The anti-epileptic drug Valproic Acid (VPA) inhibits steroidogenesis in bovine theca and granulosa cells in vitro

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC(50) 67±10 µg/ml) and LH-induced (93% suppression; IC(50) 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

Impaired mechanosensing leads to heart failure and we have previously shown that a decreased ratio of cytoplasmic to nuclear CSRP3/Muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. Here we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μM blebbistatin resulted in a ∼3 fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signaling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme-oxygenase1 (HO-1) activity with PPZII blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea pig model of angiotensin II infusion (400 ng/kg/min) over 12 weeks. Using subcellular fractionation we showed that the MLP ratio increased 88% (n = 4, P < 0.01) during compensated hypertrophy, but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01 n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signaling.

HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain

Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug-induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.

Diallyl disulfide-induced apoptosis in a breast-cancer cell line (MCF-7) may be caused by inhibition of histone de-acetylation

The health benefits of garlic have been proven by epidemiological and experimental studies. Diallyl disulphide (DADS), the major organosulfur compound found in garlic oil, is known to lower the incidence of breast cancer both in vitro and in vivo. The studies reported here demonstrate that DADS induces apoptosis in the MCF-7 breast-cancer cell line through interfering with cell-cycle growth phases in a way that increases the sub-G0 population and substantially halts DNA synthesis. DADS also induces phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane and activates caspase-3. Further studies revealed that DADS modulates the cellular levels of Bax, Bcl-2, Bcl-xL and Bcl-w in a dose-dependent manner, suggesting the involvement of Bcl-2 family proteins in DADS induced apoptosis. Histone deacetylation inhibitors (HDACi) are known to suppress cancer growth and induce apoptosis in cancer cells. Here it is shown that DADS has HDACi properties in MCF-7 cells as it lowers the removal of an acetyl group from an acetylated substrate and induces histone-4 (H4) hyper-acetylation. The data thus indicate that the HDACi properties of DADS may be responsible for the induction of apoptosis in breast cancer cells.

G-protein-coupled-receptor-mediated presynaptic inhibition in the cerebellum

Throughout the central nervous system a dominant form of inhibition of neurotransmitter release from presynaptic terminals is mediated by G-protein-coupled receptors (GPCRs). Neurotransmitter release is typically induced by action potentials (APs), but can also occur spontaneously. Presynaptic inhibition by GPCRs has been associated with modulation of voltage-dependent ion channels. However, electrophysiological recordings of spontaneous, AP-independent (so-called ‘miniature’) postsynaptic events reveal an additional, important form of GPCR-mediated presynaptic inhibition, distinct from effects on ionic conductances and consistent with a direct action on the vesicle release machinery. Recent studies suggest that such miniature events might be of physiological relevance not only in signalling but also in development. In the cerebellum, neurotransmitter release onto Purkinje cells occurs by AP-dependent and AP-independent pathways. Here, I focus on inhibitory synapses between interneurons and Purkinje cells, which are subject to strong, identifiable regulation by endogenous GPCR agonists, to consider mechanisms of GPCR-mediated presynaptic inhibition.

Inhibition of human mesangial cell proliferation by targeting T-Type calcium channels

Background: Aberrant glomerular mesangial cell (MC) proliferation is a common finding in renal diseases. T-type calcium channels (T-CaCN) play an important role in the proliferation of a number of cell types, including vascular smooth muscle cells. The hypothesis that T-CaCN may play a role in the proliferation of human MC was investigated. Methods: The presence of T-CaCN in primary cultures of human MC was examined using voltage clamping and by RT-PCR. The effect of calcium channel inhibitors, and of siRNA directed against the Cav3.2 T-CaCN isoform, on MC proliferation was assessed using the microculture tetrazolium assay and nuclear BrdU incorporation. Results: Human MC express only the Cav3.2 T-CaCN isoform. Co-incubation of MC with a T-CaCN inhibitor (mibefradil, TH1177 or Ni2+) results in a concentration-dependent attenuation of proliferation. This effect cannot be attributed to direct drug-induced cytotoxicity or apoptosis and is not seen with verapamil, an L-type channel blocker. Transfection of MC with siRNA results in knockdown of T-CaCN Cav3.2 mRNA and a clear attenuation of MC proliferation. Conclusions: These results demonstrate for the first time an important role for T-CaCN in human MC proliferation. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Non-genomic signalling of the retinoic X receptor through inhibition of Gq signalling in human platelets

