Tyrosine Kinase Receptor Type Research Articles

Diagnostic utility of phosphorylated signal transducer and activator of transcription 5 immunostaining in the diagnosis of mammary analogue secretory carcinoma of the salivary gland: A comparative study of salivary gland cancers.

Mammary analogue secretory carcinoma (MASC) with an ETS variant gene 6 (ETV6)-neurotrophic tyrosine kinase receptor type 3 (NTRK3) translocation is a newly described type of salivary gland cancer. It is known that overexpression of signal transducer and activator of transcription 5a (STAT5a) occurs in secretory carcinoma of the breast and MASC, and STAT5a expression may be related to the ETV6-NTRK3 translocation. It was hypothesized that phosphorylated signal transducer and activator of transcription 5 (p-STAT5) might be specifically expressed in MASC of the salivary gland. The expression of p-STAT5 and mammaglobin (MMG) was examined with immunohistochemistry (IHC)/immunocytochemistry (ICC) in tissue sections from 58 salivary gland cancers (8 MASCs and 50 other salivary gland cancers) and in cytological smears from 17 salivary gland cancers (7 MASCs with paired histologic samples and 10 other salivary gland cancers). p-STAT5 IHC was clearly increased in MASC versus normal salivary gland tissue and other salivary gland cancers. p-STAT5 expression was found in 7 of 8 MASCs (87.5%) and in none of the 50 other salivary gland cancers (0%) by IHC. On cytology, p-STAT5 expression was found in all cases of MASC (7 of 7 or 100%) but in none of the 10 other salivary gland cancers (0%) by ICC. The expression rate of MMG by histology and cytology was higher than that of p-STAT5 in the other salivary gland cancers. p-STAT5 might be useful as a detection marker of MASC in the differential diagnosis of salivary gland cancers, and initial screening with p-STAT5 IHC/ICC, combined with auxiliary fluorescence in situ hybridization confirmation, is a reliable, economical approach to identifying MASC of the salivary gland.

Membranous Insulin-like Growth Factor-1 Receptor (IGF1R) Expression Is Predictive of Poor Prognosis in Patients with Epidermal Growth Factor Receptor (EGFR)-Mutant Lung Adenocarcinoma.

Background:Insulin-like growth factor-1 receptor (IGF1R) is a membrane receptor-type tyrosine kinase that has attracted considerable attention as a potential therapeutic target, although its clinical significance in non-small cell lung cancer (NSCLC) is controversial. This study aimed to clarify the clinical significance of IGF1R expression in human NSCLC.Methods:IGF1R protein expression was evaluated using immunohistochemistry in 372 patients with NSCLC who underwent curative surgical resection (146 squamous cell carcinomas [SqCCs] and 226 adenocarcinomas [ADCs]). We then analyzed correlations between expression of IGF1R and clinicopathological and molecular features and prognostic significance.Results:Membranous and cytoplasmic IGF1R expression were significantly higher in SqCCs than in ADCs. In patients with SqCC, membranous IGF1R expression was associated with absence of vascular, lymphatic, and perineural invasion; lower stage; and better progression-free survival (PFS) (hazard ratio [HR], 0.586; p = .040). In patients with ADC, IGF1R expression did not have a significant prognostic value; however, in the subgroup of epidermal growth factor receptor (EGFR)-mutant ADC, membranous IGF1R expression was associated with lymphatic and perineural invasion, solid predominant histology, and higher cancer stage and was significantly associated with worse PFS (HR, 2.582; p = .009).Conclusions:Lung ADC and SqCC showed distinct IGF1R expression profiles that demonstrated prognostic significance. High membranous IGF1R expression was predictive of poor PFS in EGFR-mutant lung ADC, while it was predictive of better PFS in SqCC. These findings will help improve study design for subsequent investigations and select patients for future anti-IGF1R therapy.

