Microarray analysis of proliferative and hypertrophic growth plate zones identifies differentiation markers and signal pathways

Department of Biomedical Sciences, Cornell University, Итак, New York, United States
Bone (Impact Factor: 3.97). 01/2005; 35(6):1273-93. DOI: 10.1016/j.bone.2004.09.009
Source: PubMed


Longitudinal bone growth results from coordination of proliferation and hypertrophy of chondrocytes, calcification of the matrix, vascular invasion, and completion of endochondral bone formation in the growth plate. Although proliferative and hypertrophic chondrocytes are well characterized histomorphologically, the understanding of factors governing this transition is not fully explained. Our hypothesis was that significant differential gene expression exists between proliferative and hypertrophic chondrocytes that may provide clues to the regulation of this transition at the transcriptional level. Normal Sprague-Dawley rat growth plate chondrocytes from the proliferative zone (PZ) and hypertrophic zone (HZ) were isolated by laser capture microdissection and then subjected to microarray analysis. Confirmation of the differential expression of selected genes was done by in situ hybridization and quantitative reverse transcription (RT) polymerase chain reaction (PCR). A total of 40 transcripts showed at least twofold greater expression in the PZ compared to HZ at both 6 and 7 weeks of age, while 52 transcripts showed twofold greater expression in the HZ compared to PZ at these time points. Many of the differentially expressed genes in each zone had very high levels of expression and thus were classified as "enriched transcripts" for that zone. The PZ-enriched transcripts included fibromodulin, proline arginine-rich end leucine-rich repeat protein, lactate dehydrogenase, and enolase 1 alpha. In contrast, HZ-enriched transcripts included collagen I, protein kinase (lysine deficient 4), proteasome (prosome, macropain) activator subunit 4, prostaglandin I2 synthase, and integrin-binding sialoprotein, matrix metalloproteinase 13 (MMP13), and collagen X. Other genes were highly expressed in cells from both zones, including collagen II, aggrecan, cartilage oligomeric protein, cartilage link protein, laminin receptor, and eukaryotic translocation elongation factor. Functional classification of the PZ-enriched transcripts showed an increased percentage of genes expressed in nuclear cell cycle and transcription functions. In contrast, the HZ-enriched transcripts were more involved in extracellular structure and membrane receptor and transporter functions. Pathway analysis indicated that transforming growth factor beta and parathyroid hormone-related protein (PTHrP) pathways were important in both zones, and bone morphogenic protein pathway played a role in the HZ. It is likely that these differentially expressed genes are involved in regulation of the transition from proliferation to differentiation functions in the growth plate.

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    • "In physiological endochondral ossification, chondrocytes become hypertrophic and remove the extracellular matrix proteins by expressing MMPs and ADMTSs. Chondrocytes finally die by apoptosis and are substituted by osteoblasts [28],[29]. Recent studies disclose that OA follow a similar path to the physiological endochondral ossification: chondrocytes lose the stable phenotype and undergo terminal differentiations, as indicated by upregulation of marker genes for hypertrophy [26]. Wnt/β-catenin signaling pathway is known to drive endochondral ossifications by upregulating MMPs and ADAMTSs [30] in both physiological and pathological conditions [8]. "
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    ABSTRACT: In past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. FRZB, a soluble antagonist of Wnt signaling, has been studied in osteoarthritis (OA) animal models and OA patients as a modulator of Wnt signaling. We screened for FDA-approved drugs that induce FRZB expression and suppress Wnt/β-catenin signaling. We found that verapamil, a widely prescribed L-type calcium channel blocker, elevated FRZB expression and suppressed Wnt/β-catenin signaling in human OA chondrocytes. Expression and nuclear translocation of β-catenin was attenuated by verapamil in OA chondrocytes. Lack of the verapamil effects in LiCl-treated and FRZB-downregulated OA chondrocytes also suggested that verpamil suppressed Wnt signaling by inducing FRZB. Verapamil enhanced gene expressions of chondrogenic markers of ACAN encoding aggrecan, COL2A1 encoding collagen type II α1, and SOX9, and suppressed Wnt-responsive AXIN2 and MMP3 in human OA chondrocytes. Verapamil ameliorated Wnt3A-induced proteoglycan loss in chondrogenically differentiated ATDC5 cells. Verapamil inhibited hypertrophic differentiation of chondrocytes in the explant culture of mouse tibiae. Intraarticular injection of verapamil inhibited OA progression as well as nuclear localizations of β-catenin in a rat OA model. We propose that verapamil holds promise as a potent therapeutic agent for OA by upregulating FRZB and subsequently downregulating Wnt/β-catenin signaling.
    PLoS ONE 03/2014; 9(3):e92699. DOI:10.1371/journal.pone.0092699 · 3.23 Impact Factor
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    • "The use of DNA microarray techniques has made it possible for large-scale analysis of gene function and regulation to be conducted. DNA microarrays have been used to study skeletal development in humans and rodents [13-15]. In this study, we screened temporal changes in DEGs during early stages of TD in broiler chickens using an Affymetrix GeneChip, and verified expression patterns at different phases by qPCR. "
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    ABSTRACT: Background Tibial dyschondroplasia (TD) is a common skeletal disorder in broiler chickens. It is characterized by the presence of a non-vascularized and unmineralized cartilage in the growth plate. Previous studies have investigated differential expression of genes related to cartilage development during latter stages of TD. The aim of our study was to identify differentially expressed genes (DEGs) in the growth plate of broiler chickens, which were associated with early stage TD. We induced TD using tetramethylthiuram disulfide (thiram) for 1, 2, and 6 days and determined DEGs with chicken Affymetrix GeneChip assays. The identified DEGs were verified by quantitative polymerase chain reaction (qPCR) assays. Results We identified 1630 DEGs, with 82, 1385, and 429 exhibiting at least 2.0-fold changes (P < 0.05) at days 1, 2, and 6, respectively. These DEGs participate in a variety of biological processes, including cytokine production, oxidation reduction, and cell surface receptor linked signal transduction on day 1; lipid biosynthesis, regulation of growth, cell cycle, positive and negative gene regulation, transcription and transcription regulation, and anti-apoptosis on day 2; and regulation of cell proliferation, transcription, dephosphorylation, catabolism, proteolysis, and immune responses on day 6. The identified DEGs were associated with the following pathways: neuroactive ligand-receptor interaction on day 1; synthesis and degradation of ketone bodies, terpenoid backbone biosynthesis, ether lipid metabolism, JAK-STAT, GnRH signaling pathway, ubiquitin mediated proteolysis, TGF-β signaling, focal adhesion, and Wnt signaling on day 2; and arachidonic acid metabolism, mitogen-activated protein kinase (MAPK) signaling, JAK-STAT, insulin signaling, and glycolysis on day 6. We validated seven DEGs by qPCR. Conclusions Our findings demonstrate previously unrecognized changes in gene transcription associated with early stage TD. The DEGs we identified by microarray analysis will be used in future studies to clarify the molecular pathogenic mechanisms of TD. From these findings, potential pathways involved in early stage TD warrant further investigation.
    BMC Genomics 04/2013; 14(1):276. DOI:10.1186/1471-2164-14-276 · 3.99 Impact Factor
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    Arthritis research & therapy 04/2012; 14(2):R82. DOI:10.1186/ar3805 · 3.75 Impact Factor
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