The Role of the Histone Methyltransferase Enhancer of Zeste Homolog 2 (EZH2) in the Pathobiological Mechanisms Underlying Inflammatory Bowel Disease (IBD).

Regulatory T (Treg) cells expressing the transcription factor FOXP3 play a pivotal role in maintaining immunologic self-tolerance. We and others have shown previously that EZH2 is recruited to the FOXP3 promoter and its targets in Treg cells. To further address the role for EZH2 in Treg cellular function, we have now generated mice that lack EZH2 specifically in Treg cells (EZH2Δ/ΔFOXP3+). We find that EZH2 deficiency in FOXP3+ T cells results in lethal multiorgan autoimmunity. We further demonstrate that EZH2Δ/ΔFOXP3+ T cells lack a regulatory phenotype in vitro and secrete proinflammatory cytokines. Of special interest, EZH2Δ/ΔFOXP3+ mice develop spontaneous inflammatory bowel disease. Guided by these results, we assessed the FOXP3 and EZH2 gene networks by RNA sequencing in isolated intestinal CD4+ T cells from patients with Crohn's disease. Gene network analysis demonstrates that these CD4+ T cells display a Th1/Th17-like phenotype with an enrichment of gene targets shared by FOXP3 and EZH2. Combined, these results suggest that the inflammatory milieu found in Crohn's disease could lead to or result from deregulation of FOXP3/EZH2-enforced T cell gene networks contributing to the underlying intestinal inflammation.

Krüppel-like factor KLF10 regulates transforming growth factor receptor II expression and TGF-ß signaling in CD8+ T lymphocytes.

KLF10 has recently elicited significant attention as a transcriptional regulator of transforming growth factor-ß1 (TGF-ß1) signaling in CD4(+) T cells. In the current study, we demonstrate a novel role for KLF10 in the regulation of TGF-ß receptor II (TGF-ßRII) expression with functional relevance in antiviral immune response. Specifically, we show that KLF10-deficient mice have an increased number of effector/memory CD8(+) T cells, display higher levels of the T helper type 1 cell-associated transcription factor T-bet, and produce more IFN-γ following in vitro stimulation. In addition, KLF10(-/-) CD8(+) T cells show enhanced proliferation in vitro and homeostatic proliferation in vivo. Freshly isolated CD8(+) T cells from the spleen of adult mice express lower levels of surface TGF-ßRII (TßRII). Congruently, in vitro activation of KLF10-deficient CD8(+) T cells upregulate TGF-ßRII to a lesser extent compared with wild-type (WT) CD8(+) T cells, which results in attenuated Smad2 phosphorylation following TGF-ß1 stimulation compared with WT CD8(+) T cells. Moreover, we demonstrate that KLF10 directly binds to the TGF-ßRII promoter in T cells, leading to enhanced gene expression. In vivo viral infection with Daniel's strain Theiler's murine encephalomyelitis virus (TMEV) also led to lower expression of TGF-ßRII among viral-specific KLF10(-/-) CD8(+) T cells and a higher percentage of IFN-γ-producing CD8(+) T cells in the spleen. Collectively, our data reveal a critical role for KLF10 in the transcriptional activation of TGF-ßRII in CD8(+) T cells. Thus, KLF10 regulation of TGF-ßRII in this cell subset may likely play a critical role in viral and tumor immune responses for which the integrity of the TGF-ß1/TGF-ßRII signaling pathway is crucial.

Krüppel-like factor KLF10 deficiency predisposes to colitis through colonic macrophage dysregulation.

Krüppel-like factor (KLF)-10 is an important transcriptional regulator of TGF-ß1 signaling in both CD8(+) and CD4(+) T lymphocytes. In the present study, we demonstrate a novel role for KLF10 in the regulation of TGFßRII expression with functional relevance in macrophage differentiation and activation. We first show that transfer of KLF10(-/-) bone marrow-derived macrophages into wild-type (WT) mice leads to exacerbation of experimental colitis. At the cell biological level, using two phenotypic strategies, we show that KLF10-deficient mice have an altered colonic macrophage phenotype with higher frequency of proinflammatory LyC6(+)MHCII(+) cells and a reciprocal decrease of the anti-inflammatory LyC6(-)MHCII(+) subset. Additionally, the anti-inflammatory CD11b(+)CX3CR1(hi) subset of colonic macrophages is significantly decreased in KLF10(-/-) compared with WT mice under inflammatory conditions. Molecularly, CD11b(+) colonic macrophages from KLF10(-/-) mice exhibit a proinflammatory cytokine profile with increased production of TNF-α and lower production of IL-10 in response to LPS stimulation. Because KLF10 is a transcription factor, we explored how this protein may regulate macrophage function. Consequently, we analyzed the expression of TGFßRII expression in colonic macrophages and found that, in the absence of KLF10, macrophages express lower levels of TGFßRII and display an attenuated Smad-2 phosphorylation following TGF-ß1 stimulation. We further show that KLF10 directly binds to the TGFßRII promoter in macrophages, leading to enhanced gene expression through histone H3 acetylation. Collectively, our data reveal a critical role for KLF10 in the epigenetic regulation of TGFßRII expression in macrophages and the acquisition of a "regulatory" phenotype that contributes to intestinal mucosal homeostasis.

