Intracellular signalling pathways regulating the adaptation of skeletal muscle to exercise and nutritional changes

The focus of the present review is to assimilate current knowledge concerning the differing signalling transduction cascades that control muscle mass development and affect skeletal muscle phenotype following exercise or nutritional uptake. Effects of mechanical loading on protein synthesis are discussed. Muscle growth control is regulated by the interplay of growth promoting and growth suppressing factors, which act in concert. Much emphasis has been placed on understanding how increases in the rate of protein synthesis are induced in skeletal muscle during the adaptive process. One key point to emerge is that protein synthesis following resistance exercise or increased nutrient availability is mediated through changes in signal transduction involving the phosphorylation of mTOR and sequential activation of downstream targets. On the other hand, AMPK activation plays an important role in the inhibition of protein synthesis by suppressing the function of multiple translation regulators of the mTOR signalling pathway in response to cellular energy depletion and low metabolic conditions. The effects of exercise and/or nutritional uptake on the activation of signalling molecules that regulate protein synthesis are highlighted, providing a better understanding of the molecular changes in the cell.

Crosstalk between developmental and tumour-specific signalling pathways : kallikrein-related serine peptidases and nodal in prostrate cancer

Prostate cancer is an important male health issue. The strategies used to diagnose and treat prostate cancer underscore the cell and molecular interactions that promote disease progression. Prostate cancer is histologically defined by increasingly undifferentiated tumour cells and therapeutically targeted by androgen ablation. Even as the normal glandular architecture of the adult prostate is lost, prostate cancer cells remain dependent on the androgen receptor (AR) for growth and survival. This project focused on androgen-regulated gene expression, altered cellular differentiation, and the nexus between these two concepts. The AR controls prostate development, homeostasis and cancer progression by regulating the expression of downstream genes. Kallikrein-related serine peptidases are prominent transcriptional targets of AR in the adult prostate. Kallikrein 3 (KLK3), which is commonly referred to as prostate-specific antigen, is the current serum biomarker for prostate cancer. Other kallikreins are potential adjunct biomarkers. As secreted proteases, kallikreins act through enzyme cascades that may modulate the prostate cancer microenvironment. Both as a panel of biomarkers and cascade of proteases, the roles of kallikreins are interconnected. Yet the expression and regulation of different kallikreins in prostate cancer has not been compared. In this study, a spectrum of prostate cell lines was used to evaluate the expression profile of all 15 members of the kallikrein family. A cluster of genes was co-ordinately expressed in androgenresponsive cell lines. This group of kallikreins included KLK2, 3, 4 and 15, which are located adjacent to one another at the centromeric end of the kallikrein locus. KLK14 was also of interest, because it was ubiquitously expressed among the prostate cell lines. Immunohistochemistry showed that these 5 kallikreins are co-expressed in benign and malignant prostate tissue. The androgen-regulated expression of KLK2 and KLK3 is well-characterised, but has not been compared with other kallikreins. Therefore, KLK2, 3, 4, 14 and 15 expression were all measured in time course and dose response experiments with androgens, AR-antagonist treatments, hormone deprivation experiments and cells transfected with AR siRNA. Collectively, these experiments demonstrated that prostatic kallikreins are specifically and directly regulated by the AR. The data also revealed that kallikrein genes are differentially regulated by androgens; KLK2 and KLK3 were strongly up-regulated, KLK4 and KLK15 were modestly up-regulated, and KLK14 was repressed. Notably, KLK14 is located at the telomeric end of the kallikrein locus, far away from the centromeric cluster of kallikreins that are stimulated by androgens. These results show that the expression of KLK2, 3, 4, 14 and 15 is maintained in prostate cancer, but that these genes exhibit different responses to androgens. This makes the kallikrein locus an ideal model to investigate AR signalling. The increasingly dedifferentiated phenotype of aggressive prostate cancer cells is accompanied by the re-expression of signalling molecules that are usually expressed during embryogenesis and foetal tissue development. The Wnt pathway is one developmental cascade that is reactivated in prostate cancer. The canonical Wnt cascade regulates the intracellular levels of β-catenin, a potent transcriptional co-activator of T-cell factor (TCF) transcription factors. Notably, β-catenin can also bind to the AR and synergistically stimulate androgen-mediated gene expression. This is at the expense of typical Wnt/TCF target genes, because the AR:β-catenin and TCF:β-catenin interactions are mutually exclusive. The effect of β-catenin on kallikrein expression was examined to further investigate the role of β-catenin in prostate cancer. Stable knockdown of β-catenin in LNCaP prostate cancer cells attenuated the androgen-regulated expression of KLK2, 3, 4 and 15, but not KLK14. To test whether KLK14 is instead a TCF:β-catenin target gene, the endogenous levels of β-catenin were increased by inhibiting its degradation. Although KLK14 expression was up-regulated by these treatments, siRNA knockdown of β-catenin demonstrated that this effect was independent of β-catenin. These results show that β-catenin is required for maximal expression of KLK2, 3, 4 and 15, but not KLK14. Developmental cells and tumour cells express a similar repertoire of signalling molecules, which means that these different cell types are responsive to one another. Previous reports have shown that stem cells and foetal tissues can reprogram aggressive cancer cells to less aggressive phenotypes by restoring the balance to developmental signalling pathways that are highly dysregulated in cancer. To investigate this phenomenon in prostate cancer, DU145 and PC-3 prostate cancer cells were cultured on matrices pre-conditioned with human embryonic stem cells (hESCs). Soft agar assays showed that prostate cancer cells exposed to hESC conditioned matrices had reduced clonogenicity compared with cells harvested from control matrices. A recent study demonstrated that this effect was partially due to hESC-derived Lefty, an antagonist of Nodal. A member of the transforming growth factor β (TGFβ) superfamily, Nodal regulates embryogenesis and is re-expressed in cancer. The role of Nodal in prostate cancer has not previously been reported. Therefore, the expression and function of the Nodal signalling pathway in prostate cancer was investigated. Western blots confirmed that Nodal is expressed in DU145 and PC-3 cells. Immunohistochemistry revealed greater expression of Nodal in malignant versus benign glands. Notably, the Nodal inhibitor, Lefty, was not expressed at the mRNA level in any prostate cell lines tested. The Nodal signalling pathway is functionally active in prostate cancer cells. Recombinant Nodal treatments triggered downstream phosphorylation of Smad2 in DU145 and LNCaP cells, and stably-transfected Nodal increased the clonogencity of LNCaP cells. Nodal was also found to modulate AR signalling. Nodal reduced the activity of an androgen-regulated KLK3 promoter construct in luciferase assays and attenuated the endogenous expression of AR target genes including prostatic kallikreins. These results demonstrate that Nodal is a novel example of a developmental signalling molecule that is reexpressed in prostate cancer and may have a functional role in prostate cancer progression. In summary, this project clarifies the role of androgens and changing cellular differentiation in prostate cancer by characterising the expression and function of the downstream genes encoding kallikrein-related serine proteases and Nodal. Furthermore, this study emphasises the similarities between prostate cancer and early development, and the crosstalk between developmental signalling pathways and the AR axis. The outcomes of this project also affirm the utility of the kallikrein locus as a model system to monitor tumour progression and the phenotype of prostate cancer cells.

