Defying DNA Double-Strand Break-Induced Death during Prophase I Meiosis by Temporal TAp63α Phosphorylation Regulation in Developing Mouse Oocytes

The dichotomy in DNA damage sensitivity of developing mouse oocytes during female germ line development is striking. Embryonic oocytes withstand hundreds of programmed DNA double-strand breaks (DSBs) required for meiotic recombination. Postnatal immature oocytes fail to tolerate even a few DSBs induced by gamma radiation treatment. TAp63α, a p53 family member, undergoes phosphorylation and mediates postnatal immature oocyte death following gamma radiation treatment, which is thought important for germ line quality maintenance. Whether prenatal meiotic oocytes tolerate DNA DSBs simply because they lack TAp63α expression is not clear. We found a significant number of oocytes in newborn mice initiate TAp63α expression and simultaneously carry meiotic DNA DSBs. However, the risk of premature death appears unlikely, because newborn oocytes strongly abate TAp63α phosphorylation induction and resist normally lethal doses of ionizing radiation damage. A calyculin A-sensitive Ser/Thr phosphatase activity downregulates TAp63α phosphorylation and ATM kinase mediates phosphorylation. Possible alterations in the relative balance of these counteracting activities during development may first temper TAp63α phosphorylation and death induction during meiotic DNA DSB repair and recombination, and afterward, implement germ line quality control in later stages. Insights into inherent DNA DSB resistance mechanisms in newborn oocytes may help prevent infertility in women in need of radiation or chemotherapy.     

p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.Subject terms: Mechanisms of disease, Tumour-suppressor proteins     

CDK5RAP2 loss-of-function causes premature cell senescence via the GSK3β/β-catenin-WIP1 pathway

Developmental disorders characterized by small body size have been linked to CDK5RAP2 loss-of-function mutations, but the mechanisms underlying which remain obscure. Here, we demonstrate that knocking down CDK5RAP2 in human fibroblasts triggers premature cell senescence that is recapitulated in Cdk5rap2^an/an mouse embryonic fibroblasts and embryos, which exhibit reduced body weight and size, and increased senescence-associated (SA)-β-gal staining compared to Cdk5rap2^+/+ and Cdk5rap2^+/an embryos. Interestingly, CDK5RAP2-knockdown human fibroblasts show increased p53 Ser15 phosphorylation that does not correlate with activation of p53 kinases, but rather correlates with decreased level of the p53 phosphatase, WIP1. Ectopic WIP1 expression reverses the senescent phenotype in CDK5RAP2-knockdown cells, indicating that senescence in these cells is linked to WIP1 downregulation. CDK5RAP2 interacts with GSK3β, causing increased inhibitory GSK3β Ser9 phosphorylation and inhibiting the activity of GSK3β, which phosphorylates β-catenin, tagging β-catenin for degradation. Thus, loss of CDK5RAP2 decreases GSK3β Ser9 phosphorylation and increases GSK3β activity, reducing nuclear β-catenin, which affects the expression of NF-κB target genes such as WIP1. Consequently, loss of CDK5RAP2 or β-catenin causes WIP1 downregulation. Inhibition of GSK3β activity restores β-catenin and WIP1 levels in CDK5RAP2-knockdown cells, reducing p53 Ser15 phosphorylation and preventing senescence in these cells. Conversely, inhibition of WIP1 activity increases p53 Ser15 phosphorylation and senescence in CDK5RAP2-depleted cells lacking GSK3β activity. These findings indicate that loss of CDK5RAP2 promotes premature cell senescence through GSK3β/β-catenin downregulation of WIP1. Premature cell senescence may contribute to reduced body size associated with CDK5RAP2 loss-of-function.Subject terms: Senescence, Diseases     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

A Function for Phosphatidylinositol 3-Kinase β (p85α-p110β) in Fibroblasts during Mitogenesis: Requirement for Insulin- and Lysophosphatidic Acid-Mediated Signal Transduction

We have previously shown that phosphatidylinositol 3-kinase α (PI 3-Kα) (p85α-p110α) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185–9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kβ (p85α-p110β) during mitogenesis. By using antibodies specific to p110β we showed that PI 3-Kβ is expressed in NIH 3T3 cells. PI 3-Kβ and PI 3-Kα have common features: PI 3-Kβ is tightly associated with a protein serine kinase that phosphorylates p85α, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kβ was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kα was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110β into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kβ plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kα and PI 3-Kβ, suggesting that Ras uses other effectors for DNA synthesis.     

