Pengcui Li

Decreased histone deacetylase 4 is associated with human osteoarthritis cartilage degeneration by releasing histone deacetylase 4 inhibition of runt-related transcription factor-2 and increasing osteoarthritis-related genes: a novel mechanism of human osteoarthritis cartilage degeneration

IntroductionTo investigate if decreased histone deacetylase 4 (HDAC4) is associated with human osteoarthritis (OA) cartilage degeneration by releasing HDAC4 inhibition of runt-related transcription factor-2 (Runx2) resulting in increase of OA cartilage degeneration-related genes.MethodsThe mRNA and protein levels of HDAC4, Runx2, matrix metalloproteinase (MMP)-13, Indian hedgehog (Ihh) and type X collagen were detected by performing real-time PCR (RT-PCR), western blotting and immunohistochemistry on specimens from human OA and normal cartilage. To further explore the mechanism of regulation of Runx2 and OA-related genes by HDAC4, changes in these OA-related genes were further quantified by RT-PCR after overexpression of HDAC4 and knockdown of HDAC4 by siRNA. Runx2 and MMP-13 promoter activities were measured by dual luciferase assays.ResultsThe levels of HDAC4 in the cartilage from OA patients and healthy 40- to 60-year-old donors were decreased to 31% and 65% compared with specimens from 20- to 40-year-old healthy donors, respectively (P <0.05). Decreased HDAC4 was associated with increased Runx2 and other OA-related genes in human OA cartilage, specifically: MMP-13, Ihh and type X collagen. Exogenous HDAC4 decreased the mRNA levels of Runx2, MMP1, MMP3, MMP-13, type X collagen, Ihh, ADAMTS-4 and -5, and increased the mRNA of type II collagen. In addition, the data also shows that overexpression of HDAC4 not only decreased the expression of interleukin (IL)-1β, Cox2 and iNos and increased the expression of aggrecan, but also partially blocked the effect of IL-1β on expression of catabolic events in human OA chondrocytes. HDAC4 also inhibited Runx2 promoter activity and MMP13 promotor activity in a dose-dependent manner. In contrast, inhibition of HDAC4 by TSA drug had an opposite effect.ConclusionsOur study is the first to demonstrate that decreased HDAC4 contributes, at least in part, to the pathogenesis of OA cartilage degeneration. Thus, HDAC4 may have chondroprotective properties by inhibiting Runx2 and OA-related genes.

MicroRNA-1 regulates chondrocyte phenotype by repressing histone deacetylase 4 during growth plate development

MicroRNAs (miRs) are noncoding RNAs (17‐25 nt) that control translation and/or mRNA degradation. Using Northern blot analysis, we identified that miR‐1 is specifically expressed in growth plate cartilage in addition to muscle tissue, but not in brain, intestine, liver, or lung. We obtained the first evidence that miR‐1 is highly expressed in the hypertrophic zone of the growth plate, with an 8‐fold increase compared with the proliferation zone; this location coincides with the Ihh and Col X expression regions in vivo. MiR‐1 significantly induces chondrocyte proliferation and differentiation. We further identified histone deacetylase 4 (HDAC4) as a target of miR‐1. HDAC4 negatively regulates chondrocyte hypertrophy by inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. MiR‐1 inhibits both endogenous HDAC4 protein by 2.2‐fold and the activity of a reporter gene bearing the 3‘‐untranslated region (UTR) of HDAC4 by 3.3‐fold. Conversely, knockdown of endogenous miR‐1 relieves the repression of HDAC4. Deletion of the miR‐1 binding site in HDAC4 3‘‐UTR or mutated miR‐1 abolishes miR‐1‐mediated inhibition of the reporter gene activity. Overexpression of HDAC4 reverses miR‐1 induction of chondrocyte differentiation markers Col X and Ihh. HDAC4 inhibits Runx2 promoter activity in a dosage‐dependent manner. Thus, miR‐1 plays an important role in the regulation of the chondrocyte phenotype during the growth plate development via direct targeting of HDAC4. —Li, P., Wei, X., Guan, Y., Chen, Q., Zhao, T., Sun, C., Wei, L. MicroRNA‐1 regulates chondrocyte phenotype by repressing histone deacetylase 4 during growth plate development. FASEB J. 28, 3930‐3941 (2014). www.fasebj.org

Attenuation of cartilage pathogenesis in post-traumatic osteoarthritis (PTOA) in mice by blocking the stromal derived factor 1 receptor (CXCR4) with the specific inhibitor, AMD3100.

