Multiple Cellular Signaling Pathways Research Articles

Suppressing MicroRNA-30b by Estrogen Promotes Osteogenesis in Bone Marrow Mesenchymal Stem Cells.

MicroRNAs (miRNAs) have been widely demonstrated to interact with multiple cellular signaling pathways and to participate in a wide range of physiological processes. Estradiol-17β (E2) is the most potent and prevalent endogenous estrogen that plays a vital role in promoting bone formation and reducing bone resorption. Currently, little is known about the regulation of miRNAs in E2-induced osteogenic differentiation. In the present study, the primary bone marrow mesenchymal stem cells from rats (rBMSCs) were isolated and incubated with E2, followed by miRNA profiling. The microarray showed that 29 miRNAs were differentially expressed in response to E2 stimulation. Further verification by real-time reverse-transcriptase polymerase chain reaction revealed that E2 enhanced the expression of let-7b and miR-25 but suppressed the miR-30b expression. Moreover, a gain-of-function experiment confirmed that miR-30b negatively regulated the E2-induced osteogenic differentiation. These data suggest an important role of miRNAs in osteogenic differentiation.

Bruton’s tyrosine kinase (BTK) inhibitors in treating cancer: a patent review (2010-2018)

ABSTRACTIntroduction: Bruton’s tyrosine kinase (BTK) plays a critical role in the regulation of survival, proliferation, activation and differentiation of B-lineage cells. It participates by regulating multiple cellular signaling pathways, including B cell receptor and FcR signaling cascades. BTK is abundantly expressed and constitutively active in the pathogenesis of B cell hematological malignancies, as well as several autoimmune diseases. Therefore, BTK is considered as an attractive target for treatment of B-lineage lymphomas, leukemias, and some autoimmune diseases. Many industry and academia efforts have been made to explore small molecular BTK inhibitors.Areas covered: This review aims to provide an overview of the patented BTK inhibitors for the treatment of cancer from 2010 to 2018.Expert opinion: BTK inhibitors attract much interest for their therapeutic potential in the treatment of cancers and autoimmune diseases, especially for B cell hematological malignancies. In 2013, ibrutinib was approved by the FDA as the first-in-class BTK inhibitors for the treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), and now it is also undergoing clinical evaluation for other indications in either single or combined therapy. It is clear that BTK inhibitors can provide a promising clinical benefit in treating B-lineage lymphomas and leukemias.

Roles of FGF8 subfamily in embryogenesis and oral‑maxillofacial diseases (Review).

Fibroblast growth factors(FGFs) are diffusible polypeptides released by a variety of cell types. FGF8 subfamily members regulate embryonic development processes through controlling progenitor cell growth and differentiation, and are also functional in adults in tissue repair to maintain tissue homeostasis. FGF8 family members exhibit unique binding affinities with FGF receptors and tissue distribution patterns. Increasing evidence suggests that, by regulating multiple cellular signaling pathways, alterations in the FGF8 subfamily are involved in craniofacial development, odontogenesis, tongue development and salivary gland branching morphogenesis. Aberrant FGF signaling transduction, caused by mutations as well as abnormal expression or isoform splicing, plays an important role in the development of oral diseases. Targeting FGF8 subfamily members provides a new promising strategy for the treatment of oral diseases. The aim of this review was to summarize the aberrant regulations of FGF8 subfamily members and their potential implications in oral‑maxillofacial diseases.

Potential Mechanisms of Action of Curcumin for Cancer Prevention: Focus on Cellular Signaling Pathways and miRNAs.

Despite significant progressions in treatment modalities over the last decade, either cancer incidence or mortality is continuously on the rise throughout the world. Current anticancer agents display limited efficacy, accompanied by severe side effects. In order to improve therapeutic outcomes in patients with cancer, it is crucial to identify novel, highly efficacious pharmacological agents. Curcumin, a hydrophobic polyphenol extracted from turmeric, has gained increasing attention due to its powerful anticancer properties. Curcumin can inhibit the growth, invasion and metastasis of various cancers. The anticancer mechanisms of curcumin have been extensively studied. The anticancer effects of curcumin are mainly mediated through its regulation of multiple cellular signaling pathways, including Wnt/β-catenin, PI3K/Akt, JAK/STAT, MAPK, p53 and NF-ĸB signaling pathways. Moreover, curcumin also orchestrates the expression and activity of oncogenic and tumor-suppressive miRNAs. In this review, we summarized the regulation of these signaling pathways by curcumin in different cancers. We also discussed the modulatory function of curcumin in the downregulation of oncogenic miRNAs and the upregulation of tumor-suppressive miRNAs. An in-depth understanding of the anticancer mechanisms of curcumin will be helpful for developing this promising compound as a therapeutic agent in clinical management of cancer.

