Noboru Ashida

Myostatin Regulates Cardiomyocyte Growth Through Modulation of Akt Signaling

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine–threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

IKKβ regulates essential functions of the vascular endothelium through kinase-dependent and -independent pathways

Vascular endothelium provides a selective barrier between the blood and tissues, participates in wound healing and angiogenesis, and regulates tissue recruitment of inflammatory cells. Nuclear factor (NF)-κB transcription factors are pivotal regulators of survival and inflammation, and have been suggested as potential therapeutic targets in cancer and inflammatory diseases. Here we show that mice lacking IKKβ, the primary kinase mediating NF-κB activation, are smaller than littermates and born at less than the expected Mendelian frequency in association with hypotrophic and hypovascular placentae. IKKβ-deleted endothelium manifests increased vascular permeability and reduced migration. Surprisingly, we find that these defects result from loss of kinase-independent effects of IKKβ on activation of the serine-threonine kinase, Akt. Together, these data demonstrate essential roles for IKKβ in regulating endothelial permeability and migration, as well as an unanticipated connection between IKKβ and Akt signalling.

Differential Signaling for MCP-1-Dependent Integrin Activation and Chemotaxis

Abstract: Transmigration of monocytes to the subendothelial space is the initial step in atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein‐1 (MCP‐1), we investigated the roles of MAPK in THP‐1 cells, a monocytic cell line. MCP‐1 stimulated β1 integrin‐dependent, but not β2 integrin‐dependent cell adhesion in a time‐dependent manner. MCP‐1‐mediated cell adhesion was inhibited by a MEK inhibitor, but not by a p38‐MAPK inhibitor. By contrast, MCP‐1‐mediated chemotaxis was inhibited by the p38‐MAPK inhibitor, but not by the MEK inhibitor. These data indicate that ERK is responsible for integrin activation and that p38‐MAPK is responsible for chemotaxis mediated by MCP‐1. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades, leading to integrin activation and chemotaxis induced by MCP‐1 in THP‐1 cells.

AMAP1 as a negative-feedback regulator of nuclear factor-κB under inflammatory conditions.

NF-κB is a major transcriptional factor regulating many cellular functions including inflammation; therefore, its appropriate control is of high importance. The detailed mechanism of its activation has been well characterized, but that of negative regulation is poorly understood. In this study, we showed AMAP1, an Arf-GTPase activating protein, as a negative feedback regulator for NF-κB by binding with IKKβ, an essential kinase in NF-κB signaling. Proteomics analysis identified AMAP1 as a binding protein with IKKβ. Overexpression of AMAP1 suppressed NF-κB activity by interfering the binding of IKKβ and NEMO, and deletion of AMAP1 augmented NF-κB activity. The activation of NF-κB induced translocation of AMAP1 to cytoplasm from cell membrane and nucleus, which resulted in augmented interaction of AMAP1 and IKKβ. These results demonstrated a novel role of AMAP1 as a negative feedback regulator of NF-κB, and presented it as a possible target for anti-inflammatory treatments.

Aspirin augments the expression of Adenomatous Polyposis Coli protein by suppression of IKKβ.

Aspirin has been widely used as analgesic, antipyretic and anti-inflammatory medicine for long. In addition to these traditional effects, clinical studies suggest that aspirin can protect against cancer, but its mechanism has not been explored. To unveil it, we identified the proteins up- or down-regulated after incubation with aspirin by using proteomics analysis with Nano-flow LC/MALDI-TOF system. Interestingly, the analysis identified the protein of Adenomatous Polyposis Coli (APC) as one of the most up-regulated protein. APC regulates cell proliferation or angiogenesis, and is widely known as a tumor-suppressing gene which can cause colorectal cancer when it is mutated. Western blots confirmed this result, and real-time PCR indicated it is transcriptionally regulated. We further tried to elucidate the molecular mechanism with focusing on IKKβ. IKKβ is the essential kinase in activation of nuclear factor-kappa B (NF-κB), major transcriptional factors that regulate genes responsible for inflammation or immune response. Previous reports indicated that aspirin specifically inhibits IKKβ activity, and constitutively active form of IKKβ accelerates APC loss. We found that aspirin suppressed the expression of IKKβ, and the deletion of IKKβ by siRNA increases the expression of APC in HEK294 cells. Finally, we observed similar effects of aspirin in human umbilical vein endothelial cells. Taken together, these results reveal that aspirin up-regulates the expression of APC via the suppression of IKKβ. This can be a mechanism how aspirin prevents cancer at least in part, and a novel link between inflammatory NF-κB signaling and cancer.

Deletion of IκB‐Kinase β in Smooth Muscle Cells Induces Vascular Calcification Through β‐Catenin–Runt‐Related Transcription Factor 2 Signaling

Background Vascular calcification was previously considered as an advanced phase of atherosclerosis; however, recent studies have indicated that such calcification can appear in different situations. Nevertheless, there has been a lack of mechanistic insight to explain the difference. For example, the roles of nuclear factor‐κB, a major regulator of inflammation, in vascular calcification are poorly explored, although its roles in atherosclerosis were well documented. Herein, we investigated the roles of nuclear factor‐κB signaling in vascular calcification. Methods and Results We produced mice with deletion of IKKβ, an essential kinase for nuclear factor‐κB activation, in vascular smooth muscle cells (VSMCs; KO mice) and subjected them to the CaCl2‐induced aorta injury model. Unexpectedly, KO mice showed more calcification of the aorta than their wild‐type littermates, despite the formers suppressed nuclear factor‐κB activity. Cultured VSMCs from the aorta of KO mice also showed significant calcification in vitro. In the molecular analysis, we found that Runt‐related transcription factor 2, a transcriptional factor accelerating bone formation, was upregulated in cultured VSMCs from KO mice, and its regulator β‐catenin was more activated with suppressed ubiquitination in KO VSMCs. Furthermore, we examined VSMCs from mice in which kinase‐active or kinase‐dead IKKβ was overexpressed in VSMCs. We found that kinase‐independent function of IKKβ is involved in suppression of calcification via inactivation of β‐catenin, which leads to suppression of Runt‐related transcription factor 2 and osteoblast marker genes. Conclusions IKKβ negatively regulates VSMC calcification through β‐catenin–Runt‐related transcription factor 2 signaling, which revealed a novel function of IKKβ on vascular calcification.