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Specific and effective accumulation of nanoparticles within tumors is highly crucial for precise cancer diagnosis and treatment. Therefore, spatiotemporally manipulating the aggregation of small gold nanoparticles (AuNPs) in a tumor microenvironment is of great significance for enhancing the diagnostic and therapeutic efficacy of tumors. Herein, we reported a novel furin enzyme/acidic pH synergistically triggered small AuNP aggregation strategy for activating the photoacoustic (PA) imaging and photothermal (PTT) functions of AuNPs in vivo. Smart gold nanoparticles decorated with furin-cleavable RVRR (Arg-Val-Arg-Arg) peptides (Au-RRVR) were rationally designed and fabricated. Both in vitro and in vivo experiments demonstrated that such Au-RRVR nanoparticles could be simultaneously induced by furin and acidic pH to form large aggregates within tumorous tissue resulting in improved tumor accumulation and retention, which can further activate the PA and PTT effect of AuNPs for sensitive imaging and efficient therapy of tumors. Thus, we believe that this dual-stimuli-responsive aggregation system may offer a universal platform for effective cancer diagnosis and treatment.

Background Signal transducer and activator of transcription 3 (STAT3) has been shown to upregulate gene transcription during tumorigenesis. However, how STAT3 initiates transcription remains to be exploited. This study is to reveal the role of CREPT (cell cycle-related and elevated-expression protein in tumours, or RPRD1B) in promoting STAT3 transcriptional activity. Methods BALB/c nude mice, CREPT overexpression or deletion cells were employed for the assay of tumour formation, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing. Results We demonstrate that CREPT, a recently identified oncoprotein, enhances STAT3 transcriptional activity to promote tumorigenesis. CREPT expression is positively correlated with activation of STAT3 signalling in tumours. Deletion of CREPT led to a decrease, but overexpression of CREPT resulted in an increase, in STAT3-initiated tumour cell proliferation, colony formation and tumour growth. Mechanistically, CREPT interacts with phosphorylated STAT3 (p-STAT3) and facilitates p-STAT3 to recruit p300 to occupy at the promoters of STAT3-targeted genes. Therefore, CREPT and STAT3 coordinately facilitate p300-mediated acetylation of histone 3 (H3K18ac and H3K27ac), further augmenting RNA polymerase II recruitment. Accordingly, depletion of p300 abolished CREPT-enhanced STAT3 transcriptional activity. Conclusions We propose that CREPT is a co-activator of STAT3 for recruiting p300. Our study provides an alternative strategy for the therapy of cancers related to STAT3.

Besides their original regulating roles in the brain, spinal cord, retina, and peripheral nervous system for mediating fast excitatory synaptic transmission, glutamate receptors consisting of metabotropic glutamate receptors (GluRs) and ionotropic glutamate receptors (iGluRs) have emerged to have a critical role in the biology of cancer initiation, progression, and metastasis. However, the precise mechanism underpinning the signal transduction mediated by ligand-bound GluRs is not clearly elucidated. Here, we show that iGluRs, GluR1 and GluR2, are acetylated by acetyltransferase CREB-binding protein upon glutamate stimulation of cells, and are targeted by lysyl oxidase-like 2 for deacetylation. Acetylated GluR1/2 recruit β-arrestin1/2 and signal transducer and activator of transcription 3 (STAT3) to form a protein complex. Both β-arrestin1/2 and STAT3 are subsequently acetylated and activated. Simultaneously, activated STAT3 acetylated at lysine 685 translocates to mitochondria to upregulate energy metabolism-related gene transcription. Our results reveal that acetylation-dependent formation of GluR1/2–β-arrestin1/2–STAT3 signalosome is critical for glutamate-induced cell proliferation.

Background and aims STAT3, a member of the signal transducer and activator of transcription (STAT) family, is strongly associated with liver injury, inflammation, regeneration, and hepatocellular carcinoma development. However, the signals that regulate STAT3 activity are not completely understood. Approach and results Here we characterize CREB/ATF bZIP transcription factor CREBZF as a critical regulator of STAT3 in the hepatocyte to repress liver regeneration. We show that CREBZF deficiency stimulates the expression of the cyclin gene family and enhances liver regeneration after partial hepatectomy. Flow cytometry analysis reveals that CREBZF regulates cell cycle progression during liver regeneration in a hepatocyte-autonomous manner. Similar results were observed in another model of liver regeneration induced by intraperitoneal injection of carbon tetrachloride (CCl4 ). Mechanistically, CREBZF potently associates with the linker domain of STAT3 and represses its dimerization and transcriptional activity in vivo and in vitro. Importantly, hepatectomy-induced hyperactivation of cyclin D1 and liver regeneration in CREBZF liver-specific knockout mice was reversed by selective STAT3 inhibitor cucurbitacin I. In contrast, adeno-associated virus-mediated overexpression of CREBZF in the liver inhibits the expression of the cyclin gene family and attenuates liver regeneration in CCl4 -treated mice. Conclusions These results characterize CREBZF as a coregulator of STAT3 to inhibit regenerative capacity, which may represent an essential cellular signal to maintain liver mass homeostasis. Therapeutic approaches to inhibit CREBZF may benefit the compromised liver during liver transplantation.

Summary The mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cellular innate immunity, metabolism, and senescence. FK506-binding protein 12 (FKBP12) inhibits mTOR kinase activity via direct association. The FKBP12-mTOR association can be strengthened by the immunosuppressant rapamycin, but the underlying mechanism remains elusive. We show here that the FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle. FKBP12 is acetylated on the lysine cluster (K45/K48/K53) by CREB-binding protein (CBP) in mammalian cells in response to nutrient treatment. Acetyl-FKBP12 associates with CBP acetylated Rheb. Rapamycin recruits SIRT2 with a high affinity for FKBP12 association and deacetylation. SIRT2-deacetylated FKBP12 then switches its association from Rheb to mTOR. Nutrient-activated mTOR phosphorylates IRF3S386 for the antiviral response. In contrast, rapamycin strengthening FKBP12-mTOR association blocks mTOR antiviral activity by recruiting SIRT2 to deacetylate FKBP12. Hence, on/off mTOR activity in response to environmental nutrients relies on FKBP12 acetylation and deacetylation status in mammalian cells., Graphical abstract, Highlights • FKBP12-mTOR association is tightly regulated by an acetylation–deacetylation cycle • SIRT2 is responsible for FKBP12 deacetylation • Acetylation of Rheb is indispensable to mTOR activation • mTOR phosphorylates IRF3 S386 for type-I interferon gene expression, Biochemistry; Protein; Molecular biology

