Fascin1 suppresses RIG-I-like receptor signaling and interferon-ß production by associating with IκB kinase ϵ (IKKϵ) in colon cancer.

Fascin1 is an actin-bundling protein involved in cancer cell migration and has recently been shown also to have roles in virus-mediated immune cell responses. Because viral infection has been shown to activate immune cells and to induce interferon-ß expression in human cancer cells, we evaluated the effects of fascin1 on virus-dependent signaling via the membrane- and actin-associated protein RIG-I (retinoic acid-inducible gene I) in colon cancer cells. We knocked down fascin1 expression with shRNA retrovirally transduced into a DLD-1 colon cancer and L929 fibroblast-like cell lines and used luciferase reporter assays and co-immunoprecipitation to identify fascin1 targets. We found that intracellular poly(I·C) transfection to mimic viral infection enhances the RIG-I/MDA5 (melanoma differentiation-associated gene 5)-mediated dimerization of interferon regulatory factor 3 (IRF-3). The transfection also significantly increased the expression levels of IRF-7, interferon-ß, and interferon-inducible cytokine IP-10 in fascin1-deleted cells compared with controls while significantly suppressing cell growth, migration, and invasion. We also found that fascin1 constitutively interacts with IκB kinase ϵ (IKKϵ) in the RIG-I signaling pathway. In summary, we have identified fascin1 as a suppressor of the RIG-I signaling pathway associating with IκB kinase ϵ in DLD-1 colon cancer cells to suppress immune responses to viral infection.

Targeting KRAS-mutant pancreatic cancer through simultaneous inhibition of KRAS, MEK, and JAK2.

The Kirsten rat sarcoma (KRAS) oncogene was considered "undruggable" until the development of sotorasib, a KRASG12C selective inhibitor that shows favorable effects against lung cancers. MRTX1133, a novel KRASG12D inhibitor, has shown promising results in basic research, although its effects against pancreatic cancer are limited when used alone. Therefore, there is an urgent need to identify effective drugs that can be used in combination with KRAS inhibitors. In this study, we found that administration of the KRAS inhibitors sotorasib or MRTX1133 upregulated STAT3 phosphorylation and reactivated ERK through a feedback reaction. The addition of the MEK inhibitor trametinib and the JAK2 inhibitor fedratinib successfully reversed this effect and resulted in significant growth inhibition in vitro and in vivo. Analyses of sotorasib- and MRTX1133-resistant cells showed that trametinib plus fedratinib reversed the resistance to sotorasib or MRTX1133. These findings suggest that the JAK2-mediated pathway and reactivation of the MAPK pathway may play key roles in resistance to KRAS inhibitors in pancreatic cancers. Accordingly, simultaneous inhibition of KRAS, MEK, and JAK2 could be an innovative therapeutic strategy against KRAS-mutant pancreatic cancer.

RhoA G17E/Vav1 Signaling Induces Cancer Invasion via Matrix Metalloproteinase-9 in Gastric Cancer.

BACKGROUND: RAS homolog family member A (RhoA), a member of the Rho family of small GTPases, and Vav1, a guanine nucleotide exchange factor for Rho family GTPases, have been reported to activate pathways related to the actin cytoskeleton and regulation of cell shape, attachment, and motility. The interaction between these molecules in lymphoma is involved in malignant signaling, but its function in epithelial malignancy is unknown. Here, we investigated the malignant signal of mutant RhoA in gastric cancer and demonstrated the potential of RhoA G17E/Vav1 as a therapeutic target for diffuse gastric cancer. METHODS: The RhoA mutants R5W, G17E, and Y42C were retrovirally transduced into the gastric cancer cell line MKN74. The stably transduced cells were used for morphology, proliferation, and migration/invasion assays in vitro. MKN74 cells stably transduced with ectopic wild-type RhoA and mutant RhoA (G17E) were used in a peritoneal xenograft assay. RESULTS: The RhoA mutations G17E and Y42C induced morphological changes in MKN74. G17E induced Vav1 expression at the mRNA and protein levels and promoted the migration and invasion of MKN74. An RNA interference assay of Vav1 revealed that RhoA G17E enhanced cancer cell invasion via Vav1. Furthermore, immunoprecipitation revealed that Vav1 and RhoA G17E specifically bind and function together through matrix metalloproteinase -9. In a peritoneal xenograft model of nude mice, RhoA G17E promoted peritoneal dissemination, whereas Vav1 knockdown suppressed it. CONCLUSION: Overall, our findings indicate that RhoA G17E is associated with Vav1 and promoted cancer invasion via matrix metalloproteinase -9 in gastric cancer cells. Thus, RhoA G17E/Vav1 signaling in diffuse gastric cancer may be a useful therapeutic target.

Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells.

BACKGROUND: Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling. METHODS: We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling. RESULTS: Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells. CONCLUSION: In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.

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-1ß and IL-18 in the pathophysiology of RA has been well established. IL-1ß 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-1ß 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-1ß 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.

Drug sensitivity profile of minor KRAS mutations in colorectal cancer using mix culture assay: The effect of AMG-510, a novel KRAS G12C selective inhibitor, on colon cancer cells is markedly enhanced by the combined inhibition of MEK and BCL-XL.

Colorectal cancer with a Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) gene mutation is considered to be resistant to anti-EGFR agents. G12D is the most common KRAS mutation in colorectal cancer, followed by G12V and G13D. According to clinical and basic research data, patients with colorectal cancer exhibiting G12D and G12V KRAS mutations are resistant to anti-EGFR agents; however, this is not true of G13D and other minor mutations, which are still not well understood. The current study focused on minor KRAS mutations (G12A, G12C, G12S, Q61H and A146T) and evaluated whether these were resistant to anti-EGFR antibodies using a mix culture assay. The results demonstrated that all KRAS mutations, including minor mutations, were resistant to two anti-EGFR agents: Cetuximab and panitumumab. The combined effect of MEK and BCL-XL inhibition on colorectal cancer cells with KRAS minor mutations were subsequently evaluated. The combined effect of MEK and BCL-XL inhibitors was confirmed in all KRAS minor mutations. The sensitivity of AMG510, a novel KRAS G12C selective inhibitor, was also assessed. The mix culture assay revealed that AMG510 selectively exerted an antitumor effect on colon cancer cells with a G12C KRAS mutation. The combination of MEK and BCL-XL inhibition markedly enhanced the effect of AMG510 in colon cancer cells. The current study suggested that AMG510 may have potential clinical use in combination with MEK and BCL-XL inhibitors in the treatment of patients with colorectal cancer exhibiting the G12C KRAS mutation.

Tail-suspended mice lacking calponin H1 experience decreased bone loss.

Calponin h1 (CNh1) is an actin-binding protein originally isolated from vascular smooth muscle and has been reported to suppress bone formation. We are therefore curious how CNh1 is involved in bone loss that is caused by space flight in microgravity. We assessed the effects of tail suspension (TS) in C57BL/6J wild (CN+/+) and CNh1-deleted (CN-/-) mice to elucidate the role of CNh1 in bone loss under weightless conditions. Bone mineral density (BMD) of tibiae was measured by single energy X-ray absorptiometry, and bone volume fraction (BV/TV), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were measured by bone histomorphometry. BMD, BV/TV, MAR, and BFR/BS were lower in CN+/+ mice with TS than in those without. In the CN-/- group, however, the decrease in each of these parameters by TS was ameliorated. Decreases in serum osteocalcin levels by TS in CN+/+ mice were attenuated in CN-/- mice. Furthermore, urinary deoxypyridinolin (DPD), an indicator of bone resorption, was increased in CN+/+ mice following TS, but not in CN-/- mice. In transfection experiments, the degree of induction of bone formation markers, alkaline phosphatase (ALP) activity and bone morphogenetic protein (BMP)-4 mRNA expression, under stimulation with BMP-2, was lower in MC3T3-E1 mouse osteoblast-like cells expressing CNh1 than that in mock transfected cells. Notably, the BMP-2-induced ALP activity was decreased by CNh1 expression, which was partially rescued by treatment with the Rho kinase inhibitor Y27632. Taken together, these results indicate that CNh1 is responsible for weightlessness-induced bone loss in part through Rho signaling pathway.

