Novel Pathway For Regulation Research Articles

Microbial Indole Metabolites Provide a Novel Pathway for Regulation of Intestinal Homeostasis

Inflammatory bowel disease (IBD) is a multifactorial chronic condition that result in numerous perturbations in the gastrointestinal mucosa. IBD is characterized by the continual breakdown of the intestinal epithelial barrier leading to an inappropriate immune response towards intestinal microbiota. This exposure promotes inflammation and continued mucosal injury, though the pathogenesis of the disease remains unknown. There is currently significant interest in understanding the role of gut microbiota in IBD. Loss of commensal bacteria and their metabolites may play a role in homeostasis of the intestinal epithelia. Microbes benefit the host through the local synthesis of numerous metabolites. One such metabolite is indole. Indoles are gut microbiota‐derived tryptophan metabolites that are abundant in the healthy mammalian gut and positively influence intestinal health. Using an unbiased metabolomics profiling approach, we identified a selective reduction in microbiota‐derived indole metabolites in active murine colitis. We have developed HPLC‐based technologies to validate these results and quantify indole and indole‐derived metabolites within murine and human colitis samples. We show that exposure of intestinal epithelial cells to indole metabolites induces genes important in formation and maintenance of intestinal barrier. For instance, the anti‐inflammatory cytokine IL‐10 functions through binding to the IL‐10 receptor alpha subunit (IL‐10R1), and colitis is strongly associated with the induction of the epithelial IL‐10R1. Our results have revealed a prominent induction of IL‐10R1 mRNA and protein expression following treatment of intestinal epithelial cells with indole metabolites. We also show a potential role for selective indole metabolites, particularly indole‐3‐propionic acid (IPA), to alleviate disease severity and decrease inflammatory mediators in models of murine colitis. Together, these data suggest that microbial‐derived indoles play a central role in mucosal homeostasis. Based on these findings and ongoing studies, we show that microbial‐derived indole metabolites inhibit inflammation‐induced damage and promote mucosal homeostasis. These studies elucidate a novel role for microbial metabolites in innate immune responses during intestinal inflammation using in vitro and in vivo models. This work will provide new insight and identify possible avenues to utilize indoles as therapeutic approaches for treating IBD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

N6-methyladenosine contributes to cellular phenotype in a genetically-defined model of breast cancer progression.

The mRNA modification N6-methyladenosine (m6A) is involved in many post-transcriptional regulatory processes including mRNA stability and translational efficiency. However, it is also imperative to correlate these processes with phenotypic outputs during cancer progression. Here we report that m6A levels are significantly decreased in genetically-defined immortalized and oncogenically-transformed human mammary epithelial cells (HMECs), as compared with their primary cell predecessor. Furthermore, the m6A methyltransferase (METTL3) is decreased and the demethylase (ALKBH5) is increased in the immortalized and transformed cell lines, providing a possible mechanism for this basal change in m6A levels. Although the immortalized and transformed cells showed lower m6A levels than their primary parental cell line, overexpression of METTL3 and METTL14, or ALKBH5 knockdown to increase m6A levels in transformed cells increased proliferation and migration. Remarkably, these treatments had little effect on the immortalized cells. Together, these results suggest that m6A modification may be downregulated in immortalized cells as a brake against malignant progression. Finally, we found that m6A levels in the immortalized and transformed cells increased in response to hypoxia without corresponding changes in METTL3, METTL14 or ALKBH5 expression, suggesting a novel pathway for regulation of m6A levels under stress.

Sequences enriched in Alu repeats drive nuclear localization of long RNAs in human cells.

Long noncoding RNAs (lncRNAs) are emerging as key players in multiple cellular pathways1, but their modes of action, and how those are dictated by sequence remain elusive. lncRNAs tend to be enriched in the nuclear fraction, whereas most mRNAs are overtly cytoplasmic2, although several studies have found that hundreds of mRNAs in various cell types are retained in the nucleus3,4. It is thus conceivable that some mechanisms that promote nuclear enrichment are shared between lncRNAs and mRNAs. In order to identify elements that can force nuclear localization in lncRNAs and mRNAs we screened libraries of short fragments tiled across nuclear RNAs, which were cloned into the untranslated regions of an efficiently exported mRNA. The screen identified a short sequence derived from Alu elements and bound by HNRNPK that increases nuclear accumulation. We report that HNRNPK binding to C-rich motifs outside Alu elements is also associated with nuclear enrichment in both lncRNAs and mRNAs, and this mechanism is conserved across species. Our results thus detail a novel pathway for regulation of RNA accumulation and subcellular localization that has been co-opted to regulate the fate of transcripts that integrated Alu elements.

