The role of MAPK during the activation of NK cells

Although many clinical trials investigated the use of IL-2, IL-12, and LAK in adoptive immunotherapy to treat cancer, only limited clinical success has been achieved. Better understanding of the intracellular processes that IL-2 and IL-12 utilize to generate LAK and other functions in NK cells is necessary to improve this mode of therapy. IL-2 and IL-12 stimulate extracellular signal-regulated protein kinase (ERK) and p38 MAPK in mitogen-activated T lymphocytes. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK Kinase (MKK)/ERK and/or p38 MAPK pathways are necessary for IL-2 or IL-12 to activate NK cells. We established that IL-2 activates MKK1/2/ERK pathway in freshly isolated human NK cells without any prior stimulation. Furthermore, we determined that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK activity, IFN-γ secretion, and CD25 and CD69 expression. Treatment of NK cells with a specific inhibitor of MKK1/2 PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four activation parameters. Although activation of ERK was not detected in NK cells immediately after IL-12 stimulation, IL-12-induced functional activation was inhibited by the MKK1/2 inhibitor, as well. In contrast to what was observed by others in T lymphocytes, activation of p38 MAPK by IL-2 was not detected in NK cells. Additionally, a specific inhibitor of p38 MAPK (SB203850) did not inhibit IL-2-activated NK functions. These data reveal selective signaling differences between NK cells and T lymphocytes. Collectively, the data support that the MKK/ERK pathway plays a critical positive regulatory role in NK cells during activation by IL-2 and IL-12. ^

Temporal analysis of PI-3kinase and MAPK signal transduction during skeletal muscle overload

Skeletal muscles can adapt to increased mechanical forces (or loading) by increasing the size and strength of the muscle. Knowledge of the molecular mechanisms by which muscle responds to increased loading may lead to the discovery of novel treatment strategies for muscle wasting and frailty. The objective of this research was to examine the temporal associations between the activation of specific signaling pathway intermediates and their potential upstream regulator(s) in response to increased muscle loading. Previous work has demonstrated that focal adhesion kinase (FAK) activity is increased in overloaded hypertrophying skeletal muscle. Thus FAK is a candidate for transducing the loading stimulus in skeletal muscle, potentially by activating phosphatidylinositol 3-kinase (PI3K) and members of the mitogen-activated protein kinase (MAPK) family. However, it was unknown if muscle overload would result in activation of PI3K or the MAPKs. Thus, this work seeks to characterized the temporal response of (1) MAPK phosphorylation (including Erk 2, p38 MAPK and JNK), (2) PI3K activity, and (3) FAK tyrosine phosphorylation in response to 24 hours of compensatory overload in the rat soleus and plantaris muscles. In both muscles, overload resulted in transient Increases in the phosphorylation state of Erk2 and JNK, which peaked within the first hour of overload and returned to baseline thereafter. In contrast, p38 MAPK phosphorylation remained elevated throughout the entire 24-hour overload period. Moreover, overload increased PI3K activity only, in the plantaris and only at 12 hours. Moreover, 24 hours of overload induced a significant increase in total protein content in the plantaris but not the soleus. Thus an increase in total muscle protein content within the 24-hour loading period was observed only in muscle exhibiting increased PI3K activity. Surprisingly, FAK tyrosine phosphorylation was not increased during the overload period in either muscle, indicating that PI3K activation and increased MAPK phosphorylation were independent of increased FAK tyrosine phosphorylation. In summary, increased PI3K activity and sustained elevation of p38 MAPK phosphorylation were associated with muscle overload, identifying these pathways as potential mediators of the early hypertrophic response to skeletal muscle overload. This suggests that stimuli or mechanisms that activate these pathways may reduce/minimize muscle wasting and frailty. ^

