Transcription Factors Fos Research Articles

AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae

Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae.

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

A Phenotypic Assay to Identify Chikungunya Virus Inhibitors Targeting the Nonstructural Protein nsP2

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for an acute infection of abrupt onset, characterized by high fever, polyarthralgia, myalgia, headaches, chills, and rash. In 2006, CHIKV was responsible for an epidemic outbreak of unprecedented magnitude in the Indian Ocean, stressing the need for therapeutic approaches. Since then, we have acquired a better understanding of CHIKV biology, but we are still missing active molecules against this reemerging pathogen. We recently reported that the nonstructural nsP2 protein of CHIKV induces a transcriptional shutoff that allows the virus to block cellular antiviral response. This was demonstrated using various luciferase-based reporter gene assays, including a trans-reporter system where Gal4 DNA binding domain is fused to Fos transcription factor. Here, we turned this assay into a high-throughput screening system to identify small molecules targeting nsP2-mediated shutoff. Among 3040 molecules tested, we identified one natural compound that partially blocks nsP2 activity and inhibits CHIKV replication in vitro. This proof of concept suggests that similar functional assays could be developed to target other viral proteins mediating a cellular shutoff and identify innovative therapeutic molecules.

(−)-Epigallocatechin-3-gallate induces apoptosis in human endometrial adenocarcinoma cells via ROS generation and p38 MAP kinase activation

(−)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been shown to inhibit carcinogenesis of various tumor types. The aim of this study was to elucidate the antiproliferative potential of EGCG and its mechanism in human endometrial cancer cells (Ishikawa cells) and primary endometrial adenocarcinoma cells. The antiproliferative effect of EGCG was evaluated by cell viability assay. Apoptosis was measured by annexin/propidium iodide staining. Reactive oxygen species (ROS) generation was measured by using 2’,7’-dichlorofluorescin diacetate dye. Expression of mitogen-activated protein kinases, proliferation and apoptotic markers were measured by immunoblot analysis. EGCG was found to inhibit proliferation in Ishikawa as well as in primary endometrial adenocarcinoma cells and effectively down-regulated the expression of proliferation markers, i.e., estrogen receptor α, progesterone receptor, proliferating cell nuclear antigen and cyclin D1. EGCG also decreased the activation of ERK and downstream transcription factors fos and jun. EGCG caused apoptotic cell death accompanied by up-regulation of proapoptotic Bax and down-regulation of antiapoptotic protein Bcl2. EGCG induced the cleavage of caspase-3 and poly(ADP-ribose) polymerase, the hallmark of apoptosis. EGCG significantly induced the ROS generation as well as p38 activation in Ishikawa cells, which appeared to be a critical mediator in EGCG-induced apoptosis. The apoptotic effect of EGCG and the p38 activation were blocked by pretreatment of cells with the ROS scavenger N-acetylcysteine. EGCG reduced the glutathione levels, which might be responsible for enhanced ROS generation causing oxidative stress in endometrial cancer cells. Taken together, these results suggest that EGCG inhibits cellular proliferation via inhibiting ERK activation and inducing apoptosis via ROS generation and p38 activation in endometrial carcinoma cells.

Fra-1 Promotes Breast Cancer Chemosensitivity by Driving Cancer Stem Cells from Dormancy

Fra-1 is a member of the Fos transcription factor family that is highly expressed in multiple cancers, playing important roles in transformation, proliferation, and metastasis. In this study, we observed an inverse correlation between the expression of Fra-1 in human stage II breast cancer tissues and the corresponding level of clinical chemoresistance. Extending these findings in vitro, we found that knockdown of Fra-1 in breast tumor cells was sufficient to confer resistance to doxorubicin and cyclophosphamide, whereas enhanced Fra-1 expression could render these cells chemosensitive. The tumor cell side population, which is enriched for cancer stem cells, was found to be associated with chemoresistance. Increased side population fractions were detected among tumor cell lines subjected to Fra-1 knockdown. In contrast, enhanced expression of Fra-1 was correlated with a decreased side population fraction, and significantly, this finding was recapitulated in vivo, where tumors with enhanced expression of Fra-1 were found to have blunted growth. Tumor cells subjected to Fra-1 knockdown grew faster and were larger in size. Taken together, our findings suggest that Fra-1 may be an important prognostic marker for breast cancer therapy.

