Collagen type X expression and chondrocyte hypertrophic differentiation during OA and OS development.

Chondrocyte hypertrophy and the expression of its specific marker, the collagen type X gene (COL10A1), constitute key terminal differentiation stages during endochondral ossification in long bone development. Mutations in the COL10A1 gene are known to cause schmid type metaphyseal chondrodysplasia (SMCD) and spondyloepiphyseal dyschondrodysplasia (SMD). Moreover, abnormal COL10A1 expression and aberrant chondrocyte hypertrophy are strongly correlated with skeletal diseases, notably osteoarthritis (OA) and osteosarcoma (OS). Throughout the progression of OA, articular chondrocytes undergo substantial changes in gene expression and phenotype, including a transition to a hypertrophic-like state characterized by the expression of collagen type X, matrix metalloproteinase-13, and alkaline phosphatase. This state is similar to the process of endochondral ossification during cartilage development. OS, the most common pediatric bone cancer, exhibits characteristics of abnormal bone formation alongside the presence of tumor tissue containing cartilaginous components. This observation suggests a potential role for chondrogenesis in the development of OS. A deeper understanding of the shifts in collagen X expression and chondrocyte hypertrophy phenotypes in OA or OS may offer novel insights into their pathogenesis, thereby paving the way for potential therapeutic interventions. This review systematically summarizes the findings from multiple OA models (e.g., transgenic, surgically-induced, mechanically-loaded, and chemically-induced OA models), with a particular focus on their chondrogenic and/or hypertrophic phenotypes and possible signaling pathways. The OS phenotypes and pathogenesis in relation to chondrogenesis, collagen X expression, chondrocyte (hypertrophic) differentiation, and their regulatory mechanisms were also discussed. Together, this review provides novel insights into OA and OS therapeutics, possibly by intervening the process of abnormal endochondral-like pathway with altered collagen type X expression.

Ddx5 participates in regulation of Col10a1 expression and chondrocyte hypertrophic differentiation in vitro.

BACKGROUND AND AIMS: The type X collagen gene (Col10a1), is a specific molecular marker of hypertrophic chondrocytes during endochondral ossification. Col10a1 expression is known to be influenced by many regulators. In this study, we aim to investigate how DEAD-box helicase 5 (DDX5), a potential binding factor for Col10a1 enhancer, may play a role in Col10a1 expression and chondrocyte hypertrophic differentiation in vitro. METHODS: The potential binding factors of the 150-bp Col10a1 cis-enhancer were identified with the hTFtarget database. The expression of DDX5 and COL10A1 was detected by quantitative real-time PCR (qRT-PCR) and Western blot in chondrogenic ATDC5 and MCT cell models with or without Ddx5 knockdown or overexpression. Dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) were performed to determine the interaction between DDX5 and the Col10a1 enhancer. The effect and mechanism of DDX5 on chondrocyte differentiation and maturation was evaluated by alcian blue, alkaline phosphatase (ALP), and alizarin red staining in ATDC5 cell lines with stable knockdown of Ddx5. RESULTS: DDX5 was identified as a potential binding factor for the Col10a1 enhancer. The expression of DDX5 in hypertrophic chondrocytes was higher than that in proliferative chondrocytes. Knockdown of Ddx5 decreased, while overexpression of Ddx5 slightly increased COL10A1 expression. DDX5 promotes the enhancer activity of Col10a1 as demonstrated by dual-luciferase reporter assay, and the ChIP experiment suggests a direct interaction between DDX5 and the Col10a1 enhancer. Compared to the control (NC) group, we observed weaker alcian blue and ALP staining intensity in the Ddx5 knockdown group of ATDC5 cells cultured both for 7 and 14 days. Whereas weaker alizarin red staining intensity was only found in the Ddx5 knockdown group of cells cultured for 7 days. Meanwhile, knockdown of Ddx5 significantly reduced the level of runt-related transcription factor 2 (RUNX2) in related ATDC5 cells examined. CONCLUSIONS: Our results suggest that DDX5 acts as a positive regulator for Col10a1 expression and may cooperate with RUNX2 together to control Col10a1 expression and promote the proliferation and maturation of chondrocytes.

CircCRIM1 suppresses osteosarcoma progression via sponging miR146a-5p and targeting NUMB.

