Standardized monitoring of cytomegalovirus-specific immunity can improve risk stratification of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation.

Recurrence of cytomegalovirus reactivation remains a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation. Monitoring cytomegalovirus-specific cellular immunity using a standardized assay might improve the risk stratification of patients. A prospective multicenter study was conducted in 175 intermediate- and high-risk allogeneic hematopoietic stem cell transplant recipients under preemptive antiviral therapy. Cytomegalovirus-specific cellular immunity was measured using a standardized IFN-γ ELISpot assay (T-Track® CMV). Primary aim was to evaluate the suitability of measuring cytomegalovirus-specific immunity after end of treatment for a first cytomegalovirus reactivation to predict recurrent reactivation. 40/101 (39.6%) patients with a first cytomegalovirus reactivation experienced recurrent reactivations, mainly in the high-risk group (cytomegalovirus-seronegative donor/cytomegalovirus-seropositive recipient). The positive predictive value of T-Track® CMV (patients with a negative test after the first reactivation experienced at least one recurrent reactivation) was 84.2% in high-risk patients. Kaplan-Meier analysis revealed a higher probability of recurrent cytomegalovirus reactivation in high-risk patients with a negative test after the first reactivation (hazard ratio 2.73; p=0.007). Interestingly, a post-hoc analysis considering T-Track® CMV measurements at day 100 post-transplantation, a time point highly relevant for outpatient care, showed a positive predictive value of 90.0% in high-risk patients. Our results indicate that standardized cytomegalovirus-specific cellular immunity monitoring may allow improved risk stratification and management of recurrent cytomegalovirus reactivation after hematopoietic stem cell transplantation. This study was registered at www.clinicaltrials.gov as #NCT02156479.

Clinical validation of a novel enzyme-linked immunosorbent spot assay-based in vitro diagnostic assay to monitor cytomegalovirus-specific cell-mediated immunity in kidney transplant recipients: a multicenter, longitudinal, prospective, observational study.

Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T-Track® CMV, a novel IFN-γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate-risk renal transplant recipients. CMV-CMI, CMV viral load, and clinical complications were monitored over 6 months post-transplantation. Ninety-five percent and 88-92% ELISpot assays were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65-specific response was ninefold higher in patients with self-clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T-Track® CMV is a highly sensitive IFN-γ ELISpot assay, suitable for the immunomonitoring of CMV-seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity.

BACKGROUND: In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions. METHODS: Objective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay. RESULTS: Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3-CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive. CONCLUSION: The combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients.

BACKGROUND: Uncontrolled cytomegalovirus (CMV) replication in immunocompromised solid-organ transplant recipients is a clinically relevant issue and an indication of impaired CMV-specific cell-mediated immunity (CMI). Primary aim of this study was to assess the suitability of the immune monitoring tool T-Track® CMV to determine CMV-reactive CMI in a cohort of hemodialysis patients representative of patients eligible for renal transplantation. Positive and negative agreement of T-Track® CMV with CMV serology was examined in 124 hemodialysis patients, of whom 67 (54%) revealed a positive CMV serostatus. Secondary aim of the study was to evaluate T-Track® CMV performance against two unrelated CMV-specific CMI monitoring assays, QuantiFERON®-CMV and a cocktail of six class I iTAg™ MHC Tetramers. RESULTS: Positive T-Track® CMV results were obtained in 90% (60/67) of CMV-seropositive hemodialysis patients. In comparison, 73% (45/62) and 77% (40/52) positive agreement with CMV serology was achieved using QuantiFERON®-CMV and iTAg™ MHC Tetramer. Positive T-Track® CMV responses in CMV-seropositive patients were dominated by pp65-reactive cells (58/67 [87%]), while IE-1-responsive cells contributed to an improved (87% to 90%) positive agreement of T-Track® CMV with CMV serology. Interestingly, T-Track® CMV, QuantiFERON®-CMV and iTAg™ MHC Tetramers showed 79% (45/57), 87% (48/55) and 93% (42/45) negative agreement with serology, respectively, and a strong inter-assay variability. Notably, T-Track® CMV was able to detect IE-1-reactive cells in blood samples of patients with a negative CMV serology, suggesting either a previous exposure to CMV that yielded a cellular but no humoral immune response, or TCR cross-reactivity with foreign antigens, both suggesting a possible protective immunity against CMV in these patients. CONCLUSION: T-Track® CMV is a highly sensitive assay, enabling the functional assessment of CMV-responsive cells in hemodialysis patients prior to renal transplantation. T-Track® CMV thus represents a valuable immune monitoring tool to identify candidate transplant recipients potentially at increased risk for CMV-related clinical complications.

