The Role And Mechanisms Of ALK5 Signaling In Articular Cartilage Homeostasis And The Development Of Arthritis

Osteoarthritis（OA）is the most common degenerative joint disease.The primary characteristic of OA includes progressive fibrillation and degradation of articular cartilage,synovial inflammation and hyperplasia,subchondral bone sclerosis and osteophyte formation,which lead to movement-associated pain,joint stiffness and eventually impaired mobility and disability.However,the exact pathophysiology and mechanisms underlying OA are not fully understood,and currently there is no effective therapeutic intervention to decelerate OA progression.Articular cartilage is a specialized,avascular,aneural connective tissue covering bone surfaces within synovial joints that protects the underlying bones from biomechanical dama ge and friction during joint loading.The extracellular matrix（ECM）of articular cartilage mainly contains type II collagen（Col II）and Aggrecan（ACAN）,which are degraded by specific collagenases such as matrix metalloproteinase 13（MMP13）,MMP9,and MM P3 and aggrecanases such as a disintegrin and metalloproteinase with thrombospondin motif 4（ADAMTS4）and ADAMTS5 under pathological conditions including OA.Chondrocyte is the only cell population present in articular cartilage,which is responsible for m aintaining the homeostasis of articular cartilage by synthesizing and degrading matrix components in response to environmental cues such as growth factors,cytokines,and biomechanical loading,etc.Failure of chondrocytes to maintain the homeostasis of articular cartilage and/or to repair its damage will lead to articular cartilage degradation,the key feature of OA.A variety of molecules and signaling pathways such as hypoxia-inducible factor-1α/-2α,Wnt/β-Catenin and fibroblast growth factor signaling play critical roles in the cartilage homeostasis and development of OA.However the molecular mechanisms underlying the pathogenesis of OA are still not clearly clarified.Understanding the precise molecular mechanisms that regulate articular cartilage homeostasis and the development of OA is extremely important for the development of effective biological treatment for OA.Recently,the role of transforming growth factor-β（TGF-β）signaling in the regulation of cartilage homeostasis has received increasing and specific attention.In canonical TGF-β-Smad signaling,the TGF-βligands initiate signaling cascades by binding to the TGF-βtype II receptor（TGFβRII）first,then TGFβRII and TGF-βtype I receptor（also called activing receptor-like kinases 5,ALK5）assemble to form a heteromeric complex on the cell membranes.Upon ligands binding,constitutively actived TGFβRII receptor phosphorylates ALK5.The activated（phosphorylated）ALK5 subsequently recruits and phosphorylates receptor-regulated Smads（R-Smads,Smad2 and Smad3）,which then form a heteromeric complex with the one common Smad（co-Smad,Smad4）.The heteromeric complex translocated into the nucleus to regulate the expression of TGF-βtarget genes.In addition,TGF-βcan also activate non-Smad signaling pathways such as mitogen-activating protein kinases（MAPKs）,phosphatidylinositol 3 kinase（PI3K）/Akt and small GTPases.Genetic manipulation of TGF-βpathway components demonstrates that dysregulation of TGF-βsignaling induces development of OA.Transgenic overexpression of dominant-negative TGFβRII（dn-TGFβRII）in skeletal tissue or chondrocyte-specific Tgfbr2 deletion in postnatal mice,which reduces the TGF-βsignaling at the TGFβRII level,results in up-regulation of MMP13 and ADAMTS5 expressions in articular cartilage,eventually leads to a progressive OA-like phenotype in mice.Similarly,conventional or chondrocyte-specific Smad3 knockout mice also develop degenerative joint disease resembling human OA characterized by decreased Col II and ACAN protein levels,and increased MMP13 and type X collagen protein levels in articular cartilage tissue.Based on the above evidences,it suggests that TGF-β/TGFβRII-Smad3signaling axis in chondrocytes plays a vital