SDF‐1 was found to infiltrate cartilage, decrease proteoglycan content, and increase MMP‐13 activity after joint trauma. In this study, we tested the hypothesis that interference of the SDF‐1/CXCR4 signaling pathway via AMD3100 can attenuate pathogenesis in a mouse model of PTOA. We also tested the predictive and confirmatory power of fluorescence molecular tomography (FMT) for cartilage assessment. AMD3100 was continuously delivered via mini‐osmotic pumps. The extent of cartilage damage after AMD3100 or PBS treatment was assessed by histological analysis 2 months after PTOA was induced by surgical destabilization of the medial meniscus (DMM). Biochemical markers of PTOA were assessed via immunohistochemistry and in vivo fluorescence molecular tomography (FMT). Regression analysis was used to validate the predictive power of FMT measurements. Safranin‐O staining revealed significant PTOA damage in the DMM/PBS mice, while the DMM/AMD3100 treated mice showed a significantly reduced response with minimal pathology. Immunohistochemistry showed that AMD3100 treatment markedly reduced typical PTOA marker expression in chondrocytes. FMT measurements showed decreased cathepsins and MMP activity in knee joints after treatment. The results demonstrate that AMD3100 treatment attenuates PTOA. AMD3100 may provide a viable and expedient option for PTOA therapy given the drugs FDA approval and well‐known safety profile.

Intra-Articular Injection of Cross-Linked Hyaluronic Acid-Dexamethasone Hydrogel Attenuates Osteoarthritis: An Experimental Study in a Rat Model of Osteoarthritis

Cross-linked hyaluronic acid hydrogel (cHA gel) and dexamethasone (Dex) have been used to treat knee osteoarthritis (OA) in clinical practice owing to their chondroprotective and anti-inflammatory effects, respectively. The aim of the present study was to compare the treatment effects of the cHA gel pre-mixed with/without Dex in a surgery-induced osteoarthritis model in rats. Anterior cruciate ligament transection (ACLT) surgery was performed on the right knee of rats to induce OA. Male 2-month-old Sprague-Dawley rats were randomly divided into five groups (n = 10/per group): (1) ACLT + saline; (2) ACLT + cHA gel; (3) ACLT + cHA-Dex (0.2 mg/mL) gel; (4) ACLT + cHA-Dex (0.5 mg/mL) gel; (5) Sham + saline. Intra-joint injections were performed four weeks after ACLT in the right knee. All animals were euthanized at 12 weeks post-surgery. Cartilage damage and changes in the synovial membrane were assessed by micro X-ray, Indian ink articular surface staining, Safranin-O/Fast Green staining, immunohistochemistry, hematoxylin and eosin staining of the synovial membrane, and quantitative reverse transcription-polymerase chain reaction for changes in gene expression. Micro X-ray revealed that the knee joint treated with the cHA-Dex gel was wider than those treated with cHA gel alone or saline. The cHA-Dex gel group had less Indian ink staining (indicator of cartilage fibrillation) than the cHA gel or saline injection groups. Safranin-O/Fast Green staining indicated that increased proteoglycan staining and less cartilage damage were found in the cHA-Dex gel group compared with the cHA gel or saline injection groups. Quantification of histology findings from saline, cHA gel, cHA-Dex (0.2 mg/mL) gel, cHA-Dex (0.5 mg/mL) gel, and sham groups were 5.84 ± 0.29, 4.50 ± 0.87, 3.00 ± 1.00, 2.00 ± 0.48, and 0.30 ± 0.58 (p < 0.05), respectively. A strong staining of type II collagen was found in both the cHA-Dex gel groups compared with saline group or cHA alone group. Similar result was found for the mRNA level of aggrecan and opposite result for type X collagen. Hematoxylin and eosin staining in the synovial membrane showed less synovial lining cell layers and reduced inflammatory cell infiltration in cHA-Dex gel-treated animals compared with saline or cHA only groups. Altogether, cHA-Dex gel has better chondroprotective and anti-inflammatory effects in rat surgery-induced osteoarthritis than cHA alone.

