Rho/Rho-associated kinase-II signaling mediates disassembly of epithelial apical junctions

Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin-dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rho-associated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

IFN-alpha priming results in a gain of proinflammatory function by IL-10: implications for systemic lupus erythematosus pathogenesis

Interleukin-10 is a predominantly anti-inflammatory cytokine that inhibits macrophage and dendritic cell function, but can acquire proinflammatory activity during immune responses. We investigated whether type I IFNs, which are elevated during infections and in autoimmune diseases, modulate the activity of IL-10. Priming of primary human macrophages with low concentrations of IFN-alpha diminished the ability of IL-10 to suppress TNF-alpha production. IFN-alpha conferred a proinflammatory gain of function on IL-10, leading to IL-10 activation of expression of IFN-gamma-inducible, STAT1-dependent genes such as IFN regulatory factor 1, IFN-gamma-inducible protein-10 (CXCL10), and monokine induced by IFN-gamma (CXCL9). IFN-alpha priming resulted in greatly enhanced STAT1 activation in response to IL-10, and STAT1 was required for IL-10 activation of IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma expression in IFN-alpha-primed cells. In control, unprimed cells, IL-10 activation of STAT1 was suppressed by constitutive activity of protein kinase C and Src homology 2 domain-containing phosphatase 1. These results demonstrate that type I IFNs regulate the balance between IL-10 anti- and proinflammatory activity, and provide insight into molecular mechanisms that regulate IL-10 function. Gain of IL-10 proinflammatory functions may contribute to its pathogenic role in autoimmune diseases characterized by elevated type I IFN levels, such as systemic lupus erythematosus.     

FAK-MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse

Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase-mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A.     

Conformational dynamics coupled to protonation equilibrium at the CuA site of Thermus thermophilus: insights into the origin of thermostability

An extremely slow pH dependent conformational equilibrium between a valence-delocalized and a valence-trapped species of the dinuclear CuA domain of cytochrome c oxidase from Thermus thermophilus has been identified and characterized using UV-visible absorption, circular dichroism, time-resolved fluorescence and electron paramagnetic resonance spectroscopy as well as by stopped-flow kinetic techniques. The results indicated that the nature of this pH dependent conformation change in the CuA domain in the Thermus protein was distinctly different from that observed in the mesophilic analogue from Paracoccus denitrificans and in the engineered CuA domain in azurin. pH jump kinetic studies suggested existence of a fast deprotonation equilibrium followed by slow conformational change in the protein, which is contrary to that observed in the case of the analogous protein from P. denitrificans. Continuous-flow electrospray mass spectral studies on H/D exchange in the TtCuA showed that approximately 75% of the protons are exchanged within the dead-time of the experiment supporting fast proton transfer kinetics in the protein. Analysis of temperature dependence of the kinetics of the conformational transition showed that the rigidity of the protein structure decreases with increase in temperature. The results indicated that though the rate of proton transfer at individual sites in the protein could be very fast, the conformational change that requires simultaneous breaking of several interactions in a segment of the structure might be slow in the thermostable protein.     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.