS 21680 effectively increased cell survival, proliferation, and insulin secretion in TM-treated cells. The antiapoptotic effects were possibly mediated through Bcl-2 upregulation. Our western blotting results indicated that A2aR effectively downregulated p-eIF2α/eIF2α, XBP-1, XBP-1s, BIP, and CHOP levels, whereas GADD34 was upregulated. Altogether, the present study revealed that A2aR signaling through PKA/Akt/CREB mediators alleviated TM cytotoxicity effects in MIN6 beta cells. Thus, the stimulation of this receptor was seen as a new approach to control ER stress in the PBC cells.     

The expression of a disintegrin and metalloproteinase with thrombospondin motifs 4 in human macrophages is inhibited by the anti-atherogenic cytokine transforming growth factor-β and requires Smads, p38 mitogen-activated protein kinase and c-Jun

Atherosclerosis is an inflammatory disorder of the vasculature that is orchestrated by the action of cytokines. Macrophages play a prominent role in all stages of this disease, including foam cell formation, production of reactive oxygen species, modulation of the inflammatory response and the regulation of the stability of atherosclerotic plaques. The role of the matrix metalloproteinase family in the control of plaque stability is well established. A disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) family has been implicated in several diseases and the expression of ADAMTS-4 in macrophages of atherosclerotic lesions has suggested a potential role for this protease in atherosclerosis. However, the action of cytokines on the expression of ADAMTS-4 in macrophages is poorly understood. We have investigated here the effect of transforming growth factor-β (TGF-β) on ADAMTS-4 expression in macrophages along with the regulatory mechanisms underlying its actions. Consistent with the anti-atherogenic role of TGF-β, this cytokine decreased the expression of ADAMTS-4 mRNA and protein in human macrophages. Transient transfection assays showed that the -100 to +10 promoter region contained the minimal TGF-β response elements. Small-interfering RNA-mediated knockdown revealed a critical role for Smads, p38 mitogen-activated protein kinase and c-Jun in the action of TGF-β on ADAMTS-4 mRNA expression. These studies show for the first time that TGF-β inhibits the expression of ADAMTS-4 in human macrophages and identifies the signalling pathways underlying this response. The inhibition of macrophage ADAMTS-4 expression is likely to contribute to the anti-atherogenic, plaque stabilisation action of TGF-β.