Translation Stress Regulates Ribosome Synthesis and Cell Proliferation.

Ribosome and protein synthesis are major metabolic events that control cellular growth and proliferation. Impairment in ribosome biogenesis pathways and mRNA translation is associated with pathologies such as cancer and developmental disorders. Processes that control global protein synthesis are tightly regulated at different levels by numerous factors and linked with multiple cellular signaling pathways. Several of these merge on the growth promoting factor c-Myc, which induces ribosome biogenesis by stimulating Pol I, Pol II, and Pol III transcription. However, how cells sense and respond to mRNA translation stress is not well understood. It was more recently shown that mRNA translation stress activates c-Myc, through a specific induction of E2F1 synthesis via a PI3Kδ-dependent pathway. This review focuses on how this novel feedback pathway stimulates cellular growth and proliferation pathways to synchronize protein synthesis with ribosome biogenesis. It also describes for the first time the oncogenic activity of the mRNA, and not the encoded protein.

GDE5 inhibition accumulates intracellular glycerophosphocholine and suppresses adipogenesis at a mitotic clonal expansion stage.

Mammalian glycerophosphodiesterases (GDEs) were recently shown to be involved in multiple cellular signaling pathways. This study showed that decreased GDE5 expression results in accumulation of intracellular glycerophosphocholine (GPC), showing that GDE5 is actively involved in GPC/choline metabolism in 3T3-L1 adipocytes. Using 3T3-L1 adipocytes, we further studied the biological significance of GPC/choline metabolism during adipocyte differentiation. Inhibition of GDE5 suppressed the formation of lipid droplets, which is accompanied by the decreased expression of adipocyte differentiation markers. We further showed that the decreased GDE5 expression suppressed mitotic clonal expansion (MCE) of preadipocytes. Decreased expression of CTP: phosphocholine cytidylyltransferase (CCTβ), a rate-limiting enzyme for phosphatidylcholine (PC) synthesis, is similarly able to inhibit MCE and PC synthesis; however, the decreased GDE5 expression resulted in accumulation of intracellular GPC but did not affect PC synthesis. Furthermore, we showed that mRNAs of proteoglycans and transporters for organic osmolytes are significantly upregulated and that intracellular amino acids and urea levels are altered in response to GDE5 inhibition. Finally, we showed that reduction of GDE5 expression increased lactate dehydrogenase release from preadipocytes. These observations indicate that decreased GDE5 expression can suppress adipocyte differentiation not through the PC pathway but possibly by intracellular GPC accumulation. These results provide insight into the roles of mammalian GDEs and their dependence upon osmotic regulation by altering intracellular GPC levels.

Ginsenoside Rg1 Prevents Doxorubicin-Induced Cardiotoxicity through the Inhibition of Autophagy and Endoplasmic Reticulum Stress in Mice

Ginsenoside Rg1, a saponin that is a primary component of ginseng, has been demonstrated to protect hearts from diverse cardiovascular diseases with regulating multiple cellular signal pathways. In the present study, we investigated the protective role of ginsenoside Rg1 on doxorubicin-induced cardiotoxicity and its effects on endoplasmic reticulum stress and autophagy. After pre-treatment with ginsenoside Rg1 (50 mg/kg i.g.) for 7 days, male C57BL/6J mice were intraperitoneally injected with a single dose of doxorubicin (6 mg/kg) every 3 days for four injections. Echocardiographic and pathological findings showed that ginsenoside Rg1 could significantly reduce the cardiotoxicity induced by doxorubicin. Ginsenoside Rg1 significantly inhibited doxorubicin-induced formation of autophagosome. At the same time, ginsenoside Rg1 decreased the doxorubicin-induced cardiac microtubule-associated protein-light chain 3 and autophagy related 5 expression. Ginsenoside Rg1 can reduce endoplasmic reticulum dilation caused by doxorubicin. Compared with the doxorubicin group, the expression of cleaved activating transcription factor 6 and inositol-requiring enzyme 1 decreased in group ginsenoside Rg1. Treatment with ginsenoside Rg1 reduces the expression of TIF1 and increases the expression of glucose-regulated protein 78. In the ginsenoside Rg1 group, the expression of p-P70S6K, c-Jun N-terminal kinases 1 and Beclin1 declined. These results indicate that ginsenoside Rg1 may improve doxorubicin-induced cardiac dysfunction by inhibiting endoplasmic reticulum stress and autophagy.

ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening

Necrosis is a key factor in myocardial injury during cardiac pathological processes, such as myocardial infarction (MI), ischemia/reperfusion (I/R) injury and heart failure. Increasing evidence suggests that several aspects of necrosis are programmed and tightly regulated, so targeting the necrosis process has become a new trend for myocardial protection. Multiple cellular signaling pathways have been implicated in necrotic cell death, such as the death receptor-mediated extrinsic and mitochondrial intrinsic pathways. However, the precise mechanisms underlying myocardial necrosis remain unclear. In this study, we showed that apoptosis repressor with caspase recruitment domain (ARC) participated in the mitochondrial intrinsic pathway and inhibited myocardial necrosis by preventing the opening of the mitochondrial permeability transition pore (mPTP). ARC attenuated necrotic cell death triggered by exposure to 500 μM hydrogen peroxide (H2O2) in the cardiomyocyte cell line H9c2. In mice, ARC ameliorated myocardial necrosis, reduced the myocardial infarct size and improved long-term heart function during I/R injury. Mechanistically, it has been shown that the inhibition of necrosis by ARC was dependent on its mitochondrial localization and that ARC prevented the opening of mPTP by targeting CypD, the main regulator of mPTP. In addition, ARC expression was negatively regulated by the transcription factor p53 at the transcriptional level during the necrosis process. These findings identified the novel role of ARC in myocardial necrosis and delineated the p53-ARC-CypD/mPTP necrosis pathway during ischemia- and oxidative stress-induced myocardial damage, which can provide a new strategy for cardiac protection.

Recent advances in endotoxin tolerance.

Endotoxin tolerance is defined as a reduced capacity of a cell to respond endotoxin (lipopolysaccharide, LPS) challenge after an initial encounter with endotoxin in advance. The body becomes tolerant to subsequent challenge with a lethal dose of endotoxin and cytokines release and cell/tissue damage induced by inflammatory reaction are significantly reduced in the state of endotoxin tolerance. The main characteristics of endotoxin tolerance are downregulation of inflammatory mediators such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and C-X-C motif chemokine 10 (CXCL10) and upregulation of anti-inflammatory cytokines such as IL-10 and transforming growth factor β (TGF-β). Therefore, endotoxin tolerance is often regarded as the regulatory mechanism of the host against excessive inflammation. Endotoxin tolerance is a complex pathophysiological process and involved in multiple cellular signal pathways, receptor alterations, and biological molecules. However, the exact mechanism remains elusive up to date. To better understand the underlying cellular and molecular mechanisms of endotoxin tolerance, it is crucial to investigate the comprehensive cellular signal pathways, signaling proteins, cell surface molecules, proinflammatory and anti-inflammatory cytokines, and other mediators. Endotoxin tolerance plays an important role in reducing the mortality of sepsis, endotoxin shock, and other endotoxin-related diseases. Recent reports indicated that endotoxin tolerance is also related to other diseases such as cystic fibrosis, acute coronary syndrome, liver ischemia-reperfusion injury, and cancer. The aim of this review is to discuss the recent advances in endotoxin tolerance mainly based on the cellular and molecular mechanisms by outline the current state of the knowledge of the involvement of the toll-like receptor 4 (TLR4) signaling pathways, negative regulate factor, microRNAs, apoptosis, chromatin modification, and gene reprogramming of immune cells in endotoxin tolerance. We hope to provide a new idea and scientific basis for the rational treatment of endotoxin-related diseases such as endotoxemia, sepsis, and endotoxin shock clinically.

