When cells are exposed to death ligands such as TRAIL, a fraction undergoes apoptosis and a fraction survives; if surviving cells are re-exposed to TRAIL, fractional killing is once again observed. Therapeutic antibodies directed against TRAIL receptors also cause fractional killing, even at saturating concentrations, limiting their effectiveness. Fractional killing arises from cell-to-cell fluctuations in protein levels (extrinsic noise), but how this results in a clean bifurcation between life and death remains unclear. In this paper, we identify a threshold in the rate and timing of initiator caspase activation that distinguishes cells that live from those that die; by mapping this threshold, we can predict fractional killing of cells exposed to natural and synthetic agonists alone or in combination with sensitizing drugs such as bortezomib. A phenomenological model of the threshold also quantifies the contributions of two resistance genes (c-FLIP and Bcl-2), providing new insight into the control of cell fate by opposing pro-death and pro-survival proteins and suggesting new criteria for evaluating the efficacy of therapeutic TRAIL receptor agonists.

Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

Identifying factors responsible for variation in drug response is essential for the effective use of targeted therapeutics. We profiled signaling pathway activity in a collection of breast cancer cell lines before and after stimulation with physiologically relevant ligands, which revealed the variability in network activity among cells of known genotype and molecular subtype. Despite the receptor-based classification of breast cancer subtypes, we found that the abundance and activity of signaling proteins in unstimulated cells (basal profile), as well as the activity of proteins in stimulated cells (signaling profile), varied within each subtype. Using a partial least squares regression approach, we constructed models that significantly predicted sensitivity to 23 targeted therapeutics. This analysis identified key proteins that could serve as biomarkers of drug sensitivity. For example, one model showed that the response to the growth factor receptor ligand heregulin effectively predicted the sensitivity of cells to drugs targeting the cell survival pathway mediated by PI3K (phosphoinositide 3-kinase) and Akt; whereas the abundance of Akt or the mutational status of the enzymes in the pathway did not. Thus, basal and signaling protein profiles may yield new biomarkers and enable the identification of appropriate therapies in cancers characterized by similar functional dysregulation of signaling networks.

BackgroundThe Fibroblast Growth Factor (FGF) pathway is driving various aspects of cellular responses in both normal and malignant cells. One interesting characteristic of this pathway is the biphasic nature of the cellular response to some FGF ligands like FGF2. Specifically, it has been shown that phenotypic behaviors controlled by FGF signaling, like migration and growth, reach maximal levels in response to intermediate concentrations, while high levels of FGF2 elicit weak responses. The mechanisms leading to the observed biphasic response remains unexplained.ResultsA combination of experiments and computational modeling was used to understand the mechanism behind the observed biphasic signaling responses. FGF signaling involves a tertiary surface interaction that we captured with a computational model based on Ordinary Differential Equations (ODEs). It accounts for FGF2 binding to FGF receptors (FGFRs) and heparan sulfate glycosaminoglycans (HSGAGs), followed by receptor-phosphorylation, activation of the FRS2 adapter protein and the Ras-Raf signaling cascade. Quantitative protein assays were used to measure the dynamics of phosphorylated ERK (pERK) in response to a wide range of FGF2 ligand concentrations on a fine-grained time scale for the squamous cell lung cancer cell line H1703. We developed a novel approach combining Particle Swarm Optimization (PSO) and feature-based constraints in the objective function to calibrate the computational model to the experimental data. The model is validated using a series of extracellular and intracellular perturbation experiments. We demonstrate that in silico model predictions are in accordance with the observed in vitro results.ConclusionsUsing a combined approach of computational modeling and experiments we found that competition between binding of the ligand FGF2 to HSGAG and FGF receptor leads to the biphasic response. At low to intermediate concentrations of FGF2 there are sufficient free FGF receptors available for the FGF2-HSGAG complex to enable the formation of the trimeric signaling unit. At high ligand concentrations the ligand binding sites of the receptor become saturated and the trimeric signaling unit cannot be formed. This insight into the pathway is an important consideration for the pharmacological inhibition of this pathway.

