The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB includeIL8,CXCL1, andCXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.
Neurons and glia share a mutual dependence in establishing a functional relationship, and none is more evident than the process by which axons control myelination. Here, we identify LRR and Ig domain-containing, Nogo receptor-interacting protein (LINGO-1) as a potent axonal inhibitor of oligodendrocyte differentiation and myelination that is regulated by nerve growth factor and its cognate receptor TrkA in a dose-dependent manner. Whereas LINGO-1 expressed by oligodendrocyte progenitor cells was previously identified as an inhibitor of differentiation, we demonstrate that axonal expression of LINGO-1 inhibits differentiation with equal potency. Disruption of LINGO-1 on either cell type is sufficient to overcome the inhibitory action and promote differentiation and myelination, independent of axon diameter. Furthermore, these results were recapitulated in transgenic mice overexpressing the full length LINGO-1 under the neuronal promoter synapsin. Myelination was greatly inhibited in the presence of enforced axonal LINGO-1. The implications of these results relate specifically to the development of potential therapeutics targeting extrinsic growth factors that may regulate the axonal expression of modulators of oligodendrocyte development.
NG2 cells (polydendrocytes) comprise an abundant glial population that is widely and uniformly distributed throughout the developing and mature CNS and are identified by the expression of the NG2 proteoglycan at the cell surface. Although recent electrophysiological studies suggest that they are capable of receiving signals from axon terminals, other studies, based on the finding that the NG2 molecule itself induces growth cone collapse, have led to a widely held speculation that NG2 cells themselves also repel and inhibit growing axons. In this study, we have examined the effects of rat NG2 cells on growing hippocampal and neocortical axons in vitro and in vivo. NG2 cells did not repel growing axons but promoted their growth in vitro, and axonal growth cones formed extensive contacts with NG2 cells both in vitro and in the developing corpus callosum. Punctate immunoreactivity for fibronectin and laminin was found to be colocalized with NG2 on the surface of NG2 cells. Altering the level of cell surface NG2 expression had no effect on the growth-promoting effects of NG2 cells on growing axons. Thus, our study indicates that NG2 cells are not inhibitory to growing axons but provide an adhesive substrate for axonal growth cones and promote their growth even in the presence of elevated levels of the NG2 proteoglycan. These findings suggest a novel role for NG2 cells in facilitating axonal growth during development and regeneration.
DNA damage triggers complex cellular responses in eukaryotic cells, including initiation of DNA repair and activation of cell cycle checkpoints. In addition to inducing cell cycle arrest, checkpoint also has been suggested to modulate a variety of other cellular processes in response to DNA damage. In this study, we present evidence showing that the cellular function of xeroderma pigmentosum group A (XPA), a major nucleotide excision repair (NER) factor, could be modulated by checkpoint kinase ataxia-telangiectasia mutated and Rad3-related (ATR) in response to UV irradiation. We observed the apparent interaction and colocalization of XPA with ATR in response to UV irradiation. We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation. The Ser196 of XPA was identified as a biologically significant residue to be phosphorylated in vivo. The XPA-deficient cells complemented with XPA-S196A mutant, in which Ser196 was substituted with an alanine, displayed significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Moreover, substitution of Ser196 with aspartic acid for mimicking the phosphorylation of XPA increased the cell survival to UV irradiation. Taken together, our results revealed a potential physical and functional link between NER and the ATRdependent checkpoint pathway in human cells and suggested that the ATR checkpoint pathway could modulate the cellular activity of NER through phosphorylation
Classic studies recognize two functionally segregated macroglial cell types in the central nervous system (CNS), namely astrocytes and oligodendrocytes. A third macroglial cell type has now been identified by its specific expression of the NG2 chondroitin sulphate proteoglycan (NG2-glia). These NG2-glia exist abundantly in both grey and white matter of the mature CNS and are almost as numerous as astrocytes. It is well established that NG2-glia give rise to oligodendrocytes. However, the majority of NG2-glia in the adult CNS proliferate very slowly and are nonmotile. Both astrocytes and NG2-glia display a stellate morphology and express ion channels and receptors to neurotransmitters used by neurons. Both types of glia make intimate contacts with neurons in grey and white matter, and their functional differences and similarities are only beginning to be unravelled. Recent observations emphasize the need to examine the relationship between astrocytes and NG2-glia, and address the question of whether they represent overlapping or two distinct glial cell populations. To be of any relevance, this classification must relate to specific functions in the neural network. At present, the balance of evidence is that NG2-glia and astrocytes are functionally segregated populations.
RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.
Nucleotide excision repair (NER) is a repair pathway that removes a variety of bulky DNA lesions in both prokaryotic and eukaryotic cells. The perturbation of DNA helix structure caused by the oxidative intrastrand lesions could render them good substrates for the NER pathway. Here we employed Escherichia coli NER enzymes, i.e., UvrA, UvrB, and UvrC, to examine the incision efficiency of duplex DNA carrying three different oxidative intrastrand cross-link lesions, that is, G[8-5]C, G[8-5m]mC, and G[8-5m]T, and two dithymine photoproducts, namely, the cis,syn-cyclobutane pyrimidine dimer (T[c,s]T) and the pyrimidine(6-4)pyrimidone product (T[6-4]T). Our results showed that T[6-4]T was the best substrate for UvrA binding, followed by G[8-5]C, G[8-5m]mC, and G[8-5m]T, and then by T[c,s]T. The efficiencies of the UvrABC incisions of these lesions were consistent with their UvrA binding affinities: the stronger the binding to UvrA, the higher the rate of incision. In addition, flanking DNA sequences appeared to have little effect on the binding affinity of UvrA for G[8-5]C as AG[8-5]CA was only slightly preferred over CG[8-5]CG. Consistently, these two sequences exhibited almost no difference in incision rates. Furthermore, we investigated the thermal stability of dodecameric duplexes containing G[8-5m]mC or G[8-5m]T, and our results revealed that these two lesions destabilized the duplex, due to an increase in the free energy for duplex formation at 37 degrees C, by approximately 5.4 and 3.6 kcal/mol, respectively. The destabilizations to the DNA helix caused by those lesions, for the most part, are correlated with the binding affinities of UvrA and incision rates of UvrABC. Taken together, the results from this study suggest that oxidative intrastrand lesions might be substrates for NER enzymes in vivo.
Human XPA is an important DNA damage recognition protein in nucleotide excision repair (NER). We previously observed that XPA binds to DNA lesion as a homodimer (1). Herein we report that XPA recognized undamaged DNA doublestrand/ single-strand (ds-ssDNA) junctions containing ssDNA branches with binding affinity (K d = 49.1±5.1 nM) much higher than its ability to bind to DNA damage. The recognized DNA junction structures include Y-shape junction (with both 3′-and 5′-ssDNA branches), 3′-overhang junction (with a 3′-ssDNA branch), and 5′-overhang junction (with a 5′-ssDNA branch). Using gel filtration chromatography and gel mobility shift assays, we showed that the highly efficient binding appeared to be carried out by the XPA monomer and that the binding was largely independent of RPA. Furthermore, XPA efficiently bound to six-nucleotide mismatched DNA bubble substrates with or without DNA adducts including C8 guanine adducts of AF, AAF, and AP, and the T[6,4] T photo products. Using a set of defined DNA substrates with varying degrees of DNA bending, we also found that the XPC-HR23B complex recognized DNA bending, whereas neither XPA nor the XPA-RPA complex could bind to bent DNA. We propose that besides DNA damage recognition, XPA may also play a novel role in stabilizing, via its high affinity to ds-ssDNA junctions, the DNA strand opening surrounding the lesion for stable formation of pre-incision NER intermediates. Our results provide a plausible mechanistic interpretation for the indispensable requirement of XPA for both global genome and transcription-coupled repairs. Since ds-ssDNA junctions are common intermediates in many DNA metabolic pathways, the additional potential role of XPA in cellular processes is discussed.
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