Temporal phosphoproteomics to investigate the mechanotransduction of vascular smooth muscle cells in response to cyclic stretch

Vascular smooth muscle cells (VSMCs) are exposed to mechanical cyclic stretch in vivo, which play important roles in maintenance of vascular homeostasis and regulation of pathological vascular remodeling. Reversible protein phosphorylation is crucial for intracellular signaling transduction. However, the dynamic phosphorylated profile induced by cyclic stretch in VSMCs is still unclear. Using the stable isotope labeling by amino acid in cell culture, VSMCs were labeled and exposed to 10% physiological cyclic stretch in vitro at 1.25 Hz for 0 min, 15 min, 30 min, 1 h and 6 h, respectively. Using TiO₂ beads and liquid chromatography tandem mass spectrometry, the temporal phosphoproteomic profiles in response to cyclic stretch were then detected. Bioinformatics analysis including fuzzy c-means clustering, functional classifications, and Ingenuity Pathway Analysis were applied to further reveal the potential mechanotranduction networks. The results indicated that protein kinase C (PKCs) family, Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and Akt may participate in cyclic-stretch induced VSMC functions. Cyclic stretch repressed the expression of ROCK1, while it had no significant effect on the phosphorylation of PKCα/βII, PKCζ/λ and PKCδ/θ. PKCθ was activated first at short time-phase (15 min and 30 min), and again at long time-phase (6 h, 12 h and 24 h). The activation of p-PKCμ was immediate and short-term, similar to p-Akt. Our present in vitro work hence revealed that cyclic stretch activates complex mechanotransduction networks, suggesting that novel mechanoresponsive molecules, i.e., PKCθ, PKCμ, and ROCK1, may participate in the mechanotransduction and modulation VSMC functions.     

Predicting cortisol stress responses in older individuals: influence of serotonin receptor 1A gene (HTR1A) and stressful life events

Considerable variability in the activity of the hypothalamus-pituitary-adrenal (HPA) axis in response to stress has been found in quantitative genetic studies investigating healthy individuals suggesting that at least part of this variance is due to genetic factors. Since the HPA axis is regulated by a neuronal network including amygdala, hippocampus, prefrontal cortex as well as brainstem circuits, the investigation of candidate genes that impact neurotransmitter systems related to these brain regions might further elucidate the genetic underpinnings of the stress response. However, aside from genetic risk factors, past stressful life events might also result in long-term adjustments of HPA axis reactivity. Here, we investigated the effects of the − 1019 G/C polymorphism in the HTR1A gene encoding the serotonin (5-HT) receptor 1A (5-HT_1A) and stressful life events experienced during childhood and adolescence on changes in cortisol levels in response to the Trier Social Stress Test (TSST) in a sample of healthy older adults (N = 97). Regression analyses revealed a significant effect of HTR1A genotype with the G allele being associated with a less pronounced stress response. In addition, an inverse relationship between past stressful life events and cortisol release but no gene × environment interaction was detected. The results further underscore the crucial role of functional serotonergic genetic variation as well as stressful events during critical stages of development on the acute stress response later in life.     

Kamolonol suppresses angiotensin II-induced stress fiber formation and cellular hypertrophy through inhibition of Rho-associated kinase 2 activity

