Ca2+‐Inactivated K+ Current is Modulated by Endothelin‐1 in B‐16 Murine Melanoma Cells

It is well established that endothelin‐1 (ET‐1) plays a role in differentiation and proliferation in a variety of cells such as fibroblasts and human melanoma cells via a receptor‐mediated mechanism. However, whether ET‐1 modulates ion channel activity in these cell types is still unknown. In this report, we recorded the voltage‐dependent outward K⁺ current in cultured B16 melanoma cells using the patch‐clamp technique. Biophysical and pharmacological properties of the K⁺ current, and the effect of ET‐1 on the K⁺ current were investigated. When cells were loaded with a Ca²⁺‐chelating agent (EGTA or BAPTA), the K⁺ current amplitude gradually increased with time after establishment of the whole cell configuration. Replacement of Ca²⁺ with Co²⁺ in the extracellular medium caused no significant modulation of the K⁺ current amplitude. Addition of BaCl₂ or quinidine to the extracellular solution reduced the K⁺ current amplitude, whereas the K⁺ current was insensitive to tetraethylammonium. ET‐1 (10 nM) reversibly decreased the K⁺ current amplitude and accelerated the decay of the K⁺ current. The ET‐1‐induced inhibitory effect displayed no desensitization following repeated ET‐1 application. Pretreatment with pertussis toxin (PTX) or perfusion of cells with the protein kinase C (PKC) inhibitor H‐7 abolished the inhibitory effect of ET‐1 on the K⁺ current. We conclude that the outward K⁺ current recorded in murine B‐16 melanoma cells represents a Ca²⁺‐inactivated K⁺ current, and that the inhibitory effect of ET‐1 on the K⁺ current may reveal a novel mechanism to control the differentiation and proliferation of melanoma cells.     

KIF1A mediates axonal transport of BACE1 and identification of independently moving cargoes in living SCG neurons

Neurons rely heavily on axonal transport to deliver materials from the sites of synthesis to the axon terminals over distances that can be many centimetres long. KIF1A is the neuron‐specific kinesin with the fastest reported anterograde motor activity. Previous studies have shown that KIF1A transports a subset of synaptic proteins, neurofilaments and dense‐core vesicles. Using two‐colour live imaging, we showed that beta‐secretase 1 (BACE1)‐mCherry moves together with KIF1A‐GFP in both the anterograde and retrograde directions in superior cervical ganglions (SCG) neurons. We confirmed that KIF1A is functionally required for BACE1 transport by using KIF1A siRNA and a KIF1A mutant construct (KIF1A‐T312M) to impair its motor activity. We further identified several cargoes that have little or no co‐migration with KIF1A‐GFP and also move independently from BACE1‐mCherry. Together, these findings support a primary role for KIF1A in the anterograde transport of BACE1 and suggest that axonally transported cargoes are sorted into different classes of carrier vesicles in the cell body and are transported by cargo‐specific motor proteins through the axon.      

Thrombin stimulates RPE cell motility by PKC‐ζ‐ and NF‐κB‐dependent gene expression of MCP‐1 and CINC‐1/GRO chemokines

Retinal pigment epithelial cells (RPE) are the major cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR), which involves the epithelial‐mesenchymal transition, proliferation, and directional migration of transformed RPE cells to the vitreous upon RPE exposure to serum components, thrombin among them. Although the aqueous humor and vitreous of PVR patients contain high levels of chemokines, their possible involvement in PVR development has not been explored. We here analyzed the effect of thrombin on chemokine gene expression and its correlation with RPE cell migration using rat RPE cells in culture as a model system. We demonstrated that thrombin induces RPE cell migration through the dose‐dependent stimulation of MCP1 and GRO expression/release, and the autocrine activation of CXCR‐2 and CCR‐2 chemokine receptors. Whereas inhibition of CXCR2 by Sb‐225002 and of CCR2 by Rs‐504393 partially prevented hirudin‐sensitive cell migration, the joint inhibition of these receptors abolished thrombin effect, suggesting the contribution of distinct but coincident mechanisms. Thrombin effects were not modified by Ro‐32‐0432 inhibition of conventional/novel PKC isoenzymes or by the MAPkinase pathway inhibitor U0126. MCP1 and GRO expression/secretion, and cell migration were completely prevented by the inhibitory PKC‐ζ pseudosubstrate and by the nuclear factor‐kappa B (NF‐κB) inhibitor BAY11‐7082, but not by wortmannin inhibition of PI3K. Results show that signaling pathways leading to RPE cell migration differ from the MEK–ERK–PI3K‐mediated promotion RPE of cell proliferation, both of which concur at the activation of PKC‐ζ. J. Cell. Biochem. © 2010 Wiley‐Liss, Inc.     

