Osteoarthritis (OA) is the most common musculoskeletal disorder and the only effective treatment is surgical joint replacement. MicroRNAs (miRNA) are a class of non-coding RNAs regulating gene expression. Role of specific miRNAs in OA pathogenesis is yet to be defined. Our preliminary results showed that Hsa-MIR-323B-5P (miR-323b- 5p), with no known function, was downregulated several fold in IL-1?-stimulated chondrocytes. In silico analysis identified Sonic Hedgehog (SHH) mRNA as a target of miR-323b-5p. SHH signaling is associated with the expression of MMP-13 and cartilage degeneration and inhibition of SHH attenuates the severity of OA. Butea monosperma (Lam) is widely distributed in India and water extract of Butea monosperma flowers (BME) is used to treat arthritis. Our preliminary results show that IL-1? stimulates the expression of SHH in OA chondrocytes. Of note, BME modulated the SHH signaling genes expression and blocked the SHH-induced expression of MMP-13 in cartilage explants and that miR-323b-5p inhibited SHH protein expression by directly targeting coding region in the mRNA. We propose to test the cartilage protective activity of BME in vitro and in vivo using well described assays and a preclinical animal model of OA. Our basic hypothesis is that "BME suppresses the IL-1?-induced cartilage catabolic effects in OA via post-transcriptional regulation of SHH protein expression by modulating the expression of miR-323b-5p in human chondrocytes". A corollary of this hypothesis is that "bioactive constituents of BME may exert their cartilage protective effects in OA by modulating the expression of specific miRNAs that negatively regulate the expression of SHH and other catabolic factors in vivo".
Specific Aim -1: Determine (a) the effect of BME on IL-1?- induced expression of SHH and its receptor PTCH1;and (b) the effect of BME on the SHH-induced expression of PTCH1, GLI-1, and MMP-13 in human OA chondrocytes and cartilage explants in vitro. Using microarray profiling we will also (c) identify additional miRNAs whose expression is modulated by IL-1? in human OA chondrocytes and bioinformatically determine if additional miRNAs target SHH mRNA;and (d) validate the interactions of newly identified additional miRNAs with SHH mRNA using reporter assays.
Specific Aim -2: We will analyze the effect of IL-1? on the mRNA expression profile (Transcriptome) in chondrocytes with altered miR-323b-5p expression and determine whether the genes with altered expression are also targets of miR-323b-5p.
Specific Aim -3: Using a rabbit model of OA we will characterize the expression profile of SHH and of the miRNAs in the joints during disease induction and progression. The articular cartilage will be evaluated macroscopically and histologically. Synovial fluid and serum will be analyzed for (a) levels of inflammatory cytokines (IL-1?, TNF-?, IL-6);(b) expression of secreted MMP-2, -9,-13;and (c) in animals given two different doses of BME and Isobutrin we will determine the levels of BME constituents (Butein, Butrin, Isobutrin) by LC/MS. We will also examine the effect of BME and Isobutrin consumption on the expression levels of miR-323b-5p, MMP-13 and SHH mRNA and protein in the joints and correlate with disease induction and progression.
Safe and effective treatment of Osteoarthritis (OA) remains an unmet clinical need that can be addressed by developing less expensive oral therapies. We propose using plant-derived orally bioavailable agents with little or no toxicity to block or inhibt the cartilage degradation in OA. Studies in this project will investigate the mechanism of cartilage/chondroprotection by Butea monosperma flower extract (BME). BME is widely used in Ayurveda for the treatment of arthritis and other inflammatory diseases in India. Our studies will focus on the role of specific microRNAs in cartilage homeostasis and will test whether BME inhibit cytokine-induced down-regulation of specific microRNAs that target the expression of Sonic Hedgehog (SHH) mRNA in human cartilage cells. SHH has recently been identified as a catabolic factor in OA. The proposed studies are highly relevant to the goals of the NIH and may be beneficial to the society because they focus on developing a novel and safe therapy for OA.
|Haseeb, Abdul; Makki, Mohammad Shahidul; Haqqi, Tariq M (2014) Modulation of ten-eleven translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) expression, ?-Ketoglutarate (?-KG), and DNA hydroxymethylation levels by interleukin-1? in primary human chondrocytes. J Biol Chem 289:6877-85|