No abstract

Purpose Expression of the ΔN isoform of p63 (ΔNp63) is a diagnostic marker highly specific for lung squamous cell carcinoma (SCC). We previously found that Syntaxin Binding Protein 4 (STXBP4) regulates ΔNp63 ubiquitination, suggesting that STXBP4 may also be a SCC biomarker. To address this issue, we investigated the role of STXBP4 expression in SCC biology and the impact of STXBP4 expression on SCC prognosis. Experimental design We carried out a clinicopathological analysis of STXBP4 expression in 87 lung SCC patients. Whole transcriptome analysis using RNA-seq was performed in STXBP4-positive and STXBP4-negative tumors of lung SCC. Soft agar assay and xenograft assay were performed using overexpressing or knockdown SCC cells. Results Significantly higher levels of STXBP4 expression were correlated with accumulations of ΔNp63 in clinical lung SCC specimens (Spearman’s rank correlation ρ=0.219). Notably, STXBP4-positive tumors correlated with three important clinical parameters: T factor (P<0.001), disease stage (P=0.030) and pleural involvement (P=0.028). Whole transcriptome sequencing followed by pathway analysis indicated that STXBP4 is involved in functional gene networks that regulate cell growth, proliferation, cell death and survival in cancer. Platelet-Derived Growth Factor Receptor alpha (PDGFRα) was a key downstream mediator of STXBP4 function. In line with this, shRNA mediated STXBP4 and PDGFRA knockdown suppressed tumor growth in soft agar and xenograft assays. Conclusions STXBP4 plays a crucial role in driving SCC growth and is an independent prognostic factor for predicting worse outcome in lung SCC. These data suggest that STXBP4 is a relevant therapeutic target for patients with lung SCC.

The plasmodium Physarum polycephalum exhibits periodic cycles of cytoplasmic streaming in association with those of contraction and relaxation movement. In the present study, we injected Calcium Green dextran as a fluorescent Ca2+ indicator into the thin-spread living plasmodium. We found changes in the [Ca2+]i (intracellular concentration of Ca2+), which propagated in a wave-like form in its cytoplasm. The Ca2+ waves were also detected when we used Fura dextran which detected [Ca2+]i by the ratio of two wavelengths. We prepared the plasmodial fragment from the thin-spread and found that the cycles of the contraction-relaxation movement was so synchronized that the measurement of its area provided an indication of the movement. We observed that [Ca2+]i also synchronized in the entire fragment and that the relaxation ensued upon the reduction in [Ca2+]i. We suggest that the Ca2+ wave generated periodically is one of the major factors playing a crucial role in the relaxation of P. polycephalum.

The expression of A3B in breast cancer was higher than in non-cancerous tissues and was related to the lymph node metastasis and nuclear grade, which are reliable aggressive phenotype markers in breast cancer. Evaluation of A3B expression in tumor may be a marker for breast cancer with malignant potential.

Molecular mechanisms underlying migration of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (SPC) were analyzed in light of the hypothesis that remodeling of the actin cytoskeleton should be involved. After SPC stimulation, mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38) and p42/44 MAPK (p42/44), were found to be phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44. Pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 phosphorylation and VSMC migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed migration. Complete inhibition was achieved with the addition of SB-203580. After SPC stimulation, the actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated filopodia, i.e., magunapodia. The peripheral actin bundles consisted of alpha- and beta-actin, but magunapodia consisted exclusively of beta-actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphological data confirmed the regulation of actin remodeling and suggest that VSMCs migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway.

We successfully synthesized full-length and the mutant Physarum myosin and heavy meromyosin (HMM) constructs associated with Physarum regulatory light chain and essential light chain (PhELC) using Physarum myosin heavy chain in Sf-9 cells, and examined their Ca(2+)-mediated regulation. Ca(2+) inhibited the motility and ATPase activities of Physarum myosin and HMM. The Ca(2+) effect is also reversible at the in vitro motility of Physarum myosin. We demonstrated that full-length myosin increases the Ca(2+) inhibition more effectively than HMM. Furthermore, Ca(2+) did not affect the motility and ATPase activities of the mutant Physarum myosin with PhELC that lost Ca(2+)-binding ability. Therefore, we conclude that PhELC plays a critical role in Ca(2+)-dependent regulation of Physarum myosin.

To explore the precise mechanisms of the inhibitory effects of blebbistatin, a potent inhibitor of myosin II, on smooth muscle contraction, we studied the blebbistatin effects on the mechanical properties and the structure of contractile filaments of skinned (cell membrane permeabilized) preparations from guinea pig taenia cecum. Blebbistatin at 10 microM or higher suppressed Ca(2+)-induced tension development at any given Ca(2+) concentration but had little effects on the Ca(2+)-induced myosin light chain phosphorylation. Blebbistatin also suppressed the 10 and 2.75 mM Mg(2+)-induced, "myosin light chain phosphorylation-independent" tension development at more than 10 microM. Furthermore, blebbistatin induced conformational change of smooth muscle myosin (SMM) and disrupted arrangement of SMM and thin filaments, resulting in inhibition of actin-SMM interaction irrespective of activation with Ca(2+). In addition, blebbistatin partially inhibited Mg(2+)-ATPase activity of native actomyosin from guinea pig taenia cecum at around 10 microM. These results suggested that blebbistatin suppressed skinned smooth muscle contraction through disruption of structure of SMM by the agent.

Abstract. We prepared a cell-populated collagen-gel fiber including GbaSM-4 cells derived from the basilar artery of guinea pigs. This fiber tended to be a differentiated contractile phenotype in electron-microscope observations. Sphingosylphosphorylcholine (SPC) can induce contraction of the fiber (EC 50 = 0.70 ± 0.05 µM), and blebbistatin can inhibit the SPC-induced contraction (IC 50 = 22.8 ± 1.26 µM). Phosphorylation of the 20 kD myosin light chain (MLC20) significantly increased in GbaSM-4 cells provided with 1 µM SPC (P<0.05), which was maintained in the presence of 1 to 100 µM blebbistatin. These results suggest that vascular smooth muscle can relax even if MLC20 is phosphorylated.