MUTATIONS IN STAT3 ASSOCIATED WITH HUMAN HYPER IgE SYNDROME ENHANCE NFκB AND MAPK-MEDIATED GENE EXPRESSION

Hyper IgE syndrome (HIES) is a multisystem disorder resulting in bone and immune system abnormalities. It is associated with mutations in STAT3, which disrupt protein domains responsible for transcriptional function. Patients with HIES display osteoporosis and enhanced inflammatory cytokine production similar to hematopoietic Stat3-deficient mice. Since osteoclast and inflammatory cytokine genes are NFκB targets, these observations indicate a possible deregulation of NFκB signaling in both mice and humans with STAT3-deficiency. Here, we sought to examine the role of STAT3 in the regulation of NFκB-mediated gene expression through analysis of three HIES STAT3 point mutations in both hematopoietic and non- hematopoietic cells. We found that IL-6-induced tyrosine phosphorylation of STAT3 was partially or completely abrogated by HIES mutations in the transactivation domain (V713L) or SH2 domain (V637M), respectively, in both hematopoietic and non- hematopoietic cells. By contrast, IL-6-induced tyrosine phosphorylation of an HIES mutant in the STAT3 DNA-binding domain (R382W) was intact. The R382W and V713L mutants significantly reduced IL-6-dependent STAT3 transcriptional activity in reporter gene assays. Moreover, the R382W and V637M mutants significantly diminished IL-6-responsive expression of the endogenous STAT3 target gene, Socs3, as assessed by quantitative real-time PCR (qPCR) in the RAW macrophage cell line. These observations indicate the HIES mutants dominantly suppress the transcriptional activity of wild type STAT3, albeit to varying degrees. All three HIES mutants enhanced LPS-induced expression of the NFκB target genes IL6 (IL-6), Cxcl10 (IP- 10), and Tnf (TNFα) in RAW cells, as indicated by qPCR. Furthermore, overexpression of wild type STAT3 in Stat3-deficient murine embryonic fibroblasts significantlyreduced LPS-stimulated expression of IL6, Cxcl10, and IL12p35. In addition, in aprimary murine osteoclast differentiation assay, a STAT3-specific SH2 domain inhibitor led to significantly increased levels of osteoclast-specific gene expression. These results suggest that STAT3 serves as a negative regulator of NFκB-mediated gene expression, and furthermore imply that STAT3 mutations associated with HIES contribute to the osteopenia and inflammation observed in HIES patients.

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage.

BACKGROUND: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. PRINCIPAL FINDINGS: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2. SIGNIFICANCE: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.

A model of the roles of essential kinases in the induction and expression of late long-term potentiation.

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca(2+)]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, -burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

Role and Regulation of EphA2 in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cancer cause of death in the US. Gemcitabine is the first-line therapy for this disease, but unfortunately it shows only very modest benefit. The focus of the current study was to investigate the role and regulation of EphA2, a receptor tyrosine kinase expressed in PDAC, to further understand this disease and identify new therapeutic targets. The role of EphA2 was determined in PDAC by siRNA mediated silencing. In combination with gemcitabine, silencing of EphA2 caused a dramatic increase in apoptosis even in highly resistant cells in vitro. Furthermore, EphA2 silencing was found to be useful in 2 orthotopic models in vivo: 1) shRNA-pretreated Miapaca-2 cells, and 2) in vivo delivery of siRNA to established MPanc96 tumors. Silencing of EphA2 alone reduced tumor growth in Miapaca-2 cells. In MPanc96, only the combination treatment of gemcitabine plus siEphA2 significantly reduced tumor growth, as well as the number of lung and liver metastases. Taken together, these observations support EphA2 as a target for combination therapies for PDAC. The regulation of EphA2 was further explored with a focus on the role of Ras. K-Ras activating mutations are the most important initiating event in PDAC. We demonstrated that Ras regulates EphA2 expression through activation of MEK2 and phosphorylation of ERK. Downstream of ERK, silencing of the transcription factor AP-1 subunit c-Jun or inhibition of the ERK effector RSK caused a decrease in EphA2 expression, supporting their roles in this process. Further examination of Ras/MEK/ERK pathway modulators revealed that PEA-15, a protein that sequesters ERK to the cytoplasm, inhibited expression of EphA2. A significant inverse correlation between EphA2 and PEA-15 levels was observed in mouse models of PDAC. In cells where an EGFR inhibitor reduced phospho-Erk, expression of EphA2 was also reduced, indicating that changes in EphA2 levels may allow monitoring the effectiveness of anti-Ras/MEK/ERK therapies. In conclusion, EphA2 levels may be a good prognostic factor for anti-EGFR/anti-Ras therapies, and EphA2 itself is a relevant target for the development of new therapies.