Cartilage oligomeric matrix protein and hyaluronic acid are sensitive serum biomarkers for early cartilage lesions in the knee joint

Abstract The purpose of this study was to evaluate the relationship between five previously established serum osteoarthritis biomarkers and the severity of cartilage lesions in the knee. Cartilage damage (classified according to the Outerbridge scoring system) and serum concentrations of cartilage oligomeric matrix protein (COMP), collagen type II C-telopeptide (CTX-II), matrix metalloproteinase-3 (MMP-3), collagen type III N-propeptide, (PIIINP), and hyaluronic acid (HA) were determined in 79 patients who underwent knee arthroscopy or total knee replacement. HA and COMP concentrations were significantly higher in the Outerbridge score 1 and 2 groups, respectively. These results suggest that serum COMP and HA concentrations can be used to predict early cartilage lesions in the knee.

Mitogen-activated protein kinase p38 induces HDAC4 degradation in hypertrophic chondrocytes

Histone deacetylase 4 (HDAC4) is a critical negative regulator for chondrocyte hypertrophy by binding to and inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. It is unclear how HDAC4 expression and stability are regulated during growth plate development. We report here that inhibition of mitogen-activated protein kinase (MAPK) p38 by dominant negative p38 or p38 inhibitor prevents HDAC4 degradation. Mutation of a potential caspase-2 and 3 cleavage site Asp289 stabilizes HDAC4 in chondrocytes. In contrast, constitutively active MAPK kinase 6 (constitutive activator of p38) transgenic mice exhibit decreased HDAC4 content in vivo. We also observed that p38 stimulates caspase-3 activity in chondrocytes. Inhibition of p38 or caspases reduced HDAC4 degradation. HDAC4 inhibited Runx2 promoter activity in a dose-dependent manner and caspase inhibitors further enhanced this inhibition by preventing HDAC4 degradation. Overall, these results demonstrate that p38 promotes HDAC4 degradation by increasing caspase-mediated cleavage, which releases Runx2 from a repressive influence of HDAC4 and promotes the chondrocyte hypertrophy and bone formation.

Identification of α1-Antitrypsin as a Potential Candidate for Internal Control for Human Synovial Fluid in Western Blot.

Western blot of synovial fluid has been widely used for osteoarthritis (OA) research and diagnosis, but there is no ideal loading control for this purpose. Although β-actin is extensively used as loading control in western blot, it is not suitable for synovial fluid because it is not required in synovial fluid as a cytoskeletal protein. A good loading control for synovial fluid in OA studies should have unchanged content in synovial fluids from normal and OA groups, because synovial fluid protein content can vary with changes in synovial vascular permeability with OA onset. In this study, we explore the potential of using α1-antitripsin (A1AT) as loading control for OA synovial fluid in western blot. A1AT level is elevated in inflammatory conditions such as rheumatoid arthritis (RA). Unlike RA, OA is a non-inflammation disease, which does not induce A1AT. In this study, we identified A1AT as an abundant component of synovial fluid by Mass Spectrometry and confirmed that the level of A1AT is relative constant between human OA and normal synovial fluid by western blot and ELISA. Hence, we proposed that A1AT may be a good loading control for western blot in human OA synovial fluid studies provided that pathological conditions such as RA or A1AT deficiency associated liver or lung diseases are excluded.

MicroRNA‐134 inhibits osteosarcoma angiogenesis and proliferation by targeting the VEGFA/VEGFR1 pathway

