Olfactory receptor (OR)-associated events are mediated by well-conserved components in the olfactory epithelium, including olfactory G-protein (Golf), adenylate cyclase III (ACIII), and olfactory marker protein (OMP). The expression of ORs has recently been observed in non-olfactory tissues where they are involved in monitoring extracellular chemical cues. The large number of OR genes and their sequence similarities illustrate the need to find an effective and simple way to detect non-olfactory OR-associated events. In addition, expression profiles and physiological functions of ORs in non-olfactory tissues are largely unknown. To overcome limitations associated with using OR as a target protein, this study used OMP with Golf and ACIII as targets to screen for potential OR-mediated sensing systems in non-olfactory tissues. Here, we show using western blotting, real-time PCR, and single as well as double immunoassays that ORs and OR-associated proteins are co-expressed in diverse tissues. The results of immunohistochemical analyses showed OMP (+) cells in mouse heart and in the following cells using the corresponding marker proteins c-kit, keratin 14, calcitonin, and GFAP in mouse tissues: interstitial cells of Cajal of the bladder, medullary thymic epithelial cells of the thymus, parafollicular cells of the thyroid, and Leydig cells of the testis. The expression of ORs in OMP (+) tissues was analyzed using a refined microarray analysis and validated with RT-PCR and real-time PCR. Three ORs (olfr544, olfr558, and olfr1386) were expressed in the OMP (+) cells of the bladder and thyroid as shown using a co-immunostaining method. Together, these results suggest that OMP is involved in the OR-mediated signal transduction cascade with olfactory canonical signaling components between the nervous and endocrine systems. The results further demonstrate that OMP immunohistochemical analysis is a useful tool for identifying expression of ORs, suggesting OMP expression is an indicator of potential OR-mediated chemoreception in non-olfactory systems.

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Chronic exposure to TGFβ, a frequent occurrence for tumor cells in the tumor microenvironment, confers more aggressive phenotypes on cancer cells by promoting their invasion and migration while at the same time increasing their resistance to the growth-inhibitory effect of TGFβ. In this study, a transdifferentiated (TD) A549 cell model, established by chronically exposing A549 cells to TGFβ, showed highly invasive phenotypes in conjunction with attenuation of Smad-dependent signaling. We show that Snail protein, the mRNA expression of which strongly correlates with a poor prognosis in lung cancer patients, was highly stable in TD cells after TGFβ stimulation. The increased protein stability of Snail in TD cells correlated with elevated inhibitory phosphorylation of GSK3β, resulting from the high Akt activity. Notably, integrin β3, whose expression was markedly increased upon sustained exposure to TGFβ, was responsible for the high Akt activity as well as the increased Snail protein stability in TD cells. Consistently, clinical database analysis on lung cancer patients revealed a negative correlation between overall survival and integrin β3 mRNA levels. Therefore, we suggest that the integrin β3-Akt-GSK3β signaling axis plays an important role in non-canonical TGFβ signaling, determining the invasive properties of tumor cells chronically exposed to TGFβ.

While metastasis, the main cause of lung cancer-related death, has been extensively studied, the underlying molecular mechanism remains unclear. A previous clinicogenomic study revealed that expression of N-acetylgalactosaminyltransferase (GalNAc-T14), is highly inversely correlated with recurrence-free survival in those with non-small cell lung cancer (NSCLC). However, the underlying molecular mechanism(s) has not been determined. Here, we showed that GalNAc-T14 expression was positively associated with the invasive phenotype. Microarray and biochemical analyses revealed that HOXB9, the expression of which was increased in a GalNAc-T14-dependent manner, played an important role in metastasis. GalNAc-T14 increased the sensitivity of the WNT response and increased the stability of the β-catenin protein, leading to induced expression of HOXB9 and acquisition of an invasive phenotype. Pharmacological inhibition of β-catenin in GalNAc-T14-expressing cancer cells suppressed HOXB9 expression and invasion. A meta-analysis of clinical genomics data revealed that expression of GalNAc-T14 or HOXB9 was strongly correlated with reduced recurrence-free survival and increased hazard risk, suggesting that targeting β-catenin within the GalNAc-T14/WNT/HOXB9 axis may be a novel therapeutic approach to inhibit metastasis in NSCLC.

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Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR.

We investigated the effects of two antimicrobial peptides (AMPs) isolated from Scolopendra subspinipes mutilans on neutrophil activity. Stimulation of mouse neutrophils with the two AMPs elicited chemotactic migration of the cells in a pertussis toxin-sensitive manner. The two AMPs also stimulated activation of ERK and Akt, which contribute to chemotactic migration of neutrophils. We found that AMP-stimulated neutrophil chemotaxis was blocked by a formyl peptide receptor (FPR) 1 antagonist (cyclosporin H); moreover the two AMPs stimulated the chemotactic migration of FPR1-expressing RBL-2H3 cells but not of vector-expressing RBL-2H3 cells. We also found that the two AMPs stimulate neutrophil migration in vivo, and that this effect is blocked in FPR1-deficient mice. Taken together, our results suggest that the two AMPs stimulate neutrophils, leading to chemotactic migration through FPR1, and the two AMPs will be useful for the study of FPR1 signaling and neutrophil activation. [BMB Reports 2016; 49(9): 520-525]