Abstract 3120: Tumor-infiltrating lymphocyte profiling in hepatitis B virus pre-S2 mutant-positive hepatocellular carcinoma

Abstract Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), the leading cause of cancer-related deaths worldwide. Pre-S2 mutant is well demonstrated as a HBV oncoprotein that disturbs multiple cellular signaling pathways, leading to HCC development. HCC patients with pre-S2 mutant are recognized as a high-risk population for postoperative recurrence. Plentiful evidences reveal that tumor infiltrating lymphocytes (TILs) play an essential role in the stepwise progression of HCC development, and are closely associated with clinical outcomes. However, to date, the role of pre-S2 mutant in regulation of TILs distribution in HCC remains totally unknown. In this study, we collected plasma and paraffin-embedded liver samples from HBV-related HCC patients. The plasma samples were used for detection of the pre-S2 mutant with polymerase chain reaction-based method. As a result, the patients were divided into two groups: the pre-S2 mutant-positive and -negative groups. The paraffin-embedded liver samples were used for examination of the distribution of specific population of TILs with fluorescent immunohistochemistry staining. We analyzed 6 cases of HBV-related HCC samples, half of which were detected as pre-S2 mutant-positive. The distribution level of the cytotoxic CD3+CD8+ TILs in HCCs was examined. We found that patients with pre-S2 mutant had a considerably lower density of cytotoxic CD3+CD8+ TILs in HCC tissues than patients without pre-S2 mutant. The mean ± SEM of cytotoxic CD3+CD8+ TILs density was 3.74 ± 0.60 and 6.66 ± 0.66 in patients with and without pre-S2 mutant, respectively (p value=0.031). Our result suggests that pre-S2 mutant may have a role in regulation of TILs distribution in HBV-related HCCs. Systematic evaluation of specific population of TILs may help guide development of novel strategies for prognosis and/or therapies of HCC, especially for the HBV-related pre-S2 mutant-positive high-risk patient population. Citation Format: Chiao-Fang Teng, Woei-Cherng Shyu, Long-Bin Jeng. Tumor-infiltrating lymphocyte profiling in hepatitis B virus pre-S2 mutant-positive hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3120.

Control of Neuronal Ryanodine Receptor-Mediated Calcium Signaling by Calsenilin.

Calsenilin is a calcium ion (Ca2+)-binding protein involved in regulating the intracellular concentration of Ca2+, a second messenger that controls multiple cellular signaling pathways. The ryanodine receptor (RyR) amplifies Ca2+ signals entering the cytoplasm by releasing Ca2+ from endoplasmic reticulum (ER) stores, a process termed calcium-induced calcium release (CICR). Here, we describe a novel mechanism, in which calsenilin controls the activity of neuronal RyRs. We show calsenilin co-localized with RyR2 and 3 in the ER of mouse hippocampal and cortical neurons using immunocytochemistry. The underlying protein-protein interaction between calsenilin and the RyR was determined in mouse central nervous system (CNS) neurons using immunoprecipitation studies. The functional relevance of this interaction was assayed with single-channel electrophysiology. At low physiological Ca2+ concentrations, calsenilin binding to the cytoplasmic face of neuronal RyRs decreased the RyR's open probability, while calsenilin increased the open probability at high physiological Ca2+ concentrations. This novel molecular mechanism was studied further at the cellular level, where faster release kinetics of caffeine-induced Ca2+ release were measured in SH-SY5Y neuroblastoma cells overexpressing calsenilin. The interaction between calsenilin and neuronal RyRs reveals a new regulatory mechanism and possibly a novel pharmacological target for the control of Ca2+ release from intracellular stores.

Ssu72 phosphatase is involved in immunometabolism

Abstract Reversible protein phosphorylation is the primary type of various post-translational modifications in multiple cellular signaling pathways of eukaryotic cell. Dynamic alterations of phosphorylation state of many cellular signaling-mediated proteins including adipogenesis and thermogenesis regulate their molecular and cellular fates. Ssu72 is dual protein phosphatase that can act upon tyrosine or serine/threonine residues and transcription/RNA-processing factor. Recently, we found that Ssu72 expressed in adipose tissue, especially strongly in BAT, indicating that the potential involvement of Ssu72 in regulating BAT function. In this aspect, we generated conditional knock out mice which Ssu72 is deleted specifically in adipose tissue and found that the deficiency of Ssu72 leads to BAT dysfunction and abnormal thermoregulation compared to wild type mice. Moreover, we observed not only dramatically reduction of macrophage population but also defective M2 macrophage generation in Ssu72-deficient BAT. Notably, it has been reported that IL-4-induced M2 activation of macrophages is required for adaptive thermogenesis in mice. Thus, we further generated Ssu72-deleted macrophage mouse model. This study includes the physiological relevance of Ssu72 loss-of-function in macrophage during metabolic and thermogenic responses.