Chondrocytes regulate the composition of cartilage extracellular matrix in response to mechanical signals, but the intracellular pathways involved in mechanotransduction are still being defined. Mitogen-activated protein kinase (MAPK) pathways are activated by static and dynamic compression of cartilage, which simultaneously induce intratissue fluid flow, pressure gradients, cell, and matrix deformation. First, to determine whether cell and matrix deformation alone could induce MAPK activation, we applied dynamic shear to bovine cartilage explants. Using Western blotting, we measured ERK1/2 and p38 activation at multiple time points over 24 h. Distinct activation time courses were observed for different MAPKs: a sustained 50% increase for ERK1/2 and a delayed increase in p38 of 180%. We then investigated the role of MAPK activation in mechano-induced chondrocyte gene expression. Cartilage explants were preincubated with inhibitors of ERK1/2 and p38 activation before application of 1-24 h of three distinct mechanical stimuli relevant to in vivo loading (50% static compression, 3% dynamic compression at 0.1 Hz, or 3% dynamic shear at 0.1 Hz). mRNA levels of selected genes involved in matrix homeostasis were measured using realtime PCR and analyzed by k-means clustering to characterize the time-and load-dependent effects of the inhibitors. Most genes examined required ERK1/2 and p38 activation to be regulated by these loading regimens, including matrix proteins aggrecan and type II collagen, matrix metalloproteinases MMP13, and ADAMTS5, and transcription factors downstream of the MAPK pathway, c-Fos, and c-Jun. Thus, we demonstrated that the MAPK pathway is a central conduit for transducing mechanical forces into biological responses in cartilage.In vitro models of chondrocyte mechanobiology relevant to physiological loading of cartilage in vivo have shown that biosynthesis and gene transcription of the major matrix proteins in cartilage are regulated by distinct tissue deformations (reviewed in Ref. 1). Application of static compression to intact cartilage explants is inhibitory to proteoglycan and type II collagen production and transcription (2-7), whereas dynamic loading regimens, including dynamic compression and dynamic shear deformation, generally enhance matrix protein biosynthesis and transcription (4, 8 -13). Differences in intracellular signaling between normal and osteoarthritic chondrocytes following mechanical loading (14 -16) have led to speculation that altered mechanotransduction may be a cause or result of the development of osteoarthritis following injury. Therefore, understanding the pathway(s) by which chondrocytes transduce mechanical forces into biological responses is of great importance to the treatment of osteoarthritis and to cartilage tissue engineering. The mitogen-activated protein kinase (MAPK) 5 pathway has been implicated in chondrocyte mechanotransduction responses. In cartilage explants, static compression can induce the phosphorylation of extracellular signal-regulated kinases ...

Objective. To compare the response of chondrocytes and cartilage matrix to injurious mechanical compression and treatment with interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣), by characterizing proteins lost to the medium from cartilage explant culture.Methods. Cartilage explants from young bovine stifle joints were treated with 10 ng/ml of IL-1␤ or 100 ng/ml of TNF␣ or were subjected to uniaxial, radiallyunconfined injurious compression (50% strain; 100%/ second strain rate) and were then cultured for 5 days. Pooled media were subjected to gel-based separation (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and analysis by liquid chromatography tandem mass spectrometry, and the data were analyzed by Spectrum Mill proteomics software, focusing on protein identification, expression levels, and matrix protein proteolysis.Results. More than 250 proteins were detected, including extracellular matrix (ECM) structural proteins, pericellular matrix proteins important in cell-cell interactions, and novel cartilage proteins CD109, platelet-derived growth factor receptor-like, angiopoietin-like 7, and adipocyte enhancer binding protein 1. IL-1␤ and TNF␣ caused increased release of chitinase 3-like protein 1 (CHI3L1), CHI3L2, complement factor B, matrix metalloproteinase 3, ECM-1, haptoglobin, serum amyloid A3, and clusterin. Injurious compression caused the release of intracellular proteins, including Grp58, Grp78, ␣4-actinin, pyruvate kinase, and vimentin. Injurious compression also caused increased release and evidence of proteolysis of type VI collagen subunits, cartilage oligomeric matrix protein, and fibronectin.Conclusion. Overload compression injury caused a loss of cartilage integrity, including matrix damage and cell membrane disruption, which likely occurred through strain-induced mechanical disruption of cells and matrix. IL-1␤ and TNF␣ caused the release of proteins associated with an innate immune and stress response by the chondrocytes, which may play a role in host defense against pathogens or may protect cells against stress-induced damage.Osteoarthritis (OA) is characterized by cartilage degeneration, which results from an imbalance between matrix synthesis and matrix degradation. Development of OA secondary to traumatic joint injury occurs in ϳ15-75% of patients over followup periods of 14-22 years, equivalent to an average relative risk or odds ratio of between 3 and 20 of developing OA postinjury (for review, see ref. 1). Moreover, corrective surgery has little or no impact on the risk of developing OA following traumatic joint injury (1). In vitro models of joint injury have helped to understand the contribution of

