The RXFP3 receptor is functionally associated with cellular responses to oxidative stress and DNA damage.

DNA damage response (DDR) processes, often caused by oxidative stress, are important in aging and -related disorders. We recently showed that G protein-coupled receptor (GPCR) kinase interacting protein 2 (GIT2) plays a key role in both DNA damage and oxidative stress. Multiple tissue analyses in GIT2KO mice demonstrated that GIT2 expression affects the GPCR relaxin family peptide 3 receptor (RXFP3), and is thus a therapeutically-targetable system. RXFP3 and GIT2 play similar roles in metabolic aging processes. Gaining a detailed understanding of the RXFP3-GIT2 functional relationship could aid the development of novel anti-aging therapies. We determined the connection between RXFP3 and GIT2 by investigating the role of RXFP3 in oxidative stress and DDR. Analyzing the effects of oxidizing (H2O2) and DNA-damaging (camptothecin) stressors on the interacting partners of RXFP3 using Affinity Purification-Mass Spectrometry, we found multiple proteins linked to DDR and cell cycle control. RXFP3 expression increased in response to DNA damage, overexpression, and Relaxin 3-mediated stimulation of RXFP3 reduced phosphorylation of DNA damage marker H2AX, and repair protein BRCA1, moderating DNA damage. Our data suggests an RXFP3-GIT2 system that could regulate cellular degradation after DNA damage, and could be a novel mechanism for mitigating the rate of age-related damage accumulation.

Indoxyl Sulfate and p-Cresyl Sulfate Promote Vascular Calcification and Associate with Glucose Intolerance.

BACKGROUND: Protein-bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (PCS) have been associated with cardiovascular morbidity and mortality in patients with CKD. However, direct evidence for a role of these toxins in CKD-related vascular calcification has not been reported. METHODS: To study early and late vascular alterations by toxin exposure, we exposed CKD rats to vehicle, IS (150 mg/kg per day), or PCS (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure). We also performed unbiased proteomic analyses of arterial samples coupled to functional bioinformatic annotation analyses to investigate molecular signaling events associated with toxin-mediated arterial calcification. RESULTS: Long-term exposure to either toxin at serum levels similar to those experienced by patients with CKD significantly increased calcification in the aorta and peripheral arteries. Our analyses revealed an association between calcification events, acute-phase response signaling, and coagulation and glucometabolic signaling pathways, whereas escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. Additional metabolic linkage to these pathways revealed that IS and PCS exposure engendered a prodiabetic state evidenced by elevated resting glucose and reduced GLUT1 expression. Short-term exposure to IS and PCS (before calcification had been established) showed activation of inflammation and coagulation signaling pathways in the aorta, demonstrating that these signaling pathways are causally implicated in toxin-induced arterial calcification. CONCLUSIONS: In CKD, both IS and PCS directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis.

Cell cycle-dependent O-GlcNAc modification of tobacco histones and their interaction with the tobacco lectin.

The Nicotiana tabacum agglutinin or Nictaba is a nucleocytoplasmic lectin that is expressed in tobacco after the plants have been exposed to jasmonate treatment or insect herbivory. Nictaba specifically recognizes GlcNAc residues. Recently, it was shown that Nictaba is interacting in vitro with the core histone proteins from calf thymus. Assuming that plant histones - similar to their animal counterparts - undergo O-GlcNAcylation, this interaction presumably occurs through binding of the lectin to the O-GlcNAc modification present on the histones. Hereupon, the question was raised whether this modification also occurs in plants and if it is cell cycle dependent. To this end, histones were purified from tobacco BY-2 suspension cells and the presence of O-GlcNAc modifications was checked. Concomitantly, O-GlcNAcylation of histone proteins was studied. Our data show that similar to animal histones plant histones are modified by O-GlcNAc in a cell cycle-dependent fashion. In addition, the interaction between Nictaba and tobacco histones was confirmed using lectin chromatography and far Western blot analysis. Collectively these findings suggest that Nictaba can act as a modulator of gene transcription through its interaction with core histones.

Tobacco BY-2 cells expressing fission yeast cdc25 bypass a G2/M block on the cell cycle.

The mitotic inducer gene from Schizosaccharomyces pombe, Spcdc25, was used as a tool to investigate regulation of G2/M in higher plants using the BY-2 (Nicotiana tabacum) cell line as a model. Spcdc25-expressing BY-2 cells exhibited a reduced mitotic cell size through a shortening of the G2 phase. The cells often formed isodiametric double files both in BY-2 cells and in cell suspensions derived from 35S::Spcdc25 tobacco plants. In Spcdc25-expressing cells, the tobacco cyclin-dependent kinase, NtCDKB1, showed high activity in early S phase, S/G2 and early M phase, whereas in empty vector cells CDKB1 activity was transiently high in early S phase but thereafter remained lower. Spcdc25-expressing cells also bypassed a block on G2/M imposed by the cytokinin biosynthetic inhibitor lovastatin (LVS). Surprisingly, cytokinins were at remarkably low levels in Spcdc25-expressing cells compared with the empty vector, explaining why these cells retained mitotic competence despite the presence of LVS. In conclusion, synchronised Spcdc25-expressing BY-2 cells divided prematurely at a small cell size, and they exhibited premature, but sustained, CDKB1 activity even though endogenous cytokinins were virtually undetectable.

Identification of four adenosine kinase isoforms in tobacco By-2 cells and their putative role in the cell cycle-regulated cytokinin metabolism.

Adenosine kinase (ADK), a key enzyme in the regulation of the intracellular level of adenosine is also speculated to be responsible for the conversion of cytokinin ribosides to their respective nucleotides. To elucidate the role of ADK in the cytokinin metabolism of tobacco BY-2 cells (Nicotiana tabacum cv. "Bright Yellow-2"; TBY-2), we have identified and characterized the full-length cDNAs encoding four ADK isoforms of N. tabacum and determined their catalytic properties. The four TBY-2 ADK isoforms (designated 1S, 2S, 1T, and 2T) display a high affinity for both adenosine (Km 1.88-7.30 microm) and three distinct types of cytokinin ribosides: isopentenyladenosine; zeatin riboside; and dihydrozeatin riboside (Km 0.30-8.71 microm). The Vmax/Km values suggest that ADK2S exhibits in vitro an overall higher efficiency in the metabolism of cytokinin ribosides than the other three isoforms. The expression pattern of NtADK genes is modulated significantly during the cell cycle. We suggest that the increased transcript accumulation of NtADK coupled to an increased ADK activity just prior to mitosis is associated with a very active cytokinin metabolism at that phase of the cell cycle of synchronized TBY-2 cells.

Jasmonic acid prevents the accumulation of cyclin B1;1 and CDK-B in synchronized tobacco BY-2 cells.

Jasmonic acid (JA) plays a crucial role in plant fertility and defense responses. It exerts an inhibitory effect on plant growth when applied exogenously. This effect seems to be somehow related to a negative regulation of cell cycle progression in the meristematic tissues. In this report, we focus on the molecular events that occur during JA-induced G2 arrest. We demonstrate that JA prevents the accumulation of B-type cyclin-dependent kinases and the expression of cyclin B1;1, which are both essential for the initiation of mitosis. This feature suggests the existence of an early G2 checkpoint that is affected by JA.