N-myristoylation is a crucial signaling and pathogenic modification process that confers hydrophobicity to cytosolic proteins. Although different large-scale approaches have been applied, a large proportion of myristoylated proteins remain to be identified. EZH2 is overexpressed in lung cancer cells and exerts oncogenic effects via its intrinsic methyltransferase activity. Using a well-established click chemistry approach, we found that EZH2 can be modified by myristoylation at its N-terminal glycine in lung cancer cells. Hydrophobic interaction is one of the main forces driving or stabilizing liquid-liquid phase separation (LLPS), raising the possibility that myristoylation can modulate LLPS by mediating hydrophobic interactions. Indeed, myristoylation facilitates EZH2 to form phase-separated liquid droplets in lung cancer cells and in vitro. Furthermore, we provide evidence that myristoylation-mediated LLPS of EZH2 compartmentalizes its non-canonical substrate, STAT3, and activates STAT3 signaling, ultimately resulting in accelerated lung cancer cell growth. Thus, targeting EZH2 myristoylation may have significant therapeutic efficacy in the treatment of lung cancer. Altogether, these observations not only extend the list of myristoylated proteins, but also indicate that hydrophobic lipidation may serve as a novel incentive to induce or maintain LLPS.

T helper 17 (Th17)‐cell differentiation triggered by interleukin‐6 (IL‐6) via STAT3 activation promotes inflammation in inflammatory bowel disease (IBD) patients. However, leukemia inhibitory factor (LIF), an IL‐6 family cytokine, restricts inflammation by blocking Th17‐cell differentiation via an unknown mechanism. Here, we report that microbiota dysregulation promotes LIF secretion by intestinal epithelial cells (IECs) in a mouse colitis model. LIF greatly activates STAT4 phosphorylation on multiple SPXX elements within the C‐terminal transcription regulation domain. STAT4 and STAT3 act reciprocally on both canonical cis‐inducible elements (SIEs) and noncanonical “AGG” elements at different loci. In lamina propria lymphocytes (LPLs), STAT4 activation by LIF blocks STAT3‐dependent Il17a/Il17f promoter activation, whereas in IECs, LIF bypasses the extraordinarily low level of STAT4 to induce YAP gene expression via STAT3 activation. In addition, we found that the administration of LIF is sufficient to restore microbiome homeostasis. Thus, LIF effectively inhibits Th17 accumulation and promotes repair of damaged intestinal epithelium in inflamed colon, serves as a potential therapy for IBD.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. Significance: Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells. See related commentary by Wiley, p. 3193

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src–cell membrane association–dissociation and catalytic activation–inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation. Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825–38. ©2018 AACR.

Geminin expression is essential for embryonic development and the maintenance of chromosomal integrity. In spite of this protective role, geminin is also frequently overexpressed in human cancers and the molecular mechanisms underlying its role in tumor progression remain unclear. The histone deacetylase HDAC3 modulates transcription factors to activate or suppress transcription. Little is known about how HDAC3 specifies substrates for modulation among highly homologous transcription factor family members. Here, we have demonstrated that geminin selectively couples the transcription factor forkhead box O3 (FoxO3) to HDAC3, thereby specifically facilitating FoxO3 deacetylation. We determined that geminin-associated HDAC3 deacetylates FoxO3 to block its transcriptional activity, leading to downregulation of the downstream FoxO3 target Dicer, an RNase that suppresses metastasis. Breast cancer cells depleted of geminin or HDAC3 exhibited poor metastatic potential that was attributed to reduced suppression of the FoxO3-Dicer axis. Moreover, elevated levels of geminin, HDAC3, or both together with decreased FoxO3 acetylation and reduced Dicer expression were detected in aggressive human breast cancer specimens. These results underscore a prominent role for geminin in promoting breast cancer metastasis via the enzyme-substrate-coupling mechanism in HDAC3-FoxO3 complex formation.

Irisin, a newly identified myokine, is critical to modulating body metabolism and biological homeostasis. However, whether irisin protects the skeletal muscles against metabolic stresses remains unknown. In this study, we determine the effect of irisin on high glucose and fatty acid-induced damages using irisin-overexpressed mouse C2C12 (irisin-C2C12) myoblasts and skeletal muscle from irisin-injected mice. Compared with empty vector-transfected control C2C12 cells, irisin overexpression resulted in a marked increase in cell viability and decrease in apoptosis under high-glucose stress. Progression of the cell cycle into the G2/M phase in the proliferative condition was also observed with irisin overexpression. Furthermore, glucose uptake, glycogen accumulation, and phosphorylation of AMPKα/insulin receptor (IR) β-subunit/Erk1/2 in response to insulin stimulation were enhanced by irisin overexpression. In irisin-C2C12 myoblasts, these responses of phosphorylation were preserved under palmitate treatment, which induced insulin resistance in the control cells. These effects of irisin were reversed by inhibiting AMPK with compound C. In addition, high glucose-induced suppression of the mitochondrial membrane potential was also prevented by irisin. Moreover, suppression of IR in irisin-C2C12 myoblasts by cotransfection of shRNA against IR also mitigated the effects of irisin while not affecting AMPKα phosphorylation. As an in vivo study, soleus muscles from irisin-injected mice showed elevated phosphorylation of AMPKα and Erk1/2 and glycogen contents. Our results indicate that irisin counteracts the stresses generated by high glucose and fatty acid levels and irisin overexpression serves as a novel approach to elicit cellular protection. Furthermore, AMPK activation is a crucial factor that regulates insulin action as a downstream target.

