Effects of Doxorubicin on Extracellular Matrix Regulation in Primary Cardiac Fibroblasts from Mice.

OBJECTIVE: Doxorubicin (DOX) is a highly effective chemotherapeutic used to treat many adult and pediatric cancers. However, its use is limited due to a dose-dependent cardiotoxicity, which can lead to lethal cardiomyopathy. In contrast to the extensive research efforts on toxic effects of DOX in cardiomyocytes, its effects and mechanisms on cardiac extracellular matrix (ECM) homeostasis and remodeling are poorly understood. In this study, we examined the potential effects of DOX on cardiac ECM to further our mechanistic understanding of DOX-induced cardiotoxicity. RESULTS: DOX-induced significant down-regulation of several ECM related genes in primary cardiac fibroblasts, including Adamts1, Adamts5, Col4a1, Col4a2, Col5a1, Fbln1, Lama2, Mmp11, Mmp14, Postn, and TGFß. Quantitative proteomics analysis revealed significant global changes in the fibroblast proteome following DOX treatment. A pathway analysis using iPathwayGuide of the differentially expressed proteins revealed changes in a list of biological pathways that involve cell adhesion, cytotoxicity, and inflammation. An apparent increase in Picrosirius red staining indicated that DOX-induced an increase in collagen production in cardiac primary fibroblasts after 3-day treatment. No significant changes in collagen organization nor glycoprotein production were observed.

Expression and purification of a cleavable recombinant fortilin from Escherichia coli for structure activity studies.

Complications related to atherosclerosis account for approximately 1 in 4 deaths in the United States and treatment has focused on lowering serum LDL-cholesterol levels with statins. However, approximately 50% of those diagnosed with atherosclerosis have blood cholesterol levels within normal parameters. Human fortilin is an anti-apoptotic protein and a factor in macrophage-mediated atherosclerosis and is hypothesized to protect inflammatory macrophages from apoptosis, leading to subsequent cardiac pathogenesis. Fortilin is unique because it provides a novel drug target for atherosclerosis that goes beyond lowering cholesterol and utilization of a solution nuclear magnetic resonance (NMR) spectroscopy, structure-based drug discovery approach requires milligram quantities of pure, bioactive, recombinant fortilin. Here, we designed expression constructs with different affinity tags and protease cleavage sites to find optimal conditions to obtain the quantity and purity of protein necessary for structure activity relationship studies. Plasmids encoding fortilin with maltose binding protein (MBP), 6-histidine (6His) and glutathione-S-transferase (GST), N- terminal affinity tags were expressed and purified from Escherichia coli (E. coli). Cleavage sites with tobacco etch virus (TEV) protease and human rhinovirus (HRV) 3C protease were assessed. Despite high levels of expression of soluble protein, the fusion constructs were resistant to proteinases without the inclusion of amino acids between the cleavage site and N-terminus. We surveyed constructs with increasing lengths of glycine/serine (GGS) linkers between the cleavage site and fortilin and found that inclusion of at least one GGS insert led to successful protease cleavage and pure fortilin with conserved binding to calcium as measured by NMR.

Bioactive recombinant human oncostatin M for NMR-based screening in drug discovery.

Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Recently, elevated OSM levels have been found in the serum of COVID-19 patients in intensive care units. Multiple anti-OSM therapeutics have been investigated, but to date no OSM small molecule inhibitors are clinically available. To pursue a high-throughput screening and structure-based drug discovery strategy to design a small molecule inhibitor of OSM, milligram quantities of highly pure, bioactive OSM are required. Here, we developed a reliable protocol to produce highly pure unlabeled and isotope enriched OSM from E. coli for biochemical and NMR studies. High yields (ca. 10 mg/L culture) were obtained in rich and minimal defined media cultures. Purified OSM was characterized by mass spectrometry and circular dichroism. The bioactivity was confirmed by induction of OSM/OSM receptor signaling through STAT3 phosphorylation in human breast cancer cells. Optimized buffer conditions yielded 1H, 15N HSQC NMR spectra with intense, well-dispersed peaks. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 µM. These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening.

Center of Biomedical Research Excellence in Matrix Biology: Building Research Infrastructure, Supporting Young Researchers, and Fostering Collaboration.

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

Epithelial Smad4 Deletion Up-Regulates Inflammation and Promotes Inflammation-Associated Cancer.

Background & Aims: Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor ß (TGFß) family signaling through SMAD4 in colonic epithelial cells. Methods: The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFß1 and bone morphogenetic protein 2. Results: Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis-associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis-associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFß signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions: TGFß suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082.

cFLIP critically modulates apoptotic resistance in epithelial-to-mesenchymal transition.