Abstract 770: E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation

Abstract The fibroblast growth factor (FGF) signaling pathway comprises 18 ligands and 4 FGF receptor subtypes, FGFR1, 2, 3 and 4, which are known as receptor-type tyrosine kinases corresponding to those ligands. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. Genetic abnormalities (gene fusion, mutation and amplification) of FGFRs are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, drug resistance, etc. These abnormalities have also been reported to be involved in different cancer types including lung, breast, endometrial, gastric, and bladder cancer so far. Therefore, FGFRs are considered as one of potential targets for cancer therapy. E7090 is an orally available, selective and potent inhibitor of FGFR1, 2 and 3 tyrosine kinase activities. E7090 displayed inhibition of FGFR1, 2, and 3 kinase activities with the IC50 values of approximately 1 nmol/L. E7090 also inhibited the growth of SNU-16, human gastric cancer cell line harboring FGFR2 amplification with an IC50 value of 3 nmol/L. This activity was about 60-fold stronger than that against VEGF-stimulated HUVEC growth. Kinase profiling assay consisting of 93 kinases including non-receptor and serine/threonine kinases, also demonstrated that E7090 inhibited limited kinases including FGFR-1, -2 and -3. In addition, E7090 inhibited proliferation of human cancer cell lines harboring various types of FGFRs gene abnormalities such as amplification, mutation, or translocation in vitro, which are confirmed by the inhibition of FGFR signaling. E7090 also showed significant antitumor activities on various human xenografts harboring FGFRs gene abnormalities in a dose dependent manner and demonstrated tumor shrinkage at several doses in some models. Furthermore, pharmacodynamics analysis revealed that E7090 inhibited phosphorylation of FGFRs in SNU-16 xenograft tumors in a dose-dependent manner. Overall, the in vitro and in vivo studies confirm that E7090 is a potent and selective FGFRs inhibitor, showing promising antitumor activities with wider therapeutic windows in preclinical cancer models harboring FGFRs gene abnormalities. E7090 has been advanced through preclinical development and we disclose here the first details of its preclinical profile. Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Setsuo Funasaka, Satoshi Nagao, Naoko Hata Sugi, Hiroko Kuramochi, Katsuyuki Ishikawa, Kiyoshi Okamoto, Yukinori Minoshima, Takayuki Nakagawa, Yusuke Nakatani, Yuki Karoji, Isao Ohashi, Yoshinobu Yamane, Keigo Tanaka, Toshimi Okada, Tomohiro Matsushima, Junji Matsui, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka. E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 770. doi:10.1158/1538-7445.AM2015-770

Erratum] Effects of glucose-dependent insulinotropic polypeptide receptor knockout and a high-fat diet on cognitive function and hippocampal gene expression in mice.

RACHAEL R. LENNOX, CHARLOTTE MOFFETT, DAVID W. PORTER, NIGEL IRWIN, VICTOR A. GAULT and PETER R. FLATT Mol Med Rep 12:[Related article:] 1544–1548, 2015; DOI: 10.3892/mmr.2015.3447 After the publication of the article, the authors noted that there was an error regarding the author list. RACHAEL R. LENNOX and CHARLOTTE MOFFETT should actually be listed as RACHAEL LENNOX and R. CHARLOTTE MOFFETT. We apologize for the mistake and possible misunderstanding. The correct author list is as follows: RACHAEL LENNOX, R. CHARLOTTE MOFFETT, DAVID W. PORTER, NIGEL IRWIN, VICTOR A. GAULT and PETER R. FLATT.

Genetic association of the transcription of neuroplasticity-related genes and variation in stress-coping style.

IntroductionStress coping has been defined as the cognitive and behavioral efforts made to conquer, endure, or decrease external and internal demands and the conflicts between them. It has two main elements: the control or modification of the person–environment relationship causing the stress (i.e., problem-focused coping) and/or regulation of stressful feelings (i.e., emotion-focused coping). Research suggests that the expressions of brain-derived neurotrophic factor (BDNF) and neurotrophic tyrosine kinase receptor type 2 (NTRK2) play important roles in brain adaptation to investigate stress. To clarify the genetic basis of stress coping, we investigated the association of stress-coping strategies and social adaptation with single-nucleotide polymorphisms (SNPs) involved in neural plasticity, anxiety, and depression.MethodsIn 252 healthy controls (94 women; 158 men), we measured and estimated the stress-coping style using the Lazarus-type stress-coping inventory, ego aptitude scale (EAS), and social adaptation self-evaluation scale (SASS). We investigated one SNP of BDNF (rs6265, Val/Met) and five SNPs of NTRK2 (rs11140800, rs1187286, rs1867283, rs1147198, and rs10868235).ResultsWe observed significant associations between BDNF and emotion-focused strategies, seeking social support, self-control, and distancing. We also found significant associations between NTRK2 and cognitive strategies, problem-solving, confrontive- coping, seeking social support, distancing and positive reappraisal. Significant associations were also found between BDNF and critical attitudes and between NTRK2 and all seven ego-related factors on the EAS. In the SASS, the minor allele rs1867283 of NTRK2 had a significantly higher score than the heterozygote.ConclusionsThese findings may provide insights into the partial effects of genetic mutations in BDNF and NTRK2 on stress tolerance and personality.

