Ring finger protein 138 inhibits transcription factor C/EBPα protein turnover leading to differentiation arrest in acute myeloid leukemia.

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited ß-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML.

RING finger E3 ligase, RNF138 inhibits osteoblast differentiation by negatively regulating Runx2 protein turnover.

A few ubiquitin ligases have been shown to target Runx2, the key osteogenic transcription factor and thereby regulate bone formation. The regulation of Runx2 expression and function are controlled both at the transcriptional and posttranslational levels. Really interesting new gene (RING) finger ubiquitin ligases of which RNF138 is a member are important players in the ubiquitin-proteasome system, contributing to the regulation of protein turnover and cellular processes. Here, we demonstrated that RNF138 negatively correlated with Runx2 protein levels in osteopenic ovariectomized rats which implied its role in bone loss. Accordingly, RNF138 overexpression potently inhibited osteoblast differentiation of mesenchyme-like C3H10T1/2 as well primary rat calvarial osteoblast (RCO) cells in vitro, whereas overexpression of catalytically inactive mutant RNF138Δ18-58 (lacks RING finger domain) had mild to no effect. Contrarily, RNF138 depletion copiously enhanced endogenous Runx2 levels and augmented osteogenic differentiation of C3H10T1/2 as well as RCOs. Mechanistically, RNF138 physically associates within multiple regions of Runx2 and ubiquitinates it leading to its reduced protein stability in a proteasome-dependent manner. Moreover, catalytically active RNF138 destabilized Runx2 which resulted in inhibition of its transactivation potential and physiological function of promoting osteoblast differentiation leading to bone loss. These findings underscore the functional involvement of RNF138 in bone formation which is primarily achieved through its modulation of Runx2 by stimulating ubiquitin-mediated proteasomal degradation. Thus, our findings indicate that RNF138 could be a promising novel target for therapeutic intervention in postmenopausal osteoporosis.

TORC1 mediated regulation of mitochondrial integrity and calcium ion homeostasis by Wat1/mLst8 in S. pombe.

The mTOR complexes play a fundamental role in mitochondrial biogenesis and cellular homeostasis. Wat1, an ortholog of mammalian Lst8 is an important component of TOR complex and is essential for the regulation of downstream signaling. Earlier we reported the role of Wat1 in oxidative stress response. Here, we have shown that the abrogation of wat1 causes respiratory defects and mitochondrial depolarization that leads to a decrease in ATP production. The confocal and electron microscopy in wat1Δ cells revealed the fragmented mitochondrial morphology implying its role in mitochondrial fission. Furthermore, we also showed its role in autophagy and the maintenance of calcium ion homeostasis. Additionally, tor2-287 mutant cells also exhibit defects in mitochondrial integrity indicating the TORC1-dependent involvement of Wat1 in the maintenance of mitochondrial homeostasis. The interaction studies of Wat1 and Tor2 with Por1 and Mmm1 proteins revealed a plausible cross-talk between mitochondria and endoplasmic reticulum through the Mitochondria-associated membranes (MAM) and endoplasmic reticulum-mitochondria encounter structure (ERMES) complex, involving TORC1. Taken together, this study demonstrates the involvement of Wat1/mLst8 in harmonizing various mitochondrial functions, redox status, and Ca2+ homeostasis.

AIP4 regulates adipocyte differentiation by targeting C/EBPα for ubiquitin-mediated proteasomal degradation.

Adipogenesis, that is, the formation of terminally differentiated adipocytes is intricately regulated by transcription factors where CCAAT/enhancer binding protein alpha (C/EBPα) plays a key role. In the current study, we demonstrate that E3 ubiquitin ligase AIP4 negatively regulates C/EBPα protein stability leading to reduced adipogenesis. While AIP4 overexpression in 3T3-L1 cells preadipocytes inhibited lipid accumulation when treated with differentiation inducing media (MDI), AIP4 depletion was sufficient to partially promote lipid accumulation even in the absence of MDI. Mechanistically, overexpression of AIP4 inhibited protein levels of both ectopically expressed as well as endogenous C/EBPα while catalytically inactive AIP4 failed. On the contrary, AIP4 depletion profoundly enhanced endogenous C/EBPα protein levels. The observation that AIP4 levels decrease with concomitant increase in C/EBPα levels during adipocyte differentiation further indicated that AIP4 negatively regulates C/EBPα levels. We further show that AIP4 physically interacts with C/EBPα and ubiquitinates it leading to its proteasomal degradation. AIP4 promoted K48-linked ubiquitination of C/EBPα while catalytically inactive AIP4-C830A failed. Taken together, our data demonstrate that AIP4 inhibits adipogenesis by targeting C/EBPα for ubiquitin-mediated proteasome degradation.