Chromatin composition alterations and the critical role of MeCP2 for epigenetic silencing of progesterone receptor-B gene in endometrial cancers.

OBJECTIVE: To understand the epigenetic mechanism underlying the PR-B gene silencing in endometrial cancer (EC) cells, we compared the chromatin composition between transcriptionally active and silenced PR-B genes in EC cell lines and cancer tissues. METHODS: Chromatin Immunoprecipitation (ChIP) assay was performed to measure MBD occupancy and histone acetylation/methylation in transcriptionally active and silenced PR-B genes. PR-B-positive/-negative, as well as epigenetic inhibitor-treated/-untreated EC cells were used as study models. Real-time polymerase chain reaction (PCR) and Western blot analysis were applied to measure the mRNA and protein levels of PR-B, MBD, and histones. RESULTS: A close association among PR-B methylation, MBD binding and PR-B gene silencing was observed. Treatment with epigenetic inhibitors led to dynamic changes in the PR-B chromatin composition and gene expression. Increased H3/H4 acetylation and H3-K4 methylation, and decreased H3-K9 methylation were found to be associated with re-activation of silenced PR-B genes. MeCP2 knockdown resulted in a decreased MeCP2 binding to PR-B genes and an increased PR-B expression. ChIP analysis of MeCP2 binding to PR-B genes in the PR-B-positive/-negative EC samples confirmed the significant role of MeCP2 in PR-B silencing. CONCLUSION: PR-B gene expression is regulated by a concerted action of epigenetic factors including DNA methylation, MBD binding, and histone modifications. MeCP2 occupancy of PR-B genes plays a critical role in PR-B gene silencing. These findings enriched our knowledge of the epigenetic regulation of PR-B expression in EC, and suggested that the epigenetic re-activation of PR-B could be explored as a potential strategy to sensitize the PR-B-negative endometrial cancers to progestational therapy.

Differential coupling of KLF10 to Sin3-HDAC and PCAF regulates the inducibility of the FOXP3 gene.

Inducible gene expression, which requires chromatin remodeling on gene promoters, underlies the epigenetically inherited differentiation program of most immune cells. However, chromatin-mediated mechanisms that underlie these events in T regulatory cells remain to be fully characterized. Here, we report that inducibility of FOXP3, a key transcription factor for the development of T regulatory cells, depends upon Kruppel-like factor 10 (KLF10) interacting with two antagonistic histone-modifying systems. We utilized chromatin immunoprecipitation, genome-integrated reporter assays, and functional domain KLF10 mutant proteins, to characterize reciprocal interactions between this transcription factor and either the Sin3-histone deacetylase complex or the histone acetyltransferase, p300/CBP-associated factor (PCAF). We characterize a Sin3-interacting repressor domain on the NH2 terminus of KLF10, which works to limit the activating function of this transcription factor. Indeed, inactivation of this Sin3-interacting domain renders KLF10 able to physically associate with PCAF as to induce FOXP3 gene transcription. We show that this biochemical data derived from studying our genome-integrated reporter cell system are recapitulated in primary murine lymphocytes. Collectively, these results advance our understanding of how a single transcription factor, namely KLF10, functions as a toggle to integrate antagonistic signals regulating FOXP3 and, thus, immune activation.

Exome sequencing identifies a novel FOXP3 mutation in a 2-generation family with inflammatory bowel disease.

