CDK1 interacts with RARγ and plays an important role in treatment response of acute myeloid leukemia

Alterations in cell cycle pathways and retinoic acid signaling are implicated in leukemogenesis. However, little is known about the roles of cyclin-dependent kinases (CDKs) in treatment response of leukemia. In this study, we observed that CDK1 expression was significantly higher in bone marrow from 42 patients with acute myeloid leukemia (AML) at recurrence than that at first diagnosis (p = 0.04). AML patients had higher level of nuclear CDK1 in their leukemic blasts tended to have poorer clinical outcome compared with those with lower levels. We showed that CDK1 function is required for all-trans retinoic acid (ATRA) to achieve the optimal effect in U-937 human leukemic cells. CDK1 modulates the levels of P27^kip and AKT phosphorylation in response to ATRA treatment. Further, we show, for the first time, that RARγ in concert with ATRA regulates protein levels of CDK1 and its subcellular localization. The regulation of the subcellular content of CDK1 and RARγ by ATRA is an important process for achieving an effective response in treatment of leukemia. RARγ and CDK1 form a reciprocal regulatory circuit in the nucleus and influence the function and protein stability of each other and the level of P27^kip protein. In addition, expression of wee1 kinase and Cdc25A/C phosphatases also coincide with CDK1 expression and its subcellular localization in response to ATRA treatment. Our study reveals a novel mechanism by which CDK1 and RARγ coordinate with ATRA to influence cell cycle progression and cellular differentiation.     

Substance P plays an important role in cell adhesion molecule 1-mediated nerve–pancreatic islet α cell interaction

Autonomic neurons innervate pancreatic islets of Langerhans and maintain blood glucose homeostasis by regulating hormone levels. We previously showed that cell adhesion molecule 1 (CADM1) mediated the attachment and interaction between nerves and aggregated pancreatic islet α cells. In this study, we cocultured αTC6 cells, a murine α cell line, with mouse superior cervical ganglion (SCG) neurons. The oscillation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) was observed in 27% and 14% of αTC6 and CADM1-knockdown αTC6 cells (αTC6^siRNA-CADM1 cells) in aggregates, respectively, within 1 min after specific SCG nerve stimulation with scorpion venom. In αTC6^siRNA-CADM1 cells, the responding rate during 3 min after SCG nerve stimulation significantly increased compared with that within 1 min, whereas the increase in the responding rate was not significantly different in αTC6 cells. This indicated that the response of αTC6 cells according to nerve stimulation occurred more rapidly and effectively than that of αTC6^siRNA-CADM1 cells, suggesting CADM1 involvement in promoting the interaction between nerves and α cells and among α cells. In addition, because we found that neurokinin (NK)-1 receptors, which are neuropeptide substance P receptors, were expressed to a similar extent by both cells, we investigated the effect of substance P on nerve–α cell interaction. Pretreatment with CP99,994 (0.1 μg/ml), an NK-1 receptor antagonist, reduced the responding rate of both cells, suggesting that substance P released from stimulated neurites was a mediator to activate αTC6 cells. In addition, α cells that were attached to neurites in a CADM1-mediated manner appeared to respond effectively to neurite activation via substance P/NK-1 receptors.     

NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells

We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated β-galactosidase activity and expression of p53, transforming growth factor (TGF)-β1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, β-galactosidase, TGF-β1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.     

C-terminal di-leucine motif of dopamine D₁ receptor plays an important role in its plasma membrane trafficking

The dopamine D₁ receptor (D₁R), a G protein-coupled receptor, plays a critical role in regulating blood pressure through its actions on renal hemodynamics and epithelial ion transport, which are highly linked to its intracellular trafficking. In this study, we generated a series of C-terminal mutants of D₁R that were tagged with or without enhanced yellow fluorescent protein, and analyzed the consequences of these mutants on the plasma membrane trafficking of D₁R and cyclic AMP response to D₁R stimulation. D₁R with mutations within the endocytic recycling signal (amino acid residues 360–382) continued to be functional, albeit decreased relative to wild-type D₁R. Mutation of the palmitoylation site (347C>S) of D₁R did not impair its trafficking to the plasma membrane, but abolished its ability to increase cyclic AMP accumulation. In contrast, replacement of di-leucines (344–345L>A) by alanines resulted in the retention of D₁R in the early endosome, decreased its glycosylation, and prevented its targeting to the plasma membrane. Our studies suggest that di-L motif at the C-terminus of D₁R is critical for the glycosylation and cell surface targeting of D₁R.     

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.     

On the role of Wnt/β-catenin signaling in stem cells

Background

Stem cells are mainly characterized by two properties: self-renewal and the potency to differentiate into diverse cell types. These processes are regulated by different growth factors including members of the Wnt protein family. Wnt proteins are secreted glycoproteins that can activate different intracellular signaling pathways.

