Axonal Odorant Receptors Mediate Axon Targeting.

In mammals, odorant receptors not only detect odors but also define the target in the olfactory bulb, where sensory neurons project to give rise to the sensory map. The odorant receptor is expressed at the cilia, where it binds odorants, and at the axon terminal. The mechanism of activation and function of the odorant receptor at the axon terminal is, however, still unknown. Here, we identify phosphatidylethanolamine-binding protein 1 as a putative ligand that activates the odorant receptor at the axon terminal and affects the turning behavior of sensory axons. Genetic ablation of phosphatidylethanolamine-binding protein 1 in mice results in a strongly disturbed olfactory sensory map. Our data suggest that the odorant receptor at the axon terminal of olfactory neurons acts as an axon guidance cue that responds to molecules originating in the olfactory bulb. The dual function of the odorant receptor links specificity of odor perception and axon targeting.

Raf kinase inhibitor protein mediates myocardial fibrosis under conditions of enhanced myocardial oxidative stress.

Fibrosis is a hallmark of maladaptive cardiac remodelling. Here we report that genome-wide quantitative trait locus (QTL) analyses in recombinant inbred mouse lines of C57BL/6 J and DBA2/J strains identified Raf Kinase Inhibitor Protein (RKIP) as genetic marker of fibrosis progression. C57BL/6 N-RKIP-/- mice demonstrated diminished fibrosis induced by transverse aortic constriction (TAC) or CCl4 (carbon tetrachloride) treatment compared with wild-type controls. TAC-induced expression of collagen Iα2 mRNA, Ki67+ fibroblasts and marker of oxidative stress 8-hydroxyguanosine (8-dOHG)+ fibroblasts as well as the number of fibrocytes in the peripheral blood and bone marrow were markedly reduced in C57BL/6 N-RKIP-/- mice. RKIP-deficient cardiac fibroblasts demonstrated decreased migration and fibronectin production. This was accompanied by a two-fold increase of the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2), the main transcriptional activator of antioxidative proteins, and reduced expression of its inactivators. To test the importance of oxidative stress for this signaling, C57BL/6 J mice were studied. C57BL/6 J, but not the C57BL/6 N-strain, is protected from TAC-induced oxidative stress due to mutation of the nicotinamide nucleotide transhydrogenase gene (Nnt). After TAC surgery, the hearts of Nnt-deficient C57BL/6 J-RKIP-/- mice revealed diminished oxidative stress, increased left ventricular (LV) fibrosis and collagen Iα2 as well as enhanced basal nuclear expression of Nrf2. In human LV myocardium from both non-failing and failing hearts, RKIP-protein correlated negatively with the nuclear accumulation of Nrf2. In summary, under conditions of Nnt-dependent enhanced myocardial oxidative stress induced by TAC, RKIP plays a maladaptive role for fibrotic myocardial remodeling by suppressing the Nrf2-related beneficial effects.

Raf Kinase Inhibitor Protein Preferentially Promotes TLR3-Triggered Signaling and Inflammation.

Raf kinase inhibitor protein (RKIP) protects against host immunological responses in nematodes and Drosophila Whether RKIP functions in innate immune responses in mammals remains unknown. In this article, we report that RKIP preferentially regulates the TLR3-mediated immune response in macrophages. RKIP deficiency or silencing significantly decreases polyinosinic:polycytidylic acid [Poly(I:C)]-induced IFN-ß, IL-6, and TNF-α production without affecting the counterpart induced by LPS or CpG. Compared with their wild-type counterparts, RKIP-deficient mice produce less IFN-ß, IL-6, and TNF-α in serum and display decreased lethality upon peritoneal Poly(I:C) plus d-galactosamine injection. Mechanistically, RKIP interacts with TBK1 and promotes the Poly(I:C)-induced TANK-binding kinase 1/IRF3 activation. Simultaneously, RKIP enhances the Poly(I:C)-induced interaction between TGF-ß-activated kinase 1 and MAPK kinase 3 (MKK3), thus promoting MKK3/6 and p38 activation. We further demonstrated that Poly(I:C) treatment, but not LPS treatment, induces RKIP phosphorylation at S109. This action is required for RKIP to promote TANK-binding kinase 1 activation, as well as the interaction between TGF-ß-activated kinase 1 and MKK3, which lead to activation of the downstream IRF3 and p38, respectively. Therefore, RKIP acts as a positive-feedback regulator of the TLR3-induced inflammatory response and may be a potential therapeutic target for inflammatory disease.

