SOCS domain targets ECM assembly in lung fibroblasts and experimental lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease defined by a progressive decline in lung function due to scarring and accumulation of extracellular matrix (ECM) proteins. The SOCS (Suppressor Of Cytokine Signaling) domain is a 40 amino acid conserved domain known to form a functional ubiquitin ligase complex targeting the Von Hippel Lindau (VHL) protein for proteasomal degradation. Here we show that the SOCS conserved domain operates as a molecular tool, to disrupt collagen and fibronectin fibrils in the ECM associated with fibrotic lung myofibroblasts. Our results demonstrate that fibroblasts differentiated using TGFß, followed by transduction with the SOCS domain, exhibit significantly reduced levels of the contractile myofibroblast-marker, α-SMA. Furthermore, in support of its role to retard differentiation, we find that lung fibroblasts expressing the SOCS domain present with significantly reduced levels of α-SMA and fibrillar fibronectin after differentiation with TGFß. We show that adenoviral delivery of the SOCS domain in the fibrotic phase of experimental lung fibrosis in mice, significantly reduces collagen accumulation in disease lungs. These data underscore a novel function for the SOCS domain and its potential in ameliorating pathologic matrix deposition in lung fibroblasts and experimental lung fibrosis.

Hypoxia-inducible factor-1 alpha expression is induced by IL-2 via the PI3K/mTOR pathway in hypoxic NK cells and supports effector functions in NKL cells and ex vivo expanded NK cells.

Natural killer (NK) cells are cytotoxic innate lymphocytes that are specialized to kill tumor cells. NK cells are responsive to the primary cytokine IL-2 in the tumor microenvironment (TME), to activate its effector functions against tumors. Despite their inherent ability to kill tumor cells, dysfunctional NK cells observed within advanced solid tumors are associated with poor patient survival. Hypoxia in the TME is a major contributor to immune evasion in solid tumors that could contribute to impaired NK cell function. HIF-1α is a nodal regulator of hypoxia in driving the adaptive cellular responses to changes in oxygen concentrations. Whether HIF-1α is expressed in hypoxic NK cells in the context of IL-2 and whether its expression regulates NK cell effector function are unclear. Here, we report that freshly isolated NK cells from human peripheral blood in hypoxia could not stabilize HIF-1α protein coincident with impaired anti-tumor cytotoxicity. However, ex vivo expansion of these cells restored HIF-1α levels in hypoxia to promote antitumor cytotoxic functions. Similarly, the human NK cell line NKL expressed HIF-1α upon IL-2 stimulation in hypoxia and exhibited improved anti-tumor cytotoxicity and IFN-γ secretion. We found that ex vivo expanded human NK cells and NKL cells required the concerted activation of PI3K/mTOR pathway initiated by IL-2 signaling in combination with hypoxia for HIF-1α stabilization. These findings highlight that HIF-1α stabilization in hypoxia maximizes NK cell effector function and raises the prospect of NK cells as ideal therapeutic candidates for solid tumors.

Fibronectin assembly regulates lumen formation in breast acini.

Fibronectin (FN) is an extracellular matrix (ECM) glycoprotein that self-assembles into FN fibrils, forming a FN matrix contributing to the stiffness of the ECM. Stromal FN stiffness in cancer has been shown to impact epithelial functions such as migration, cancer metastasis, and epithelial-to-mesenchymal transition. The role of the FN matrix of epithelial cells in driving such processes remains less well understood and is the focus of this study. Hypoxia, defined by low oxygen tension (<5%) is one of the hallmarks of tumor microenvironments impacting fibril reorganization in stromal and epithelial cells. Here, using the MCF10 breast epithelial progression series of cell lines encompassing normal, preinvasive, and invasive states, we show that FN fibril formation decreases during hypoxia, coinciding with a decrease in migratory potential of these cells. Conversely, we find that FN fibril disruption during three-dimensional acinar growth of normal breast cells resulted in acinar luminal filling. Our data also demonstrates that the luminal filling upon fibril disruption in untransformed MCF10A cells results in a loss of apicobasal polarity, characteristic of pre-invasive and invasive breast cell lines MCF10AT and MCF10 DCIS.com. Overall this is the first study that relates fibril-mediated changes in epithelial cells as critical players in lumen clearing of breast acini and maintenance of the untransformed growth characteristic.

HIFα independent mechanisms in renal carcinoma cells modulate divergent outcomes in fibronectin assembly mediated by hypoxia and CoCl2.

