Combination of retinoids and narrow-band ultraviolet B inhibits matrix metalloproteinase 13 expression in HaCaT keratinocytes and a mouse model of psoriasis

Matrix metalloproteinase13 (MMP13) can be released by keratinocytes and fibroblasts and involved in the pathogenesis of skin disorders. Retinoic acid derivative drugs include tazarotene and acitretin. Tazarotene/acitretin and narrow-band ultraviolet B (NB-UVB) irradiation are common treatment options for psoriasis. However, their impact on MMP13 expression in the context of psoriasis has yet to be determined. The expression of MMP13 was analyzed in patients with psoriasis. The effects of tazarotene/acitretin and NB-UVB on MMP13 expression were also investigated in a mouse model of psoriasis. Human HaCaT keratinocytes were exposed to acitretin or NB-UVB and then assayed for cell proliferation and MMP13 expression levels. We showed that patients with psoriasis had increased levels of MMP13 protein in skin lesions and serum samples. Exposure to acitretin and NB-UVB irradiation alone or in combination led to reduction of cell proliferation and MMP13 expression in HaCaT cells. Consistently, tazarotene treatment or NB-UVB irradiation attenuated imiquimod-induced psoriasis-like dermatitis and decreased MMP13 expression in a mouse model. Based on these from HaCaT keratinocytes cells and animal experiments, we suggest that tazarotene/acitretin and NB-UVB irradiation can inhibit the expression of MMP13 in HaCaT keratinocytes and psoriasis mouse models. Blockade of MMP13 activity may have therapeutic potential in improving symptoms of psoriasis.

The impact of hypoestrogenism and occlusal function on MMP1, MMP8 and MMP13 expression in the odontogenic region in rats

Background: The impact of estrogen deficiency and occlusion in the matrix metalloproteinases (MMPs) expression in dental tissues has not yet been elucidated. Objective: To evaluate the influence of estrogen deficiency and occlusal hypofunction and hyperfunction on the gene expression of MMP1, MMP8 and MMP13 in the odontogenic region of teeth in continuous growth, in the murine model. Material and methods: Rats (Wistar Hannover lineage) were divided into two groups according to the intervention received: Hypoestrogenism Group - ovariectomy surgery and Control Group - fictitious surgery. Occlusal hypofunction and hyperfunction conditions were also established in all animals (each animal presented both conditions). After euthanasia, the hemimandibles were removed to evaluate the gene expression through real time PCR. T-test was used to compare the mean differences between groups (P<0.05). Results: There was no statistically significant difference of the relative gene expression of MMP1, MMP8 and MMP13 between the hypoestrogenism and control groups (P>0.05). A statistically significant difference of the relative MMP13 expression between the occlusal hypofunction and hyperfunction tooth was observed (P=0.03). In the hypoestrogenism group, MMP13 was overexpressed in hypofunction tooth (P=0.045). Conclusion: Occlusal function affects MMP13 expression in the odontogenic region, in murine model.

Effect of retinoic acid combined with narrow-band ultraviolet B on matrix metalloprotein 13（MMP13）in psoriasis

Matrix metalloproteinase 13 (MMP13) is a zinc-containing endopeptidase secreted by keratinocytes and skin fibroblasts and participates in many inflammatory diseases. Drugs for retinoic acid include tazarotene and acitretin. Tazarotene/acitretin and narrow-band ultraviolet B (NB-UVB) irradiation are used as a general treatment for psoriasis. However, their impact on MMP13 expression has yet to be determined. In this study, we measured the expression of MMP13 in patients with psoriasis, and investigated the effects of tazarotene and/or NB-UVB on MMP13 expression in a mouse model of psoriasis. After exposure to acitretin and/or NB-UVB, immortalized human HaCaT keratinocytes were analyzed for viability and MMP13 expression. Our results showed that MMP13 protein levels increased in skin lesions and serum samples in patients with psoriasis. Treatment with acitretin and NB-UVB irradiation alone or in combination suppressed cell viability and MMP13 expression in HaCaT cells. Consistently, tazarotene treatment and/or NB-UVB irradiation attenuated imiquimod-induced psoriasis-like dermatitis and inhibited MMP13 expression in a mouse model. Taken together, these results indicate that tazarotene/acitretin and NB-UVB irradiation can inhibit the expression of MMP13 in keratinocytes and psoriasis mouse models. Targeting MMP13 may represent a promising therapeutic strategy against psoriasis.

Intervening up-regulated SLC7A5 could mitigate inflammatory mediator by mTOR-P70S6K signal in rheumatoid arthritis synoviocytes.