Scope of review

Here we summarize our current knowledge on the role of Wnt/β-catenin signaling with respect to these two main features of stem cells.

Major conclusions

A particular focus is given on the function of Wnt signaling in embryonic stem cells. Wnt signaling can also improve reprogramming of somatic cells towards iPS cells highlighting the importance of this pathway for self-renewal and pluripotency. As an example for the role of Wnt signaling in adult stem cell behavior, we furthermore focus on intestinal stem cells located in the crypts of the small intestine.

General significance

A broad knowledge about stem cell properties and the influence of intrinsic and extrinsic factors on these processes is a requirement for the use of these cells in regenerative medicine in the future or to understand cancer development in the adult. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

A vacuolar β-glucosidase homolog that possesses glucose-conjugated abscisic acid hydrolyzing activity plays an important role in osmotic stress responses in Arabidopsis

The phytohormone abscisic acid (ABA) plays a critical role in various physiological processes, including adaptation to abiotic stresses. In Arabidopsis thaliana, ABA levels are increased both through de novo biosynthesis and via β-glucosidase homolog1 (BG1)-mediated hydrolysis of Glc-conjugated ABA (ABA-GE). However, it is not known how many different β-glucosidase proteins produce ABA from ABA-GE and how the multiple ABA production pathways are coordinated to increase ABA levels. Here, we report that a previously undiscovered β-glucosidase homolog, BG2, produced ABA by hydrolyzing ABA-GE and plays a role in osmotic stress response. BG2 localized to the vacuole as a high molecular weight complex and accumulated to high levels under dehydration stress. BG2 hydrolyzed ABA-GE to ABA in vitro. In addition, BG2 increased ABA levels in protoplasts upon application of exogenous ABA-GE. Overexpression of BG2 rescued the bg1 mutant phenotype, as observed for the overexpression of NCED3 in bg1 mutants. Multiple Arabidopsis bg2 alleles with a T-DNA insertion in BG2 were more sensitive to dehydration and NaCl stress, whereas BG2 overexpression resulted in enhanced resistance to dehydration and NaCl stress. Based on these observations, we propose that, in addition to the de novo biosynthesis, ABA is produced in multiple organelles by organelle-specific β-glucosidases in response to abiotic stresses.     

Substrate specificity plays an important role in uncoupling the catalytic and scaffolding activities of rat testis DNA topoisomerase IIα

Abstract

Topoisomerase II (topo II) is a dyadic enzyme found in all eukaryotic cells. Topo II is involved in a number of cellular processes related to DNA metabolism, including DNA replication, recombination and the maintenance of genomic stability. We discovered a correlation between the development of postnatal testis and increased binding of topo IIα to the chromatin fraction. We used this observation to characterize DNA-binding specificity and catalytic properties of purified testis topo IIα. The results indicate that topo IIα binds a substrate containing the preferred site with greater affinity and, consequently, catalyzes the conversion of form I to form IV DNA more efficiently in contrast to substrates lacking such a site. Interestingly, topo IIα displayed high-affinity and cooperativity in binding to the scaffold associated region. In contrast to the preferred site, however, high-affinity binding of topo IIα to the scaffold-associated region failed to result in enhanced catalytic activity. Intriguingly, competition assays involving scaffold-associated region revealed an additional DNA-binding site within the dyadic topo IIα. These results implicate a dual role for topo IIα in vivo consistent with the notion that its sequestration to the chromatin might play a role in chromosome condensation and decondensation during spermatogenesis.     

β-catenin plays a central role in setting up the head organizer in hydra

In an adult hydra the head organizer, located in the hypostome, is constantly active in maintaining the structure of the animal in the context of its steady state tissue dynamics. Several Wnt genes, TCF, and elevated levels of β-catenin are expressed in the hypostome as well as during the formation of a new organizer region in developing buds suggesting they play a role in the organizer. Transgenic hydra were generated in which a modified hydra β-catenin gene driven by an actin promoter is continuously expressed at a high level throughout the animal. These animals formed heads and secondary axes in multiple locations along the body column. Transplantation experiments indicate they have a high and stable level of head organizer activity throughout the body columns. However, none of the Wnt genes are expressed in the body columns of these transgenic animals. Further, in alsterpaullone-treated animals, which results in a transient rise in head organizer activity throughout the body column, the time of expression of the Wnt genes is much shorter than the time of the elevated level of head inducing activity. These results for the first time provide direct functional evidence that β-catenin plays a crucial role in the maintenance and activity of the head organizer and suggest that Wnt ligands may be required only for the initiation but not in maintenance of the organizer in Hydra.