Bisphenol-A Abrogates Proliferation and Differentiation of C2C12 Mouse Myoblasts via Downregulation of Phospho-P65 NF-κB Signaling Pathway.

Previous studies showed that bisphenol-A (BPA), a monomer of polycarbonate plastic, is leached out and contaminated in foods and beverages. This study aimed to investigate the effects of BPA on the myogenesis of adult muscle stem cells. C2C12 myoblasts were treated with BPA in both proliferation and differentiation conditions. Cytotoxicity, cell proliferation and differentiation, antioxidant activity, apoptosis, myogenic regulatory factors (MRFs) gene expression, and mechanism of BPA on myogenesis were examined. C2C12 myoblasts exposed to 25-50 µM BPA showed abnormal morphology, expressing numerous and long cytoplasmic extensions. Cell proliferation was inhibited and was accumulated in subG1 and S phases of the cell cycle, subsequently leading to apoptosis confirmed by nuclear condensation and the expression of apoptosis markers, cleaved caspase-9 and caspase-3. In addition, the activity of antioxidant enzymes, catalase, superoxide dismutase, and glutathione peroxidase was significantly decreased. Meanwhile, BPA suppressed myoblast differentiation by decreasing the number and size of multinucleated myotubes via the modulation of MRF gene expression. Moreover, BPA significantly inhibited the phosphorylation of P65 NF-κB in both proliferation and differentiation conditions. Altogether, the results revealed the adverse effects of BPA on myogenesis leading to abnormal growth and development via the inhibition of phospho-P65 NF-κB.

Styrene Oxide Caused Cell Cycle Arrest and Abolished Myogenic Differentiation of C2C12 Myoblasts.

Contaminations of chemicals in foods and drinks are raising public concerns. Among these, styrene, a monomer for plastic production, receives increasing interest due to its ability to leach from the packaging and contaminate in foods and drinks causing many health problems. The present study was designed to investigate the effects of styrene monomer (STR) and its metabolite styrene oxide (STO) on C2C12 myoblast proliferation and differentiation. Based on an MTT assay, both STR and STO showed no cytotoxic effect at 10-100 µM. However, at 50-100 µM STO, but not STR, significantly inhibited cell proliferation. The STO-treated cells were accumulated in S-phase of cell cycles as revealed by flow cytometry. The antioxidant enzyme (catalase and superoxide dismutase) activities and the gene expressing these enzymes of the arrested cells were decreased and ultimately led to nuclear condensation and expression of apoptotic markers such as cleaved caspase-3 and-9, but not cleaved caspase-8. In addition, STO significantly suppressed myogenic differentiation by decreasing both the number and size of differentiated myotubes. Biochemical analysis showed attenuations of total protein synthesis and myosin heavy chain (MHC) protein expression. In conclusion, a metabolite of styrene, STO, leached from plastic packaging of foods and beverages suppressed both myoblast proliferation and differentiation, which would affect skeletal muscle development and regeneration.

Diarylheptanoid 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one enhances C2C12 myoblast differentiation by targeting membrane estrogen receptors and activates Akt-mTOR and p38 MAPK-NF-κB signaling axes.

Diarylheptanoid, 1-(4-hydroxyphenyl)-7-phenyl(6E)-6-hepten-3-one (HPPH), has been reported to enhance myoblast differentiation via estrogen receptor (ER). However, the underlying signaling pathway promising this action remains unknown. The present study thus aimed to investigate the signaling pathway of HPPH that enhances myoblast differentiation. Confluence C2C12 myoblasts were induced to differentiate in the absence or presence of HPPH (10 nM). Differentiation markers (myosin heavy chain (MHC) and myogenin) and other signaling molecules implicated in myogenic differentiation were analyzed by immunostaining and western blotting methods. To identify the location of ER and the signaling molecules, specific inhibitors were applied targeting these molecules. Nuclear factor-κB (NF-κB) DNA binding activity was measured using the electrophoresis mobility shift assay. The results showed that HPPH enhanced myoblast differentiation by increasing MHC and myogenin levels, number, and size as well as the fusion index of myotubes. These actions occurred via membrane ER. Several MAPK proteins were activated at the early stage of differentiation. However, only Akt and p38 MAPK, but not ERK, were implicated in these effects. The underlying signaling molecules of Akt to enhance myogenic differentiation by HPPH, at least in part, were mTOR/P70S6K and GSK-3ß. On the other hand, the downstream signaling molecule of p38 MAPK was NF-κB. Our results suggested that HPPH enhanced myogenic differentiation by binding with membrane ER, which in turn recruited multiple axes including Akt-mTOR-P70S6K, Akt-GSK-3ß, and p38 MAPK-NF-κB.