Functional impairment of CMV-reactive cellular immunity during pregnancy.

Cytomegalovirus (CMV) is the most common congenital viral infection. Mother-to-child transmission can cause severe child disability. Intact CMV-specific cell-mediated immunity (CMI) was shown to prevent uncontrolled replication in healthy individuals. This study aimed to determine whether CMV-specific CMI is impaired in pregnant women, thus potentially increasing the overall risk for active CMV replication and transmission. CMV-specific CMI in peripheral blood of 60 pregnant women was determined using T-Track® CMV for detection of IE-1 and pp65-reactive effector cells by IFN-γ ELISpot, and compared to the CMV-IgG and -IgM serostatus. CMV-specific CMI was detected in 65% of CMV-seropositive pregnant women. Five percent of CMV-IgG seronegative women showed IE-1- but not pp65-reactive cells. The overall number of CMV-reactive cells in pregnant women was significantly lower compared to a matched non-pregnant control group (P < 0.001). No significant difference in CMV-specific CMI was detected in the course of the three trimesters of pregnancy of CMV-IgG seropositive women. Postpartum (median days postnatal = 123), IE-1- and pp65-specific CMI remained significantly lower than in the non-pregnant control group (P < 0.001 and 0.0032, respectively). Functional analysis of CMV-reactive immune cells using T-Track® CMV therefore suggests a systemic down-regulation of CMV-specific CMI in pregnant women. Further studies are needed to investigate whether this may be indicative of a higher susceptibility to CMV reactivation or transmission. J. Med. Virol. 89:324-331, 2017. © 2016 Wiley Periodicals, Inc.

Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function.

Signal transducer and activator of transcription STAT5 is essential for the regulation of proliferation and survival genes. Its activity is tightly regulated through cytokine signaling and is often upregulated in cancer. We showed previously that the deacetylase inhibitor trichostatin A (TSA) inhibits STAT5-mediated transcription by preventing recruitment of the transcriptional machinery at a step following STAT5 binding to DNA. The mechanism and factors involved in this inhibition remain unknown. We now show that deacetylase inhibitors do not target STAT5 acetylation, as we initially hypothesized. Instead, they induce a rapid increase in global histone acetylation apparently resulting in the delocalization of the bromodomain and extra-terminal (BET) protein Brd2 and of the Brd2-associated factor TBP to hyperacetylated chromatin. Treatment with the BET inhibitor (+)-JQ1 inhibited expression of STAT5 target genes, supporting a role of BET proteins in the regulation of STAT5 activity. Accordingly, chromatin immunoprecipitation demonstrated that Brd2 is associated with the transcriptionally active STAT5 target gene Cis and is displaced upon TSA treatment. Our data therefore indicate that Brd2 is required for the proper recruitment of the transcriptional machinery at STAT5 target genes and that deacetylase inhibitors suppress STAT5-mediated transcription by interfering with Brd2 function.

Signal transducer and activator of transcription STAT5 is recruited to c-Myc super-enhancer.

