MicroRNA-532-3p Regulates Pro-Inflammatory Human THP-1 Macrophages by Targeting ASK1/p38 MAPK Pathway.

Inflammation is a complex biological process which alters the normal physiological function of the immune system resulting in an abnormal microenvironment that leads to several clinical complications. The process of inflammation is mediated through various intracellular signaling factors inside the cells. Apoptosis signal-regulating kinase 1 (ASK1) is an inflammation-derived kinase that controls the activation of other family of kinases such as p38 mitogen-activated protein kinases (p38 MAPKs), which mediates various the inflammatory processes. In this study, we cultured THP-1 macrophage cells to undergo inflammatory proliferation with LPS (1 µg/ml) and TNFα (10 ng/ml) stimulation. Initial in silico analysis was utilized to predict novel microRNAs (miRNAs) that target ASK1 signaling and its expression levels in LPS and TNFα stimulated THP-1 cells were estimated. Among the miRNAs, miR-532-3p showcased the highest binding affinity towards ASK1 kinase. We witnessed that transient transfection of miR-532-3p diminished the levels of ASK1 and downstream phosphorylation/translocation of p38 MAPK. Furthermore, direct targeting of ASK1 resulted in regulation of uncontrolled release of cytokines (TNFα, IL-6, and IL-23) and chemokines (GM-CSF and MIP-2α). Overall, we suggest that miR-532-3p attenuates the pro-inflammatory nature of macrophages by targeting ASK1/p38 MAPK signaling pathway and can be used as a molecular intervention for treating inflammatory diseases.

miR-506-3p alleviates uncontrolled osteoclastogenesis via repression of RANKL/NFATc1 signaling pathway.

Bone erosion is the major cause of deformities in autoimmune disease conditions such as osteoporosis and rheumatoid arthritis. Aberrant receptor activator of nuclear factor kappa B ligand (RANKL) secretion in bone disorders have been implicated to promote uncontrolled osteoclast differentiation through the regulation of nuclear factor of activated T cells 1 (NFATc1) transcription factor. This phenomenon is governed by several molecular factors including microRNAs, which are under-expressed during disease progression. This report focuses on elucidating the molecular mechanism of miR-506-3p towards the RANKL/NFATc1 pathway. miR-506-3p showed high binding affinity towards NFATc1 (ΔG = -22.4 kcal/mol). Bone marrow-derived macrophages (BMMs) isolated from rats stimulated with RANKL (100 ng/ml) showed active expression of NFATc1 which differentiated into mature osteoclasts. Moreover, NFATc1 activation resulted in downstream secretion of various bone resorptive enzymes (cathepsin K, carbonic anhydrase II, tartarate acid phosphatase, and matrix metalloproteinase 9) which lead to active bone resorption. However, transfection of miR-506-3p resulted in selective repression of NFATc1 inside the cells. This further resulted in the diminished release of bone resorptive enzymes that were essential for the degradation of the bone. Overall, we predict that miR-506-3p can be used as a molecular intervention for RANKL/NFATc1 mediated osteoclastogenesis.

Berberine encapsulated PEG-coated liposomes attenuate Wnt1/ß-catenin signaling in rheumatoid arthritis via miR-23a activation.

Bone erosion is a debilitating pathological process of osteopathic disorder like rheumatoid arthritis (RA). Current treatment strategies render low disease activity but with disease recurrence. To find an alternative, we designed this study with an aim to explore the underlying therapeutic effect of PEGylated liposomal BBR (PEG-BBR) against Wnt1/ß-catenin mediated bone erosion in adjuvant-induced arthritic (AA) rat model and fibroblast-like synoviocytes (FLS) with reference to microRNA-23a (miR-23a) activity. Our initial studies using confocal microscopy and Near-Infrared Imaging (NIR) showed successful internalization of PEG-BBR and PEG-miR-23a in vitro and in vivo respectively and was retained till 48 h. The preferential internalization of PEG-BBR into the inflamed joint region significantly reduced the gene and protein level expression of major Wnt1 signaling mediators and reduced bone erosion in rats. Moreover, PEG-BBR treatment in FLS cells attenuated the gene and protein expression levels of FZD4, LRP5, ß-catenin, and Dvl-1 through the induction of CYLD. Furthermore, inhibition of these factors resulted in reduced bone loss and increased calcium retainability by altering the RANKL/OPG axis. PEG-BBR treatment markedly inhibited the expression of LRP5 protein on par with the DKK-1 (LRP5/Wnt signaling inhibitor) and suppressed the transcriptional activation of ß-catenin inside the cells. We further witnessed that miR-23a altered the expression levels of LRP5 through RNA interference. Overall, our findings endorsed that miR-23a possesses a multifaceted therapeutic efficiency like berberine in RA pathogenesis and can be considered as a potential candidate for therapeutic targeting of Wnt1/ß-catenin signaling in RA disease condition.

MiR-145-5p mitigates dysregulated Wnt1/ß-catenin signaling pathway in rheumatoid arthritis.

