Distinct Roles of Glycogen Synthase Kinase (GSK)-3�� and GSK-3�� in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and α-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3β and down-regulation of β-catenin, whereas it stimulated GSK-3α expression only modestly. The promoter region of GSK-3β was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3β potently induced CM differentiation, that of GSK-3α induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3β abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3α facilitated CM differentiation in MSCs. Although GSK-3β is found in both the cytosol and nucleus in MSCs, GSK-3α is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3β failed to stimulate CM differentiation. Down-regulation of β-catenin mediates GSK-3β-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3α knockdown. These results suggest that GSK-3α and GSK-3β have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3β in the cytosol induces CM differentiation of MSCs through down-regulation of β-catenin. In contrast, GSK-3α in the nucleus inhibits CM differentiation through down-regulation of c-Jun.     

Lithium Inhibits Tumorigenic Potential of PDA Cells through Targeting Hedgehog-GLI Signaling Pathway

Hedgehog signaling pathway plays a critical role in the initiation and development of pancreatic ductal adenocarcinoma (PDA) and represents an attractive target for PDA treatment. Lithium, a clinical mood stabilizer for mental disorders, potently inhibits the activity of glycogen synthase kinase 3β (GSK3β) that promotes the ubiquitin-dependent proteasome degradation of GLI1, an important downstream component of hedgehog signaling. Herein, we report that lithium inhibits cell proliferation, blocks G1/S cell-cycle progression, induces cell apoptosis and suppresses tumorigenic potential of PDA cells through down-regulation of the expression and activity of GLI1. Moreover, lithium synergistically enhances the anti-cancer effect of gemcitabine. These findings further our knowledge of mechanisms of action for lithium and provide a potentially new therapeutic strategy for PDA through targeting GLI1.     

The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.     

CDK5RAP2 loss-of-function causes premature cell senescence via the GSK3β/β-catenin-WIP1 pathway

Developmental disorders characterized by small body size have been linked to CDK5RAP2 loss-of-function mutations, but the mechanisms underlying which remain obscure. Here, we demonstrate that knocking down CDK5RAP2 in human fibroblasts triggers premature cell senescence that is recapitulated in Cdk5rap2^an/an mouse embryonic fibroblasts and embryos, which exhibit reduced body weight and size, and increased senescence-associated (SA)-β-gal staining compared to Cdk5rap2^+/+ and Cdk5rap2^+/an embryos. Interestingly, CDK5RAP2-knockdown human fibroblasts show increased p53 Ser15 phosphorylation that does not correlate with activation of p53 kinases, but rather correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses the senescent phenotype in CDK5RAP2-knockdown cells, indicating that senescence in these cells is linked to WIP1 downregulation. CDK5RAP2 interacts with GSK3β, causing increased inhibitory GSK3β Ser9 phosphorylation and inhibiting the activity of GSK3β, which phosphorylates β-catenin, tagging β-catenin for degradation. Thus, loss of CDK5RAP2 decreases GSK3β Ser9 phosphorylation and increases GSK3β activity, reducing nuclear β-catenin, which affects the expression of NF-κB target genes such as WIP1. Consequently, loss of CDK5RAP2 or β-catenin causes WIP1 downregulation. Inhibition of GSK3β activity restores β-catenin and WIP1 levels in CDK5RAP2-knockdown cells, reducing p53 Ser15 phosphorylation and preventing senescence in these cells. Conversely, inhibition of WIP1 activity increases p53 Ser15 phosphorylation and senescence in CDK5RAP2-depleted cells lacking GSK3β activity. These findings indicate that loss of CDK5RAP2 promotes premature cell senescence through GSK3β/β-catenin downregulation of WIP1. Premature cell senescence may contribute to reduced body size associated with CDK5RAP2 loss-of-function.Subject terms: Senescence, Diseases     

Apoptosis and autophagy have opposite roles on imatinib-induced K562 leukemia cell senescence

Imatinib, the anti-Abl tyrosine kinase inhibitor used as first-line therapy in chronic myeloid leukemia (CML), eliminates CML cells mainly by apoptosis and induces autophagy. Analysis of imatinib-treated K562 cells reveals a cell population with cell cycle arrest, p27 increase and senescence-associated beta galactosidase (SA-β-Gal) staining. Preventing apoptosis by caspase inhibition decreases annexin V-positive cells, caspase-3 cleavage and increases the SA-β-Gal-positive cell population. In addition, a concomitant increase of the cell cycle inhibitors p21 and p27 is detected emphasizing the senescent phenotype. Inhibition of apoptosis by targeting Bim expression or overexpression of Bcl2 potentiates senescence. The inhibition of autophagy by silencing the expression of the proteins ATG7 or Beclin-1 prevents the increase of SA-β-Gal staining in response to imatinib plus Z-Vad. In contrast, in apoptotic-deficient cells (Bim expression or overexpression of Bcl2), the inhibition of autophagy did not significantly modify the SA-β-Gal-positive cell population. Surprisingly, targeting autophagy by inhibiting ATG5 is accompanied by a strong SA-β-Gal staining, suggesting a specific inhibitory role on senescence. These results demonstrate that in addition to apoptosis and autophagy, imatinib induced senescence in K562 CML cells. Moreover, apoptosis is limiting the senescent response to imatinib, whereas autophagy seems to have an opposite role.     