role in articular cartilage homeostasis and the development of OA.However,the role of TGF-βsignaling in articular cartilage maintenance at the level of ALK5 is not well understood.ALK5 is expressed in murine and human articular cartilage,and its expression level decreases in both aging and OA articular cartilage.In vitro overexpression of ALK5 in articular chondrocytes increases the m RNA expression of Aggrecan,while knockdown of Alk5 in articular chondrocytes leads to elevated mRNA expression of Mmp13.Consistently,intraperitoneal injection of ALK5 inhibitor SB-505124 induces proteoglycan loss in the superficial to middle zones of articular cartilage in mice.12 These results indicate that ALK5may play an important role in promoting anabolic metabolism and inhibiting catabolic metabolism in chondrocytes.However,in vivo genetic data about the role of ALK5 signaling in articular cartilage homeostasis and OA development are still lacking.In this study,we utilized mice with inducible deletion of Alk5 specifically in chondrocytes at postal stages to elucidate the in vivo role of ALK5 signaling in articular cartilage homeostasis and OA development.We reveal that TGF-β/ALK5 signaling is involved in maintaining articular cartilage homeostasis in vivo,partly by upregulating the expression of Proteoglycan 4（PRG4）through PKA-CREB signaling in articular chondrocytes.Proteoglycan 4 proteins（PRG4）,also called lubricin or superficial zone proteins（SZP）,is expressed by superficial zone chondrocytes at the articular cartilage surface.Superficial zone is the first line of defense against OA initiation because of its surface location,unique composition,and functions.It has 2–4 layers of flat cells expressing unique molecules,such as PRG4 and tenascin C,and contains a fine network of collagen fibrils that are oriented horizontally and parallel to the articular surface.Its multifaceted roles include,but are not limited to,producing lubricant proteins,harboring cartilage stem/progenitor cells and resistin g shear stresses.The superficial zone cells express TGF-βisoforms and receptors,TGF-βstrongly enhance synthesis of PRG4 and stimulate expression of cartilage matrix molecules,such as Collagen II and Aggrecan,in the superficial layer cells in micromass culture.The superficial cell cultures used in these studies represent stem/progenitor characteristics.Thus TGF-βwould play a role as a stimulator of differentiation of the stem/progenitor cells into articular chondrocytes.However,in vivo genetic data about the role of superficial zone TGF-β/ALK5 signaling in and OA development are still lacking.In this study,we utilized mice with inducible deletion of Alk5specifically in superficial zone cells at postal stages to elucidate the in vivo role of ALK5signaling in superficial zone function and OA development.Our study has revealed that ALK5signaling is critical for maintaining the superficial zone of articular cartilage and preventing osteoarthritis initiation,therefore,therapeutic agents specifically targeting cartilage surface ALK5 should be considered as a novel direction for OA treatment.Methods:PartⅠInducible cartilage-specific deletion of Alk5 gene results in a progressive Osteoarthritis-like phenotype in mice1.Postnatal cartilage-specific inducible deletion of Alk5 in mice:Alk5^flox/flox;Col2α1-CreER^T2(Alk5^Col2ERT2)and Alk5^flox/flox（Cre-Negative）mice were intraperitoneally injected with tamoxifen（1 mg/10 g body weight,daily for 5 days）starting at 2 weeks to specifically inactivate ALK5 signaling in a tamoxifen-inducible and chondrocyte-specific manner（hereafter referred to as Alk5 cKO mice）.Immunohistochemistry（IHC）analysis was performed to detect the expression of ALK5 and pSmad3 in articular cartilage of 2-month-old Alk5 cKO mice and Cre-Negative mice.2.Knee joint samples were dissected from 2-,3-and 6-month-old mice.Safranin O/Fast green staining was performed to detect morphological changes of articular cartilage,osteophyte and subchondral bone.An Osteoarthritis Research Society