Aspergillus nidulans hypA regulates morphogenesis through the secretion pathway

Aspergillus nidulans hypA encodes a predicted 1474 amino acid, 161.9 kDa cytoplasmic peptide. Strains with hypA1 and hypA6 alleles are wild type at 28 degrees C but have wide, slow-growing hyphae and thick walls at 42 degrees C. hypA1 and hypA6 have identical genetic lesions. hypA1 and hypA6 restrictive phenotypes have statistically similar morphometry, and strains with either allele can conidiate at 42 degrees C. hypA deletion strains require osmotic support and have aberrant morphology, but produce viable spores at 28 degrees C. hypA has full-length orthologs in filamentous fungi and yeasts and a 200 amino acid region with similarity to sequences in plants and animals. The Saccharomyces cerevisiae hypA ortholog is TRS120, a regulatory subunit in the TRAPP II complex that mediates traffic through the Golgi equivalent. Enzyme secretion is reduced in hypA1 cells at 42 degrees C. Endomembranes and cytoplasmic actin arrays in hypA1 have weak polarity at 42 degrees C and cytoplasmic microtubules have reduced number and normal distribution.     

Electroacupuncture regulates TRPM7 expression through the trkA/PI3K pathway after cerebral ischemia-reperfusion in rats

Recently, it was demonstrated that TRPM7 is an essential mediator of anoxia-induced neuronal death. Meanwhile, nerve growth factor (NGF) is known to have survival and neuroprotective effects by interacting with the high affinity neurotrophin receptor, tropomyosin-related kinase A (trkA). In the present study, we found that electroacupuncture (EA) treatment could up-regulate trkA expression after focal cerebral ischemia in rats. At the same time, EA therapy obviously decreased the high expression of TRPM7 induced by ischemia. Using K252a to inhibit trkA, we found that the EA-mediated down-regulation of TRPM7 was significantly suppressed in rats subjected to cerebral ischemia. TrkA can utilize two distinct signaling pathways: the phosphatidylinositol 3-kinase (PI3K) pathway and the extracellular signal-related kinase (ERK) pathway. We found that the effect of EA on TRPM7 was also inhibited by a PI3K inhibitor, while an ERK inhibitor had no effect. Taken together, our findings suggest that EA can reverse the ischemia-induced increase of TRPM7 levels through the trkA-PI3K pathway.     

The Src tyrosine kinase pathway regulates thecal CYP17 expression and androstenedione secretion

In order to evaluate the role of Src tyrosine kinase in thecal cell steroidogenesis, a pharmacological approach was utilized by treating enriched populations of mouse ovarian theca-interstitial cells in vitro with a direct Src kinase inhibitor, PP2. Inhibition of Src with PP2 increased both basal and forskolin-stimulated androstenedione secretion, and increased cytochrome P450 17-alpha hydroxylase-lyase (CYP17) promoter activity and steady state mRNA. PP2 did not change thecal levels of StAR mRNA. Inhibition of mitogen-activated protein kinase kinase, a downstream regulator of Src activity, using PD98059 also increased forskolin-stimulated secretion of androstenedione above forskolin alone, but had no effect on basal secretion of androstenedione. Src inhibition increased mitogen-activated protein kinase phosphatase-1 protein and decreased phosphorylation of SF-1, which correlated with increased CYP17 promoter activity and mRNA levels. These results implicate Src tyrosine kinase in the regulation of CYP17 and thecal androgen secretion.     

EGFR signaling pathway negatively regulates PSA expression and secretion via the PI3K-Akt pathway in LNCaP prostate cancer cells

Epidermal growth factor (EGF) and its receptor (EGFR) are involved in hormone-refractory growth and poor prognosis of a subgroup of human prostate cancer. In this communication, we investigated the regulation of PSA by the EGFR signaling pathway using LNCaP C-81 prostate cancer cells. Administration of EGF stimulated the growth of LNCaP C-81 cells, however, PSA expression and secretion were suppressed. An EGFR inhibitor, AG1478, abrogated the PSA suppression effect by EGF, in concurrence with the suppression of tyro-phosphorylation levels of EGFR. Interestingly, the AR level was also decreased in EGF-treated LNCaP C-81 cells. Moreover, LY294002, but not PD98059, inhibited the PSA and AR suppression effect by EGF in concurrence with the suppression of phosphorylation levels of Akt. In conclusion, our results strongly suggest the existence of a novel androgen-independent PSA regulatory mechanism, i.e., the EGFR signaling pathway negatively regulates PSA expression which may be induced by the alteration of AR expression via the PI3K-Akt pathway in LNCaP C-81 cells.     

Transforming growth factor-beta regulates stearoyl coenzyme A desaturase expression through a Smad signaling pathway.

The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism.     

Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway

Invadopodia are invasive protrusions with proteolytic activity uniquely found in tumor cells. Cortactin phosphorylation is a key step during invadopodia maturation, regulating Nck1 binding and cofilin activity. The precise mechanism of cortactin-dependent cofilin regulation and the roles of this pathway in invadopodia maturation and cell invasion are not fully understood. We provide evidence that cortactin-cofilin binding is regulated by local pH changes at invadopodia that are mediated by the sodium-hydrogen exchanger NHE1. Furthermore, cortactin tyrosine phosphorylation mediates the recruitment of NHE1 to the invadopodium compartment, where it locally increases the pH to cause the release of cofilin from cortactin. We show that this mechanism involving cortactin phosphorylation, local pH increase, and cofilin activation regulates the dynamic cycles of invadopodium protrusion and retraction and is essential for cell invasion in 3D. Together, these findings identify a novel pH-dependent regulation of cell invasion.     

