Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling

Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 +/- 10.3%, and type III, 271.7 +/- 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.     

Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts

Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin''s GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.Podosomes are specialized transient actin-containing adhesion structures (11, 14, 37, 60) that are found in highly motile cells, such as osteoclasts, macrophages, dendritic cells, transformed metastatic cells, and v-src-transformed cells (37, 43), where they are thought to play important roles in cellular migration and invasion (34). In resorbing osteoclasts on bone, podosomes are concentrated within the sealing zone, a beltlike actin-rich structure that is important for adhesion and which delineates the resorptive region of the cell known as the ruffled border. Unlike focal adhesions, which are relatively stable structures (11, 60), the assembly and disassembly of podosomes occurs within minutes (t_1/2 = 2 to 4 min) and involves the recruitment and activation of integrins, signaling proteins and scaffolding proteins (11, 14, 35, 47, 60). However, the mechanisms of action of key signaling proteins involved in podosome assembly and disassembly are only partially understood.The focal adhesion kinase Pyk2 has been linked to the proliferation, migration, and activity of a variety of mesenchymal, epithelial, and hematopoietic cell types. Several groups, including our own, have reported the importance of Pyk2 in podosome belt organization, cell spreading, and bone-resorbing activity in osteoclasts (18, 26, 31, 40, 65, 66). Pyk2 is recruited to activated β₂ and β₃ integrins (9, 20) at adhesion sites and is autophosphorylated at Y402 (17, 47, 50) via an intermolecular trans-acting mechanism (46). Although Pyk2 is partially activated by integrin-induced Ca²⁺ signaling (20, 50), the induction of Pyk2''s full catalytic activity requires the binding of Src via its SH2 domain to autophosphorylated Pyk2 Y402 and the subsequent phosphorylation of Pyk2 at functionally distinct sites, including Y579, Y580, and Y881 (17, 31, 46). The binding of Src to phosphorylated Pyk2, which leads to the formation of a multiprotein signaling complex at adhesion sites (17, 40, 50), is critical for Pyk2 activity, as demonstrated by the fact that Pyk2 phosphorylation and activity are significantly reduced in osteoclasts derived from Src^−/− mice (17, 40). Src^−/− osteoclasts also exhibit decreased motility (50) and decreased bone-resorbing activity (40, 54, 59), and we recently demonstrated that Src promotes both podosome formation and disassembly, as well as actin flux into existing podosomes and the organization of podosomes into a peripheral belt in osteoclasts (15).We have also demonstrated that the GTP-hydrolyzing protein dynamin-2, which is ubiquitously expressed and well known for its role in endocytosis (53), regulates actin remodeling in the podosomes of osteoclasts and Rous sarcoma virus-transformed baby hamster kidney cells (43). In addition, a dynamin-2 mutant that binds GTP with reduced affinity (dynK44A) (12) decreased the flux of actin into podosomes (43) and disrupted podosome belt formation in osteoclasts, thereby affecting osteoclast migration and bone-resorbing activity (8). The dynamin proteins, of which there are three homologous isoforms (3), contain several protein domains: a GTP-hydrolyzing domain (GTPase), a plextrin homology domain that mediates binding to phosphoinositides, a GTPase effector domain (GED), and a C-terminal proline-rich domain (PRD) (38, 45, 55) through which dynamin binds a number of functionally diverse SH3-containing molecules, such as Src, cortactin, Grb2, and N-Wasp (1, 7, 27, 39, 58). We previously reported that dynamin-2 partially colocalizes and associates with the E3-ubiquitin ligase Cbl within the podosome belt/sealing zone of osteoclasts, as well as in SYF cells, which lack the Src family kinases Src, Yes, and Fyn, and in HEK 293 cells that stably express the vitronectin receptor (293VnR) (8). Protein complexes containing dynamin-2 and Cbl, which are both substrates of Src (1, 2, 23, 50, 56), were disrupted in the presence of activated Src and stabilized in the absence of Src (8), demonstrating a key role of Src in regulating the formation of signaling complexes in osteoclasts downstream of integrins.In the present study, we sought to determine whether dynamin, which regulates podosome actin dynamics and bone resorption in osteoclasts, also associates with Pyk2 and/or regulates Pyk2''s activities in osteoclasts. We report here that dynamin associates with Pyk2 and promotes the dephosphorylation of Pyk2 Y402 and that catalytically active Src promotes both dynamin''s association with Pyk2 and the dynamin-induced dephosphorylation of Pyk2 Y402, resulting, in turn, in the decreased binding of Src to Pyk2. Thus, we propose that dynamin regulates podosome dynamics and osteoclast bone-resorbing activity by promoting the disassembly of the Pyk2-Src-Cbl complex that is formed in osteoclasts downstream of β₃ integrin activation.     

Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage

In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.     

Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage

Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET¹ with Renilla luciferase (RLuc) and coelenterazine h (CLZ, λ_em ∼ 475 nm) and BRET² with the substrate coelenterazine 400a (CLZ400A substrate, λ_em = 395 nm) as the respective donors. For BRET¹ the acceptor is yellow fluorescent protein (YFP) (λ_em ∼ 535 nm), a mutant of green fluorescent protein (GFP), and for BRET² it is GFP² (λ_em ∼ 515 nm). It is not clear from previous studies which of these systems has superior signal-to-background characteristics. Here we directly compared BRET¹ and BRET² by placing two different protease-specific cleavage sequences between the donor and acceptor domains. The intact proteins simulate protein-protein association. Proteolytic cleavage of the peptide linker simulates protein dissociation and can be detected as a change in the BRET ratios. Complete cleavage of its target sequence by thrombin changed the BRET² ratio by a factor of 28.9 ± 0.2 (relative standard deviation [RSD], n = 3) and changed the BRET¹ ratio by a factor of 3.05 ± 0.07. Complete cleavage of a caspase-3 target sequence resulted in the BRET ratio changes by factors of 15.45 ± 0.08 for BRET² and 2.00 ± 0.04 for BRET¹. The BRET² assay for thrombin was 2.9 times more sensitive compared with the BRET¹ version. Calculated detection limits (blank signal + 3σ_b, where σ_b = standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET¹ and 15 pM (0.0005 U) thrombin with BRET². The results presented here suggest that BRET² is a more suitable system than BRET¹ for studying protein-protein interactions and as a potential sensor for monitoring protease activity.     

Structure-Based Rational Design of a Phosphotriesterase

In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z-CVP and E-CVP hydrolysis, respectively, eliminating stereospecificity.Synthetic organophosphate pesticides (OPs) can cause acute neurotoxicity in insects and humans as a result of their inhibition of acetylcholinesterase at the nerve synapse (15). The >90% identical bacterial phosphotriesterases (PTEs) from Pseudomonas diminuta (oph; PTE_Pd) (5) and Agrobacterium radiobacter (opdA; PTE_Ar) (9) efficiently catalyze the hydrolysis of a broad range of OPs, effectively detoxifying them. This has led to the commercialization of PTE_Ar as a free-enzyme bioremediant (14) and its use in treating OP poisoning in animal studies (1). However, not all OPs are efficiently turned over by the PTEs. For instance, despite having a reasonably reactive leaving group (Fig. ​(Fig.1),1), the turnover of chlorfenvinphos (CVP) by PTE_Ar was not detected in a previous study (9).Open in a separate windowFIG. 1.Structures of the leaving groups of all the substrates discussed in this work. (a) 3,5,6-Trichloro-2-pyridinol for methyl chlorpyrifos oxon; (b) 4-nitrophenol for methyl paraoxon and methyl parathion; (c) 2,2-dichloroethenol for dichlorvos; (d) Z/E-2-chloro-1-(2,4-dichlorophenyl)ethanol for E/Z-CVP; (e) 4-methoxyphenol for EPO.     