Low­dose trametinib and Bcl­xL antagonist have a specific antitumor effect in KRAS­mutated colorectal cancer cells.

KRAS­mutant colorectal cancer (CRC) is a highly malignant cancer with a poor prognosis, however specific therapies targeting KRAS mutations do not yet exist. Anti­epidermal growth factor receptor (EGFR) agents, including cetuximab and panitumumab, are effective for the treatment of certain patients with CRC. However, these anti­EGFR treatments have no effect on KRAS­mutant CRC. Therefore, new therapeutic strategies targeting KRAS­mutant CRC are urgently needed. To clarify the direct effect of KRAS gene mutations, the present study transduced mutant forms of the KRAS gene (G12D, G12V and G13D) into CACO­2 cells. A drug­screening system (Mix Culture assay) was then applied, revealing that the cells were most sensitive to the MEK inhibitor trametinib among tested drugs, Cetuximab, Panitumumab, Regorafenib, Vemurafenib, BEZ­235 and Palbociclib. Trametinib suppressed phosphorylated ERK (p­ERK) expression and inhibited the proliferation of KRAS­mutant CACO­2 cells. However, low­dose treatment with trametinib also increased the expression of the anti­apoptotic protein Bcl­xL in a dose­dependent manner, leading to drug resistance. To overcome the resistance of KRAS­mutant CRC to apoptosis, the combination of trametinib and the Bcl­xL antagonist ABT263 was assessed by in vitro and in vivo experiments. Compared with the effects of low­dose trametinib monotherapy, combination treatment with ABT263 had a synergistic effect on apoptosis in mutant KRAS transductants in vitro. Furthermore, in vivo combination therapy using low­dose trametinib and ABT263 against a KRAS­mutant (G12V) xenograft synergistically suppressed growth, with an increase in apoptosis compared with the effects of trametinib monotherapy. These data suggest that a low dose of trametinib (10 nM), rather than the usual dose of 100 nM, in combination with ABT263 can overcome the resistance to apoptosis induced by Bcl­xL expression, which occurs concurrently with p­ERK suppression in KRAS­mutant cells. This strategy may represent a promising new approach for treating KRAS­mutant CRC.

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-1beta (pro-IL-1beta) 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-1beta and IL-18, and substantial reduction of interferon-gamma (IFN-gamma) expression, in ASC-deficient mice without apparent upregulation of other cytokines, including IL-10 and tumor necrosis factor-alpha. These results as a whole indicate that ASC exerts considerable influence on the host defense, acting through IL-1beta/IL-18 and subsequent IFN-gamma production, which in turn contributes to continuous chronic inflammatory response and consequent reduction of H. pylori colonization.

A splice variant of ASC regulates IL-1beta 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-1beta 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.

Survival pathway of cholangiocarcinoma via AKT/mTOR signaling to escape RAF/MEK/ERK pathway inhibition by sorafenib.

Cholangiocarcinoma (CCC) is a strongly aggressive malignancy for which surgical resection is the only potential curative therapy. Sorafenib, a multikinase inhibitor of the RAF/MEK/ERK pathway, is a molecular-targeted drug that is approved for hepatocellular carcinoma (HCC) but not for CCC. The differences in signaling pathway characteristics under sorafenib treatment between HCC (HLF, Huh7, PLC/PRF/5) and CCC (RBE, YSCCC, Huh28) cell lines were therefore investigated using cell proliferation, western blotting, and apoptosis analyses. Sorafenib inhibited cell growth significantly less in CCC cells than in HCC cells, with lower suppression of ERK phosphorylation. Significantly decreased AKT Ser473 phosphorylation in HCC cells, and conversely enhanced phosphorylation of AKT Ser473 and mTORC2 in CCC cells, were observed with sorafenib treatment. Disassembly of the mTORC2 complex in RBE cells with siRNA targeting Rictor resulted in the downregulation of AKT Ser473 phosphorylation and enhanced apoptosis presumably via increased FOXO1, which consequently suppressed RBE cell proliferation. Phosphorylation of mTORC1 and autophagy were not influenced by sorafenib in CCC cells. Simultaneous administration of everolimus to suppress activated mTORC1 in RBE cells revealed that combined everolimus and sorafenib treatment under mTORC2 disassembly could enhance growth inhibition through the suppression of both sorafenib- and everolimus-dependent AKT Ser473 phosphorylation in addition to the inhibition of mTORC1 phosphorylation. Prevention of escape by AKT/mTOR signaling from the RAF/MEK/ERK pathway in sorafenib treatment by suppressing mTORC2 activity may lead to promising new approaches in CCC therapy.

ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication.

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.

Methylation of ASC/TMS1, a proapoptotic gene responsible for activating procaspase-1, in human colorectal cancer.

ASC/TMS1, a proapoptotic activator of procaspase-1, was reported to be aberrantly methylated in human breast cancer. We found that ASC was methylated in three of five human colon cancer cell lines lacking ASC protein expression. Demethylation treatment of these cell lines lacking ASC with 5-aza-2'-deoxycytidine partially restored ASC expression. Methylated ASC was also detected in six of ten colorectal cancer tissues. Although clear down-regulation of ASC in the whole region of a tumor tissue was hardly observed by immunostaining with anti-ASC mAb, complete suppression of ASC was identified in a minor population of the colorectal tumor cells. The biological significance of ASC methylation inducible ASC suppression in colorectal cancer will be discussed.

Clinical significance of apoptosis-associated speck-like protein containing a caspase recruitment domain in oral squamous cell carcinoma.

OBJECTIVES: To assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression in oral squamous cell carcinoma (OSCC) and analyze its clinical and pathological significance. STUDY DESIGN: ASC expression was studied using immunohistochemistry in 119 OSCCs patients. The relationships between ASC expression and clinical and pathological parameters were statistically analyzed. In addition, the relationships between ASC expression and cell differentiation [IVL (involcrin) expression] and apoptosis (TUNEL [TdT-mediated dUTP nick end labeling] positive cell number) were investigated. RESULTS: ASC expression showed significant correlations with parameters including clinical tumor stage, mode of invasion, and histological differentiation, and had a significant impact on survival of OSCC. The distribution of ASC correlated well with that of IVL. ASC expression was significantly correlated with the TUNEL-positive cell number. CONCLUSIONS: Lower ASC expression correlates with clinical and pathological malignancy and, consequently, poor prognosis of OSCC. ASC has a close association with cell differentiation and apoptosis.

Calponin h1-S175T point mutation enhances resistance to actin cytoskeleton perturbation in human cancer cells.

To investigate Calponin h1 (CNh1) regions responsible for suppressing cancer cell motility, the interaction between CNh1 and actin in HeLa cells was examined. First, it was observed that the actin binding of CNh1 depends on the calponin repeat 1 (CNR 1), region more than the actin binding site region. Next, point mutantions were generated at S175 and/or T184 in CNR1, substrates of protein kinase C, and it was observed by cellular immunostaining that the actin binding of CNh1 depends on 175th amino acid. Furthermore, the point mutation S175T exhibited more resistance to actin rearrangement by cytochalasin D and PDBu than intact CNh1, suppressing cell motility induced by PDBu. This result indicates that S175T may be an effective target for new cancer treatments.

Hyaluronan deficiency in tumor stroma impairs macrophage trafficking and tumor neovascularization.

Despite the importance of stromal cells in tumor progression, our overall understanding of the molecular signals that regulate the complex cellular interactions within tumor stroma is limited. Here, we provide multiple lines of evidence that tumor-associated macrophages (TAM) preferentially traffic to stromal areas formed within tumors in a manner dependent on a hyaluronan (HA)-rich tumor microenvironment. To address the role of stroma-derived HA in macrophage recruitment, we disrupted the HA synthase 2 (Has2) gene in stromal fibroblasts using conditional gene targeting. The Has2 null fibroblasts showed severe impairment in recruiting macrophages when inoculated with tumor cells into nude mice, which shows the contribution of stroma-derived HA in intratumoral macrophage mobilization. Furthermore, a deficiency in stromal HA attenuated tumor angiogenesis and lymphangiogenesis concomitantly with impaired macrophage recruitment. Taken together, our results suggest that stromal HA serves as a microenvironmental signal for the recruitment of TAMs, which are key regulatory cells involved in tumor neovascularization.