Analgesia via blockade of NGF/TrkA signaling does not influence fracture healing in mice.

Abatement of fracture-related pain is important in patient welfare. However, the frequently used non-steroidal anti-inflammatory drugs are considered to impair fracture healing through blockade of cyclooxygenase-2. An alternative for fracture-related pain treatment may be blockade of nerve growth factor (NGF)/neurotrophic tyrosine kinase receptor type 1 (TrkA) signaling. Because the effect of blocking this signal-pathway on bone healing has not been extensively investigated, we addressed this issue by applying neutralizing antibodies that target NGF and TrkA, respectively, in a mouse fracture model. Mice with a knock-in for human TrkA underwent femur osteotomy and were randomly allocated to phosphate-buffered-saline, anti-NGF-antibody, or anti-TrkA-antibody treatment. The analgesic effect of the antibodies was determined from the activity and the ground reaction force of the operated limb. The effect of antibody administration on fracture healing was assessed by histomorphometry, micro-computed tomography, and biomechanics. NGF/TrkA-signaling blockade had no negative effect on fracture healing as callus formation and maturation were not altered. Mice treated with anti-TrkA antibody displayed significantly greater activity on post-operative day 2 compared to PBS treatment indicating effective analgesia. Our data indicate, that blockade of NGF/TrkA signaling via specific neutralizing antibodies for pain reduction during fracture healing does not influence fracture healing.

The neurotrophic receptor Ntrk2 directs lymphoid tissue neovascularization during Leishmania donovani infection.

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80hiCD11bloCD11c+ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.

Teratogenic effects of pyridoxine on the spinal cord and dorsal root ganglia of embryonic chickens

Our understanding of the role of somatosensory feedback in regulating motility during chicken embryogenesis and fetal development in general has been hampered by the lack of an approach to selectively alter specific sensory modalities. In adult mammals, pyridoxine overdose has been shown to cause a peripheral sensory neuropathy characterized by a loss of both muscle and cutaneous afferents, but predominated by a loss of proprioception. We have begun to explore the sensitivity of the nervous system in chicken embryos to the application of pyridoxine on embryonic days 7 and 8, after sensory neurons in the lumbosacral region become post-mitotic. Upon examination of the spinal cord, dorsal root ganglion and peripheral nerves, we find that pyridoxine causes a loss of neurotrophic tyrosine kinase receptor type 3-positive neurons, a decrease in the diameter of the muscle innervating nerve tibialis, and a reduction in the number of large diameter axons in this nerve. However, we found no change in the number of Substance P or calcitonin gene-related peptide-positive neurons, the number of motor neurons or the diameter or axonal composition of the femoral cutaneous nerve. Therefore, pyridoxine causes a peripheral sensory neuropathy in embryonic chickens largely consistent with its effects in adult mammals. However, the lesion may be more restricted to proprioception in the chicken embryo. Therefore, pyridoxine lesion induced during embryogenesis in the chicken embryo can be used to assess how the loss of sensation, largely proprioception, alters spontaneous embryonic motility and subsequent motor development.

Rat BMSCs initiate retinal endogenous repair through NGF/TrkA signaling

Müller cells can completely repair retinal injury by acting as endogenous stem/progenitor cells in lower-order vertebrates. However, a safe and effective approach to activate progenitor potential of retinal Müller cells in higher-order vertebrates, which rarely re-enter the cell cycle, is a bottleneck problem. In the present study, Royal College of Surgeon's (RCS) rats were subjected to rat bone marrow mesenchymal stem cells (rBMSCs) subretinal space transplantation. Electroretinography (ERG) recordings showed that the b-wave amplitudes and ONL thicknesses statistically increased after transplantation. The number of Müller cells expressing proliferative, stem/progenitor and neuronal markers significantly increased after rBMSCs transplantation in vivo or after co-culturing with rBMSCs in vitro. The cultured rBMSCs could secrete nerve growth factor (NGF). In addition, we confirmed that NGF or NGF-neutralizing antibody could activate or depress Müller cells dedifferentiation, both in vivo and in vitro. Furthermore, Müller cells expressing high levels of the NGF receptor neurotrophic tyrosine kinase receptor type 1 (TrkA) were observed in the retinas of rats transplanted with rBMSCs. Moreover, the protein expression of downstream elements of NGF/TrkA signaling, such as p-PI3K, p-Akt and p-CREB, increased in Müller cells in the retinas of rBMSCs-treated rats in vivo or in Müller cells co-cultured with rBMSCs in vitro. Blocking TrkA with K-252a reduced the number of dedifferentiated Müller cells and the expression of NGF/TrkA signaling in vitro. Thus, rBMSCs might initiate endogenous regenerative mechanisms, which may constitute a new therapeutic strategy for retinal dystrophic diseases.

