Michiko Takeoka

Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells

Calponin h1 (CNh1) is an actin-binding protein that is expressed mainly in smooth muscle cells and is known to regulate smooth muscle contraction. Recently, re-expression of CNh1 in leiomyosarcoma cell lines is reported to suppress cell proliferation and tumorigenicity. However, little is known about the associated cellular structural and functional changes. Since CNh1 is also detected in normal fibroblasts, we hypothesised that CNh1 would also inhibit cell proliferation of the fibrosarcoma cells, HT1080, in which CNh1 is suppressed. An expression vector of human CNh1 complementary DNA was transfected into human HT1080 cells by a calcium-phosphate precipitation method. CNh1-transfected cells exhibited a flattened morphology with organised actin filaments, a significant decrease in cell motility and enhancement in adhesion to fibronectin in association with an increase in integrin alpha5beta1 expression. Anchorage-independent growth and tumorigenicity in nude mice were suppressed in the CNh1-transfected cells. Our results suggest that CNh1 may have a role as a tumour suppressor in human fibrosarcoma by influencing cytoskeletal activities.

Comparisons of oxygen transport between Tibetan and Han residents at moderate altitude

To compare the difference for oxygen transport between Tibetans living for generations at high altitude and acclimatized Han newcomers to high altitude, we measured the ventilatory, circulatory, metabolic, and gas exchange functions during exercise in 17 Tibetans and 19 Hans at the altitude of 3417 m. At maximal effort, the Tibetans, compared with the Hans, showed higher O 2 consumption ( V ˙ o 2 max ) (2.54±0.1 versus 2.10±0.1 liters min −1 , p p −1 , p P −1 ) was higher than that in the Hans (68.0±3.9 liters min −1 ), but the ventilatory equivalent was equal. The anaerobic threshold for absolute O 2 consumption ( V ˙ o 2 AT ) for the Tibetans and Hans was 1.91±0.1 versus 1.45±0.1 liters min −1 , respectively ( p 2 consumption (in % V ˙ o 2 max ) was 76.2±1.5 versus 70.5±2.2, respectively ( p V ˙ o 2 max correlated well with maximal cardiac output. No difference was found in the maximal heart rate between the two groups. Arterial oxygen desaturation from rest to exercise in the Hans (15.3%; 90.3–76.5%) was greater than that in the Tibetans (9.2%; 90.1–81.8%). We conclude that the lifelong exposure to an hypoxic environment has resulted in an increase in oxygen transport and improved aerobic exercise performance.

A Splice Variant of ASC Regulates IL-1β Release and Aggregates Differently from Intact ASC

The apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) is involved in apoptosis and innate immunity and is a major adaptor molecule responsible for procaspase-1 activation. ASC mRNA is encoded by three exons: exons 1 and 3 encode a pyrin domain (PYD) and caspase recruit domain (CARD), respectively, and exon 2 encodes a proline and glycine-rich (PGR) domain. Here, we identified a variant ASC protein (vASC) lacking the PGR domain that was smaller than full length ASC (fASC) derived from fully transcribed mRNA and searched for differences in biochemical and biological nature. Both fASC and vASC were found to activate procaspase-1 to a similar degree, but the efficiency of IL-1β excretion was significantly higher for vASC. There was also a marked structural difference observed in the fibrous aggregates formed by fASC and vASC. These results suggest that although the PGR domain is dispensable for procaspase-1 activation, it plays an important role in the regulation of the molecular structure and activity of ASC.

Impaired Arterial Pressure Regulation During Exercise Due to Enhanced Muscular Vasodilatation in Calponin Knockout Mice

Calponin is known to be an actin binding protein in smooth muscle, inhibiting actomyosin ATPase activity in vitro. We previously reported that α‐adrenergic vasoconstriction in calponin knockout (KO) mice was reduced compared with that in wild‐type C57BL/6J (WT) mice and, as a compensation, arterial baroreflex sensitivity in KO mice was enhanced at rest. In the present study, we assessed arterial pressure regulation in WT and KO mice during graded treadmill exercise at 5, 10, and 15 m min‐1. Mean arterial pressure (MAP) in KO mice fluctuated more than that in WT mice at every speed of exercise with two‐fold higher variances (P < 0.001). The baroreflex sensitivity (ΔHR/ΔMAP) in WT mice (n= 6), determined from the heart rate response (δHR) to spontaneous change in MAP (δMAP), was ‐5.1 ± 0.6 beats min‐1 mmHg‐1 (mean ±s.e.m.) at rest and remained unchanged at ‐5.0 ± 0.9 beats min‐1 mmHg‐1 during exercise (P < 0.01), while that in KO mice (n= 6) was ‐9.9 ± 1.7 beats min‐1 mmHg‐1 at rest, significantly higher than that in WT mice (P < 0.001), and was reduced to ‐4.7 ± 0.4 beats min‐1 mmHg‐1 during exercise (P < 0.01), not significantly different from that in WT mice. In another experiment, we measured muscle blood flow (MBF) in the thigh by laser‐Doppler flowmetry, electromyogram (EMG), and MAP during voluntary locomotion in KO (n= 7) and WT (n= 7) mice. Muscle vascular conductance, MBF/MAP, started to increase immediately after locomotion, judged from EMG, and reached 50 % of the maximum after the time of 2.3 ± 0.2 s in KO mice, shorter than 5.8 ± 0.6 s in WT mice (P < 0.001). Prior administration of α‐adrenergic blockade (phentolamine) shortened the time in WT mice to that in KO mice (P < 0.001), but did not shorten the time in KO mice. Thus, impaired MAP regulation in KO mice during exercise was caused by a blunted muscle vascular α‐adrenergic contractile response and by the attenuated HR response to spontaneous change in MAP due to reduced baroreflex sensitivity.

