A stimulus-contingent positive feedback loop enables IFN-ß dose-dependent activation of pro-inflammatory genes.

Type I interferons (IFN) induce powerful antiviral and innate immune responses via the transcription factor, IFN-stimulated gene factor (ISGF3). However, in some pathological contexts, type I IFNs are responsible for exacerbating inflammation. Here, we show that a high dose of IFN-ß also activates an inflammatory gene expression program in contrast to IFN-λ3, a type III IFN, which elicits only the common antiviral gene program. We show that the inflammatory gene program depends on a second, potentiated phase in ISGF3 activation. Iterating between mathematical modeling and experimental analysis, we show that the ISGF3 activation network may engage a positive feedback loop with its subunits IRF9 and STAT2. This network motif mediates stimulus-specific ISGF3 dynamics that are dependent on ligand, dose, and duration of exposure, and when engaged activates the inflammatory gene expression program. Our results reveal a previously underappreciated dynamical control of the JAK-STAT/IRF signaling network that may produce distinct biological responses and suggest that studies of type I IFN dysregulation, and in turn therapeutic remedies, may focus on feedback regulators within it.

Four guiding principles for effective trainee-led STEM community engagement through high school outreach.

To address ongoing academic achievement gap, there is a need for more school-university partnerships promoting early access to STEM education. During summer 2020, members of our institute initiated QBio-EDGE (Quantitative Biology-Empowering Diversity and Growth in Education), an outreach program for high schools in Los Angeles. In the hope of contributing to increasing diversity in academia, QBio-EDGE aims to make STEM education more accessible for students from historically excluded communities by exposing them to scientific research and diverse scientist role models. This program is led by early career researchers (ECRs), i.e., undergraduate, graduate, and postdoctoral researchers. In our first year, the outreach activities took place during virtual learning, presenting challenges and opportunities within the program development. Here, we provide a practical guide outlining our outreach efforts, key factors we considered in the program development, and hurdles we overcame. Specifically, we describe how we assembled our diverse team, how we established trusting partnerships with participating schools, and how we designed engaging student-centered, problem-based classroom modules on quantitative biology and computational methods applications to understand living systems. We also discuss the importance of increased institutional support. We hope that this may inspire researchers at all career stages to engage with local schools by participating in science outreach, specifically in quantitative and computational fields. We challenge institutions to actively strengthen these efforts.

Muscadine grape skin extract can antagonize Snail-cathepsin L-mediated invasion, migration and osteoclastogenesis in prostate and breast cancer cells.

To develop new and effective chemopreventive agents against bone metastasis, we assessed the effects of muscadine grape skin extract (MSKE), whose main bioactive component is anthocyanin, on bone turnover, using prostate and breast cancer cell models overexpressing Snail transcription factor. MSKE has been shown previously to promote apoptosis in prostate cancer cells without affecting normal prostate epithelial cells. Snail is overexpressed in prostate and breast cancer, and is associated with increased invasion, migration and bone turnover/osteoclastogenesis. Cathepsin L (CatL) is a cysteine cathepsin protease that is overexpressed in cancer and involved in bone turnover. Snail overexpression in prostate (LNCaP, ARCaP-E) and breast (MCF-7) cancer cells led to increased CatL expression/activity and phosphorylated STAT-3 (pSTAT-3), compared to Neo vector controls, while the reverse was observed in C4-2 (the aggressive subline of LNCaP) cells with Snail knockdown. Moreover, CatL expression was higher in prostate and breast tumor tissue compared to normal tissue. MSKE decreased Snail and pSTAT3 expression, and abrogated Snail-mediated CatL activity, migration and invasion. Additionally, Snail overexpression promoted osteoclastogenesis, which was significantly inhibited by the MSKE as effectively as Z-FY-CHO, a CatL-specific inhibitor, or osteoprotegerin, a receptor activator of nuclear factor kappa B ligand (RANKL) antagonist. Overall, these novel findings suggest that Snail regulation of CatL may occur via STAT-3 signaling and can be antagonized by MSKE, leading to decreased cell invasion, migration and bone turnover. Therefore, inhibition using a natural product such as MSKE could potentially be a promising bioactive compound for bone metastatic cancer.

Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte-endothelial cell signaling and JNK activation.

