Activated Protein Kinase Signaling Pathway Research Articles

Prevalence of Sequence Variants in the RAS-Mitogen Activated Protein Kinase Signaling Pathway in Pre-Adolescent Children With Hypertrophic Cardiomyopathy

Most cases of apparently idiopathic hypertrophic cardiomyopathy (HCM) in children are caused by mutations in cardiac sarcomere protein genes. HCM also commonly occurs as an associated feature in some patients with disorders caused by mutations in genes encoding components of the RAS-mitogen activated protein kinase (MAPK) signaling pathway. Although diagnosis of these disorders is based on typical phenotypic features, the dysmorphic manifestations can be subtle and therefore overlooked. The aim of this study was to determine the prevalence of mutations in RAS-MAPK genes in preadolescent children with idiopathic HCM. Seventy-eight patients diagnosed with apparently nonsyndromic HCM aged ≤13 years underwent clinical and genetic evaluation. The entire protein coding sequence of 9 genes implicated in Noonan syndrome and related conditions (PTPN11, SOS1, HRAS, KRAS, NRAS, BRAF, RAF1, MAP2K1, and MAP2K2), together with CBL (exons 8 and 9) and SHOC2 (4A>G), were screened for mutations. Five probands (6.4%) carried novel sequence variants in SOS1 (2 individuals), BRAF, MAP2K1, and MAP2K2. Structural and molecular data suggest that these variants may have functional significance. Nine cardiac sarcomere protein genes were screened also; 2 individuals also had mutations in MYBPC. This study reports novel and potentially pathogenic sequence variants in genes of the RAS-MAPK pathway, suggesting that genetic lesions promoting signaling dysregulation through RAS contribute to disease pathogenesis or progression in children with HCM.

Inhibitory effects of diallyl disulfide on the production of inflammatory mediators and cytokines in lipopolysaccharide-activated BV2 microglia

Diallyl disulfide (DADS), a main organosulfur component responsible for the diverse biological effects of garlic, displays a wide variety of internal biological activities. However, the cellular and molecular mechanisms underlying DADS' anti-inflammatory activity remain poorly understood. In this study, therefore, the anti-inflammatory effects of DADS were studied to investigate its potential therapeutic effects in lipopolysaccharide (LPS)-stimulated BV2 microglia. We found that pretreatment with DADS prior to treatment with LPS significantly inhibited excessive production of nitric oxide (NO) and prostaglandin E2 (PGE2) in a dose-dependent manner. The inhibition was associated with down-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression. DADS also attenuated the production of pro-inflammatory cytokines and chemokines, including interleukin-1β (IL-1β), tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (MCP-1) by suppressing the expression of mRNAs for these proteins. The mechanism underlying this protective effect might be related to the inhibition of nuclear factor-kappaB, Akt and mitogen-activated protein kinase signaling pathway activation in LPS-stimulated microglial cells. These findings indicated that DADS is potentially a novel therapeutic candidate for the treatment of various neurodegenerative diseases.

Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2

The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment.

Gene Expression Analysis of the Human Astrocytoma Cell after Aβ25-35 Stimulation Followed by Ibuprofen Administration