Objective. To evaluate the effects of injurious compression on the biosynthesis of lubricin at different depths within articular cartilage and to examine alterations in structure and function of the articular surface following mechanical injury.Methods. Bovine cartilage explants were subdivided into level 1, with intact articular surface, and level 2, containing middle and deep zone cartilage. Following mechanical injury, lubricin messenger RNA (mRNA) levels were monitored by quantitative reverse transcriptase-polymerase chain reaction, and soluble or cartilage-associated lubricin protein was analyzed by Western blotting and immunohistochemistry. Cartilage morphology was assessed by histologic staining, and tissue functionality was assessed by friction testing.Results. Two days after injury, lubricin mRNA expression was up-regulated ϳ3-fold for level 1 explants and was down-regulated for level 2 explants. Lubricin expression in level 1 cartilage returned to control levels after 6 days in culture. Similarly, lubricin protein synthesis and secretion increased in response to injury for level 1 explants and decreased for level 2 cartilage. Histologic staining revealed changes in the articular surface of level 1 explants following injury, with respect to glycosaminoglycan and collagen content. Injured level 1 explants displayed an increased coefficient of friction relative to controls. Conclusion. Our findings indicate that increased lubricin biosynthesis appears to be an early transient response of surface-layer cartilage to injurious compression. However, distinct morphologic changes occur with injury that appear to compromise the frictional properties of the tissue.Osteoarthritis (OA) is characterized by the degeneration of articular cartilage, leading to matrix fibrillation, fissuring, and the development of lesions. In the final stages of the disease, erosion of cartilage leads to painful bone-on-bone contact. The etiology of OA is complex and involves multiple biochemical, biomechanical, and genetic factors in addition to aging (1-3). Cartilage injury in young individuals is a prominent predisposing factor leading to increased risk of the subsequent development of OA (4,5) and, as such, represents a discrete pathologic event. Damage to the meniscus or ligaments sustained during traumatic joint injury causes instability, subjecting articular cartilage to abnormal biomechanical forces and resulting in the release of mediators of inflammation (6). Several animal models of OA are thus based on the observation that joint instability, i.e., via anterior cruciate ligament transaction or perturbation of the meniscus (7), results in the rapid onset of articular cartilage degeneration with an OA-like phenotype. The initial events following joint injury are thought to be crucial, since surgical interventions to restore joint stability do not seem to reduce the risk of developing posttraumatic OA (8).

BackgroundSoluble growth factors present in the microenvironment play a major role in tumor development, invasion, metastasis, and responsiveness to targeted therapies. While the biochemistry of growth factor-dependent signal transduction has been studied extensively in individual cell types, relatively little systematic data are available across genetically diverse cell lines.ResultsWe describe a quantitative and comparative dataset focused on immediate-early signaling that regulates the AKT (AKT1/2/3) and ERK (MAPK1/3) pathways in a canonical panel of well-characterized breast cancer lines. We also provide interactive web-based tools to facilitate follow-on analysis of the data. Our findings show that breast cancers are diverse with respect to ligand sensitivity and signaling biochemistry. Surprisingly, triple negative breast cancers (TNBCs; which express low levels of ErbB2, progesterone and estrogen receptors) are the most broadly responsive to growth factors and HER2amp cancers (which overexpress ErbB2) the least. The ratio of ERK to AKT activation varies with ligand and subtype, with a systematic bias in favor of ERK in hormone receptor positive (HR+) cells. The factors that correlate with growth factor responsiveness depend on whether fold-change or absolute activity is considered the key biological variable, and they differ between ERK and AKT pathways.ConclusionsResponses to growth factors are highly diverse across breast cancer cell lines, even within the same subtype. A simple four-part heuristic suggests that diversity arises from variation in receptor abundance, an ERK/AKT bias that depends on ligand identity, a set of factors common to all receptors that varies in abundance or activity with cell line, and an “indirect negative regulation” by ErbB2. This analysis sets the stage for the development of a mechanistic and predictive model of growth factor signaling in diverse cancer lines. Interactive tools for looking up these results and downloading raw data are available at http://lincs.hms.harvard.edu/niepel-bmcbiol-2014/.