Fibronectin (FN) is a core matrix protein that assembles to form a dynamic cellular scaffold, frequently perturbed during oncogenic transformation. Tumor hypoxia, characterized by low oxygen concentrations in the microenvironment of most solid tumors has been shown to accelerate FN assembly in fibroblasts and cancer-associated fibroblasts, cell types that produce abundant amounts of FN protein. Nevertheless, FN matrix regulation in epithelial cancer cells during hypoxia remains less well defined. In this study we investigate the assembly of the FN matrix during hypoxia in renal cancer epithelial cells, the cells of origin of renal cell carcinoma (RCC). We show that hypoxia (1% O2) specifically increases matrix disassembly and increases migratory propensity in renal cancer cells. However, HIFα stabilization using hypoxia mimetics, does not recapitulate the effect of hypoxia on FN matrix reorganization or cell migration. Using a combination of knockdown and inhibitor-based approaches, our work characterizes the signaling events that mediate these two disparate changes on the matrix and explores its functional significance on chemotactic cell migration. Our study systematically reexamines the role of hypoxia mimetics as experimental substitutes for hypoxia and provides new findings on HIFα stabilization and the FN matrix in the context of renal cancer.

Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells.

Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5ß1. Integrin α5ß1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell migration and metastasis. While immunostaining and microscopy methods help visualize FN incorporation into fibrils, with each fibril being at least 3 µm in length, the first study that developed a method to biochemically fractionate FN to quantify fibril incorporated FN was published by Jean Schwarzbauer's group in 1996. Our protocol was adapted from the original publication, and has been tested on multiple cell types including as shown here in MCF10A mammary epithelial and Caki-1 renal cancer epithelial cells. Using two detergent extractions, cellular FN is separated into detergent insoluble or fibril incorporated FN and soluble FN or unincorporated fractions. To determine whether fibrillogenesis utilizes a recycled pool of FN, we have used a Biotin labeled FN (FN-Biotin) recycling assay, that has been modified from a previous study. Using a combination of the recycling assay and deoxycholate fractionation methods, one can quantitatively demonstrate the extent of fibrillogenesis in cells under different experimental conditions and determine the source of FN for fibrillogenesis.

TGF-ß triggers rapid fibrillogenesis via a novel TßRII-dependent fibronectin-trafficking mechanism.

Fibronectin (FN) is a critical regulator of extracellular matrix (ECM) remodeling through its availability and stepwise polymerization for fibrillogenesis. Availability of FN is regulated by its synthesis and turnover, and fibrillogenesis is a multistep, integrin-dependent process essential for cell migration, proliferation, and tissue function. Transforming growth factor ß (TGF-ß) is an established regulator of ECM remodeling via transcriptional control of ECM proteins. Here we show that TGF-ß, through increased FN trafficking in a transcription- and SMAD-independent manner, is a direct and rapid inducer of the fibrillogenesis required for TGF-ß-induced cell migration. Whereas TGF-ß signaling is dispensable for rapid fibrillogenesis, stable interactions between the cytoplasmic domain of the type II TGF-ß receptor (TßRII) and the FN receptor (α5ß1 integrin) are required. We find that, in response to TGF-ß, cell surface-internalized FN is not degraded by the lysosome but instead undergoes recycling and incorporation into fibrils, a process dependent on TßRII. These findings are the first to show direct use of trafficked and recycled FN for fibrillogenesis, with a striking role for TGF-ß in this process. Given the significant physiological consequences associated with FN availability and polymerization, our findings provide new insights into the regulation of fibrillogenesis for cellular homeostasis.

Altering the Proteoglycan State of Transforming Growth Factor ß Type III Receptor (TßRIII)/Betaglycan Modulates Canonical Wnt/ß-Catenin Signaling.

Hyperactive Wnt/ß-catenin signaling is linked to cancer progression and developmental abnormalities, making identification of mechanisms controlling Wnt/ß-catenin signaling vital. Transforming growth factor ß type III receptor (TßRIII/betaglycan) is a transmembrane proteoglycan co-receptor that exists with or without heparan and/or chondroitin sulfate glycosaminoglycan (GAG) modifications in cells and has established roles in development and cancer. Our studies here demonstrate that TßRIII, independent of its TGFß co-receptor function, regulates canonical Wnt3a signaling by controlling Wnt3a availability through its sulfated GAG chains. Our findings revealed, for the first time, opposing functions for the different GAG modifications on TßRIII suggesting that Wnt interactions with the TßRIII heparan sulfate chains result in inhibition of Wnt signaling, likely via Wnt sequestration, whereas the chondroitin sulfate GAG chains on TßRIII promote Wnt3a signaling. These studies identify a novel, dual role for TßRIII/betaglycan and define a key requirement for the balance between chondroitin sulfate and heparan sulfate chains in dictating ligand responses with implications for both development and cancer.