The effects of protein kinase C inhibitors on the contents of several transcription and apoptosis modulating factors in the spinal cords of rats with diabetic neuropathy

We studied changes in the contents of the transcription factors NF-kB and Fos, the apoptosis regulating factors bax and bcl-xl, and a marker of astroglial activity, viz., the glial fibrillar acidic protein, in the spinal cords of rats with experimental streptozotocin-induced diabetes. We found that shifts in the levels of these factors after induction of diabetes may be partially prevented by administration of the protein kinase C (PKC) inhibitors chelerythrine hydrochloride and rottlerin. Our data show that these substances are promising drugs for the correction of diabetic neuropathy. PKC isoenzymes contribute to pathogenesis of diabetic neuropathy differently; therefore, the development of selective inhibitors for specific PKC isoenzymes may improve their efficacy and decrease the probability of the appearance of side effects.

Ubiquitous [Na+]i/[K+]i-Sensitive Transcriptome in Mammalian Cells: Evidence for Ca2+i-Independent Excitation-Transcription Coupling

Stimulus-dependent elevation of intracellular Ca2+ ([Ca2+]i) affects the expression of numerous genes – a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na+]i trigger c-Fos expression via a novel Ca2+ i-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na+]i/[K+]i-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na+]i and reduce [K+]i, cells were treated for 3 hrs with the Na+,K+-ATPase inhibitor ouabain or placed for the same time in the K+-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p<0.0001; R2>0.62). Among these Na+ i/K+ i-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca2+]i, we performed identical experiments in Ca2+-free media supplemented with extracellular and intracellular Ca2+ chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na+ i/K+ i-sensitive genes. Among the ubiquitous Na+ i/K+ i-sensitive genes whose expression was regulated independently of the presence of Ca2+ chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca2+-depleted cells. Overall, our findings indicate that Ca2+ i-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na+]i/[K+]i ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders.

Mono-(2-ethylhexyl)-phthalate (MEHP) affects ERK-dependent GDNF signalling in mouse stem-progenitor spermatogonia

Many commercial and household products such as lubricants, cosmetics, plastics, and paint contain phthalates, in particular bis-(2-ethyhexyl)-phthalate (DEHP). As a consequence, phthalates have been found in a number of locations and foods (streambeds, household dust, bottled water and dairy products). Epidemiological and animal studies analysing phthalate exposure in males provide evidence of degradation in sperm quality, associated to an increase in the incidence of genital birth defects and testicular cancers. In the testis, spermatogenesis is maintained throughout life by a small number of spermatogonial stem cells (SSCs) that self-renew or differentiate to produce adequate numbers of spermatozoa. Disruption or alteration of SSC self-renewal induce decreased sperm count and sperm quality, or may potentially lead to testicular cancer. GDNF, or glial cell-line-derived neurotrophic factor, is a growth factor that is essential for the self-renewal of SSCs and continuous spermatogenesis. In the present study, the SSC-derived cell line C18-4 was used as a model for preliminary assessment of the effects of mono-(2-ethylhexyl)-phthalate (MEHP, main metabolite of DEHP) on spermatogonial stem cells. Our data demonstrate that MEHP disrupts one of the known GDNF signalling pathways in these cells. MEHP induced a decrease of C18-4 cell viability in a time- and dose-dependent manner, as well as a disruption of ERK1/2 activation but not of SRC signalling. As a result, we observed a decrease of expression of the transcription factor FOS, which is downstream of the GDNF/ERK1/2 axis in these cells. Taken together, our data suggest that MEHP exposure affects SSC proliferation through inhibition of specific signalling molecules.

AP-1 Transcription Factors, Mucin-Type Molecules and MMPs Regulate the IL-11 Mediated Invasiveness of JEG-3 and HTR-8/SVneo Trophoblastic Cells

This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo cells.

Transcriptional regulation of Profilin during wound closure in Drosophila larvae

Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. Here, we show that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. We found that JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, we show that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. Our results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized.

Transcription factor co-localization patterns affect human cell type-specific gene expression.