Background: The molecular events leading to the development of sporadic late-onset Alzheimer's disease (AD) have not been defined. A number of mechanism for the protective effects of non-steroidal anti-inflammatory drugs (NSAIDs) in AD have been proposed. We investigated the ibuprofen effect of global gene expression on the amyloid-β25-35 (Aβ25-35)-stim ulated human astrocytoma cell. Methods: U373MG, a human astrocytoma cell line, was incubated with 25 μM of aggregated Aβ25-35 or aggregated Aβ25-35 plus 100 μM ibuprofen at 37°C for 24 hours. Cells treated with ibuprofen alone were used as the negative control. Differential gene expression analysis was carried out with the Illumina human whole genome microarray. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was also done to validate the gene expression changes. After Welch's t-test, the significant subset of outlier genes were identified by an expression change cut-off 1.5 fold, p<0.05. Kyoto Encyclopedia of Genes and Genomes database was used for cellular signaling pathway analysis. Results: A total of 371 differentially expressed genes were identified from 16,692 detectable signals in Aβ25-35 peptide stimulated U373MG cells182 up-regulated genes with 21 biological pathways including biosynthesis of steroid, peroxisome proliferator-activated receptor signaling pathway and focal adhesion and 189 down-regulated genes with 14 biological pathways including transforming growth factor-beta signaling pathway, axon guidance and mitogen activated protein kinase signaling pathway. Ibuprofen suppressed the up-regulated expression of immunity/inflammation (especially, SERPINE1), signal pathway, metabolism and cancer-related genes. The expression of microarray data was confirmed by real-time RT-PCR. Conclusion: Aggregated Aβ25-35 induces expression of widespread transcriptional alterations, namely 21 functional groups 182 up-regulated genes and 14 functional groups 189 down-regulated genes in U373MG cells. Ibuprofen, a commonly used NSAID, suppressed Aβ25-35-induced increase of global changes in transcription of sets of genes especially immunity/inflammation, signal pathway, metabolism and cancer-related genes.

Polygoni Rhizoma Inhibits Inflammatory Response through Inactivation of Nuclear Factor‐kappaB and Mitogen Activated Protein Kinase Signaling Pathways in RAW264.7 Mouse Macrophage Cells

The objective of this study was to determine the antiinflammatory effects of Polygoni Rhizoma (PR), an Oriental medicinal herb, in interleukin 1 beta (IL-1β) and lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. PR significantly reduced the production of pro-inflammatory cytokines such as IL-6, tumor necrosis factor alpha (TNF-α) and pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase 2 (COX-2) and prostaglandin E₂ (PGE₂) even at a concentration of 1 µg/mL in the cells. In addition, PR inhibited the transcriptional activity of NF-κB as well as the degradation and phosphorylation of inhibitory kappa B alpha (IκBα). Furthermore, PR suppressed the phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase 1/2 (JNK1/2) in IL-1β and LPS-treated RAW264.7. The results suggest that PR exerts an antiinflammatory property by inhibiting iNOS, COX-2, TNF-α and IL-6 production in association with inactivation of the NF-κB and MAPK signaling pathways in RAW 264.7 cells.

Therapy of B-cell malignancies by anti–HLA-DR humanized monoclonal antibody, IMMU-114, is mediated through hyperactivation of ERK and JNK MAP kinase signaling pathways

AbstractA humanized IgG4 anti–HLA-DR monoclonal antibody (IMMU-114), engineered to avoid side effects associated with complement activation, was examined for binding and cytotoxicity on leukemia, lymphoma, and multiple myeloma cell lines and chronic lymphocytic leukemia (CLL) patient specimens, followed by evaluation of the effects of IMMU-114 on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. HLA-DR was expressed on the majority of these cells at markedly higher levels than CD20, CD22, and CD74. IMMU-114 was toxic to mantle cell lymphoma, CLL, acute lymphoblastic leukemia, hairy cell leukemia, non-Hodgkin lymphoma (including rituximab-resistant), and multiple myeloma cell lines, and also patient CLL cells. IMMU-114 induced disease-free survival in tumor-bearing SCID mice with early-stage disease and in models that are relatively resistant to anti-CD20 monoclonal antibodies. Despite positive staining, acute myelogenous leukemic cells were not killed by IMMU-114. The ability of IMMU-114 to induce activation of ERK and JNK signaling correlated with cytotoxicity and differentiates the mechanism of action of IMMU-114 from monoclonal antibodies against CD20 and CD74. Thus, antigen expression is not sufficient for cytotoxicity; antibody-induced hyperactivation of ERK and JNK mitogen activated protein kinase signaling pathways are also required.

