Mohammad Shahidul Makki

Modulation of Ten-Eleven Translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) Expression, α-Ketoglutarate (α-KG), and DNA Hydroxymethylation Levels by Interleukin-1β in Primary Human Chondrocytes

Background: Cytosine hydroxymethylation in the genomic DNA controls gene expression. Results: Treatment of primary chondrocytes with IL-1β alone or in conjunction with TNF-α inhibited TET1 and IDH expression and suppressed cytosine hydroxymethylation. Conclusion: IL-1β and TNF-α decrease DNA hydroxymethylation by suppressing the expression and activity of TET1 and IDH. Significance: Pro-inflammatory cytokines likely control gene expression by modulating the 5-hmC levels. 5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1–3 (TET1–3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1β and TNF-α are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1β and TNF-α on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be ∼0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1β alone or in combination with TNF-α. We observed a dramatic (10–20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2–3-fold) in TET3 expression in chondrocytes stimulated with IL-1β and TNF-α. IL-1β and TNF-α significantly suppressed the activity and expression of IDHs, which correlated with the reduced α-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1β and TNF-α, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.

MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes.

Hedgehog (HH) signaling has recently been associated with cartilage degradation in osteoarthritis (OA). Because interleukin‐1β (IL‐1β) has been implicated as a principal instigator of OA, we sought to determine whether IL‐1β induces the expression of sonic HH (SHH) and its regulation by microRNAs (miRNAs) in human chondrocytes.

MicroRNA-9 Promotion of Interleukin-6 Expression by Inhibiting Monocyte Chemoattractant Protein–Induced Protein 1 Expression in Interleukin-1β–Stimulated Human Chondrocytes

Enhanced expression of interleukin‐6 (IL‐6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein–induced protein 1 (MCPIP‐1) is a novel posttranscriptional regulator of IL‐6 expression and is targeted by microRNA‐9 (miR‐9). We investigated the expression of MCPIP‐1 in OA cartilage and explored whether targeting of MCPIP‐1 by miR‐9 contributes to enhanced IL‐6 expression in OA.

miR-139 modulates MCPIP1/IL-6 expression and induces apoptosis in human OA chondrocytes

IL-6 is an inflammatory cytokine and its overexpression plays an important role in osteoarthritis (OA) pathogenesis. Expression of IL-6 is regulated post-transcriptionally by MCPIP1. The 3′ untranslated region (UTR) of MCPIP1 mRNA harbors a miR-139 ‘seed sequence’, therefore we examined the post-transcriptional regulation of MCPIP1 by miR-139 and its impact on IL-6 expression in OA chondrocytes. Expression of miR-139 was found to be high in the damaged portion of the OA cartilage compared with unaffected cartilage from the same patient and was also induced by IL-1β in OA chondrocytes. Inhibition of miR-139 decreased the expression of IL-6 mRNA by 38% and of secreted IL-6 protein by 40%. However, overexpression of miR-139 increased the expression of IL-6 mRNA by 36% and of secreted IL-6 protein by 56%. These data correlated with altered expression profile of MCPIP1 in transfected chondrocytes. Studies with a luciferase reporter construct confirmed the interactions of miR-139 with the ‘seed sequence’ located in the 3′ UTR of MCPIP mRNA. Furthermore, miR-139 overexpression increased the catabolic gene expression but expression of anabolic markers remained unchanged. Overexpression of miR-139 also induced apoptosis in OA chondrocytes. Importantly, we also discovered that IL-6 is a potent inducer of miR-139 expression in OA chondrocytes. These findings indicate that miR-139 functions as a post-transcriptional regulator of MCPIP1 expression and enhances IL-6 expression, which further upregulates miR-139 expression in OA chondrocytes. These results support our hypothesis that miR-139-mediated downregulation of MCPIP1 promotes IL-6 expression in OA. Therefore, targeting miR-139 could be therapeutically beneficial in the management of OA.

Histone Deacetylase Inhibitor Vorinostat (SAHA) Suppresses IL-1β–Induced Matrix Metallopeptidase-13 Expression by Inhibiting IL-6 in Osteoarthritis Chondrocyte

Osteoarthritis (OA) is the most common whole-joint disease and is characterized by progressive loss of the cartilage matrix. Matrix metallopeptidase-13 (MMP-13) is a highly active and an abundantly expressed protease in OA cartilage and chondrocytes and degrades type II collagen and proteoglycans. We investigated the mechanism of MMP-13 suppression by histone deacetylase inhibitor vorinostat (SAHA). OA chondrocytes were obtained from knee cartilage after enzymatic digestion and treated with IL-1β in the absence or presence of various histone deacetylase inhibitors. Gene expression was quantified using quantitative RT-PCR. Protein expression and chromatin modifications were determined by Western immunoblotting using specific antibodies. The effect of IL-6 on the expression of MMP-13 was determined by treating chondrocytes with recombinant IL-6 or by IL6 knockdown using IL6-specific siRNA. We found that SAHA is a potent suppressor of IL-1β-induced MMP-13, tumor necrosis factor-α, and other catabolic marker expression in OA chondrocytes. Interestingly, SAHA rescued the COL2A1 and ACAN expression in OA chondrocytes that was down-regulated by IL-1β. Of importance is our finding that IL-6-stimulated MMP-13 expression was independent of IL-1β stimulation and was blocked by SAHA, suggesting that SAHA inhibits IL-6 signaling in OA chondrocytes. Taken together, our results suggest that SAHA could be used as a therapeutic agent for the management of OA.

