Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an <i>in vitro</i> model of CNS tuberculosis

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB

Primary cilia elongation in response to interleukin-1 mediates the inflammatory response

Primary cilia are singular, cytoskeletal organelles present in the majority of mammalian cell types where they function as coordinating centres for mechanotransduction, Wnt and hedgehog signalling. The length of the primary cilium is proposed to modulate cilia function, governed in part by the activity of intraflagellar transport (IFT). In articular cartilage, primary cilia length is increased and hedgehog signaling activated in osteoarthritis (OA). Here, we examine primary cilia length with exposure to the quintessential inflammatory cytokine interleukin-1 (IL-1), which is up-regulated in OA. We then test the hypothesis that the cilium is involved in mediating the downstream inflammatory response. Primary chondrocytes treated with IL-1 exhibited a 50 % increase in cilia length after 3 h exposure. IL-1-induced cilia elongation was also observed in human fibroblasts. In chondrocytes, this elongation occurred via a protein kinase A (PKA)-dependent mechanism. G-protein coupled adenylate cyclase also regulated the length of chondrocyte primary cilia but not downstream of IL-1. Chondrocytes treated with IL-1 exhibit a characteristic increase in the release of the inflammatory chemokines, nitric oxide and prostaglandin E2. However, in cells with a mutation in IFT88 whereby the cilia structure is lost, this response to IL-1 was significantly attenuated and, in the case of nitric oxide, completely abolished. Inhibition of IL-1-induced cilia elongation by PKA inhibition also attenuated the chemokine response. These results suggest that cilia assembly regulates the response to inflammatory cytokines. Therefore, the cilia proteome may provide a novel therapeutic target for the treatment of inflammatory pathologies, including OA

Coordinated Translocation of Mammalian Gli Proteins and Suppressor of Fused to the Primary Cilium

Intracellular transduction of Hedgehog (Hh) signals in mammals requires functional primary cilia. The Hh signaling effectors, the Gli family of transcription factors, and their negative regulator, Suppressor of Fused (Sufu), accumulate at the tips of cilia; however, the molecular mechanism regulating this localization remains elusive. In the current study, we show that the ciliary localization of mammalian Gli proteins depends on both their N-terminal domains and a central region lying C-terminal to the zinc-finger DNA-binding domains. Invertebrate Gli homologs Ci and Tra1, when over-expressed in ciliated mouse fibroblasts, fail to localize to the cilia, suggesting the lack of a vertebrate-specific structural feature required for ciliary localization. We further show that activation of protein kinase A (PKA) efficiently inhibits ciliary localization of Gli2 and Gli3, but only moderately affects the ciliary localization of Gli1. Interestingly, variants of Gli2 mimicking the phosphorylated or non-phosphorylated states of Gli2 are both localized to the cilia, and their ciliary localizations are subjected to the inhibitory effect of PKA activation, suggesting a likely indirect mechanism underlying the roles of PKA in Gli ciliary localization. Finally, we show that ciliary localization of Sufu is dependent on ciliary-localized Gli proteins, and is inhibited by PKA activation, suggesting a coordinated mechanism for the ciliary translocation of Sufu and Gli proteins

A Novel Signaling Pathway Mediated by the Nuclear Targeting of C-Terminal Fragments of Mammalian Patched 1

Background: Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure. Principal Findings: In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1. Conclusions: Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings sugges

Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles

Confinement bifurcation by magnetic compression on TUMAN-3

INTRODUCTION Experiments on magnetic compression on different tokamaks have shown superadiabatic increases in the plasma parameters On TUMAN-3 the both enhancement and degradation of the confinement were observed in the minor radius magnetic compression experiments In this paper the results of the new study of the L-H transition triggered by magnetic compression are presented. The feature of the recent experiments was small compression ratio what allowed reducing power input P ∂B/∂t during compression phase. The mechanism of the radial electric field E r origin during magnetic compression is proposed to account for the L-H transition