Abstract B73: To sleep or die: Cell fate after chemotherapy

Abstract NOTE: This abstract was not presented at the conference. The DNA damage signaling network can be considered a computational device in which the input is the type and extent of the DNA damage and the output is cell cycle arrest and repair, permanent cell cycle arrest or cell death. A signaling network of protein kinases makes these cellular decisions, but how the cell commits to these different outcomes is unclear. To understand how these cell fates are determined we have generated a network level understanding of how this cellular decision process works through the synergistic application of experimental and computational methods. This project builds upon the work of Lauffenburger and Yaffe, who successfully monitored and modeled the activity of pro-death and pro-survival signal transduction pathways in response to EGF, insulin and TNFα. As was the case for the cytokine induced apoptotic decision, cellular signaling space has been probed using a combination of cues. In this case, Doxorubicin induced DNA damage in combination with TNF drives populations of cells to different levels of senescence and apoptosis. To understand the population dynamics and to be able to explore heterogeneous sub-populations of cells we have monitored our cellular results at the level of individual cells. Through the use of single cell methods we have been able to capture the cellular information processing by the DNA damage response, cell cycle machinery, apoptosis machinery and the stress/mitogen activated protein kinases. Using flow cytometry we have monitored the response of the U2-OS osteosarcoma cell line to doxorubicin treatment in combination with TNFa treatment. We quantitatively measured the proliferation, cell cycle arrest kinetics and apoptosis levels. Concomitantly, we have used high-content, fixed cell immuno-fluorescence of protein level, protein localization and phosphorylation state of 30 signaling proteins within the cell. We monitored the dynamics of signaling by taking multiple timepoints over the course of 96 hours. The intracellular signaling measurements and the cellular outcomes were selected because they provide a broad overview of the multiple regulatory pathways that are likely to be affected by the DNA damage response, including progression through various stages of the cell cycle, cell cycle arrest, initiation of DNA repair, survival and stress responses, chromatin remodeling and transcriptional regulation. A 4 hour pulse of 2 µM Dox induces a pulse of apoptosis with the surviving cells entering DNA damage induced cellular senescence. A 4 hour pulse of 10 µM Dox induces apoptosis. Adding saturating TNFα to these treatment increases the amount of apoptosis, but the 2 µM Dox + TNFα treatment still leads to senescence. Using single cell assays we capture the dynamics of the emergence of these various cell states. Principle Component Analysis of the response measurements has identified latent variables that correspond to proliferation, apoptosis and senescence. The ability of these unsupervised methods to identify biological relevant axes in cellular outcome space is striking. Partial Least Squares Regression of the signaling data against the response data identifies the anticipated components of the DNA damage response signaling pathways (e.g. gH2AX, p53), cell cycle machinery (e.g. Cyclin B, p21), apoptosis machinery (e.g. Bcl-XL) that regulate the cellular decision to apoptose or senesce. Interestingly, the regression analysis also identifies roles for the stress/mitogen activated protein kinase signaling pathways in modulating the cellular response to DNA damage. In particular, we have found that the activities of Erk, p38, Jnk and Akt contribute to the cell fate decision. Armed with this network understanding of how intracellular signaling defines phenotypic outcome we are capable of engineering the fate of heterogeneous cell populations. Such controlled modulation of cell fate could prove invaluable in a clinical or engineering situation. Citation Information: Cancer Res 2009;69(23 Suppl):B73.

The Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Single Oral Doses of CH4987655 in Healthy Volunteers: Target Suppression Using a Biomarker