Histone deacetylase inhibitor vorinostat (SAHA, MK0683) perturb miR-9-MCPIP1 axis to block IL-1β-induced IL-6 expression in human OA chondrocytes

ABSTRACT Aim of the Study: High levels of IL-6 are believed to contribute to osteoarthritis (OA) pathogenesis. The expression of IL-6 is regulated post-transcriptionally by the miR-9-MCPIP-1 axis in chondrocytes. Vorinostat (SAHA) inhibits the IL-6 expression in OA chondrocytes. We investigated whether SAHA suppresses the expression of IL-6 by perturbing the miR-9-MCPIP1 axis in OA chondrocytes under pathological conditions. Materials and Methods: OA chondrocytes were isolated by enzymatic digestion and treated with IL-1β in the absence or presence of SAHA. Genes and protein expression levels were determined by TaqMan assays and Western blotting, respectively. Secreted IL-6 was quantified by enzyme linked immunosorbent assay (ELISA). MCPIP1 promoter deletion mutants were generated by polymerase chain reaction (PCR). Promoter recruitment of transcription factors was determined by ChIP. Nuclear run-on was employed to measure the ongoing transcription. siRNA-mediated knockdown of the CEBPα expression was employed for loss of function studies. Results: Expression of MCPIP1 was high in SAHA treated OA chondrocytes but expression of IL-6 mRNAs and secreted IL-6 were reduced by ~70%. SAHA suppressed the expression of miR-9 but enhanced the activity of the MCPIP1 promoter localized to a 156bp region which also harbors the binding site for CEBPα. Treatment with SAHA enhanced the recruitment of CEBPα to the MCPIP1 promoter. Ectopically expressed CEBPα enhanced the promoter activity and the expression of MCPIP1 while siRNA-mediated knockdown of CEBPα inhibited the expression of MCPIP1. Conclusions: Taken together our data indicate that SAHA-mediated suppression of the IL-6 expression is achieved through increased recruitment of CEBPα to the MCPIP1 promoter and by relieving the miR-9-mediated inhibition of MCPIP1 expression in OA chondrocytes.

MicroRNA-9 promotes IL-6 expression by inhibiting MCPIP1 expression in IL-1β-stimulated human chondrocytes

Enhanced expression of interleukin‐6 (IL‐6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein–induced protein 1 (MCPIP‐1) is a novel posttranscriptional regulator of IL‐6 expression and is targeted by microRNA‐9 (miR‐9). We investigated the expression of MCPIP‐1 in OA cartilage and explored whether targeting of MCPIP‐1 by miR‐9 contributes to enhanced IL‐6 expression in OA.

Deep sequencing and analyses of miRNAs, isomiRs and miRNA induced silencing complex (miRISC)-associated miRNome in primary human chondrocytes

MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1β treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5′ and 3′ ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1β, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to IL-1β treatment. These findings will provide an insight to the miRNA-mediated control of gene expression in the pathogenesis of OA.

An effective and efficient method of transfecting primary human chondrocytes in suspension

Human chondrocytes accumulate an ECM-rich matrix by secreting matrix macromolecules during monolayer culture, which makes them difficult to transfect efficiently. Here we report a non-viral based protocol to transfect the primary human chondrocytes with high efficiency in suspension. Chondrocyte cultures were digested using Pronase and Collagenase and transfected in suspension. Transfection efficiencies of more than 80% were achieved routinely using the protocol described. The viability of siRNA transfected or un-transfected chondrocytes was not affected and resulted in 80-90% knockdown of the target mRNA levels. This protocol may be useful in gene knockdown, and ectopic overexpression studies in chondrocytes.

MicroRNA-9 Promotion of Interleukin-6 Expression by Inhibiting Monocyte Chemoattractant Protein-Induced Protein 1 Expression in Interleukin-1β-Stimulated Human Chondrocytes: MicroRNA-9 PROMOTION OF IL-6 EXPRESSION IN HUMAN CHONDROCYTES

Enhanced expression of interleukin‐6 (IL‐6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein–induced protein 1 (MCPIP‐1) is a novel posttranscriptional regulator of IL‐6 expression and is targeted by microRNA‐9 (miR‐9). We investigated the expression of MCPIP‐1 in OA cartilage and explored whether targeting of MCPIP‐1 by miR‐9 contributes to enhanced IL‐6 expression in OA.