Kamolonol (7-[[(1R,2R,4R,4aS,5R,8aS)-4-hydroxy-1,2,4a,5-tetramethyl-6-oxo-3,4,5,7,8,8a-hexahydro-2H-naphthalen-1-yl]methoxy]chromen-2-one) is a sesquiterpene coumarin and an active component of gum extracts from Ferulaassafoetida. The aim of this study was to investigate the anti-fibrotic and anti-cellular hypertrophic effects of kamolonol, and further to explore its possible mechanism. Kamolonol (3–30 μM) significantly inhibited stress fiber formation induced by angiotensin II (Ang II) in rat heart-derived H9c2 cells. Furthermore, kamolonol (3–30 μM) showed a potent inhibitory effect on Ang II-induced cellular hypertrophy in H9c2 cells. Next, a Rho-associated kinase (ROCK) activity was measured because actin stress fiber formation and/or cellular hypertrophy are usually induced by the activation of ROCK. Rho-associated kinase 2 (ROCK2) studies using a time-resolved fluorescence resonance energy transfer (TR-FRET) showed that kamolonol possesses a potent ROCK2 inhibitory activity with IC₅₀ values of 2.27 μM, and has an ATP-competitive inhibitory mode. In validation study, pretreatment of kamolonol (3–30 μM) for 2 h decreased the Ang II-induced phosphorylation of myosin phosphatase 1 (MYPT1) and myosin light chain 2 (MLC2). Taken together, these results indicate that kamolonol suppresses Ang II-induced stress fiber formation and cellular hypertrophy, and propose that one mechanism underlying these anti-fibrotic and anti-cellular hypertrophic effects involves inhibition of the ROCK-MLC pathway.     

Central nervous system activities of two diterpenes isolated from Aloysia virgata

Using the guide of a competitive assay for the benzodiazepine binding site in the γ-aminobutyric acid type A receptor (GABA_A), two active diterpenes were isolated from the aerial parts of Aloysia virgata (Ruíz & Pavón) A.L. Jussieu var. platyphylla (Briquet) Moldenke. These compounds, identified as (16R)-16,17,18-trihydroxyphyllocladan-3-one (1) and (16R)-16,17-dihydroxyphyllocladan-3-one (2) on the basis of spectral data, competitively inhibited the binding of [³H]-FNZ to the benzodiazepine binding site with K_i ± S.E.M. values of 56 ± 19 μM and 111 ± 13 μM, respectively. The behavioral actions of these diterpenes, intraperitoneally (i.p.) administered in mice, were examined in the plus-maze, holeboard, locomotor activity and light/dark tests. Compound 1 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the holeboard test (the number of head dips at 0.3 mg/kg and 3 mg/kg, the rears at 1 mg/kg and the time spent head-dipping at 3 mg/kg), in the plus-maze assay (the percentage of open arm entries at 1 mg/kg) and in the light/dark test (the time in light and the number of transitions at 1 mg/kg). Compound 2 augmented the number of rearings in the holeboard apparatus (at 0.3 mg/kg and 1 mg/kg) and the locomotor activity (at 1 mg/kg). These results reveal the presence of neuroactive compounds in Aloysia virgata.     

HY251, a novel decahydrocyclopenta[a]indene analog, from Aralia continentalis induces apoptosis via down-regulation of AR expression in human prostate cancer LNCaP cells

In the course of screening for a novel anticancer drug candidate, we previously isolated HY251 with the molecular structure of 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[a]indene-3,3a,7a,8a-tetraol from the roots of Aralia continentalis. The current study was designed to evaluate the detailed mechanisms of apoptotic induction of HY251 in androgen-sensitive prostate cancer LNCaP cells. TUNEL assay and Western blot analysis revealed an appreciable apoptotic induction in LNCaP cells treated with 95 μM of HY251 for 24 h. This apoptotic induction is also associated with cytochrome c release from mitochondria which, in turn, resulted in the activation of caspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, we found that HY251 significantly inhibited the expression levels of androgen receptor (AR) and prostate-specific antigen (PSA) in a time-dependent manner, as well as abrogated up-regulation of AR and PSA genes with and without androgen. Therefore, we suggest that HY251, a novel androgen antagonist, may be a potent cancer chemotherapeutic candidate for the treatment of both androgen-sensitive and hormone-refractory prostate cancer.     

Expression and purification of non-glycosylated Trypanosoma brucei transferrin receptor in insect cells

The transferrin receptor of the parasite Trypanosoma brucei is a heterodimeric protein complex encoded by the 2 expression site-associated genes (ESAGs) 6 and 7. ESAG6 is a heterogeneously glycosylated protein of 50-60 kDa modified by a glycosylphosphatidylinositol anchor at the C-terminus, while ESAG7 is a 40-42 kDa glycoprotein carrying an unmodified C-terminus. In order to determine whether glycosylation is necessary for dimer formation and ligand binding, the receptor was expressed in insect cells in the presence of tunicamycin. When insect cells were infected with recombinant ESAG6/ESAG7 double expressor baculovirus and grown in the presence of tunicamycin, non-glycosylated forms of ESAG6 and ESAG7 of 46 and 36 kDa, respectively, were synthesized. The non-glycosylated ESAG6 and ESAG7 were capable of forming a heterodimer and of binding transferrin. This results shows that glycosylation is not necessary for synthesis of a functional T. brucei transferrin receptor.     

