Oxidative inhibition of the vascular Na+-K+ pump via NADPH oxidase-dependent β1-subunit glutathionylation: Implications for angiotensin II-induced vascular dysfunction

Glutathionylation of the Na⁺-K⁺ pump’s β₁-subunit is a key molecular mechanism of physiological and pathophysiological pump inhibition in cardiac myocytes. Its contribution to Na⁺-K⁺ pump regulation in other tissues is unknown, and cannot be assumed given the dependence on specific β-subunit isoform expression and receptor-coupled pathways. As Na⁺-K⁺ pump activity is an important determinant of vascular tone through effects on [Ca²⁺]_i, we have examined the role of oxidative regulation of the Na⁺-K⁺ pump in mediating angiotensin II (Ang II)-induced increases in vascular reactivity. β₁-subunit glutathione adducts were present at baseline and increased by exposure to Ang II in rabbit aortic rings, primary rabbit aortic vascular smooth muscle cells (VSMCs), and human arterial segments. In VSMCs, Ang II-induced glutathionylation was associated with marked reduction in Na⁺-K⁺ATPase activity, an effect that was abolished by the NADPH oxidase inhibitory peptide, tat-gp91ds. In aortic segments, Ang II-induced glutathionylation was associated with decreased K⁺-induced vasorelaxation, a validated index of pump activity. Ang II-induced oxidative inhibition of Na⁺-K⁺ ATPase and decrease in K⁺-induced relaxation were reversed by preincubation of VSMCs and rings with recombinant FXYD3 protein that is known to facilitate deglutathionylation of β₁-subunit. Knock-out of FXYD1 dramatically decreased K⁺-induced relaxation in a mouse model. Attenuation of Ang II signaling in vivo by captopril (8 mg/kg/day for 7 days) decreased superoxide-sensitive DHE levels in the media of rabbit aorta, decreased β₁-subunit glutathionylation, and enhanced K⁺-induced vasorelaxation. Ang II inhibits the Na⁺-K⁺ pump in VSMCs via NADPH oxidase-dependent glutathionylation of the pump’s β₁-subunit, and this newly identified signaling pathway may contribute to altered vascular tone. FXYD proteins reduce oxidative inhibition of the Na⁺-K⁺ pump and may have an important protective role in the vasculature under conditions of oxidative stress.     

PrPC regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP^SC) has been studied in depth, the physiological role of PrP^C remains elusive and controversial. PrP^C is a cell‐surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP^C in animals and in cellular models. In this article, we present PrP^C‐dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP^C over‐expression enhances cell proliferation and cell cycle re‐entrance after serum stimulation, while PrP^C silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP^C in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP^C in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP^C over‐expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT‐Cdc42‐N‐WASP‐dependent pathway.

     

Characterization of Tiki,a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn²⁺/Co²⁺ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.     

Wnt and BMP signaling pathways co-operatively induce the differentiation of multiple myeloma mesenchymal stem cells into osteoblasts by upregulating EMX2

Osteoblast differentiation, defined as the process whereby a relatively unspecialized cell acquires the specialized features of an osteoblast, is directly linked to multiple myeloma (MM) bone disease. Wnt and bone morphogenetic protein (BMP) are proved to be implicated in the pathological or defective osteoblast differentiation process. This study aims to test the involvement of Wnt, bone morphogenetic proteins (BMP) pathways, and empty spiracles homeobox 2 (EMX2) in osteoblast differentiation and MM development. Initially, differentially expressed genes in bone marrow mesenchymal stem cells (MSCs) from MM patients and healthy donors were identified using microarray-based gene expression profiling. The functional role of Wnt and BMP in MM was determined. Next, we focused on the co-operative effects of Wnt and BMP on calcium deposition, alkaline phosphatase (ALP) activity, the number of mineralized nodules, and osteocalcin (OCN) content in MSCs. The expression patterns of Wnt and BMP pathway–related genes, EMX2 and osteoblast differentiation-related factors were determined to assess their effects on osteoblast differentiation. Furthermore, regulation of Wnt and BMP in ectopic osteogenesis was also investigated in vivo. An integrated genomic screen suggested that Wnt and BMP regularly co-operate to regulate EMX2 and affect MM. EMX2 was downregulated in MSCs. The activated Wnt and BMP resulted in more calcium salt deposits, mineralized nodules, and a noted increased in ALP activity and OCN content by upregulating EMX2, leading to induced differentiation of MSCs into osteoblasts. Collectively, this study demonstrated that Wnt and BMP pathways could co-operatively stimulate differentiation of MSCs into osteoblasts and inhibit MM progression, representing potential targets for MM treatment.     

Evidence for calpains in cancer metastasis

Metastatic dissemination represents the final stage of tumor progression as well as the principal cause of cancer-associated deaths. Calpains are a conserved family of calcium-dependent cysteine proteinases with ubiquitous or tissue-specific expression. Accumulating evidence indicates a central role for calpains in tumor migration and invasion via participating in several key processes, including focal adhesion dynamics, cytoskeletal remodeling, epithelial-to-mesenchymal transition, and apoptosis. Activated after the increased intracellular calcium concentration () induced by membrane channels and extracellular or intracellular stimuli, calpains induce the limited cleavage or functional modulation of various substrates that serve as metastatic mediators. This review covers established literature to summarize the mechanisms and underlying signaling pathways of calpains in cancer metastasis, making calpains attractive targets for aggressive tumor therapies.     

