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CD95: A Question of Life or Death

death becomes you by Esther Kirby

Image: Flickr - Esther Kirby

Programmed cell death (apoptosis) is essential to maintain homeostasis within living organisms and is controlled by a variety of intra- and extra-cellular signals. Activation of the death receptor CD95 (also known as Fas or Apo-1) by its physiological ligand, CD95 ligand (Fas ligand), leads to apoptosis in many tissues and is especially important in the immune system.

Resistance to apoptosis is also key to the survival of malignant cells but the role of CD95 in cancer progression is complex: although the down-regulation of CD95 that is frequently observed in tumour cells could contribute to their survival, complete loss of CD95 is rare in human cancers. On the other hand, many cancer cells express large quantities of CD95 and are highly sensitive to CD95-mediated apoptosis in vitro. Cancer patients often have high levels of CD95 ligand, suggesting that CD95 could perhaps promote the growth of tumours through non-apoptotic activities. Scientists from the University of Chicago and Northwestern University Feinberg School of Medicine have further investigated the role of CD95 in several human cancer cell lines and in mouse models of liver and ovarian cancer. Cancer cells – regardless of their sensitivity to CD95-mediated apotosis – were found to produce CD95 ligand and to depend on constitutive activity of CD95 for optimal growth. In the mouse models, deletion of CD95 reduced both the incidence and size of tumours. In further studies, the tumour promoting activity of CD95 was shown to be mediated by pathways involving JNK and Jun but not caspase-8.

The study, which is published in Nature, suggests that, paradoxically, reducing rather than enhancing activity of the death receptor CD95 may be an effective way to control the growth and proliferation of cancer cells. Further research is needed to understand the switch between signalling ‘die’ and ‘grow’, but eventually soluble CD95 or antibodies against CD95 ligand could find a role in the treatment of cancer.


New Approach to Cystic Fibrosis

Cystic fibrosis (CF) results from a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in impaired transport of chloride and bicarbonate ions. Patients with CF have thickened mucus, accompanied by inflammation, which affects the lungs and organs of the intestinal tract. Although the disease has received much scientific attention, current treatments only manage the symptoms and affected individuals continue to suffer from reduced life expectancy.

A new study from researchers at University of California, San Diego School of Medicine, has now identified defects in signalling mediated by peroxisome proliferator-activated receptor-γ (PPAR-γ) that contribute to disease symptoms. Examining colonic epithelial cells and whole lung tissue from CFTR-deficient mice, the team found reduced expression of genes that are normally activated by PPAR-γ. Lipidomic analysis of the colonic epithelial cells suggested that the defect resulted in part from reduced amounts of the endogenous PPAR-γ ligand, 15-keto-prostaglandin E2 (15-keto-PGE2). The researchers were able to partially restore gene expression by treating the mice with rosiglitazone, a PPAR-γ agonist used in the treatment of diabetes, reducing the severity of disease.

PPar pathway

PPAR-α and -γ Signalling; Image:Mark Somoza


Rosiglitazone had no effect on chloride secretion in the colon, but increased expression of carbonic anhydrases 2 and 4 (Car2 and Car4) resulting in increased bicarbonate secretion and reduced mucus retention.

The study, published in Nature Medicine, suggests that levels of 15-keto-PGE2 could provide a marker for patients who might benefit from treatment with a PPAR-γ agonist.


Drug Discovery with Zebrafish

Zebrafish

Image: Wikipedia - Azul

Scientists at Vanderbilt University have previously used zebrafish embryos to identify compounds that interfere with signalling pathways involved in early development – pathways that also play a role in many disease processes. One of these compounds, dorsomorphin, was shown to block bone morphogenetic protein (BMP) signalling, a pathway that is involved in bone and cartilage formation and that has also been linked to anaemia and inflammatory responses. Subsequent studies showed that dorsomorphin also blocked the vascular endothelial growth factor (VEGF) type-2 receptor and disrupted angiogenesis.