The EML4-ALK oncogene: targeting an essential growth driver in human cancer.

Targeting of essential growth drivers represents an ideal approach to cancer treatment. To identify such molecules in clinical specimens, we developed a highly sensitive functional screening system based on the preparation of retroviral cDNA expression libraries. By screening such a library of lung adenocarcinoma with a focus formation assay, we discovered the EML4-ALK fusion-type oncogene. A small chromosomal inversion thus leads to fusion of the amino-terminal portion of the microtubule-associated protein EML4 to the intracellular kinase domain of ALK, a receptor-type protein tyrosine kinase. Constitutive dimerization of EML4-ALK mediated by a dimerization motif of EML4 results in kinase activation. Specific inhibitors of the kinase activity of ALK have been developed as therapeutic drugs for EML4-ALK–positive lung cancer, three of which (crizotinib, ceritinib, and alectinib) have already been approved for clinical use. An overall clinical response rate of 93.5% for alectinib has shown that agents that target essential growth drivers can become magic bullets for cancer treatment.

Functional Analysis of Kinase-Activating Fusions in Ph-like Acute Lymphoblastic Leukemia

Introduction: Ph-like or BCR-ABL1-like B-progenitor acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by a gene expression profile similar to BCR-ABL1 ALL. The prevalence of Ph-like ALL rises from 10% in standard risk childhood ALL to over 25% in young adults. Next-generation sequencing of Ph-like ALL identified a variety of alterations involving kinase or cytokine receptor genes, including rearrangement, sequence mutation and copy number alterations. Chromosomal rearrangements in about one-third of Ph-like ALL cases create fusion genes of a variety of 5’ partners that involve ABL1-class genes (ABL1, ABL2, CSF1R and PDGFRB) or activate JAK family members (JAK2, TYK2, IL2RB) that are potentially amenable to treatment with ABL1-class or JAK-class tyrosine kinase inhibitors (TKIs). Notably, ABL2 (Abelson-related gene, ARG), a homolog of ABL1, has rarely been identified as a rearrangement partner in ALL. CSF1R (encoding the macrophage colony stimulating receptor) regulates the differentiation of macrophages, and is not normally expressed in lymphocytes. Likewise, rearrangements involving the JAK family member TYK2, the beta chain of the interleukin 2 cytokine receptor (IL2RB), and the neurotrophic tyrosine kinase receptor type 3 (NTRK3), have not been previously described in leukemia. The goals of this study were to assess the role of these kinase alterations in leukemogenesis, to determine the activation of signaling pathways, and to investigate the efficacy of TKIs.Methods: Kinase fusions were expressed in interleukin-3 dependent Ba/F3 cells, and co-expressed with the dominant negative isoform of IKAROS (IK6) in interleukin-7 dependent Arf-/- mouse pre-B cells. Xenograft models of 10 Ph-like ALL tumors - ETV6-ABL1, RANBP2-ABL1, PAG1-ABL2, RCSD1-ABL2, SSBP2-CSF1R, IGH-EPOR, ETV6-NTRK3, ATF7IP-JAK2, PAX5-JAK2 and ZEB2-PDGFRB - were generated by engrafting primary human leukemia cells into NOD-SCID IL2R gamma null (NSG) mice. Activation of kinase signaling was performed using phosphoflow cytometry analysis, and sensitivity to TKIs was assessed ex vivo and in vivo.Results: All kinase fusions (PAG1-ABL2, MYH9-IL2RB, ATF7IP-JAK2, ETV6-NTRK3 or MYB-TYK2) induced cytokine-independent proliferation of Ba/F3 cells. Mice transplanted with Arf-/- pre-B cells co-expressing IK6 and either RCSD1-ABL2 or SSBP2-CSF1R developed pre-B ALL (CD43+, B220+, CD19+, BP-1+ and IgM-) with a median latency of 36 and 40 days respectively, providing evidence that ABL2 and CSF1R fusions contribute to leukemogenesis.In human leukemic cells harvested from xenograft mice we observed distinct patterns of kinase signaling activation and TKI sensitivity for the different fusions. Xenograft cells expressing ABL1-class kinase fusions showed activation of STAT5 that was inhibited with imatinib or dasatinib. Phosphorylation of CRKL, a known target of ABL1 and ABL2, was only observed in cells expressing ABL1/2 fusions. Cells harboring ATF7IP-JAK2, PAX5-JAK2 or IGH-EPOR showed phosphorylation of STAT5 that was attenuated with the JAK2 inhibitor, ruxolitinib. In contrast, cells expressing ETV6-NTRK3 signaled through the MAPK pathway with constitutive pERK1/2 that was inhibited with the ALK-inhibitor, crizotinib. This TKI response profile was confirmed by cytotoxicity assays in xenograft cells, with ABL1-class fusions being sensitive to dasatinib (IC50 range 1-2nM), whilst cases harboring ATF7IP-JAK2 or EPOR rearrangement uniquely responded to ruxolitinib with IC50 values of 500nM and 850nM respectively. Interestingly, in human leukemic cells harboring the ETV6-NTRK3 fusion we observed selective inhibition with both crizotinib and the FLT3 inhibitor, lestaurtinib. Pre-clinical studies on three xenograft models of Ph-like ALL - ETV6-ABL1, RCSD1-ABL2 and SSBP2-CSF1R – showed significantly reduced leukemic burden in dasatinib treated mice (20mg/kg/day p.o) compared to vehicle treated mice.Conclusions: These data provide important insight on new targets of rearrangement in ALL and describe the first engineered mouse models of Ph-like B-ALL. Functional modeling of these alterations is essential to improve the clinical management of Ph-like ALL by identifying patients with specific genomic lesions at diagnosis and directing them to treatment with appropriate TKIs combined with chemotherapy, analogous to current treatment for BCR-ABL1 B-ALL. DisclosuresHunger:Bristol Myers Squibb: Consultancy.