CLUH functions as a negative regulator of inflammation in human macrophages and determines ulcerative colitis pathogenesis.

Altered mitochondrial function without a well-defined cause has been documented in patients with ulcerative colitis (UC). In our efforts to understand UC pathogenesis, we observed reduced expression of clustered mitochondrial homolog (CLUH) only in the active UC tissues compared with the unaffected areas from the same patient and healthy controls. Stimulation with bacterial Toll-like receptor (TLR) ligands similarly reduced CLUH expression in human primary macrophages. Further, CLUH negatively regulated secretion of proinflammatory cytokines IL-6 and TNF-α and rendered a proinflammatory niche in TLR ligand-stimulated macrophages. CLUH was further found to bind to mitochondrial fission protein dynamin related protein 1 (DRP1) and regulated DRP1 transcription in human macrophages. In the TLR ligand-stimulated macrophages, absence of CLUH led to enhanced DRP1 availability for mitochondrial fission, and a smaller dysfunctional mitochondrial pool was observed. Mechanistically, this fissioned mitochondrial pool in turn enhanced mitochondrial ROS production and reduced mitophagy and lysosomal function in CLUH-knockout macrophages. Remarkably, our studies in the mouse model of colitis with CLUH knockdown displayed exacerbated disease pathology. Taken together, this is the first report to our knowledge explaining the role of CLUH in UC pathogenesis, by means of regulating inflammation via maintaining mitochondrial-lysosomal functions in the human macrophages and intestinal mucosa.

Ormeloxifene, a nonsteroidal antifertility drug promotes megakaryocyte differentiation in leukemia cell line K562.

Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(ß-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.

Dexamethasone activates c-Jun NH2-terminal kinase (JNK) which interacts with GR and protects it from ubiquitin-mediated degradation in NSCLC cells.

Dexamethasone-mediated pharmacological activation of the glucocorticoid receptor (GR) is widely used in the treatment regimen of hematological malignancies and solid cancers. However, DEX sensitivity towards patients primarily depends on the endogenous protein levels of GR. We observed that DEX treatment leads to an increase in GR protein levels despite inhibition of neo-protein synthesis in non-small cell lung cancer (NSCLC) cells. Mechanistically, DEX-stimulation concomitantly increased the JNK phosphorylation and GR protein levels, however the JNK stimulation preceds GR upregulation. Moreover, we also observed that DEX-mediated phosphorylation is partially mediated by upregulation in MEKK1 phosphorylation. Further, GR protein levels were significantly decreased in JNK inhibitor (JNKi, SP600125) treated cells whereas MG132 treatment restored GR levels indicating that DEX induced JNK activity regulated the GR protein levels through proteasomal-degradation pathway. Next, we showed that DEX led to JNK activation which physically interacts with GR and protects it from ubiquitination-mediated degradation. Furthermore, at basal level GR interacts with JNK in cytoplasm whereas upon DEX stimulation GR and pJNK both localized to nucleus and interact with each other. Next, we show that JNK-mediated GR stabilization affects its nuclear transcriptional functional activity in NSCLC cells. In line with these in vitro data, patient dataset analysis also shows that increased levels of both JNK and GR contributes towards better prognosis of NSCLC patients. Taken together, our data shows that DEX treatment may lead to positive feedback regulation of GR by activating JNK and thus highlights importance of GR-JNK crosstalk in NSCLC.

SOX4-mediated FBW7 transcriptional upregulation confers Tamoxifen resistance in ER+ breast cancers via GATA3 downregulation.

