Antigenic profiles of human,bovine and canine articular chondrocytes

Summary Human, bovine and canine articular chondrocytes have been shown to bear cartilage matrix, chondrocyte-specific and histocompatibility antigens. These cell-surface antigens of chondrocytes were demonstrated both simultaneously and separately either by complement-mediated cytotoxicity or by immunohistochemical reactions. The chondrocyte-specific antigens involve subsets of species-common and species-specific determinants, which are also present on the surfaces of rib and laryngeal chondrocytes. In addition to these antigens, human and calf articular chondrocytes also express unique cell-surface components that are capable of producing a blastogenic stimulation of autologous T-lymphocytes in vitro. These putative autoantigens segregated from lymphocytes in vivo could be released in trauma and in inflammatory joint diseases triggering the immune system of the host.     

Critical roles of the TGF-β type I receptor ALK5 in perichondrial formation and function, cartilage integrity, and osteoblast differentiation during growth plate development

TGF-β has been implicated in the proliferation and differentiation of chondrocytes and osteoblasts. However, the in vivo function of TGF-β in skeletal development is unclear. In this study, we investigated the role of TGF-β signaling in growth plate development by creating mice with a conditional knockout of the TGF-β type I receptor ALK5 (ALK5^CKO) in skeletal progenitor cells using Dermo1-Cre mice. ALK5^CKO mice had short and wide long bones, reduced bone collars, and trabecular bones. In ALK5^CKO growth plates, chondrocytes proliferated and differentiated, but ectopic cartilaginous tissues protruded into the perichondrium. In normal growth plates, ALK5 protein was strongly expressed in perichondrial progenitor cells for osteoblasts, and in a thin chondrocyte layer located adjacent to the perichondrium in the peripheral cartilage. ALK5^CKO growth plates had an abnormally thin perichondrial cell layer and reduced proliferation and differentiation of osteoblasts. These defects in the perichondrium likely caused the short bones and ectopic cartilaginous protrusions. Using tamoxifen-inducible Cre-ER™-mediated ALK5-deficient primary calvarial cell cultures, we found that TGF-β signaling promoted osteoprogenitor proliferation, early differentiation, and commitment to the osteoblastic lineage through the selective MAPKs and Smad2/3 pathways. These results demonstrate the important roles of TGF-β signaling in perichondrium formation and differentiation, as well as in growth plate integrity during skeletal development.     

Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia

The FGF signaling pathway plays essential roles in endochondral ossification by regulating osteoblast proliferation and differentiation, chondrocyte proliferation, hypertrophy, and apoptosis. FGF signaling is controlled by the complementary action of both positive and negative regulators of the signal transduction pathway. The Spry proteins are crucial regulators of receptor tyrosine kinase-mediated MAPK signaling activity. Sprys are expressed in close proximity to FGF signaling centers and regulate FGFR-ERK-mediated organogenesis. During endochondral ossification, Spry genes are expressed in prehypertrophic and hypertrophic chondrocytes. Using a conditional transgenic approach in chondrocytes in vivo, the forced expression of Spry1 resulted in neonatal lethality with accompanying skeletal abnormalities resembling thanatophoric dysplasia II, including increased apoptosis and decreased chondrocyte proliferation in the presumptive reserve and proliferating zones. In vitro chondrocyte cultures recapitulated the inhibitory effect of Spry1 on chondrocyte proliferation. In addition, overexpression of Spry1 resulted in sustained ERK activation and increased expression of p21 and STAT1. Immunoprecipitation experiments revealed that Spry1 expression in chondrocyte cultures resulted in decreased FGFR2 ubiquitination and increased FGFR2 stability. These results suggest that constitutive expression of Spry1 in chondrocytes results in attenuated FGFR2 degradation, sustained ERK activation, and up-regulation of p21Cip and STAT1 causing dysregulated chondrocyte proliferation and terminal differentiation.     

Secretion of inflammatory factors from chondrocytes by layilin signaling

Layilin (LAYN) is thought to be involved in reorganization of cytoskeleton structures, interacting with merlin, radixin, and talin. Also, LAYN is known to be one of the receptors for hyaluronic acid (HA).     

Mesenchyme-specific Knockout of ESET Histone Methyltransferase Causes Ectopic Hypertrophy and Terminal Differentiation of Articular Chondrocytes

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.     