Aging is characterized by a progressive loss of physiological integrity, while cancer represents one of the primary pathological factors that severely threaten human lifespan and healthspan. In clinical oncology, drug resistance limits the efficacy of most anticancer treatments, and identification of major mechanisms remains a key to solve this challenging issue. Here, we highlight the multifaceted senescence‐associated secretory phenotype (SASP), which comprises numerous soluble factors including amphiregulin (AREG). Production of AREG is triggered by DNA damage to stromal cells, which passively enter senescence in the tumor microenvironment (TME), a process that remarkably enhances cancer malignancy including acquired resistance mediated by EGFR. Furthermore, paracrine AREG induces programmed cell death 1 ligand (PD‐L1) expression in recipient cancer cells and creates an immunosuppressive TME via immune checkpoint activation against cytotoxic lymphocytes. Targeting AREG not only minimized chemoresistance of cancer cells, but also restored immunocompetency when combined with classical chemotherapy in humanized animals. Our study underscores the potential of in vivo SASP in driving the TME‐mediated drug resistance and shaping an immunosuppressive niche, and provides the proof of principle of targeting major SASP factors to improve therapeutic outcome in cancer medicine, the success of which can substantially reduce aging‐related morbidity and mortality.

Chromatin-associated fumarase (FH) affects histone methylation via its metabolic activity. However, whether this effect is involved in gene transcription remains to be clarified. In this study, we show that under glucose deprivation conditions, AMPK phosphorylates FH at Ser75, which in turn forms a complex with ATF2 and participates in promoter activation. FH-catalysed fumarate in promoter regions inhibits KDM2A demethylase activity, and thus maintains the H3K36me2 profile and facilitates gene expression for cell growth arrest. On the other hand, FH is found to be O-GlcNAcylated at the AMPK phosphorylation site; FH-ATF2-mediated downstream events are impeded by FH O-GlcNAcylation, especially in cancer cells that display robust O-GlcNAc transferase (OGT) activity. Consistently, the FH-Ser75 phosphorylation level inversely correlates with the OGT level and poor prognosis in pancreatic cancer patients. These findings uncover a previously uncharacterized mechanism underlying transcription regulation by FH and the linkage between dysregulated OGT activity and growth advantage of cancer cells under glucose deficiency.

Summary In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3 −/− mice, Stat3 is constitutively acetylated and naive CD4 + T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

Interferon-γ (IFN-γ) triggers macrophage for inflammation response by activating the intracellular JAK−STAT1 signaling. Suppressor of cytokine signaling 1 (SOCS1) and protein tyrosine phosphatases can negatively modulate IFN-γ signaling. Here, we identify a novel negative feedback loop mediated by STAT3−SOCS3, which is tightly controlled by SENP1 via de-SUMOylation of protein tyrosine phosphatase 1B (PTP1B), in IFN-γ signaling. SENP1-deficient macrophages show defects in IFN-γ signaling and M1 macrophage activation. PTP1B in SENP1-deficient macrophages is highly SUMOylated, which reduces PTP1B-induced de-phosphorylation of STAT3. Activated STAT3 then suppresses STAT1 activation via SOCS3 induction in SENP1-deficient macrophages. Accordingly, SENP1-deficient macrophages show reduced ability to resist Listeria monocytogenes infection. These results reveal a crucial role of SENP1-controlled STAT1 and STAT3 balance in macrophage polarization.

Background Histone deacetylases (HDACs) play a critical role in modulating myocardial protection and cardiomyocyte survivals. However, Specific HDAC isoforms in mediating myocardial ischemia/reperfusion injury remain currently unknown. We used cardiomyocyte-specific overexpression of active HDAC4 to determine the functional role of activated HDAC4 in regulating myocardial ischemia and reperfusion in isovolumetric perfused hearts. Methods In this study, we created myocyte-specific active HDAC4 transgenic mice to examine the functional role of active HDAC4 in mediating myocardial I/R injury. Ventricular function was determined in the isovolumetric heart, and infarct size was determined using tetrazolium chloride staining. Results Myocyte-specific overexpressing activated HDAC4 in mice promoted myocardial I/R injury, as indicated by the increases in infarct size and reduction of ventricular functional recovery following I/R injury. Notably, active HDAC4 overexpression led to an increase in LC-3 and active caspase 3 and decrease in SOD-1 in myocardium. Delivery of chemical HDAC inhibitor attenuated the detrimental effects of active HDAC4 on I/R injury, revealing the pivotal role of active HDAC4 in response to myocardial I/R injury. Conclusions Taken together, these findings are the first to define that activated HDAC4 as a crucial regulator for myocardial ischemia and reperfusion injury. Electronic supplementary material The online version of this article (10.1186/s10020-018-0037-2) contains supplementary material, which is available to authorized users.

Hyperuricemia is an independent risk factor for CKD and contributes to kidney fibrosis. In this study, we investigated the effect of EGF receptor (EGFR) inhibition on the development of hyperuricemic nephropathy (HN) and the mechanisms involved. In a rat model of HN induced by feeding a mixture of adenine and potassium oxonate, increased EGFR phosphorylation and severe glomerular sclerosis and renal interstitial fibrosis were evident, accompanied by renal dysfunction and increased urine microalbumin excretion. Administration of gefitinib, a highly selective EGFR inhibitor, prevented renal dysfunction, reduced urine microalbumin, and inhibited activation of renal interstitial fibroblasts and expression of extracellular proteins. Gefitinib treatment also inhibited hyperuricemia-induced activation of the TGF-β1 and NF-κB signaling pathways and expression of multiple profibrogenic cytokines/chemokines in the kidney. Furthermore, gefitinib treatment suppressed xanthine oxidase activity, which mediates uric acid production, and preserved expression of organic anion transporters 1 and 3, which promotes uric acid excretion in the kidney of hyperuricemic rats. Thus, blocking EGFR can attenuate development of HN via suppression of TGF-β1 signaling and inflammation and promotion of the molecular processes that reduce uric acid accumulation in the body.

Leukemia inhibitory factory (LIF), as a member of the IL-6 family, has been reported to ameliorate myocardial fibrosis and myocardial cell death. The purpose of the present study was to investigate the effect of LIF on renal fibrosis and its underlying mechanism. Our results showed, first, that LIF inhibited collagen type 1 and collagen type 3 expression induced by ANG II in NRK-49F (rat kidney fibroblast) cells and in mice with unilateral ureteral obstruction. Second, LIF induced Stat3 Tyr705phosphorylation and inhibited Stat3 Tyr705and Ser727phosphorylation induced by ANG II in NRK-49F cells. Third, LIF exerted an antirenal fibrosis effect mainly through activation of Stat3 Tyr705phosphorylation in NRK-49F cells. These effects of LIF were not observed in Stat3−/−cells. Finally, LIF-Stat3 upregulated microRNA-29c expression, and the latter downregulated collagen type 1 and collagen type 3 expression in NRK-49F cells and in mice with unilateral ureteral obstruction. In conclusion, LIF played a role in antirenal fibrosis by competitively activating Stat3 Tyr705phosphorylation, which upregulated microRNA-29c to suppress collagen expression.

Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator that catalyzes the trimethylation of H3K27 and is modulated by post-translational modifications (PTMs). However, the precise regulation of EZH2 PTMs remains elusive. We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1. We identified that PCAF interacts with and acetylates EZH2 mainly at lysine 348 (K348). Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2). Functionally, EZH2 K348 acetylation enhances its capacity in suppression of the target genes and promotes lung cancer cell migration and invasion. Further, elevated EZH2 K348 acetylation in lung adenocarcinoma patients predicts a poor prognosis. Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

Lysine acetylation is a post-translational modification known to regulate protein functions. Here we identify several acetylation sites of the influenza A virus nucleoprotein (NP), including the lysine residues K77, K113 and K229. Viral growth of mutant virus encoding K229R, mimicking a non-acetylated NP lysine residue, is severely impaired compared to wildtype or the mutant viruses encoding K77R or K113R. This attenuation is not the result of decreased polymerase activity, altered protein expression or disordered vRNP co-segregation but rather caused by impaired particle release. Interestingly, release deficiency is also observed mimicking constant acetylation at this site (K229Q), whereas virus encoding NP-K113Q could not be generated. However, mimicking NP hyper-acetylation at K77 and K229 severely diminishes viral polymerase activity, while mimicking NP hypo-acetylation at these sites has no effect on viral replication. These results suggest that NP acetylation at K77, K113 and K229 impacts multiple steps in viral replication of influenza A viruses., Post-translational modifications of influenza A virus proteins can regulate virus replication, but the effect of nucleoprotein (NP) acetylation is not known. Here, Giese et al. identify four NP lysine residues that are acetylated in infected cells and study their role in polymerase activity and virion release.

Bone metastasis is a frequent complication of breast cancer that is often accelerated by TGF-β signaling; however, little is known about how the TGF-β pathway is regulated during bone metastasis. Here we report that deleted in liver cancer 1 (DLC1) is an important regulator of TGF-β responses and osteolytic metastasis of breast cancer cells. In murine models, breast cancer cells lacking DLC1 expression exhibited enhanced capabilities of bone metastasis. Knockdown of DLC1 in cancer cells promoted bone metastasis, leading to manifested osteolysis and accelerated death in mice, while DLC1 overexpression suppressed bone metastasis. Activation of Rho-ROCK signaling in the absence of DLC1 mediated SMAD3 linker region phosphorylation and TGF-β–induced expression of parathyroid hormone–like hormone (PTHLH), leading to osteoclast maturation for osteolytic colonization. Furthermore, pharmacological inhibition of Rho-ROCK effectively reduced PTHLH production and breast cancer bone metastasis in vitro and in vivo. Evaluation of clinical breast tumor samples revealed that reduced DLC1 expression was linked to elevated PTHLH expression and organ-specific metastasis to bone. Overall, our findings define a stroma-dependent paradigm of Rho signaling in cancer and implicate Rho–TGF-β crosstalk in osteolytic bone metastasis.

Cytoplasmic STAT3, after activation by growth factors, translocates to different subcellular compartments, including nuclei and mitochondria, where it carries out different biological functions. However, the precise mechanism by which STAT3 undergoes mitochondrial translocation and subsequently regulates the tricarboxylic acid (TCA) cycle-electron transport chain (ETC) remains poorly understood. Here, we clarify this process by visualizing STAT3 acetylation in starved cells after serum reintroduction or insulin stimulation. CBP-acetylated STAT3 undergoes mitochondrial translocation in response to serum introduction or insulin stimulation. In mitochondria, STAT3 associates with the pyruvate dehydrogenase complex E1 (PDC-E1) and subsequently accelerates the conversion of pyruvate to acetyl-CoA, elevates the mitochondrial membrane potential, and promotes ATP synthesis. SIRT5 deacetylates STAT3, thereby inhibiting its function in mitochondrial pyruvate metabolism. In the A549 lung cancer cell line, constitutively acetylated STAT3 localizes to mitochondria, where it maintains the mitochondrial membrane potential and ATP synthesis in an active state.

Transforming growth factor beta (TGFβ) signaling in breast cancer is selectively associated with pulmonary metastasis. However, the underlying mechanisms remain unclear. Here we show that Bcl-3, a member of the IκB family, serves as a critical regulator in TGFβ signaling to modulate breast cancer pulmonary metastasis. Bcl-3 expression was significantly associated with metastasis-free survival in breast cancer patients. Bcl-3 deletion inhibited the migration and invasion of breast cancer cells in vitro, as well as breast cancer lung metastasis in vivo. Bcl-3 was required for the expression of downstream TGFβ signaling genes that are involved in breast cancer lung metastasis. Bcl-3 knockdown enhanced the degradation of Smad3 but not Smad2 following TGFβ treatment. Bcl-3 could bind to Smad3 and prevent the ubiquitination and degradation of Smad3 protein. These results indicate that Bcl-3 serves as a promising target to prevent breast tumor lung metastasis.

CCN family protein 2 (CCN2), also known as connective tissue growth factor, is a secreting protein that modulates multiple cellular events. We previously demonstrated the metastasis-suppressive effect of CCN2 in lung cancer cells. In this study, we investigate the role of CCN2 in anoikis, a form of programmed cell death that is critical in suppressing cancer metastasis. CCN2 binds to the epidermal growth factor receptor (EGFR) and triggers ubiquitination by inhibiting the formation of the β-pix/Cbl complex, resulting in the degradation of EGFR. Binding of CCN2 to EGFR suppresses the phosphorylation of c-Src and extracellular signal-regulated kinase but increases the expression of death-associated protein kinase, which leads to anoikis. Overall, our findings provide evidence validating the use of CCN2 as an anti-metastatic therapy in lung cancer patients, and prospect a potential therapeutic synergy between CCN2 and the anti-EGFR antibody for the treatment of lung cancer.