BMP inhibitor DMH1

BMP Inhibtor, DMH1

To identify more selective compounds, the team turned again to zebrafish embryos. It was quickly discovered that the two effects could be separated, with some compounds only affecting patterning and some only affecting angiogenesis. The former were shown to be potent and selective inhibitors of BMP signalling and the latter to be selective VEGF inhibitors.
VEGF Inhibitor

VEGF Inhibitor

As well as identifying a VEGF inhibitor that outperformed a compound that had entered phase III clinical trials, the team also discovered a BMP inhibitor, DMH1, which exclusively targets the BMP pathway. Using zebrafish embryos for structure-activity analyses allows selectivity and bioavailability to be assessed at the same time as efficacy and the team believe that zebrafish provide an attractive complementary platform for drug discovery. The potential of small molecule signalling inhibitors is often limited by off-target activities and zebrafish provide a very good model for assessing selectivity since compounds that hit multiple targets are toxic to the embryos.

The study is published in ACS Chemical Biology.


NO Good in Septic Shock

Septic shock, characterised by refractory hypotension and resulting end-organ dysfunction, is a major cause of death in intensive care units. Systemic inflammation leads to increased production of nitric oxide (NO), an important regulator of vascular tone, which was believed to play a key role in the pathogenesis of septic shock. In support of this hypothesis, early animal studies and small scale clinical trials appeared to show that nitric oxide synthase inhibitors were beneficial in septic shock. Disappointingly, however, outcome studies with the non-selective nitric oxide synthase inhibitor, tilarginine, found that treatment was associated with excess mortality in septic shock and provided no benefit in cardiogenic shock.

A team led by scientists at VIB and Ghent University have now shown that, rather than contributing to damage in septic shock, NO may instead be beneficial. When mice with septic shock induced by a lethal TNF challenge were treated with nitrite – which can serve as a source of NO – hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction and mortality were all attenuated. Nitrite also provided protection against toxicity induced by Gram-negative lipopolysaccharide, although higher doses were needed. Until recently, nitrite was believed to be an inert metabolite of NO but is now considered to be a central homeostatic molecule in NO biology and to serve as an important signalling molecule and regulator of gene expression in its own right. The protection afforded by nitrite treatment was largely abolished in mice lacking the soluble guanylate cyclase 1 subunit, an important intracellular NO receptor and signal transducer. Although the mechanisms underlying the protective effects of nitrite in septic shock are not yet fully understood, the study provides new information about the role of NO in septic shock and suggests new opportunities for treatment.

The study is published in the Journal of Experimental Medicine.


Insulin Growth Factor Signalling in Alzheimer’s Disease

Although family history and lifestyle choices play a role, ageing is recognised to be the largest single risk factor for Alzheimer’s disease. Progression of Alzheimer’s disease is not well understood but accumulation of toxic amyloid peptides in the brain is believed to be a significant contributory factor and much research has focussed on reducing levels of these peptides. Researchers led by a team at the Salk Institute have now asked whether slowing the ageing progress might also delay the onset of Alzheimer’s disease. The insulin/insulin growth factor (IGF) signalling (IIS) pathway regulates stress resistance, and reduction of IIS has been shown to increase lifespan in worms, flies, and mice. Although reduced IGF signalling extends the life span of mice, IGF-1 infusion has also been shown to protect mice against amyloid toxicity. To address this apparent paradox, the team crossed mice that model Alzheimer’s disease with long-lived mice that have reduced IGF signalling. The animals were found to be protected from Alzheimer’s disease-like symptoms, including behavioural impairment, neuroinflammation, and neuronal loss. Although the mice continued to produce amyloid peptides, these were found to form densely packed, ordered plaques, suggesting that hyper-aggregation of more toxic soluble amyloid oligomers may explain, at least in part, the protection conferred by reduced IGF signalling.

Although previous studies have shown that IGF-1 infusion protects mice against amyloid toxicity, the growth hormone secretagogue MK-677 (ibutamoren mesylate), a potent inducer of IGF-1 secretion, was ineffective at slowing the rate of progression of Alzheimer’s disease in human patients.

IIS reduction has been found to correlate with longevity in humans – some very long-lived people have defects in components of IIS – and the present study, which is published in the journal Cell, suggests that reduction in IGF-1 signalling may be a promising strategy for the treatment of Alzheimer’s disease.


Mapping Phosphorylation in Cancer

Phosphorylation is a key mechanism for regulation of protein activity and the phosphorylation of tyrosine residues, in particular, is important in signalling pathways. Aberrant phosphorylation has been observed in many cancers and has driven the development of kinase inhibitors that have utility in a number of cancer subtypes. Since abnormal activation of signalling pathways is a common feature that accompanies tumour initiation and progression, methods to assess signalling pathway status of cancer tissue relative to normal could provide important insights.