Objective: The disruption of metabolic events and changes to nutrient and oxygen availability due to sustained inflammation in RA increases the demand of bioenergetic and biosynthetic processes within the damaged tissue. The current study aimed to understand the molecular mechanisms of SLC7A5 (amino acid transporter) in synoviocytes of RA patients.Methods: Synovial tissues were obtained from OA and RA patients. Fibroblast-like synoviocytes (FLS) were isolated and SLC7A5 expression was examined by using RT-qPCR, immunofluorescence and Western blotting. RNAi and antibody blocking treatments were used to knockdown SLC7A5 expression or to block its transporter activities. mTOR activity assay and MMPs expression levels were monitored in RA FLS under amino acid deprivation or nutrient rich conditions. Results: RA FLS displayed significantly upregulated expression of SLC7A5 compared to OA FLS. Cytokine IL-1β was found to play a crucial role in up-regulating SLC7A5 expression via NF-κB pathway. Intervening SLC7A5 expression with RNAi or blocking its function by monoclonal antibody ameliorated MMP3 and MMP13 protein expression. Conversely, up regulation of SLC7A5 or tryptophan supplementation enhanced mTOR-P70S6K signals which promoted the protein translation of MMP3 and MMP13 in RA FLS.Conclusion: Activated NF-κB pathway up-regulates SLC7A5, which enhances mTOR-P70S6K activity and MMP3 and MMP13 expression in RA FLS.

High-Throughput Identification of MiR-145 Targets in Human Articular Chondrocytes

MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

High through-put identification of miR-145 targets in human articular chondrocytes

ABSTRACTObjectiveMicroRNAs play a key role in biological processes, including cartilage development and homeostasis and are dysregulated in many diseases, including osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs. This information is critical if miR-145 is to be considered a target for cartilage repair.MethodsRIP-seq (RNA-immunoprecipitation plus HT-sequencing) of miRISC (miRNA-induced silencing complex) was performed in HACs overexpressing miR-145 to identify miR-145 direct targets. The motif discovery method cWords was used to assess enrichment on miR-145 seed matches in the identified targets. RT-qPCR, Western (immuno-)blot and luciferase assays were used to validate miRNA-target interactions.ResultsMiR-145 overexpression affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3’UTR or, more commonly, the coding region.We also demonstrate that miR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion using specific siRNAs lead to MMP13 up-regulation, suggesting that miR-145-mediated DUSP6 depletion contributes to the effect of miR-145 on MMP13 expression.ConclusionWe demonstrate that miR-145 directly targets several genes in primary chondrocytes including those involved in the expression of the extracellular matrix and inflammation. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

Matrix-targeted Nanoparticles for MMP13 RNA Interference Blocks Post-Traumatic Osteoarthritis

Osteoarthritis (OA) is a debilitating and prevalent chronic disease, but there are no approved disease modifying OA drugs (DMOADs), only pharmaceuticals for pain management. OA progression, particularly for post-traumatic osteoarthritis (PTOA), is associated with inflammation and enzymatic degradation of the extracellular matrix. In particular, Matrix Metalloproteinase 13 (MMP13) breaks down collagen type 2 (CII), a key structural component of cartilage extracellular matrix, and consequently, matrix degradation fragments perpetuate inflammation and a degenerative cycle that leads to progressive joint pathology. Here, we tested targeted delivery of endosome-escaping, MMP13 RNA interference (RNAi) nanoparticles (NPs) as a DMOAD. The new targeting approach pursued here deviates from the convention of targeting specific cell types (e.g., through cell surface receptors) and instead leverages a monoclonal antibody (mAbCII) that targets extracellular CII that becomes uniquely accessible at early OA focal defects. Targeted mAbCII-siNPs create an in situ NP depot for retention and potent activity within OA joints. The mAbCII-siNPs loaded with MMP13 siRNA (mAbCII-siNP/siMMP13) potently suppressed MMP13 expression (95% silencing) in TNFα-stimulated chondrocytes in vitro, and the targeted mAbCII-siNPs had higher binding to trypsin-damaged porcine cartilage than untargeted control NPs. In an acute mechanical injury mouse model of PTOA, mAbCII-siNP/siMMP13 achieved 80% reduction in MMP13 expression (p = 0.00231), whereas a non-targeted control achieved only 55% silencing. In a more severe, PTOA model, weekly mAbCII-siNP/siMMP13 long-term treatment provided significant protection of cartilage integrity (0.45+/− .3 vs 1.6+/−.5 on the OARSI scale; p=0.0166), and overall joint structure (1.3+/−.6 vs 2.8+/−.2 on the Degenerative Joint Disease scale; p<0.05). Intra-articular mAbCII-siNPs better protected articular cartilage (OARSI score) relative to either single or weekly treatment with the clinical gold stand steroid treatment methylprednisolone. Finally, multiplexed gene expression analysis of 254 inflammation-related genes showed that MMP13 inhibition suppressed clusters of genes associated with tissue restructuring, angiogenesis (associated with synovial inflammation and thickening), innate immune response, and proteolysis. This work establishes the new concept of targeting unique local extracellular matrix signatures to sustain retention and increase delivery efficacy of biologics with intracellular activity and also validates the promise of MMP13 RNAi as a DMOAD in a clinically-relevant therapeutic context. Abstract Figure:PTOA targeted delivery of MMP13 siRNA to block disease progressionThe top left schematic illustrates the progression (left to right) from healthy knee joint, through inflammation induction following traumatic injury, to cartilage loss and degenerative joint disease (including synovial response). Degradation of cartilage enhances inflammation, inducing a degenerative cycle (middle right). The bottom of the graphic illustrates the concept of the matrix targeted nanocarriers for enhanced retention and activity of MMP13 siRNA at sites of cartilage injury.