Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation

Treatment of murine myoblasts, myotubes and tumour cells with a tumour-produced lipid mobilizing factor (LMF), caused a concentration-dependent stimulation of protein synthesis, within a 24 h period. There was no effect on cell number or [3H] thymidine incorporation, but a similar concentration-dependent stimulation of 2-deoxyglucose uptake. LMF produced an increase in intracellular cyclic AMP levels, which was linearly (r2 = 0.973) related to the increase in protein synthesis. The effect of LMF was attenuated by the adenylate cyclase inhibitor MDL12330A, and was additive with the stimulation produced by forskolin. Both propranolol (10 μM) and the specific β3-adrenergic receptor antagonist SR 59230A (10-5M), significantly reduced the stimulation of protein synthesis induced by LMF. Protein synthesis was also increased by 69% (P = 0.006) in soleus muscles of mice administered LMF, while there was a 26% decrease in protein degradation (P = 0.03). While LMF had no effect on the lysosomal enzymes, cathepsins B and L, there was a decrease in proteasome activity, as determined both by the 'chymotrypsin-like' enzyme activity, as well as expression of proteasome α-type subunits, determined by Western blotting. These results show that in addition to its lipid-mobilizing activity LMF also increases protein accumulation in skeletal muscle both by an increase in protein synthesis and a decrease in protein catabolism. © 2001 Cancer Research Campaign.

Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.

Effect of zinc-alpha 2-glycoprotein (ZAG) on expression of uncoupling proteins in skeletal muscle and adipose tissue

The plasma protein zinc-α2-glycoprotein (ZAG) has been shown to be identical with a lipid mobilizing factor capable of inducing loss of adipose tissue in cancer cachexia through an increased lipid mobilization and utilization. The ability of ZAG to induce uncoupling protein (UCP) expression has been determined using in vitro models of adipose tissue and skeletal muscle. ZAG induced a concentration-dependent increase in the expression of UCP-1 in primary cultures of brown, but not white, adipose tissue, and this effect was attenuated by the β3-adrenergic receptor (β3-AR) antagonist SR59230A. A 6.5-fold increase in UCP-1 expression was found in brown adipose tissue after incubation with 0.58 μM ZAG. ZAG also increased UCP-2 expression 3.5-fold in C2C12 murine myotubes, and this effect was also attenuated by SR59230A and potentiated by isobutylmethylxanthine, suggesting a cyclic AMP-mediated process through interaction with a β3-AR. ZAG also produced a dose-dependent increase in UCP-3 in murine myotubes with a 2.5-fold increase at 0.58 μM ZAG. This effect was not mediated through the β3-AR, but instead appeared to require mitogen activated protein kinase. These results confirm the ability of ZAG to directly influence UCP expression, which may play an important role in lipid utilization during cancer cachexia. © 2004 Elsevier Ireland Ltd. All rights reserved.

Microarray analysis of expression of cell death-associated genes in spinal cord cells with cyclic tensile strain

Previous studies have described alterations in gene expression following spinal cord injury, but this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile strain on cultured spinal cord cells from E15 Sprague-Dawley rats. Microarray analysis of gene expression and categorization of identified genes were performed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. The application of cyclic tensile strain reduced the viability of cultured spinal cord cells significantly in a dose- and time-dependent manner. GO analysis identified candidate genes related to apoptosis (44) and to response to stimulus (17). KEGG analysis identified changes in the expression levels of 12 genes of the mitogen-activated protein kinase (MAPK) signaling pathway, which were confirmed to be upregulated and validated by RT-PCR analysis. Spinal cord cells undergo cell death in response to cyclic tensile strain, which were dose- and time-dependent, with upregulation of various genes, in particular of the MAPK pathway.