Uncovering BRCA1-regulated signalling pathways by microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

The effect of silicate ions on proliferation, osteogenic differentiation and cell signalling pathways (WNT and SHH) of bone marrow stromal cells

Silicon (Si) is a trace element, which plays an important role in human bone growth. Si has been incorporated into biomaterials for bone regeneration in order to improve their osteogenic potential, both in vitro and in vivo. Little is known, however, as to how Si ions elicit their biological response on bone-forming cells. The aim of this study was to investigate the effect of Si ions on the proliferation, differentiation, bone-related gene expression and cell signalling pathways of bone marrow stromal cells (BMSCs) by comparing the BMSC responses to different concentrations of NaCl and Na2SiO3, while taking into account and excluding the effect of Na ions. Our study showed that Si ions at a concentration of 0.625 mM significantly enhanced the proliferation, mineralization nodule formation, bone-related gene expression (OCN, OPN and ALP) and bone matrix proteins (ALP and OPN) of BMSCs. Furthermore, Si ions at 0.625 mM could counteract the effect of the WNT inhibitor (W.I.) cardamonin on the osteogenic genes expression, (OPN, OCN and ALP), WNT and SHH signalling pathway-related genes in BMSCs. These results suggest that Si ions by themselves play an important role in regulating the proliferation and osteogenic differentiation of BMSCs, with the involvement of WNT and SHH signalling pathways. Our study provides evidence to explain possible molecular mechanisms whereby Si ions released from Si-containing biomaterials can acquire enhanced bioactivity at desired concentration.

Stromal upregulation of lateral epithelial adhesions : gene expression analysis of signalling pathways in prostate epithelium

BACKGROUND: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process. METHODS: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model. RESULTS: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1). CONCLUSIONS: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

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.

