IGF-1 induces human myotube hypertrophy by increasing cell recruitment

Insulin-like growth factor-1 (IGF-1) has been shown in rodents (i) in vivo to induce muscle fiber hypertrophy and to prevent muscle mass decline with age and (ii) in vitro to enhance the proliferative life span of myoblasts and to induce myotube hypertrophy. In this study, performed on human primary cultures, we have shown that IGF-1 has very little effect on the proliferative life span of human myoblasts but does delay replicative senescence. IGF-1 also induces hypertrophy of human myotubes in vitro, as characterized by an increase in the mean number of nuclei per myotube, an increase in the fusion index, and an increase in myosin heavy chain (MyHC) content. In addition, muscle hypertrophy can be triggered in the absence of proliferation by recruiting more mononucleated cells. We propose that IGF-1-induced hypertrophy can involve the recruitment of reserve cells in human skeletal muscle.     

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Considering the well-known antioxidant properties of statins, it seems important to assess their impact on major markers of oxidative stress (superoxide anion radical, nitric oxide, and index of lipid peroxidation) to compare the antioxidative potentials of atorvastatin and simvastatin during the different degrees of hyperhomocysteinemia (HHcy) in rats. This study was conducted on adult male Wistar albino rats (n = 90; 4 weeks old; 100 ± 15 g body mass) in which HHcy was achieved by dietary manipulation. For 4 weeks, the animals were fed with one of the following diets: standard rodent chow, diet enriched in methionine with no deficiency in B vitamins (folic acid, B6, and B12), or diet enriched in methionine and deficient in B vitamins (folic acid, B6, and B12). At the same time, animals were treated with atorvastatin at doses of 3 mg/kg/day i.p. or simvastatin at doses of 5 mg/kg/day i.p. Levels of superoxide anion radical and TBARS were significantly decreased by administration of simvastatin in normal and high-homocysteine (Hcy) groups (p < 0.05). At 4 weeks after feeding with purified diets, the concentrations of the GSH, CAT, and SOD antioxidants were significantly affected among all groups (p < 0.05). Our results indicated that statin therapy had variable effects on the redox status in hyperhomocysteinemic rats, and simvastatin demonstrated stronger antioxidant effects than did atorvastatin.     

Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes

Mesenchymal cells can differentiate into osteoblasts, adipocytes, myoblasts, or chondroblasts. Whether mesenchymal cells that have initiated differentiation along one lineage can transdifferentiate into another is largely unknown. Using 3T3-F442A preadipocytes, we explored whether extracellular signals could redirect their differentiation from adipocyte into osteoblast. 3T3-F442A cells expressed receptors and Smads required for bone morphogenetic protein (BMP) signaling. BMP-2 increased proliferation and induced the early osteoblast differentiation marker alkaline phosphatase, yet only mildly affected adipogenic differentiation. Retinoic acid inhibited adipose conversion and cooperated with BMP-2 to enhance proliferation, inhibit adipogenesis, and promote early osteoblastic differentiation. Expression of BMP-RII together with BMP-RIA or BMP-RIB suppressed adipogenesis of 3T3-F442A cells and promoted full osteoblastic differentiation in response to retinoic acid. Osteoblastic differentiation was characterized by induction of cbfa1, osteocalcin, and collagen I expression, and extracellular matrix calcification. These results indicate that 3T3-F442A preadipocytes can be converted into fully differentiated osteoblasts in response to extracellular signaling cues. Furthermore, BMP and retinoic acid signaling cooperate to stimulate cell proliferation, repress adipogenesis, and promote osteoblast differentiation. Finally, BMP-RIA and BMP-RIB induced osteoblast differentiation and repressed adipocytic differentiation to a similar extent.     

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGβ/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGβ/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFβ/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.     