Retinoid X receptors (RXRs) are important transcriptional nuclear hormone receptors, acting as either homodimers or the binding partner for at least one fourth of all the known human nuclear receptors. Functional nongenomic effects of nuclear receptors are poorly understood; however, recently peroxisome proliferator-activated receptor (PPAR) gamma, PPARbeta, and the glucocorticoid receptor have all been found active in human platelets. Human platelets express RXRalpha and RXRbeta. RXR ligands inhibit platelet aggregation and TXA(2) release to ADP and the TXA(2) receptors, but only weakly to collagen. ADP and TXA(2) both signal via the G protein, Gq. RXR rapidly binds Gq but not Gi/z/o/t/gust in a ligand-dependent manner and inhibits Gq-induced Rac activation and intracellular calcium release. We propose that RXR ligands may have beneficial clinical actions through inhibition of platelet activation. Furthermore, our results demonstrate a novel nongenomic mode for nuclear receptor action and a functional cross-talk between G-protein and nuclear receptor signaling families.

Histone H4 gene expression and morphological changes on shoot apices of strawberry (Fragaria x ananassa Duch.) during floral induction

To investigate flower induction in June-bearing strawberry plants, morphological changes in shoot apices and Historic H4 expression in the central zone during flower initiation were observed. Strawberry plants were placed under flower inducible, short-day conditions (23 degrees C/17 degrees C, 10 h day length) for differing number of days (8, 16, 20, 24 or 32 days) and then these plants were transferred to non-inducible, long-day conditions (25 degrees C/20 degrees C, 14 h day length). The shoot apices of plants placed under short-day conditions for 8 days were flat, similar to shoot apices of plants in the vegetative phase of development, and Histone H4 was not expressed in the central zone during the experimental period. On the other hand, the shoot apices of plants placed under short-day conditions for 16 days remained flat, similar to shoot apices of plants placed under short-day conditions for 8 days, but Histone H4 was expressed in the central zone at the end of the short-day treatment. Morphological changes in the shoot apices of these plants were observed 8 days after the change in day-length. These plants developed differentiated flower organs after they were grown for another 30 days under long-day conditions. These results indicate that changes in the expression pattern of the Histone H4 gene occur before morphological changes during flower induction and that the expression of the gene in the central zone can be used as one of the indicators of the flowering process in strawberries. (c) 2006 Elsevier B.V. All rights reserved.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis

Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development. (C) 2004 Elsevier Inc. All rights reserved.

Oxidation affects the flow-induced aggregation of low density lipoprotein and its inhibition by albumin

We investigated whether oxidation alters the self-aggregation of low density lipoprotein (LDL) and the inhibition of such aggregation by albumin. Incubation with copper for different durations produced mildly, moderately, and highly oxidised LDL (having, respectively, ca. 60, 300 and 160 nmol lipid hydroperoxides/mg protein, and electrophoretic mobilities 1.2, 2.6 and 4.4 times that of native LDL). The rate of flow-induced aggregation was the same for native, mildly oxidised and moderately oxidised LDL, but decreased for highly oxidised LDL. The inhibitory effect of albumin (40 mg/ml) on aggregation was reduced by mild oxidation and further reduced by moderate or severe oxidation. The net result of the two effects was that in the presence of albumin, moderately oxidised LDL had the highest rate of aggregation and native the lowest. The reduction in the anti-aggregatory effect of albumin provides a new mechanism by which LDL oxidation might enhance net aggregation in vivo. (C) 2003 Elsevier B.V. All rights reserved.