CH4987655 (RO4987655) is an orally active and highly selective small-molecule MEK inhibitor. It potently inhibits mitogen-activated protein kinase signaling pathway activation and tumor cell growth, with an in vitro IC(50) of 5.2 nmol/L for inhibition of MEK1/2. Single-agent oral administration of CH4987655 resulted in complete tumor regressions in xenograft models. All 40 subjects received a single oral dose followed by 72 hrs of pharmacokinetic, pharmacodynamic, and safety/tolerability assessments. The pharmacodynamics were measured by changes in phosphorylated extracellular signal-regulated kinase (pERK) levels in a surrogate tissue peripheral blood mononuclear cells ex vivo stimulated by PMA. Doses of 0.5, 1, 2, 3, and 4 mg were safe and well tolerated. No clinically significant safety event was observed. A total of 26 adverse events (n = 15) were reported: 21 mild, 5 moderate, and none severe. Moderate adverse events were experienced by one subject at 1 mg (autonomic nervous system imbalance) and three subjects at 4 mg (diarrhea, abdominal pain, autonomic nervous system and acne). CH4987655 was rapidly absorbed with a t(max) of approximately 1 h. Exposures were dose proportional from 0.5 to 4 mg. The disposition was biphasic with a terminal t(1/2) of approximately 25 hr. Intersubject variability was low, 9% to 23% for C(max) and 14% to 25% for area-under-the-curve (AUC). pERK inhibition was exposure dependent and was greater than 80% inhibition at higher doses. The pharmacokinetic-pharmacodynamic relationship was characterized by an inhibitory E(max) model (E(max) approximately 100%; IC(50) 40.6 ng/mL) using nonlinear mixed-effect modeling. A significant extent of pERK inhibition was achieved for a single dose that was considered to be safe and well tolerated in healthy volunteers.

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG −/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.

Regulation of apoptosis in tumor cells by p38 mitogen-activated protein kinases signal transduction

p38 mitogen- activated protein kinases signaling pathway is an important way of Mitogen- ac-tivated protein kinases( MAPK) superfamily ,which can be activated by diverse extracellular stimuli and plays a central role in cell apoptosis process. p38MAPK is involved in apoptosis of malignant tumor by activating caspase family members,regulating activities of Bel-2 family members,activating p53,participating in Fas/FasL pathway and so on. This paper provides a brief review of p38MAPK,including structural characteristics,activity regulation and it' s role in apoptosis of malignant tumor. Key words: Neoplasms; Apoptosis; p38MAPK

Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis

A complex relationship exists between autophagy and apoptosis, but the regulatory mechanisms underlying their interactions are largely unknown. We conducted a systematic study of Drosophila melanogaster cell death–related genes to determine their requirement in the regulation of starvation-induced autophagy. We discovered that six cell death genes—death caspase-1 (Dcp-1), hid, Bruce, Buffy, debcl, and p53—as well as Ras–Raf–mitogen activated protein kinase signaling pathway components had a role in autophagy regulation in D. melanogaster cultured cells. During D. melanogaster oogenesis, we found that autophagy is induced at two nutrient status checkpoints: germarium and mid-oogenesis. At these two stages, the effector caspase Dcp-1 and the inhibitor of apoptosis protein Bruce function to regulate both autophagy and starvation-induced cell death. Mutations in Atg1 and Atg7 resulted in reduced DNA fragmentation in degenerating midstage egg chambers but did not appear to affect nuclear condensation, which indicates that autophagy contributes in part to cell death in the ovary. Our study provides new insights into the molecular mechanisms that coordinately regulate autophagic and apoptotic events in vivo.

Ginsenoside Rg1 activated CaMKIIα mediated extracellular signal-regulated kinase/mitogen activated protein kinase signaling pathway1

We carried out this study to investigate the effect of ginsenoside Rg1 on the extracellular signal-regulated kinase/mitogen activated protein kinase (ERK/ MAPK) pathway for understanding its effect on synaptic platicity.Western blotting and immunostaining were used to examine the phosphorylation of ERK1/2, CaMKIIalpha and cAMP response element binding protein (CREB) in PC12 cells and synaptosomes. The confocal microscopy and fluorescent indicator Fluo-3 was applied to observe the intracellular calcium ion flux.The phosphorylation of ERK1/2 in PC12 cells and synaptosomes incubated with Rg1 was increased and reached maximum at 4 min. Rg1 also promoted the transient enhancement of upstream calcium ion and activated CaMKIIalpha, which reached maximum at 2 min. CREB, the downstream protein, was phosphorylated within 8 min in PC12 cells after being incubated with Rg1. Moreover, KN93 partially inhibited the activation of ERK1/2, and PD98059 also partially blocked the phosphorylation of CREB.Rg1 activated ERK/MAPK pathway by CaMKIIalpha, and the activation of CREB was not only dependent on ERK induced by Rg1, which may provide an explanation for the effect of Rg1 on long-term potentiation.