Design, synthesis, and biological activity of novel Magmas inhibitors

Magmas (mitochondria associated, granulocyte-macrophage colony stimulating factor signaling molecule), is a highly conserved and essential gene, expressed in all cell types. We designed and synthesized several small molecule Magmas inhibitors (SMMI) and assayed their effects on proliferation in yeast. We found that the most active compound 9 inhibited growth at the 4 μM scale. This compound was shown by fluorometric titration to bind to Magmas with a K_d = 33 μM. Target specificity of the lead compound was established by demonstrating direct binding of the compound to Magmas and by genetic studies. Molecular modeling suggested that the inhibitor bound at the predicted site in Magmas.     

Chronological expression of Wnt target genes Ccnd1, Myc, Cdkn1a, Tfrc, Plf1 and Ramp3

The canonical Wnt pathway regulates several biological processes including development, cell growth and proliferation via consecutive gene regulation. A high number of target genes of the Wnt pathway has been identified, but the chronological order of target gene expression is still elusive. This order is supposed to be crucial for the controlled course of events downstream of the activated Wnt pathway. Here we present the expression chronologies of the target genes Ccnd1 (encoding for cyclin D1), Myc (c-Myc), Cdkn1a (p21^CIP1/WAF1), Tfrc (Transferrin receptor 1), Plf1 (Proliferin-1) and Ramp3 (Receptor activity-modifying protein 3) in C57MG cells after stimulation with Wnt-3a. We discriminated between immediate (below 1 h), early (between 1 and 6 h), intermediate (between 6 and 12 h) and late (after 12 h) targets. According to this classification Myc and Tfrc belong to the immediate target genes; Ccnd1, Plf1 and Ramp3 are early target genes and Cdkn1a is an intermediate target gene.     

Elevated expression of urotensin II and its receptor in diethylnitrosamine-mediated precancerous lesions in rat liver

Urotensin II (UII) is a somatostatin-like peptide involved in cell proliferation and in tumor biology. To explore the role of liver-derived UII in the pathogenesis of precancerous liver lesions in rat, we investigated the expression of UII and its receptor, UT, in diethylnitrosamine (DEN)-induced precancerous liver lesions and the effects of UII on cell proliferation by hepatic oval cells. Radioimmunoassay, RT-PCR, immunohistochemistry and western blot were used in this study. Compared with untreated controls, rats treated with DEN showed increased UII content by 47.7% in plasma and by 164.9% in liver tissue (all P < 0.01). The expression of UII protein and of both UT mRNA and protein was significantly enhanced in the liver of treated rats. Western blot analysis revealed that the expression of phosphorylated protein kinase C (p-PKC) and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) was increased in the liver of treated animals. Treatment with UII (10⁻¹⁰-10⁻⁶ M) for 24 h significantly increased number of cultured hepatic oval cells (at 10⁻⁹-10⁻⁸ M). However, during the pre-incubation with calphostin C (inhibitor of PKC) or PD98059 (inhibitor of MEK), the proliferation was decreased by 40.1% and 25.4% respectively (both P < 0.05). In DEN-induced precancerous liver lesions, the UII/UT system was up-regulated, which may contribute to the pathogenesis of liver cancer through a PKC- or ERK1/2-dependent pro-mitogenic pathway in an autocrine/paracrine manner.     