International（OARSI）recommended subjective scoring system was used to quantify the histologic grading of cartilage degeneration in mice.3.Quantitative real-time polymerase chain reaction（qRT-PCR）,Western blotting（WB）and IHC analysis were performed to detect expressions of ECM related genes in articular cartilage of Alk5 cKO mice and Cre-Negative mice.4.Knee joint samples were dissected from 2-month-old mice,TUNEL assay and cleaved caspase 3 IHC were performed to detect the apoptosis in articular cartilage of Alk5 cKO mice and Cre-Negative mice.5.Knee joint samples were dissected from 3-month-old mice,and hematoxylin and eosin staining（H&E）was performed to detect morphological changes of synovium.6.Detection the effect of ALK5 signaling on the protein expression of PRG4 in chondrocytes.1)IHC analysis was performed to detect expressions of PRG4 in in articular cartilage of2-month-old Alk5 cKO mice and Cre-Negative mice.2)qRT-PCR analysis was performed to detect the mRNA expression of Prg4 in articular chondrocytes of Alk5 cKO mice and Cre-Negative mice.3)qRT-PCR analysis was performed to detect the mRNA expression of Prg4 in articular chondrocytes treated with increasing concentrations of ALK5 inhibitor SB-505124（0.1,0.5 and1μM）for 24 hours or treated with 1μM SB-505124 for different periods of time（6,12 and 24hours）.7.Detection the role of ALK5 signaling in TGF-β1-induced PRG4 expression in articular chondrocytes.1)qRT-PCR and WB were performed to detect the expression of PRG4 in articular chondrocytes treated with increasing concentrations of TGF-β1（5,10 and 20 ng/ml）for 24hours or treated with 10 ng/ml TGF-β1 for different periods of time（6,12 and 24 hours）.2)Articular chondrocytes were pretreated with 1μM SB-505124 for 30 minutes,followed by an additional 24 hours incubation of 10 ng/ml TGF-β1,1μM SB-505124,or a combination of both.q RT-PCR and WB were performed to detect the expression pf PRG4.3)Cre-Negative and Alk5-deficient articular chondrocytes were treated with 10 ng/ml TGF-β1 for 24 hours.qRT-PCR was performed to detect the mRNA expression of Prg4.4)Articular chondrocytes were transfected with constitutively activated ALK5（CA-ALK5）expression plasmid or pcDNA3.1/Myc empty vector.12 hours after transfection,cells were treated with 1μM SB-505124 for another 12 hours.qRT-PCR was performed to detect the Prg4m RNA expression.8.Detection the role of PKA-CREB signaling in TGF-β1/ALK5-induced PRG4 expression in articular chondrocytes.1)Articular chondrocytes were pretreated with 10μM PKA inhibitor H89 for 30 minutes,followed by an additional 24 hours incubation with 10ng/ml TGF-β1,H89,or a combination of both.q RT-PCR and WB were performed to detect the expression of PRG4.2)Articular chondrocytes were pretreated with 10μM H89 or 1μM SB-505124 for 30minutes,followed by an additional 30 minutes incubation of 10 ng/ml TGF-β1,10μM H89/1μM SB-505124,or a combination of both.WB was performed to detect the pCREB and pSmad3protein expressions.3)Cre-Negative or Alk5-deficient articular chondrocytes were treated with TGF-β1 for 30minutes.WB was performed to detect the pCREB protein expression.4)IHC analysis was performed to detect the expression of pCREB in articular cartilage of2-month-old Alk5 cKO mice and Cre-Negative mice.5)Articular chondrocytes were pretreated with CBP-CREB interaction inhibitor for 30minutes,followed by an additional 24 hours incubation with TGF-β1,CBP-CREB interaction inhibitor,or a combination of both.q RT-PCR and WB were performed to detect the expression of PRG4.6)Alk5-deficient articular chondrocytes were treated with increasing concentrations of PKA/CREB agonist forskolin for 24 hours.q RT-PCR was performed to detect Prg4 m RNA expression.PartⅡThe role and mechanisms of ALK5 signaling in articular cartilage superficial zone cells phenotypic expression and the development of arthritis1.Postnatal superficial zone cells-specific inducible deletion of Alk5 in miceAlk5^flox/flox;Prg4^{GFPCre ERT2/+}(Alk5^prg4ERT2)and Alk5^flox/flox（Cre-Negative）mice were