BACKGROUND: c-Myc has been proposed as a putative target gene of signal transducer and activator of transcription 5 (STAT5). No functional STAT5 binding site has been identified so far within the c-Myc gene locus, therefore a direct transcriptional regulation by STAT5 remains uncertain. c-Myc super-enhancer, located 1.7 Mb downstream of the c-Myc gene locus, was recently reported as essential for the regulation of c-Myc gene expression by hematopoietic transcription factors and bromodomain and extra-terminal (BET) proteins and for leukemia maintenance. c-Myc super-enhancer is composed of five regulatory regions (E1-E5) which recruit transcription and chromatin-associated factors, mediating chromatin looping and interaction with the c-Myc promoter. RESULTS: We now show that STAT5 strongly binds to c-Myc super-enhancer regions E3 and E4, both in normal and transformed Ba/F3 cells. We also found that the BET protein bromodomain-containing protein 2 (BRD2), a co-factor of STAT5, co-localizes with STAT5 at E3/E4 in Ba/F3 cells transformed by the constitutively active STAT5-1*6 mutant, but not in non-transformed Ba/F3 cells. BRD2 binding at E3/E4 coincides with c-Myc transcriptional activation and is lost upon treatment with deacetylase and BET inhibitors, both of which inhibit STAT5 transcriptional activity and c-Myc gene expression. CONCLUSIONS: Our data suggest that constitutive STAT5 binding to c-Myc super-enhancer might contribute to BRD2 maintenance and thus allow sustained expression of c-Myc in Ba/F3 cells transformed by STAT5-1*6.

Inhibition of interleukin-3- and interferon- α-induced JAK/STAT signaling by the synthetic α-X-2',3,4,4'-tetramethoxychalcones α-Br-TMC and α-CF3-TMC.

The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2',3,4,4'-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.

Enhancing the anti-inflammatory activity of chalcones by tuning the Michael acceptor site.

Inflammatory signaling pathways orchestrate the cellular response to infection and injury. These pathways are known to be modulated by compounds that alkylate cysteinyl thiols. One class of phytochemicals with strong thiol alkylating activity is the chalcones. In this study we tested fourteen chalcone derivatives, α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH), for their ability to modulate inflammatory responses, as monitored by their influence on heme oxygenase-1 (HO-1) activity, inducible nitric oxide synthase (iNOS) activity, and cytokine expression levels. We confirmed that the transcriptional activity of Nrf2 was activated by α-X-TMCs while for NF-κB it was inhibited. For most α-X-TMCs, anti-inflammatory activity was positively correlated with thiol alkylating activity, i.e. stronger electrophiles (X = CF3, Br and Cl) being more potent. Notably, this correlation did not hold true for the strongest electrophiles (X = CN and NO2) which were found to be ineffective as anti-inflammatory compounds. These results emphasize the idea that chemical fine-tuning of electrophilicity is needed to achieve and optimize desired therapeutic effects.

LMX1B is essential for the maintenance of differentiated podocytes in adult kidneys.

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini.

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.

Comparison of Cytomegalovirus-Specific Immune Cell Response to Proteins versus Peptides Using an IFN-γ ELISpot Assay after Hematopoietic Stem Cell Transplantation.

Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8+ counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.

The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes.

LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-kappaB target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-kappaB activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-kappaB activity by TNF-alpha synergizes induction of NF-kappaB target genes by LMX1B. Chromatin immunoprecipitation demonstrated that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized kappaB site, and that LMX1B recruitment correlates with increased NF-kappaB DNA association. IL-6 promoter-reporter assays showed that the kappaB site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-kappaB target genes is affected in the kidney of Lmx1b(-/-) knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-kappaB target genes in cooperation with nuclear p50/p65 NF-kappaB.

In vivo identification of novel STAT5 target genes.

STAT5A and STAT5B proteins belong to the family of signal transducers and activators of transcription. They are encoded by two separate genes with 91% identity in their amino acid sequences. Despite their high degree of conservation, STAT5A and STAT5B exert non-redundant functions, resulting at least in part from differences in target gene activation. To better characterize the differential contribution of STAT5A and STAT5B in gene regulation, we performed single or double knockdown of STAT5A and STAT5B using small interfering RNA. Subsequent gene expression profiling and RT-qPCR analyses of IL-3-stimulated Ba/F3-beta cells led to the identification of putative novel STAT5 target genes. Chromatin immunoprecipitation assays analyzing the corresponding gene loci identified unusual STAT5 binding sites compared to conventional STAT5 responsive elements. Some of the STAT5 targets identified are upregulated in several human cancers, suggesting that they might represent potential oncogenes in STAT5-associated malignancies.