EGF is highly expressed in hepatocellular carcinoma (HCC) and promotes motility of HCC cells via fibronectin.

Better understanding of metastasis process would allow for the development of effective approaches to treat hepatocellular carcinoma (HCC). Recent literature has highlighted the fundamental role of interaction between tumor cells and their microenvironment components in tumor metastasis. Aberrant expression of epidermal growth factor (EGF) induces highly malignant HCC, and activated EGF/EGFR signaling is correlated with an aggressive phenotype and intrahepatic metastasis. Thus, EGF in the tumor microenvironment may influence the behavior of HCC cells. In this study, for the first time, we studied the expression of EGF in HCCs, and the potential role of EGF in the motility of HCC cells and the underlying mechanisms. It was demonstrated that EGF was highly expressed in HCCs and positively associated with higher tumor grade. In addition, EGF promoted the migration and invasion of HCC cells mainly via induction of fibronectin (FN) in vitro. Mechanistically, EGF simultaneously increased the nuclear translocation and PKC mediated phosphorylation of p65 which could bind to the -356 bp to -259 bp fragment of FN promoter, leading to a markedly increased activity of FN promoter in HCC cells. These results highlight the potential role of EGF in promoting HCC metastasis, demonstrate a novel pathway for regulation of FN expression and provide potential targets for HCC prevention and treatment.

Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes

Kidney disease has been linked to dysregulated signaling via PKC in kidney cells such as podocytes. PKCα is a conventional isoform of PKC and a well-known binding partner of β-catenin, which promotes its degradation. β-Catenin is the main effector of the canonical Wnt pathway and is critical in cell adhesion. However, whether other PKC isoforms interact with β-catenin has not been studied systematically. Here we demonstrate that PKCϵ-deficient mice, which develop proteinuria and glomerulosclerosis, display lower β-catenin expression compared with PKC wild-type mice, consistent with an altered phenotype of podocytes in culture. Remarkably, β-catenin showed a reversed subcellular localization pattern: Although β-catenin exhibited a perinuclear pattern in undifferentiated wild-type cells, it predominantly localized to the nucleus in PKCϵ knockout cells. Phorbol 12-myristate 13-acetate stimulation of both cell types revealed that PKCϵ positively regulates β-catenin expression and stabilization in a glycogen synthase kinase 3β-independent manner. Further, β-catenin overexpression in PKCϵ-deficient podocytes could restore the wild-type phenotype, similar to rescue with a PKCϵ construct. This effect was mediated by up-regulation of P-cadherin and the β-catenin downstream target fascin1. Zebrafish studies indicated three PKCϵ-specific phosphorylation sites in β-catenin that are required for full β-catenin function. Co-immunoprecipitation and pulldown assays confirmed PKCϵ and β-catenin as binding partners and revealed that ablation of the three PKCϵ phosphorylation sites weakens their interaction. In summary, we identified a novel pathway for regulation of β-catenin levels and define PKCϵ as an important β-catenin interaction partner and signaling opponent of other PKC isoforms in podocytes.

Microbiota‐Derived Tryptophan Metabolites Provide a Novel Pathway for Regulation of Mucosal Barrier Function