The ING1a Tumor Suppressor Regulates Endocytosis to Induce Cellular Senescence Via the Rb-E2F Pathway

The INhibitor of Growth (ING) proteins act as type II tumor suppressors and epigenetic regulators, being stoichiometric members of histone acetyltransferase and histone deacetylase complexes. Expression of the alternatively spliced ING1a tumor suppressor increases >10-fold during replicative senescence. ING1a overexpression inhibits growth; induces a large flattened cell morphology and the expression of senescence-associated β-galactosidase; increases Rb, p16, and cyclin D1 levels; and results in the accumulation of senescence-associated heterochromatic foci. Here we identify ING1a-regulated genes and find that ING1a induces the expression of a disproportionate number of genes whose products encode proteins involved in endocytosis. Intersectin 2 (ITSN2) is most affected by ING1a, being rapidly induced >25-fold. Overexpression of ITSN2 independently induces expression of the p16 and p57^KIP2 cyclin-dependent kinase inhibitors, which act to block Rb inactivation, acting as downstream effectors of ING1a. ITSN2 is also induced in normally senescing cells, consistent with elevated levels of ING1a inducing ITSN2 as part of a normal senescence program. Inhibition of endocytosis or altering the stoichiometry of endosome components such as Rab family members similarly induces senescence. Knockdown of ITSN2 also blocks the ability of ING1a to induce a senescent phenotype, confirming that ITSN2 is a major transducer of ING1a-induced senescence signaling. These data identify a pathway by which ING1a induces senescence and indicate that altered endocytosis activates the Rb pathway, subsequently effecting a senescent phenotype.     

Lithium Chloride Dependent Glycogen Synthase Kinase 3 Inactivation Links Oxidative DNA Damage,Hypertrophy and Senescence in Human Articular Chondrocytes and Reproduces Chondrocyte Phenotype of Obese Osteoarthritis Patients

Introduction

Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy.

Methods

In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2'',7''-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (γH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining.

Results

In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in γH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10.

Conclusions

In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response.     

Upregulation of miR-760 and miR-186 Is Associated with Replicative Senescence in Human Lung Fibroblast Cells

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) down-regulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the α subunit of CK2 (CK2α) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the CK2α 3′-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for CK2α downregulation. The four miRNAs increased senescence-associated β-galactosidase (SA-β-gal) staining, p53 and p21^Cip1/WAF1 expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. CK2α over-expression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through CK2α downregulation-dependent ROS generation.     

Absence of PKC-Alpha Attenuates Lithium-Induced Nephrogenic Diabetes Insipidus

Lithium, an effective antipsychotic, induces nephrogenic diabetes insipidus (NDI) in ∼40% of patients. The decreased capacity to concentrate urine is likely due to lithium acutely disrupting the cAMP pathway and chronically reducing urea transporter (UT-A1) and water channel (AQP2) expression in the inner medulla. Targeting an alternative signaling pathway, such as PKC-mediated signaling, may be an effective method of treating lithium-induced polyuria. PKC-alpha null mice (PKCα KO) and strain-matched wild type (WT) controls were treated with lithium for 0, 3 or 5 days. WT mice had increased urine output and lowered urine osmolality after 3 and 5 days of treatment whereas PKCα KO mice had no change in urine output or concentration. Western blot analysis revealed that AQP2 expression in medullary tissues was lowered after 3 and 5 days in WT mice; however, AQP2 was unchanged in PKCα KO. Similar results were observed with UT-A1 expression. Animals were also treated with lithium for 6 weeks. Lithium-treated WT mice had 19-fold increased urine output whereas treated PKCα KO animals had a 4-fold increase in output. AQP2 and UT-A1 expression was lowered in 6 week lithium-treated WT animals whereas in treated PKCα KO mice, AQP2 was only reduced by 2-fold and UT-A1 expression was unaffected. Urinary sodium, potassium and calcium were elevated in lithium-fed WT but not in lithium-fed PKCα KO mice. Our data show that ablation of PKCα preserves AQP2 and UT-A1 protein expression and localization in lithium-induced NDI, and prevents the development of the severe polyuria associated with lithium therapy.     