BackgroundCellular development requires the precise control of gene expression states. Transcription factors are involved in this regulatory process through their combinatorial binding with DNA. Information about transcription factor binding sites can help determine which combinations of factors work together to regulate a gene, but it is unclear how far the binding data from one cell type can inform about regulation in other cell types.ResultsBy integrating data on co-localized transcription factor binding sites in the K562 cell line with expression data across 38 distinct hematopoietic cell types, we developed regression models to describe the relationship between the expression of target genes and the transcription factors that co-localize nearby. With K562 binding sites identifying the predictors, the proportion of expression explained by the models is statistically significant only for monocytic cells (p-value< 0.001), which are closely related to K562. That is, cell type specific binding patterns are crucial for choosing the correct transcription factors for the model. Comparison of predictors obtained from binding sites in the GM12878 cell line with those from K562 shows that the amount of difference between binding patterns is directly related to the quality of the prediction. By identifying individual genes whose expression is predicted accurately by the binding sites, we are able to link transcription factors FOS, TAF1 and YY1 to a sparsely studied gene LRIG2. We also find that the activity of a transcription factor may be different depending on the cell type and the identity of other co-localized factors.ConclusionOur approach shows that gene expression can be explained by a modest number of co-localized transcription factors, however, information on cell-type specific binding is crucial for understanding combinatorial gene regulation.

Hole-in-One Mutant Phenotypes Link EGFR/ERK Signaling to Epithelial Tissue Repair in Drosophila

BackgroundEpithelia act as physical barriers protecting living organisms and their organs from the surrounding environment. Simple epithelial tissues have the capacity to efficiently repair wounds through a resealing mechanism. The known molecular mechanisms underlying this process appear to be conserved in both vertebrates and invertebrates, namely the involvement of the transcription factors Grainy head (Grh) and Fos. In Drosophila, Grh and Fos lead to the activation of wound response genes required for epithelial repair. ERK is upstream of this pathway and known to be one of the first kinases to be activated upon wounding. However, it is still unclear how ERK activation contributes to a proper wound response and which molecular mechanisms regulate its activation.Methodology/Principal FindingsIn a previous screen, we isolated mutants with defects in wound healing. Here, we describe the role of one of these genes, hole-in-one (holn1), in the wound healing process. Holn1 is a GYF domain containing protein that we found to be required for the activation of several Grh and Fos regulated wound response genes at the wound site. We also provide evidence suggesting that Holn1 may be involved in the Ras/ERK signaling pathway, by acting downstream of ERK. Finally, we show that wound healing requires the function of EGFR and ERK signaling.Conclusions/SignificanceBased on these data, we conclude that holn1 is a novel gene required for a proper wound healing response. We further propose and discuss a model whereby Holn1 acts downstream of EGFR and ERK signaling in the Grh/Fos mediated wound closure pathway.

A Protein Epitope Signature Tag (PrEST) Library Allows SILAC-based Absolute Quantification and Multiplexed Determination of Protein Copy Numbers in Cell Lines

Mass spectrometry-based proteomics increasingly relies on relative or absolute quantification. In relative quantification, stable isotope based methods often allow mixing at early stages of sample preparation, whereas for absolute quantification this has generally required recombinant expression of full length, labeled protein standards. Here we make use of a very large library of Protein Epitope Signature Tags (PrESTs) that has been developed in the course of the Human Protein Atlas Project. These PrESTs are expressed recombinantly in E. coli and they consist of a short and unique region of the protein of interest as well as purification and solubility tags. We first quantify a highly purified, stable isotope labeling of amino acids in cell culture (SILAC)-labeled version of the solubility tag and use it determine the precise amount of each PrEST by its SILAC ratios. The PrESTs are then spiked into cell lysates and the SILAC ratios of PrEST peptides to peptides from endogenous target proteins yield their cellular quantities. The procedure can readily be multiplexed, as we demonstrate by simultaneously determining the copy number of 40 proteins in HeLa cells. Among the proteins analyzed, the cytoskeletal protein vimentin was found to be most abundant with 20 million copies per cell, while the transcription factor and oncogene FOS only had 6000 copies. Direct quantification of the absolute amount of single proteins is possible via a SILAC experiment in which labeled cell lysate is mixed both with the heavy labeled solubility tag and with the corresponding PrEST. The SILAC-PrEST combination allows accurate and streamlined quantification of the absolute or relative amount of proteins of interest in a wide variety of applications.