Epigenetic Regulation of GDF2 Suppresses Anoikis in Ovarian and Breast Epithelia.

Anoikis, a cell death mechanism triggered upon cell-matrix detachment, is regarded as a physiological suppressor of metastasis that can be regulated by a diverse array of signals. The protein encoded by GDF2 is BMP9 and is a member of the bone morphogenetic protein family and the transforming growth factor (TGF) ß superfamily with emerging yet controversial roles in carcinogenesis. In an attempt to identify the function of growth and differentiation factor 2 (GDF2) in epithelial systems, we examined the signaling machinery that is involved and cell fate decisions in response to GDF2 in ovarian and breast epithelia. We find that GDF2 can robustly activate the SMAD1/5 signaling axis by increasing complex formation between the type I receptor serine threonine kinases activin receptor-like kinase (ALK) 3 and ALK6 and the type II receptor serine threonine kinase BMPRII. This activation is independent of cross talk with the SMAD2-transforming growth factor ß pathway. By activating SMAD1/5, epithelial cells regulate anchorage-independent growth by increasing anoikis sensitivity that is dependent on GDF2's ability to sustain the activation of SMAD1/5 via ALK3 and ALK6. Consistent with a role for GDF2 in promoting anoikis susceptibility, the analysis of cell lines and patient data suggests epigenetic silencing of GDF2 in cancer cell lines and increased promoter methylation in patients. These findings collectively indicate an antimetastatic role for GDF2 in ovarian and breast cancer. The work also implicates loss of GDF2 via promoter methylation-mediated downregulation in promotion of carcinogenesis with significant relevance for the use of epigenetic drugs currently in clinical trials.

SUMO Ubc9 enzyme as a viral target.

Viruses alter specific host cell targets to counteract possible defense mechanisms aimed at eliminating infectivity and viral propagation. The SUMO conjugating enzyme Ubc9 functions as a hub for protein sumoylation, whilst also providing an interactive surface for sumoylated proteins through noncovalent interactions. The targeting of Ubc9 by viruses and viral proteins is thus highly beneficial for the disruption of both protein modification and protein-protein interaction mechanisms with which proteins increase their functional repertoire in cells. This review explores some of the clever mechanisms adopted by viruses to deregulate Ubc9, influence effector pathways and positively impact viral persistence consequently.

BC-box protein domain-related mechanism for VHL protein degradation.

The tumor suppressor VHL (von Hippel-Lindau) protein is a substrate receptor for Ubiquitin Cullin Ring Ligase complexes (CRLs), containing a BC-box domain that associates to the adaptor Elongin B/C. VHL targets hypoxia-inducible factor 1α to proteasome-dependent degradation. Gam1 is an adenoviral protein, which also possesses a BC-box domain that interacts with the host Elongin B/C, thereby acting as a viral substrate receptor. Gam1 associates with both Cullin2 and Cullin5 to form CRL complexes targeting the host protein SUMO enzyme SAE1 for proteasomal degradation. We show that Gam1 protein expression induces VHL protein degradation leading to hypoxia-inducible factor 1α stabilization and induction of its downstream targets. We also characterize the CRL-dependent mechanism that drives VHL protein degradation via proteasome. Interestingly, expression of Suppressor of Cytokine Signaling (SOCS) domain-containing viral proteins and cellular BC-box proteins leads to VHL protein degradation, in a SOCS domain-containing manner. Our work underscores the exquisite ability of viral domains to uncover new regulatory mechanisms by hijacking key cellular proteins.

Single-step affinity purification for fungal proteomics.

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

Copy number suppressors of the Aspergillus nidulans nimA1 mitotic kinase display distinctive and highly dynamic cell cycle-regulated locations.