DNA polymerase η (Polη) implements translesion DNA synthesis but has low fidelity in replication. We have previously shown that Polη plays an important role in the genesis of nontargeted mutations at undamaged DNA sites in cells exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Here, we report that MNNG-induced Polη expression in an interferon regulatory factor 1 (IRF1)-dependent manner in human cells. Mutagenesis analysis showed that four critical residues (Arg-82, Cys-83, Asn-86, and Ser-87) located in the IRF family conserved DNA binding domain-helix α3 were involved in DNA binding and POLH transactivation by IRF1. Furthermore, Polη up-regulation induced by IRF1 was responsible for the increase of mutation frequency in a SupF shuttle plasmid replicated in the MNNG-exposed cells. Interestingly, IRF1 was acetylated by the histone acetyltransferase CBP in these cells. Lys → Arg substitution revealed that Lys-78 of helix α3 was the major acetylation site, and the IRF1-K78R mutation partially inhibited DNA binding and its transcriptional activity. Thus, we propose that IRF1 activation is responsible for MNNG-induced Polη up-regulation, which contributes to mutagenesis and ultimately carcinogenesis in cells.

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

Accumulation of both interstitial myofibroblasts and excessive production of extracellular matrix proteins is a common pathway contributing to chronic kidney disease. In a number of tissues, activation of STAT3 (signal transducer and activator of transcription 3) increases expression of multiple profibrotic genes. Here, we examined the effect of a STAT3 inhibitor, S3I-201, on activation of renal interstitial fibroblasts and progression of renal fibrosis. Treatment of cultured rat renal interstitial fibroblasts with S3I-201 inhibited their activation, as evidenced by dose- and time-dependent blockade of alpha-smooth muscle actin and fibronectin expression. In a mouse model of renal interstitial fibrosis induced by unilateral ureteral obstruction, STAT3 was activated, and administration of S3I-201 attenuated both this activation and extracellular matrix protein deposition following injury. S3I-201 reduced infiltration of the injured kidney by inflammatory cells and suppressed the injury-induced expression of fibronectin, alpha-smooth muscle actin, and collagen type-1 proteins, as well as the expression of multiple cytokines. Furthermore, S3I-201 inhibited proliferation and induced apoptosis preferentially in renal interstitial fibroblasts of the obstructed kidney. Thus, our results suggest that increased STAT3 activity mediates activation of renal interstitial fibroblasts and the progression of renal fibrosis. Inhibition of STAT3 signaling with S3I-201 may hold therapeutic potential for fibrotic kidney diseases.

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. In this study, we investigated the role of TGase-1 in cell survival and the survival signaling pathways regulated by TGase-1 in RPTC following oxidant injury. Exposure of RPTC to hydrogen peroxide (H2O2) resulted in apoptosis and an increase in TGase activity. Inhibition of TGase activity with monodansylcadervine (MDC), a TGase inhibitor, or knockdown of TGase-1 with small interference (si)RNA enhanced apoptosis and decreased cell survival in H2O2-treated RPTC. Conversely, overexpression of TGase-1 rendered RPTC more resistant to H2O2toxicity and MDC treatment blocked this response. Concurrent with RPTC apoptosis, phosphorylation of AKT, signal transducer and activator of transcription-3 (STAT3), and glucogen synthase kinase-3β (GSK-3β) were observed. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated H2O2-induced cell death and increased GSK-3β activity by dephosphorylation at serine 9. Furthermore, treatment with GSK-3β inhibitors reduced H2O2-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore, we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury.

We have shown that NADPH oxidase NOX5-S is overexpressed in Barrett's esophageal adenocarcinoma (EA) cells and may contribute to the progression from Barrett's esophagus (BE) to EA presumably by increasing cell proliferation and decreasing apoptosis (Fu X, Beer DG, Behar J, Wands J, Lambeth D, Cao W. J Biol Chem 281: 20368–20382, 2006). The mechanism(s) of NOX5-S overexpression in EA, however, is not fully understood. In SEG1 EA cells we found that acid treatment significantly increased platelet-activating factor (PAF) production, which in turn markedly increased NOX5-S expression and hydrogen peroxide (H2O2) production. Knockdown of NOX5-S by NOX5-S small interfering RNA (siRNA) blocked PAF-dependent H2O2production. PAF-dependent induction of NOX5-S expression and H2O2production were significantly decreased by the MAPK kinase 1 inhibitor PD-98059, by the cytosolic phospholipase A2(cPLA2) inhibitor AACOCF3, and by STAT5 downregulation with STAT5 siRNA. PAF significantly increased the phosphorylation of ERK1/2 MAPK, cPLA2, and STAT5. Using inhibitors, we demonstrated that PAF-induced STAT5 phosphorylation depends on activation of ERK1/2 MAPK and cPLA2, whereas PAF-induced cPLA2phosphorylation was associated with activation of ERK1/2 MAPK. Given that STAT5 bound to the c-sis-inducible element (TTCTGGTAA) of the NOX5-S promoter, overexpression of STAT5 significantly increased NOX5-S promoter activity. We conclude that acid-induced NOX5-S expression and H2O2production is mediated in part by production of PAF in SEG1 EA cells, and that PAF-induced increase in NOX5-S expression depends on sequential activation of ERK MAP kinases, cPLA2, and STAT5 in these cells.

Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.

Among the 22 fibroblast growth factors (FGFs), FGF21 has now emerged as a key metabolic regulator. However, the mechanism whereby FGF21 mediates its metabolic actions per se remains largely unknown. Here, we show that FGF21 represses mammalian target of rapamycin complex 1 (mTORC1) and improves insulin sensitivity and glycogen storage in a hepatocyte-autonomous manner. Administration of FGF21 in mice inhibits mTORC1 in the liver, whereas FGF21-deficient mice display pronounced insulin-stimulated mTORC1 activation and exacerbated hepatic insulin resistance (IR). FGF21 inhibits insulin- or nutrient-stimulated activation of mTORC1 to enhance phosphorylation of Akt in HepG2 cells at both normal and IR condition. TSC1 deficiency abrogates FGF21-mediated inhibition of mTORC1 and augmentation of insulin signaling and glycogen synthesis. Strikingly, hepatic βKlotho knockdown or hepatic hyperactivation of mTORC1/ribosomal protein S6 kinase 1 abrogates hepatic insulin-sensitizing and glycemic-control effects of FGF21 in diet-induced insulin-resistant mice. Moreover, FGF21 improves methionine- and choline-deficient diet-induced steatohepatitis. Conclusions: FGF21 acts as an inhibitor of mTORC1 to control hepatic insulin action and maintain glucose homeostasis, and mTORC1 inhibition by FGF21 has the therapeutic potential for treating IR and type 2 diabetes. (Hepatology 2016;64:425-438)