Analysis of kinase gene expression in the frontal cortex of suicide victims: Implications of fear and stress

Suicide is a serious public health issue that results from an interaction between multiple risk factors including individual vulnerabilities to complex feelings of hopelessness, fear, and stress. Although kinase genes have been implicated in fear and stress, including the consolidation and extinction of fearful memories, expression profiles of those genes in the brain of suicide victims are less clear. Using gene expression microarray data from the Online Stanley Genomics Database 1 and a quantitative PCR, we investigated the expression profiles of multiple kinase genes including the calcium calmodulin-dependent kinase (CAMK), the cyclin-dependent kinase, the mitogen-activated protein kinase (MAPK), and the protein kinase C (PKC) in the prefrontal cortex (PFC) of mood disorder patients died with suicide (N = 45) and without suicide (N = 38). We also investigated the expression pattern of the same genes in the PFC of developing humans ranging in age from birth to 49 year (N = 46). The expression levels of CAMK2B, CDK5, MAPK9, and PRKCI were increased in the PFC of suicide victims as compared to non-suicide controls (false discovery rate, FDR-adjusted p < 0.05, fold change >1.1). Those genes also showed changes in expression pattern during the postnatal development (FDR-adjusted p < 0.05). These results suggest that multiple kinase genes undergo age-dependent changes in normal brains as well as pathological changes in suicide brains. These findings may provide an important link to protein kinases known to be important for the development of fear memory, stress associated neural plasticity, and up-regulation in the PFC of suicide victims. More research is needed to better understand the functional role of these kinase genes that may be associated with the pathophysiology of suicide

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

Activation of forkhead box O transcription factors by oncogenic BRAF promotes p21cip1-dependent senescence

Oncogene-induced senescence (OIS) is a potent tumor-suppressive mechanism that is thought to come at the cost of aging. The Forkhead box O (FOXO) transcription factors are regulators of life span and tumor suppression. However, whether and how FOXOs function in OIS have been unclear. Here, we show a role for FOXO4 in mediating senescence by the human BRAFV600E oncogene, which arises commonly in melanoma. BRAFV600E signaling through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase resulted in increased reactive oxygen species levels and c-Jun NH 2 terminal kinase-mediated activation of FOXO4 via its phosphorylation on Thr223, Ser226, Thr447, and Thr451. BRAFV600E-induced FOXO4 phosphorylation resulted in p21cip1-mediated cell senescence independent of p16 ink4a or p27kip1. Importantly, melanocyte-specific activation of BRAFV600E in vivo resulted in the formation of skin nevi expressing Thr223/Ser226-phosphorylated FOXO4 and elevated p21cip1. Together, these findings support a model in which FOXOs mediate a trade-off between cancer and aging.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

The Effects of Nicotine on the Regulation of Neuronal Alpha7 Nicotinic Acetylcholine Receptors and Intracellular Signalling Pathways

Nicotine, the addictive compound of tobacco products, exerts its effects in the brain by binding to neuronal acetylcholine nicotinic receptors (nAChRs). The aim of the present study was to increase the knowledge of nicotine s complex effects, the focus being on homomeric alpha7-nAChRs that are widely expressed in the brain. Nicotinic regulation of differential signalling molecules including transcriptional regulators was also studied. We found that the number of alpha7-nAChRs is increased in specific brain regions in mice, in a time-dependent manner after chronic oral nicotine administration. Our results suggest that in addition to alpha4beta2-nAChRs, the other major nAChR subtype expressed in the brain, the number of alpha7-nAChRs is affected by chronic presence of nicotine. We suggest that when studying the long-term effects of nicotine, the duration on administration is of great importance. Next, we observed that nicotine exposure induces accumulation of cAMP in cell cultures expressing nAChRs. Furthermore, nicotine-induced alpha7-nAChR upregulation was potentiated by treatments enhancing cAMP-signalling, suggesting a role for cAMP in the upregulation process. Protein kinase C (PKC) was found essential for the basal regulation of alpha7-nAChR number. The nicotine-evoked alpha7-nAChR upregulation could be further increased by PKC overexpression. Thirdly, the effects of nicotine on dopamine and cAMP regulated phosphoprotein (DARPP-32) were characterised in rat brain. The results show that DARPP-32 is regulated by both acute and long-term nicotine treatment in the striatal subdivisions. The effect of acute nicotine is dose-dependent and the three striatal regions display differential sensitivities to nicotine. Chronic nicotine is also able to regulate DARPP-32 signalling with prominent effect seen in the nucleus accumbens (NAc), suggesting a role for DARPP-32 in the mediation of long-term effects of nicotine. Finally, the regulation of transcription factors Elk-1 and FosB/deltaFosB by nicotine was investigated. We found that Elk-1 is activated by acute nicotine selectively in the NAc core and hippocampal area CA1, whereas acute nicotine does not affect FosB/deltaFosB. Long-term intermittent or continuous nicotine increases the level of total Elk-1 in the same brain regions as acute nicotine. FosB/deltaFosB is also affected by chronic nicotine. Thus, similarly to other drugs of abuse, nicotine regulates transcriptional regulators Elk-1 and FosB/deltaFosB. These results bring further support for a common mechanism underlying the development of addiction. Nicotine s positive effects on learning and memory might involve the transcription factor Elk-1 based on the changes seen in the hippocampus, the key area in cognitive functions.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Hormonal modulation of riboflavin carrier protein secretion by immature rat Sertoli cells in culture