Immunohistochemical analysis of RTKs expression identified HER3 as a prognostic indicator of gastric cancer.

Standard treatment in Japan for the 13th Japanese Gastric Cancer Association stage II/III advanced gastric cancer is postoperative adjuvant S-1 administration after curative surgery. High expression of receptor type tyrosine kinases (RTKs) has repeatedly represented poor prognosis for cancers. However it has not been demonstrated whether RTKs have prognostic relevance for stage II/III gastric cancer with standard treatment. Tumor tissues were obtained from 167 stage II/III advanced gastric cancer patients who underwent curative surgery and received postoperative S-1 chemotherapy from 2000 to 2010. Expression of the RTKs including EGFR, HER2, HER3, IGF-1R, and EphA2 was analyzed using immunohistochemistry (IHC). Analysis using a multivariate proportional hazard model identified the most significant RTKs that represented independent prognostic relevance. When tumor HER3 expression was classified into IHC 1+/2+ (n = 98) and IHC 0 (n = 69), the cumulative 5-year Relapse Free Survival (5y-RFS) was 56.5 and 82.9%, respectively (P = 0.0034). Significant prognostic relevance was similarly confirmed for IGF-1R (P = 0.014), and EGFR (P = 0.030), but not for EphA2 or HER2 expression. Intriguingly, HER3 expression was closely correlated with IGF-1R (P < 0.0001, R = 0.41), and EphA2 (P < 0.0001, R = 0.34) expression. Multivariate proportional hazard model analysis identified HER3 (IHC 1+/2+) (HR; 1.53, 95% CI, 1.11–2.16, P = 0.0078) as the sole RTK that was a poor prognostic factor independent of stage. Of the 53 patients who recurred, 40 patients (75.5%) were HER3-positive. Thus, of the RTKs studied, HER3 was the only RTK identified as an independent prognostic indicator of stage II/III advanced gastric cancer with standard treatment.

Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation.

Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation.