Vascular endothelial growth factor (VEGF) A and vascular endothelial growth factor receptor 1 (VEGFR1) signaling is crucial for angiogenesis and progression of osteosarcoma (OS). However, the regulation of the VEGF/VEGFR1 expression is still unclear in OS. Here, we show lower levels of miRNA‐134 (miR‐134) in OS tissues and cells. Induction of miR‐134 overexpression significantly reduced the proliferation of Saos‐2 cells and their secretion of pro‐angiogenic factors, but increased the frequency of apoptotic Saos‐2 cells. Treatment with conditioned medium from the cells transfected with miR‐134 reduced the tube formation in human umbilical vein endothelial cells, which was abrogated by a combination of VEGF and conditioned medium. Furthermore, miR‐134 significantly inhibited the growth of implanted OS tumors in vivo and attenuated the VEGFA and VEGFR1 expression and angiogenesis in the tumors. In addition, higher levels of VEGFA and VEGFR1 were detected and miR‐134 inhibited the expression of VEGFA and VEGFR1 in Saos‐2 cells and OS tumors. Bioinformatic analysis indicated that the 3′‐UTR of VEGFA and VEGFR1 contained the motif for miR‐134 binding. Co‐transfection with the luciferase reporter containing the wild‐type, but not the mutant, of the 3′‐UTR of VEGFA or VEGFR1 together with miR‐134 decreased the luciferase activity in Saos‐2 cells. Finally, miR‐134 dramatically inhibited AKT activation and proliferating cell nuclear antigen expression in Saos‐2 cells. Collectively, these findings indicate that miR‐134 is a potential tumor suppressor by targeting VEGFA/VEGFR1 signaling to attenuate the progression and angiogenesis in OS. Therefore, miR‐134 may be a novel biomarker for the prognosis of OS and a target for the design of new therapies for OS.

Blockade of hypoxia-induced CXCR4 with AMD3100 inhibits production of OA-associated catabolic mediators IL-1β and MMP-13

Binding of the chemokine stromal cell-derived factor-1 (SDF-1) to its receptor C-X-C chemokine receptor type 4 (CXCR4) results in receptor activation and the subsequent release of matrix metalloproteinases (MMPs) that contribute to osteoarthritis (OA) cartilage degradation. As hypoxia is a defining feature of the chondrocyte microenvironment, the present study investigated the possible mechanism through which SDF-1 induces cartilage degradation under hypoxic conditions. To do this, OA chondrocyte cultures and patient tissue explants pretreated with the CXCR4 inhibitor, AMD3100 were incubated with SDF-1. It was identified that hypoxic conditions significantly elevated the expression of CXCR4 in osteoarthritic chondrocytes relative to normoxic conditions. Furthermore, SDF-1 elevated MMP-13 mRNA levels and proteinase activity. It also elevated the mRNA and protein levels of runt-related transcription factor 2, and induced the release of glycosaminoglycans and the inflammatory cytokine, interleukin-1β. By contrast, such changes did not occur to an appreciable degree in cells that were pretreated with AMD3100. The results of the present study demonstrate that even under hypoxic conditions, where CXCR4 expression is significantly elevated in chondrocytes, AMD3100 effectively blocks this receptor and protects chondrocytes from OA-induced catabolism, suggesting that the successful inhibition of CXCR4 may be an effective approach for OA treatment.

Knockdown Indian Hedgehog (Ihh) does not delay Fibular Fracture Healing in genetic deleted Ihh mice and pharmaceutical inhibited Ihh Mice

The objective of this study was to determine if Ihh is required for fracture healing. Fibular fracture was created in adult Col2a1-CreERT2; Ihhfl/fl mice. Ihhfl/fl mice received Tamoxifen (TM) to delete Ihh. WT mice received Cyclopamine to inhibit Hh pathway. Callus tissue properties and Ihh pathway were analyzed at 1, 2, and 3 weeks post-fracture by X-ray, micro-CT, mechanical test, RT-PCR and immunohistochemistry. Deleted Ihh was evidenced by the occurrence of growth plate closure in the Ihhfl/fl mice by X-ray 3 weeks after TM treatment. All mice showed fracture healing at 3 weeks post-operation. Histology analysis indicated that, compared to the control, cartilage area was less in fracture sites from Ihh deficient animals by either genetic deletion or drug inhibition at 1 and 2 weeks post-fracture. Ihh immunostaining and its mRNA level were diminished in the fracture callus in Ihh reduced mice. There was no significant difference in BV/TV, BMD and mechanical test. Interruption to Ihh pathway by either genetic or pharmaceutical approach didn’t affect fibular fracture healing in these mice. This surprised finding implicates that the deleted Ihh does not affect fracture healing in this model.