Growth Signal Transduction Research Articles

Global Gene Expression Analysis of Reactive Stroma in Prostate Cancer

Marked reactive stroma formation, designated as grade 3 reactive stroma, is associated with poor outcome in clinically localized prostate cancer. To understand the biological processes and signaling mechanisms underlying the formation of such reactive stroma, we carried out microarray gene expression analysis of laser-captured reactive stroma and matched normal stroma. Seventeen cases of reactive stroma grade 3 cancer were used to laser-capture tumor and normal stroma. Expression analysis was carried out using Agilent 44K arrays. Up-regulation of selected genes was confirmed by quantitative reverse transcription-PCR. Expression data was analyzed to identify significantly up- and down-regulated genes, and gene ontology analysis was used to define pathways altered in reactive stroma. A total of 544 unique genes were significantly higher in the reactive stroma and 606 unique genes were lower. Gene ontology analysis revealed significant alterations in a number of novel processes in prostate cancer reactive stroma, including neurogenesis, axonogenesis, and the DNA damage/repair pathways, as well as evidence of increases in stem cells in prostate cancer reactive stroma. Formation of reactive stroma in prostate cancer is a dynamic process characterized by significant alterations in growth factor and signal transduction pathways and formation of new structures, including nerves and axons.

Effective screening method of agonistic diabodies based on autocrine growth

Agonistic diabodies that mimic the function of natural ligands are expected to increase the value of therapeutic antibodies. We have developed a method that detects agonistic diabodies based on their ability to transduce growth signals through receptors, thereby permitting cytokine-independent growth of BaF/3-derived cytokine-dependent cells. Retrovirus-mediated expression of the diabody in cytokine-dependent cells was followed by selection of clones for growth in the absence of cytokine. A diabody library derived from splenocytes of human Mpl immunized mice was constructed. Infection of cells with viral particles led to the isolation of over 500 autonomously growing clones whose cultured supernatants showed agonistic activities against Mpl. Genome-integrated diabodies were cloned; representative clone AB317 showed agonistic activities as potent as a natural ligand and cross-reactive against mouse Mpl.

A novel WD-40 repeat protein WDR26 suppresses H 2O 2-induced cell death in neural cells

WD-40 repeat proteins play important roles in a variety of cellular functions, such as cell growth, proliferation, apoptosis and intracellular signal transduction. We previously identified a novel member of this family, WDR26. To examine the biological function of WDR26, we used hWDR26 plasmids and antisense phosphorothioate oligodeoxynucleotides (asODNs) against WDR26 to examine its role in response to oxidative stress in human SH-SY5Y neuroblastoma cells. Our results showed that H 2O 2 at 0.5 mM substantially induced cell death and significantly up-regulated the WDR26 expression, and WDR26 over-expression in turn strongly suppressed H 2O 2-induced cell death. Moreover, asODNs markedly inhibited the de novo biosynthesis of WDR26, which contributed to enhanced cell death induced by H 2O 2. Finally, we found that WDR26 over-expression also down-regulated the transcriptional activity of AP-1 during H 2O 2-induced SH-SY5Y cell death. Taken together, these results indicated that WDR26 was up-regulated by oxidative stress and played a key role in H 2O 2-induced SH-SY5Y cell death, which may be mediated by the down-regulation of AP-1 transcriptional activity.

Phenotypic variation meets systems biology

A report of the 18th Annual Growth Factor and Signal Transduction Symposium, Ames, USA, 11-14 June 2009.

BMP-7 protects mesangial cells from injury by polymeric IgA

Bone morphogenetic protein-7 (BMP-7) is a potential therapeutic agent for acute and chronic renal diseases. Here we found that addition of polymeric IgA, isolated from patients with IgA nephropathy, increased the synthesis of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and fibronectin in cultured human mesangial cells, effects blunted by BMP-7. When mesangial cells were cultured with both polymeric IgA and BMP-7 there was an increase in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The activation of NF kappaB and TNF-alpha synthesis induced by polymeric IgA or TNF-alpha were downregulated by BMP-7 or rosiglitazone. BMP-7 inhibited TNF-alpha release from polymeric IgA-stimulated mesangial cells by activation of PPAR-gamma but suppressed TGF-beta release by mechanisms independent of PPAR-gamma. The expression of inhibitory Smad6 and 7 was increased whereas the expression of active Smad2 and 3 was reduced in these mesangial cells by BMP-7. Our study shows that BMP-7 ameliorates IgA nephropathy-derived polymeric IgA-induced TNF-alpha and TGF-beta synthesis in human mesangial cells through multiple mechanisms involving inhibitory Smads and PPAR-gamma.

