Molecular mechanisms of signal transducer and activator of transcription (STAT) protein function in hematopoiesis

Hematopoietic growth factors play important roles in regulating blood cell growth and development in vivo. In this work, we investigated the signaling mechanisms of two growth factors with clinical significance, erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF). Epo is essential for the survival, proliferation and differentiation of red blood cell progenitors, while G-CSF plays an important role in controlling mature neutrophil production. To identify which amino acid(s) and/or motif in EpoR is responsible for cell survival, wild type or mutant EpoR isoforms were transfected into the growth factor-dependent 32D cell line. Proliferation and apoptosis assays demonstrated that an EpoR isoform that lacks intracellular tyrosine residues and is truncated after 321 amino acids in the cytoplasmic tail (EpoR 1-321) mediates Epo-dependent cell survival. Furthermore, in absence of fetal calf serum (FCS), Epo signaling through wild type or mutant receptors supported anti-apoptosis, but not proliferation during 72 hours in response to Epo. To investigate the signaling pathway by which EpoR regulates cell survival, a dominant negative Stat5b (dnStat5b) isoform was generated and coexpressed with EpoR in stable cell lines. Expression of dnStat5b causes a significant induction of apoptosis in the presence of Epo in cells expressing EpoR 1-321, indicating that Stat5 is essential for survival signaling through tyrosine independent sequences in the EpoR. In a second project to investigate G-CSF signaling, we studied mechanisms by which G-CSF regulates the expression of PU.1, an important transcription factor in myeloid and B cell development. We demonstrated, by immunoblot and real time RT-PCR, that PU.1 is induced by G-CSF ex vivo as well as in vivo. To test whether G-CSF signaling through Stat3 is required for PU.1 regulation, the upstream region of the PU.1 gene was analyzed for potential Stat3 binding motifs. Four potential sites were identified; chromatin immunoprecipitations demonstrated that G-CSF activated Stat3 binds to 3 of the 4 binding motifs. In addition, PU.1 induction by G-CSF was completely abrogated in bone marrow from hematopoietic conditional Stat3 knockout mice. These results indicate an important role for Stat3 in G-CSF-dependent PU.1 gene regulation. Collectively, our works demonstrate that Stat protein play important and diverse roles in hematopoietic growth factor signaling. ^

Significance of signal transducer and activator of transcription 3 during multistage mouse skin carcinogenesis

Signal transducer and activator of transcription 3 (Stat3) is a signaling molecule that transduces signal from cell surface receptors, itself translocates into the nucleus, binds to consensus promoter sequences and activates gene transcription. Here, we showed that Stat3 is constitutively activated in both premalignant tumors (papillomas) and squamous cell carcinomas of mouse skin that is induced by topical treatment with an initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Additional data demonstrated that epidermal growth factor signaling contributes to the activation of Stat3 in this model. Using mice where Stat3 function is abrogated in keratinocytes via the Cre-LoxP system (K5Cre.Stat3 flox/flox), we demonstrated that Stat3 is required for de novo carcinogenesis since Stat3 deficiency leads to a complete abrogation of skin tumor development induced by DMBA and TPA. We subsequently showed that Stat3 plays a role in both the initiation and promotion stages of carcinogenesis. During initiation, Stat3 functions as an anti-apoptotic molecule for maintaining the survival of DNA-damaged keratinocyte stem cells. During promotion, Stat3 functions as a critical regulator for G1 to S phase cell cycle progression to confer selective clonal expansion of initiated cells into papillomas. On the other hand, using transgenic mice over-expressing a constitutively dimerized form of Stat3 (Stat3C) in keratinocytes (K5.Stat3C), we revealed a role for Stat3 in tumor progression. After treatment with DMBA and TPA, K5.Stat3C transgenic mice developed skin tumors with a shorter latency when 100% bypassed the premalignant stage and became carcinoma in situ. Histological and immunohistochemical analysis revealed these tumors as highly vascularized and poorly differentiated. More strikingly, these tumors exhibited invasion into surrounding mesenchymal tissue, some of which metastasized into lung. The tumor-mesenchymal front was characterized by partial loss of E-cadherin and elevation of vimentin, markers characterizing epithelial-mesenchymal transition. On the other hand, inhibition of Stat3 via a decoy oligonucleotide led to a significant reduction of tumor size in approximately 50% of all papillomas tested. In conclusion, we demonstrated that Stat3 plays a critical in all three stages (initiation, promotion and progression) of skin carcinogenesis, and it may potentially become a good target for cancer prevention and anti-cancer therapy. ^

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Molecular interactions and signal transfer between sensory rhodopsin-I and its cognate transducer