SummaryOrigin recognition complex 2 (ORC2), a subunit of the ORC, is essential for DNA replication initiation in eukaryotic cells. In addition to a role in DNA replication initiation at the G1/S phase, ORC2 has been shown to localize to the centromere during the G2/M phase. Here, we show that ORC2 is modified by small ubiquitin-like modifier 2 (SUMO2), but not SUMO1, at the G2/M phase of the cell cycle. SUMO2-modification of ORC2 is important for the recruitment of KDM5A in order to convert H3K4me3 to H3K4me2, a “permissive” histone marker for α-satellite transcription at the centromere. Persistent expression of SUMO-less ORC2 led to reduced α-satellite transcription and impaired pericentric heterochromatin silencing, which resulted in re-replication of heterochromatin DNA. DNA re-replication eventually activated the DNA damage response, causing the bypass of mitosis and the formation of polyploid cells. Thus, ORC2 sustains genomic stability by recruiting KDM5A to maintain centromere histone methylation in order to prevent DNA re-replication.

Stat3 and its isoforms belong to a family of cytoplasmic transcription factors that affect the synthesis of various proteins. Caspases are cysteinyl-aspartate proteases that function under apoptotic and non-apoptotic conditions. We now report that, in addition to transcriptional splicing, Stat3 fragmentation can be mediated by caspases. Caspase activation in DU145 cells was achieved by staurosporine (STS) exposure, and Western analysis revealed a reduction in full-length Stat3 (fl-Stat3) expression that was caspase-mediated. This proteolytic relationship was further studied by exposing purified Stat3 protein to a mixture of active caspases under cell-free conditions. This demonstrated that caspases directly cleaved Stat3 and Stat3 cleavage was accompanied by the apparent formation of cleavage fragment(s). Stat3 cleavage fragments, reflecting multiple caspase cleavage sites, also were observed in vitro following STS exposure in DU145 cells and in HEK293T cells transfected to express Stat3 truncation mutants. The impact of cleavage on Stat3 transcriptional activity next was assessed and revealed that cleavage of fl-Stat3 was accompanied by reductions in Stat3-DNA binding, Stat3-driven reporter protein (luciferase) activity, and the expression of selected Stat3-dependent genes. Further, reduced Stat3 expression correlated with increased sensitivity to apoptotic stimuli. In concomitant experiments, reporter activity was assessed in Stat3 truncation mutant-expressing HEK293T cells and revealed that, under non-apoptotic conditions, expression of different Stat3 fragments induced differential effects on Stat3-driven luciferase activity. These findings demonstrate that fl-Stat3 undergoes proteolytic processing by caspases that reduces its expression and leads to the formation of cleavage fragments that may modulate Stat3 transcriptional activity.

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.

The cytoplasmic region of human epidermal growth factor receptor (EGFR) contains an intrinsic tyrosine kinase (697-955) followed by a 231-residue-long COOH-terminal tail (C-tail), which contains multiple tyrosine residues. To examine the role of the EGFR C-tail in signal transducer and activator of transcription (STAT) activation, a series of EGFR C-tail truncations were constructed. Transient transfection of 293 cells with EGFR lacking the C-tail, i.e. Y974DeltaEGFR or Y992DeltaEGFR, led to EGF-independent or constitutive STAT activation, whereas EGF-dependent STAT activation was restored with truncations made COOH-terminal to the next tyrosine residue, i.e. EGFR-Y1045Delta. Transfection with the-truncated form EGFR-Y954Delta resulted in the loss of STAT activation, suggesting that the sequence between Tyr(974) and Tyr(954) is essential for STAT activation. Phosphopeptide competition analysis revealed multiple tyrosine residues within the C-tail that can act as the docking sites for both Stat1 and Stat3. A region that negatively regulated STAT activation was also identified, extending from Tyr(1114) to Glu(1172), consistent with the ability of this region to recruit a suppressor of cytokine signaling factors SOCS1 and SOCS3. When cotransfected with the full-length EGFR, but not Y992DeltaEGFR, SOCS1 or SOCS3 inhibited STAT activation by EGF in 293 cells. This suggests that both SOCS1 and SOCS3 can negatively regulate EGFR activation, presumably by inducing ubiquitination-dependent EGFR degradation upon ligand binding. These findings may therefore offer clues to how the EGF receptor C-tail regulates STAT activity.

Background GPRC5A is a retinoic acid inducible gene that is preferentially expressed in lung tissue. Gprc5a– knockout mice develop spontaneous lung cancer, indicating Gprc5a is a lung tumor suppressor gene. GPRC5A expression is frequently suppressed in majority of non-small cell lung cancers (NSCLCs), however, elevated GPRC5A is still observed in a small portion of NSCLC cell lines and tumors, suggesting that the tumor suppressive function of GPRC5A is inhibited in these tumors by an unknown mechanism. Methods In this study, we examined EGF receptor (EGFR)-mediated interaction and tyrosine phosphorylation of GPRC5A by immunoprecipitation (IP)-Westernblot. Tyrosine phosphorylation of GPRC5A by EGFR was systematically identified by site-directed mutagenesis. Cell proliferation, migration, and anchorage-independent growth of NSCLC cell lines stably transfected with wild-type GPRC5A and mutants defective in tyrosine phosphorylation were assayed. Immunohistochemical (IHC) staining analysis with specific antibodies was performed to measure the total and phosphorylated GPRC5A in both normal lung and lung tumor tissues. Result We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y350, at the C-terminal tail of GPRC5A. EGF induced phosphorylation of GPRC5A, which disrupted GPRC5A-mediated suppression on anchorage-independent growth of NSCLC cells. On contrary, GPRC5A-4 F, in which the four tyrosine residues have been replaced with phenylalanine, was resistant to EGF-induced phosphorylation and maintained tumor suppressive activities. Importantly, IHC analysis with anti-Y317/Y320-P sites showed that GPRC5A was non-phosphorylated in normal lung tissue whereas it was highly tyrosine-phosphorylated in NSCLC tissues. Conclusion GPRC5A can be inactivated by receptor tyrosine kinase via tyrosine phosphorylation. Thus, targeting EGFR can restore the tumor suppressive functions of GPRC5A in lung cancer.