Dectin-1 Positive Dendritic Cells Expand after Infection with Leishmania major Parasites and Represent Promising Targets for Vaccine Development.

Resistant mouse strains mount a protective T cell-mediated immune response upon infection with Leishmania (L.) parasites. Healing correlates with a T helper (Th) cell-type 1 response characterized by a pronounced IFN-γ production, while susceptibility is associated with an IL-4-dependent Th2-type response. It has been shown that dermal dendritic cells are crucial for inducing protective Th1-mediated immunity. Additionally, there is growing evidence that C-type lectin receptor (CLR)-mediated signaling is involved in directing adaptive immunity against pathogens. However, little is known about the function of the CLR Dectin-1 in modulating Th1- or Th2-type immune responses by DC subsets in leishmaniasis. We characterized the expression of Dectin-1 on CD11c+ DCs in peripheral blood, at the site of infection, and skin-draining lymph nodes of L. major-infected C57BL/6 and BALB/c mice and in peripheral blood of patients suffering from cutaneous leishmaniasis (CL). Both mouse strains responded with an expansion of Dectin-1+ DCs within the analyzed tissues. In accordance with the experimental model, Dectin-1+ DCs expanded as well in the peripheral blood of CL patients. To study the role of Dectin-1+ DCs in adaptive immunity against L. major, we analyzed the T cell stimulating potential of bone marrow-derived dendritic cells (BMDCs) in the presence of the Dectin-1 agonist Curdlan. These experiments revealed that Curdlan induces the maturation of BMDCs and the expansion of Leishmania-specific CD4+ T cells. Based on these findings, we evaluated the impact of Curdlan/Dectin-1 interactions in experimental leishmaniasis and were able to demonstrate that the presence of Curdlan at the site of infection modulates the course of disease in BALB/c mice: wild-type BALB/c mice treated intradermally with Curdlan developed a protective immune response against L. major whereas Dectin-1-/- BALB/c mice still developed the fatal course of disease after Curdlan treatment. Furthermore, the vaccination of BALB/c mice with a combination of soluble L. major antigens and Curdlan was able to provide a partial protection from severe leishmaniasis. These findings indicate that the ligation of Dectin-1 on DCs acts as an important checkpoint in adaptive immunity against L. major and should therefore be considered in future whole-organism vaccination strategies.

Deacetylase activity is required for recruitment of the basal transcription machinery and transactivation by STAT5.

The signal transducer and activator of transcription STAT5 plays a major role in the cellular response to cytokines, but the mechanism by which it activates transcription remains poorly understood. We show here that deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate) prevent induction of endogenous STAT5 target genes, implying that a deacetylase activity is required for that process. Microarray analyses revealed that this requirement is common to all STAT5 target genes. Using chromatin immunoprecipitation, we show that, following STAT5 DNA binding, deacetylase inhibitors block transcription initiation by preventing recruitment of the basal transcription machinery. This inhibition is not due to effects on histone H3 and H4 acetylation or chromatin remodeling within the promoter region. This novel mechanism of transactivation by STAT5 provides a rationale for the use of deacetylase inhibitors for therapeutic intervention in STAT5-associated cancers.

Role of transcription factors in podocytes.