Modulatory roles of microRNAs in the regulation of different signalling pathways in large bowel cancer stem cells

There are emerging data to suggest that microRNAs (miRNAs) have significant roles in regulating the function of normal cells and cancer stem cells (CSCs). This review aims to analyse the roles of miRNAs in the regulation of colon CSCs through their interaction with various signalling pathways. Studies showed a large number of miRNAs that are reported to be deregulated in colon CSCs. However, few of the studies available were able to outline the function of miRNAs in colon CSCs and uncover their signalling pathways. From those miRNAs, which are better described, miR-21 followed by miR-34, miR-200 and miR-215 are the most reported miRNAs to have roles in colon CSC regulation. In particular, miRNAs have been reported to regulate the stemness features of colon CSCs mainly via Wnt/B-catenin and Notch signalling pathways. Additionally, miRNAs have been reported to act on processes involving CSCs through cell cycle regulation genes and epithelial-mesenchymal transition. The relative paucity of data available on the significance of miRNAs in CSCs means that new studies will be of great importance to determine their roles and to identify the signalling pathways through which they operate. Such studies may in future guide further research to target these genes for more effective cancer treatment. miRNAs were shown to regulate the function of cancer stem cells in large bowel cancer by targeting a few key signalling pathways in cells.

Glucose-potentiated chemotaxis in human vascular smooth muscle is dependent on cross-talk between the P13K and MAPK signalling pathways

Atheroma formation involves the movement of vascular smooth muscle cells (VSMC) into the subendothelial space. The aim of this study was to determine the involvement of PI3K and MAPK pathways and the importance of cross-talk between these pathways, in glucose-potentiated VSMC chemotaxis to serum factors. VSMC chemotaxis occurred in a serum gradient in 25 mmol/L glucose (but not in 5 mmol/L glucose) in association with increased phosphorylation (activation) of Akt and ERK1/2 in PI3K and MAPK pathways, respectively. Inhibitors of these pathways blocked chemotaxis, as did an mTOR inhibitor. VSMC expressed all class IA PI3K isoforms, but microinjection experiments demonstrated that only the p110beta isoform was involved in chemotaxis. ERK1/2 phosphorylation was reduced not only by MAPK pathway inhibitors but also by PI3K and mTOR inhibitors; when PI3K was inhibited, ERK phosphorylation could be induced by microinjected activated Akt, indicating important cross-talk between the PI3K and ERK1/2 pathways. Glucose-potentiated phosphorylation of molecules in the p38 and JNK MAPK pathways inhibited these pathways but did not affect chemotaxis. The statin, mevinolin, blocked chemotaxis through its effects on the MAPK pathway. Mevinolin-inhibited chemotaxis was restored by farnesylpyrophosphate but not by geranylgeranylpyrophosphate; in the absence of mevinolin, inhibition of farnesyltransferase reduced ERK phosphorylation and blocked chemotaxis, indicating a role for the Ras family of GTPases (MAPK pathway) under these conditions. In conclusion, glucose sensitizes VSMC to serum, inducing chemotaxis via pathways involving p110beta-PI3K, Akt, mTOR, and ERK1/2 MAPK. Cross-talk between the PI3K and MAPK pathways is necessary for VSMC chemotaxis under these conditions.

Haplotype association analysis of genes within the WNT signalling pathways in diabetic nephropathy

Background: Renal interstitial fibrosis and glomerular sclerosis are hallmarks of diabetic nephropathy (DN) and several studies have implicated members of the WNT pathways in these pathological processes. This study comprehensively examined common genetic variation within the WNT pathway for association with DN.

Methods: Genes within the WNT pathways were selected on the basis of nominal significance and consistent direction of effect in the GENIE meta-analysis dataset. Common SNPs and common haplotypes were examined within the selected WNT pathway genes in a white population with type 1 diabetes, discordant for DN (cases: n = 718; controls: n = 749). SNPs were genotyped using Sequenom or Taqman assays. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Correction for multiple testing was performed by either permutation testing or using false discovery rate.

Results: A logistic regression model including collection centre, duration of diabetes, and average HbA1c as covariates highlighted three SNPs in GSK3B (rs17810235, rs17471, rs334543), two in DAAM1 (rs1253192, rs1252906) and one in NFAT5 (rs17297207) as being significantly (P< 0.05) associated with DN, however these SNPs did not remain significant after correction for multiple testing. Logistic regression of haplotypes, with ESRD as the outcome, and pairwise interaction analyses did not yield any significant results after correction for multiple testing.

Conclusions: These results indicate that both common SNPs and common haplotypes of WNT pathway genes are not strongly associated with DN. However, this does not completely exclude these or the WNT pathways from association with DN, as unidentified rare genetic or copy number variants could still contribute towards the genetic architecture of DN.© 2013 Kavanagh et al.; licensee BioMed Central Ltd.

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.