CircCRIM1 (hsa_circ_0002346) is a circular RNA derived from gene CRIM1 (the cysteine rich transmembrane BMP regulator 1 circRNAs) by back-splicing. Recent studies have suggested the diverse function of CircCRIM1 in the tumorigenesis of multiple malignancies, including osteosarcoma (OS). Here, we investigated the role and mechanism of circCRIM1 during OS progression. Differentially expressed circRNAs (including circCRIM1) in OS and human osteoblast (hFOB1.19) cell lines were selected by searching the circRNA expression microarray dataset of GSE96964. The expression levels of circCRIM1 and its sponging miRNAs and target genes were examined by RT-qPCR. The effects of circCRIM1 on the proliferation, migration, and invasion of OS cells were investigated by in vitro gain of function experiments. The in vivo function of circCRIM1 on OS was evaluated by measuring the subcutaneous and in situ tumor growth in nude mice. In addition, dual-luciferase reporter assay and in situ hybridization (FISH) were performed to explore the underlying mechanisms of circCRIM1 and its sponging miRNAs and target genes in OS. CircCRIM1 is downregulated in human OS cell lines and predominantly presents in the cytoplasm as demonstrated by RT-qPCR and FISH assays. Overexpression of circCRIM1 suppressed the migration, invasion, proliferation of OS cells in vitro and OS tumor growth in vivo. Mechanistically, we identified miR146a-5p as a sponge miRNA of circCRIM1 through bioinformatic prediction and confirmed their interaction and colocalization via reporter gene assay and FISH analysis. This interaction leads to increase expression of the downstream target gene NUMB, which will cause inhibition of the Notch signal pathway. We further demonstrated that miR146a-5p overexpression could reverse the antitumor effect induced by circCRIM1 in OS cells. Our results support that circCRIM1 acts as a tumor suppressor in OS by sponging miR146a-5p and its downstream target NUMB.

Stat5a promotes Col10a1 gene expression during chondrocyte hypertrophic differentiation.

OBJECTIVES: Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) expression via interaction with Col10a1 promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of Col10a1 cis-enhancer, in controlling Col10a1 gene expression and chondrocyte hypertrophic differentiation. METHODS: The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation. RESULTS: The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1. CONCLUSIONS: Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.

Mef2a is a positive regulator of Col10a1 gene expression during chondrocyte maturation.

BACKGROUND: The type X collagen gene (Col10a1) is a signature gene of hypertrophic chondrocytes that are known as the main engine of long bone growth. Multiple transcription factors (TFs), including myocyte enhancer factor 2A (Mef2a), have previously been identified by in silico analysis as potential Col10al gene regulators. OBJECTIVES: In this study, we aimed to investigate the correlation between Mef2a and Col10a1 expression and the possible effects on chondrocyte proliferation and hypertrophic differentiation in vitro. METHODS: First, Mef2a expression in proliferating and hypertrophic chondrocytes were detected by quantitative real-time PCR (qRT-PCR) and Western blotting in two chondrocytic models, ATDC5 and MCT cells, as well as in mouse chondrocytes in situ. Transfection with Mef2a small interfering fragments or Mef2a overexpression plasmids in the above chondrocytic models were performed to determine how Mef2a knockdown or overexpression may influence Col10a1 expression. The binding between Mef2a and its putative binding site within the 150 bp Col10a1 cis-enhancer which was evaluated by the dual luciferase reporter assay. The effect of Mef2a on chondrocyte differentiation was determined by examining the chondrogenic marker gene expression by qRT-PCR and by alcian blue, alkaline phosphatase (ALP), and alizarin red staining of the ATDC5 cells stably knocked down by Mef2a. RESULTS: The expression of Mef2a in hypertrophic chondrocytes was significantly higher than that in proliferative chondrocytes in both chondrocytic models as well as in mouse chondrocytes in situ. Interference with Mef2a caused decreased Col10a1 expression, while overexpression of Mef2a upregulated Col10a1. The result of the dual luciferase reporter assay showed that Mef2a enhanced Col10a1 gene enhancer activity via its putative Mef2a binding site. For the staining of ATDC5 stable cell lines, although no significant differences were seen in ALP staining, significantly weaker alcian blue staining intensity was noticed in Mef2a knockdown stable cell lines compared to the control cells at day 21, while slightly weaker alizarin red staining was seen in the stable cell lines at days 14 and 21. Correspondingly, we detected decreased runt-related transcription factor 2 (Runx2), increased SRY-box transcription factor 9 (Sox9), as well as differential expression of other chondrogenic markers in ATDC5 stable cell lines compared with the controls. CONCLUSIONS: In conclusion, our results support that Mef2a upregulates Col10a1 expression possibly by interaction with its cis-enhancer. Altered levels of Mef2a affects the expression of chondrogenic marker genes, such as Runx2 and Sox9, but may only play an insignificant role during chondrocyte proliferation and maturation.