We report here that a protein species with biochemical and immunological similarity with chicken egg riboflavin carrier protein (RCP) is synthesized and secreted by immature rat Sertoli cells in culture. When quantitated by a specific heterologous radioimmunoassay, optimal concentrations of FSH (25 ng/ml) brought about 3-fold stimulation of RCP secretion. FSH, in the presence of testosterone (10−6 M) brought about 6-fold stimulation of secretion of RCP over the control cultures which were maintained in the absence of these two factors. The aromatase inhibitor (1,4,6-androstatrien-3,17-dione) curtailed 85% of the enhanced secretion of RCP, suggesting that the hormonal stimulation is mediated through in situ synthesized estrogen and this could be confirmed with exogenous estradiol-17 β which brought about 3 — fold enhancement of secretion of RCP at a concentration of 10−6 M. When tamoxifen (10 μM) was added along with FSH and testosterone, there was 75% decrease in the enhanced secretion of RCP. Addition of this anti-estrogen together with exogenous estradiol resulted in 55% decrease in elevated levels of RCP. Cholera toxin (1 μg/ml) and 8-bromo-cyclic AMP (0.5 mM) mimicked the action of FSH on the secretion of RCP thus suggesting that FSH stimulation of RCP production may be mediated through cyclic AMP. These findings suggest that estrogen mediates RCP induction in hormonally stimulated sertoli cells presumably to function as the carrier of riboflavin to the developing germ cells through blood-testis barrier in rodents.

Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro

Adult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [S-35]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1 alpha and PKR

Background: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1 alpha (ck1 alpha) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results: Serine 232 of NS5A is known to be phosphorylated by human ck1 alpha. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1 alpha has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1 alpha has been identified from the model and these are found to be conserved well in the ck1 family. ck1 alpha - substrate peptide complex has also been used to understand the structural basis of association between ck1 alpha and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions: The substrate interacting residues in ck1 alpha have been identified using the structural model of kinase substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Cyclic nucleotide signaling in intestinal epithelia: getting to the gut of the matter

The intestine is the primary site of nutrient absorption, fluid-ion secretion, and home to trillions of symbiotic microbiota. The high turnover of the intestinal epithelia also renders it susceptible to neoplastic growth. These diverse processes are carefully regulated by an intricate signaling network. Among the myriad molecules involved in intestinal epithelial cell homeostasis are the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP). These cyclic nucleotides are synthesized by nucleotidyl cyclases whose activities are regulated by extrinsic and intrinsic cues. Downstream effectors of cAMP and cGMP include protein kinases, cyclic nucleotide gated ion channels, and transcription factors, which modulate key processes such as ion-balance, immune response, and cell proliferation. The web of interaction involving the major signaling pathways of cAMP and cGMP in the intestinal epithelial cell, and possible cross-talk among the pathways, are highlighted in this review. Deregulation of these pathways occurs during infection by pathogens, intestinal inflammation, and cancer. Thus, an appreciation of the importance of cyclic nucleotide signaling in the intestine furthers our understanding of bowel disease, thereby aiding in the development of therapeutic approaches.