Influence of hypoxia and pulmonary air embolism on lung injury in perfused rat lungs.

We investigated the influence of low oxygen ventilation, air-bubble infusion into the pulmonary artery and their synergistic effect on pulmonary hemodynamics and microvascular permeability in isolated perfused rat lungs. Pulmonary arterial pressure was significantly increased by 70 min of ventilation with 3% O2 (hypoxia, group H); by 0.2-ml air-bubble infusion (pulmonary air embolism, group AE), and by 0.2-ml air-bubble infusion and 70 min of 3% O2 ventilation (hypoxia and pulmonary air embolism, group H & AE) compared with that of a control group (0.2 ml saline infusion, group C). Neither total (TPR) nor arterial (Ra) pulmonary vascular resistance in group H showed any difference compared to control values. TPR and Ra in groups AE and H & AE were significantly higher than those in group C. However, there was no significant difference in TPR or Ra between groups AE and H & AE. The pulmonary capillary fluid filtration coefficient, dry lung to wet lung weight ratio and white blood cell count in the perfusate of group H were not changed, while those of the groups AE and H & AE were significantly increased compared to those of controls. However, there was no significant difference in these values between groups AE and H & AE. Since hypoxia did not damage isolated perfused rat lungs, as determined by hemodynamics and permeability, nor enhance lung injury caused by air embolism, it was suggested that air embolism contributed more to high-altitude lung injury than low oxygen.

Effects of high altitudes on finger cooling test in Japanese and Tibetans at Qinghai Plateau

The influences of both hypobaric hypoxia and cold on peripheral circulation were studied using the finger cooling test (measurement of the decrease in finger temperature, measured at the dorsal surface of the finger, during immersion of the hand in 0° C water for 20 min) at Qinghai Plateau. The same test was carried out at simulated altitudes in a 25° C climatic chamber to separate the hypobaric hypoxia influence from that of cold. In Japanese subjects at Qinghai Plateau there was a significant difference between finger skin temperatures (FSTs) during 20 min of 0° C water immersion at altitudes of 2260 m and 4860 m by ANOVA. Mean finger skin temperature during the 20-min immersion (5–20 min, MST) measured at 4860 m was significantly lower than that at 2260 m. In Tibetan subjects, there was also a significant difference between FSTs at 2260 m and at 4860 m by ANOVA. MST at 4860 m tended to be lower than that at 2260 m. In the 25° C climatic chamber, there was a significant difference between FSTs of Japanese expedition members at 2000 m and at 4000 m by ANOVA. MST was higher at 4000 m than at 2000 m, contrary to the data obtained in Qinghai. In conclusion, the higher skin temperature in response to local cold immersion, which would have been caused by stronger hypobaric hypoxia, must have been masked by the lower ambient temperature.

Role of ASC in the mouse model of Helicobacter pylori infection.

Apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC) is an adaptor molecule activating caspase-1 that stimulates pro-interleukin-1β (pro-IL-1β) and pro-IL-18, two pro-inflammatory cytokines with critical functions in host defense against a variety of pathogens. In this study, we investigated the role of ASC in the host defense against Helicobacter pylori utilizing ASC-deficient mice. Mice were orally inoculated with H. pylori; bacterial load, degree of gastritis, and mucosal levels of inflammatory cytokines were analyzed and compared with those obtained from wild-type mice. We found more prominent H. pylori colonization in ASC-deficient mice, as revealed by colony-forming unit counts. Both groups of mice developed gastritis; however, ASC-deficient mice showed significant attenuation of inflammation despite high H. pylori colonization. ELISA, immunohistochemistry, and quantitative RT-PCR analyses revealed complete suppression of IL-1β and IL-18, and substantial reduction of interferon-γ (IFN-γ) expression, in ASC-deficient mice without apparent upregulation of other cytokines, including IL-10 and tumor necrosis factor-α. These results as a whole indicate that ASC exerts considerable influence on the host defense, acting through IL-1β/IL-18 and subsequent IFN-γ production, which in turn contributes to continuous chronic inflammatory response and consequent reduction of H. pylori colonization.