GPRC5A is a G-protein–coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a−/− mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a−/− mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a−/− MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a−/− mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non–small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801–14. ©2015 AACR.

Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 4353–63. ©2014 AACR.

Background & Aims Constitutive activation of the transcription factors nuclear factor κB (NF-κB) and STAT3 is involved in the development and progression of human colorectal cancer (CRC). Little is known about how these factors become activated in cancer cells. We investigated whether microRNA miR-221 and miR-222 regulate NF-κB and signal transducer and activator of transcription 3 (STAT3) activation in human CRC cell lines. Methods CRC cell lines (HCT116 and RKO) were transfected with miR-221 or miR-222 mimics or inhibitors. The activity levels of NF-κB and STAT3 were measured in dual luciferase reporter assays. We used immunoblot and real-time polymerase chain reaction analyses to measure protein and messenger RNA (mRNA) levels. Cells were analyzed by proliferation, viability, and flow cytometry analyses. Mice were given injections of azoxymethane, followed by dextran sodium sulfate, along with control lentivirus or those expressing mRNAs that bind miR-221 and miR-222 (miR-221/miR-222 sponge). The levels of miR-221 and miR-222 as well as RelA , STAT3 , and PDLIM2 mRNAs were measured in 57 paired CRC and adjacent nontumor tissues from patients. Results In CRC cell lines, mimics of miR-221 and miR-222 activated NF-κB and STAT3, further increasing expression of miR-221 and miR-222. miR-221 and miR-222 bound directly to the coding region of RelA mRNA, increasing its stability. miR-221 and miR-222 also reduced the ubiquitination and degradation of the RelA and STAT3 proteins by binding to the 3′ untranslated region of PDLIM2 mRNA (PDLIM2 is a nuclear ubiquitin E3 ligase for RelA and STAT3). Incubation of CRC cells with miR-221 and miR-222 inhibitors reduced their proliferation and colony formation compared with control cells. In mice with colitis, injection of lentiviruses expressing miR-221/miR-222 sponges led to formation of fewer tumors than injection of control lentiviruses. Human CRC tissues had higher levels of miR-221 and miR-222 than nontumor colon tissues; increases correlated with increased levels of RelA and STAT3 mRNAs. Levels of PDLIM2 mRNA were lower in CRC than nontumor tissues. Conclusions In human CRC cells, miR-221 and miR-222 act in a positive feedback loop to increase expression levels of RelA and STAT3. Antagonism of miR-221 and miR-222 reduces growth of colon tumors in mice with colitis.

SummaryThe ubiquitin-proteasome and autophagy-lysosome systems are major proteolytic pathways, whereas function of the Ub-independent proteasome pathway is yet to be clarified. Here, we investigated roles of the Ub-independent REGγ-proteasome proteolytic system in regulating metabolism. We demonstrate that mice deficient for the proteasome activator REGγ exhibit dramatic autophagy induction and are protected against high-fat diet (HFD)-induced liver steatosis through autophagy. Molecularly, prevention of steatosis in the absence of REGγ entails elevated SirT1, a deacetylase regulating autophagy and metabolism. REGγ physically binds to SirT1, promotes its Ub-independent degradation, and inhibits its activity to deacetylate autophagy-related proteins, thereby inhibiting autophagy under normal conditions. Moreover, REGγ and SirT1 dissociate from each other through a phosphorylation-dependent mechanism under energy-deprived conditions, unleashing SirT1 to stimulate autophagy. These observations provide a function of the REGγ proteasome in autophagy and hepatosteatosis, underscoring mechanistically a crosstalk between the proteasome and autophagy degradation system in the regulation of lipid homeostasis.

Context: Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or multiple organ failure after blood transfusion. Objective: We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived plasma on neutrophil morphology, migration and phagocytosis. Materials and methods: We evaluated the production of reactive oxygen species (ROS) and the expression of non-muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils. Results: Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk) in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton protein involved in immune cell migration and morphological change, was also significantly upregulated in neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis induced by aged PRBC-derived plasma. Conclusion: Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.

Regulatory T (Treg) cells suppress autoimmune disease, and impaired Treg cell function is associated with rheumatoid arthritis. Here we demonstrate that forkhead box P3 (FOXP3) transcriptional activity and, consequently, Treg cell suppressive function are regulated by phosphorylation at Ser418 in the C-terminal DNA-binding domain. In rheumatoid arthritis-derived Treg cells, the Ser418 site was specifically dephosphorylated by protein phosphatase 1 (PP1), whose expression and enzymatic activity were induced in the inflamed synovium by tumor necrosis factor α (TNF-α), leading to impaired Treg cell function. Moreover, TNF-α-induced Treg cell dysfunction correlated with increased numbers of interleukin-17 (IL-17)(+) and interferon-γ (IFN-γ)(+)CD4(+) T cells within the inflamed synovium in rheumatoid arthritis. Treatment with a TNF-α-specific antibody restored Treg cell function in subjects with rheumatoid arthritis, which was associated with decreased PP1 expression and increased FOXP3 phosphorylation in Treg cells. Thus, TNF-α controls the balance between Treg cells and pathogenic TH17 and TH1 cells in the synovium of individuals with rheumatoid arthritis through FOXP3 dephosphorylation.

Thrombin, a G protein-coupled receptor agonist, induced a biphasic expression of cyclin D1 in primary vascular smooth muscle cells. Although both phases of cyclin D1 expression require binding of the newly identified cooperative complex, NFATc1·STAT-3, to its promoter, the second phase, which is more robust, depends on NFATc1-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT-3. In addition, STAT-3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires NFATc1-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT-3 by overexpression of acetylation null STAT-3 mutant led to the loss of the late phase of cyclin D1 expression. EMSA analysis and reporter gene assays revealed that NFATc1·STAT-3 complex binding to the cyclin D1 promoter led to an enhanceosome formation and facilitated cyclin D1 expression. In the early phase of its expression, cyclin D1 is localized mostly in the cytoplasm and influenced cell migration. However, during the late and robust phase of its expression, cyclin D1 is translocated to the nucleus and directed cell proliferation. Together, these results demonstrate for the first time that the dual function of cyclin D1 in cell migration and proliferation is temperospatially separated by its biphasic expression, which is mediated by cooperative interactions between NFATc1 and STAT-3.