The majority of microbial species within the human body contribute an important role to human health. Mucosal surfaces act as a primary barrier against microbial toxins and invaders, while commensal bacteria work in a dynamic and intimate interaction with the epithelium to influence host cellular and immune responses. Indole, a tryptophan metabolite produced by anaerobic bacteria, is known for its intercellular signal activity and serves as an aryl hydrocarbon receptor (AHR) ligand. AHR is a ligand‐dependent transcription factor activated by a variety of synthetic and biological molecules. The physiological function of AHR includes roles in immune regulation and mucosal barrier function. Further, IL‐10 is an anti‐inflammatory cytokine that inhibits production of numerous pro‐inflammatory mediators in various cell types. This cytokine functions through binding to the IL‐10 receptor alpha subunit (IL‐10R1). An unbiased metabolomic profile of serum from healthy and colitic mice identified significant decreases in several microbial‐derived indole derivatives (e.g. indoleacetate, indolepropionate, 5‐OH‐indoleacetate). Given that colitis is strongly associated with the induction of the epithelial IL‐10R1, we hypothesized that indole‐containing tryptophan metabolites regulate epithelial IL‐10R1 expression. Human intestinal epithelial cells were exposed to indole derivatives and examined for expression of IL‐10R1. These studies revealed a prominent induction of IL‐10R1 mRNA and protein expression following treatment with indole derivatives. Such results were explained, at least in part, by the activation of AHR. Indeed, IL‐10R1 expression was abolished when indole derivatives were administered to AHR nuclear translocator (ARNT) knockdown cells. Likewise, the AHR inhibitor, CH‐223191, significantly reduced IL‐10R1 expression in cells exposed to indole derivatives. Furthermore, ongoing work has shown a potential role for indole metabolites in alleviating disease severity in a murine model of colitis. Together, these results provide strong evidence for a novel role of tryptophan‐indole derivatives in anti‐inflammatory pathways mediated by IL‐10 signaling in intestinal epithelia. The discovery of indole metabolites as AHR ligands provides new insight to our understanding of the host‐microbial axis during inflammatory processes. These studies also identify possible avenues for incorporating the indole‐AHR pathway as novel therapies for mucosal disease.

G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involved in several stages of the cell cycle, including the entry and exit from mitosis, and cytokinesis. Furthermore, it has an essential role in the regulation of DNA replication. Together with cyclin A, PLK1 also promotes CDH1 phosphorylation to trigger its ubiquitination and degradation, allowing cell cycle progression. The PLK1 levels in different type of tumors are very high compared to normal tissues, which is consistent with its role in promoting proliferation. Therefore, several PLK1 inhibitors have been developed and tested for the treatment of cancer. Here, we further analyzed PLK1 degradation and found that cytoplasmic PLK1 is ubiquitinated and subsequently degraded by the SCFβTrCP/proteasome. This procedure is triggered when heat shock protein (HSP) 90 is inhibited with geldanamycin, which results in misfolding of PLK1. We also identified CDK1 as the major kinase involved in this degradation. Our work shows for the first time that HSP90 inhibition arrests cell cycle progression at the G1/S transition. This novel mechanism inhibits CDH1 degradation through CDK1-dependent PLK1 destruction by the SCFβTrCP/proteasome. In these conditions, CDH1 substrates do not accumulate and cell cycle arrests, providing a novel pathway for regulation of the cell cycle at the G1-to-S boundary.-Giráldez, S., Galindo-Moreno, M., Limón-Mortés, M. C., Rivas, A. C., Herrero-Ruiz, J., Mora-Santos, M., Sáez, C., Japón, M. Á., Tortolero, M., Romero, F. G1/S phase progression is regulated by PLK1 degradation through the CDK1/βTrCP axis.

Brief Report: Stress‐Inducible Nuclear Protein 1 Regulates Matrix Metalloproteinase 13 Expression in Human Articular Chondrocytes

ObjectiveNuclear protein 1 (Nupr1) is a stress‐inducible protein that is involved in gene transcription. The present study was undertaken to determine whether chondrocytes express Nupr1 and whether Nupr1 regulates matrix metalloproteinase 13 (MMP‐13) expression.MethodsParaffin‐embedded cartilage sections from normal human and osteoarthritic (OA) cartilage were immunostained using anti‐Nupr1 antibody. To measure Nupr1 expression, total RNA was isolated from joint tissue obtained 8 weeks after surgery from young (12‐week‐old) and older (12‐month‐old) mice that underwent destabilization of the medial meniscus (DMM) to induce OA. Human chondrocytes were stimulated with 1–10 ng/ml interleukin‐1β (IL‐1β), 25 μM tert‐butyl‐hydroperoxide (tBHP), or 2 μM thapsigargin, and Nupr1 expression was analyzed by quantitative polymerase chain reaction. In addition, chondrocytes were transfected with small interfering RNA to knock down Nupr1 expression and then stimulated overnight with IL‐1β. After incubation, the conditioned medium was collected and MMP levels measured.ResultsIncreased Nupr1 immunostaining was noted in human OA cartilage compared to normal cartilage. Expression was also increased in joint tissue from 12‐month‐old mice that underwent DMM surgery compared to sham‐operated controls. Stimulation of chondrocytes with IL‐1β induced a 2‐fold increase in Nupr1 messenger RNA (mRNA) within 1 hour, with the increase peaking to 4‐fold at 6 hours. Treatment of chondrocytes with tBHP to induce oxidative stress increased Nupr1 mRNA expression by >2‐fold; treatment with thapsigargin to induce endoplasmic reticulum stress did not produce a similar effect. Knockdown of Nupr1 inhibited IL‐1β–mediated induction of MMP‐13.ConclusionNupr1 is expressed in cartilage, and its levels are increased in OA. Nupr1 expression is required for IL‐1β–mediated expression of MMP‐13. These findings provide evidence of a novel pathway for regulation of IL‐1β–mediated production of MMPs in chondrocytes.