Next-generation sequencing-based miRNA expression analysis in Parp1-deficient embryonic stem cell-derived exosomes

Poly (ADP-ribose) polymerase family, member 1 (Parp1) has pleiotropic and disparate functions in multiple cellular signaling pathways through post-translational protein modification. It contributes to the regulation of various cellular processes, including DNA damage repair, cell death, and cell differentiation, genetically or epigenetically. Meanwhile, the functions of Parp1 in intercellular signaling remain to be established. To examine the functions of Parp1 in intercellular signaling, we examined microRNA (miRNA) regulation in exosomes derived from Parp1-deficient (Parp1−/−) embryonic stem (ES) cells. The percentages of miRNAs among total RNAs, including small RNAs such as miRNAs, snRNAs, snoRNAs, tRNAs, exonic RNAs, and intronic RNAs, in Parp1+/+ and Parp1−/− ES cell-derived exosomes were 8.2% and 3.5%, respectively. Overall, 329 distinct miRNAs exhibited ≥2-fold changes (118 upregulated; 211 downregulated). The upregulated miRNAs targeted 810 candidate genes, and the downregulated miRNAs targeted 716 candidate genes. Pathway analyses revealed that the upregulated miRNAs were significantly associated with five pathways including MAPK signaling cascades (p < 0.05), indicating that the target genes in these pathways were suppressed in Parp1−/− ES cells. In quantitative analyses of miRNA expression, miR365-3p, let-7a-5p, miR196b-5p, miR203-3p, miR98-5p, and miR146a-5p were increased by ≥ 2-fold in Parp1−/− ES cell-derived exosomes. Gene ontology enrichment analyses revealed that the upregulated miRNAs were significantly annotated for growth and stress-related cell signaling and cell communication (p < 0.05). Parp1 deficiency in ES cells led to inhibition of cell–cell communication, possibly by intercellular signal transduction, suggesting that Parp1 functions extracellularly by regulating exosomal miRNAs.

TSC1 and TSC2 regulate cilia length and canonical Hedgehog signaling via different mechanisms

Primary cilia are sensory organelles that coordinate multiple cellular signaling pathways, including Hedgehog (HH), Wingless/Int (WNT) and Transforming Growth Factor-β (TGF-β) signaling. Similarly, primary cilia have been implicated in regulation of mTOR signaling, in which Tuberous Sclerosis Complex proteins 1 and 2 (TSC1/2) negatively regulate protein synthesis by inactivating the mTOR complex 1 (mTORC1) at energy limiting states. Here we report that TSC1 and TSC2 regulate Smoothened (SMO)-dependent HH signaling in mouse embryonic fibroblasts (MEFs). Reduced SMO-dependent expression of Gli1 was demonstrated in both Tsc1−/− and Tsc2−/− cells, and we found that Tsc1 is required for TGF-β induced phosphorylation of SMAD2/3 and subsequent expression of the HH signaling effector and transcription factor GLI2. Hedgehog signaling was restored in Tsc1−/− cells after exogenous expression of Gli2, whereas rapamycin restored HH signaling in Tsc2−/− cells. Furthermore, we observed that Tsc1−/− MEFs display significantly elongated cilia, whereas cilia in Tsc2−/− MEFs were shorter than normal. The elongated cilium phenotype of Tsc1−/− MEFs is likely due to increased mTORC1-dependent autophagic flux observed in these cells, as both the autophagic flux and the cilia length phenotype was restored by rapamycin. In addition, ciliary length control in Tsc1−/− MEFs was also influenced by reduced expression of Gli2, which compromised expression of Wnt5a that normally promotes cilia disassembly. In summary, our results support distinct functions of Tsc1 and Tsc2 in cellular signaling as the two genes affect ciliary length control and HH signaling via different mechanisms.

Loss of Neuroglobin Expression Alters Cdkn1a/Cdk6-Expression Resulting in Increased Proliferation of Neural Stem Cells.