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1–TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase β plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.

Free-fatty acids (FFAs) are well-characterized factor for causing production of inflammatory factors and insulin resistance in adipocytes. Using cultured adipocytes, we demonstrate that FFAs can activate endoplasmic reticulum (ER) stress pathway by examination of ER stress sensor activation and marker gene expression. Chemical chaperone tauroursodeoxycholic acid (TUDCA) can reduce FFA-induced adipocyte inflammation and improve insulin signaling whereas overexpression of spliced X-box protein 1 (XBP-1s) only attenuates FFA-induced inflammation. PKR-like eukaryotic initiation factor 2α kinase (PERK) is one of the three major ER stress sensor proteins and deficiency of PERK alleviates FFA-induced inflammation and insulin resistance. The key downstream target of FFA-induced ER stress is IκB kinase β (IKKβ), a master kinase for regulating expression of inflammatory genes. Deficiency of PERK attenuates FFA-induced activation of IKKβ and deficiency of IKKβ alleviates FFA-induced inflammation and insulin resistance. Consistently, overexpression of IKKβ in 3T3-L1 CAR adipocytes causes inflammation and insulin resistance. In addition, IKKβ overexpression has profound effect on adipocyte lipid metabolism, including inhibition of lipogenesis and promotion of lipolysis. Furthermore, increased endogenous IKKβ expression and activation is also observed in isolated primary adipocytes from mice injected with lipids or fed on high-fat diet (HFD) acutely. These results indicate that ER stress pathway is a key mediator for FFA-induced inflammation and insulin resistance in adipocytes with PERK and IKKβ as the critical signaling components.

To maintain intracellular redox homeostasis, genes encoding many antioxidants and detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis antioxidant responsive elements (AREs) in their promoter regions. Nrf2 is the critical transcription factor responsible for ARE-dependent transcription. We and others have previously demonstrated that Nrf2 is targeted for ubiquitin-mediated degradation by Keap1 in a redox-sensitive manner through modifications of distinct cysteine residues of Keap1. Here, we report that p300/CBP directly acetylates Nrf2 in response to arsenite-induced stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.

Protein tyrosine phosphorylation plays a central role in cell-signaling and is a focus of biomedical studies and cancer therapy. However, it is still challenging to identify or characterize the coordinated changes of many candidate proteins of one particular pathway or multiple pathways simultaneously. Antibody array is a recently developed approach applied for differential analysis of multiple protein posttranslational modification events in mammalian cells. It is based on the highly specific recognition between the immobilized antibodies on the array and their specific target proteins in a high-throughput screening format. Here we have described in detail two methods for differential analysis of protein tyrosine phosphorylation in cells by (1) using a single fluorescent protein capture format on membrane array and (2) a competitive protein capture method on glass surface array.

Precise spatial and temporal regulation of cell adhesion and de-adhesion is critical for dynamic lymphocyte migration. Although a great deal of information has been learned about integrin lymphocyte function–associated antigen (LFA)-1 adhesion, the mechanism that regulates efficient LFA-1 de-adhesion from intercellular adhesion molecule (ICAM)-1 during T lymphocyte migration is unknown. Here, we show that nonmuscle myosin heavy chain IIA (MyH9) is recruited to LFA-1 at the uropod of migrating T lymphocytes, and inhibition of the association of MyH9 with LFA-1 results in extreme uropod elongation, defective tail detachment, and decreased lymphocyte migration on ICAM-1, without affecting LFA-1 activation by chemokine CXCL-12. This defect was reversed by a small molecule antagonist that inhibits both LFA-1 affinity and avidity regulation, but not by an antagonist that inhibits only affinity regulation. Total internal reflection fluorescence microscopy of the contact zone between migrating T lymphocytes and ICAM-1 substrate revealed that inactive LFA-1 is selectively localized to the posterior of polarized T lymphocytes, whereas active LFA-1 is localized to their anterior. Thus, during T lymphocyte migration, uropodal adhesion depends on LFA-1 avidity, where MyH9 serves as a key mechanical link between LFA-1 and the cytoskeleton that is critical for LFA-1 de-adhesion.

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains a major obstacle in the successful treatment of cancer patients [1, 2]. In this regard, one of the major determinants of apoptosis sensitivity in cancer cells is the transcription factor nuclear factor kappa B (NFκB). Constitutive expression of NF-κB has been implicated in decreasing apoptosis in cancer cell lines [3]. NF-κB is an inducible transcription factor required for the upregulation of a large number of genes in response to inflammation, viral and bacterial infection, cell survival, cell adhesion, inflammation, differentiation, growth, and stress stimuli [4, 5]. Genes that are responsive to NF-κB activation include a variety of cytokines, cell adhesion molecules, acute phase response proteins, and apoptotic and anti-apoptotic proteins. It is believed that this reprogramming of gene expression is essential for cell survival during physiologic crisis situations. Active NF-κB is a dimer comprised of various members of the Rel family of proteins, and some form of NF-κB is expressed in most cell types. In unstimulated cells NF-κB is retained in the cytoplasm in an inactive form bound to a family of inhibitory proteins known as IκB (inhibitor of κB). Activation of NF-κB requires the phosphorylation and degradation of I-κB, which allows the NF-κB dimer to translocate to the nucleus [6, 7]. NF-κB can be activated by several signaling cascades and is subject to multiple levels of regulation. Considerable progress has been made in the identification of kinases that phosphorylate IκB and target it for subsequent degradation. In addition to its critical role in re-programming gene expression in response to infection and other stresses, NF-κB also mediates cell survival signals, protecting cells from apoptosis [8–10]. For example, cells derived from NF-κB p65 knockout mice are significantly more sensitive to TNFαinduced cytotoxicity than normal cells. Specificity to NF-κB was demonstrated by transfecting p65 into the knockout cells that reversed the cytotoxicity [11, 12]. Similar effects were also observed when NF-κB activation was ablated with an IκB dominantnegative mutant. Cells with compromised NF-κB activation are also more vulnerable to other proapoptotic signals such as ionizing radiation and cancer chemotherapeutic agents [13]. NF-κB has also been implicated in downregulating apoptosis in cncer cell lines that constitutively express elevated NF-κB activity by the observation that ablation of NF-κB activity by a variety of means induced apoptosis [3]. Many of the target genes that are activated by NF-κB are critical to the