Diabetic polyneuropathy: an update

To examine current issues in the diagnosis and treatment of diabetic polyneuropathy. Diabetic neuropathies are common and rising in prevalence with the global burden of type 2 diabetes. Polyneuropathy is also emerging as a complication of impaired glucose tolerance, without frank diabetes. Ideas about the pathogenesis of diabetic polyneuropathy have evolved, and some new pathogenic mechanisms are being considered. These include roles for direct insulin signaling on neurons and axons that transduce growth signals, actions of the cleaved C-peptide product of the insulin prohormone, abnormal signaling by advanced glycation endproducts on neuronal and glial receptors and activation of poly (ADP-ribose) polymerase in microvessels and neurons. Manipulation of these pathways may offer new therapeutic approaches. Ideas about neuronal targets in the treatment of neuropathic pain have also advanced emphasizing abnormal sodium and calcium channel signaling. Consideration of diabetic polyneuropathy as a unique neurodegenerative condition has generated interest in new pathways involved in its development. A new round of clinical trials that address its pathogenesis may be welcome, as recent attempts have been largely disappointing. In the interim, several forms of therapy for neuropathic pain are available.

Intracellular Delivery of Glutathione S-transferase-fused Proteins into Mammalian Cells by Polyethylenimine-Glutathione Conjugates

The glutathione S-transferase (GST)-fused protein expression system has been extensively used to generate a large quantity of proteins and has served for functional analysis in vitro. In this study, we developed a novel approach for the efficient intracellular delivery of GST-fused proteins into living cells to expand their usefulness up to in vivo use. Since protein cationization techniques are powerful strategies for efficient intracellular uptake by adsorptive-mediated endocytosis, GST-fused proteins were cationized by forming a complex with a polycationic polyethylenimine (PEI)-glutathione conjugate. On screening of protein transduction, optimized PEI-glutathione conjugate for protein transduction was characterized by a partly oligomerized mixture of PEI with average molecular masses of 600 (PEI600) modified with multiple glutathiones, which could have sufficient avidity for GST. Furthermore, enhanced endosomal escape of transduced GST-fused proteins was observed when they were delivered with a glutathione-conjugated PEI600 derivative possessing a hydroxybutenyl moiety. These results were confirmed by both intracellular confocal imaging of GST-fused green fluorescent protein and activation of an endogenous growth signal transduction pathway by a GST-fused constitutively active mutant of a kinase protein. These PEI-glutathione conjugates seem to be convenient molecular tools for protein transduction of widely used GST-fused proteins.

Disrupting actions of bisphenol A and malachite green on growth hormone receptor gene expression and signal transduction in seabream

Environmental estrogen could mimic natural estrogens thereby disrupting the endocrine systems of human and animals. The actions of such endocrine disruptors have been studied mainly on reproduction and development. However, estrogen could also affect the somatotropic axis via multiple targets such as growth hormone (GH). In the present study, two endocrine disruptors were chosen to investigate their effects on the expression level and signal transduction of growth hormone receptor (GHR) in fish. Using real-time PCR, it was found that exposure to both the estrogenic (bisphenol A) and anti-estrogenic (malachite green) compounds could attenuate the expression levels of GHR1 and GHR2 in black seabream (Acanthopagrus schlegeli) hepatocytes. The expression level of IGF-I, the downstream effector of GHR activation in the liver, was decreased by bisphenol A but not by malachite green. Luciferase reporter assay of the beta-casein promoter was used to monitor GHR signaling in transfected cells. In the fish liver cell line Hepa-T1, both GHR1 and GHR2 signaling were attenuated by bisphenol A and malachite green. This attenuation could only occur in the presence of estrogen receptor, indicating that these agents probably produce their actions via the estrogen receptor. Results of the present study demonstrated that estrogenic or anti-estrogenic compounds could down-regulate the somatotropic axis in fish by affecting both the gene expression and signaling of GHR. In view of the increasing prevalence of these compounds in the environment, the impact on fish growth and development both in the wild and in aquaculture would be considerable.

Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-beta and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93A resolution. The polypeptide chain adopts the typical alphabetaalpha PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.

Visualization of growth signal transduction cascades in living cells with genetically encoded probes based on Förster resonance energy transfer

Fluorescence probes based on the principle of Förster resonance energy transfer (FRET) have shed new light on our understanding of signal transduction cascades. Among them, unimolecular FRET probes containing fluorescence proteins are rapidly increasing in number because these genetically encoded probes can be easily loaded into living cells and allow simple acquisition of FRET images. We have developed probes for small GTPases, tyrosine kinases, serine-threonine kinases and phosphoinositides. Images obtained with these probes have revealed that membrane protrusions such as nascent lamellipodia or neurites provide an active signalling platform in the growth factor-stimulated cells.