P2Y6 receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 & CXCL8 mRNA expression in respiratory epithelium.

One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-ß, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (ß-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y6R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca2+ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y6 purinergic receptors.

Nitric oxide signalling is involved in diarylheptanoid-induced increases in femoral arterial blood flow in ovariectomized rats.

The mechanisms by which the hexane extract of Curcuma comosa increases femoral blood flow (FBF) in ovariectomized rats are not known. Thus, the aim of the present study was to investigate the acute effects and modes of action of the diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3), a phyto-oestrogen isolated from C. comosa, on FBF in ovariectomized rats. On Day 7 after ovariectomy, rats were injected once intra-arterially with D3 (100, 200, 400 and 800 µg/kg), 17ß-oestradiol (E2; 1, 2, 4 and 8 µg/kg) or vehicle. In some experiments, rats were injected with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) 120 min after 800 µg/kg D3 or 4 µg/kg E2. In other experiments, rats were injected with 10 mg/kg L-NAME, 900 µg/kg 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 900 µg/kg ICI 182 780 30 min prior to the injection of 800 µg/kg D3 or 4 µg/kg E2. Mean arterial blood pressure (mABP) and FBF were recorded using a pressure transducer and a laser Doppler flow meter, respectively. Both D3 and E2 dose-dependently increased FBF without changing mABP or heart rate. The EC(50) at 120 min for D3 and E2 was 195.8 and 1.8 µg/kg, respectively. In addition, D3 and E2 dose-dependently decreased femoral vascular resistance (FVR). The EC(50) of D3 was about 100-fold greater than that of E2. The effects of D3 and E2 on FBF and FVR were diminished by intravenous injection of 10 mg/kg l-NAME. Furthermore, 30 min pretreatment with L-NAME (10 mg/kg), ODQ (900 µg/kg) or ICI 182 780 (900 µg/kg) blocked the effects of D3 and E2 on FBF and FVR. The results of the present study suggest that the phyto-oestrogen D3 increases FBF in ovariectomized rats via oestrogen receptor and nitric oxide-guanylyl cyclase signalling, which, in turn, relaxes femoral vascular resistance.

Leukaemia inhibitory factor is expressed in rat gastrocnemius muscle after contusion and increases proliferation of rat L6 myoblasts via c-Myc signalling.

1. Leukaemia inhibitory factor (LIF) has been shown to have an important role during muscle regeneration. The regenerative capacity of muscles after contusion injury in LIF-knockout mice is impaired compared with that of wild-type mice. 2. To clarify whether LIF modulates muscle regeneration by regulating myogenic precursor cell activity, we studied LIF expression and myogenic precursor cell activity in gastrocnemius muscles from Wistar rats at various times after contusion injury using immunohistochemistry and the direct effect of LIF on a rat myoblast cell line (L6). 3. After contusion injury, transient upregulation of the mRNA expression of LIF, LIF receptors and signal transducer and activator of transcription (STAT) 3, downstream of LIF and involved in enhanced cell proliferation, was observed. A marked increase in LIF protein in the cytosol of damaged myofibres was strongly correlated with a significant increase in the number of myogenic precursor cells (MyoD-positive cells) by 12 h after contusion. In addition, coexpression of LIF and MyoD protein in control and injured muscles after contusion injury from 3 h to 7 days was evident. 4. Treatment of L6 cells with LIF (1 ng/mL) in serum-free medium enhanced proliferation (bromodeoxyuridine incorporation) by 24 h. This was accompanied by increased expression of c-Myc protein within 12 h and was abolished by short interference RNA against c-Myc mRNA. 5. Together, the results of the present study suggest that LIF acts via paracrine and autocrine actions to regulate myogenic precursor cell activity during muscle regeneration after contusion injury and that the proliferative effect of LIF on L6 cells occurs via c-Myc signalling.

Role of Na+ /H+ exchanger 3 in the acidification of the male reproductive tract and male fertility.

1. Male fertility is a complex process that is dependent on sex hormones and the normal function of the reproductive organs. Defects of these organs result in abnormal sperm production and function, which, in turn, lead to infertility. 2. Spermatozoa released from the testis are unable to move and fertilize with eggs. These features, known as sperm maturation, are acquired during their transit through the epididymis. 3. Among several processes that take place in the epididymis, absorption and acidification of the luminal fluid are essential for sperm maturation, sperm storage and fertility. Currently, the mechanism by which acidification occurs in the epididymis is still not fully understood. 4. The epididymis is fully equipped with the proteins required for acid/base transport, such as Na(+) /H(+) exchanger 3 (NHE3, SLC9A3), vacuolar-type adenosine triphosphatase (V-ATPase) and various isoforms of enzyme carbonic anhydrase (CA). 5. Most studies, so far, have focused on the role of V-ATPase on H(+) secretion and acidification of the epididymis. The involvement of NHE3 in creating the acidic environment of the epididymal spermatozoa receives little attention. 6. This review presents evidence for and discusses the role of NHE3 in the acidification of the male reproductive tract and its requirement for male fertility.

