Increased sensitivity to apoptosis induced by methotrexate is mediated by JNK

Spurlock, Charles F.; Aune, Zachary T.; Tossberg, John T.; Collins, Patrick L.; Aune, Jessica P.; Huston, Joseph W.; Crooke, Philip S.; Olsen, Nancy J.; Aune, Thomas M.

doi:10.1002/art.30457

Cited by 51 publications

(46 citation statements)

References 61 publications

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“…The statistical analysis also suggests a time-to-therapy effect whose functional PBMC characterization was explored for the two active treatments (MS versus MTX at day 34, Fig. 1d, Supplementary Table S1 and Supplementary Data S2) highlighting 3 major functional areas: MTX’s distinctive nucleic acid metabolism and regulation of transcription , in line with known effects of the drug8; lymphocyte activation and differentiation in both treatments, also in line with MTX immunomodulatory effects9; and wound healing related processes, peculiar to MS. Wound healing (EMT, type2 ref.…”

Section: Resultsmentioning

confidence: 98%

Systemic Wound Healing Associated with local sub-Cutaneous Mechanical Stimulation

Nardini¹,

Devescovi²,

Liu³

et al. 2016

Sci Rep

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Degeneration is a hallmark of autoimmune diseases, whose incidence grows worldwide. Current therapies attempt to control the immune response to limit degeneration, commonly promoting immunodepression. Differently, mechanical stimulation is known to trigger healing (regeneration) and it has recently been proposed locally for its therapeutic potential on severely injured areas. As the early stages of healing consist of altered intra- and inter-cellular fluxes of soluble molecules, we explored the potential of this early signal to spread, over time, beyond the stimulation district and become systemic, to impact on distributed or otherwise unreachable injured areas. We report in a model of arthritis in rats how stimulations delivered in the subcutaneous dorsal tissue result, over time, in the control and healing of the degeneration of the paws’ joints, concomitantly with the systemic activation of wound healing phenomena in blood and in correlation with a more eubiotic microbiome in the gut intestinal district.

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Section: Resultsmentioning

confidence: 98%

Systemic Wound Healing Associated with local sub-Cutaneous Mechanical Stimulation

Nardini¹,

Devescovi²,

Liu³

et al. 2016

Sci Rep

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“…Previously we have shown that sub-micromolar concentrations of MTX induces transcription of JUN in a homogeneous T cell population (Jurkat T cells) by DHFR-dependent depletion of BH4 resulting in uncoupling of NOS and corresponding increased production of reactive oxygen species (ROS) and JNK activation (20). Therefore, we employed this model system to investigate MTX-induction of TP53, CDKN1A, RANGAP1 and CHEK2 .…”

Section: Resultsmentioning

confidence: 99%

“…MTX-mediated JNK activation results in induction of pro-apoptotic target genes and increased sensitivity to 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.…”

Section: Introductionmentioning

confidence: 99%

Methotrexate increases expression of cell cycle checkpoint genes via JNK activation

Spurlock

Tossberg²,

Fuchs

et al. 2012

Arthritis & Rheumatism

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Objective To assess defects in expression of critical cell cycle checkpoint genes and proteins in subjects with rheumatoid arthritis relative to presence or absence of methotrexate medication and assess the role of Jun N-terminal kinase in methotrexate induction of these genes. Methods Flow cytometry analysis was used to quantify changes in intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) was employed to determine changes in cell cycle checkpoint target genes. Results RA subjects express lower baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and total JNK protein. MAPK9, TP53, CDKN1A, and CDKN1B mRNA expression, but not CHEK2, and RANGAP1, is higher in patients on low-dose MTX therapy. Further, JNK levels inversely correlate with CRP levels in RA patients. In tissue culture, MTX induces expression of both p53 and p21 by JNK2 and JNK1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 are not induced by MTX. MTX also induces ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocks these MTX-mediated effects. Conclusions Our findings support the notion that MTX restores some, but not all of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells by a JNK dependent pathway.

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“…Immunoblotting was performed as described (9). Whole cell lysates were resolved by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes.…”

Section: Methodsmentioning

confidence: 99%

Methotrexate Inhibits NF‐κB Activity Via Long Intergenic (Noncoding) RNA–p21 Induction

Spurlock

Tossberg

Matlock

et al. 2014

Arthritis & Rheumatology

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131

104

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Objective. We sought to determine interrelationships among expression of lincRNA-p21, a long intergenic non-coding RNA, activity of NF-κB, and responses to methotrexate in rheumatoid arthritis (RA) by analyzing patient samples and cell culture models. Methods. Expression levels of long non-coding RNAs and messenger RNAs were determined by quantitative reverse transcription-polymerase chain reaction. Western blotting and flow cytometry were used to quantify levels of intracellular proteins. Intracellular NF-κB was determined using an NF-κB luciferase reporter plasmid. Results. RA patients expressed reduced basal levels of lincRNA-p21 and increased basal levels of phosphorylated p65 (RelA), a marker of NF-κB activation. RA subjects not receiving MTX expressed lower levels of lincRNA-p21 and higher levels of phosphorylated p65 compared to RA subjects receiving low-dose MTX. In cell culture using primary cells and transformed cell lines, we found that MTX induced lincRNA-p21 through a DNA-PKcs-dependent mechanism. Deficiencies of PRKDC mRNA levels in RA subjects were also corrected by MTX, in vivo. Further, MTX lowered NF-κB activity in TNF-α treated cells through a DNA-PKcs-dependent mechanism via induction of lincRNA-p21. Finally, we found that depressed levels of TP53 and lincRNA-p21 increased NF-κB activity in cell lines. Decreased levels of lincRNA-p21 did not alter NFKB1 or RELA transcripts. Rather, lincRNA-p21 physically bound to RELA mRNA. Conclusion. Our findings support a model whereby depressed levels of lincRNA-p21 in RA contribute to increased NF-κB activity. MTX decreases basal levels of NF-κB activity by increasing lincRNA-p21 through a DNA-PKcs dependent mechanism.

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Increased sensitivity to apoptosis induced by methotrexate is mediated by JNK

Cited by 51 publications

References 61 publications

Systemic Wound Healing Associated with local sub-Cutaneous Mechanical Stimulation

Systemic Wound Healing Associated with local sub-Cutaneous Mechanical Stimulation

Methotrexate increases expression of cell cycle checkpoint genes via JNK activation

Methotrexate Inhibits NF‐κB Activity Via Long Intergenic (Noncoding) RNA–p21 Induction

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