Fibroblast-like synoviocytes (FLS) lining the arthritic synovial joint region have been implicated to be a key player in bone remodeling. The uncontrolled proliferation of this cell subtype is strictly regulated by various molecular elements including microRNAs (miRNAs). The Wnt1/ß-catenin signaling pathway plays a crucial role in the survival of FLS cells. This study explores the underlying mechanism of miR-145-5p towards the Wnt1/ß-catenin pathway. MiR-145-5p depicted a strong binding affinity towards frizzled class receptor 4 (FZD4) 3' UTR, a key receptor complex essential for recognizing circulating Wnt1 molecules. Adjuvant induced arthritic fibroblast-like synoviocytes (AA-FLS) isolated from rats stimulated with Wnt1 (10 ng/ml) elicited active Wnt1/ß-catenin signaling. Transfection of miR-145-5p mimic (50 pmol) to AA-FLS stimulated with Wnt1 elicited reduced expression levels of various factors of Wnt1/ß-catenin signaling including low-density lipoprotein receptor-related protein 5 (LRP5), dishevelled segment polarity protein 1 (Dvl1) and ß-catenin transcription factor. Moreover, pro-inflammatory cytokines (TNFα, IL-1ß, IL-6 and IL-23) were regulated compared to the diseased groups. Furthermore, miR-145-5p counterbalanced the levels of receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) at the cellular level, essential for bone remodeling. Hence, we suggest that miR-145-5p regulates the survival/proliferation of FLS cells in RA disease condition through attenuation of Wnt1/ß-catenin signaling.

Berberine coated mannosylated liposomes curtail RANKL stimulated osteoclastogenesis through the modulation of GSK3ß pathway via upregulating miR-23a.

Drug-induced microRNAs manifest significant therapeutic approaches; however, such progress in the treatment of osteopathic disorders including osteoporosis and rheumatoid arthritis still remains obscure. Contrarily, non-specific drug delivery, at high doses, increases the risk of side effects and reduces drug therapeutic efficacy. Accordingly, the present study was designed to examine the therapeutic effect of berberine coated mannosylated liposomes (ML-BBR) on RANKL (100 ng/ml) stimulated bone marrow-derived monocytes/macrophages (BMMs) via altering miR-23a expression. Initial studies using confocal microscopy showed successful internalization of ML-BBR in RANKL stimulated BMMs. Treatment with ML-BBR abrogated the increased osteoclast formation in BMM cells via inhibiting phosphorylated glutathione synthase kinase beta (p-GSK3ß) mediated NFATc1 activation. Consequently, ML-BBR also attenuated the expression of bone-degrading enzymes (TRAP, cathepsin K and MMP-9) thereby inhibiting the bone resorptive activity of osteoclasts. Moreover, ML-BBR induced the expression levels of miR-23a at the gene level, which in turn attenuated GSK3ß/p-GSK3ß expression as confirmed via blotting analysis. Further miR-23a inhibition of the GSK3ß phosphorylation was confirmed using luciferase reporter assay. Comparatively, LY2090314 (GSK3ß inhibitor) treatment inhibited the protein level expression of GSK3ß/p-GSK3ß. However, LY2090314 treatment induced a basal level expression of miR-23a owing to the suggestion that ML-BBR has an influential role in upregulating miR-23a level to inhibit GSK-3ß phosphorylation. Cumulatively, our findings endorsed that preferential internalization of ML-BBR by BMMs effectively modulated the RANKL/p-GSK3ß pathway and curtailed the osteoclast-mediated bone erosion possibly through post-transcriptional gene silencing via miR-23a.

Berberine modulates ASK1 signaling mediated through TLR4/TRAF2 via upregulation of miR-23a.

The current study was designed to explore the underlying therapeutic effect of berberine (BBR), an alkaloid compound against LPS (1 µg/ml)/TNFα (10 ng/ml) mediated apoptosis signal-regulating kinase 1 (ASK1) signaling in RAW 264.7 macrophages and adjuvant-induced arthritic synovial macrophages (AA-SM) with relation to miR-23a levels. LPS and TNFα stimulation abrogated the expression of miR-23a resulting in TLR4/TRAF2 mediated ASK1 activation and downstream phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). BBR (25-75 µM) treatment ameliorated the gene expression levels of TLR4, TRAF2, TNFα, IL-6, and IL-23 through the upregulation of miR-23a. Subsequently, BBR suppressed the levels of TLR4/TRAF2 mediated phosphorylation of ASK1/p38 and attenuated the expression of various pro-inflammatory cytokines (TNFα, IL-6 & IL-23) in RAW 264.7 macrophages and AA-SM cells. BBR was able to counteract these factors through activation of miR-23a levels in LPS/TNFα stimulated RAW 264.7 macrophages and AA-SM cells. NQDI1 (30 µM) treatment inhibited ASK1 activation resulting in basal levels of miR-23a, owing to the conclusion that ASK1 activation downregulates miR-23a levels inside the cells. Overall, our current findings predict that BBR is a potential candidate for therapeutic targeting of TLR4/TRAF2 mediated ASK1 activation in inflammatory and in RA pathogenesis possibly through post-transcriptional gene silencing via upregulation of miR-23a.

MicroRNAs and bioactive compounds on TLR/MAPK signaling in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune mediated joint disease with severe complications affecting 1% of the population worldwide. Although the exact mechanism underlying the aggravation of RA remains unknown, its occurrence can lead to joint degradation and functional disability. Recent evidences have shown that the aberrant expression of microRNAs (miRNAs) play a prominent role in the furtherance of RA. Over the last decade, various intensive studies have validated different microRNAs to be good candidates for diagnostic purposes and for monitoring the disease progression in various inflammatory diseases. A deeper understanding of the molecular mechanism through which miRNAs amplify the production of inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-17), pro-inflammatory mediators, growth factors and MMPs will act as potential therapeutic targets. More importantly, several studies have briefly reported the crucial role of TLR dependent MAPK signaling pathway, which mediates the pathological features of RA. In this review, we summarize the recent findings and provide a detailed report of the molecular mechanism of microRNA along with the role of TLR/MAPK signaling pathway in RA. However, the major aim of this review is to correlate the aberrantly expressed microRNAs in TLR/MAPK pathway with various well reported bioactive compounds that can modulate these signaling pathways in rheumatoid arthritis. Targeting miRNA expression using specific bioactive compounds might be a potent and an effective target in RA treatment by suppressing the TLR/MAPK pathway.