磁纳米颗粒标记脂肪干细胞向软骨分化及体外MRI示踪的实验研究

目的:探讨磁纳米颗粒(magnetic iron oxide particles,MIOP)体外标记脂肪间充质干细胞(ASCs)向软骨分化及MRI示踪的可行性。方法:从小鼠脂肪组织中分离培养、扩增脂肪间充质干细胞(ASCs),流式鉴定细胞表型后,分别采用不同浓度(25μg/mL,50μg/mL)的MIOP标记ASCs并向软骨细胞诱导分化。普鲁士蓝染色和透射电镜(TEM)鉴定细胞内磁纳米铁颗粒分布情况,应用3.0T MRI体外检测标记软骨细胞MRI信号。结果:从脂肪组织中可以分离获得大量高表达CD90、CD105、Sca-1的ASCs,不同浓度(25μg/mL,50μg/mL)的MIOP与ASCs共同孵育24小时后,普鲁士蓝染色发现ASCs随MIOP浓度的增加,蓝染程度逐渐加深且标记的ASCs可以向软骨细胞分化;TEM证实细胞内分布大量的黑色纳米铁颗粒。体外MRI T2序列证实随着MIOP浓度(25μg/mL,50μg/mL)的增加MRI信号值逐渐减低且具有统计学差异(P0.05)。结论:MIOP可以标记ASCs向软骨分化,体外应用MRI可以对其进行示踪。     

Intracellular modulation of signaling pathways by annexin A6 regulates terminal differentiation of chondrocytes

Annexin A6 (AnxA6) is highly expressed in hypertrophic and terminally differentiated growth plate chondrocytes. Rib chondrocytes isolated from newborn AnxA6-/- mice showed delayed terminal differentiation as indicated by reduced terminal differentiation markers, including alkaline phosphatase, matrix metalloproteases-13, osteocalcin, and runx2, and reduced mineralization. Lack of AnxA6 in chondrocytes led to a decreased intracellular Ca(2+) concentration and protein kinase C α (PKCα) activity, ultimately resulting in reduced extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activities. The 45 C-terminal amino acids of AnxA6 (AnxA6(1-627)) were responsible for the direct binding of AnxA6 to PKCα. Consequently, transfection of AnxA6-/- chondrocytes with full-length AnxA6 rescued the reduced expression of terminal differentiation markers, whereas transfection of AnxA6-/- chondrocytes with AnxA6(1-627) did not or only partially rescued the decreased mRNA levels of terminal differentiation markers. In addition, lack of AnxA6 in matrix vesicles, which initiate the mineralization process in growth plate cartilage, resulted in reduced alkaline phosphatase activity and Ca(2+) and inorganic phosphate (P(i)) content and the inability to form hydroxyapatite-like crystals in vitro. Histological analysis of femoral, tibial, and rib growth plates from newborn mice revealed that the hypertrophic zone of growth plates from newborn AnxA6-/- mice was reduced in size. In addition, reduced mineralization was evident in the hypertrophic zone of AnxA6-/- growth plate cartilage, although apoptosis was not altered compared with wild type growth plates. In conclusion, AnxA6 via its stimulatory actions on PKCα and its role in mediating Ca(2+) flux across membranes regulates terminal differentiation and mineralization events of chondrocytes.     

Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Osteoarthritis (OA) is characterized by cartilage degradation. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. The aim of this study was to analyze, by a proteomic approach, the changes that are characteristic of OA chondrocytes, and to identify new OA-related proteins. Chondrocytes were isolated from the cartilage of ten OA patients undergoing joint replacement and ten donors with no history of joint disease. Whole-cell proteins were resolved by 2-DE and stained with SYPRO Ruby. Protein expression patterns of 2-DE gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA-related proteins were identified by MALDI-TOF or MALDI-TOF/TOF MS. The results were validated for ANXA1, GSTO1, GRP78, and HSP90beta in cells by Western blotting and in tissue cartilage by immunohistochemistry. Results showed an average of 700 protein spots that were present in the 2-DE gels. Compared to normal chondrocytes, 19 protein spots were found to be significantly increased in OA cells (ratio OA:N> or =2.0, p<0.05), whereas nine were decreased in OA chondrocytes (ratio OA:N< or =0.5, p<0.05). Three stress response proteins were increased (HSP90beta, GRP78, and GRP94) and three proteins involved in glycolysis were decreased (enolase, glyceraldehyde 3-phosphate dehydrogenase, and fructose biphosphate aldolase). Functionally, almost all proteins could be classified as proteins involved in cellular metabolism (33%), structure (21%), or protein targeting (21%).