Dynamic expression pattern of the growth hormone receptor during early development of the Chilean flounder

The entire cDNA sequence of the growth hormone receptor (GHR) of the Chilean flounder ( Paralichthys. adspersus) was cloned by RT-PCR and RNA ligase rapid amplification of 5′ and 3′ends. The deduced amino acid sequence contains 641 residues and codes for the GHR1 form. The receptor includes all the structural domains and motifs responsible for its interaction with the growth hormone and growth signal transduction. Sequence comparison revealed 95 and 88% identity with other flat fish such as the Japanese flounder and Atlantic halibut respectively, but decreased to 41% with the GHR of other teleosts such as salmon. In addition we performed a phylogenetic analysis of this receptor in teleosts. RT-PCR experiments were performed to study the expression of GHR1 mRNA in different tissues of juvenile fish, detecting the transcripts in all tissues investigated with higher expressions in the liver, brain and gonads. Additionally, using whole-mount in situ hybridization in larvae stages, we observed an on and off GHR1 mRNA expression pattern. This novel finding evidences that during early development of a teleost, GHR1 is transiently expressed in somites, a source of muscle, bone and spinal chord precursors cells, suggesting a relevant role of GH in fish development. GHR1 was also temporally detected in the notochord, intestines, brain and retinal layers, before its ubiquitous establishment.

Prostate expression of endothelins and their receptors in rat growth

Endothelins (ETs) have been shown to have mitogenic effects on prostate cells in vitro. To study a relationship between ETs and prostate growth, we investigated expression of ET-1 and ET-3 and their receptors ETA and ETB in the dorsolateral (DLP) and ventral (VP) prostate lobes of rats aged 3 weeks, 3 months, and 22 months. The weight ratio of each lobe to the body was not different at 3 weeks but increased more in the DLP than in the VP at 3 months; the ratio did not change at 22 months. The density of the major receptor ETA, measured by radio-labelled ET-1 binding to prostate membrane particulates, was highest at 3 weeks in both prostate lobes and decreased thereafter. mRNA expression levels of ETA, ETB, ET-1, and ET-3 in the DLP and ETA in the VP, as assessed by relative multiplex reverse transcription-polymerase chain reaction, were highest at 3 weeks, and no significant change was seen in mRNA expression of ET-converting enzyme-1. The highest expression of ETA and the ligands at 3 weeks, followed by dramatic growth at 3 months in the DLP, suggests a role of ETA-mediated signal transduction in prostate growth of rats and possibly of humans.

RNA interference of LRRK2–microarray expression analysis of a Parkinson’s disease key player

The protein leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease (PD). Mutations in the LRRK2 gene account for up to 10% of all autosomal dominant forms of familiar and for approximately 1-3% of sporadic PD patients. Although the LRRK2 protein has many functional domains like a leucine-rich repeat domain, a Roc-GTPase domain, a kinase domain of the tyrosine kinase-like subfamily and multiple protein interaction domains (armadillo, ankyrin, WD40), the exact biological role of LRRK2 in the human brain is elusive. To gain more insight into the biological function of this protein, we monitored the changes in the expression profiles of SH-SY5Y cells, a dopaminergic neuroblastoma cell line, induced by a depletion of LRRK2 levels by RNA interference (RNAi) with Affymetrix U133 Plus 2.0 microarrays. A total of 187 genes were differentially regulated by at least a 1.5-fold change with 94 transcripts being upregulated and 93 transcripts being downregulated compared to scrambled control siRNA transfected cells. Key players of the interaction networks were independently verified by qRT-PCR. The differentially expressed gene products are involved in axonal guidance, nervous system development, cell cycle, cell growth, cell differentiation, cell communication, MAPKKK cascade, and Ras protein signal transduction. Defined gene expression networks will now serve to look more closely for candidates affected by LRRK2 reduction and how they might be altered in other forms of familial or sporadic PD.

Mutational analysis of the FXNPXY motif within LDL receptor-related protein 1 (LRP1) reveals the functional importance of the tyrosine residues in cell growth regulation and signal transduction

LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.

The DRE/DREF transcriptional regulatory system: a master key for cell proliferation

