In the broad sense, defects in DNA damage responses have been linked to infertility, cardiovascular disease, and metabolic syndrome (47)

In the broad sense, defects in DNA damage responses have been linked to infertility, cardiovascular disease, and metabolic syndrome (47). to Urocanic acid cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway. Keywords:Rheumatoid Arthritis (RA), Disease-Modifying Antirheumatic Drugs (DMARDS), Methotrexate (MTX), Apoptosis, p53, p21, Jun N-terminal kinase (JNK), Tetrahydrobiopterin (BH4), Reactive Oxygen Urocanic acid Species (ROS) == INTRODUCTION == Rheumatoid arthritis is the most common serious autoimmune disease affecting 1.3 million people in the United States (1). Often characterized by bone erosion and cartilage destruction through chronic inflammation, RA is a multisystem disorder affecting synovial spaces between small and large joints. Accounting for 250,000 hospitalizations and 9 million clinic visits each year, costs associated with RA represent approximately 1% of the U.S. gross domestic product (2). While initially developed as a chemotherapeutic, methotrexate (MTX) has been the mainstay for RA treatment since the 1980s (3-7). Once-weekly administration of 7.5 to 25 milligrams yields optimal clinical outcomes, compared to the 5000 mg/week dosage LGR3 used in the treatment of malignancy (7,8). MTX is a potent, competitive inhibitor of dihydrofolate reductase (DHFR) (9-11) resulting in decreased tetrahydrofolate levels, and inhibition of de novo purine and pyrimidine synthesis leading to cell cycle arrest (8,12). However, mechanisms of action surrounding low-dose, once weekly MTX may differ significantly from high dose therapy with MTX. Since supplementation with low doses of folic acid, 1-5 mg/day, in RA patients does not attenuate clinical efficacy of MTX, the anti-inflammatory actions of low-dose MTX may stem from alternative pathways (8,13,14). Although the etiology of RA is incompletely understood, T lymphocytes from subjects with RA exhibit loss of genomic integrity and deficiencies in specific proteins that repair DNA damage and induce cell cycle arrest and apoptosis. Specifically, reduced expression of ataxia telangiectasia (AT) mutated (ATM), a critical component of DNA damage repair and activation of p53-dependent cell cycle arrest and apoptosis, p53 itself, checkpoint kinase 2, which also phosphorylates p53, and cyclin kinase inhibitors, p21 and p27, contribute to these defects in RA (15-19). We recently found that Jun-N-terminal kinase (JNK), a MAP kinase, is activated by MTX through production of reactive oxygen species (ROS) due to uncoupling of nitric oxide synthase (NOS) arising from MTX-dependent inhibition of DHFR, which blocks reduction of dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4). MTX-mediated JNK activation results in induction of pro-apoptotic target genes and increased sensitivity to Urocanic acid apoptosis (20). Since JNKs, members of the MAP kinase family of proteins, also directly phosphorylate p53 leading to its increased accumulation and activity (21-23) we hypothesized that JNKs, or MAPKs in general, may also be deficient in RA and that MTX therapy may correct, not only JNK deficiency, but also deficiencies in critical regulators of cell cycle checkpoints. Here, we show that RA lymphocytes exhibit a Urocanic acid selective deficiency inMAPK9(JNK2), but not other MAPK transcripts.MAPK9, TP53, CDKN1A, andCDKN1Btranscript levels, along with total JNK protein levels, Urocanic acid are significantly lower in RA subjects compared to healthy control subjects (CTRL). Further,MAPK9, TP53, CDKN1A, andCDKN1Btranscript levels, but notCHEK2andRANGAP1, are elevated in RA subjects taking MTX compared to RA subjects not taking MTX. In cell culture models, MTX directly induces increased expression of p53, p21, and p27, but notCHEK2andRANGAP1. We hypothesize that therapeutic activity of MTX may arise, in part, from its ability to restore expression levels of key proteins required for cell cycle checkpoint.