The Cytosolic Adaptor AP‐1A Is Essential for the Trafficking and Function of Niemann‐Pick Type C Proteins

Niemann‐Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over‐accumulation of low‐density lipoprotein‐derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three‐hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP‐1 via its acidic/di‐leucine motif. Consequently, a nonfunctional AP‐1A cytosolic complex resulted in a typical NPC‐like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ‐cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP‐1A is essential for the lysosomal targeting and function of NPC1 and NPC2.     

Distribution of transferrin and transferrin receptor of the eype 1 in the process of formation of the rat eye retina in early postnatal ontogenesis

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type 1 (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layer (in the neuroblast layer-NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retina layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retina is discussed.     

Clinorotation upregulates inducible nitric oxide synthase by inhibiting AP‐1 activation in human umbilical vein endothelial cells

Alterations of nitric oxide contribute to post‐flight orthostatic intolerance. The aim of this study was to investigate the changes of inducible nitric oxide synthase (iNOS) and the mechanisms underlying regulation of iNOS by simulated microgravity in human umbilical vein endothelial cells (HUVECs). Clinorotation, a simulated‐model of microgravity, increased iNOS expression and promoter activity in HUVECs. The transactivations of NF‐κB and AP‐1 were suppressed by 24 h clinorotation. A key role for AP‐1, but not NF‐κB in the regulation of iNOS was shown. (1) PDTC, a NF‐κB inhibitor, had no effect on clinorotation upregulation of iNOS. (2) SP600125, a JNK‐specific inhibitor, which resulted in inhibition of AP‐1 activity, enhanced the iNOS expression and promoter activity in clinorotation. (3) Overexpression of AP‐1 remarkably attenuated the upregulation effect of clinorotation. These findings indicate that clinorotation upregulates iNOS in HUVECs by a mechanism dependent on suppression of AP‐1, but not NF‐κB. These results support a key role for AP‐1 in the signaling of postflight orthostatic intolerance. J. Cell. Biochem. 107: 357–363, 2009. © 2009 Wiley‐Liss, Inc.     

AGAP1/AP‐3‐dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M₅ is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M₅ and the AP‐3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP‐3 to M₅. Interaction with AGAP1 and activity of AP‐3 were required for the endocytic recycling of M₅ in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP‐3 or abrogation of AGAP1–M₅ interaction in vivo decreased the magnitude of presynaptic M₅‐mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor‐recycling pathway that may underlie mechanisms of G‐protein‐coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.     

Long non‐coding RNA SNHG16 promotes proliferation and inhibits apoptosis of diffuse large B‐cell lymphoma cells by targeting miR‐497‐5p/PIM1 axis

The aberrant expression and dysfunction of long non‐coding RNAs (lncRNAs) have been identified as critical factors governing the initiation and progression of different human cancers, including diffuse large B‐cell lymphoma (DLBCL). LncRNA small nucleolar RNA host gene 16 (SNHG16) has been recognized as a tumour‐promoting factor in various types of cancer. However, the biological role of SNHG16 and its underlying mechanism are still unknown in DLBCL. Here we disclosed that SNHG16 was overexpressed in DLBCL tissues and the derived cell lines. SNHG16 knockdown significantly suppressed cell proliferation and cell cycle progression, and it induced apoptosis of DLBCL cells in vitro. Furthermore, silencing of SNHG16 markedly repressed in vivo growth of OCI‐LY7 cells. Mechanistically, SNHG16 directly interacted with miR‐497‐5p by acting as a competing endogenous RNA (ceRNA) and inversely regulated the abundance of miR‐497‐5p in DLBCL cells. Moreover, the proto‐oncogene proviral integration site for Moloney murine leukaemia virus 1 (PIM1) was identified as a novel direct target of miR‐497‐5p. SNHG16 overexpression rescued miR‐497‐5p‐induced down‐regulation of PIM1 in DLBCL cells. Importantly, restoration of PIM1 expression reversed SNHG16 knockdown‐induced inhibition of proliferation, G0/G1 phase arrest and apoptosis of OCI‐LY7 cells. Our study suggests that the SNHG16/miR‐497‐5p/PIM1 axis may provide promising therapeutic targets for DLBCL progression.