Novel Therapeutic Approach for Neurogenic Erectile Dysfunction: Effect of Neurotrophic Tyrosine Kinase Receptor Type 1 Monoclonal Antibody

BackgroundErectile dysfunction (ED) is a major health issue in aged populations, and neurogenic ED is particularly difficult to treat. Novel therapeutic approaches are needed for treatment of neurogenic ED of peripheral origin. ObjectiveTo investigate the therapeutic effects of a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) on erectile function and sexual behavior in a rat model of cavernous nerve injury (CNI). Design, setting, and participantsIn one experiment, 84 male rats were randomly assigned to seven groups. The groups underwent either CNI or sham surgery, subsequent injection into the major pelvic ganglion (IMPG) of phosphate-buffered saline (PBS), an immunoglobulin G (IgG) control, or TrkA-mAb, and then intracavernosal (IC) injection of either PBS or varying TrkA-mAb concentrations immediately after surgery and then 1 wk later. Erectile function was assessed and histologic/molecular analyses were performed at 6 wk after surgery. In a second experiment, 36 male rats were randomly divided into three groups. The groups underwent CNI or sham surgery and then IC injection of PBS, IgG, or TrkA-mAb immediately after surgery and for 5 wk thereafter. At 6 wk after surgery, the performance of the rats in sexual behavior tests was videotaped. InterventionCNI or sham surgery; IMPG of PBS, IgG, or TrkA-mAb; IC injection of PBS or TrkA-mAb. Outcome measurements and statistical analysisThe intracavernous pressure response to cavernous nerve electrostimulation was measured and midpenile cross-sections were histologically examined. Western blotting (WB) of cavernous tissue protein was performed. Rats were assessed for chasing, mounting, intromission, and ejaculation behaviors during sexual behavior tests. The data were analyzed using one-way analysis of variance followed by the Tukey-Kramer t test. Results and limitationsRecovery of erectile function of varying degrees was observed in the TrkA-mAb groups. TrkA-mAb treatment significantly suppressed tyrosine hydroxylase–positive nerve fibers in the corpus cavernosum and enhanced neuronal nitric oxide synthase–positive fibers in the dorsal nerve. The ratio of smooth muscle to collagen in the corpus cavernosum was significantly improved in TrkA-mAb treatment groups compared to PBS vehicle and IgG control groups. WB confirmed these biological changes. There was a nonsignificant increase in the average number of intromissions and ejaculations in the TrkA-mAb group. The study limitations include small sample size, variability in sexual behavior, lack of data on the neuromuscular mechanism involved, and lack of information of the role of neurotrophins or cytokines in regeneration. ConclusionsTrkA-mAb successfully inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on ED and sexual behavior disorder in a rat model of CNI. Patient summaryThis report provides strong evidence that a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on erectile dysfunction and sexual behavior disorder in a rat model of cavernous nerve injury. The results raise the possibility that human patients with neurogenic erectile dysfunction may respond to TrkA-mAb in a manner that parallels the response seen in our rodent study.

Growth Factor Midkine Deteriorates Cadiac Hypertrophy Via Epidermal Growth Factor Receptor Signaling: A Novel Mediator of Cardio-renal Interaction

Background: Midkine (MK), a 13-kDa heparin-binding growth factor, has shown to be up-regulated in injured renal tubule, and released into circulation. We recently reported that cardiac-specific overexpression of MK exacerbates cardiac hypertrophy in mice. However, it remains unknown by which mechanisms MK affects on heart. Although receptors for MK are thought to be receptor-type tyrosine kinase, specific receptor for MK has not been determined. Here we show that MK acts by activating the epidermal growth factor receptor (EGFR) and its downstream signaling and leads to cardiac hypertrophy. Methods and Results: MK phosphorylated EGFR (Tyr1068) on neonatal rat cardiomyocytes. MK also promoted phosphorylation of ERK1/2 and Akt, induced fetal cardiac gene expression, and led to cardiomyocyte hypertrophy. Pre-treatment with EGFR inhibitors attenuated MK-induced ERK1/2 and Akt phosphorylation, and induction of fetal cardiac gene, as well as cardiomyocyte hypertrophy in neonatal rat cardiomyocytes. EGFR silencing by siRNA also attenuated MK-induced cardiomyocyte hypertrophy. Next, we examine whether the action of MK mediates EGFR transactivation using TAPI-2, a metalloproteinase inhibitor, to inhibit EGFR transactivation. Pre-treatment with TAPI-2 did not affect MK-induced elevated cardiac fetal gene activity and activation of EGFR, suggesting that MK may directly phosphorylated EGFR rather than mediating EGFR transactivation. For in vivo study, subtotal nephrectomy was created with MK systemic knockout (MK-KO) mice and littermate wild-type (WT) mice. Subtotal nephrectomy induced cardiac hypertrophy and poor survival rate in WT mice, whereas cardiac hypertrophy and survival rate was improved in MK-KO mice compared with WT mice. Conclusions: We demonstrated for the first time that EGFR signaling plays a crucial role in MK-induced cardiac hypertrophy, and MK is thought to be one of key molecule mediating cardio-renal interaction.