intraperitoneally injected with tamoxifen（1 mg/10 g body weight,daily for 10 days）starting at20 days to specifically inactivate ALK5 signaling in a tamoxifen-inducible and superficial zone cells-specific manner（hereafter referred to as Alk5 pcKO mice）.Immunohistochemistry（IHC）analysis was performed to detect the expression of ALK5 and pSmad3 in articular cartilage superficial zone of 34-day-old Alk5 pcKO mice and Cre-Negative mice.2.Knee joint samples were dissected from 4-and 8-month-old mice.Safranin O/Fast green staining was performed to detect age-associated morphological changes of articular cartilage.3.The destabilization of the medial meniscus（DMM）induced OA model was made in right knee joints of 2-mold-old male Cre-Negative and Alk5 pcKO mice.Sham surgery was performed on left knee joints per animal with medial capsulotomy only.Mice were kept for an additional 8weeks after surgery and joint samples were harvested for histological analysis.Safranin O/Fast green staining was performed to detect surgically induced morphological changes of articular cartilage.4.Knee joint samples were dissected from 4-month-old mice.H&E staining of femoral articular cartilage was performed to detect the number of superficial zone cells in Alk5 pcKO mice and Cre-Negative.5.Mice received a daily intraperitoneal injection of 50mg/kg Edu for 5 times starting at P11.Knee joint samples were dissected from 2-month-old mice.Edu and TUNEL assays were performed to detect the Edu-labeled cells and apoptosis cells in articular cartilage of Alk5 pcKO mice and Cre-Negative mice,respectively.6.Isolation and testing of superficial zone cells（SFZ cells）7.MTT assay and morphological analysis were performed to detect the role of TGF-β1/ALK5 signaling on the proliferation of SFZ cells.8.q RT-PCR and IHC analysis were performed to detect the role of TGF-β1/ALK5 signaling on the markers gene expression of SFZ cells.9.q RT-PCR analysis and Alcian blue staining were performed to detect the role of ALK5signaling on SFZ cell chondrogenic differentiation potential.Results:PartⅠInducible cartilage-specific deletion of Alk5 gene results in a progressive Osteoarthritis-like phenotype in mice1.IHC results showed that the numbers of ALK5-and pSmad3-positive cells in the articular cartilage were significantly decreased in Alk5 cKO mice compared with that in Cre-Negative mice,which demonstrated that Alk5 was effectively deleted in articular cartilage.2.Safranin O/Fast green staining results showed that from age 2 to 6 months,a progressive Osteoarthritis-like phenotype became apparent in Alk5 cKO mice,including loss of proteoglycan content,increased hypertrophic chondrocytes,loss of articular cartilage tissue,osteophyte formation and subchondral sclerosis.3.Chondrocyte-specific knockout of Alk5 gene significantly increased the expressions of collagen X,MMP13 and ADAMTS5,while decreased the expressions of aggrecan and Col 2.4.TUNEL-and cleaved caspase 3-positive cells were markedly increased in articular cartilage of Alk5 c KO mice compared with those in Cre-Negative mice.5.H&E staining showed that synovial hyperplasia was apparent in Alk5 c KO mice.6.Reduced expression of PRG4 in articular cartilage of Alk5 cKO mice1)IHC results revealed that the PRG4-positive cells in articular cartilage were significantly decreased in Alk5 cKO mice compared with that in Cre-Negative mice.2)qRT-PCR results showed that the m RNA expression of Prg4 was downregulated in Alk5-deficient articular chondrocytes.3)qRT-PCR results showed SB-505124 significantly decreased the m RNA expression of Prg4 in a dose-and time-dependent manner.7.TGF-β1 induces the expression of PRG4 via ALK5 signaling in articular chondrocytes.1)qRT-PCR and WB results showed that TGF-β1 robustly induced the PRG4 m RNA and protein expressions in a dose-and time-dependent manner2)Deletion of Alk5 or ALK5 inhibitor attenuated the TGF-β1-induced PRG4 expression in articular chondrocyte.3)Constitutively Activated ALK5（CA-ALK5）directly induced the Prg4 mRNA expression in articular