AIM: Tamoxifen-mediated endocrine therapy has been standard treatment for ER+ breast cancers; however, majority of them acquire resistance leading to disease relapse. Although numerous substrates of E3 ligase FBW7 are known, only a handful of factors that regulate FBW7 expression and function are reported. In particular, there remains a lack of in-depth understanding of FBW7 transcriptional regulation. MATERIALS AND METHODS: Luciferase reporter assay was performed after cloning full length and truncated FBW7 promoters followed by Chromatin immunoprecipitation assay to validate binding of SOX4 on FBW7 promoter. Transcriptional regulation of FBW7 by SOX4 and their biological consequences with respect to ER+ breast cancer was then evaluated using immunoblotting and other cell based assays. KEY FINDINGS: SOX4 positively regulates FBW7 at transcriptional level by binding to three putative SOX4 biding sites within 3.1 kb long FBW7 promoter. Analysis of publicly available RNAseq datasets also showed a positive correlation between SOX4 and FBW7 mRNA in cancer cell lines and patient samples. qPCR and Immunoblotting confirmed that transiently or stably expressed SOX4 induced both endogenous FBW7 mRNA and protein levels. Our findings further demonstrated that increased levels of SOX4 and FBW7 in MCF7 mammospheres promoted cancer stemness and tumor cell dormancy. We further showed that both MCF7 mammospheres and MCFTAMR cells had elevated SOX4 levels which apparently enhanced FBW7 to potentiate GATA3 degradation leading to enhanced stemness, tumor dormancy and Tamoxifen resistance in MCF7TAMR as well as patients with ER+ breast cancers. SIGNIFICANCE: Targeting SOX4-FBW7-GATA3 axis may overcome tamoxifen resistance in ER+ breast cancers.

Proteomic analysis of TGFß-induced A549 secretome identifies putative regulators of epithelial-mesenchymal transition.

Imparting epithelial to mesenchymal transition (EMT) during cellular transformation, a major driving force behind tumor progression, is one of the notorious oncogenic activities of transforming growth factor ß (TGFß); however, the secretary factors released during TGFß-induced EMT that may have role in potentiating EMT and tumor progression are poorly known. This study was undertaken to identify such secreted protein factors from TGFß-induced A549 cells cultured in serum-free chemically defined medium (FreestyleTM ) using Matrix Assisted Laser Desorption Ionization-Time of flight/Time of flight (MALDI-TOF/TOF) mass spectrometry. We identified some of the potential factors such as ESR, ANXA2, ALDH1A, TGFß-induced protein ig-h3, and PAI-1 that were not only secreted but some were also elevated in TGFß-induced A549 cells. Interestingly, these factors are widely reported to play crucial role in EMT induction and progression, which not only validates our findings but also opens avenues for further investigation, if upon secretion they act exogenously through certain receptors to potentiate cellular signaling involved in EMT induction and tumor progression.

Estradiol overcomes adiponectin-resistance in diabetic mice by regulating skeletal muscle adiponectin receptor 1 expression.

Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.

CDK2-instigates C/EBPα degradation through SKP2 in Acute myeloid leukemia.

Transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPα) regulates myelopoiesis by coupling growth arrest with differentiation of myeloid progenitors. Mutations in one or both alleles are observed in 10-14% AML cases that render C/EBPα functionally inactive. Besides, antagonistic protein-protein interactions also impair C/EBPα expression and function. In recent independent studies, we showed that CDK2 and SKP2 downregulated C/EBPα expression in an ubiquitin-dependent proteasome degradation manner leading to differentiation block in AML. Here, we demonstrate that CDK2-instigated C/EBPα downregulation is actually mediated by SKP2. Mechanistically, we show that CDK2 stabilizes SKP2 by phosphorylating it at Ser64 and thereby potentiates C/EBPα ubiquitination and subsequent degradation in AML cells. Immunoblot experiments showed that CDK2 inhibition downregulated SKP2 levels and concomitantly enhanced C/EBPα levels in myeloid cells. We further show that while CDK2 promoted C/EBPα ubiquitination and inhibited its protein levels, negatively affected its transactivation potential and DNA binding ability, simultaneous SKP2 depletion abrogated CDK2-promoted ubiquitination and restored C/EBPα expression and function. Taken together, these findings consolidate that CDK2 potentiates SKP2-mediated C/EBPα degradation in AML and targeting CDK2-SKP2 axis can be harnessed for therapeutic benefit in AML. Hypothetical model depicts that SKP2-mediated C/EBPα proteasomal degradation is reinforced by CDK2. CDK2 phopshorylates SKP2 leading to its enhanced stabilization which in turn exaggerates C/EBPα degradation leading to differentiation arrest in AML.

FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1.

Glycogen synthase kinase 3ß (GSK3ß), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3ß inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3ß target. We demonstrate that GSK3ß phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte-macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF-induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3ß-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. IMPLICATIONS: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3ß inhibition and further substantiates potential of GSK3ß as a therapeutic target in AML.

Chebulinic acid inhibits MDA-MB-231 breast cancer metastasis and promotes cell death through down regulation of SOD1 and induction of autophagy.