In the quest to unravel its functional significance, neuroglobin (Ngb), a brain-specific neuroprotective protein, has recently been proposed as an actor in neurodevelopment. As neural stem cells (NSCs) are fundamental during brain development, the present study aimed at investigating the role of Ngb in the growth and proliferation of NSCs by comparing an Ngb-floxed (Ngbfl-)NSC line, equivalent to the wild-type cellular situation, with an in-house created Ngb knockout (NgbKO-)NSC line. NgbKO-NSCs were characterized by an increased growth and proliferation capacity in vitro, supported by RNA sequencing and western blot results reporting the downregulation of Cdkn1a and the upregulation of Cdk6, both enhancing the cell cycle. Based on additional gene ontology enrichment and pathway analyses, we hypothesize that the loss of Ngb affects multiple cellular signaling pathways with the most important being the Akt-Tp53 axis.

Repeated restraint stress promotes hippocampal neuronal cell ciliogenesis and proliferation in mice

Stress severely disturbs physiological and mental homeostasis which includes adult neurogenesis in hippocampus. Neurogenesis in hippocampus is a key feature to adapt to environmental changes and highly regulated by multiple cellular signaling pathways. The primary cilium is a cellular organelle, which acts as a signaling center during development and neurogenesis in adult mice. However, it is not clear how the primary cilia are involved in the process of restraint (RST) stress response. Using a mouse model, we examined the role of primary cilia in repeated and acute RST stress response. Interestingly, RST stress increased the number of ciliated cells in the adult hippocampal dentate gyrus (DG). In our RST model, cell proliferation in the DG also increased in a time-dependent manner. Moreover, the analysis of ciliated cells in the hippocampal DG with cell type markers indicated that cells that were ciliated in response to acute RST stress are neurons. Taken together, these findings suggest that RST stress response is closely associated with an increase in the number of ciliated neurons and leads to an increase in cell proliferation.

The desumoylating enzyme sentrin-specific protease 3 contributes to myocardial ischemia reperfusion injury

Sentrin-specific protease 3 (SENP3), a member of the desumoylating enzyme family, is known as a redox sensor and could regulate multiple cellular signaling pathways. However, its implication in myocardial ischemia reperfusion (MIR) injury is unclear. Here, we observed that SENP3 was expressed and upregulated in the mouse heart depending on reactive oxygen species (ROS) production in response to MIR injury. By utilizing siRNA-mediated cardiac specific gene silencing, SENP3 knockdown was demonstrated to significantly reduce MIR-induced infarct size and improve cardiac function. Mechanistic studies indicated that SENP3 silencing ameliorated myocardial apoptosis mainly via suppression of endoplasmic reticulum (ER) stress and mitochondrial-mediated apoptosis pathways. By contrast, adenovirus-mediated cardiac SENP3 overexpression significantly exaggerated MIR injury. Further molecular analysis revealed that SENP3 promoted mitochondrial translocation of dynamin-related protein 1 (Drp1) in reperfused myocardium. In addition, mitochondrial division inhibitor-1 (Mdivi-1), a pharmacological inhibitor of Drp1, significantly attenuated the exaggerated mitochondrial abnormality and cardiac injury by SENP3 overexpression after MIR injury. Taken together, we provide the first direct evidence that SENP3 upregulation pivotally contributes to MIR injury in a Drp1-dependent manner, and suggest that SENP3 suppression may hold therapeutic promise for constraining MIR injury.

Expression and Subcellular Localization of the Kaposi's Sarcoma-Associated Herpesvirus K15P Protein during Latency and Lytic Reactivation in Primary Effusion Lymphoma Cells.