SRI is unique among known photoreceptors in that it produces opposite signals depending on the color of light stimuli. Absorption of orange light (587 nm) triggers an attractant response by the cell, whereas absorption of orange light followed by near-UV light (373 run) triggers a repellent response. Using behavioral mutants that exhibit aberrant color-sensing ability, we tested a two-conformation equilibrium model, using FRET and EPR spectroscopy. The essence of the model applied to SRI-HtrI is that the complex exists in a metastable two-conformer equilibrium which is shifted in one direction by orange light absorption (producing an attractant signal) and in the opposite direction by a second UV-violet photon (producing a repellent signal). First, by FRET we found that the E-F cytoplasmic loop of SRI moves toward the RAMP domain of the HtrI transducer during the formation of the orange-light activated signaling state of the complex. This is the first localization of a change in the physical relationship between the receptor and transducer subunits of the complex and provides a structural property of the two proposed conformers that we can monitor. Second, EPR spectra of a spin label probe at this cytoplasmic position showed shifts in the dark in the mutants toward shorter or longer EF loop-RAMP distances, explaining their behavior in terms of their mutations causing pre-stimulus shifts into one or the other conformer. ^ Next, we applied a novel electrophysiological method for monitoring the directionality of proton movement during photoactivation of SRI, to investigate the process of proton transfer in the photoactive site from the chromophore to proton acceptors on both the wildtype and aberrant color-response mutants. We observed an unexpected and critical difference in the two signaling conformations of the SRI-HtrI complex. The finding is that the vectoriality (i.e. movement away or toward the cytoplasm) of the light-induced proton transfer from the chromophore to the protein is opposite in formation of the two conformations. Retinylidene proton transfer is a common critical process in rhodopsins and these results are the first to show differences in vectoriality in a rhodopsin receptor, and to demonstrate functional importance of the direction of proton transfer. ^

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Effect of the Wake of the Transducer Supports on the Ultrasonic Anemometer Measurements

The wake produced by the structural supports of the ultrasonic anemometers (UAs)causes distortions in the velocity field in the vicinity of the sonic path. These distortions are measured by the UA, inducing errors in the determination of the mean velocity, turbulence intensity, spectrum, etc.; basic parameters to determine the effect of wind on structures. Additionally, these distortions can lead to indefinition in the calibration function of the sensors (Cuerva et al., 2004). Several wind tunnel tests have been dedicated to obtaining experimental data, from which have been developed fit models to describe and to correct these distortions (Kaimal, 1978 and Wyngaard, 1985). This work explores the effect of a vortex wake generated by the supports of an UA, on the measurement of wind speed done by this instrument. To do this, the Von Karman¿s vortex street potential model is combined with the mathematical model of the measuring process carried out by UAs developed by Franchini et al. (2007). The obtained results are the correction functions of the measured wind velocity, which depends on the geometry of the sonic anemometer and aerodynamic conditions. These results have been validated with the ones obtained in a wind tunnel test done on a single path UA, especially developed for research. The supports of this UA have been modified in order to reproduce the conditions of the theoretical model. Good agreements between experimental and theoretical results have been found.

Integrated phased array transducer for on-board structural health monitoring

Permanently bonded onto a structure, an integrated Phased Array (PhA II) transducer that can provide reliable electromechanical connection with corresponding sophisticated miniaturized ?all in one? SHM electronic device installed directly above it, without need for any interface cabling, during all aerospace structure lifecycle phases and for a huge variety of real harsh service environments of structures to be monitored is presented. This integrated PhA II transducer [1], as a key component of the PAMELA SHM? (Phased Array Monitoring for Enhanced Life Assessment) system, has two principal tasks at the same time, reliably transceive elastic waves in real aerospace service environments and serves as a reliable sole carrier or support for associated integrated on-board SHM electronic device attached above. The PhA II transducer successfully accomplished both required task throughout extensive test campaigns which included low to high temperature tests, temperature cycling, mechanical loading, combined thermo- mechanical loading and vibration resistance, etc. both with and without SHM device attached above due to RTCA DO-160F.

Simulation, construction and characterization of a piezoelectric transducer using rexolite as acoustic matching

This article describes the simulation and characterization of an ultrasonic transducer using a new material called Rexolite to be used as a matching element. This transducer was simulated using a commercial piezoelectric ceramic PIC255 at 8 MHz. Rexolite, the new material, presents an excellent acoustic matching, specially in terms of the acoustic impedance of water. Finite elements simulations were used in this work. Rexolite was considered as a suitable material in the construction of the transducer due to its malleability and acoustic properties, to validate the simulations a prototype transducer was constructed. Experimental measurements were used to determine the resonance frequency of the prototype transducer. Simulated and experimental results were very similar showing that Rexolite may be an excellent matching, particularly for medical applications.