Calponin h1 Suppresses Tumor Growth of Src‐induced Transformed 3Y1 Cells in Association with a Decrease in Angiogenesis

Calponin h1 (CNhl) is a basic actin‐binding protein that is abundantly expressed in smooth muscle cells and involved in smooth muscle contraction by inhibiting actomyosin MgATPase. In recent studies, CNhl was noted to suppress cell proliferation and tumorigenicity in leiomyosarcoma and tumor growth in fibrosarcoma cell lines. To further investigate the function of CNhl as a tumor suppressor, we transfected the human CNhl gene into a v‐src‐transformed rat fibroblast cell line SR–3Y1. The volume of the tumors derived from one randomly selected CNh1‐transfectant (C1) in nude mice was reduced to 34.1% of that from a randomly selected vector transfectant (VI). A similar tendency was observed in another independent pair (C2, V2). Pathological analysis showed a significant decrease in the number of mitotic cells in the CNh1‐transfectants. Further, a marked reduction in the number of vessels in the CNhl‐transfectant was observed. DNA synthesis under conditions without serum was significantly reduced in the CNhl‐transfectant (C1) compared with the control transfectant (VI), while no significant difference was seen in the cellular growth in the presence of 10% serum. A slight but significant reduction in in vitro cellular motility in the CNhl‐transfectant was also observed. While the suppression of growth potential and cell motility by CNhl transfer was significant but partial, a marked reduction in vascular endothelial growth factor (VEGF) mRNA and the secretion of VEGF protein was observed in the CNhl‐transfectant. These results suggest that CNhl plays a role as tumor suppressor in SR–3Y1 mainly by decreasing VEGF expression and angiogenesis in vivo and partially through reducing cellular proliferative potential and cell motility.

Suppression of peritoneal dissemination through protecting mesothelial cells from retraction by cancer cells

In a previous study, we demonstrated that calponin h1 suppressed tumor growth of transformed cells and that the peritonitis carcinomatosa induced by mouse B16‐F10 melanoma (F10) cells was more extensive in calponin h1‐deficient (CN−/−) mice with fragility of mesothelial (MS) cells than in their calponin h1‐wild (CN+/+) counterparts. In our study, we assessed the therapeutic effect of calponin h1 on peritoneal dissemination. F10 cells were overlaid on the cultured CN+/+ or CN−/− MS cells and the effect of calponin h1 on retraction of MS cells was evaluated. Then, an adenoviral vector with the calponin h1 gene (AdGFP‐CN) inserted was constructed and was applied to CN−/− MS cells or CN−/− mouse peritoneum to investigate its suppressive effect on the peritoneal dissemination caused by F10 cells. Greater retraction and invasion of F10 cells were observed in CN−/− MS than in CN+/+ cells in vitro, while down‐regulation of calponin h1 was observed in CN+/+ MS cells prior to the invasion of F10 cells. Infecting CN−/− MS cells with AdGFP‐CN prevented their retraction and the invasion of F10 cells. Peritoneal dissemination was prominently suppressed in AdGFP‐CN‐infected CN−/− mice, and the survival of those mice was significantly prolonged. Thus, calponin h1 functioned to protect host MS cells from the invasion of F10 cells.

ASC plays a role in the priming phase of the immune response to type II collagen in collagen-induced arthritis

Although rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, the role of IL-1β and IL-18 in the pathophysiology of RA has been well established. IL-1β and IL-18 are generated via cleavage of their pro-forms in the presence of the apoptosis-associated speck-like protein containing a caspase recruit domain (ASC), a known adaptor protein that activates procaspase-1. As such, we investigated the involvement of ASC in the progression of murine collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) using ASC-deficient (ASC−/−) and wild-type (ASC+/+) mice. Analyses were performed by immunohistochemistry for tissues and ELISA for sera. We observed an increase in the expression of ASC, as well as IL-1β and IL-18, in the joints of CIA DBA mice, which indicated that ASC is involved in disease development. Next, we demonstrated that the infiltration of inflammatory cells and cartilage/bone destruction in CIA knee joints were significantly increased in ASC+/+ mice compared with ASC−/− mice. No such differences were noted in ASC+/+ and ASC−/− CAIA mice. In terms of cytokine expression in knee joints, IL-1β and IL-18 were depressed in ASC-deficient CIA mice compared with wild-type mice, but were similarly expressed in CAIA joints in both mice groups. Taken together, we can conclude that ASC is involved in the development of CIA and plays a role in the priming phase of the immune response to type II collagen.