Decreased pro‐inflammatory cytokines and increased CCR7 expression on T‐lymphocyte subsets are predictive of response to extracorporeal photopheresis in patients with GvHD

Decreased pro‐inflammatory cytokines and increased CCR7 expression on T‐lymphocyte subsets are predictive of response to extracorporeal photopheresis in patients with GvHD

Timothy grass pollen extract‐induced gene expression and signalling pathways in airway epithelial cells

Grass pollen allergy is one of the most common allergies worldwide and airborne allergens are the major cause of allergic rhinitis. Airway epithelial cells (AECs) are the first to encounter and respond to aeroallergens and are therefore interesting targets for the development of new therapeutics. Our understanding of the epithelial contribution to immune responses is limited as most studies focus on only a few individual genes or proteins. To describe in detail the Timothy grass pollen extract (GPE)-induced gene expression in AECs. NCI-H292 cells were exposed to GPE for 24 h, and isolated RNA and cell culture supernatants were used for microarray analysis and multiplex ELISA, respectively. Eleven thousand and seven hundred fifty-eight transcripts were affected after exposure to GPE, with 141 genes up-regulated and 121 genes down-regulated by more than threefold. The gene ontology group cell communication was among the most prominent categories. Network analysis revealed that a substantial part of regulated genes are related to the cytokines IL-6, IL-8, IL-1A, and the transcription factor FOS. After analysing significantly regulated signalling pathways, we found, among others, epidermal growth factor receptor 1, IL-1, Notch-, and Wnt-related signalling members. Unexpectedly, we found Jagged to be down-regulated and an increased release of IL-12, in line with a more Th1-biased response induced by GPE. Our data show that the stimulation of AECs with GPE results in the induction of a broad response on RNA and protein level by which they are able to affect the initiation and regulation of local immune responses. Detailed understanding of GPE-induced genes and signalling pathways will allow us to better define the pathogenesis of the allergic response and to identify new targets for treatment.

EGF signaling regulates the proliferation of intestinal stem cells in Drosophila

Precise control of somatic stem cell proliferation is crucial to ensure maintenance of tissue homeostasis in high-turnover tissues. In Drosophila, intestinal stem cells (ISCs) are essential for homeostatic turnover of the intestinal epithelium and ensure epithelial regeneration after tissue damage. To accommodate these functions, ISC proliferation is regulated dynamically by various growth factors and stress signaling pathways. How these signals are integrated is poorly understood. Here, we show that EGF receptor signaling is required to maintain the proliferative capacity of ISCs. The EGF ligand Vein is expressed in the muscle surrounding the intestinal epithelium, providing a permissive signal for ISC proliferation. We find that the AP-1 transcription factor FOS serves as a convergence point for this signal and for the Jun N-terminal kinase (JNK) pathway, which promotes ISC proliferation in response to stress. Our results support the notion that the visceral muscle serves as a functional 'niche' for ISCs, and identify FOS as a central integrator of a niche-derived permissive signal with stress-induced instructive signals, adjusting ISC proliferation to environmental conditions.

PPARG Regulates Gonadotropin-Releasing Hormone Signaling in LbetaT2 Cells In Vitro and Pituitary Gonadotroph Function In Vivo in Mice1

Peroxisome proliferators-activated receptor gamma (PPARG) ligands improve insulin sensitivity in type 2 diabetes and polycystic ovarian syndrome (PCOS). Despite clinical studies showing normalization of pituitary responsiveness to gonadotropin-releasing hormone (GnRH) in patients with PCOS, the precise role of PPARG in regulating the hypothalamic-pituitary-gonadal axis remains unclear. In the present study, we tested the hypothesis that the PPARG agonist rosiglitazone has a direct effect on the pituitary. In mouse LbetaT2 immortalized gonadotrophs, rosiglitazone treatment inhibited GnRH stimulation of the stress kinases p38MAPK and MAPKs/JNKs, but did not alter activation of ERKs, both in the presence and absence of activin. Furthermore, p38MAPK signaling was critical for both Lhb and Fshb promoter activity, and rosiglitazone suppressed the GnRH-mediated induction of Lhb and Fshb mRNA. Depletion of PPARG using a lentivirally encoded short hairpin RNA abolishes the effect of rosiglitazone to suppress activation of JNKs and induction of the transcription factors EGR1 and FOS as well as the gonadotropin genes Lhb and Fshb. Lastly, we show conditional knockout of Pparg in pituitary gonadotrophs caused an increase in luteinizing hormone levels in female mice, a decrease in follicle-stimulating hormone in male mice, and a fertility defect characterized by reduced litter size. Taken together, our data support a direct role for PPARG in modulating pituitary function in vitro and in vivo.