Transmembrane domain of EphA1 receptor forms dimers in membrane-like environment

Eph receptor tyrosine kinases (RTKs) are activated by a ligand-mediated dimerization in the plasma membrane and subjected to clusterization at a high local density of receptors and their membrane-anchored ligands. Interactions between transmembrane domains (TMDs) were recognized to assist to the ligand-binding extracellular domains in the dimerization of some RTKs, whereas a functional role of Eph-receptor TMDs remains unknown. We have studied a propensity of EphA1-receptor TMDs (TMA1) to self-association in membrane-mimetic environment. Dimerization of TMA1 in SDS environment was revealed by SDS-PAGE and confirmed by FRET analysis of the fluorescently labeled peptide (K_d = 7.2 ± 0.4 μM at 1.5 mM SDS). TMA1 dimerization was also found in 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes (ΔG = −15.4 ± 0.5 kJ/mol). Stability of TMA1 dimers is comparable to the reported earlier stability of TMD dimers of fibroblast growth factor receptor 3 and tenfold weaker than the stability of TMD dimers of glycophorin A possessing high propensity to dimerization. Our results suggest that EphA1-receptor TMD contribute to the dimerization-mediated receptor activation. An assumed role of the TMD interactions is the efficient signal transduction due to TMD-driving mutual orientation of kinase domains in dimers, while a relatively low force of the TMD interactions does not prevent a ligand-controlled regulation of the receptor dimerization.     

D-Lys-GHRP-6 antagonizes the effect of unacylated but not of acylated ghrelin on the growth of HECa10 murine endothelial cells

Recent studies demonstrate that ghrelin can be an endogenous regulator of angiogenesis. We studied direct effects of human acylated (hAG) and unacylated (hUAG) ghrelin, as well as of rat acylated ghrelin (rAG) on the growth of HECa10 murine endothelial cells. Ghrelin was applied separately or together with D-Lys³-GHRP-6, which is commonly used as an antagonist of ghrelin receptor type 1a – GHS-R1a. The growth of HECa10 cells was assessed with Mosmann and in selected study conditions also with BrdU and TUNEL methods. Both hAG and hUAG (10⁻⁵ M to 10⁻¹² M) inhibited the growth of HECa10 cells in 24 h and 72 h cultures. Similarly, rAG decreased the growth of the cells after 24 h (10⁻⁷ M and 10⁻¹¹ M), and after 72 h (10⁻⁷ M, 10⁻⁸ M and 10⁻¹¹ M). Unexpectedly, D-Lys³-GHRP-6 itself also inhibited the growth of these cells at 10⁻⁴ to 10⁻⁶ M in 24 h, 48 h (dose–response effect) and 72 h cultures. D-Lys³-GHRP-6 did not modify the inhibitory effect of rAG. However, D-Lys³-GHRP-6 at the concentration of 10⁻⁴ M diminished, abolished or even reversed the inhibitory effect of hUAG in 72 h culture and this was dependent on ghrelin concentrations. These data indicate that both AG and UAG have antiangiogenic properties at least at the level of endothelial growth, through decreased metabolic activity of the cells or stimulation of apoptosis. D-Lys³-GHRP-6 (inhibitor of GHS-R1a) seems not to be an appropriate antagonist in this experimental condition. Similar effects of these substances on HECa10 cells suggest that they are not mediated by GHS-R1a.     

Potentially functional polymorphisms in ATG10 are associated with risk of breast cancer in a Chinese population

Autophagy is a cellular process directed at recycling of cellular proteins and removal of intracellular microorganisms, which is important for balancing sources of energy at critical times in development and in response to nutrient stress. It has been reported to be a critical process in cancer initiation and progression. We hypothesized that genetic variants in critical genes of autophagy may be involve in the development of breast cancer. Thus, we systematically screened 14 potentially functional polymorphisms in six autophagy-related genes (ATG3, ATG5, ATG7, ATG10, and ATG12 and LC3) that are core components in autophagosome formation. We conducted a case-control study including 1064 breast cancer cases and 1073 cancer-free controls to evaluate the associations of these variants with breast cancer risk. We found that rs1864182 and rs10514231 in ATG10 were significantly associated with a decreased risk of breast cancer [odds ratios (OR) = 0.77, 95% confidence interval (CI): 0.61–0.96, P = 0.023; and OR = 0.75, 95% CI: 0.59–0.93, P = 0.010, respectively]. Similar protective effects for both loci were observed between subgroups stratified by ages at diagnosis/recruitment, menarche and first live birth, and status of menopause, estrogen receptor (ER) and progesterone receptor (PR). These results suggest that genetic variants in ATG10 may implicate with breast cancer susceptibility in Chinese population. Further large and functional studies are needed to confirm our findings.     