Despite a wealth of information on structural proteins, comparatively little is known on the transcriptional regulation of podocyte structure and function. In this review we will highlight those transcription factors which, by gene inactivation or classical transgenic experiments, have been shown to be essential for podocytes or probably will turn out to be so. The tumor suppressor protein WT1 is not only indispensable for the initial stages of kidney development, but also very likely maintains the integrity of the fully differentiated podocyte. In the kidney, the LIM homeodomain transcription factor LMX1B is specifically synthesized in podocytes, and mutations in LMX1B lead to nail-patella syndrome and the associated nephropathy. Other transcription factors such as hypoxia-inducible factors and PAX2 are likely to play a role in podocytes, whereas the significance of others, e.g. of POD1 and CITED2, is more speculative at this point.

Assessing HDAC Function in the Regulation of Signal Transducer and Activator of Transcription 5 (STAT5) Activity Using Chromatin Immunoprecipitation (ChIP).

Transcriptional activation by STAT5 is repressed by deacetylase inhibitors. Investigating the role of deacetylases (HDACs) in STAT5-mediated transcription implies the analysis of molecular events taking place at the chromatin level. We describe here two alternative methods of chromatin immunoprecipitation that allow the characterization of chromatin modifications ensuing STAT5 activation and its inhibition by deacetylase inhibitors, in particular changes in histone acetylation, in histone occupancy, and in the association/dissociation of transcription factors and other chromatin-associated factors.

Chromatin acetylation and remodeling at the Cis promoter during STAT5-induced transcription.

The signal transducer and activator of transcription STAT5 plays a major role in cytokine-induced expression of genes involved in cell proliferation and survival. Although several STAT5 partners have been identified, the molecular events taking place at the promoter level upon STAT5 recruitment have not yet been characterized in great detail. Using chromatin immunoprecipitation and accessibility assays, we characterized histone acetylation and chromatin remodeling events occurring during transcriptional activation of the endogenous murine Cis gene, a STAT5 target gene, in response to IL-3. We found that STAT5 binding in vivo is associated with low histone H3 and H4 acetylation levels in the proximity of the STAT5 binding sites. STAT5 recruitment also results in chromatin reorganization over that promoter region. These events (STAT5 binding, histone acetylation and chromatin remodeling) are not sufficient for transcriptional activation, which requires a non-histone protein deacetylase. These data reveal novel implications of STAT5 in chromatin regulation during cytokine-induced transcription, thus contributing to a better understanding of the mechanism of transcriptional activation by STAT5.

The Chemopreventive Phytochemical Moringin Isolated from Moringa oleifera Seeds Inhibits JAK/STAT Signaling.

Sulforaphane (SFN) and moringin (GMG-ITC) are edible isothiocyanates present as glucosinolate precursors in cruciferous vegetables and in the plant Moringa oleifera respectively, and recognized for their chemopreventive and medicinal properties. In contrast to the well-studied SFN, little is known about the molecular pathways targeted by GMG-ITC. We investigated the ability of GMG-ITC to inhibit essential signaling pathways that are frequently upregulated in cancer and immune disorders, such as JAK/STAT and NF-κB. We report for the first time that, similarly to SFN, GMG-ITC in the nanomolar range suppresses IL-3-induced expression of STAT5 target genes. GMG-ITC, like SFN, does not inhibit STAT5 phosphorylation, suggesting a downstream inhibitory event. Interestingly, treatment with GMG-ITC or SFN had a limited inhibitory effect on IFNα-induced STAT1 and STAT2 activity, indicating that both isothiocyanates differentially target JAK/STAT signaling pathways. Furthermore, we showed that GMG-ITC in the micromolar range is a more potent inhibitor of TNF-induced NF-κB activity than SFN. Finally, using a cellular system mimicking constitutive active STAT5-induced cell transformation, we demonstrated that SFN can reverse the survival and growth advantage mediated by oncogenic STAT5 and triggers cell death, therefore providing experimental evidence of a cancer chemopreventive activity of SFN. This work thus identified STAT5, and to a lesser extent STAT1/STAT2, as novel targets of moringin. It also contributes to a better understanding of the biological activities of the dietary isothiocyanates GMG-ITC and SFN and further supports their apparent beneficial role in the prevention of chronic illnesses such as cancer, inflammatory diseases and immune disorders.