The phosphotriesterase gene opdA in Agrobacterium radiobacter P230 is transposable

We report a transposase gene (tnpA) upstream of the opdA phosphotriesterase gene of Agrobacterium radiobacter P230, as well as inverted repeats indicative of insertion sequences, flanking the two genes. Both the tnpA gene and the inverted repeats resemble the Tn610 transposon from Mycobacterium fortuitum. Two additional putative open reading frames separate opdA and tnpA with inferred translation products with similarity to two proteins encoded on the Geobacillus stearothermophilus IS5376 transposon. To test the proposition that these genes were contained on a transposon, an artificial composite transposon was constructed. This artificial transposon was then delivered into Escherichia coli DH10beta cells. Transposition was demonstrated by the presence of opdA on the E. coli chromosome and confirmation of insertion by inverse polymerase chain reaction. The data presented suggest a possible role of transposition in the distribution of the opd/opdA genes across a wide range of soil bacteria.     

Invertebrate responses to the management of genetically modified herbicide-tolerant and conventional spring crops. I. Soil-surface-active invertebrates

The effects of herbicide management of genetically modified herbicide-tolerant (GMHT) beet, maize and spring oilseed rape on the abundance and diversity of soil-surface-active invertebrates were assessed. Most effects did not differ between years, environmental zones or initial seedbanks or between sugar and fodder beet. This suggests that the results may be treated as generally applicable to agricultural situations throughout the UK for these crops. The direction of the effects was evenly balanced between increases and decreases in counts in the GMHT compared with the conventional treatment. Most effects involving a greater capture in the GMHT treatments occurred in maize, whereas most effects involving a smaller capture were in beet and spring oilseed rape. Differences between GMHT and conventional crop herbicide management had a significant effect on the capture of most surface-active invertebrate species and higher taxa tested in at least one crop, and these differences reflected the phenology and ecology of the invertebrates. Counts of carabids that feed on weed seeds were smaller in GMHT beet and spring oilseed rape but larger in GMHT maize. In contrast, collembolan detritivore counts were significantly larger under GMHT crop management.     

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.     

Molecular complexes that contain both c-Cbl and c-Src associate with Golgi membranes

Cbl is an adaptor protein that is phosphorylated and recruited to several receptor and non-receptor tyrosine kinases upon their activation. After binding to the activated receptor, Cbl plays a key role as a kinase inhibitor and as an E3 ubiquitin ligase, thereby contributing to receptor down-regulation and internalization. In addition, Cbl translocates to intracellular vesicular compartments following receptor activation. We report here that Cbl also associates with Golgi membranes. Confocal immunofluorescence staining of Cbl in a variety of unstimulated cells, including CHO cells, revealed a prominent perinuclear colocalization of Cbl and a Golgi marker. Both the prominent Cbl staining and the Golgi marker were dispersed by brefeldin A. Subcellular fractionation of CHO cells demonstrated that about 10% of Cbl is stably associated with membranes, and that Golgi-enriched membrane fractions produced by isopycnic density centrifugation and free-flow electrophoresis are also enriched in Cbl, relative to other membrane fractions. The membrane-bound Cbl was hyperphosphorylated and it co-immunoprecipitated with endogenous Src. By immunofluorescence, some Src colocalized with Cbl and Golgi markers, and Src, like Cbl, was present in the Golgi-enriched fraction prepared by sequential density centrifugation and free-flow electrophoresis. Transfection of an activated form of Src, but not wild-type Src, increased the amount of Src that co-immunoprecipitated with Cbl, and increased the intensity of Cbl staining on the Golgi. This result, together with the increased tyrosine phosphorylation of the membrane-associated Cbl, suggests that Golgi-associated Cbl could be part of a molecular complex that contains activated Src. The localization and interaction of Src and Cbl at the Golgi and the regulation of the interaction of Cbl with Golgi membrane suggest that this complex may contribute to the regulation of Golgi function.