A Novel Pathway for Regulation of Insulin Secretion by Fractalkine and CX3CR1 System (LB772)

Fractalkine (FKN) (CX3CL1) and its receptor CX3CR1 mediate cell‐to‐cell interactions in different tissues. Here, we demonstrate that the FKN/CX3CR1 system represents a novel regulatory mechanism for pancreatic islet beta cell function and insulin secretion. CX3CR1 KO mice exhibit glucose intolerance with normal insulin sensitivity, due to a marked beta cell defect in glucose and GLP1‐stimulated insulin secretion. The defect in insulin secretion was also observed in vitro in isolated islets from CX3CR1 KO mice. In vivo administration of FKN improved glucose tolerance with an increase in insulin secretion. In vitro treatment of islets with FKN increased intracellular Ca2+ level and potentiated insulin secretion. The KO islets exhibited reduced expression of a set of genes which are necessary for the fully functional, differentiated beta cell state, whereas, treatment of WT islets with FKN leads to increased expression of these genes. Lastly, expression of FKN in islets was decreased by aging and HFD/obesity, suggesting that decreased fractalkine/CX3CR1 signaling could be a mechanism underlying beta cell dysfunction in type 2 diabetes.

Control of Outflow Resistance by Soluble Adenylyl Cyclase

Abstract Glaucoma is a leading cause of blindness in the United States affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure, even in patients with normal tension glaucoma. Typically, this is accomplished by reducing the rate of aqueous flow by limiting aqueous production or enhancing drainage using drugs and surgery. Whereas these strategies are effective in diminishing vision loss, some patients continue to lose vision and many discontinue use of their medications because of undesirable side effects. Drugs known to be effective in altering conventional outflow have for the most part been abandoned from modern clinical practice due to undesirable side effects. Identification of new drugs that could enhance conventional outflow, would offer additional options in the treatment of glaucoma and ocular hypertension. To this end, our laboratory has recently uncovered a novel pathway for regulation of conventional outflow by the ciliary body. This pathway is dependent on soluble adenylyl cyclase, an enzyme that catalyzes the generation of cyclic adenosine 3',5' monophosphate (cAMP) in response to bicarbonate.

Abstract 2969: A Novel pathway for regulation of ANXA2 expression in lung cancer cell metastasis by SETDB1/SMAD3 repressor complex.

Abstract Aberrant histone methylation contributes to tumor development and progression by histone methyltransferase. SETDB1 is an H3K9 methyltransferase that contributes to the epigenetic silencing of target genes in cancer cells, but the role of SETDB1 in the progression of tumorigenesis remains largely unknown. We show here that H3K9Me2 and SETDB1 are down-regulated in metastatic lung cancer cell lines. Immunohistochemistry examination of the lung carcinoma tissue microarray revealed that expression of SETDB1 is significant decreased in metastasis status. Ectopic expression of SETDB1 in highly invasive lung cancer cells suppressed cancer cell migration, invasion and filopodia formation. In contrast, knockdown of endogenous SETDB1 in non-invasive lung cancer cells by RNA interference increases cell migration and invasion in vitro. Mechanistic investigations suggested that SETDB1 is specifically down-regulated by miR-29b in metastatic lung cancer cells. Ectopic miR-29b expression in non-invasive lung cancer cells increases cell invasion ability. Furthermore, we also establish a causal role for the SETDB1/SMAD3 complex in driving metastasis and identify ANXA2 as a critical target in this process among numerous target genes. These findings suggest a novel pathway regulated by SETDB1/SMAD3 complex in lung cancer metastasis. Citation Format: Tsai-Yu Tzeng, Ming-Ta Hsu. A Novel pathway for regulation of ANXA2 expression in lung cancer cell metastasis by SETDB1/SMAD3 repressor complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2013-2969

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca(2+) binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33-45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430-435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969-1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca(2+) saturation. A simple theory was developed to account for this fact.