A group of US researchers has now conducted a detailed analysis of tyrosine phosphorylation states in lung cancer compared to normal tissue. The work documents a large set of sites that are differentially phosphorylated in the cancer tissue, immediately providing a number of drug target candidates. Taking the study a step further, the group has developed computational methodology to identify signalling pathways where phosphorylation activity is strongly correlated with the lung cancer phenotype.

The EGFR signalling pathway. Pink indicates higher phosphorylation in tumour samples, while green indicates higher phosphorylation in normal tissue samples. Full details can be found in PLoSone.

The EGFR signalling pathway. Pink indicates higher phosphorylation in tumour samples, while green indicates higher phosphorylation in normal tissue samples. Full details can be found in PLoSone.

The results provide a highly predictive set of signatures that reliably distinguish each lung cancer from normal. Since the signatures identify proteins and pathways where phosphorylation should be inhibited, the methodology highlights potential new targets for drug discovery.

The study is published in the journal PLoSone.


Norepinephrine Boosts Memory in Down’s Syndrome Model

Down’s syndrome is a chromosomal disorder caused by the presence of all (trisomy 21) or part of an extra copy of chromosome 21. The consequences of the extra genetic material are very variable and the condition is associated with a combination of physical and mental characteristics. Although there is no specific treatment for Down’s syndrome, many of the physical health problems that are associated with the condition can be successfully managed. There are, however, currently no treatments for the memory deficits which hinder learning and delay development. People with Down’s syndrome find it difficult to use spatial and contextual information to form new memories, a process that depends on the hippocampus, but are much better at capturing sensory memories that are coordinated by the amygdala. Down’s syndrome is also associated with an increased incidence of dementia, including Alzheimer’s disease, which may be linked to an extra copy of the gene encoding the amyloid producing APP in some people with the condition.

Researchers at Stanford University School of Medicine and the University of California have now suggested a possible treatment for the neurological manifestations of Down’s syndrome. The team carried out experiments in a well established mouse model of Down’s syndrome. The mice, which have three copies of a fragment of mouse chromosome 16, show abnormal responses in behavioural tests of contextual learning such as conditioned fear learning. The mice also did not build nests when placed in a strange environment, unlike wild type animals. When the team examined the mice, they found significant neurodegeneration in the locus coeruleus region of the brain – an area that also degenerates in the brains of people with Down’s syndrome. The locus coeruleus supplies the hippocampus with the neurotransmitter, norepinephrine (noradrenaline), and when the genetically engineered mice were treated with L-DOPS (L-threo-dihydroxyphenylserine), a prodrug of norepinephrine and epinephrine which can cross the blood-brain barrier, they behaved much more like normal animals in both fear conditioning tests and nest building activities. Direct examination of neurons in the hippocampus of the genetically altered mice showed that these cells responded well to norepinephrine.

Degeneration in the locus coeruleus also occurs in other dementias, including Alzheimer’s disease, and mice with three copies of the gene expressing APP had fewer neurons producing norepinephrine than those with just two copies.

The authors hope that early intervention with agents targeting the norepinephrine system could lead to improvements in cognitive function in children with Down’s syndrome. Since improvements were seen even in the presence of established neurodegeneration in the genetically engineered mice, such agents may also have a role in restoring function in older individuals with Down’s syndrome and in Alzheimer’s disease sufferers. Previous studies of drug treatments for Down’s syndrome have focused on the neurotransmitter acetylcholine, which also acts at the hippocampus. Based on the new findings, the researchers suggest that the ideal treatment approach for improving cognition in people with Down’s syndrome will likely enhance both norepinephrine and acetylcholine signalling.

The study is published in Science Translational Medicine.