Satellite cell activity in muscle regeneration after contusion in rats.

1. The role of satellite cells in muscle growth during development is well documented, but the involvement of these cells in muscle repair after contusion is less well known. In the present study, we investigated the time-course of satellite cell activity (from 3h to 7days) after contusion of rat gastrocnemius muscle using specific molecular markers for immunofluorescence and real-time polymerase chain reaction (PCR). 2. Inflammation of the injured muscle occurred within 6h, followed by disintegration of the damaged myofibres within 12h. Newly formed myofibres appeared by Day 7. 3. The number of MyoD-positive nuclei (activated satellite cells) in the injured muscle was significantly increased by 6h, reaching a maximum by 12h after contusion. However, the number of MyoD-positive nuclei decreased towards control levels by Day 7. Changes in the number of bromodeoxyuridine-labelled nuclei (proliferating satellite cells) paralleled the changes seen in the number of MyoD-positive nuclei. Conversely, expression of myogenin protein was not apparent until Day 3 and increased further by Day 7. Colabelling of MyoD and myogenin was seen in only a few cells. 4. The time-course of MyoD mRNA expression corresponded with MyoD protein expression. However, there were two peaks in myogenin mRNA expression: 6h and Day 7 after contusion. The second peak coincided with upregulation of myostatin mRNA levels. 5. The results of the present study suggest that contusion activates a homogeneous population of satellite cells to proliferate within 3days, followed by differentiation to form new myofibres. The latter may be regulated, in part, by myostatin.

Serum antibodies and cytokines in C4-deficient mice and their responses to exercise.

Psychological stress is believed to be one of the predisposing factors for systemic lupus erythematosus (SLE), whereas physical stress such as exercise has never been reported to be related. We measured the circulating levels of antibodies (IgM, IgG, anti-dsDNA IgG), Th1 (IFN-gamma), Th2 (IL-4, IL-6), and of pro-inflammatory (TNF-alpha, IL-1beta) and anti-inflammatory (TGF-beta) cytokines of C4(-l-) female mice at rest, after acute exercise and after exercise training, using an antibody-capture ELISA. Prior to the exercise, the C4(-l-) mice had higher levels of IgG and anti-dsDNA IgG but lower levels of IFN-gamma, IL-1beta, IL-6 and IL-4 than wild-type C57BL/6 (B6) mice. A single bout of exercise to exhaustion increased serum IgG, TNF-alpha, IL-1beta and TGF-beta in the B6 mice but only TGF-beta in the C4(-l-) mice was increased. We conclude that exhaustive or moderate exercise has no effect on the levels of serum antibodies and cytokines and is thus unlikely to promote the onset of SLE.

Activity and subcellular trafficking of the sodium-coupled choline transporter CHT is regulated acutely by peroxynitrite.

Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 4 min. SIN-1 significantly reduced V(max) of choline uptake without altering the K(m). This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.

GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes.

Spermatogonial stem cells (SSCs) are essential for spermatogenesis, and these adult tissue stem cells balance self-renewal and differentiation to meet the biological demand of the testis. The developmental dynamics of SSCs are controlled, in part, by factors in the stem cell niche, which is located on the basement membrane of seminiferous tubules situated among Sertoli cells. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), and disruption of GDNF expression results in spermatogenic defects and infertility. The GDNF signals through a receptor complex that includes GDNF family receptor alpha1 (GFRA1), which is thought to be expressed by SSCs. However, expression of GFRA1 on SSCs has not been confirmed by in vivo functional assay, which is the only method that allows definitive identification of SSCs. Therefore, we fractionated mouse pup testis cells based on GFRA1 expression using magnetic activated cell sorting. The sorted and depleted fractions of GFRA1 were characterized for germ cell markers by immunocytochemistry and for stem cell activity by germ cell transplantation. The GFRA1-positive cell fraction coeluted with other markers of SSCs, including ITGA6 and CD9, and was significantly depleted of KIT-positive cells. The transplantation results confirmed that a subpopulation of SSCs expresses GFRA1, but also that the stem cell pool is heterogeneous with respect to the level of GFRA1 expression. Interestingly, POU5F1-positive cells were enriched nearly 15-fold in the GFRA1-selected fraction, possibly suggesting heterogeneity of developmental potential within the stem cell pool.