Lixisenatide improves recognition memory and exerts neuroprotective actions in high-fat fed mice

The metabolic benefits of lixisenatide as an anti-diabetic agent are recognized but potential extra-pancreatic effects of this glucagon-like peptide-1 (GLP-1) mimetic in the brain are less well known. This study examines actions within the hippocampus following chronic 40-day peripheral administration of lixisenatide to high-fat fed mice with established obesity, insulin resistance and impaired cognition. Lixisenatide (50nmol/kg bw, twice-daily) resulted in marked improvements in glycemic status, insulin secretion and insulin sensitivity. Examination of pancreatic tissue revealed decreased islet area, increased islet number, and increased insulin content, with no evidence of pancreatic inflammation. Lixisenatide improved recognition memory during a novel object recognition task and this was associated with up-regulation of hippocampal expression of neurotrophic tyrosine kinase receptor type 2 (NTRK2) and mammalian target of rapamycin (mTOR) genes involved in modulating synaptic plasticity and long-term potentiation. Lixisenatide also enhanced progenitor cell proliferation and increased the number of immature neurons in the hippocampal dentate gyrus. These data indicate that lixisenatide is not only a new efficacious drug for treatment of diabetes but it also exerts favorable neuroprotective effects, reversing memory impairment in obesity-diabetes. Further clinical studies are necessary to fully assess potential beneficial actions of lixisenatide in the hippocampus and cognition in man.

Good proteins gone bad: brain-derived neurotrophic factor and its receptors in endometriotic implants and the role of estrogen

Previously we described brain-derived neurotrophic factor (BDNF) and its receptors in the eutopic endometrium. BDNF and its high affinity receptor, neurotrophic tyrosine kinase receptor type 2 (NTRK2), induce nerve growth, differentiation, and maintenance, but also activate the proliferation, adhesion, and angiogenesis pathways; each central to endometriosis. Thus, our objective was to determine the effect of hormones on BDNF in the uterus and ectopic endometriosis lesions.

Evidence-based modeling of mode-of-action for functional ingredients influencing Alzheimer’s disease through neurotrophin pathway

Background : Brain-derived neurotrophic factor (BDNF) is the most widely expressed member of the neurotrophin family in the human brain and is crucially involved in the development of neural circuits, modulation of synaptic plasticity, and regulation of cognitive functions, including learning and memory. Many studies have shown the association of altered BDNF levels with neurodegenerative and neuropsychiatric disorders. However, BDNF is not able to cross the blood-brain barrier and, thus, its delivery to the nervous system is a challenge. Therefore, functional diets with the ability to induce production of BDNF in the brain may offer an alternative route. The objective of this study was three-fold: first, to find out diets that are causally linked to the agonistic activity of BDNF in the neurotrophin signaling pathway; second and mainly, to investigate mode-of-action of these functional diets through systems-based mechanistic modeling in the context of Alzheimer’s disease; and third, to demonstrate the proof-of-concept application of systems biology methods, that are well established in the pharmaceutical sector, to the emerging field of functional food. Methods : In the first step, two cause-and-effect models of BDNF signaling in two states, i.e. normal state and Alzheimer’s disease state, were constructed using published knowledge in scientific literature and pathway databases. A “differential model analysis” between the two states was performed by which mechanistic mode-of-action of BDNF in neurotrophin signaling pathway could be explained with a high molecular resolution in both normal and disease states. The BDNF mode-of-action model was further validated using the “biomarker￾guided validation” approach. In the second step, scientific evidence on the effect of various functional diets on BDNF levels and BDNF-related biological processes or outcomes was harvested from biomedical literature using a disease-specific semantic search. This information was then added to the mechanistic model of BDNF mode-of-action and used to substantiate the mode-of-action model. Results : The differential model analysis resulted in a mechanistic mode-of-action model for the effector BDNF signaling pathway through NTRK receptors (Neurotrophic tyrosine kinase receptor type 2) in neurons. The model revealed an amyloid-mediated neurotrophin switch mechanism by which the amyloid-beta protein competitively blocks BDNF-NTRK2 downstream signaling under Alzheimer’s conditions, thereby “switching” the entire pathway from its normal state with neuroprotective effect to the disease state with a strong push towards neuron apoptosis. This hypothetical switch mechanism was validated by expressed biomarkers as well as empirical data obtained from experimentation of BDNF mimetics in animal models. Several functional diets were found in the literature that showed agonistic effects on the effector BDNF pathway. These effects are exerted through increased levels of BDNF and subsequently, activating the BDNF survival pathway, which leads to similar observations that have been made with BDNF mimetics in animal models. Conclusions : To our knowledge, this is the first study to investigate mode-of-action of functional foods using systems-based modeling approaches. Moreover, such models can answer the question how functional diets can possibly act at the molecular level and interfere with the disease mechanism. Using scientific evidence supporting such models, there is a possibility to introduce new functional formulations by combining functional ingredients of these diets. Keywords : evidence-based modeling, mode-of-action, functional ingredient, BDNF, Alzheimer’s disease