Relaxed Skeletal Muscle Crossbridges Containing Dephosphorylated Regulatory Light Chain are Better Oriented Than Phosphorylated Ones

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca2+ binding, such as phosphorylation of myosin RLC also controls contraction of vertebrate striated muscle [1]. Electron Paramagnetic Resonance (EPR) studies using nucleotide-analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC [2]. The slower ATPase activity in the dephosphorylated moiety has been defined as a new super-relaxed state (SRX) [1]. It can be observed in both skeletal [3] and cardiac muscle fibers [4]. Given the importance of the SRX which suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (1) relaxed cross-bridges are statistically better ordered in muscle where the RLC is dephosphorylated compared to phosphorylated RLC; (2) during relaxation, phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (3) during isometric contraction, phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones.1. Cooke, R. Biophys Rev, 2011. 3(1): p. 33-45.2. Naber et al. Biophysical Soc 55-th Annual Meeting, 2011: p. 26.3. Stewart, M.A., et al. Proc Natl Acad Sci USA, 2010. 107(1): p. 430-5.4. Hooijman, et al, Biophys J, 2011. 100(8): p. 1969-76.

Thermogenic Activation Induces FGF21 Expression and Release in Brown Adipose Tissue

FGF21 is a novel metabolic regulator involved in the control of glucose homeostasis, insulin sensitivity, and ketogenesis. The liver has been considered the main site of production and release of FGF21 into the blood. Here, we show that, after thermogenic activation, brown adipose tissue becomes a source of systemic FGF21. This is due to a powerful cAMP-mediated pathway of regulation of FGF21 gene transcription. Norepinephrine, acting via β-adrenergic, cAMP-mediated, mechanisms and subsequent activation of protein kinase A and p38 MAPK, induces FGF21 gene transcription and also FGF21 release in brown adipocytes. ATF2 binding to the FGF21 gene promoter mediates cAMP-dependent induction of FGF21 gene transcription. FGF21 release by brown fat in vivo was assessed directly by analyzing arteriovenous differences in FGF21 concentration across interscapular brown fat, in combination with blood flow to brown adipose tissue and assessment of FGF21 half-life. This analysis demonstrates that exposure of rats to cold induced a marked release of FGF21 by brown fat in vivo, in association with a reduction in systemic FGF21 half-life. The present findings lead to the recognition of a novel pathway of regulation the FGF21 gene and an endocrine role of brown fat, as a source of FGF21 that may be especially relevant in conditions of activation of thermogenic activity.

Adipocyte expression of the glucose-dependent insulinotropic polypeptide receptor involves gene regulation by PPARγ and histone acetylation

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that exerts insulinotropic and growth and survival effects on pancreatic β-cells. Additionally, there is increasing evidence supporting an important role for GIP in the regulation of adipocyte metabolism. In the current study we examined the molecular mechanisms involved in the regulation of GIP receptor (GIPR) expression in 3T3-L1 cells. GIP acted synergistically with insulin to increase neutral lipid accumulation during progression of 3T3-L1 preadipocytes to the adipocyte phenotype. Both GIPR protein and mRNA expression increased during 3T3-L1 cell differentiation, and this increase was associated with upregulation of nuclear levels of sterol response element binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor γ (PPARγ), as well as acetylation of histones H3/H4. The PPARγ receptor agonists LY171883 and rosiglitazone increased GIPR expression in differentiated 3T3-L1 adipocytes, whereas the antagonist GW9662 ablated expression. Additionally, both PPARγ and acetylated histones H3/H4 were shown to bind to a region of the GIPR promoter containing the peroxisome proliferator response element (PPRE). Knockdown of PPARγ in differentiated 3T3-L1 adipocytes, using RNA interference, reduced GIPR expression, supporting a functional regulatory role. Taken together, these studies show that GIP and insulin act in a synergistic manner on 3T3-L1 cell development and that adipocyte GIPR expression is upregulated through a mechanism involving interactions between PPARγ and a GIPR promoter region containing an acetylated histone region.

