Role of Rho small GTPases in meniscus cells

We previously reported that mechanical stretch regulates Sry‐type HMG box (SOX) 9‐dependent α1(II) collagen (COL2A1) expression in inner meniscus cells. This study examined the role of the small Rho guanosine 5' triphosphatase Rac1 and Rho‐associated kinase (ROCK) in the regulation of stretch‐induced SOX9 gene expression in cultured human inner meniscus cells. COL2A1 and SOX9 gene expression was assessed by real‐time PCR after application of uni‐axial cyclic tensile strain (CTS) in the presence or absence of ROCK and Rac1 inhibitors. The subcellular localization of SOX9 and the Rac1 effector cyclic AMP response element‐binding protein (CREB), the phosphorylation state of SOX9, Rac1 activation, and the binding of CREB to the SOX9 promoter were assessed. CTS increased the expression of COL2A1 and SOX9, which was suppressed by inhibition of Rac1. ROCK inhibition enhanced COL2A1 and SOX9 gene expression in the absence of CTS. CTS stimulated the nuclear translocation and phosphorylation of SOX9, and increased Rac1 activation. CTS also increased the binding of CREB to the SOX9 promoter. The results suggest that mechanical stretch‐dependent upregulation of SOX9 by CREB in inner meniscus cells depends on the antagonistic activities of ROCK and Rac1. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1479–1486, 2014.     

Screening of the COL2A1 mutation in idiopathic osteonecrosis of the femoral head

Idiopathic osteonecrosis of the femoral head (idiopathic ONFH) is an ischemic disorder resulting in necrosis of the subchondral bone of the femoral head. COL2A1 mutations, including c.3508G>A, have been reported to be involved in its etiology. However, the etiological role of COL2A1 mutations in idiopathic ONFH remains controversial, because the pathology of idiopathic ONFH is ischemic necrosis, not epiphyseal dysplasia usually seen in the diseases caused by COL2A1 mutations. The purpose of this study is to examine whether COL2A1 mutations have causal relation with idiopathic ONFH or not. We recruited 1,451 Japanese patients with idiopathic ONFH, including steroid‐, alcohol‐, and neither steroid nor alcohol‐associated (neither‐associated) ONFH. The diagnosis was based on the criteria of the Japanese Research Committee on idiopathic ONFH of the Ministry of Health, Labour and Welfare. By whole‐exome sequencing, entire COL2A1 coding regions and flanking introns were analyzed in 49 neither‐associated ONFH patients. In addition, the c.3508G>A mutation of COL2A1 was checked in all idiopathic ONFH patients using the invader assay. Whole‐exome sequencing did not detect any COL2A1 mutations in the 49 patients. The c.3508G>A mutation was not found in any of the 1,451 patients. In conclusion, COL2A1 is unlikely to cause idiopathic ONFH. Epiphyseal dysplasia of the femoral head caused by COL2A1 mutations may radiographically mimic idiopathic ONFH. COL2A1 mutations should prompt clinical re‐evaluation of the patient's phenotype. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:768–774, 2017.

     

Mechanical stretch enhances COL2A1 expression on chromatin by inducing SOX9 nuclear translocalization in inner meniscus cells

The meniscus plays an important role in controlling the biomechanics of the knee. However, the mechanical stress‐related response in meniscus cells remains unclear. We investigated mechanical stretch‐regulated gene expression in human meniscus cells. Human inner and outer meniscus cells were prepared from the inner and outer halves of the lateral meniscus. The gene expressions of Sry‐type HMG box (SOX) 9 and α1(II) collagen (COL2A1) were assessed by real‐time PCR analyses after cyclic tensile strain (CTS) treatment (0.5 Hz, 5% stretch). The localization and phosphorylation of SOX9 were evaluated by immunohistochemical and Western blot (WB) analyses. Chromatin immunoprecipitation (IP) analysis was performed to assess the stretch‐related protein–DNA complex formation between SOX9 and the COL2A1 enhancer on chromatin. Type II collagen deposition and SOX9 production were detected only in inner menisci. CTS treatments increased expression of the COL2A1 and SOX9 genes in inner meniscus cells, but not in outer meniscus cells. In addition, CTS treatments stimulated nuclear translocalization and phosphorylation of SOX9 in inner meniscus cells. Chromatin IP analyses revealed that CTS increased the association between SOX9 and its DNA‐binding site, included in the COL2A1 enhancer, on chromatin. Our results indicate that inner and outer meniscus cells have different properties in mechanical stretch‐induced COL2A1 expression. In inner meniscus cells, mechanical stretch may have an essential role in the epigenetic regulation of COL2A1 expression. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:468–474, 2012     

Hypoxia‐inducible factor 3‐alpha expression is associated with the stable chondrocyte phenotype