Activation of GSNOR transcription by NF-κB negatively regulates NGF-induced PC12 differentiation

S-nitrosoglutathione reductase (GSNOR) is the key enzyme controlling the intracellular levels of S-nitrosoglutathione and S-nitrosothiols. GSNOR has been implicated in many biological processes, such as the cardiovascular and respiratory systems. However, the role of GSNOR, the sole brain alcohol dehydrogenase, in the nervous systems is still largely a mystery. Here we report that GSNOR was induced during the PC12 neuronal differentiation. Luciferase assays indicated that the region of -88bp to -73bp of the GSNOR promoter encodes an essential responsive sequence to nerve growth factor (NGF). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays revealed NF-κB binds to this essential sequence, which demonstrates that GSNOR can be activated by NF-κB in response to NGF. Blocking either the neurotrophic tyrosine kinase receptor type 1 (TrkA receptor) for NGF or the downstream MEK1/2 pathway inhibited the increase of GSNOR. In contrast to usual neurogenic signals in response to NGF, GSNOR negatively regulated neurite growth; overexpression of GSNOR significantly decreased the percent of differentiated cells, and knockdown of GSNOR promoted the differentiation. To our knowledge, this is the first time the transcriptional mechanism of GSNOR in neuronal differentiation has been explored. We have defined a novel role of GSNOR in neurite development and provide molecular insights into the control of neurite growth.

Phosphorylation of Cyclin-dependent Kinase 5 (Cdk5) at Tyr-15 Is Inhibited by Cdk5 Activators and Does Not Contribute to the Activation of Cdk5

Cdk5 is a member of the cyclin-dependent kinase (Cdk) family. In contrast to other Cdks that promote cell proliferation, Cdk5 plays a role in regulating various neuronal functions, including neuronal migration, synaptic activity, and neuron death. Cdks responsible for cell proliferation need phosphorylation in the activation loop for activation in addition to binding a regulatory subunit cyclin. Cdk5, however, is activated only by binding to its activator, p35 or p39. Furthermore, in contrast to Cdk1 and Cdk2, which are inhibited by phosphorylation at Tyr-15, the kinase activity of Cdk5 is reported to be stimulated when phosphorylated at Tyr-15 by Src family kinases or receptor-type tyrosine kinases. We investigated the activation mechanism of Cdk5 by phosphorylation at Tyr-15. Unexpectedly, however, it was found that Tyr-15 phosphorylation occurred only on monomeric Cdk5, and the coexpression of activators, p35/p25, p39, or Cyclin I, inhibited the phosphorylation. In neuron cultures, too, the activation of Fyn tyrosine kinase did not increase Tyr-15 phosphorylation of Cdk5. Further, phospho-Cdk5 at Tyr-15 was not detected in the p35-bound Cdk5. In contrast, expression of active Fyn increased p35 in neurons. These results indicate that phosphorylation at Tyr-15 is not an activation mechanism of Cdk5 but, rather, indicate that tyrosine kinases could activate Cdk5 by increasing the protein amount of p35. These results call for reinvestigation of how Cdk5 is regulated downstream of Src family kinases or receptor tyrosine kinases in neurons, which is an important signaling cascade in a variety of neuronal activities.