The Aspergillus nidulans NIMA kinase is essential for mitosis and is the founding member of the conserved NIMA-related kinase (Nek) family of protein kinases. To gain insight into NIMA function, a copy number suppression screen has been completed that defines three proteins termed MCNA, MCNB, and MCNC (multi-copy-number suppressor of nimA1 A, B, and C). All display a distinctive and dynamic cell cycle-specific distribution. MCNC has weak similarity to Saccharomyces cerevisiae Def1 within a shared CUE-like domain. MCNC, like Def1, is a cytoplasmic protein with slow mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its deletion causes polarization defects and a small colony phenotype. MCNC enters nuclei during mitosis. In contrast, MCNB is a nuclear protein displaying increased nuclear levels as cells progress through interphase but is lost from nuclei at mitosis. MCNB is highly related to the Schizosaccharomyces pombe forkhead transcription factor Sep1 and is likely a transcriptional activator of nimA. Most surprisingly, MCNA, a protein restricted to the aspergilli and pathogenic systemic dimorphic fungi (the Eurotiomycetes), defines a nuclear body located near nucleoli at the nuclear periphery of G(2) nuclei. During progression through mitosis, the MCNA body is excluded from nuclei. Cytoplasmic MCNA bodies then diminish during early stages of interphase, and single MCNA bodies are formed within nuclei as interphase progresses. Three sites of MCNA phosphorylation were mapped and mutated to implicate proline-directed phosphorylation in the equal segregation of MCNA during the cell cycle. The data indicate all three MCN proteins likely have cell cycle functions.

Ataxin-1 fusion partners alter polyQ lethality and aggregation.

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.

Stress responses of PML nuclear domains are ablated by ataxin-1 and other nucleoprotein inclusions.

The polyglutamine diseases are characterized by expansion of triplet CAG repeats that encode polyglutamine tracts in otherwise unrelated proteins. One plausible explanation for the neurodegeneration of these disorders proposes that inclusions of such proteins sequester other significant nuclear proteins in inactive form. The present study shows that PML protein is sequestered by inclusions of the pathogenic mutant form of the polyglutamine protein ataxin-1 and that this sequestration removes from the nucleus the free 0.2-1 microm diameter PML nuclear domains (PML-NDs), together with at least one of their many cargo proteins (Sp100). The present study demonstrates that this sequestration can be effected equally by another nuclear protein, RED, which lacks a polyglutamine tract, but expresses a polar zipper repeat. The sequestered PML-NDs no longer respond to stress signals (heat shock or ionizing radiation) to which they are normally sensitive. In both cases, there is independent evidence that the cells initiate other responses to their injury (nuclear translocation of heat shock protein or generation of gamma-H2AX-rich nuclear foci, respectively). The data thus provide strong evidence that multiple species of nuclear inclusion functionally sequester PML-NDs. This mechanism is likely to distort cellular responses to injury of many different types.

A novel antioxidant and antiapoptotic role of omeprazole to block gastric ulcer through scavenging of hydroxyl radical.

The mechanism of the antiulcer effect of omeprazole was studied placing emphasis on its role to block oxidative damage and apoptosis during ulceration. Dose-response studies on gastroprotection in stress and indomethacin-induced ulcer and inhibition of pylorus ligation-induced acid secretion indicate that omeprazole significantly blocks gastric lesions at lower dose (2.5 mg/kg) without inhibiting acid secretion, suggesting an independent mechanism for its antiulcer effect. Time course studies on gastroprotection and acid reduction also indicate that omeprazole almost completely blocks lesions at 1 h when acid inhibition is partial. The severity of lesions correlates well with the increased level of endogenous hydroxyl radical (*OH), which when scavenged by dimethyl sulfoxide causes around 90% reduction of the lesions, indicating that *OH plays a major role in gastric damage. Omeprazole blocks stress-induced increased generation of *OH and associated lipid peroxidation and protein oxidation, indicating that its antioxidant role plays a major part in preventing oxidative damage. Omeprazole also prevents stress-induced DNA fragmentation, suggesting its antiapoptotic role to block cell death during ulceration. The oxidative damage of DNA by *OH generated in vitro is also protected by omeprazole or its analogue, lansoprazole. Lansoprazole when incubated in a *OH-generating system scavenges *OH to produce four oxidation products of which the major one in mass spectroscopy shows a molecular ion peak at m/z 385, which is 16 mass units higher than that of lansoprazole (m/z 369). The product shows no additional aromatic proton signal for aromatic hydroxylation in (1)H NMR. The product absorbing at 278 nm shows no alkaline shift for phenols, thereby excluding the formation of hydroxylansoprazole. The product is assigned to lansoprazole sulfone formed by the addition of one oxygen atom at the sulfur center following attack by the *OH. Thus, omeprazole plays a significant role in gastroprotection by acting as a potent antioxidant and antiapoptotic molecule.