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma

Delineating the mechanism(s) of BDNF/TrkB mediated proliferation in Neuroblastoma Timothy Christopher Graham, B.S. Supervisory Professor: Patrick Zweidler-McKay, MD/PhD Neuroblastoma is the most common extra-cranial solid tumor in children, arising from neural crest precursor cells. The neurotrophin receptors (TrkA/B/C) have been implicated as important prognostic markers, linking the biology of the tumor to patient outcome. High expression of TrkA and TrkC receptors have been linked to favorable biological features and high patient survival, while TrkB is expressed in unfavorable, aggressive tumors. Several studies suggest that high levels and activation of TrkB by its ligand brain-derived neurotrophic factor (BDNF) stimulates tumor cell survival, proliferation, and chemoresistance. However, little is known about the molecular mechanisms that regulate proliferation. The TrkB signaling pathway in neuroblastoma cells has been difficult to evaluate due to the loss of TrkB expression when the cells are used in vitro. Here we determined the role of proximal signaling pathways downstream of TrkB on neuroblastoma proliferation. By analyzing a panel of neuroblastoma cell lines, we found that the SMS-KCN cells express detectable levels of protein and mRNA levels of TrkB as analyzed by western, RT-PCR, and surface expression by flow cytometry. By the addition of exogenous human recombinant BDNF, we showed that activation of TrkB is important in the proliferation of the cells and can be repressed by inhibiting TrkB kinase function. By BDNF stimulation and use of specific kinase inhibitors, the common pathways involving PLCg, PI3K/AKT, and MAPK were initially investigated in addition to PI3K/MTOR and FYN pathways. We demonstrate for the first time that Fyn plays a critical role in TrkB mediated proliferation in neuroblastoma. Constitutively active and over-expressed Fyn reduced neuroblastoma proliferation, as measured by PCNA expression. Knockdown of Fyn by shRNA was shown to cooperate with activated TrkB for an enhanced proliferative response. Although TrkB activation has been implicated in the proliferation of neuroblastoma cells, little is known about its effects on cell cycle regulation. Protein levels of pRB, CDK2, CDK4, CDC25A, cyclin D1, and cyclin E were analyzed following BDNF stimulation. We found that BDNF mediated activation of TrkB induces multiple common proximal signaling pathways including the anti-proliferative Fyn pathway and drives cell cycle machinery to enhance the proliferation of neuroblastoma cells.

The Role of Acidic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor 4 in High Grade Serous Carcinoma

