The Journal of biological chemistry
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Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxin-resistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4-8 weeks after onset of diabetes. ⋯ Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Na(v)1.3 (TTX-S) and Na(v) 1.7 (TTX-S) and decreases in the expression of Na(v) 1.6 (TTX-S) and Na(v)1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Na(v) 1.6 and In Na(v)1.8 increased in response to diabetes. addition, increased tyrosine phosphorylation of Na(v)1.6 and Na(v)1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.
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Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(-/-) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(+/+) cells. ⋯ The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(-/-) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2.
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The presence of a collagenous protein (ColQ) characterizes the collagen-tailed forms of acetylcholinesterase and butyrylcholinesterase at vertebrate neuromuscular junctions which is tethered in the synaptic basal lamina. ColQ subunits, differing mostly by their signal sequences, are encoded by transcripts ColQ-1 and ColQ-1a, which are differentially expressed in slow and fast twitch muscles in mammals. Two distinct promoters, pColQ-1 and pColQ-1a, were isolated from the upstream sequences of human COLQ gene; they showed muscle-specific expression and were activated by myogenic transcriptional elements in cultured myotubes. ⋯ The slow fiber type-specific expression of ColQ-1 was abolished by the mutation of an NFAT element in pColQ-1. Moreover, both the ColQ promoters contained N-box element that was responsible for the synapse-specific expression of ColQ transcripts. These results explain the specific expression patterns of collagen-tailed acetylcholinesterase in slow and fast muscle fibers.
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To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. ⋯ All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.
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Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and mediates binding and internalization of pathogens such as virus (human immunodeficiency virus, hepatitis C), bacteria (Mycobacterium), fungi, and parasites. DC-SIGN expression in vivo is primarily restricted to interstitial dendritic cells (DC) and certain tissue macrophages. We now report that leukemic THP-1 cells, widely used as a model for monocyte-macrophage differentiation, express very low basal levels of DC-SIGN and that DC-SIGN expression in THP-1 cells is regulated during differentiation. ⋯ DC-SIGN up-regulation required a functional JAK-STAT signaling pathway, was inhibited in the presence of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha), and conferred THP-1 cells with increased pathogen recognition and T cell stimulatory capabilities. The up-regulation of DC-SIGN on THP-1 cells resembles its inducible expression on monocytes and macrophages, where DC-SIGN expression is also induced by IL-4/IL-13 and negatively regulated by TNF-alpha, LPS, and vitamin D(3). These results point to THP-1 cells as a useful cellular system to characterize the pathogen-binding capabilities of DC-SIGN and to dissect the molecular mechanisms that control its regulated and tissue-specific expression in myeloid dendritic cells, and the results suggest that DC-SIGN constitutes a marker for both DC and alternatively activated macrophages.