OBJECTIVES: Inflammatory bowel disease (IBD) is heritable, but a total of 163 variants commonly implicated in IBD pathogenesis account for only 25% of the heritability. Rare, highly penetrant genetic variants may also explain mendelian forms of IBD and some of the missing heritability. To test the hypothesis that rare loss-of-function mutations can be causative, we performed whole exome sequencing (WES) on 5 members of a 2-generation family of European ancestry presenting with an early-onset and atypical form of IBD. METHODS: WES was performed for all of the 5 family members; the mother and 3 male offspring were affected, whereas the father was unaffected. Mapping, annotation, and filtering criteria were used to reduce candidate variants. For functional testing we performed forkhead box P3 (FOXP3) staining and a T-cell suppression assay. RESULTS: We identified a novel missense variant in exon 6 of the X-linked FOXP3 gene. The c.694A>C substitution in FOXP3 results in a cysteine-to-glycine change at the protein position 232 that is completely conserved among all vertebrates. This variant (heterozygous in the mother and hemizygous in all 3 affected sons) did not impair FOXP3 protein expression, but significantly reduced the ability of the host's T regulatory cells to suppress an inappropriate autoimmune response. The variant results in a milder immune dysregulation, polyendocrinopathy, enteropathy, and X-linked phenotype with early-onset IBD. CONCLUSIONS: Our study illustrates the successful application of WES for making a definitive molecular diagnosis in a case of multiply affected families, with atypical IBD-like phenotype. Our results also have important implications for disease biology and disease-directed therapeutic development.

Polycomb antagonizes p300/CREB-binding protein-associated factor to silence FOXP3 in a Kruppel-like factor-dependent manner.

Inducible gene expression underlies the epigenetically inherited differentiation program of most immune cells. We report that the promoter of the FOXP3 gene possesses two distinct functional states: an "off state" mediated by the polycomb histone methyltransferase complex and a histone acetyltransferase-dependent "on state." Regulating these states is the presence of a Kruppel-like factor (KLF)-containing Polycomb response element. In the KLF10(-/-) mouse, the FOXP3 promoter is epigenetically silenced by EZH2 (Enhancer of Zeste 2)-mediated trimethylation of Histone 3 K27; thus, impaired FOXP3 induction and inappropriate adaptive T regulatory cell differentiation results in vitro and in vivo. The epigenetic transmittance of adaptive T regulatory cell deficiency is demonstrated throughout more than 40 generations of mice. These results provide insight into chromatin remodeling events key to phenotypic features of distinct T cell populations.

Hypomethylation and Overexpression of Th17-Associated Genes is a Hallmark of Intestinal CD4+ Lymphocytes in Crohn's Disease.

BACKGROUND: The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated. MATERIALS AND METHODS: Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells. RESULTS: CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites. CONCLUSIONS: The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells.

The pathogen recognition receptor NOD2 regulates human FOXP3+ T cell survival.

The expression of pathogen recognition receptors in human FOXP3+ T regulatory cells is established, yet the function of these receptors is currently obscure. In the process of studying the function of both peripheral and lamina propria FOXP3+ lymphocytes in patients with the human inflammatory bowel disease Crohn's disease, we observed a clear deficiency in the quantity of FOXP3+ lymphocytes in patients with disease-associated polymorphisms in the pathogen recognition receptor gene NOD2. Subsequently, we determined that the NOD2 ligand, muramyl dipeptide (MDP), activates NF-kappaB in primary human FOXP3+ T cells. This activation is functionally relevant, as MDP-stimulated human FOXP3+ T cells are protected from death receptor Fas-mediated apoptosis. Importantly, apoptosis protection was not evident in MDP-stimulated FOXP3+ T cells isolated from a patient with the disease-associated polymorphism. Thus, we propose that one function of pathogen recognition receptors in human T regulatory cells is the protection against death receptor-mediated apoptosis in a Fas ligand-rich environment, such as that of the inflamed intestinal subepithelial space.

Uric Acid Expression in Carotid Atherosclerotic Plaque and Serum Uric Acid Are Associated With Cerebrovascular Events.