Background: High grade serous carcinoma whether ovarian, tubal or primary peritoneal, continues to be the most lethal gynecologic malignancy in the USA. Although combination chemotherapy and aggressive surgical resection has improved survival in the past decade the majority of patients still succumb to chemo-resistant disease recurrence. It has recently been reported that amplification of 5q31-5q35.3 is associated with poor prognosis in patients with high grade serous ovarian carcinoma. Although the amplicon contains over 50 genes, it is notable for the presence of several members of the fibroblast growth factor signaling axis. In particular acidic fibroblast growth factor (FGF1) has been demonstrated to be one of the driving genes in mediating the observed prognostic effect of the amplicon in ovarian cancer patients. This study seeks to further validate the prognostic value of fibroblast growth receptor 4 (FGFR4), another candidate gene of the FGF/FGFR axis located in the same amplicon. The emphasis will be delineating the role the FGF1/FGFR4 signaling axis plays in high grade serous ovarian carcinoma; and test the feasibility of targeting the FGF1/FGFR4 axis therapeutically. Materials and Methods: Spearman and Pearson correlation studies on data generated from array CGH and transcriptome profiling analyses on 51 microdissected tumor samples were used to identify genes located on chromosome 5q31-35.3 that showed significant correlation between DNA and mRNA copy numbers. Significant correlation between FGF1 and FGFR4 DNA copy numbers was further validated by qPCR analysis on DNA isolated from 51 microdissected tumor samples. Immunolocalization and quantification of FGFR4 expression were performed on paraffin embedded tissue samples from 183 cases of high-grade serous ovarian carcinoma. The expression was then correlated with clinical data to assess impact on survival. The expression of FGF1 and FGFR4 in vitro was quantified by real-time PCR and western blotting in six high-grade serous ovarian carcinoma cell lines and compared to those in human ovarian surface epithelial cells to identify overexpression. The effect of FGF1 on these cell lines after serum starvation was quantified for in vitro cellular proliferation, migration/invasion, chemoresistance and survival utilizing a combination of commercially available colorimetric, fluorometric and electrical impedance assays. FGFR4 expression was then transiently silenced via siRNA transfection and the effects on response to FGF1, cellular proliferation, and migration were quantified. To identify relevant cellular pathways involved, responsive cell lines were transduced with different transcription response elements using the Cignal-Lenti reporter system and treated with FGF1 with and without transient FGFR4 knock down. This was followed by western blot confirmation for the relevant phosphoproteins. Anti-FGF1 antibodies and FGFR trap proteins were used to attempt inhibition of FGF mediated phenotypic changes and relevant signaling in vitro. Orthotopic intraperitoneal tumors were established in nude mice using serous cell lines that have been previously transfected with luciferase expressing constructs. The mice were then treated with FGFR trap protein. Tumor progression was then followed via bioluminescent imaging. The FGFR4 gene from 52 clinical samples was sequenced to screen for mutations. Results: FGFR4 DNA and mRNA copy numbers were significantly correlated and FGFR4 DNA copy number was significantly correlated with that of FGF1. Survival of patients with high FGFR4 expressing tumors was significantly shorter that those with low expression(median survival 28 vs 55 month p< 0.001) In a multivariate cox regression model FGFR expression significantly increased risk of death (HR 2.1, p<0.001). FGFR4 expression was significantly higher in all cell lines tested compared to HOSE, OVCA432 cell line in particular had very high expression suggesting amplification. FGF1 was also particularly overexpressed in OVCA432. FGF1 significantly increased cell survival after serum deprivation in all cell lines. Transient knock down of FGFR4 caused significant reduction in cell migration and proliferation in vitro and significantly decreased the proliferative effects of FGF1 in vitro. FGFR1, FGFR4 traps and anti-FGF1 antibodies did not show activity in vitro. OVCA432 transfected with the cignal lenti reporter system revealed significant activation of MAPK, NFkB and WNT pathways, western blotting confirmed the results. Reverse phase protein array (RPPA) analysis also showed activation of MAPK, AKT, WNT pathways and down regulation of E Cadherin. FGFR trap protein significantly reduced tumor growth in vivo in an orthotopic mouse model. Conclusions: Overexpression and amplification of several members of the FGF signaling axis present on the amplicon 5q31-35.3 is a negative prognostic indicator in high grade serous ovarian carcinoma and may drive poor survival associated with that amplicon. Activation of The FGF signaling pathway leads to downstream activation of MAPK, AKT, WNT and NFkB pathways leading to a more aggressive cancer phenotype with increased tumor growth, evasion of apoptosis and increased migration and invasion. Inhibition of FGF pathway in vivo via FGFR trap protein leads to significantly decreased tumor growth in an orthotopic mouse model.

THE MECHANISM OF TUMORIGENESIS IN THE IMMORTALIZED HUMAN PANCREATIC CELL LINES: CELL CULTURE MODELS OF HUMAN PANCREATIC CANCER

