Lactic acid induces HSPA1A expression through ERK1/2 activation.

Heat shock protein (HSP) A1A protects cells from various stressors. The concentrated liquid of the traditional Japanese rice black vinegar Kurozu increased HSPA1A expression in normal rat liver RLN-10 cells. Lactic acid, the primary component of concentrated Kurozu, induced HSPA1A expression in a concentration-dependent manner. Induction with 4 m m lactic acid increased HSPA1A expression by three times compared with that in the absence of lactic acid. The induction was inhibited by staurosporine or a selective MEK1/2 inhibitor (SL327). The phosphorylation of ERK1/2 was increased by lactic acid. These results suggest that lactic acid induces HSPA1A expression by activating ERK1/2. As well as lactate, 3,5-dihydroxybenzoic acid (DHBA), a ligand for G protein-coupled receptor 81 (GPR81), also induced HSPA1A at lower concentrations than lactate. The increased effect of DHBA on HSPA1A expression as compared with lactate may be related to the higher affinity of DHBA for GPR81 than of lactate.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza.

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

VEGFR-1 Regulates EGF-R to Promote Proliferation in Colon Cancer Cells.

The relationship between epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) pathways in tumor growth is well established. EGF induces VEGF production in cancer cells, and the paracrine VEGF activates vascular endothelial cells to promote tumor angiogenesis and thus supports tumor cell growth in an angiogenesis-dependent manner. In this study, we found angiogenesis-independent novel crosstalk between the VEGF and the EGF pathways in the regulation of colon cancer cell proliferation. Stimulation of colon cancer cells with VEGF-A and placental growth factor (PlGF) activated VEGF receptor-1 (VEGFR-1) and increased proliferation activity in an autocrine EGF/EGF receptor (EGF-R)-dependent manner. Mechanistically, VEGFR-1 interacted with and stabilized EGF-R, leading to increased EGF-R protein levels and prolonged its expression on cell surface plasma membrane. In contrast, VEGFR-1 blockade by a neutralizing antibody and an antagonistic peptide of VEGFR-1 suppressed the complex formation of VEGFR-1 and EGF-R and decreased EGF-R expression via a lysosome-dependent pathway, resulting in the suppression of proliferation activity. Our results indicated that VEGFR-1 regulated EGF-R expression to promote proliferation activity in a cell-autonomous-dependent manner.

VEGF pathway-targeting drugs induce evasive adaptation by activation of neuropilin-1/cMet in colon cancer cells.

Anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are important treatments for a number of human malignancies, including colorectal cancers. However, there is increasing evidence that VEGF/VEGF-R inhibitors promote the adaptive and evasive resistance of tumor cells to the therapies. The mechanism by which the cancer cells become resistant remains unclear. One potential mechanism is that VEGF/VEGF-R blockers directly act on tumor cells independently of anti-angiogenic effects. In this study, the direct effects of an anti-VEGF antibody (bevacizumab) and a VEGF-R tyrosine kinase inhibitor (sunitinib) on the evasive adaptation of colon cancer cells were compared. HCT116 and RKO human colon cancer cell lines were chronically exposed (3 months) to bevacizumab or sunitinib in vitro to establish bevacizumab- and sunitinib-adapted cells, respectively. Transwell migration and invasion assays, western blotting, reverse transcription-quantitative polymerase chain reaction, co-immunoprecipitation analysis, cell survival assays and ELISAs were conducted to analyze the adapted cells. Compared with the control vehicle-treated cells, the two cell models exhibited increased migration and invasion activities to different degrees and through different mechanisms. The bevacizumab-adapted cells, but not in the sunitinib-adapted cells, exhibited redundantly increased expression levels of VEGF/VEGF-R family members, including VEGF-A, placental growth factor, VEGF-C, VEGF-R1 and VEGF-R3. In addition, the phosphorylation levels of VEGF-R1 and VEGF-R3 were increased in the bevacizumab-adapted cells compared with the control cells. Thus, the inhibition of VEGF-R1 and VEGF-R3 decreased the evasive activities of the cells, suggesting that they remained dependent on redundant VEGF/VEGF-R signaling. By contrast, the sunitinib-adapted cells exhibited increased neuropilin-1 (NRP1) expression levels compared with the control cells. In the sunitinib-adapted cells, NRP1 interacted with phosphorylated cMet, and the cMet activation was dependent on NRP1. Thus, NRP1 or cMet blockade suppressed the evasive activation of the sunitinib-adapted cells. These results suggest that the sunitinib-adapted cells switched from a VEGF-R-dependent pathway to an alternative NRP1/cMet-dependent one. The findings of the present study indicate that VEGF/VEGF-R inhibitors directly act on colon cancer cells and activate their evasive adaptation via different mechanisms.

