No abstract

Cell adhesion is essential for proper tissue architecture and function in multicellular organisms. Cell adhesion molecules not only maintain tissue integrity but also possess signaling properties that contribute to diverse cellular events such as cell growth, survival, differentiation, polarity, and migration; however, the underlying molecular basis remains poorly defined. Here we identify that the cell adhesion signal initiated by the tight-junction protein claudin-6 (CLDN6) regulates nuclear receptor activity. We show that CLDN6 recruits and activates Src-family kinases (SFKs) in second extracellular domain-dependent and Y196/200-dependent manners, and SFKs in turn phosphorylate CLDN6 at Y196/200. We demonstrate that the CLDN6/SFK/PI3K/AKT axis targets the AKT phosphorylation sites in the retinoic acid receptor γ (RARγ) and the estrogen receptor α (ERα) and stimulates their activities. Interestingly, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors. We propose that a similar link between diverse cell adhesion and nuclear receptor signalings coordinates a wide variety of physiological and pathological processes.

Genetically encoded Forster resonance energy transfer (FRET)based biosensors have been developed for the visualization of signaling molecule activities. Currently, most of them are comprised of cyan and yellow fluorescent proteins (CFP and YFP), precluding the use of multiple FRET biosensors within a single cell. Moreover, the FRET biosensors based on CFP and YFP are incompatible with the optogenetic tools that operate at blue light. To overcome these problems, here, we have developed FRET biosensors with red-shifted excitation and emission wavelengths. We chose mKOκ and mKate2 as the favorable donor and acceptor pair by calculating the Forster distance. By optimizing the order of fluorescent proteins and modulatory domains of the FRET biosensors, we developed a FRET biosensor backbone named "Booster". The performance of the protein kinase A (PKA) biosensor based on the Booster backbone (Booster-PKA) was comparable to that of AKAR3EV, a previously developed FRET biosensor comprising CFP and YFP. For the proof of concept, we first showed simultaneous monitoring of activities of two protein kinases with Booster-PKA and ERK FRET biosensors based on CFP and YFP. Second, we showed monitoring of PKA activation by Beggiatoa photoactivated adenylyl cyclase, an optogenetic generator of cyclic AMP. Finally, we presented PKA activity in living tissues of transgenic mice expressing Booster-PKA. Collectively, the results demonstrate the effectiveness and versatility of Booster biosensors as an imaging tool in vitro and in vivo.

Prostaglandin E2 (PGE2) promotes tumor progression through evasion of antitumor immunity. In stark contrast to cyclooxygenase-dependent production of PGE2, little is known whether PGE2 secretion is regulated within tumor tissues. Here, we show that VEGF-dependent release of thromboxane A2 (TXA2) triggers Ca2+ transients in tumor cells, culminating in PGE2 secretion and subsequent immune evasion in the early stages of tumorigenesis. Ca2+ transients caused cPLA2 activation and triggered the arachidonic acid cascade. Ca2+ transients were monitored as the surrogate marker of PGE2 secretion. Intravital imaging of BrafV600E mouse melanoma cells revealed that the proportion of cells exhibiting Ca2+ transients is markedly higher in vivo than in vitro. The TXA2 receptor was indispensable for the Ca2+ transients in vivo, high intratumoral PGE2 concentration, and evasion of antitumor immunity. Notably, treatment with a VEGF receptor antagonist and an anti-VEGF antibody rapidly suppressed Ca2+ transients and reduced TXA2 and PGE2 concentrations in tumor tissues. These results identify the VEGF–TXA2 axis as a critical promoter of PGE2-dependent tumor immune evasion, providing a molecular basis underlying the immunomodulatory effect of anti-VEGF therapies. Significance: This study identifies the VEGF–TXA2 axis as a potentially targetable regulator of PGE2 secretion, which provides novel strategies for prevention and treatment of multiple types of malignancies.

Since hepatitis C virus (HCV) is thought to enter into host hepatocytes using the same cellular pathways regardless of the genotypes, the host factors are promising targets to prevent and treat HCV infection. Human occludin (hOCLN) is one representative entry factor, and its second extracellular loop (EC2) contributes to the species selectivity of HCV-susceptibility. However, the exact function of hOCLN during HCV entry remains unknown, and no hOCLN-targeting antibodies or synthetic drugs that prevent and treat HCV infection have yet been developed. Here we generated the anti-hOCLN-EC2 monoclonal antibody (mAb) 67-2, and demonstrated that it efficiently inhibited HCV infection in the HCV-permissive human cell line Huh7.5.1. We also showed, using three different culture systems of Huh7.5.1 cells, that this novel mAb is accessible to OCLN from the basolateral side of hepatocytes but not from the apical side. In addition, our Western blot analyses indicated that the established 67-2 mAb reacted not only with hOCLN but also with mouse OCLN, strongly suggesting that 67-2 does not recognize the human-specific amino acids in OCLN-EC2. Moreover, we revealed that the anti-hOCLN-EC2 mAb 67-2 showed no adverse effects on cell viability or the barrier function of tight junctions.