Akt2 and acid ceramidase cooperate to induce cell invasion and resistance to apoptosis

Both Akt 2 and acid ceramidase (ASAH1) are found aberrantly overexpressed in cancer cells, but whether these two enzymes cooperate to induce malignant transformation is not known. We found that in immortalized, non-transformed cells, ectopic co-expression of Akt2 and ASAH1 is significantly more effective than expression of each gene alone at inducing cell invasion and at conferring resistance to apoptosis. Consistent with these observations, siRNA-mediated depletion of both Akt2 and ASAH1 is much more potent than depleting each alone at inhibiting cell viability/proliferation and cell invasion. Furthermore, pharmacological inhibitors of Akt (TCN or MK-2206) and ASAH1 (B13) synergize to inhibit cell viability/proliferation, and combinations of these drugs are more effective than single-agent treatments at inhibiting cell invasion. Taken together, the results suggest that these two enzymes cooperate to induce malignant transformation and warrant further preclinical studies to evaluate the potential of combining inhibitors of Akt and ASAH1 to treat cancer.     

Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells

The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3–4 days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21–28 days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1⁺ clusters are formed. Almost all cells in PDX1⁺ clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7–14 days, then the number of insulin⁺ cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.     

CaMKII activates ASK1 and NF-kappaB to induce cardiomyocyte hypertrophy

Ca2+/calmodulin-dependent protein kinase (CaMK) is an important downstream target of Ca2+ in the hypertrophic signaling pathways. We previously showed that the activation of apoptosis signal-regulating kinase 1 (ASK1) or NF-kappaB is sufficient for cardiomyocyte hypertrophy. Infection of isolated neonatal cardiomyocytes with an adenoviral vector expressing CaMKIIdelta3 (AdCaMKIIdelta3) induced the activation of ASK1, while KN93, an inhibitor of CaMKII, inhibited phenylephrine-induced ASK1 activation. Overexpression of CaMKIIdelta3 induced characteristic features of in vitro cardiomyocyte hypertrophy. Infection of cardiomyocytes with an adenoviral vector expressing a dominant negative mutant of ASK1 (AdASK(KM)) inhibited the CaMKIIdelta3-induced hypertrophic responses. Overexpression of CaMKIIdelta3 increased the kappaB-dependent promoter/luciferase activity and induced IkappaBalpha degradation. Coinfection with AdCaMKIIdelta3 and AdASK(KM), and pre-incubation with KN93 attenuated CaMKIIdelta3- and phenylephrine-induced NF-kappaB activation, respectively. Expression of a degradation resistant mutant of IkappaBalpha inhibited CaMKIIdelta3-induced hypertrophic responses. These results indicate that CaMKIIdelta3 induces cardiomyocyte hypertrophy mediated through ASK1-NF-kappaB signal transduction pathway.     

DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients’ sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.     

Cdc42 and noncanonical Wnt signal transduction pathways cooperate to promote cell polarity

Scratch-induced disruption of cultured monolayers induces polarity in front row cells that can be visualized by spatially localized polymerization of actin at the front of the cell and reorientation of the centrosome/Golgi to face the leading edge. We previously reported that centrosomal reorientation and microtubule polarization depend on a Cdc42-regulated signal transduction pathway involving activation of the Par6/aPKC complex followed by inhibition of GSK-3beta and accumulation of the adenomatous polyposis coli (APC) protein at the plus ends of leading-edge microtubules. Using monolayers of primary rodent embryo fibroblasts, we show here that dishevelled (Dvl) and axin, two major components of the Wnt signaling pathway are required for centrosome reorientation and that Wnt5a is required for activation of this pathway. We conclude that disruption of cell-cell contacts leads to the activation of a noncanonical Wnt/dishevelled signal transduction pathway that cooperates with Cdc42/Par6/aPKC to promote polarized reorganization of the microtubule cytoskeleton.     

Nitroalkenes induce rat aortic smooth muscle cell apoptosis via activation of caspase-dependent pathways

Nitroalkene derivatives of nitro-linoleic acid (LNO₂) and nitro-oleic acid (OA-NO₂) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Complex N-glycan number and degree of branching cooperate to regulate cell proliferation and differentiation

The number of N-glycans (n) is a distinct feature of each glycoprotein sequence and cooperates with the physical properties of the Golgi N-glycan-branching pathway to regulate surface glycoprotein levels. The Golgi pathway is ultrasensitive to hexosamine flux for the production of tri- and tetra-antennary N-glycans, which bind to galectins and form a molecular lattice that opposes glycoprotein endocytosis. Glycoproteins with few N-glycans (e.g., TbetaR, CTLA-4, and GLUT4) exhibit enhanced cell-surface expression with switch-like responses to increasing hexosamine concentration, whereas glycoproteins with high numbers of N-glycans (e.g., EGFR, IGFR, FGFR, and PDGFR) exhibit hyperbolic responses. Computational and experimental data reveal that these features allow nutrient flux stimulated by growth-promoting high-n receptors to drive arrest/differentiation programs by increasing surface levels of low-n glycoproteins. We have identified a mechanism for metabolic regulation of cellular transition between growth and arrest in mammals arising from apparent coevolution of N-glycan number and branching.     