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Antiangiogenic agent sunitinib induces epithelial to mesenchymal transition and accelerates motility of colorectal cancer cells.

Although vascular endothelial growth factor receptor (VEGF-R)-targeted antiangiogenic agents are important treatment for a number of human malignancies, there is accumulating evidence that the therapies may promote disease progression, such as invasion and metastasis. How tumors become to promote their evasiveness remains fully uncertain. One of possible mechanisms for the adaptation may be a direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. To elucidate a direct effect of VEGF-R-targeting drug (sunitinib), we established a human colorectal cancer cell model adapted to sunitinib. The sunitinib-conditioned cells showed a significant increase in cellular motility and migration activities, compared to the vehicle-treated control cells. Consistent with the phenotype, the sunitinib-conditioned cells decreased the expression levels of E-cadherin (an epithelial marker), while significantly increased the levels of Slug and Zeb1 (mesenchymal markers). Expression profiles of VEGF-R in the sunitinib-conditioned cells showed that only neuropilin-1 (NRP1) expression was significantly increased among all VEGF-R tested. Blockade of NRP1 using its antagonist clearly repressed the migration activation in sunitinib-conditioned cells, but not in the control cells. These results suggest that inhibition of VEGF-R on colorectal cancer cells can drive the epithelial-mesenchymal transition, leading to activation of cell motility in an NRP1-dependent manner. J. Med. Invest. 64: 250-254, August, 2017.

Regorafenib induces adaptive resistance of colorectal cancer cells via inhibition of vascular endothelial growth factor receptor.

Recently, inhibition of tumor angiogenesis has become an important anti-cancer therapy. Tumor angiogenesis is regulated by multiple signaling pathways, including VEGF and VEGF receptor (VEGF-R), FGF and FGF receptor (FGF-R), and PDGF and PDGF receptor (PDGF-R) pathways. Thus, the antiangiogenic agents, such as regorafenib, simultaneously target those receptors on vascular endothelial cells. In addition to endothelial cells, cancer cells express the three receptors, suggesting that the antiangiogenic inhibitors affect tumor cells. In fact, we previously demonstrated that regorafenib directly acted on human colorectal cancer cells and accelerated their apoptosis resistance and migration capability. Thus, we here elucidated how regorafenib induced the malignant phenotypes in colorectal cancer cells. To identify the responsible receptor among the regorafenib-targeting proangiogenic receptors, we examined the effects of a potent selective inhibitor for VEGF-R, FGF-R or PDGF-R on apoptosis resistance and migration capability. We clarified that blockade of VEGF-R, but not FGF-R and PDGF-R, induced the malignant phenotypes. We confirmed that blocking of VEGF ligands derived from colorectal cancer cells also induced the phenotypes. These results suggest that regorafenib progressed the malignancy via prevention of autocrine and paracrine VEGF signaling in colorectal cancer cells. J. Med. Invest. 64: 262-265, August, 2017.

Capric Acid Up-Regulates UCP3 Expression without PDK4 Induction in Mouse C2C12 Myotubes.

Uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle are key regulators of the glucose and lipid metabolic processes that are involved in insulin resistance. Medium-chain fatty acids (MCFAs) have anti-obesogenic effects in rodents and humans, while long-chain fatty acids (LCFAs) cause increases in body weight and insulin resistance. To clarify the beneficial effects of MCFAs, we examined UCP3 and PDK4 expression in skeletal muscles of mice fed a MCFA- or LCFA-enriched high-fat diet (HFD). Five-week feeding of the LCFA-enriched HFD caused high body weight gain and induced glucose intolerance in mice, compared with those in mice fed the MCFA-enriched HFD. However, the amounts of UCP3 and PDK4 transcripts in the skeletal muscle of mice fed the MCFA- or LCFA-enriched HFD were similar. To further elucidate the specific effects of MCFAs, such as capric acid (C10:0), on lipid metabolism in skeletal muscles, we examined the effects of various FAs on expression of UCP3 and PDK4, in mouse C2C12 myocytes. Although palmitic acid (C16:0) and lauric acid (C12:0) significantly induced expression of both UCP3 and PDK4, capric acid (C10:0) upregulated only UCP3 expression via activation of peroxisome proliferator-activated receptor-δ. Furthermore, palmitic acid (C16:0) disturbed the insulin-induced phosphorylation of Akt, while MCFAs, including lauric (C12:0), capric (C10:0), and caprylic acid (C12:0), did not. These results suggest that capric acid (C10:0) increases the capacity for fatty acid oxidation without inhibiting glycolysis in skeletal muscle.

UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.

Effects of dietary soy protein on skeletal muscle volume and strength in humans with various physical activities.

BACKGROUND: In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities. METHODS: We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study. RESULTS: Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation. CONCLUSIONS: The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading.

The malignant progression effects of regorafenib in human colon cancer cells.

A number of anti-angiogenic drugs targeting vascular endothelial growth factor receptors (VEGF-R) have developed and enabled significant advances in cancer therapy including colorectal cancer. However, acquired resistance to the drugs occurs, leading to disease progression, such as invasion and metastasis. How tumors become the resistance and promote their malignancy remains fully uncertain. One of possible mechanisms for the resistance and the progression may be the direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. We investigated here the direct effect of a VEGF-R-targeting agent, regorafenib, which is the first small molecule inhibitor of VEGF-Rs for the treatment of patients with colorectal cancer, on phenotype changes in colon cancer HCT116 cells. Treatment of cells with regorafenib for only 2 days activated cell migration and invasion, while vehicle-treated control cells showed less activity. Intriguingly, chronic exposure to regorafenib for 90 days dramatically increased migration and invasion activities and induced a resistance to hypoxia-induced apoptosis. These results suggest that loss of VEGF signaling in cancer cells may induce the acquired resistance to VEGF/VEGF-R targeting therapy by gaining two major malignant phenotypes, apoptosis resistance and activation of migration/invasion.

Flavones Inhibit LPS-Induced Atrogin-1/MAFbx Expression in Mouse C2C12 Skeletal Myotubes.

Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented the decrease in C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy.

A novel myogenic function residing in the 5' non-coding region of Insulin receptor substrate-1 (Irs-1) transcript.

BACKGROUND: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs). RESULTS: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes. CONCLUSIONS: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

Chronic exposure of VEGF inhibitors promotes the malignant phenotype of colorectal cancer cells.

VEGF-targeting anti-angiogenic drugs have enabled significant advances in cancer therapy. However, acquired resistance to VEGF-targeting drugs occurs, leading to disease progression. How tumors become the resistance remains fully uncertain. One of possible mechanisms for the resistance may be the direct effect of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGF-R). We investigated here the direct effect of chronic VEGF inhibition on phenotype changes in cancer cells. To chronically inhibit cancer cell-derived VEGF, human colon cancer HCT116 cells were chronically exposed (3 months) to anti-VEGF neutralizing monoclonal antibody (HCT/mAb cells, blockade of VEGF alone) or VEGF-R tyrosine kinase inhibitor foretinib (HCT/fore cells, blockade of all VEGF family). HCT/mAb cells redundantly increased VEGF family member (VEGF, PlGF, VEGF-B, VEGF-R1 and VEGF-R2) and induced a resistance to hypoxia-induced apoptosis. By contrast, HCT/fore cells did not show the redundant increase in VEGF family member, but significantly increased a VEGF-independent pro-angiogenic factor FGF-2. HCT/fore cells showed increased migration and invasion activities in addition to a resistance to hypoxia-induced apoptosis. The resistance to apoptosis was significantly suppressed by inhibition of hypoxia-inducible factor-1α in HCT/mAb cells, but not in HCT/fore cells. These findings suggest that chronic inhibition of VEGF/VEGF-R accelerates malignant phenotypes of colon cancer cells. J. Med. Invest. 62: 75-79, February, 2015.

N-myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle.

A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.

Ubiquitin ligase Cbl-b and obesity-induced insulin resistance.

Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.

Isoflavones derived from soy beans prevent MuRF1-mediated muscle atrophy in C2C12 myotubes through SIRT1 activation.

Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)-α treatment. In C2C12 myotubes, TNF-α treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NFκB, a downstream target of the TNF-α signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF-α in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.

Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b.

Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.

Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells.

BACKGROUND: Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. METHODS: To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. RESULTS: Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. CONCLUSIONS: Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.