CircNRIP1 acts as a sponge of miR-1200 to suppress osteosarcoma progression via upregulation of MIA2.

Circular RNAs (circRNAs), a class of non-coding RNAs, play an essential role in embryo development and carcinogenesis, circNRIP1 was recently identified to promote development of multiple human cancers. This study investigated the role of circNRIP1 in osteosarcoma (OS) cells and the potential mechanisms relating to the sponging miRNAs and their target genes. OS cell lines and normal human osteoblasts were grown for qRT-PCR analysis of circNRIP1 expression and functions of circNRIP1 expression in OS cell proliferation, migration, and invasion in vitro. Bioinformatics analysis was then performed to predict the sponge miRNA of circNRIP1 and the target gene, which was confirmed by using the dual-luciferase reporter assay. The in vivo functions of circNRIP1 was evaluated in OS cell xenograft models, while levels of relevant marker genes were examined using immunohistochemistry. CircNRIP1 was mainly localized in OS cell cytoplasm and significantly lower in OS cell lines than in normal human osteoblasts. CircNRIP1 overexpression significantly inhibited OS cell proliferation, migration, and invasion in vitro. miR-1200 was predicted as the sponge miRNA of circNRIP1 and directly interacted with circNRIP1 confirmed by the dual-luciferase reporter assay. Moreover, miR-1200 overexpression significantly alleviated the inhibitory effect of circNRIP1 on OS cells. A protein-coding gene MIA2 was identified as the miR-1200 targeting gene and reversely associated with miR-1200 expression in OS cells. Increase in MIA2 expression in a murine OS cell xenograft model was associated with circNRIP1 expression in inhibition of OS cell xenograft growth in vivo. These data support the circNRIP1 OS-suppressive role by sponge of miR-1200 expression and in turn to upregulate MIA2 expression.

Effect of iron supplementation on the expression of hypoxia-inducible factor and antioxidant status in rats exposed to high-altitude hypoxia environment.

Iron and oxygen are essential substance for cellular activity in body tissues. Hypoxia-inducible factors (HIFs) can respond to available oxygen changes in the cellular environment and regulate the transcription of a series of target genes. The study was conducted to investigate the effects of iron supplementation on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and antioxidant status in rats exposed to high-altitude hypoxia environment. Forty rats were divided into control (CON), hypobaric hypoxia (HH), and hypobaric hypoxia plus ferrous sulfate (FeSO4) (9.93 mg/kg body weight (BW)/day) (HFS) and hypobaric hypoxia plus iron glycinate chelate (Fe-Gly) (11.76 mg/kg BW/day) (HFG) groups. Results showed that Fe-Gly effectively alleviated weight loss and intestinal mucosa damage induced by hypobaric hypoxia, whereas FeSO4 aggravated hypobaric hypoxia-induced weight loss, liver enlargement, spleen atrophy, and intestinal damage. Iron supplementation decreased liver superoxide dismutase (T-SOD) and catalase (CAT) activity (P < 0.01) and increased iron concentration in the liver compared to HH group (P < 0.001). Moreover, Fe-Gly upregulated liver transferrin expression in messenger RNA (mRNA) level (P < 0.05) and downregulated serum erythropoietin (EPO) concentration (P < 0.01) and liver HIF-1α expression level (P < 0.05 in mRNA level; P < 0.001 in protein level) compared to HH group. The study indicated that FeSO4 supplementation at high altitudes aggravated the oxidative damage of tissues and organs that could be mediated through production of malondialdehyde (MDA) and inhibition antioxidant enzyme activities. Fe-Gly can protect hypobaric hypoxia-induced tissues injury. Moreover, iron supplementation at high altitudes affected HIF-1α-mediated regulating expression of targeting genes such as EPO and transferrin. The study highlights that iron supplementation under hypobaric hypoxia environment has possible limitation, and efficient supplementation form and dosage need careful consideration.