New therapy with XLQ® to suppress chronic prostatitis through its anti-inflammatory and antioxidative activities

Chronic prostatitis is a common urological disease. The etiology of this disease and effective therapy for its treatment are yet to be elucidated. We investigated the functions of XLQ^® in chronic nonbacterial prostatitis using a complete Freund's adjuvant-induced rat model. Prostates and blood samples were collected for further evaluation after oral gavage with XLQ ^® or a vehicle for 4 weeks. The results showed that XLQ ^® significantly decreased the prostate index, ameliorated the histopathologic changes, and reduced CD3+ and CD45+ cell infiltration in the prostate stroma. Further study showed that XLQ ^® suppressed the expression of proinflammatory cytokines, such as interleukin (IL)-1β, IL-2, IL-6, IL-17A, monocyte chemoattractant protein-1, and tumor necrosis factor-α. XLQ ^® showed a strong antioxidant capacity by enhancing the activities of antioxidative enzymes (e.g., total superoxide dismutase, catalase, and glutathione peroxidase) and decreasing the level of lipid peroxidation products (malondialdehyde). Moreover, XLQ ^® can suppress the activation of nuclear factor-κB and P38-mitogen-activated protein kinase signaling pathways. In summary, XLQ ^® has affirmative effects on chronic prostatitis, which could be attributed to its anti-inflammatory and antioxidative capacities. On the basis of these results, XLQ ^® can be developed as an effective and safe therapy for chronic prostatitis.     

The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat

The barley Mla locus confers multiple resistance specificities to the obligate fungal biotroph, Blumeria (= Erysiphe) graminis f. sp. hordei. Interspersed within the 240 kb Mla complex are three families of resistance gene homologs (RGHs). Probes from the Mla-RGH1 family were used to identify three classes of cDNAs. The first class is predicted to encode a full-length CC-NBS-LRR protein and the other two classes contain alternatively spliced, truncated variants. Utilizing a cosmid that contains a gene corresponding to the full-length candidate cDNA, two single-cell expression assays were used to demonstrate complementation of AvrMla6-dependent, resistance specificity to B. graminis in barley and wheat. The first of these assays was also used to substantiate previous genetic data that the Mla6 allele requires the signaling pathway component, Rar1, for function. Computational analysis of MLA6 and the Rar1-independent, MLA1 protein reveals 91.2% identity and shows that the LRR domain is subject to diversifying selection. Our findings demonstrate that highly related CC-NBS-LRR proteins encoded by alleles of the Mla locus can dictate similar powdery mildew resistance phenotypes yet still require distinct downstream signaling components.     

Bmpr1a signaling plays critical roles in palatal shelf growth and palatal bone formation

Cleft palate, including submucous cleft palate, is among the most common birth defects in humans. While overt cleft palate results from defects in growth or fusion of the developing palatal shelves, submucous cleft palate is characterized by defects in palatal bones. In this report, we show that the Bmpr1a gene, encoding a type I receptor for bone morphogenetic proteins (Bmp), is preferentially expressed in the primary palate and anterior secondary palate during palatal outgrowth. Following palatal fusion, Bmpr1a mRNA expression was upregulated in the condensed mesenchyme progenitors of palatal bone. Tissue-specific inactivation of Bmpr1a in the developing palatal mesenchyme in mice caused reduced cell proliferation in the primary and anterior secondary palate, resulting in partial cleft of the anterior palate at birth. Expression of Msx1 and Fgf10 was downregulated in the anterior palate mesenchyme and expression of Shh was downregulated in the anterior palatal epithelium in the Bmpr1a conditional mutant embryos, indicating that Bmp signaling regulates mesenchymal-epithelial interactions during palatal outgrowth. In addition, formation of the palatal processes of the maxilla was blocked while formation of the palatal processes of the palatine was significantly delayed, resulting in submucous cleft of the hard palate in the mutant mice. Our data indicate that Bmp signaling plays critical roles in the regulation of palatal mesenchyme condensation and osteoblast differentiation during palatal bone formation.     

Molecular basis of guanine nucleotide dissociation inhibitor activity of human neuroglobin by chemical cross-linking and mass spectrometry

Oxidized human neuroglobin (Ngb), a heme protein expressed in the brain, has been proposed to act as a guanine nucleotide dissociation inhibitor (GDI) for the GDP-bound form of the heterotrimeric G protein alpha-subunit (Galpha(i)). Here, to elucidate the molecular mechanism underlying the GDI activity of Ngb, we used an glutathione-S-transferase pull-down assay to confirm that Ngb competes with G-protein betagamma-subunits (Gbetagamma) for binding to Galpha(i), and identified the Galpha(i)-binding site in Ngb by chemical cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and sulfo-N-hydroxysuccinimide, coupled with mass spectrometry (MS). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis for tryptic peptides derived from the cross-linked Ngb-Galpha(i) complex revealed several binding regions in Ngb. Furthermore, MALDI-TOF/TOF MS analysis of the cross-linked Ngb and Galpha(i) peptides, together with the MS/MS scoring method, predicted cross-linking between Glu60 (Ngb) and Ser206 (Galpha(i)), and between Glu53 (Ngb) and Ser44 (Galpha(i)). Because Ser206 of Galpha(i) is located in the region that contacts Gbetagamma, binding of Ngb could facilitate the release of Gbetagamma from Galpha(i). Binding of Ngb to Galpha(i) would also inhibit the exchange of GDP for GTP, because Ser44 (Galpha(i)) is adjacent to the GDP-binding site and Glu53 (Ngb), which is cross-linked to Ser44 (Galpha(i)), could be located close to GDP. Thus, we have identified, for the first time, the sites of interaction between Ngb and Galpha(i), enabling us to discuss the functional significance of this binding on the GDI activity of Ngb.