TGF-beta triggers rapid fibrillogenesis via a Novel T beta RII-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 β (TGF-β) is an established regulator of ECM remodeling via transcriptional control of ECM proteins. Here we show that TGF-β, through increased FN trafficking in a transcription- and SMAD-independent manner, is a direct and rapid inducer of the fibrillogenesis required for TGF-β–induced cell migration. Whereas TGF-β signaling is dispensable for rapid fibrillogenesis, stable interactions between the cytoplasmic domain of the type II TGF-β receptor (TβRII) and the FN receptor (α5β1 integrin) are required. We find that, in response to TGF-β, cell surface–internalized FN is not degraded by the lysosome but instead undergoes recycling and incorporation into fibrils, a process dependent on TβRII. These findings are the first to show direct use of trafficked and recycled FN for fibrillogenesis, with a striking role for TGF-β 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

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

IB properties and lethality of ataxin-1 fusion proteins.

<p>Figure 2A. Distribution of DsRed1-E5-PML and dual transfected ataxin-1 fusions. LH panel; puncta of DsRed1-E5-PML reveal heterogeneous red/green fluorescence (arrowed) in a HeLa transfectant. Ne denotes nuclear envelope. Scale bar: 8 microns. RH panel; cytoplasmic enrichment of aged DsRed1-E5-ataxin in dual transfectants. Green and red indicates the wavelengths scanned in each micrograph, with DAPI co-stain. Scale bar: 15 microns. Figure 2B. Red/green fluorescence in single and co-transfectants of ataxin-1 fusion proteins. LH panel; GFP-ataxin-1; Middle panel; DsRed1-E5-ataxin-1; RH panel; dual transfectants. Figure 2C. Enriched red fluorescence in co-transfectants occurs in the region indicated. Figure 2D. Dye uptake shows increased lethality of DsRed1-E5-ataxin-1. Increased DAPI uptake is seen in DsRed1-E5-ataxin-1 versus GFP-ataxin-1 and Dual transfectants. Figure 2E. Cell proliferation assay shows reduced viability in DsRed1-E5-ataxin-1 transfectants. MTT based assay of DsRed1-E5 (Ds), GFP (G) and dual (x2) transfectants, comparing ataxin-1 expressing both normal [Q30] and expanded polyQ repeats [Q82]. Figure 2F. PML-NDs are sequestered by IBs of DsRed1-E5-ataxin-1. Histogram of red/green fluorescence across a DsRed1-E5-ataxin-1 transfectant, stained with N19. Spikes of red fluorescence (darker line) denote PML-NDs tethered to IBs in a single nucleus. Figure 2G. PML/DsRed1-E5-ataxin-1 sequestration. Scale bar: 8 microns. Inset; ataxin-1/PML sequestration captured by immunofluorescent staining of endogenous PML (red) with GFP-ataxin-1. Scale bar: 15 microns. Figure 2H. Mobility of DsRed1-E5-ataxin-1 IBs revealed by time-lapsed confocal microscopy. 9-section Z series were collected every 5 minutes for an hour. The four frames shown indicate fusion events over twenty minutes in a single optical section. Multiple fusion events occur within the boxed region. The arrowed IB also tracks towards this region. The disordered fluorescence at the extremities of the nucleus corresponds to cytoplasmic material.</p

Green/Red Fluorescence ratios in IBs of DsRed1-E5-ataxin-1 IBs.

<p>Equatorial line scans through IBs were used to generate g/r ratios. Mean g/r fluorescence ratios are shown for three pairs of IBs (in separate nuclei). Fluorescence intensities were measured at .05/.06 micron intervals. SD: standard deviation.</p

Expression patterns of ataxin-1 fusion proteins.

<p>Figure 1A. Distribution of ataxin-1 and ataxin-1-GFP. Upper panel; untagged ataxin-1 stained with 1C2 with a DAPI merge; lower panel, GFP-ataxin-1 with DAPI merge. Figure 1B. DsRed1-E5-ataxin-1 emits both red (r) and green (g) fluorescence. The merged red green and red/DAPI fluorescence micrographs are also shown. An orthogonal slice through a reticular IB seeded by DsRed1-E5-ataxin is shown lower right. Scale bars: 8 microns. Figure 1C. IBs of DsRed1-E5-ataxin express protein of comparable age. Synchronous peaks and troughs of red/green fluorescence emitted by IBs of DsRed1-E5-ataxin in shared nuclei. IBs are ranked according to maturity; with high g/r ratios indicating immature protein. Figure 1D. Synchronous seeding of DsRed1-E5-ataxin IBs. HeLa nuclei were ranked according to mean g/r ratio of their IB populations (24hr after transfection). 169 IBs in 16 nuclei were included in this data set.</p

Steroid therapy in Kimura′s disease

Kimura′s disease is a rare condition and generally presents as non-tender subcutaneous swellings in the head and neck region, primarily seen in young Asian males. We report a case of Kimura′s disease, who presented to us with swelling in the head and neck region, histopathological examination confirmed the diagnosis. Investigations showed eosinophilia and raised immunoglobulin E levels. The swelling which recurred after excision subsided following short course of steroid therapy

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 1H 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

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) β 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 β 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