The mechanism of tumorigenesis in the immortalized human pancreatic cell lines: cell culture models of human pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in the world. The most common genetic lesions identified in PDAC include activation of K-ras (90%) and Her2 (70%), loss of p16 (95%) and p14 (40%), inactivation p53 (50-75%) and Smad4 (55%). However, the role of these signature gene alterations in PDAC is still not well understood, especially, how these genetic lesions individually or in combination contribute mechanistically to human pancreatic oncogenesis is still elusive. Moreover, a cell culture transformation model with sequential accumulation of signature genetic alterations in human pancreatic ductal cells that resembles the multiple-step human pancreatic carcinogenesis is still not established. In the present study, through the stepwise introduction of the signature genetic alterations in PDAC into the HPV16-E6E7 immortalized human pancreatic duct epithelial (HPDE) cell line and the hTERT immortalized human pancreatic ductal HPNE cell line, we developed the novel experimental cell culture transformation models with the most frequent gene alterations in PDAC and further dissected the molecular mechanism of transformation. We demonstrated that the combination of activation of K-ras and Her2, inactivation of p16/p14 and Smad4, or K-ras mutation plus p16 inactivation, was sufficient for the tumorigenic transformation of HPDE or HPNE cells respectively. We found that these transformed cells exhibited enhanced cell proliferation, anchorage-independent growth in soft agar, and grew tumors with PDAC histopathological features in orthotopic mouse model. Molecular analysis showed that the activation of K-ras and Her2 downstream effector pathways –MAPK, RalA, FAK, together with upregulation of cyclins and c-myc were involved in the malignant transformation. We discovered that MDM2, BMP7 and Bmi-1 were overexpressed in the tumorigenic HPDE cells, and that Smad4 played important roles in regulation of BMP7 and Bmi-1 gene expression and the tumorigenic transformation of HPDE cells. IPA signaling pathway analysis of microarray data revealed that abnormal signaling pathways are involved in transformation. This study is the first complete transformation model of human pancreatic ductal cells with the most common gene alterations in PDAC. Altogether, these novel transformation models more closely recapitulate the human pancreatic carcinogenesis from the cell origin, gene lesion, and activation of specific signaling pathway and histopathological features.

Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Collagen I modulates growth and gene expression in prostate cancer cells

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to skeletal tissue. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as extracellular matrix (ECM) components, have been implicated in the spread and propagation of prostatic carcinoma. In these studies, we have utilized the PC3 cell line, derived from a human bone metastasis, to investigate the influence of the predominant bone ECM protein, type I collagen, on prostate cancer cell proliferation and gene expression. We have also initiated the design and production of ribozymes to specific gene targets that may influence prostate cancer bone metastasis. ^ Our results demonstrate that PC3 cells rapidly adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the α1, α2 and α3 collagen binding integrin subunits. The use of antibody function blocking studies reveals that PC3 cells can utilize α2β 1 and α3β1 integrins to adhere to collagen I. Once plated on collagen I, the cells exhibit increased rates of proliferation compared with cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of proteins associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), MAP kinase (MAPK), and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. To further characterize the effect of type I collagen on prostate cancer bone metastasis, we utilized a cDNA microarray strategy to monitor type I collagen-mediated changes in gene expression. Results of this analysis revealed a gene expression profile reflecting the increased proliferation occurring on type I collagen. Microarray analysis also revealed differences in the expression of specific gene targets that may impact on prostate cancer metastasis to bone. ^ As a result of our studies on the interaction of prostate cancer cells and the skeletal ECM, we sought to develop novel molecular tools for future gene therapy of functional knockdown experiments. To this end, we developed a series of ribozymes directed against the α2 integrin and at osteopontin, a protein implicated in the metastasis of various cancers, including prostate. These ribozymes should facilitate the future study of the mechanism of prostate cancer cell proliferation, and disease progression occurring at sites of skeletal metastasis where a type I collagen-based environment predominates. ^ Together these studies demonstrate the involvement of bone ECM proteins on prostate cancer cell proliferation and suggest that they may play a significant role on the growth of prostate metastases once in the bone microenvironment. ^