Loss of Raf kinase inhibitor protein is associated with malignant progression in hepatic fibrosis.

Raf kinase inhibitory protein (RKIP), besides regulating important intracellular signaling cascades, was described to be associated with progression, metastasis and prognosis in several human neoplasms. But its role in hepatic fibrogenesis remains unclear. In the present study, we found that the absence of RKIP expression significantly enhanced the proliferation of HSC-T6 cells. Reduced RKIP expression promoted the activation of HSCs and the accumulation of collagen, as evidenced by the increases in the levels of collagen I and α-smooth muscle actin. Moreover, down-regulating RKIP expression led to severe histopathological changes and collagen accumulation in hepatic tissues of rats with liver fibrosis. Furthermore, the absence of RKIP promoted the activation of ERK/MAPK pathway in vitro and in vivo. Our findings clearly demonstrate an inverse correlation between RKIP level and the degree of the liver injury and fibrosis. Loss of RKIP may be associated with malignant progression in hepatic fibrosis.

Loss of Raf-1 kinase inhibitory protein delays early-onset severe retinal ciliopathy in Cep290rd16 mouse.

PURPOSE: Mutations in the cilia-centrosomal protein of centrosomal protein of 290 kDa (CEP290) result in severe ciliopathies, including autosomal recessive early onset childhood blindness disorder Leber congenital amaurosis (LCA). The Cep290(rd16) (retinal degeneration 16) mouse model of CEP290-LCA exhibits accumulation of CEP290-interacting protein Raf-1 kinase inhibitory protein (RKIP) prior to onset of retinal degeneration (by postnatal day P14). We hypothesized that reducing RKIP levels in the Cep290(rd16) mouse will delay or improve retinal phenotype. METHODS: We generated double mutant mice by combining the Cep290(rd16) and Rkip(ko) alleles (Cep290(rd16):Rkip(+/ko) and Cep290(rd16):Rkip(ko/ko)). Retinal function was assessed by ERG and retinal morphology and protein trafficking were assessed by histology, transmission electron microscopy (TEM), and immunofluorescence analysis. Cell death was examined by apoptosis. RESULTS: Prior to testing our hypothesis, we examined ERG and retinal morphology of Rkip(ko/ko) mice and did not find any detectable differences compared with wild-type mice. The Cep290(rd16):Rkip(+/ko) mice exhibited similar retinopathy as Cep290(rd16); however, Cep290(rd16): Rkip(ko/ko) double knockout mice demonstrated a substantial improvement (>9-fold) in photoreceptor function and structure at P18 as of Cep290(rd16) mice. We consistently detected transient preservation of photoreceptors at P18 and polarized trafficking of opsins to sensory cilia in the double mutant mice; however, retinal degeneration ensued by P30. CONCLUSIONS: Our studies implicate CEP290-RKIP pathway in CEP290-retinal degeneration and suggest that targeting RKIP levels can delay photoreceptor degeneration, assisting in extending the time-window for treating such rapidly progressing blindness disorder.

Frequent loss of RAF kinase inhibitor protein expression in acute myeloid leukemia.

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

Mice lacking the Raf-1 kinase inhibitor protein exhibit exaggerated hypoxia-induced pulmonary hypertension.