Triple-negative breast cancers (TNBC) are highly aggressive and drug resistant accounting for majority of cases with poor outcome. Purified natural compounds display substantial anticancer activity with reduced cytotoxicity providing a new avenue to combat TNBC. Chebulinic acid (CA), a polyphenol derived from the fruits of various medicinal plants has potent anticancer activity. Here, we demonstrate that CA shows significant cytotoxicity against triple negative MDA-MB-231 cells. CA exhibited cytotoxicity to MDA-MB-231 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Further, CA mitigated MDA-MB-231 cells viability and proliferation as shown by reduced live cell count, crystal violet staining, colony formation assay, soft agar assay and cell cycle analysis. Wound healing assay and trans-well migration assay demonstrated that CA significantly inhibited migration of MDA-MB-231 cells. Also reduced MMP9 expression was observed in CA-treated cells by gelatin zymography. CA negatively regulated mesenchymal characteristics of MDA-MB-231 cells demonstrated by F-actin staining and reduced expression of N-cadherin by confocal microscopy and western blot analysis. Annexin V/propidium iodide (PI) and active caspase-3 staining showed that CA was able to induce apoptosis in MDA-MB-231 cells but did not activate caspase-3. Two-dimensional gel electrophoresis based proteomic analysis demonstrated that CA regulated proteins belonging to the oxidative stress pathway, apoptotic pathway and proteins with antiproliferative activity. Western blot analysis analysis revealed that CA negatively regulated superoxide dismutase 1 (SOD1) and enhanced oxidative stress in MDA-MB-231 cells. SOD1 in-gel activity assay also showed reduced SOD1 activity upon CA treatment. Overexpression studies with GFP-LC3 and tandem tagged RFP-GFP-LC-3 also demonstrated enhanced autophagy upon CA treatment.

E3 ligase SCFSKP2 ubiquitinates and degrades tumor suppressor C/EBPα in acute myeloid leukemia.

AIM: Transcription factor CCAAT/Enhancer binding protein alpha (C/EBPα) is a key regulator of myeloid differentiation, granulopoiesis in particular. Although CEBPA mutations are found in more than 10% in AML, functional inhibition of C/EBPα protein is also widely observed in AML. Here, we sought to examine if SKP2, an aberrantly enhanced E3 ubiquitin ligase in primary AMLs inhibits C/EBPα stability to induce differentiation block. MAIN METHODS: Here we employed cell based assays such as transfections, immunoblotting, co-immunoprecipitation, luciferase and gel shift assays along with differentiation assays to investigate SKP2 regulated C/EBPα protein stability in acute myeloid leukemia. KEY FINDINGS: Here we discovered that oncogenic E3 ubiquitin ligase SCFskp2 ubiquitinates and destabilizes C/EBPα in a proteasome-dependent manner. Our data demonstrates that SKP2 physically interacts with C-terminal of C/EBPα and promotes its K48-linked ubiquitination-mediated degradation leading to its reduced transactivation potential, DNA binding ability and cellular functions. We further show that while overexpression of SKP2 inhibits both ectopic as well as endogenous C/EBPα in heterologous (HEK293T) as well as myeloid leukemia cells respectively, SKP2 depletion restores endogenous C/EBPα leading to reduced colony formation and enhanced myeloid differentiation of myeloid leukemia cells. Using Estradiol-inducible K562-C/EBPα-ER cells as yet another model of granulocytic differentiation, we further confirmed that SKP2 overexpression indeed inhibits granulocytic differentiation by mitigating C/EBPα stability. SIGNIFICANCE: Our findings identify SKP2 as a potential negative regulator of C/EBPα stability and function in AML which suggests that SKP2 can be potentially targeted in AML to restore C/EBPα and overcome differentiation block.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

Aurora kinase A-mediated phosphorylation of mPOU at a specific site drives skeletal muscle differentiation.

Aurora kinases are Ser/Thr-directed protein kinases which play pivotal roles in mitosis. Recent evidences highlight the importance of these kinases in multiple biological events including skeletal muscle differentiation. Our earlier study identified the transcription factor POU6F1 (or mPOU) as a novel Aurora kinase (Aurk) A substrate. Here, we report that Aurora kinase A phosphorylates mPOU at Ser197 and inhibit its DNA-binding ability. Delving into mPOU physiology, we find that the phospho-mimic (S197D) mPOU mutant exhibits enhancement, while the wild type or the phospho-deficient mutant shows retardation in C2C12 myoblast differentiation. Interestingly, POU6F1 depletion phenocopies S197D-mPOU overexpression in the differentiation context. Collectively, our results signify mPOU as a negative regulator of skeletal muscle differentiation and strengthen the importance of AurkA in skeletal myogenesis.