Mechanisms of TGF-beta1-induced neuronal plasticity in Aplysia

Transforming growth factor beta-1 (TGF-β1) is a cytokine and neurotrophic factor whose neuromodulatory effects in Aplysia californica were recently described. Previous results demonstrated that TGF-β1 induces long-term increases in the efficacy of sensorimotor synapses, a neural correlate of sensitization of the defensive tail withdrawal reflex. These results provided the first evidence that a neurotrophic factor regulates neuronal plasticity associated with a simple form of learning in Aplysia, and raised many questions regarding the nature of the modulation. No homologs of TGF-β had previously been identified in Aplysia, and thus, it was not known whether components of TGF-β1 signaling pathways were present in Aplysia. Furthermore, the signaling mechanisms engaged by TGF-β1 had not been identified, and it was not known whether TGF-β1 regulated other aspects of neuronal function.^ The present investigation into the actions of TGF-β1 was initiated by examining the distribution of the type II TGF-β1 receptor, the ligand binding receptor. The receptor was widely distributed in the CNS and most neurons exhibited somatic and neuritic immunoreactivity. In addition, the ability of TGF-β1 to activate the cAMP/PKA and MAPK pathways, known to regulate several important aspects of neuronal function, was examined. TGF-β1 acutely decreased cAMP levels in sensory neurons, activated MAPK and triggered translocation of MAPK to the nucleus. MAPK activation was critical for both short- and long-term regulation of neuronal function by TGF-β1. TGF-β1 acutely decreased synaptic depression induced by low frequency stimuli in a MAPK-dependent manner. This regulation may result, at least in part, from the modulation of synapsin, a major peripheral synaptic vesicle protein. TGF-β1 stimulated MAPK-dependent phosphorylation of synapsin, a process believed to regulate synaptic vesicle mobilization from reserve to readily-releasable pools of neurotransmitter. In addition to its acute effect on synaptic efficacy, TGF-β1 also induced long-term increases in sensory neuron excitability. Whereas transient exposure to TGF-β1 was not sufficient to drive short-or long-term changes in excitability, prolonged exposure to TGF-β1 induced long-term changes in excitability that depended on MAPK. The results of these studies represent significant progress toward an understanding of the role of TGF-β1 in neuronal plasticity. ^

Mechanism of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in head and neck squamous cell carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer and neuroblastoma in clinical trials. 4HPR induces growth inhibition and apoptosis in various cancer cells including head and neck squamous cell carcinoma (HNSCC) cells. 4HPR induces apoptosis by several mechanisms including increasing reactive oxygen species (ROS), or inducing mitochondrial permeability transition (MPT). 4HPR has also been shown to modulate the level of different proteins by transcriptional activation or posttranslational modification in various cellular contexts. However, the mechanism of its action is not fully elucidated. In this study, we explored the mechanism of 4HPR-induced apoptosis in HNSCC cells. ^ First, we identified proteins modulated by 4HPR by using proteomics approaches including: Powerblot western array and 2-dimensional polyacrylamide gel electrophoresis. We found that 4HPR modulated the levels of several proteins including c-Jun. Further analysis has shown that 4HPR induced activation of Activator Protein 1 (AP-1) components, c-Jun and ATF-2. We also found that 4HPR increased the level of Heat shock protein (Hsp) 70 and phosphorylation of Hsp27. ^ Second, we found that 4HPR induced prolonged activation of JNK, p38/MAPK and extracellular signal-regulated kinase (ERK). We also demonstrated that the activation of these kinases is required for 4HPR-induced apoptosis. JNK inhibitor SP600125 and siRNA against JNK1 and JNK2 suppressed, while overexpression of JNK1 enhanced 4HPR-induced apoptosis. p38/MAPK inhibitor PD169316 and MEK1/2 inhibitor PD98059 also suppressed 4HPR-induced apoptosis. We also demonstrated that activation of JNK, p38/MAPK and ERK is triggered by ROS generation induced by 4HPR. We also found that translation inhibitor, cycloheximide, suppressed 4HPR-induced apoptosis through inhibition of 4HPR-induced events (e.g. ROS generation, cytochrome c release, JNK activation and suppression of Akt). We also demonstrated that MPT is involved in 4HPR-induced apoptosis. ^ Third, we demonstrated the presence of NADPH oxidase in HNSCC 2B cells. We also found that 4HPR increased the level of the p67phox, a subunit of NADPH oxidase which participates in ROS production and apoptosis induced by 4HPR. ^ The novel insight into the mechanism by which 4HPR induces apoptosis can be used to improve design of future clinical studies with this synthetic retinoid in combination with specific MAPK modulators. ^