chondrocyte,while SB-505124 attenuated the CA-ALK5-induced Prg4m RNA expression.8.TGF-β1/ALK5 regulates Prg4 expression through the PKA-CREB pathway.1)qRT-PCR and WB results showed that PKA inhibitor H89 attenuated the TGF-β1-induced PRG4 expression in articular chondrocyte.2)WB results showed that H89 and SB-505124 attenuated the TGF-β1-induced phosphorylation of CREB in articular chondrocyte.3)WB results showed that deletion of Alk5 attenuated the TGF-β1-induced phosphorylation of CREB in articular chondrocyte.4)IHC results showed that the number of pCREB-positive cells in articular cartilage were significantly decreased in Alk5 cKO mice compared with that in Cre-Negative mice.4)qRT-PCR and WB results showed that CBP-CREB interaction inhibitor attenuated the TGF-β1-induced PRG4 expression in articular chondrocyte.5)qRT-PCR result showed that forskolin could rescue the reduced expression of Prg4 in Alk5-deficient articular chondrocytes.PartⅡThe role and mechanisms of ALK5 signaling in articular cartilage superficial zone cells phenotypic expression and the development of arthritis1.IHC results showed that the numbers of ALK5-and p Smad3-positive cells in the superficial zone were significantly decreased in Alk5 pcKO mice compared with those in Cre-Negative mice,which demonstrated that Alk5 was effectively deleted in superficial zone.2.Safranin O/Fast green staining results showed that increased aberrant hypertrophic chondrocytes in superficial and mild zone of 4-month-old Alk5 pcKO mice,and tears and defect in articular cartilage the 8-month-old Alk5 pc KO mice.3.Safranin O/Fast green staining results showed that Alk5 pcKO mice 8 weeks after DMM surgery exhibited significant cartilage damage and severe loss of proteoglycans,whereas su ch changes were significantly milder in Cre-Negative mice.4.H&E staining showed that numbers of superficial zone cells were decreased in Alk5pcKO mice compared with that in Cre-Negative mice.5.Edu assay showed that numbers of Edu-labeled long-lasting slow-cell cycle cells were decreased,while TUNEL-postive cells were markedly increased in superficial zone of Alk5pcKO mice compared with those in Cre-Negative mice.6.SFZ cells were successfully isolated from superficial zone of postnatal mice.Microscopic analysis showed that the SFZ cells displayed an elongated and fibroblastic cytoarchitecture,while chondrocytes were polygonal and epithelioid cytoarchitecture as to be expected.The SFZ cells strongly expressed mesenchymal stem cell markers including CD70,CD90 and CD105,and superficial zone cells markers including Prg4,Ets-related gene（Erg）and tenascin C,while the expression of Col2,Aggrecan（Acan）and Matrilin-1（Mat-1）was much lower in SFZ cells than chondrocytes.7.MTT and morphological analysis results showed that Alk5 ablation inhibited cell proliferation and attenuated proliferation promotion effect of TGF-β1.8.qRT-PCR result show that Alk5 ablation reduced the mRNA expressions of CD105,Prg4and tenascin C,and attenuated TGF-β1-induced gene expressions.Meanwhile,IHC result showed that Alk5 ablation reduced the protein expression PRG4 in superficial zone.9.Microscopic analysis and Alcian blue staining showed that SFZ cells were able to undergo chondrogenesis in in vitro.q RT-PCR and Alcian blue staining results showed that Alk5ablation inhibited chondrogenic differentiation potential through inhibiting the expressions of Col2 and Aggrecan,while promoted hypertrophic differentiation and accelerated matrix degradation through increasing the expressions of Col10,Mmp13 and Adamts5.Conclusion:1.Inducible chondrocyte-specific deletion of Alk5 gene leads to age-associated progressive OA-like phenotype,at least partly,via down-regulation of PKA-CREB signaling and PRG4expression in articular cartilage.2.Inducible superficial zone cells-specific deletion of Alk5 gene impaired proliferation,phenotypic expression and chondrogenic differentiation potentials of superficial zone cells,eventually accelerated age-related and surgery-induced OA development.