Overexpression of FNTB and the activation of Ras induce hypertrophy and promote apoptosis and autophagic cell death in cardiomyocytes

Farnesyltransferase (FTase) is an important enzyme that catalyses the modification of protein isoprene downstream of the mevalonate pathway. Previous studies have shown that the tissue of the heart in the suprarenal abdominal aortic coarctation (AAC) group showed overexpression of FTaseβ (FNTB) and the activation of the downstream protein Ras was enhanced. FTase inhibitor (FTI) can alleviate myocardial fibrosis and partly improve cardiac remodelling in spontaneously hypertensive rats. However, the exact role and mechanism of FTase in myocardial hypertrophy and remodelling are not fully understood. Here, we used recombinant adenovirus to transfect neonatal rat ventricular cardiomyocytes to study the effect of FNTB overexpression on myocardial remodelling and explore potential mechanisms. The results showed that overexpression of FNTB induces neonatal rat ventricular myocyte hypertrophy and reduces the survival rate of cardiomyocytes. FNTB overexpression induced a decrease in mitochondrial membrane potential and increased apoptosis in cardiomyocytes. FNTB overexpression also promotes autophagosome formation and the accumulation of autophagy substrate protein, LC3II. Transmission electron microscopy (TEM) and mCherry‐GFP tandem fluorescent‐tagged LC3 (tfLC3) showed that FNTB overexpression can activate autophagy flux by enhancing autophagosome conversion to autophagolysosome. Overactivated autophagy flux can be blocked by bafilomycin A1. In addition, salirasib (a Ras farnesylcysteine mimetic) can alleviate the hypertrophic phenotype of cardiomyocytes and inhibit the up‐regulation of apoptosis and autophagy flux induced by FNTB overexpression. These results suggest that FTase may have a potential role in future treatment strategies to limit the adverse consequences of cardiac hypertrophy, cardiac dysfunction and heart failure.     

Beta-catenin can induce hair follicle stem cell differentiation into transit-amplifying cells through c-myc activation

Hair follicle stem cells play important roles in maintaining homeostasis and skin tissue self-renewal. Transit-amplifying cells represent the transition of cells from hair follicle stem cells into differentiated epidermal cells. Thus far, the signaling pathway and the molecular biological mechanism that regulate the proliferation and differentiation of hair follicle stem cells remain unclear. In this paper, we studied the relationship between β-catenin and c-myc during the process of the differentiation of hair follicle stem cells into transit-amplifying cells. Based on our results, the expression of β-catenin can activate the nuclear gene c-myc and regulate the expression of transit-amplifying cell markers K15, K19, a6-integrin and β1-integrin, indicating that β-catenin is involved in the transformation process from hair follicle stem cells to transit-amplifying cells and suggesting that β-catenin plays an important biological role in the induction of this differentiation process.     

Glycoantigens induce human peripheral Tr1 cell differentiation with gut-homing specialization

The carbohydrate antigen (glycoantigen) PSA from an intestinal commensal bacteria is able to down-regulate inflammatory bowel disease in model mice, suggesting that stimulation with PSA results in regulatory T cell (Treg) generation. However, mechanisms of how peripheral human T cells respond and home in response to commensal antigens are still not understood. Here, we demonstrate that a single exposure to PSA induces differentiation of human peripheral CD4(+) T cells into type-Tr1 Tregs. This is in contrast to mouse models where PSA induced the production of Foxp3(+) iTregs. The human PSA-induced Tr1 cells are profoundly anergic and exhibit nonspecific bystander suppression mediated by IL-10 secretion. Most surprisingly, glycoantigen exposure provoked expression of gut homing receptors on their surface. These findings reveal a mechanism for immune homeostasis in the gut whereby exposure to commensal glycoantigens provides the requisite information to responding T cells for proper tissue localization (gut) and function (anti-inflammatory/regulatory).