CTCF regulates ataxin-7 expression through promotion of a convergently transcribed, antisense noncoding RNA

Neural networks in the spinal cord control two basic features of locomotor movements: rhythm generation and pattern generation. Rhythm generation is generally considered to be dependent on glutamatergic excitatory neurons. Pattern generation involves neural circuits controlling left-right alternation, which has been described in great detail, and flexor-extensor alternation, which remains poorly understood. Here, we use a mouse model in which glutamatergic neurotransmission has been ablated in the locomotor region of the spinal cord. The isolated in?vitro spinal cord from these mice produces locomotor-like activity-when stimulated with neuroactive substances-with prominent flexor-extensor alternation. Under these conditions, unlike in control mice, networks of inhibitory interneurons generate the rhythmic activity. In the absence of glutamatergic synaptic transmission, the flexor-extensor alternation appears to be generated by Ia inhibitory interneurons, which mediate reciprocal inhibition from muscle proprioceptors to antagonist motor neurons. Our study defines a minimal inhibitory network that is needed to produce flexor-extensor alternation during locomotion.     

Noncanonical Wnt signaling in stromal cells regulates B-lymphogenesis through interleukin-7 expression

The regulation of early B cell development and the interaction of hematopoietic precursors with stromal cells in the bone marrow (BM) are controlled by various secreted signaling molecules. Several recent studies showed Wnt signaling involved in B-lymphogenesis through stromal cells. However, the molecules modulated by Wnt signaling in stromal cells regulating B-lymphogenesis have not been identified yet. Interleukin (IL)-7 and CXC chemokine ligand (CXCL) 12 are known to be express in stromal cells, and both molecules are essential for B-lymphogenesis. In the present study, we examined the role of Wnt signaling in regulating IL-7 and CXCL12 expression and in affecting B-lymphogenesis. In mouse stromal ST2 cells, expression of IL-7 and CXCL12 mRNA was augmented by noncanonical Wnt5a. When mouse BM-derived cells were cultured on Wnt5a-overexpressing ST2 cells, an increased number of B220+/IgM- B-lymphoid precursor cells was observed. These results show that Wnt5a regulates IL-7 gene expression in stromal cells and suggest the possibility that noncanonical Wnt regulates B-lymphogenesis via IL-7 expression in stromal cells.     

N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP-WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.     

ERK pathway positively regulates the expression of Sprouty genes

Sprouty was originally identified as an inhibitor of Drosophila development-associated receptor tyrosine kinase (RTK) signaling. Although RTK signaling has been shown to induce Sprouty gene expression, the precise induction pathway downstream of RTK remains unclear. As RTK signaling pathway includes activation of extracellular signal-regulated kinases (ERKs), we have examined a correlation between activation of ERKs and induction of Sprouty gene expression. All reagents which induce the activation of ERKs induce Sprouty gene expression; these agents include not only growth factors which bind to RTK but also phorbol 12-myristate-13-acetate and active Raf-1 kinase. Furthermore, the Sprouty gene expression induced by all those agents is totally suppressed when the cells are pretreated with specific inhibitors of ERK kinase (MEK). Human tumor cells which exhibit constitutive activation of ERKs show elevated expression of Sprouty genes, which is abolished by treatment of these cells with MEK inhibitors. All these findings clearly indicate that Sprouty gene expression is positively regulated by the ERK pathway downstream of RTK.     

Particle tracking microrheology of purified gastrointestinal mucins

The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins (MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol, DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal mucins formed self‐assembled semi‐dilute networks above a certain critical mucin concentration (c*) with the viscosity (η) scaling as for MUC5AC and for MUC2, where c is the mucin concentration. Above an even higher mucin concentration threshold (c_e, the entanglement concentration) reptation occurs and there is a dramatic increase in the viscosity scaling, for MUC5AC and for MUC2. The dynamics of the self‐assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred‐fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi‐dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol–gel transition in semi‐dilute mucin solutions at neutral pH. © 2013 The Authors. Biopolymers published by Wiley Periodicals, Inc. Biopolymers 101: 366–377, 2014.     

USP7 regulates the ERK1/2 signaling pathway through deubiquitinating Raf-1 in lung adenocarcinoma

Ubiquitin-specific protease 7 (USP7) is one of the deubiquitinating enzymes (DUBs) in the ubiquitin-specific protease (USP) family. It is a key regulator of numerous cellular functions including immune response, cell cycle, DNA damage and repair, epigenetics, and several signaling pathways. USP7 acts by removing ubiquitin from the substrate proteins. USP7 also binds to a specific binding motif of substrate proteins having the [P/A/E]-X-X-S or K-X-X-X-K protein sequences. To date, numerous substrate proteins of USP7 have been identified, but no studies have been conducted using the binding motif that USP7 binds. In the current study, we analyzed putative substrate proteins of USP7 through the [P/A/E]-X-X-S and K-X-X-X-K binding motifs using bioinformatics tools, and confirmed that Raf-1 is one of the substrates for USP7. USP7 binds to the Pro-Val-Asp-Ser (PVDS) motif of the conserved region 2 (CR2) which contains phosphorylation sites of Raf-1 and decreased M1-, K6-, K11-, K27-, K33-, and K48-linked polyubiquitination of Raf-1. We further identified that the DUB activity of USP7 decreases the threonine phosphorylation level of Raf-1 and inhibits signaling transduction through Raf activation. This regulatory mechanism inhibits the activation of the ERK1/2 signaling pathway, thereby inhibiting the G2/M transition and the cell proliferation of lung adenocarcinoma cells. In summary, our results indicate that USP7 deubiquitinates Raf-1 and is a new regulator of the ERK1/2 signaling pathway in lung adenocarcinoma.Subject terms: Ubiquitylation, Non-small-cell lung cancer, Protein-protein interaction networks