BACKGROUND AND PURPOSE: Increased pulmonary vascular remodelling, pulmonary arterial pressure and pulmonary vascular resistance characterize the development of pulmonary arterial hypertension (PAH). Activation of the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK)1/2 is thought to play an important role in PAH and Raf-1 kinase inhibitor protein (RKIP), negatively regulates this pathway. This study investigated whether genetic deletion of RKIP (and hence ERK1/2 up-regulation) resulted in a pulmonary hypertensive phenotype in mice and investigated a role for RKIP in mitogen-regulated proliferative responses in lung fibroblasts. EXPERIMENTAL APPROACH: Pulmonary vascular haemodynamics and remodelling were assessed in mice genetically deficient in RKIP (RKIP-/-) after 2 weeks of either normoxia or hypoxia. Immunoblotting and immunohistochemistry were used to examine phosphorylation of Raf-1, RKIP and ERK1/2 in mouse pulmonary arteries. In vitro, RKIP inhibition of mitogen signalling was analysed in CCL39 hamster lung fibroblasts. KEY RESULTS: RKIP-/- mice demonstrated elevated indices of PAH and ERK1/2 phosphorylation compared with wild-type (WT) mice. Hypoxic RKIP-/- mice exhibited exaggerated PAH indices. Hypoxia increased phosphorylation of Raf-1, RKIP and ERK1/2 in WT mouse pulmonary arteries and Raf-1 phosphorylation in RKIP-/- mouse pulmonary arteries. In CCL39 cells, inhibition of RKIP potentiated mitogen-induced proliferation and phosphorylation of RKIP, and Raf-1. CONCLUSIONS AND IMPLICATIONS: The lack of RKIP protein resulted in a pulmonary hypertensive phenotype, exaggerated in hypoxia. Hypoxia induced phosphorylation of RKIP signalling elements in WT pulmonary arteries. RKIP inhibition potentiated mitogen-induced proliferation in lung fibroblasts. These results provide evidence for the involvement of RKIP in suppressing the development of hypoxia-induced PAH in mice.

Loss of RAF kinase inhibitor protein is a somatic event in the pathogenesis of therapy-related acute myeloid leukemias with C-RAF germline mutations.

We recently described oncogenic and anti-apoptotic C-RAF germline mutations in patients with therapy-related acute myeloid leukemia (t-AML). Activation of the RAF effector ERK was restricted to transformed cells, suggesting the requirement for cooperating events in leukemogenesis. Western blot analysis of blast cells from patients with C-RAF germline mutations revealed loss of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP). Immunohistochemistry of the patients' primary tumors revealed normal RKIP expression levels, indicating that the loss of RKIP is a somatic, t-AML-specific event. In focus formation assays, the oncogenic potential of human mutant C-RAF was strongly influenced by expression levels of RKIP. Although the number of colonies formed by C-RAF(S427G) was significantly increased by RKIP silencing, the opposite was observed after RKIP overexpression. These results show that the loss of RKIP is a functional somatic event in carriers of C-RAF germline mutations, which contributes to the development of t-AML.

Loss of Raf kinase inhibitory protein induces radioresistance in prostate cancer.

PURPOSE: External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS: The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS: RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION: The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

Mice lacking Raf kinase inhibitor protein-1 (RKIP-1) have altered sperm capacitation and reduced reproduction rates with a normal response to testicular injury.

Raf kinase inhibitor protein-1 (RKIP-1) belongs to the phosphatidyl ethanolamine-binding family of proteins (PEBP), which are highly conserved throughout evolution and widely expressed in tissues of mammalian organisms. RKIP-1 is a modulator of extracellular signal-regulated kinase (ERK), nuclear factor-kappa B (NF-kappaB), and G protein coupled receptor (GPCR) signaling cascades and is implicated as a factor in numerous physiological processes and disease states including metastasis. Testicular germ cells also express high levels of RKIP mRNA during spermatogenesis, particularly from late pachytene spermatocytes through step 15 elongate spermatids. Therefore, the sensitivity of spermatogenesis to injury was compared in wild-type and RKIP-1(-/-) mice. Unlike what has been described with tumor suppressors such as p53, RKIP-1(-/-) and wild-type mice were equally sensitive to germ cell toxicity by x-irradiation as assessed by terminal deoxynucleotidyl transferase biotin-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) positivity 9 hours after a 5 Gy exposure and testicular spermatid head counts 15.5 days after 0.5 Gy exposure. Recent findings also indicate that RKIP is a decapacitation factor receptor on sperm. The present study demonstrates that sperm from RKIP-deficient mice are precociously capacitated compared with their wild-type counterparts. Data from mating experiments indicate decreased reproduction rates between crosses of RKIP-1(-/-) male mice and either heterozygous or RKIP-1(-/-) females. Furthermore, RKIP immunolocalization of epididymal sperm supports transfer of the protein from germ cell cytoplasm to the sperm via the cytoplasmic droplet during epididymal transport. Overall, these studies indicate an important role for RKIP in reproduction as a modulator of capacitation but not in the regulation of testicular injury.