Fos expression induced by activation of NMDA and neurokinin-1 receptors in the trigeminal subnucleus caudalis in vitro: Role of protein kinases

Activity-induced neuronal plasticity is partly facilitated by the expression of the immediate-early gene c-fos and the resulting transcription factor Fos. Expression of Fos is associated with nociceptive afferent activation, but a detailed stimulation-transcription pathway for Fos expression has not yet been determined in the trigeminal system. This study utilized a novel in vitro model to determine whether Fos expression can be induced in trigeminal subnucleus caudalis by NMDA or neurokinin-1 receptor activation, and whether inhibition of intracellular kinases has any effect on Fos expression induced by activation of these receptors. Brainstems of male Wistar rats were excised and maintained in artificial cerebrospinal fluid at 37 °C. NMDA or the specific neurokinin-1 receptor agonist [Sar 9,Met(O 2) 11]-SP was applied. These agonists were subsequently tested in the presence of the protein kinase A inhibitor Rp-cAMP or protein kinase C inhibitor chelerythrine chloride. In all experiments the sodium channel blocker tetrodotoxin was used to prevent indirect neuronal activation. Brainstems were processed immunocytochemically for Fos expression, and positive cells were counted in the trigeminal subnucleus caudalis. NMDA and [Sar 9,Met(O 2) 11]-SP significantly increased Fos expression, but these increases could be prevented by chelerythrine chloride. Rp-cAMP had no effect on Fos induced by NMDA but caused a significant reduction in Fos induced by [Sar 9,Met(O 2) 11]-SP. These data demonstrate that in trigeminal subnucleus caudalis activation of either NK1 or NMDA receptors alone induces Fos expression; protein kinases A and C are involved in NK1R-induced Fos while protein kinase A is not required for NMDA receptor-induced Fos.

Protein turnover of the Wallenda/DLK kinase regulates a retrograde response to axonal injury

Regenerative responses to axonal injury involve changes in gene expression; however, little is known about how such changes can be induced from a distant site of injury. In this study, we describe a nerve crush assay in Drosophila melanogaster to study injury signaling and regeneration mechanisms. We find that Wallenda (Wnd), a conserved mitogen-activated protein kinase (MAPK) kinase kinase homologous to dual leucine zipper kinase, functions as an upstream mediator of a cell-autonomous injury signaling cascade that involves the c-Jun NH(2)-terminal kinase MAPK and Fos transcription factor. Wnd is physically transported in axons, and axonal transport is required for the injury signaling mechanism. Wnd is regulated by a conserved E3 ubiquitin ligase, named Highwire (Hiw) in Drosophila. Injury induces a rapid increase in Wnd protein concomitantly with a decrease in Hiw protein. In hiw mutants, injury signaling is constitutively active, and neurons initiate a faster regenerative response. Our data suggest that the regulation of Wnd protein turnover by Hiw can function as a damage surveillance mechanism for responding to axonal injury.

Eccentric Exercise Activates Novel Transcriptional Regulation of Hypertrophic Signaling Pathways Not Affected by Hormone Changes

Unaccustomed eccentric exercise damages skeletal muscle tissue, activating mechanisms of recovery and remodeling that may be influenced by the female sex hormone 17β-estradiol (E2). Using high density oligonucleotide based microarrays, we screened for differences in mRNA expression caused by E2 and eccentric exercise. After random assignment to 8 days of either placebo (CON) or E2 (EXP), eighteen men performed 150 single-leg eccentric contractions. Muscle biopsies were collected at baseline (BL), following supplementation (PS), +3 hours (3H) and +48 hours (48H) after exercise. Serum E2 concentrations increased significantly with supplementation (P<0.001) but did not affect microarray results. Exercise led to early transcriptional changes in striated muscle activator of Rho signaling (STARS), Rho family GTPase 3 (RND3), mitogen activated protein kinase (MAPK) regulation and the downstream transcription factor FOS. Targeted RT-PCR analysis identified concurrent induction of negative regulators of calcineurin signaling RCAN (P<0.001) and HMOX1 (P = 0.009). Protein contents were elevated for RND3 at 3H (P = 0.02) and FOS at 48H (P<0.05). These findings indicate that early RhoA and NFAT signaling and regulation are altered following exercise for muscle remodeling and repair, but are not affected by E2.