Inhibition of E2F abrogates the development of cardiac myocyte hypertrophy

Growth of the post- natal mammalian heart occurs primarily by cardiac myocyte hypertrophy. Previously, we and others have shown that a partial re- activation of the cell cycle machinery occurs in myocytes undergoing hypertrophy such that cells progress through the G(1)/ S transition. In this study, we have examined the regulation of the E2F family of transcription factors that are crucial for the G(1)/ S phase transition during normal cardiac development and the development of myocyte hypertrophy in the rat. Thus, mRNA and protein levels of E2F- 1, 3, and 4 and DP- 1 and DP- 2 were down- regulated during development to undetectable levels in adult myocytes. Interestingly, E2F- 5 protein levels were substantially up- regulated during development. In contrast, an induction of E2F- 1, 3, and 4 and the DP- 1 protein was observed during the development of myocyte hypertrophy in neonatal myocytes treated with serum or phenylephrine, whereas the protein levels of E2F- 5 were decreased with serum stimulation. E2F activity, as measured by a cyclin E promoter luciferase assay and E2F- DNA binding activity, increased significantly during the development of hypertrophy with serum and phenylephrine compared with non- stimulated cells. Inhibiting E2F activity with a specific peptide that blocks E2F- DP heterodimerization prevented the induction of hypertrophic markers ( atrial natriuretic factor and brain natriuretic peptide) in response to serum and phenylephrine, reduced the increase in myocyte size, and inhibited protein synthesis in stimulated cells. Thus, we have shown that the inhibition of E2F function prevents the development of hypertrophy. Targeting E2F function might be a useful approach for treating diseases that cause pathophysiological hypertrophic growth.

Inhibition of vascular smooth muscle cell proliferation by non-steroidal anti-inflammatory drugs

Abnormal vascular smooth muscle cell (VSMC) proliferation is known to play an important role in the pathogenesis of atherosclerosis, restenosis and instent stenosis. Recent studies suggest that salicylates, in addition to inhibiting cyclooxygenase activity, exert an antiproliferative effect on VSMC growth both in vitro and in vivo. However, whether all non-steroidal anti-inflammatory drugs (NSAID) exert similar antiproliferative effects on VSMCs, and do so via a common mechanism of action, remains unknown. In the present study, we demonstrated that the NSAIDs, aspirin, ibuprofen and sulindac induced a dose-dependent inhibition of proliferation in rat A10 VSMCs (IC50 = 1666 mumol/L, 937 mumol/L and 520 mumol/L, respectively). These drugs did not show significant cytotoxic effects as determined by LDH release assay, even at the highest concentrations tested (aspirin, 5000 mumol/L; ibuprofen, 2500 mumol/L; and sulindac, 1000 mumol/L). Flow cytometric analyses showed that a 48 h exposure of A10 VSMCs to ibuprofen (1000 mumol/L) and sulindac (750 mumol/L) led to a significant G1 arrest (from 68.7 +/- 2.0% of cells in G1 to 76.6 +/- 2.2% and 75.8 +/- 2.2%, respectively, p < 0.05). In contrast, aspirin (2500 mumol/L) failed to induce a significant G1 arrest (68.1 +/- 5.2%). Clearer evidence of a G1 block was obtained by treatment of cells with the mitotic inhibitor, nocodazole (40 ng/ml), for the final 24 h of the experiment. Under these conditions, aspirin still failed to induce a G1 arrest (from 25.9 +/- 10.9% of cells in G1 to 19.6 +/- 2.3%) whereas ibuprofen and sulindac led to a significant accumulation of cells in G1(51.8% +/- 17.2% and 54.1% +/- 10.6%, respectively, p < 0.05). These results indicate that ibuprofen and sulindac inhibit VSMC proliferation by arresting the cell cycle in the G1 phase whereas the effect of aspirin appears to be independent of any special phase of the cell cycle. Irrespective of mechanism, our results suggest that NSAIDs might be of benefit to the treatment of vascular proliferative disorders.