Cysteine Modifications: Opposing Effects on Signalling

Hydrogen sulfide (H2S) is best known for its characteristic smell of rotten eggs and for its toxicity, which is caused mainly by inhibition of mitochondrial respiration resulting from blockade of cytochrome oxidase. More recently, H2S has been established as one of a number of gaseous signalling molecules, with roles in neuromodulation, smooth muscle relaxation, inflammation, insulin release and metabolic demand. Although biochemical pathways which generate H2S have been elucidated, exactly what it does once generated has been unclear. Writing in the journal Science Signalling, a team led by researchers at Johns Hopkins University School of Medicine have now shown that H2S converts cysteine residues to thiocysteines (ie modifies the –SH group to –SSH) in many liver proteins, including actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, the cysteine residue in GAPDH that is modified to thiocysteine (Cys150) is also a target of nitrosylation by nitric oxide (NO), another gaseous signalling molecule. Nitrosylation of the cysteine residue abolishes GAPDH activity, whereas conversion to thiocysteine augments activity, suggesting that the function of some proteins could be regulated by competitive nitrosylation or sulfhydration of the same cysteine residues. The extent of sulfhydration (10-25%) is considerably higher than the degree of protein S-nitrosylation thought to occur physiologically.


New Drug Target for Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a progressive, debilitating disease characterised by increased resistance in pulmonary arteries, placing additional workload on the right ventricle of the heart. Untreated, the disease frequently results in right ventricular failure and death. Until the 1990s the only effective treatment was heart-lung transplantation. Subsequently, drug treatments that have been used include anticoagulants, calcium channel blockers, prostacylin and endothelin receptor antagonists. Whilst these drugs have demonstrated efficacy in PAH patients, delaying the need for lung transplantation, long term survival rates have not been significantly impacted.

The annual incidence of PAH is around 1-2 per million individuals, with a further 8 per million contributed by PAH associated with scleroderma. Despite the low incidence, there are approximately 100,000 PAH patients in Europe and the US.

DAPT

DAPT

Scientists at University of California–San Diego (UCSD) have now established that human pulmonary hypertension is characterised by overexpression of Notch3 in small pulmonary artery smooth muscle cells and that the severity of disease in humans and rodents correlates with the amount of Notch3 protein in the lung. In the study, published online in Nature Medicine on 25th October, the team showed that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation. Additionally, mice with pulmonary hypertension were successfully treated with a DAPT, a γ-secretase inhibitor that blocks activation of Notch3.

Notch receptor signalling is implicated in control of smooth muscle cell proliferation and maintaining smooth muscle cells in an undifferentiated state. The discovery that the Notch3 signalling pathway is crucial for the development of PAH provides a novel target for therapeutic intervention.


Still Signalling from Below the Surface

ducksCompounds acting at G-protein coupled receptors (GPCRs) form the largest class of drug molecules but, although the biochemical steps involved in GPCR signalling are known in some detail, the location of these events in space and time in living cells is still poorly understood. Scientists at Novartis reported earlier this year that S1P1 receptors bound to the ligand FTY720 continue to signal after internalisation, albeit by a different pathway from that used when the receptors are at the cell surface. Writing in the journal PLOS Biology, scientists in Italy and Germany have now reported that the thyroid-stimulating hormone (TSH) receptor continues signalling via cAMP even after internalisation. Ligand signalling through GPCRs was traditionally thought to take place exclusively at the cell membrane and involve coupling of receptors to G-proteins, activation of G-proteins and subsequent signalling via second messengers such as cAMP. Receptor internalisation was believed to contribute to signal termination by reducing the number of receptors present at the cell surface – although endocytosis was later shown to be important for receptor resensitisation. Once inside the cell, GPCRs were believed to stop signalling to second messengers, but the new study shows that the TSH receptor continues to stimulate cAMP production after internalisation.

The team isolated native, 3-D thyroid follicles from transgenic mice with ubiquitous expression of an inert fluorescent sensor for cAMP, and showed that, although the TSH receptors were rapidly internalised after ligand binding, they continued to stimulate cAMP production from inside the cells. The cAMP signalling by the internalised receptors appeared, however, to be somewhat different from that occurring at the plasma membrane since it was sustained, rather than rapidly reversible, and led to a different pattern of downstream signals. Thyroid follicles were chosen for the experiment since they are a particularly good model in which to study the spatiotemporal dynamics of cAMP signalling – they maintain the supra-cellular organisation, size, and polarisation of thyroid cells in vivo, and are a rare example of cells that are under the strict control of a GPCR and cAMP for virtually all their functions.

The study shows that GPCR signalling in a physiological setting may be more complex than previously supposed, with the sub-cellular localisation of the GPCR playing an important role in the duration and spatial pattern of downstream signals. It will be very interesting to discover whether a majority of endogenous ligands – or drug molecules – continue to signal via internalised receptors and, if so, whether intracellular signalling is inherently different from that which occurs at the cell surface.