BACKGROUND: Uric acid (UA) concentration within carotid plaque and its association with cerebrovascular events have not been detected or quantified. Systemically, serum UA is a marker of inflammation and risk factor for atherosclerosis. However, its association with carotid plaque instability and stroke pathogenesis remains unclear. In patients undergoing carotid endarterectomy, we aimed to determine whether UA is present differentially in symptomatic versus asymptomatic carotid plaques and whether serum UA is associated with cerebrovascular symptoms (stroke, transient ischemic attack, or amaurosis fugax). METHODS: Carotid atherosclerotic plaques were collected during carotid endarterectomy. The presence of UA was assessed using Gomori methenamine silver staining as well as anti-UA immunohistochemical staining and its quantity measured using an enzymatic colorimetric assay. Clinical information was obtained through a retrospective review of data. RESULTS: UA was more commonly detected in symptomatic (n=23) compared with asymptomatic (n=9) carotid plaques by Gomori methenamine silver (20 [86.9%] versus 2 [22.2%]; P=0.001) and anti-UA immunohistochemistry (16 [69.5%] versus 1 [11.1%]; P=0.004). UA concentration was higher in symptomatic rather than asymptomatic plaques (25.1 [9.5] versus 17.9 [3.8] µg/g; P=0.021). Before carotid endarterectomy, serum UA levels were higher in symptomatic (n=341) compared with asymptomatic (n=146) patients (5.9 [interquartile range, 4.6-6.9] mg/dL versus 5.2 [interquartile range, 4.6-6.2] mg/dL; P=0.009). CONCLUSIONS: The current study supports a potential role of UA as a potential tissue participant and a systemic biomarker in the pathogenesis of carotid atherosclerosis. UA may provide a mechanistic explanation for plaque instability and subsequent ischemic cerebrovascular events.

Apical scaffolding protein NHERF2 modulates the localization of alternatively spliced plasma membrane Ca2+ pump 2B variants in polarized epithelial cells.

The membrane localization of the plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expression of NHERF2 greatly enhanced the apical localization of GFP-tagged PMCA2w/b in polarized Madin-Darby canine kidney cells. GFP-PMCA2z/b was also redirected to the apical membrane by NHERF2, whereas GFP-PMCA2x/b remained exclusively basolateral. In the presence of NHERF2, GFP-PMCA2w/b co-localized with the actin-binding protein ezrin even after disruption of the actin cytoskeleton by cytochalasin D or latrunculin B. Surface biotinylation and fluorescence recovery after photobleaching experiments demonstrated that NHERF2-mediated anchorage to the actin cytoskeleton reduced internalization and lateral mobility of the pump. Our results show that the specific interaction with NHERF2 enhances the apical concentration of PMCA2w/b by anchoring the pump to the apical membrane cytoskeleton. The data also suggest that the x/b splice form of PMCA2 contains a dominant lateral targeting signal, whereas the targeting and localization of the z/b form are more flexible and not fully determined by intrinsic sequence features.

Time-restricted feeding prevents deleterious metabolic effects of circadian disruption through epigenetic control of ß cell function.

Circadian rhythm disruption (CD) is associated with impaired glucose homeostasis and type 2 diabetes mellitus (T2DM). While the link between CD and T2DM remains unclear, there is accumulating evidence that disruption of fasting/feeding cycles mediates metabolic dysfunction. Here, we used an approach encompassing analysis of behavioral, physiological, transcriptomic, and epigenomic effects of CD and consequences of restoring fasting/feeding cycles through time-restricted feeding (tRF) in mice. Results show that CD perturbs glucose homeostasis through disruption of pancreatic ß cell function and loss of circadian transcriptional and epigenetic identity. In contrast, restoration of fasting/feeding cycle prevented CD-mediated dysfunction by reestablishing circadian regulation of glucose tolerance, ß cell function, transcriptional profile, and reestablishment of proline and acidic amino acid­rich basic leucine zipper (PAR bZIP) transcription factor DBP expression/activity. This study provides mechanistic insights into circadian regulation of ß cell function and corresponding beneficial effects of tRF in prevention of T2DM.

ZNF416 is a pivotal transcriptional regulator of fibroblast mechanoactivation.

Matrix stiffness is a central regulator of fibroblast function. However, the transcriptional mechanisms linking matrix stiffness to changes in fibroblast phenotype are incompletely understood. Here, we evaluated the effect of matrix stiffness on genome-wide chromatin accessibility in freshly isolated lung fibroblasts using ATAC-seq. We found higher matrix stiffness profoundly increased global chromatin accessibility relative to lower matrix stiffness, and these alterations were in close genomic proximity to known profibrotic gene programs. Motif analysis of these regulated genomic loci identified ZNF416 as a putative mediator of fibroblast stiffness responses. Genome occupancy analysis using ChIP-seq confirmed that ZNF416 occupies a broad range of genes implicated in fibroblast activation and tissue fibrosis, with relatively little overlap in genomic occupancy with other mechanoresponsive and profibrotic transcriptional regulators. Using loss- and gain-of-function studies, we demonstrated that ZNF416 plays a critical role in fibroblast proliferation, extracellular matrix synthesis, and contractile function. Together, these observations identify ZNF416 as novel mechano-activated transcriptional regulator of fibroblast biology.