Significance of tumor-associated stroma in promotion of intratumoral lymphangiogenesis: pivotal role of a hyaluronan-rich tumor microenvironment.

Stromal cells, together with extracellular matrix components, provide a tumor microenvironment that is pivotal for cancer cell growth and progression. In our previous study using a conditional transgenic mouse model of breast cancer, the overproduction of hyaluronan, a major extracellular constituent, accelerated tumor angiogenesis through stromal cell recruitment. This finding led us to investigate the role of hyaluronan in the lymphatic vessel system. Here, we have found that microenvironmental hyaluronan promoted tumor lymphangiogenesis concurrently with the formation of stromal structures. Additionally, lymphatic vessels frequently penetrated and accumulated into stromal compartments, and up-regulation of vascular endothelial growth factor-C and -D was detected at tumor-stromal interfaces. To assess the contribution of stromal cells to lymphangiogenesis in vivo, we established tumor-associated fibroblasts from hyaluronan-overproducing mammary tumors and implanted them together with carcinoma cells from control tumors or MCF-7 human breast carcinoma cells in nude mice. Carcinoma cells grew rapidly in association with marked stromal reactions and lymphangiogenesis. Without the stromal cells, however, the tumors developed slowly with less stroma and lymphatic vessels. These findings underline the significance of tumor-associated stroma in the promotion of intratumoral lymphangiogenesis and suggest a pivotal role for the hyaluronan-rich tumor microenvironment.

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.

Enhanced baroreflex sensitivity in free-moving calponin knockout mice.

Calponin is an actin binding protein in vascular smooth muscle that modifies contractile responses. However, its role in mean arterial pressure (MAP) regulation has not been clarified. To assess this, MAP and heart rate (HR) were measured in calponin knockout (KO) mice, and the results were compared with those in wild-type (WT) mice. The measurements were performed every 100 ms during a 60-min free-moving state each day for 3 days. Mice in both groups rested during approximately 70% of the total measuring period. The mean HR during rest was significantly lower in KO mice than in WT mice but with no significant difference in MAP between the groups. The change in HR response (deltaHR) to spontaneous change in MAP (deltaMAP) varied in a wider range in KO mice with an 80% increase in the coefficient of variation for HR (P < 0.05), whereas MAP in KO mice was controlled in a narrow range similar to that in WT mice. The baroreflex sensitivity (deltaHR/deltaMAP), determined from the change in HR to the spontaneous change in MAP, was twofold higher in KO mice than that in WT mice (P < 0.01), whereas there were no significant differences in the baroreflex sensitivity determined by intravascular administration of phenylephrine and sodium nitroprusside between the two groups (P > 0.1). The MAP response to the administrated doses of phenylephrine in KO mice was reduced to one-half of that in WT mice (P < 0.01) but with no significant difference in the response to sodium nitroprusside between the groups. The differences in HR variability and the spontaneous baroreflex sensitivity between the two groups completely disappeared after carotid sinus denervation. These results suggest that the higher variability in HR for KO mice was caused by the increased spontaneous arterial baroreflex sensitivity, though not detected by the intra-arterial administration of the drug, and that the higher variability of HR may be a compensatory adaptation to the blunted alpha-adrenergic response of peripheral vessels to sympathetic nervous activity.

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 alpha-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 (Delta HR/Delta MAP) in WT mice (n = 6), determined from the heart rate response (Delta HR) to spontaneous change in MAP (Delta MAP), 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 alpha-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 alpha-adrenergic contractile response and by the attenuated HR response to spontaneous change in MAP due to reduced baroreflex sensitivity.