C-Jun N-terminal kinase/c-Jun inhibits fibroblast proliferation by negatively regulating the levels of stathmin/oncoprotein 18

The JNKs (c-Jun N-terminal kinases) are stress-activated serine/threonine kinases that can regulate both cell death and cell proliferation. We have developed a cell system to control JNK re-expression at physiological levels in JNK1/2-null MEFs (murine embryonic fibroblasts). JNK re-expression restored basal and stress-activated phosphorylation of the c-Jun transcription factor and attenuated cellular proliferation with increased cells in G1/S-phase of the cell cycle. To explore JNK actions to regulate cell proliferation, we evaluated a role for the cytosolic protein, STMN (stathmin)/Op18 (oncoprotein 18). STMN, up-regulated in a range of cancer types, plays a crucial role in the control of cell division through its regulation of microtubule dynamics of the mitotic spindle. In JNK1/2-null or c-Jun-null MEFs or cells treated with c-Jun siRNA (small interfering RNA), STMN levels were significantly increased. Furthermore, a requirement for JNK/cJun signalling was demonstrated by expression of wild-type c-Jun, but not a phosphorylation-defective c-Jun mutant, being sufficient to down-regulate STMN. Critically, shRNA (small hairpin RNA)-directed STMN down-regulation in JNK1/2-null MEFs attenuated proliferation. Thus JNK/c-Jun regulation of STMN levels provides a novel pathway in regulation of cell proliferation with important implications for understanding the actions of JNK as a physiological regulator of the cell cycle and tumour suppressor protein.

The Human COP9 Signalosome Protects Ubiquitin-conjugating Enzyme 3 (UBC3/Cdc34) from β-Transducin Repeat-containing Protein (βTrCP)-mediated Degradation

The COP9 signalosome (CSN) is an essential multisubunit complex that regulates the activity of cullin-RING ubiquitin ligases by removing the ubiquitin-like peptide NEDD8 from cullins. Here, we demonstrate that the CSN can affect other components of the ubiquitination cascade. Down-regulation of human CSN4 or CSN5 induced proteasome-mediated degradation of the ubiquitin-conjugating enzyme UBC3/Cdc34. UBC3 was targeted for ubiquitination by the cullin-RING ubiquitin ligase SCF(betaTrCP). This interaction required the acidic C-terminal extension of UBC3, which is absent in ubiquitin-conjugating enzymes of the UBCH5 family. Conversely, the UBC3 acidic domain was sufficient to impart sensitivity to SCF(betaTrCP)-mediated ubiquitination to UBCH5 enzymes. Our work indicates that the CSN is necessary to ensure the stability of selected ubiquitin-conjugating enzymes and uncovers a novel pathway of regulation of ubiquitination processes.

Fibroblast growth factor-inducible 14 (Fn14) is expressed in the lower genital tract and may play a role in amplifying inflammation during infection

TNF-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) cytokine superfamily which regulates a number of cellular responses, including inflammation and proliferation. TWEAK is primarily secreted by phagocytic cells and its receptor, fibroblast growth factor-inducible 14 (Fn14), is expressed on non-lymphoid cells, including epithelial, endothelial and mesenchymal cells. The TWEAK/Fn14 pathway is highly conserved from an evolutionary standpoint, and has been shown to play a role in tissue regeneration and inflammation in the liver, kidney, lung and skeletal muscle. We hypothesized that TWEAK/Fn14 might have a physiological role in regulating infection-induced inflammation in the lower female genital tract. To test this hypothesis, we examined expression of the receptor Fn14 in relevant cells and tissue. Receptor function was tested by treating cells with recombinant TWEAK, with and without other known proinflammatory stimuli. Flow cytometric analysis of vaginal and cervical epithelial cells revealed that Fn14 was highly expressed at the cell surface. We also detected both Fn14 and TWEAK in whole cervical tissue by RT-PCR. Treatment of vaginal and cervical epithelial cells with recombinant TWEAK led to a weak induction of the chemokine IL-8. However, TWEAK potentiated the effects of IL-1ss, the TLR2 ligand Pam(3)CysSK(4), and live Neisseria gonorrhoeae in a synergistic manner. These data reveal a novel pathway for regulation of microbial-induced inflammation in the female reproductive tract and suggest that interference with the TWEAK/Fn14 pathway might be an approach to abrogate excessive infection-induced inflammation caused by sexually transmitted pathogens.