Epithelial cancers (carcinomas) comprise the top four causes of cancer-related deaths in the United States. While overall survival has been steadily improving, therapy-resistant disease continues to present a major therapeutic challenge. Carcinomas often exploit the normal developmental program, epithelial-to-mesenchymal transition (EMT), to gain a mesenchymal phenotype associated with increased invasiveness and resistance to apoptosis. We have previously shown that an isoxazole-based small molecule, ML327, partially reverses TGF-ß-induced EMT in an immortalized mouse mammary epithelial cell line. Herein, we demonstrate that ML327 reverses much of the EMT gene expression program in cultured carcinoma cell lines. The reversal of EMT sensitizes these cancer cells to the apoptosis-inducing ligand TRAIL. This sensitization is independent of E-cadherin expression and rather relies on the downregulation of a major anti-apoptotic protein, cFLIPS. Loss of cFLIPS is sufficient to overcome resistance to TRAIL and exogenous overexpression of cFLIPS restores resistance to TRAIL-induced apoptosis despite EMT reversal with ML327. In summary, we have utilized an isoxazole-based small molecule that partially reverses EMT in carcinoma cells to demonstrate that cFLIPS critically regulates the apoptosis resistance phenotype associated with EMT.

Deleting the TGF-ß receptor in proximal tubules impairs HGF signaling.

Transforming growth factor-ß (TGF-ß) and hepatocyte growth factor (HGF) play key roles in regulating the response to renal injury but are thought to mediate divergent effects on cell behavior. However, how TGF-ß signaling alters the response to HGF in epithelia, the key site of HGF signaling in the injured kidney, is not well studied. Contrary to our expectation, we showed that deletion of the TGF-ß type II receptor in conditionally immortalized proximal tubule (PT) cells impaired HGF-dependent signaling. This reduced signaling was due to decreased transcription of c-Met, the HGF receptor, and the TGF-ß-dependent c-Met transcription and increased response to HGF in PT cells were mediated by the Notch pathway. The interactions of TGF-ß, HGF, and Notch pathways had biologically significant effects on branching morphogenesis, cell morphology, migration, and proliferation. In conclusion, epithelial TGF-ß signaling promotes HGF signaling in a Notch-dependent pathway. These findings suggest that TGF-ß modulates PT responses not only by direct effects, but also by affecting other growth factor signaling pathways.

Renal fibrosis is not reduced by blocking transforming growth factor-ß signaling in matrix-producing interstitial cells.

Transforming growth factor-ß (TGF-ß) strongly promotes renal tubulointerstitial fibrosis, but the cellular target that mediates its profibrotic actions has not been clearly identified. While in vitro data suggest that TGF-ß-induced matrix production is mediated by renal fibroblasts, the role of these cells in TGF-ß-dependent tubulointerstitial fibrosis following renal injury is not well defined. To address this, we deleted the TGF-ß type II receptor in matrix-producing interstitial cells using two different inducible Cre models: COL1A2-Cre with a mesenchymal enhancer element and tenascin-Cre that targets medullary interstitial cells, and either the mouse unilateral ureteral obstruction or the aristolochic acid renal injury model. Renal interstitial cells lacking the TGF-ß receptor had significantly impaired collagen I production, but, unexpectedly, overall tissue fibrosis was unchanged in the conditional knockouts after renal injury. Thus, abrogating TGF-ß signaling in matrix-producing interstitial cells is not sufficient to reduce fibrosis after renal injury.

Deamidation alters interactions of beta-crystallins in hetero-oligomers.

PURPOSE: Cataracts are a major cause of blindness worldwide. A potential mechanism for loss of visual acuity may be due to light scattering from disruption of normal protein-protein interactions. During aging, the lens accumulates extensively deamidated crystallins. We have previously reported that deamidation in the betaA3-crystallin (betaA3) dimer decreased the stability of the dimer in vitro. The purpose of the present study was to investigate if deamidation altered the interaction of betaA3 with other beta-crystallin subunits. METHODS: Deamidation was mimicked by replacing glutamines, Q85 and Q180, at the predicted interacting interface between betaA3 domains with glutamic acids by site-directed mutagenesis. Human recombinant wild type betaA3 or the doubly deamidated mutant betaA3 Q85E/Q180E (DM betaA3) were mixed with either betaB1- or betaB2-crystallin (betaB1 or betaB2) subunits. After incubation at increasing temperatures, hetero-oligomers were resolved from individual subunits and their molar masses determined by size exclusion chromatography with in line multiangle laser light scattering. Structural changes of hetero-oligomers were analyzed with fluorescence spectroscopy and blue-native PAGE. RESULTS: Molar masses of the hetero-oligomer complexes indicated betaA3 formed a polydispersed hetero-tetramer with betaB1 and a mondispersed hetero-dimer with betaB2. Deamidation at the interface in the betaA3 dimer decreased formation of the hetero-oligomer with betaB1 and further decreased formation of the hetero-dimer with betaB2. During thermal-induced denaturation of the deamidated betaA3 dimer, betaB1 but not betaB2 was able to prevent precipitation of betaA3. CONCLUSIONS: Deamidation decreased formation of hetero-oligomers between beta-crystallin subunits. An excess accumulation of deamidated beta-crystallins in vivo may disrupt normal protein-protein interactions and diminish the stabilizing effects between them, thus, contributing to the accumulation of insoluble beta-crystallins during aging and cataracts.