The K15P membrane protein of Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with multiple cellular signaling pathways and is thought to play key roles in KSHV-associated endothelial cell angiogenesis, regulation of B-cell receptor (BCR) signaling, and the survival, activation, and proliferation of BCR-negative primary effusion lymphoma (PEL) cells. Although full-length K15P is ∼45 kDa, numerous lower-molecular-weight forms of the protein exist as a result of differential splicing and poorly characterized posttranslational processing. K15P has been reported to localize to numerous subcellular organelles in heterologous expression studies, but there are limited data concerning the sorting of K15P in KSHV-infected cells. The relationships between the various molecular weight forms of K15P, their subcellular distribution, and how these may differ in latent and lytic KSHV infections are poorly understood. Here we report that a cDNA encoding a full-length, ∼45-kDa K15P reporter protein is expressed as an ∼23- to 24-kDa species that colocalizes with the trans-Golgi network (TGN) marker TGN46 in KSHV-infected PEL cells. Following lytic reactivation by sodium butyrate, the levels of the ∼23- to 24-kDa protein diminish, and the full-length, ∼45-kDa K15P protein accumulates. This is accompanied by apparent fragmentation of the TGN and redistribution of K15P to a dispersed peripheral location. Similar results were seen when lytic reactivation was stimulated by the KSHV protein replication and transcription activator (RTA) and during spontaneous reactivation. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the latent and lytic phases.IMPORTANCE The K15P protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to play key roles in disease, including KSHV-associated angiogenesis and the survival and growth of primary effusion lymphoma (PEL) cells. The protein exists in multiple molecular weight forms, and its intracellular trafficking is poorly understood. Here we demonstrate that the molecular weight form of a reporter K15P molecule and its intracellular distribution change when KSHV switches from its latent (quiescent) phase to the lytic, infectious state. We speculate that expression of different molecular weight forms of K15P in distinct cellular locations reflects the alternative demands placed upon the protein in the viral latent and lytic stages.

Antagonism between Hedgehog and Wnt signaling pathways regulates tumorigenicity.

The crosstalk of multiple cellular signaling pathways is crucial in animal development and tissue homeostasis, and its dysregulation may result in tumor formation and metastasis. The Hedgehog (Hh) and Wnt signaling pathways are both considered to be essential regulators of cell proliferation, differentiation and oncogenesis. Recent studies have indicated that the Hh and Wnt signaling pathways are closely associated and involved in regulating embryogenesis and cellular differentiation. Hh signaling acts upstream of the Wnt signaling pathway, and negative regulates Wnt activity via secreted frizzled-related protein 1 (SFRP1), and the Wnt/β-catenin pathway downregulates Hh activity through glioma-associated oncogene homolog 3 transcriptional regulation. This evidence suggests that the imbalance of Hh and Wnt regulation serves a crucial role in cancer-associated processes. The activation of SFRP1, which inhibits Wnt, has been demonstrated to be an important cross-point between the two signaling pathways. The present study reviews the complex interaction between the Hh and Wnt signaling pathways in embryogenesis and tumorigenicity, and the role of SFRP1 as an important mediator associated with the dysregulation of the Hh and Wnt signaling pathways.

Functional importance of PP2A regulatory subunit loss in breast cancer.

Protein phosphatase 2A (PP2A) is a family of serine/threonine phosphatases that regulate multiple cellular signalling pathways involved in proliferation, survival and apoptosis. PP2A inhibition occurs in many cancers and is considered a tumour suppressor. Deletion/downregulation of PP2A genes has been observed in breast tumours, but the functional role of PP2A subunit loss in breast cancer has not been investigated. PP2A subunit expression was examined by immunohistochemistry in human breast tumours, and by qPCR and immunoblotting in breast cancer cell lines. PP2A subunits were inhibited by shRNA, and mutant PP2A genes overexpressed, in MCF10A and MCF7 cells, and growth and signalling in standard and three-dimensional cultures were assessed. Expression of PP2A-Aα, PP2A-Bα and PP2A-B'α subunits was significantly lower in primary human breast tumours and lymph node metastases, compared to normal mammary tissue. PP2A-Aα and the regulatory subunits PP2A-Bα, -Bδ and -B'γ were also reduced in breast cancer cell lines compared to normal mammary epithelial cells. Functionally, shRNA-mediated knockdown of PP2A-Bα, -B'α and -B'γ, but not PP2A-Aα, induced hyper-proliferation and large multilobular acini in MCF10A 3D cultures, characterised by activation of ERK. Expression of a breast cancer-associated PP2A-A mutant, PP2A-Aα-E64G, which inhibits binding of regulatory subunits to the PP2A core, induced a similar hyper-proliferative phenotype. Knockdown of PP2A-Bα also induced hyper-proliferation in MCF7 breast cancer cells. These results suggest that loss of specific PP2A regulatory subunits is functionally important in breast tumourigenesis, and support strategies to enhance PP2A activity as a therapeutic approach in breast cancer.