Inflammation and damage promote monocyte adhesion to endothelium and cardiovascular disease (CVD). Elevated inflammation and increased monocyte-endothelial cell interactions represent the initial stages of vascular remodeling associated with a multitude of CVDs. Cathepsins are proteases produced by both cell types that degrade elastin and collagen in arterial walls, and are upregulated in CVD. We hypothesized that the inflammatory cytokine tumor necrosis factor alpha (TNFα) and monocyte binding would stimulate cathepsins K and V expression and activity in endothelial cells that may be responsible for initiating local proteolysis during CVD. Confluent human aortic endothelial cells were stimulated with TNFα or THP-1 monocyte co-cultures, and multiplex cathepsin zymography was used to detect changes in levels of active cathepsins K, L, S, and V. Direct monocyte-endothelial cell co-cultures stimulated with TNFα generated maximally observed cathepsin K and V activities compared to either cell type alone (n = 3, p < 0.05) by a c-Jun N-terminal kinase (JNK)-dependent manner. Inhibition of JNK with SP6000125 blocked upregulation of cathepsin K activity by 49 % and cathepsin V by 81 % in endothelial cells. Together, these data show that inflammatory cues and monocyte-endothelial cell interactions upregulate cathepsin activity via JNK signaling axis and identify a new mechanism to target toward slowing the earliest stages of tissue remodeling in CVD.

Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues.

Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer.

High Dose IFN-ß Activates GAF to Enhance Expression of ISGF3 Target Genes in MLE12 Epithelial Cells.

Interferon ß (IFN-ß) signaling activates the transcription factor complex ISGF3 to induce gene expression programs critical for antiviral defense and host immune responses. It has also been observed that IFN-ß activates a second transcription factor complex, γ-activated factor (GAF), but the significance of this coordinated activation is unclear. We report that in murine lung epithelial cells (MLE12) high doses of IFN-ß indeed activate both ISGF3 and GAF, which bind to distinct genomic locations defined by their respective DNA sequence motifs. In contrast, low doses of IFN-ß preferentially activate ISGF3 but not GAF. Surprisingly, in MLE12 cells GAF binding does not induce nearby gene expression even when strongly bound to the promoter. Yet expression of interferon stimulated genes is enhanced when GAF and ISGF3 are both active compared to ISGF3 alone. We propose that GAF may function as a dose-sensitive amplifier of ISG expression to enhance antiviral immunity and establish pro-inflammatory states.

Multipathway kinase signatures of multipotent stromal cells are predictive for osteogenic differentiation: tissue-specific stem cells.

Bone marrow-derived multipotent stromal cells (MSCs) offer great promise for regenerating tissue. Although certain transcription factors have been identified in association with tendency toward particular MSC differentiation phenotypes, the regulatory network of key receptor-mediated signaling pathways activated by extracellular ligands that induce various differentiation responses remains poorly understood. Attempts to predict differentiation fate tendencies from individual pathways in isolation are problematic due to the complex pathway interactions inherent in signaling networks. Accordingly, we have undertaken a multivariate systems approach integrating experimental measurement of multiple kinase pathway activities and osteogenic differentiation in MSCs, together with computational analysis to elucidate quantitative combinations of kinase signals predictive of cell behavior across diverse contexts. In particular, for culture on polymeric biomaterial surfaces presenting tethered epidermal growth factor, type I collagen, neither, or both, we have found that a partial least-squares regression model yields successful prediction of phenotypic behavior on the basis of two principal components comprising the weighted sums of eight intracellular phosphoproteins: phospho-epidermal growth factor receptor, phospho-Akt, phospho-extracellular signal-related kinase 1/2, phospho-heat shock protein 27, phospho-c-Jun, phospho-glycogen synthase kinase 3alpha/beta, phospho-p38, and phospho-signal transducer and activator of transcription 3. This combination provides the strongest predictive capability for 21-day differentiated phenotype status when calculated from day-7 signal measurements; day-4 and day-14 signal measurements are also significantly predictive, indicating a broad time frame during MSC osteogenesis wherein multiple pathways and states of the kinase signaling network are quantitatively integrated to regulate gene expression, cell processes, and ultimately, cell fate.

Detection of femtomole quantities of mature cathepsin K with zymography.

Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity.

Dynamic Model of Protease State and Inhibitor Trafficking to Predict Protease Activity in Breast Cancer Cells.