Radiation Induced Metabolic Alterations Associate With Tumor Aggressiveness and Poor Outcome in Glioblastoma.

Glioblastoma (GBM) is uniformly fatal with a 1-year median survival, despite best available treatment, including radiotherapy (RT). Impacts of prior RT on tumor recurrence are poorly understood but may increase tumor aggressiveness. Metabolic changes have been investigated in radiation-induced brain injury; however, the tumor-promoting effect following prior radiation is lacking. Since RT is vital to GBM management, we quantified tumor-promoting effects of prior RT on patient-derived intracranial GBM xenografts and characterized metabolic alterations associated with the protumorigenic microenvironment. Human xenografts (GBM143) were implanted into nude mice 24 hrs following 20 Gy cranial radiation vs. sham animals. Tumors in pre-radiated mice were more proliferative and more infiltrative, yielding faster mortality (p < 0.0001). Histologic evaluation of tumor associated macrophage/microglia (TAMs) revealed cells with a more fully activated ameboid morphology in pre-radiated animals. Microdialyzates from radiated brain at the margin of tumor infiltration contralateral to the site of implantation were analyzed by unsupervised liquid chromatography-mass spectrometry (LC-MS). In pre-radiated animals, metabolites known to be associated with tumor progression (i.e., modified nucleotides and polyols) were identified. Whole-tissue metabolomic analysis of pre-radiated brain microenvironment for metabolic alterations in a separate cohort of nude mice using 1H-NMR revealed a significant decrease in levels of antioxidants (glutathione (GSH) and ascorbate (ASC)), NAD+, Tricarboxylic acid cycle (TCA) intermediates, and rise in energy carriers (ATP, GTP). GSH and ASC showed highest Variable Importance on Projection prediction (VIPpred) (1.65) in Orthogonal Partial least square Discriminant Analysis (OPLS-DA); Ascorbate catabolism was identified by GC-MS. To assess longevity of radiation effects, we compared survival with implantation occurring 2 months vs. 24 hrs following radiation, finding worse survival in animals implanted at 2 months. These radiation-induced alterations are consistent with a chronic disease-like microenvironment characterized by reduced levels of antioxidants and NAD+, and elevated extracellular ATP and GTP serving as chemoattractants, promoting cell motility and vesicular secretion with decreased levels of GSH and ASC exacerbating oxidative stress. Taken together, these data suggest IR induces tumor-permissive changes in the microenvironment with metabolomic alterations that may facilitate tumor aggressiveness with important implications for recurrent glioblastoma. Harnessing these metabolomic insights may provide opportunities to attenuate RT-associated aggressiveness of recurrent GBM.

Genomic and Epigenomic Landscaping Defines New Therapeutic Targets for Adenosquamous Carcinoma of the Pancreas.

Adenosquamous cancer of the pancreas (ASCP) is a subtype of pancreatic cancer that has a worse prognosis and greater metastatic potential than the more common pancreatic ductal adenocarcinoma (PDAC) subtype. To distinguish the genomic landscape of ASCP and identify actionable targets for this lethal cancer, we applied DNA content flow cytometry to a series of 15 tumor samples including five patient-derived xenografts (PDX). We interrogated purified sorted tumor fractions from these samples with whole-genome copy-number variant (CNV), whole-exome sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analyses. These identified a variety of somatic genomic lesions targeting chromatin regulators in ASCP genomes that were superimposed on well-characterized genomic lesions including mutations in TP53 (87%) and KRAS (73%), amplification of MYC (47%), and homozygous deletion of CDKN2A (40%) that are common in PDACs. Furthermore, a comparison of ATAC-seq profiles of three ASCP and three PDAC genomes using flow-sorted PDX models identified genes with accessible chromatin unique to the ASCP genomes, including the lysine methyltransferase SMYD2 and the pancreatic cancer stem cell regulator RORC in all three ASCPs, and a FGFR1-ERLIN2 fusion associated with focal CNVs in both genes in a single ASCP. Finally, we demonstrate significant activity of a pan FGFR inhibitor against organoids derived from the FGFR1-ERLIN2 fusion-positive ASCP PDX model. Our results suggest that the genomic and epigenomic landscape of ASCP provide new strategies for targeting this aggressive subtype of pancreatic cancer. SIGNIFICANCE: These data provide a unique description of the ASCP genomic and epigenomic landscape and identify candidate therapeutic targets for this dismal cancer.