Compact duplexing for a 680-GHz radar using a waveguide orthomode transducer

A compact 680-GHz waveguide orthomode transducer (OMT) and circular horn combination has been designed, tested, and characterized in a radar transceiver's duplexer. The duplexing capability is implemented by a hybrid waveguide quasi optical solution, combining a linear polarization OMT and an external grating polarizer. Isolation between the OMT's orthogonal ports' flanges was measured with a vector network analyzer to exceed 33 dB over a >10% bandwidth between 630 and 710 GHz. Calibrated Y-factor measurements using a mixer attached to the OMT ports reveal losses through the transmit and receive paths that sum to an average of 4.7 dB of two-way loss over 660-690 GHz. This is consistent with radar sensitivity measurements comparing the new OMT/horn with a quasi-optical wire grid beam splitter. Moreover, the radar performance assessment validates the OMT as a suitable compact substitute of the wire grid for the JPL's short-range 680-GHz imaging radar.

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-γ target genes

Most of the activities of IFN-γ are the result of STAT1-mediated transcriptional responses. In this study, we show that the BRCA1 tumor suppressor acts in concert with STAT1 to differentially activate transcription of a subset of IFN-γ target genes and mediates growth inhibition by this cytokine. After IFN-γ treatment, induction of the cyclin-dependent kinase inhibitor, p21WAF1, was synergistically activated by BRCA1, whereas the IRF-1 gene was unaffected. Importantly, the differential induction of p21WAF1 was impaired in breast cancer cells homozygous for the mutant BRCA1 5382C allele. Biochemical analysis illustrated that the mechanism of this transcriptional synergy involves interaction between BRCA1 aa 502–802 and the C-terminal transcriptional activation domain of STAT1 including Ser-727 whose phosphorylation is crucial for transcriptional activation. Significantly, STAT1 proteins mutated at Ser-727 bind poorly to BRCA1, reinforcing the importance of Ser-727 in the recruitment of transcriptional coactivators by STAT proteins. These findings reveal a novel mechanism for BRCA1 function in the IFN-γ-dependent tumor surveillance system.

Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT) 1 (PIAS1) in cytokine-induced PIAS1–Stat1 interaction

STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)–phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1–Stat1 interaction has not been understood. We report here that a region near the COOH terminus of PIAS1 (amino acids 392–541) directly interacts with the NH2-terminal domain of Stat1 (amino acids 1–191). A mutant PIAS1 lacking the Stat1-interacting domain failed to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, we demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH2-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1–Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1.

Signal transducer and activator of transcription 3 in the heart transduces not only a hypertrophic signal but a protective signal against doxorubicin-induced cardiomyopathy

The signal transducer and activator of transcription (STAT) 3, a transcriptional factor downstream of several cytokines, is activated by Janus kinase families and plays a pivotal role in cardiac hypertrophy through gp130. To determine the physiological significance of STAT3 in vivo, transgenic mice with cardiac-specific overexpression of the Stat3 gene (STAT3-TG) were generated. STAT3-TG manifested myocardial hypertrophy at 12 wk of age with increased expression of the atrial natriuretic factor (ANF), β-myosin heavy chain (MHC), and cardiotrophin (CT)-1 genes. The animals were injected i.p. with 15 mg/kg doxorubicin (Dox), an antineoplastic drug with restricted use because of its cardiotoxicity. The survival rates after 10 days were 25% (5/20) for control littermates (WT), but 80% (16/20) for STAT3-TG (P < 0.01). WT showed increased expression of β-MHC and ANF mRNAs in the hearts 1 day after Dox treatment; this expression peaked at 3 days, suggesting that the WT suffered from congestive heart failure. Although the expression of these mRNAs was elevated in STAT3-TG hearts before Dox treatment, no additional increase was observed after the treatment. Dox administration significantly reduced the expression of the cardiac α-actin and Stat3 genes in WT hearts but not in STAT3-TG. These results provide direct evidence that STAT3 transduces not only a hypertrophic signal but also a protective signal against Dox-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors.

Two mechanisms for transducer adaptation in vertebrate hair cells

Deflection of the hair bundle atop a sensory hair cell modulates the open probability of mechanosensitive ion channels. In response to sustained deflections, hair cells adapt. Two fundamentally distinct models have been proposed to explain transducer adaptation. Both models support the notion that channel open probability is modulated by calcium that enters via the transduction channels. Both also suggest that the primary effect of adaptation is to shift the deflection-response [I(X)] relationship in the direction of the applied stimulus, thus maintaining hair bundle sensitivity. The models differ in several respects. They operate on different time scales: the faster on the order of a few milliseconds or less and the slower on the order of 10 ms or more. The model proposed to explain fast adaptation suggests that calcium enters and binds at or near the transduction channels to stabilize a closed conformation. The model proposed to explain the slower adaptation suggests that adaptation is mediated by an active, force-generating process that regulates the effective stimulus applied to the transduction channels. Here we discuss the evidence in support of each model and consider the possibility that both may function to varying degrees in hair cells of different species and sensory organs.