Resistin-like molecule α enhances myeloid cell activation and promotes colitis

Resistin-like molecule (Relm) alpha is a secreted protein and a hallmark signature gene for alternatively activated macrophages. Relm-alpha is highly induced by allergic inflammatory triggers and perceived to promote tissue repair. Yet the function of Relm-alpha remains unknown. We sough to determine the role of Relm-alpha in dextran sodium sulfate (DSS)-induced colonic injury. The cellular source of Relm-alpha was determined after oral DSS-induced colitis. Retnla(-/-) mice were generated, subjected to DSS treatment, and monitored for disease progression (clinical and histopathologic features). Cytokine production in the supernatants of ex vivo colon cultures, and of LPS-stimulated macrophages incubated with Relm-alpha was assessed. Relm-alpha was administered intraperitoneally, and the cellular recruitment to the peritoneum was assessed. After innate intestinal stimulation with DSS, Relm-alpha was highly expressed by eosinophils and epithelial cells. Retnla gene-targeted mice were protected from DSS-induced colitis (eg, decreased diarrhea, rectal bleeding, colon shortening, disease score, and histopathologic changes). Relm-alpha coactivated IL-6 and TNF-alpha release and inhibited IL-10 release from LPS-activated bone marrow-derived macrophages. Consistent with these finding, colon cultures of DSS-treated Retnla(-/-) mice produced decreased IL-6 and increased IL-10 ex vivo. Furthermore, Retnla(-/-) mice had substantially decreased c-Jun N-terminal kinase phosphorylation in vivo. In vivo administration of Relm-alpha initiated cellular recruitment to the peritoneum, and Relm-alpha was able to induce eosinophil chemotaxis in vitro. These findings demonstrate a central proinflammatory role for Relm-alpha in colonic innate immune responses, identifying a novel pathway for regulation of macrophage activation.

Genetic Disruption of □pdl and □pdlr Phospholipases in Mice Leads to Impaired Platelet Adhesion and Aggregation

Platelet adhesion and aggregation are critical events in thrombosis. Bioactive phospholipid LPA (lysophosphatidic acid) has been identified as an important agonist for platelet aggregation. Plasma LPA can be generated from phospholipid substrates by phospholipase A1 (PLA1) and phospholipase D (PLD). We previously identified two novel PLA1 enzymes, designated lpdl (lpd lipase) and lpdlr (lpdl related lipase). Together with phosphatidylserine phospholipase A1 (PS-PLA1), these three phospholipases form a unique PLA1 lipase subfamily. Phospholipids are important structural components of cellular membranes. Recent studies demonstrate that lipid raft microdomains on the platelet membrane contribute to platelet activation. The gathering of membrane lipid rafts is necessary for ADP-mediated platelet activation. Disruption of lipid raft results in a reduction of ADP-induced platelet aggregation. In addition, it is reported that alteration of membrane lipid composition also affects the function of platelet β3 integrin (GPIIbIIIa). Thus, phospholipases may play an important role in platelet function. However, the roles of lpdl and lpdlr in platelet activation and thrombosis are unknown. To study the function of lpdl and lpdlr, we have recently knocked out both lpdl and lpdlr genes in mice by deleting their exons I and II, including their translation start codon ATG. RT-PCR confirmed that neither lpdl nor lpdlr gene is expressed in homozygous lpdl−/− or lpdlr−/− mice but expressed in wild-type (WT) tissues, indicating successful knockout of these phospholipases. Next, optical platelet aggregometry was used to assess platelet aggregation in platelet rich plasma (PRP). Platelet aggregation was induced with 5, 10 and 20 μM ADP in pooled PRP (3×108 platelets/mL) from age- and sex-matched WT, heterozygous and homozygous mice. Our results demonstrated that both the lpdl−/− and lpdlr−/− mice have decreased platelet aggregation after ADP stimulation. We further studied thrombus formation in lpdl−/− mice using an ex vivo perfusion chamber model on collagen type I-coated rectangular glass microcapillary tubes. Our preliminary data showed that both platelet adhesion and aggregation were impaired in the lpdl−/− mice. We are repeating these experiments and are examining lpdlr−/− mice using the same perfusion chamber model. In vivo thrombosis models with intravital microscopy and the mechanisms of how lpdl and lpdlr enzymes regulate platelet activation will also be investigated. These studies could potentially identify a novel pathway for regulation of platelet function, which may lead to development of new diagnostic and/or therapeutic methods for atherothrombosis. (Drs. D. Wang and A. Reheman contribute equally to this work)