The hypoxia‐inducible factors HIF‐1α and HIF‐2α are important regulators of the chondrocyte phenotype but little is known about HIF‐3α in cartilage. The objective of this study was to characterize HIF‐3α (HIF3A) expression during chondrocyte differentiation in vitro and in native cartilage tissues. HIF3A, COL10A1, and MMP13 were quantified in mesenchymal stem cells (MSCs) and articular chondrocytes from healthy and osteoarthritic (OA) tissue in three‐dimensional cultures and in human embryonic epiphyses and adult articular cartilage. HIF3A was found to have an inverse association with hypertrophic markers COL10A1 and MMP13 in chondrogenic cells and tissues. In healthy chondrocytes, HIF3A was induced by dexamethasone and increased during redifferentiation. By comparison, HIF3A expression was extremely low in chondrogenically differentiated MSCs expressing high levels of COL10A1 and MMP13. HIF3A was also lower in redifferentiated OA chondrocytes than in healthy chondrocytes. In human embryonic epiphyseal tissue, HIF3A expression was lowest in the hypertrophic zone. Distinct splice patterns were also found in embryonic cartilage when compared with adult articular cartilage and redifferentiated chondrocytes. These in vitro and in vivo findings suggest that HIF3A levels are indicative of the hypertrophic state of chondrogenic cells and one or more splice variants may be important regulators of the chondrocyte phenotype. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1561–1570, 2015.     

Silent information regulator (Sir)T1 inhibits NF‐κB signaling to maintain normal skeletal remodeling

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF‐κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF‐κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast‐ or osteoblast‐specific SirT1 deletion using the Sirt^flox/flox mice crossed to lysozyme M‐cre and the 2.3 kb col1a1‐cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF‐κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF‐κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF‐κB. In further support of the critical role of NF‐κB signaling in bone remodeling, elevated NF‐κB activity in IκBα^+/? mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell‐autonomous manner in both osteoblasts and osteoclasts, through control of NF‐κB and bone cell differentiation. © 2013 American Society for Bone and Mineral Research.     

Potential involvement of SIRT1 in the pathogenesis of osteoarthritis through the modulation of chondrocyte gene expressions

SIRT1 has been implicated as a key factor in aging‐related diseases. Nevertheless, the role of SIRT1 in the pathogenesis of osteoarthritis (OA) is still unknown. We examined the expression of SIRT1 in cartilage samples and the effect of SIRT1 inhibition on chondrocyte gene expression changes to elucidate the role of SIRT1 in chondrocytes. SIRT1 expression was examined using cartilage samples from patients undergoing total knee arthroplasty and femoral head replacement by immunohistochemistry. The effect of SIRT1 inhibition by siRNA on chondrocyte gene expression was examined by real‐time PCR and Western blotting. SIRT1 expression was barely detectable in the severely degenerated cartilage while SIRT1 was clearly expressed in the less damaged cartilage. The inhibition of SIRT1 by siRNA induced OA‐like gene expression changes, namely the significant down‐regulation of aggrecan and up‐regulation of COL10A1 and ADAMTS‐5. Our observations suggest that SIRT1 expression decreases with development of OA and the reduction of SIRT1 in chondrocytes may cause chondrocyte hypertrophy and cartilage matrix loss. SIRT1 might play important roles in the pathogenesis of OA. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:511–515, 2011     

Platelet‐Rich Plasma Released From Polyethylene Glycol Hydrogels Exerts Beneficial Effects on Human Chondrocytes

Osteoarthritis (OA) is a debilitating joint disease resulting from chronic joint inflammation and erosion of articular cartilage. A promising biological treatment for OA is intra‐articular administration of platelet‐rich plasma (PRP). However, immediate bolus release of growth factors limits beneficial therapeutic effects of PRP, thus necessitating the demand for sustained release platforms. In this study, we evaluated the therapeutic value of PRP released from a polyethylene glycol (PEG) hydrogel on articular chondrocytes/cartilage explants derived from OA patients. Lyophilized PRP (PRGF) was encapsulated in PEG hydrogels at 10% w/v and hydrogel swelling, storage modulus and degradation and PRGF release kinetics were determined. PRGF releasate from the hydrogels was collected on day 1, 4, and 11. Encapsulation of PRGF at 10% w/v in PEG hydrogels had minimal effect on hydrogel properties. PRGF was released with an initial burst followed by sustained release until complete hydrogel degradation. Effect of PRGF releasates and bolus PRGF (1% w/v PRGF) on patient‐derived cartilage explants or chondrocytes was assessed by chondrocyte proliferation (pico‐green assay), gene expression for COL1A1, COL2A1, MMP13, COX2, and NFKB1 (real‐time polymerase chain reaction), and measurement of nitric oxide concentration (Griess’ assay). Compared to bolus PRGF, PRGF releasates enhanced chondrocyte proliferation, suppressed the expression of genes like MMP13, NFKB1, COL1A1, and COL2A1 and reduced levels of nitric oxide. Taken together, these results indicate that release of PRGF from PEG hydrogels may improve the therapeutic efficacy of PRP and merits further investigation in an animal model of OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2401–2410, 2019     

Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer

We have recently shown that a 150‐bp Col10a1 distal promoter (?4296 to ?4147 bp) is sufficient to direct hypertrophic chondrocyte‐specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem‐repeat Runx2 binding sites within the 3′‐end of this 150‐bp region (TGTGGG‐TGTGGC, ?4187 to ?4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5′‐sequence without Runx2 sites were not able to drive the cell‐specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3′‐end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150‐bp promoter abolishes its capacity to drive hypertrophic chondrocyte‐specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3′‐sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte‐specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis‐enhancer. This interaction is required but not sufficient for cell‐specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5′‐ or 3′‐sequences of this 150‐bp promoter are needed to work with Runx2 together to mediate cell‐specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation. © 2011 American Society for Bone and Mineral Research     

The AdLMP-1 transfection in two different cells; AF cells,chondrocytes as potential cell therapy candidates for disc degeneration

Summary   Background. LMP-1 is known to increase proteoglycan production through the upregulating the BMPs and it is also known that BMP-2 acts on anulus fibrosus cells and chondrocytes to increase proteoglycan production. Method. We carried out an experiment, the effect of AdLMP-1 transfection on AF cells and chondrocytes in the production of sulfated-glycosaminoglycans, mRNA expression (aggrecan, type I, II collagen, LMP-1, BMP-2, and BMP-7), and immunofluorescence staining. AF cells and chondrocytes were grown in monolayer and treated for 6 days with AdLMP1-green fluorescence protein (GFP) (10, 20, and 30 multiplicity of infection [MOI]). After 6 days, the sGAG content in the media was quantified using 1,9-dimethylmethylene blue staining. The mRNA expression was measured with real-time PCR after 20 MOI infection of AdLMP1-GFP. The each cells treated with 20 MOI infection of AdGFP was used as a control group for the mRNA expression. The each cell group was immunofluorescence stained with each antibodies in the chamber slide at 3 × 10⁴ cells/chamber. Findings. 1) The sGAG production was maximum in 20 MOI AdLMP1-GFP infection on the AdLMP-1 treatment for both of AF cells and chondrocytes. 2) The mRNA expression of aggrecan, type I collagen, type II collagen, LMP-1, BMP-2, and BMP-7 is increased in both AF cells and chondrocytes in 20 MOI AdLMP1-GFP infection. 3) On the immunofluorescence staining results, the positive immunofluorescence stained cell numbers are increased after 20 MOI AdLMP1-GFP infection concordant with upregulation of mRNA expression. Conclusions. The AdLMP-1 treatments in AF cells and chondrocytes may be useful for cell transplantation therapy in disc degeneration. Correspondence: S. Tim Yoon M.D., Ph.D., Emory Orthopaedics and Spine Center, 59 Executive Park South, Atlanta 30329, GA.     

The small GTPase Rho mediates articular chondrocyte phenotype and morphology in response to interleukin‐1α and insulin‐like growth factor‐I

Small GTPases regulate the cytoskeleton and numerous other cellular functions. In this study, the role of Rho GTPase was examined in articular chondrocytes. Chondrocytes grown in monolayer were treated with interleukin‐1α (IL‐1α), insulin‐like growth factor‐I (IGF‐I), C3 Transferase, Y27632, or transfected with Rho wild type or two constitutively active mutants of Rho (Q63L and G14V). Quantitative PCR was used to determine changes in matrix metalloproteinase‐13 (MMP‐13), collagen types IIB (COL2A1) and type I (COL1A1), aggrecan (AGG), and SOX‐9 gene expression. Affinity assays were performed to measure endogenous GTP‐bound Rho, and confocal microscopy was used to assess changes in organization of the actin cytoskeleton. IL‐1α and RhoG14V increased cytoplasmic actin stress fiber formation, which was blocked by C3 Transferase, and Y27632. IL‐1α treatment also increased Rho activity. Conversely, IGF‐I lead to formation of a cortical rim of actin and decreased Rho activity. Inhibition of Rho signaling with C3 Transferase significantly decreased Rho activity and returned IL‐1α‐induced Rho activity to a level not different from control. C3 Transferase treatment also increased mRNA expression of AGG, COL2A1, and SOX‐9, and decreased expression of MMP‐13. Expression of RhoQ63L or RhoG14V resulted in increased MMP‐13 expression; however, inhibition of Rho with Y27632 was unable to inhibit IL‐1α‐induced MMP‐13 expression. Together, these results indicate a role for increased Rho activity in mediation of chondrocyte catabolic signaling pathways. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:58–64, 2009