The coordinate expression of many cell proliferation-related genes is required for the cellular shift from the resting state into the proliferating state. One regulatory factor involved in this process, the transcription regulatory factor named DREF (DNA replication-related element-binding factor) was discovered in Drosophila and later found to have orthologues in other species including human. Drosophila DREF is a homo-dimer of a polypeptide of 709 amino acid residues, and shares about 22% identity in its amino acid sequence with the human homolog of 694 amino acid residues. The Drosophila DREF homo-dimer binds specifically to the DRE sequence (5'-TATCGATA) in the promoters of many DNA replication/ cell proliferation-related genes to activate their transcription, and the N-terminal region of DREF carries a domain for specific DRE-binding and homo-dimer formation. Ectopic expression of DREF in eye imaginal discs induces abnormal DNA synthesis, apoptosis and failure to differentiate. Conversely, expression of the dominant negative N-terminal region in larval salivary glands reduces endo-replication. Furthermore, RNA interference-mediated knockdown of DREF in vivo demonstrated its requirement for normal progression through the cell cycle and consequently for growth of imaginal discs and the endoreplicating organs. Both Drosophila and human DREF's interact genetically and physically with regulatory factors related to chromatin structures, suggesting that DREF activates the expression of proliferation-related genes through modification of the 3-D conformation of DNA. A search of the Drosophila genome database identified about 150 genes carrying DRE sequences in their promoter regions, many of which are related to reactions required for cell proliferation such as DNA replication, transcriptional regulation, cell cycle regulation, growth signal transduction and protein metabolism. Thus, DREF appears to be a master key-like factor for cell proliferation. Several differentiation-related transcription factors containing homeodomains down-regulate the function or expression of DREF by distinct mechanisms, suggesting a differentiation-coupled repression of cell proliferation via the DRE/DREF system.

E11-03: Controversy in small cell lung cancer: targeted therapy

E11-03: Controversy in small cell lung cancer: targeted therapy

The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors

The activating protein-1 (AP-1) transcription factor transduces growth signals through signal transduction pathways to the nucleus, leading to the expression of genes involved in growth and malignant transformation in many cell types. We have previously shown that overexpression of a dominant negative form of the cJun proto-oncogene, a cJun dominant negative mutant (Tam67), blocks AP-1 transcriptional activity, induces a G(1) cell cycle block and inhibits breast cancer cell growth in vitro and in vivo. We found that AP-1 blockade by Tam67 in MCF-7 breast cancer cells downregulates cyclin D1 transcriptional activity by at least two mechanisms: by suppressing transcription at the known AP-1 binding site (-934/-928) and by suppressing growth factor-induced expression through suppressing E2F activation at the E2F-responsive site (-726/-719). AP-1 blockade also led to reduced expression of E2F1 and E2F2, but not E2F4, at the mRNA and protein levels. Chromatin immunoprecipitation and supershift assays demonstrated that AP-1 blockade caused decreased binding of E2F1 protein to the E2F site in the cyclin D1 promoter. We also found that Tam67 suppressed the expression of the E2F1 dimerizing partner, DP1 and E2F-upregulated cell cycle genes (cyclins E, A, B and D3) and enhanced the expression of E2F-downregulated cell cycle genes (cyclins G(2) and I). Reduced expression of other E2F-regulated genes was also seen with AP-1 blockade and E2F suppression. Thus, the AP-1 factor regulates the expression of cyclin D and E2F (the latter in turn regulates E2F-downstream genes), leading to cell cycle progression and breast cancer cell proliferation.

Jasmonates: an update on biosynthesis, signal transduction and action in plant stress response, growth and development.

Jasmonates are ubiquitously occurring lipid-derived compounds with signal functions in plant responses to abiotic and biotic stresses, as well as in plant growth and development. Jasmonic acid and its various metabolites are members of the oxylipin family. Many of them alter gene expression positively or negatively in a regulatory network with synergistic and antagonistic effects in relation to other plant hormones such as salicylate, auxin, ethylene and abscisic acid. This review summarizes biosynthesis and signal transduction of jasmonates with emphasis on new findings in relation to enzymes, their crystal structure, new compounds detected in the oxylipin and jasmonate families, and newly found functions. Crystal structure of enzymes in jasmonate biosynthesis, increasing number of jasmonate metabolites and newly identified components of the jasmonate signal-transduction pathway, including specifically acting transcription factors, have led to new insights into jasmonate action, but its receptor(s) is/are still missing, in contrast to all other plant hormones.

Cytokinin: perception, signal transduction, and role in plant growth and development

Cytokinins are essential hormones for the proper growth and development of plants. They exert their actions through the phosphorylation of two-component signaling factors. The two-component elements in cytokinin signaling display not only overlapping, but also specific functions throughout a life cycle. These elements regulate the development of shoots, roots, and inflorescence meristems inArabidopsis; shoot meristems in rice; and nodule formation in the lotus. They are also involved in interactions between plants and pathogens. In this review, we examine the mechanism for signaling events initiated by cytokinins inArabidopsis.

Cracking the mild, difficult and fiendish codes within and downstream of the EGFR to link diagnostics and therapeutics

Over the last 45 years, I have been working on growth factors, their receptors and signal transduction mechanisms. This period has seen a tremendous growth in knowledge and technology, and all of this, together with a focus interest in oncology, has steered me along a path designed to understand growth factor signalling so that we can see how drugs that target signalling pathways might be able to control cancer. The knowledge that we already have is likely to lead to cures for many common cancers within the next 25 years.