Lysophosphatidic acid inhibits ghrelin secretion in the human gastric adenocarcinoma AGS cell line − role of mitogenic activated protein kinase signaling pathway

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor type 1a (GHS-R1a), is a 28 amino acid residue with a post-translational octanoyl modification on Ser3. Despite the biomedical interest in this hormone, the fine details of its regulation and the mechanisms controlling its secretion are largely unknown. The present study analyzes the molecular steps involved in the full lysophosphatidic acid (LPA) receptor-mediated activation of the mitogenic extracellular signal-regulated kinase (ERK) pathway and its consequent role as an inhibitor of ghrelin secretion in the gastric adenocarcinoma cell line AGS. ERK1/2 phosphorylation mediated by LPA proceeds via activation of the type 2 LPA receptor, activation of the nonreceptor tyrosine kinase c-Src, and subsequent transactivation of the epidermal growth factor receptor. Furthermore, LPA-induced ERK activation was found to be independent of matrix metalloproteinases; thus, c-Src acted as the scaffold-transactivating epidermal growth factor receptor. Finally, a correlation was observed between the mitogenic effects of LPA and ghrelin secretion in the human gastric adenocarcinoma cell line AGS. These data suggest a possible physiological role of LPA in ghrelin secretion. The relationship found between LPA and ghrelin secretion might explain the low circulating levels of ghrelin observed in obese patients, as a bona fide reflex of the energetic stores.

Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1

Nerve growth factor (NGF) induces an acute sensitization of nociceptive DRG neurons, in part, through sensitization of the capsaicin receptor TRPV1 via the high affinity trkA receptor. The mechanisms linking trkA and TRPV1 remain controversial with several candidate signaling pathways proposed. Utilizing adult rat and mouse DRG neurons and CHO cells co-expressing trkA and TRPV1, we have investigated the signaling events underlying acute TRPV1 sensitization by NGF combining biochemical, electrophysiological, pharmacological, mutational and genetic knockout approaches. Pharmacological interference with p42/p44 mitogen activated protein kinase (MAPK) or phosphoinositide-3-kinase (PI3K), but not PLC abrogated sensitization of capsaicin responses. Co-expression of TRPV1 with wild-type or Y785F (PLC signal deficient) mutant human trkA reconstituted NGF sensitization. In contrast, TRPV1 co-expressed with MAPK signaling deficient Y490A or PI3K signaling deficient Y751F trkA mutants exhibited weaker sensitization. Biochemical analysis of p42/p44 and Akt phosphorylation confirmed the specificity of pharmacological agents and trkA mutants. Finally, NGF sensitization of capsaicin responses was greatly reduced in neurons from p85α (regulatory subunit of PI3K) null mice. These data strongly suggest that PI3K and MAPK pathways, but not the PLC pathway underlie the acute sensitization of TRPV1 by NGF.

NMDA and Dopamine Converge on the NMDA-Receptor to Induce ERK Activation and Synaptic Depression in Mature Hippocampus

The formation of enduring internal representation of sensory information demands, in many cases, convergence in time and space of two different stimuli. The first conveys the sensory input, mediated via fast neurotransmission. The second conveys the meaning of the input, hypothesized to be mediated via slow neurotransmission. We tested the biochemical conditions and feasibility for fast (NMDA) and slow (dopamine) neurotransmission to converge on the Mitogen Activated Protein Kinase signaling pathways, crucial in several forms of synaptic plasticity, and recorded its effects upon synaptic transmission. We detected differing kinetics of ERK2 activation and synaptic strength changes in the CA1 for low and high doses of neurotransmitters in hippocampal slices. Moreover, when weak fast and slow inputs are given together, they converge on ERK2, but not on p38 or JNK, and induce strong short-term synaptic depression. Surprisingly, pharmacological analysis revealed that a probable site of such convergence is the NMDA receptor itself, suggesting it serves as a detector and integrator of fast and slow neurotransmission in the mature mammalian brain, as revealed by ERK2 activation and synaptic function.

Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development

Formation of the mammalian orofacial region involves multiple signaling pathways regulating sequential expression of and interaction between molecular signals during embryogenesis. The present study examined the expression patterns of members of the MAPK family in developing murine orofacial tissue. Total RNA was extracted from developing embryonic orofacial tissue during gestational days (GDs) 12-14 and used to prepare biotinylated cDNA probes, which were then denatured and hybridized to murine MAPK signaling pathways gene arrays. Expression of a number of genes involved in the (ERK1/2) cascade transiently increased in the embryonic orofacial tissue over the developmental period examined. Numerous members of the SAPK/JNK cascade were constitutively expressed in the tissue. Genes known to play a role in p38 MAPK signaling exhibited constitutive expression during orofacial development. Western blot analysis demonstrated that ERK2/1, p38, and SAPK/JNK kinases are present in embryonic orofacial tissue on each of GD 12, 13, and 14. By using phospho-specific antibodies, active ERK was shown to be temporally regulated during orofacial development. Minimal amounts of active p38 and active SAPK/JNK were detected in orofacial tissue during GDs 12-14. Our study documents specific expression patterns of genes coding for proteins belonging to the ERK1/2, p38, and SAPK/JNK MAPK families in embryonic orofacial tissue. We also demonstrate that active, phosphorylated forms of ERK1/2 only were detected in the embryonic tissue investigated, suggesting a more central role for members of this family in embryonic orofacial development.

Effects of magnetic field on MAPK signaling pathways of human retinal pigment epithelial cells bound with beads in vitro

To observe the changes of mitogen activated protein kinase (MAPK) signaling pathways of human retinal pigment epithelial cells( RPEs) bound with beads under stretch caused by the force from magnetic field in vitro. Ferric oxide microparticles, coAted with collagen, were added to dishes containing substrate-attached RPEs. After incubation, the cell layer bound with beads was laid in a magnetic field, the cells were stretched by the magnetic force. The activation status of the extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in RPEs was determined over a time course from 3 to 30 minutes with western-blot analysis. To examine the role of p38 kinase in the response to stretching, cells were grown for 30 minutes in the presence or absence of inhibitor of p38 (SB203580). The changes of the expression of active p38 kinase were observed with fluorescence staining. Total ERK, JNK, and p38 were detected in RPEs. Active ERK and p38 were detected but active JNK was not detected. Activation of ERK was unchanged during the time course. In contrast, p38 activation was barely detected in the normal cells, but this stress-activated protein kinase exhibited a robust activation after 5 minutes in the magnetic field. SB203580 blocked the p38 activation during stretch stimulation. The stretch stimulation also increased the fluorescence staining of active p38. A magnetic field can affect RPEs bound with beads. The effect may be partially through p38 signaling pathway.

Platelet-Derived Growth Factors and Receptors in the Rat Corpus Luteum: Localization and Identification of an Effect on Luteogenesis1

Platelet-derived growth factors (PDGFs) and their receptors (PDGFRs) play a vital role in regulating cell growth and angiogenesis. In this study, the expression of the family of PDGFs and PDGFRs in the ovarian corpus luteum were identified and characterized, and an effect of their activity on development of the corpus luteum revealed. Gonadotropin-stimulated immature rats were utilized as a model of induced ovulation, luteogenesis, and pseudopregnancy. Levels of ovarian mRNA for Pdgfb and Pdgfd, and their receptor, Pdgfrb, increased significantly as early as 4 h after human chorionic gonadotropin (hCG) injection in immature rats primed with equine chorionic gonadotropin (eCG). Gonadotropin regulation of Pdgfb expression was confirmed by in vitro promoter-reporter assays, which showed a 2- to 3-fold increase in Pdgfb promoter activity in response to luteinizing hormone (LH). Inhibition studies implicated protein kinase A, phosphatidylinositol 3-kinase and mitogen activated protein kinase signaling pathways in the LH-induced upregulation. In the corpus luteum, PDGFA, PDGFB, PDGFC, and PDGFRA were localized to a population of luteal parenchymal/steroidogenic cells. PDGFRB was expressed primarily in what appeared to be cells of the luteal microvasculature. Intraovarian injection of an inhibitor of PDGF receptor activity, the tyrphostin AG1295, prior to injection of hCG in eCG-primed immature rats resulted in a significant 21.86%+/-11.15% decrease in corpora lutea per treated ovary in comparison to the contralateral vehicle-injected control ovary. In addition, the treated ovary of 3 of 16 rats showed widespread hemorrhage throughout the entire ovary, indicating a possible role for PDGF receptor activity in maintenance of the ovarian vasculature.