Nuclear accumulation of glycogen synthase kinase-3 during replicative senescence of human fibroblasts

Activation of the tumor suppressor protein p53 contributes to cellular senescence. As glycogen synthase kinase-3 (GSK3) was recently found to interact with p53 and contribute to the actions of p53, this study examined whether GSK3 accumulated in the nucleus and associated with p53 in senescent cells. Compared with young and middle-aged human WI-38 fibroblasts, senescent cells were found to contain increased nuclear levels of GSK3beta, and also tended to accumulate in the nucleus the other isoform of GSK3, GSK3alpha. Co-immunoprecipitation experiments demonstrated that GSK3beta and p53 formed a complex in the nucleus. Further experiments tested whether inhibition of GSK3 altered the development of senescence using long-term treatment with the selective GSK3 inhibitor lithium. Lithium treatment reduced the senescence-associated accumulation of p53 and caused cells to enter a reversible quiescent state. These results indicate that a portion of the p53 that is activated in senescent cells is modulated by its association with GSK3beta in the nucleus, an association that is known to facilitate the actions of p53 and that may contribute to senescence.     

Overexpression of the Novel Senescence Marker β-Galactosidase (GLB1) in Prostate Cancer Predicts Reduced PSA Recurrence

Purpose

Senescence is a terminal growth arrest that functions as a tumor suppressor in aging and precancerous cells and is a response to selected anticancer compounds. Lysosomal-β-galactosidase (GLB1) hydrolyzes β-galactose from glycoconjugates and is the origin of senescence-associated β-gal activity (SA-β-gal). Using a new GLB1 antibody, senescence biology was investigated in prostate cancer (PCa) tissues.

Experimental Design

In vitro characterization of GLB1 was determined in primary prostate epithelial cell cultures passaged to replicative senescence and in therapy-induced senescence in PCa lines using chemotherapeutic agents. FFPE tissue microarrays were subjected to immunofluorescent staining for GLB1, Ki67 and HP1γ and automated quantitative imaging initially using AQUA in exploratory samples and Vectra in a validation series.

Results

GLB1 expression accumulates in replicative and induced senescence and correlates with senescent morphology and P16 (CDKN2) expression. In tissue arrays, quantitative imaging detects increased GLB1 expression in high-grade prostatic intraepithelial neoplasia (HGPIN), known to contain senescent cells, and cancer compared to benign prostate tissues (p<0.01) and senescent cells contain low Ki67 and elevated HP1γ. Within primary tumors, elevated GLB1 associates with lower T stage (p=0.01), localized versus metastatic disease (p=0.0003) and improved PSA-free survival (p=0.03). Increased GLB1 stratifies better PSA-free survival in intermediate grade PCa (0.01). Tissues that elaborate higher GLB1 display increased uniformity of expression.

Conclusion

Increased GLB1 is a valuable marker in formalin-fixed paraffin-embedded (FFPE) tissues for the senescence-like phenotype and associates with improved cancer outcomes. This protein addresses a lack of senescence markers and should be applicable to study the biologic role of senescence in other cancers.     

Potential role of senescent macrophages in radiation-induced pulmonary fibrosis

Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation in clinic with poor prognosis and limited therapeutic options. Previous results have shown that senescent cells, such as fibroblast and type II airway epithelial cell, are strongly implicated in pathology of RIPF. However, the role of senescent macrophages in the development RIPF is still unknown. In this study, we report that ionizing radiation (IR) increase cellular senescence with higher expression of senescence-associated β-galactosidase (SA-β-Gal) and senescence-specific genes (p16, p21, Bcl-2, and Bcl-xl) in irradiated bone marrow-derived monocytes/macrophages (BMMs). Besides, there’s a significant increase in the expression of pro-fibrogenic factors (TGF-β1 and Arg-1), senescence-associated secretory phenotype (SASP) proinflammatory factors (Il-1α, Il-6, and Tnf-α), SASP chemokines (Ccl2, Cxcl10, and Ccl17), and SASP matrix metalloproteinases (Mmp2, Mmp9 and Mmp12) in BMMs exposed to 10 Gy IR. In addition, the percentages of SA-β-Gal⁺ senescent macrophages are significantly increased in the macrophages of murine irradiated lung tissue. Moreover, robustly elevated expression of p16, SASP chemokines (Ccl2, Cxcl10, and Ccl17) and SASP matrix metalloproteinases (Mmp2, Mmp9, and Mmp12) is observed in the macrophages of irradiated lung, which might stimulate a fibrotic phenotype in pulmonary fibroblasts. In summary, irradiation can induce macrophage senescence, and increase the secretion of SASP in senescent macrophages. Our findings provide important evidence that senescent macrophages might be the target for prevention and treatment of RIPF.Subject terms: Senescence, Chemokines     

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.