The role of Akt activation in mouse skin tumor promotion

The importance of IGF-1/IGF-1R signaling is evident in human cancers including breast, colon, prostate, and lung which have been shown to overexpress IGF-1. Also, serum levels of IGF-1 have been identified as a risk factor for these cancers. IGF-1 has been primarily shown to mediate its mitogenic effects through signaling pathways such as MAPK and PI3K/Akt. In this regard, BK5.IGF-1 transgenic mice were generated and these mice displayed hyperplasia and hyperkeratosis in the epidermis. In addition, these mice were also found to have elevated MAPK, PI3K, and Akt activities. Furthermore, overexpression of IGF-1 in epidermis can act as a tumor promoter. BK5.IGF-1 transgenic mice developed papillomas after initiation with DMBA without further treatment with a tumor promoter such as TPA. Previous data has also shown that inhibition of the PI3K/Akt signaling pathway by the inhibitor LY294002 was able to reduce the number of tumors formed by IGF-1 mediated tumor promotion. The current studies presented demonstrate that Akt may be the critical effector molecule in IGF-1/IGF-1R mediated tumor promotion. We have found that inhibition of PI3K/Akt by LY294002 inhibits cell cycle components, particularly those associated with G1 to S phase transition including Cyclin D1, Cyclin E, E2F1, and E2F4, that are elevated in epidermis of BK5.IGF-1 transgenic mice. We have also demonstrated that Akt activation may be a central theme in early tumor promotion. In this regard, treatment with diverse tumor promoters such as TPA, okadaic acid, chrysarobin, and UVB was shown to activate epidermal Akt and its downstream signaling pathways after a single treatment. Furthermore, overexpression of Akt targeted to the basal cells of the epidermis led to hyperplasia and increased labeling index as determined by BrdU staining. These mice also had constitutively elevated levels of cell cycle components, particularly Cyclin D1, Cyclin E, E2F1, E2F4, and Mdm-2. These mice developed skin tumors following initiation with DMBA and were hypersensitive to the tumor promoting effects of TPA. Collectively, these studies provide evidence that Akt activation plays an important role in the process of mouse skin tumor promotion. ^

Studies of MEKK2 activation mechanisms and its biological role in LPS/TLR4 signaling

MEKK2 is an evolutionarily conserved mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that controls the MAPK and IKK-NF-κB pathways. The MAPK and IKK pathways are intracellular signaling networks that are crucial for the Toll-like receptor (TLR) mediated innate immunity, cellular stress and many other physiological responses. Members of the MAP3K family are central to the activation of these processes. However, the molecular mechanisms underlying stimuli-mediated MAP3K activation remain largely unknown. In this study, we identified a key phosphoserine residue, Ser-519 in MEKK2, and its equivalent site Ser-526 in MEKK3 within their activation loop whose phosphorylation are essential for their optimal activation. Mutation of this regulatory serine to an alanine severely impaired MEKK2 activation and MEKK2 signaling to its downstream targets. To demonstrate that physiological stimuli induce this serine phosphorylation, we generated an antibody that specifically recognizes the phosphorylated serine residue. We found that many, but not all, of the MAPK agonists, including the TLR ligands, growth factors, cytokines and cellular stresses, induced this regulatory serine phosphorylation in MEKK2, suggesting an involvement of MEKK2 in the activation of the MAPK cascade leading to different cellular responses. We further investigated the specific role of MEKK2 in LPS/TLR4 signaling by using MEKK2−/− mice. We found that MEKK2 was selectively required for LPS-induced ERK1/2 activation, but not JNK, p38 or NF-κB activation. We also found that MEKK2 was involved in TLR4 dependent induction of proinflammatory cytokines and LPS-induced septic shock. In conclusion, we identified a key regulatory serine residue in the activation loop of MEKK2 whose phosphorylation is a key sensor of receptor- and cellular stress-mediated signals. We also demonstrated that MEKK2 is crucial for TLR4-mediated innate immunity. ^