Long acting GLP-1 analog liraglutide ameliorates skeletal muscle atrophy in rodents.

BACKGROUND: Skeletal muscle atrophy is characterized by muscle wasting with partial or complete functional loss. Skeletal muscle atrophy severely affects the quality of life and currently, there is no available therapy except for spinal muscular atrophy. OBJECTIVE: Drug repositioning is a promising strategy that reduces cost and time due to prior availability of safety and toxicity details. Here we investigated myogenic and anti-atrophy effects of glucagon-like peptide-1 (GLP-1) analog liraglutide. METHODS: We used several in vitro atrophy models in C2C12 cells and in vivo models in Sprague Dawley rats to study Liraglutide's efficacy. Western blotting was used to assess cAMP-dependent signaling pathways specifically activated by liraglutide. Therapeutic efficacy of liraglutide was investigated by histological analysis of transverse muscle sections followed by morphometry. Myogenic capacity was investigated by immunoblotting for myogenic factors. RESULTS: Liraglutide induced myogenesis in C2C12 myoblasts through GLP-1 receptor via a cAMP-dependent complex network of signaling events involving protein kinase A, phosphoinositide 3-kinase/protein kinase B, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Liraglutide imparted protection against freeze injury, denervation, and dexamethasone -induced skeletal muscle atrophy and improved muscular function in all these models. In a therapeutic model, liraglutide restored myofibrillar architecture in ovariectomy-induced atrophy. Anti-atrophy actions of liraglutide involved suppression of atrogene expression and enhancement in expression of myogenic factors. CONCLUSION: Liraglutide imparted protection and restored myofibrillar architecture in diverse models of muscle atrophy. Given its potent anti-atrophy, and recently reported osteoanabolic effects, we propose liraglutide's clinical evaluation in skeletal muscle atrophy and musculoskeletal disorders associated with diverse pathologies.

CDK2 destabilizes tumor suppressor C/EBPα expression through ubiquitin-mediated proteasome degradation in acute myeloid leukemia.

Deregulation and functional inhibition of CCAAT-enhancer-binding protein α (C/EBPα), a key transcription factor of myeloid lineage leads to development of myeloid leukemia. In this study, we show that cyclin-dependent kinase 2 (CDK2) negatively regulates C/EBPα protein levels in myeloid leukemia cells. The overexpression of CDK2 inhibited C/EBPα both in a heterologous HEK293T and U937 myeloid leukemia cells. On the contrary, CDK2 depletion enhanced endogenous C/EBPα protein levels. CDK2 mitigated C/EBPα levels by promoting its ubiquitin-mediated proteasome degradation. We further showed that although CDK2 interacted with C/EBPα, direct interaction of CDK2 with C/EBPα is not involved in C/EBPα downregulation. CDK2-dependent phosphorylation of C/EBPα on its widely reported phosphorylatable amino acid residues is apparently not required for C/EBPα degradation by CDK2. Furthermore, our data demonstrate that CDK2-driven C/EBPα inhibition mitigates its transactivation potential and cellular functions such as ability to promote myeloid differentiation and growth arrest.

Haploinsufficient tumor suppressor Tip60 negatively regulates oncogenic Aurora B kinase.

The Aurora kinases represent a group of serine/threonine kinases which are crucial regulators of mitosis. Dysregulated Aurora kinase B (AurkB) expression, stemming from genomic amplification, increased gene transcription or overexpression of its allosteric activators, is capable of initiating and sustaining malignant phenotypes. Although AurkB level in cells is well-orchestrated, studies that relate to its stability or activity, independent of mitosis, are lacking. We report that AurkB undergoes acetylation in vitro by lysine acetyltransferases (KATs) belonging to different families, namely by p300 and Tip60. The haploinsufficient tumor suppressor Tip60 acetylates two highly conserved lysine residues within the kinase domain of AurkB which not only impinges the protein stability but also its kinase activity. These results signify a probable outcome on the increase in "overall activity" of AurkB upon Tip60 downregulation, as observed under cancerous conditions. The present work, therefore, uncovers an important functional interplay between AurkB and Tip60, frailty of which may be an initial event in carcinogenesis.