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved (P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.     

Calorie restriction influences key metabolic enzyme activities and markers of oxidative damage in distinct mouse liver mitochondrial sub-populations

Aims

The purpose of the study was to establish if enzyme activities from key metabolic pathways and levels of markers of oxidative damage to proteins and lipids differed between distinct liver mitochondrial sub-populations, and which specific sub-populations contributed to these differences.

Main methods

Male C57BL/6J mice were fed non-purified diet for one month then separated into two groups, control and calorie-restricted (CR). The two groups were fed semi-purified diet (AIN93G), with the CR group receiving 40% less calories than controls. After two months, enzyme activities and markers of oxidative damage in mitochondria were determined.

Key findings

In all mitochondrial sub-populations, enzyme activities and markers of oxidative damage, from control and CR groups, showed a pattern of M1 > M3 > M10. Higher acyl-CoA dehydrogenase (β-oxidation) and β-hydroxybutyrate dehydrogenase (ketogenesis) activities and lower carbonyl and TBARS levels were observed in M1 and M3 fractions from CR mice. ETC enzyme activities did not show a consistent pattern. In the Krebs cycle, citrate synthase and aconitase activities decreased while succinate dehydrogenase and malate dehydrogenase activities increased in the M1 mitochondria from the CR versus control mice.

Significance

CR does not produce uniform changes in enzyme activities or markers of oxidative damage in mitochondrial sub-populations, with changes occurring primarily in the heavy mitochondrial populations. Centrifugation at 10,000 g to isolate mitochondria likely dilutes the mitochondrial populations which show the greatest response to CR. Use of lower centrifugal force (3000 g or lower) may be beneficial for some studies.     

Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca^2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca^2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca^2 + increased the reduction of NADH (8%), and of cytochromes b_H (3%), c₁ (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca^2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca^2 + stimulation of oxidative phosphorylation.     

Apigenin inhibits hepatoma cell growth through alteration of gene expression patterns

Apigenin, a common plant flavonoid, has been shown to possess anti-tumor properties; however, the underlying molecular mechanisms are still not completely understood. In the present study, we investigated the effects of apigenin on human hepatoma Huh7 cell proliferation, cell cycle distribution, apoptosis, and colony formation in vitro, as well as on the tumorigenicity of Huh7 cells in vivo. To get more insight into the mechanism of apigenin action, we performed genome-wide expression profiling of apigenin-treated Huh7 cells using cDNA microarrays (Agilent Whole Human Genome Oligo Microarray) that contain 41,000 genes. Ten of the most differentially expressed genes (≧5-fold changes) were selected for further evaluation by quantitative RT-PCR (qPCR) and Western blot analyses. Notably, apigenin (5-20 μg/ml) remarkably inhibited Huh7 cell proliferation and colony formation as compared to the vehicle control, which was in a dose-dependent manner. Accompanying with the decreased growth, apigenin-treated cells showed a cell cycle arrest at G2/M phase and an increased rate of apoptosis. Moreover, the xenografts derived from Huh7 cells were significantly (p < 0.05) retarded by the delivery of apigenin (50 μg/mouse/day) relative to the control counterparts. Gene expression profile analysis revealed that 1336 genes were up-regulated and 428 genes were down-regulated by apigenin. The down-regulation of interleukin-4 receptor and ubiquitin specific protease 18 and the up-regulation of SLC27A3 and chemokine (C-C motif) receptor 2 were further confirmed by the qPCR and Western blot results. In conclusion, apigenin exhibits inhibitory effects on hepatoma cell growth, which is likely mediated through alteration of gene expression profiles.