INTRODUCTION: Cysteine cathepsins are implicated in breast cancer progression, produced by both transformed epithelial cells and infiltrated stromal cells in tumors, but to date, no cathepsin inhibitor has been approved for clinical use due to unexpected side effects. This study explores cellular feedback to cathepsin inhibitors that might yield non-intuitive responses, and uses computational models to determine underlying cathepsin-inhibitor dynamics. METHODS: MDA-MB-231 cells treated with E64 were tested by multiplex cathepsin zymography and immunoblotting to quantify total, active, and inactive cathepsins S and L. This data was used to parameterize mathematical models of intracellular free and inhibited cathepsins, and then applied to a dynamic model predicting cathepsin responses to other classes of cathepsin inhibitors that have also failed clinical trials. RESULTS: E64 treated cells exhibited increased amounts of active cathepsin S and reduced amount of active cathepsin L, although E64 binds tightly to both. This inhibitor response was not unique to cancer cells or any one cell type, suggesting an underlying fundamental mechanism of E64 preserving activity of cathepsin S, but not cathepsin L. Computational models were able to predict and differentiate between inhibitor-bound, active, and inactive cathepsin species and demonstrate how different classes of cathepsin inhibitors can have drastically divergent effects on active cathepsins located in different intracellular compartments. CONCLUSIONS: Together, this work has important implications for the development of mathematical model systems for protease inhibition in tissue destructive diseases, and consideration of preservation mechanisms by inhibitors that could alter perceived benefits of these treatment modalities.

Cathepsin Protease Inhibition Reduces Endometriosis Lesion Establishment.

Endometriosis is a gynecologic disease characterized by the ectopic presence of endometrial tissue on organs within the peritoneal cavity, causing debilitating abdominal pain and infertility. Current treatments alleviate moderate pain symptoms associated with the disorder but exhibit limited ability to prevent new or recurring lesion establishment and growth. Retrograde menstruation has been implicated for introducing endometrial tissue into the peritoneal cavity, but molecular mechanisms underlying attachment and invasion are not fully understood. We hypothesize that cysteine cathepsins, a group of powerful extracellular matrix proteases, facilitate endometrial tissue invasion and endometriosis lesion establishment in the peritoneal wall and inhibiting this activity would decrease endometriosis lesion implantation. To test this, we used an immunocompetent endometriosis mouse model and found that endometriotic lesions exhibited a greater than 5-fold increase in active cathepsins compared to tissue from peritoneal wall or eutopic endometrium, with cathepsins L and K specifically implicated. Human endometriosis lesions also exhibited greater cathepsin activity than adjacent peritoneum tissue, supporting the mouse results. Finally, we tested the hypothesis that inhibiting cathepsin activity could block endometriosis lesion attachment and implantation in vivo. Intraperitoneal injection of the broad cysteine cathepsin inhibitor, E-64, significantly reduced the number of attached endometriosis lesions in our murine model compared to vehicle-treated controls demonstrating that cathepsin proteases contribute to endometriosis lesion establishment, and their inhibition may provide a novel, nonhormonal therapy for endometriosis.

Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells.

Cathepsins are powerful proteases, once referred to as the lysosomal cysteine proteases, that have been implicated in breast cancer invasion and metastasis, but pharmaceutical inhibitors have suffered failures in clinical trials due to adverse side effects. Scientific advancement from lysosomotropic to cell impermeable cathepsin inhibitors have improved efficacy in treating disease, but off-target effects have still been problematic, motivating a need to better understand cellular feedback and responses to treatment with cathepsin inhibitors. To address this need, we investigated effects of E-64 and cystatin C, two broad spectrum cathepsin inhibitors, on cathepsin levels intra- and extracellularly in MDA-MB-231 breast cancer cells. Cathepsins S and L had opposing responses to both E-64 and cystatin C inhibitor treatments with paradoxically elevated amounts of active cathepsin S, but decreased amounts of active cathepsin L, as determined by multiplex cathepsin zymography. This indicated cellular feedback to selectively sustain the amounts of active cathepsin S even in the presence of inhibitors with subnanomolar inhibitory constant values. These differences were identified in cellular locations of cathepsins L and S, trafficking for secretion, co-localization with endocytosed inhibitors, and longer protein turnover time for cathepsin S compared to cathepsin L. Together, this work demonstrates that previously underappreciated cellular compensation and compartmentalization mechanisms may sustain elevated amounts of some active cathepsins while diminishing others after inhibitor treatment. This can confound predictions based solely on inhibitor kinetics, and must be better understood to effectively deploy therapies and dosing strategies that target cathepsins to prevent cancer progression.