Tumor necrosis factor α reversibly disrupts the blood–testis barrier and impairs Sertoli–germ cell adhesion in the seminiferous epithelium of adult rat testes

The timely restructuring of the blood-testis barrier (BTB) that facilitates the migration of preleptotene and leptotene spermatocytes from the basal to the adluminal compartment in the seminiferous epithelium of adult rat testes, which occurs at late stage VII through early stage VIII of the epithelial cycle, is a crucial cellular event of spermatogenesis. However, the regulation of BTB dynamics at the biochemical level remains elusive. In this study, tumor necrosis factor alpha (TNFalpha), a secretory product of Sertoli and germ cells in rat testes, was shown to affect junction dynamics in vivo. Following an acute administration of recombinant TNFalpha directly to adult rat testes in vivo at 0.5 and 2 mug/testis (with a body weight ~300 g), this treatment significantly and transiently disrupted the BTB. It also transiently inhibited the steady-state protein levels of occludin, zonula occludens-1, and N-cadherin, but not junction adhesion molecule-A, alpha-, and beta-catenin in testes at the BTB site as illustrated by immunoblottings, immunohistochemistry, electron microscopy, and fluorescent microscopy. This transient disruption of the BTB integrity induced by TNFalpha treatment was further demonstrated by a functional test to assess the passage of a fluorescent dye (e.g. fluorescein-5-isothiocyanate) from the systemic circulation to the adluminal compartment. Additionally, both the phosphorylated-Ser/Thr protein kinase activated by MAP kinase kinase (p-p38) and phosphorylated-externally regulated kinase (p-ERK) mitogen -activated protein kinase-signaling pathways were transiently activated. Collectively, these data coupled with the recently published in vitro studies have illustrated that the BTB is likely utilizing a novel mechanism in which localized production of TNFalpha by Sertoli and germ cells into the microenvironment at the basal compartment facilitates the timely restructuring ('opening'?) of the BTB during spermatogenesis to facilitate germ cell migration.

The Role of the Phospho-CDK2/Cyclin A Recruitment Site in Substrate Recognition

Phospho-CDK2/cyclin A, a kinase that is active in cell cycle S phase, contains an RXL substrate recognition site that is over 40 A from the catalytic site. The role of this recruitment site, which enhances substrate affinity and catalytic efficiency, has been investigated using peptides derived from the natural substrates, namely CDC6 and p107, and a bispeptide inhibitor in which the gamma-phosphate of ATP is covalently attached by a linker to the CDC6 substrate peptide. X-ray studies with a 30-residue CDC6 peptide in complex with pCDK2/cyclin A showed binding of a dodecamer peptide at the recruitment site and a heptapeptide at the catalytic site, but no density for the linking 11 residues. Kinetic studies established that the CDC6 peptide had an 18-fold lower Km compared with heptapeptide substrate and that this effect required the recruitment peptide to be covalently linked to the substrate peptide. X-ray studies with the CDC6 bispeptide showed binding of the dodecamer at the recruitment site and the modified ATP in two alternative conformations at the catalytic site. The CDC6 bispeptide was a potent inhibitor competitive with both ATP and peptide substrate of pCDK2/cyclin A activity against a heptapeptide substrate (Ki = 0.83 nm) but less effective against RXL-containing substrates. We discuss how localization at the recruitment site (KD 0.4 microm) leads to increased catalytic efficiency and the design of a potent inhibitor. The notion of a flexible linker between the sites, which must have more than a minimal number of residues, provides an explanation for recognition and discrimination against different substrates.