Butyrate inhibits IL-1β-induced inflammatory gene expression by suppression of NF-κB activity in pancreatic beta cells

Cytokine-induced beta cell dysfunction is a hallmark of type 2 diabetes (T2D). Chronic exposure of beta cells to inflammatory cytokines affects gene expression and impairs insulin secretion. Thus, identification of anti-inflammatory factors that preserve beta cell function represents an opportunity to prevent or treat T2D. Butyrate is a gut microbial metabolite with anti-inflammatory properties for which we recently showed a role in preventing interleukin-1β (IL-1β)-induced beta cell dysfunction, but how prevention is accomplished is unclear. Here, we investigated the mechanisms by which butyrate exerts anti-inflammatory activity in beta cells. We exposed mouse islets and INS-1E cells to a low dose of IL-1β and/or butyrate and measured expression of inflammatory genes and nitric oxide (NO) production. Additionally, we explored the molecular mechanisms underlying butyrate activity by dissecting the activation of the nuclear factor-κB (NF-κB) pathway. We found that butyrate suppressed IL-1β-induced expression of inflammatory genes, such as Nos2, Cxcl1, and Ptgs2, and reduced NO production. Butyrate did not inhibit IκBα degradation nor NF-κB p65 nuclear translocation. Furthermore, butyrate did not affect binding of NF-κB p65 to target sequences in synthetic DNA but inhibited NF-κB p65 binding and RNA polymerase II recruitment to inflammatory gene promoters in the context of native DNA. We found this was concurrent with increased acetylation of NF-κB p65 and histone H4, suggesting butyrate affects NF-κB activity via inhibition of histone deacetylases. Together, our results show butyrate inhibits IL-1β-induced inflammatory gene expression and NO production through suppression of NF-κB activation and thereby possibly preserves beta cell function.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

This study elucidates the role of the protein kinases p37γ and p38δ in regulating the metabolic switch that occurs in early postnatal development, revealing that they inhibit glycogen synthase 1 and glycogen metabolism. Deregulation of this mechanism results in cardiac defects and metabolic alterations which can be prevented by maternal fatty acid diet supplementation during pregnancy and lactation.     

Crystal structure of the ββα-Me type II restriction endonuclease Hpy99I with target DNA

The ββα-Me restriction endonuclease (REase) Hpy99I recognizes the CGWCG target sequence and cleaves it with unusual stagger (five nucleotide 5′-recessed ends). Here we present the crystal structure of the specific complex of the dimeric enzyme with DNA. The Hpy99I protomer consists of an antiparallel β-barrel and two β4α2 repeats. Each repeat coordinates a structural zinc ion with four cysteine thiolates in two CXXC motifs. The ββα-Me region of the second β4α2 repeat holds the catalytic metal ion (or its sodium surrogate) via Asp148 and Asn165 and activates a water molecule with the general base His149. In the specific complex, Hpy99I forms a ring-like structure around the DNA that contacts DNA bases on the major and minor groove sides via the first and second β4α2 repeats, respectively. Hpy99I interacts with the central base pair of the recognition sequence only on the minor groove side, where A:T resembles T:A and G:C is similar to C:G. The Hpy99I–DNA co-crystal structure provides the first detailed illustration of the ββα-Me site in REases and complements structural information on the use of this active site motif in other groups of endonucleases such as homing endonucleases (e.g. I-PpoI) and Holliday junction resolvases (e.g. T4 endonuclease VII).     

The Pol β-14 Dominant Negative Rat DNA Polymerase β Mutator Mutant Commits Errors during the Gap-Filling Step of Base Excision Repair in Saccharomyces cerevisiae

We demonstrated recently that dominant negative mutants of rat DNA polymerase β (Pol β) interfere with repair of alkylation damage in Saccharomyces cerevisiae. To identify the alkylation repair pathway that is disrupted by the Pol β dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol β mutants to genes known to be involved in repair of DNA alkylation damage in S. cerevisiae. We demonstrate that the rat Pol β mutants interfere with the base excision repair pathway in S. cerevisiae. In addition, expression of one of the Pol β dominant negative mutants, Pol β-14, increases the spontaneous mutation rate of S. cerevisiae whereas expression of another Pol β dominant negative mutant, Pol β-TR, does not. Expression of the Pol β-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol β-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.