Apical localization of PMCA2w/b is lipid raft-dependent.

Alternative splicing of the first intracellular loop differentially targets plasma membrane calcium ATPase (PMCA) isoform 2 to the apical or basolateral membrane in MDCK cells. To determine if the targeting is affected by lipid interactions, we stably expressed PMCA2w/b and PMCA2z/b in MDCK cells, and analyzed the PMCA distribution by confocal fluorescence microscopy and membrane fractionation. PMCA2w/b showed clear apical and lateral distribution, whereas PMCA2z/b was mainly localized to the basolateral membrane. A significant fraction of PMCA2w/b partitioned into low-density membranes associated with lipid rafts. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced lipid raft association and a striking loss of PMCA2w/b from the apical membrane, whereas the lateral localization of PMCA2z/b remained unchanged. Our data indicate that alternative splicing differentially affects the lipid interactions of PMCA2w/b and PMCA2z/b and that the apical localization of PMCA2w/b is lipid raft-dependent and sensitive to cholesterol depletion.

Cytokine and chemokine profiles in multiple myeloma; significance of stromal interaction and correlation of IL-8 production with disease progression.

Multiple myeloma (MM) is a product of interactions between tumor plasma cells and multiple cell types native to the bone marrow (BM). We have used antibody array technology to examine the proteins produced by BM stromal cells in response to stimulation by BM taken from patients diagnosed with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and MM. We observed increased production of the chemokine IL-8 by stromal cells co-cultured with supernatants from bone marrow cells of patients with active myeloma. IL-8 production is correlated with active disease and is dependent upon IL-1beta and NF-kappaB signaling. Consistent with the pro-angiogenic activity of IL-8, increased BM microvessel density (MVD) correlated with stimulation of stromal cell IL-8 production. In addition, the majority of MM cell lines and MM patient plasma cells were found to express IL-8 receptors CXCR1 and CXCR2. We conclude that stromal cell IL-8 production parallels MM disease activity, is IL-1beta induced, and correlates with bone marrow angiogenesis.

Plasma membrane Ca2+ ATPases as dynamic regulators of cellular calcium handling.

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.

Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells.

It is well known that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) acts synergistically with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (ADC) to reactivate DNA methylation-silenced genes. Moreover, in several studies, TSA was capable of inducing DNA demethylation even in the absence of ADC. Here we describe a mechanism by which HDAC inhibitors affect DNA methylation through their regulation on DNMT3B, a methyltransferase responsible for de novo DNA methylation. Using quantitative real-time PCR and Western blot analysis, we show that TSA down-regulates DNMT3B mRNA and protein expression in human endometrial cancer cells. This decrease in DNMT3B mRNA results in a significant reduction in de novo methylation activities. Further experiments indicated that TSA decreases DNMT3B mRNA stability and reduces its half-life from approximately 4 to 2.5 hours. We established that protein synthesis is required for posttranscriptional regulation, suggesting the involvement of an RNase and/or key mRNA stabilization factor(s) controlling the DNMT3B mRNA stability. Therefore, TSA may not only modify histone acetylation, but also potentially alter DNA methylation. Since the HDAC inhibitors are frequently used in epigenetic studies and are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Identification of two groups of smoldering multiple myeloma patients who are either high or low producers of interleukin-1.

Interleukin-1beta (IL-1beta) is abnormally expressed by the plasma cells obtained from myeloma patients, and it is a potent inducer of the important myeloma growth factor, IL-6. We investigated whether levels of IL-1beta biologic activity might distinguish different groups of patients with smoldering multiple myeloma (SMM). We measured the ability of IL-6 production by bone marrow stromal cells to serve as a surrogate marker for IL-1beta biologic activity. Using this IL-1beta bioassay, we found that it is sensitive at < 1 pg/ml of recombinant IL-1beta and that IL-1beta biologic activity is detectable with either mature or pro-IL-1beta-transduced myeloma cell lines. Patients with active myeloma induced quantitatively higher levels of stromal cell IL-6 production when compared with those with monoclonal gammopathy of undetermined significance (MGUS). The bioassay distinguished two groups of SMM patients, those who were high producers, similar to patients with active MM, and those who were low producers, comparable to MGUS patients. IL-1 antagonists inhibited the paracrine IL-6 production by > or = 90% in the majority of patients with an elevated IL-6 level. Based on such studies, it may be possible to predict patients that will progress to active MM and to delay or prevent this progression with IL-1 antagonists.