Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways

Abstract

In the growth plate, the reserve and perichondral zones have been hypothesized to have similar functions, but their exact functions are poorly understood. Our hypothesis was that significant differential gene expression exists between perichondral and reserve chondrocytes that may differentiate the respective functions of these two zones. Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC) and reserve zone (RZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator. Confirmation of the differential expression of selected genes was done by quantitative real time RT-PCR. A total of 8 transcripts showing high expression unique to the PC included translationallycontrolled tumor protein (Tpt1), connective tissue growth factor (Ctgf), mortality factor 4 (Morf4l1), growth arrest specific 6 (Gas6), type V procollagen (Col5a2), frizzled-related protein (Frzb), GDP dissociation inhibitor 2 (Gdi2) and Jun D proto-oncogene (Jund). In contrast, 8 transcripts showing unique high expression in the RZ included hyaluronan and proteoglycan link protein 1 (Hapln1), hemoglobin beta-2 subunit, type I procollagen (Col1a2), retinoblastoma binding protein 4 (LOC685491), Sparc related modular calcium binding 2 (Smoc2), and calpastatin (Cast). Other genes were highly expressed in cells from both PC and RZ zones, including collagen II, collagen IX, catenin (cadherin associated protein) beta 1, eukaryotic translation elongation factor, high mobility group, ribosomal protein, microtubule-associated protein, reticulocalbin, thrombospondin, retinoblastoma binding protein, carboxypeptidase E, carnitine palmitoyltransferase 1, cysteine rich glycoprotein, plexin B2 (Plxnb2), and gap junction membrane channel protein. Functional classification of the most highly expressed transcripts were analyzed, and the pathway analysis indicated that ossification, bone remodeling, and cartilage development were uniquely enriched in the PC whereas both the PC and RZ showed pathway enrichment for skeletal development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part pathways. We conclude that differential gene expression exists between the RZ and PC

Microarray Analysis of Irradiated Growth Plate Zones following Laser Microdissection Shows Later Importance of Differentially Expressed Genes during Radiorecovery

“…Previous microarray studies in our lab have suggested the importance of the ECM genes in all zones of the GP following irradiation. The ECM genes Sparc, Gas6, Fn1, Timp3, and Mmp2 and various procollagens have shown high expression levels in the RZ and PC following fractionated irradiation and have shown differential expression in irradiated compared to nonirradiated PZ and HZ following single-fraction irradiation [Zhang et al, , 2008. The differential expression of these related genes within the RZ in the current data set further supports their importance in radiorecovery.…”

“…The 24 RNA samples included the right and left RZ at 16 days, the right and left PZ at 16 days, the right and left HZ at 16 days, and the right and left PC at 16 days. Identical sets for 7 and 11 days were the focus of earlier reports [Damron et al, 2009;Zhang et al, 2008]. Each pooled sample had 30-50 ng RNA.…”

Endogenous regeneration after collagenase-induced knee joint damage in the adult newt Notophthalmus viridescens

“…The product of GAS6 is a ligand for the members of the Axl family of receptor tyrosine kinases which play a role in dedifferentiation and early blastema formation during regeneration of the adult newt forelimb 28. Additionally, GAS6 has been shown to negatively regulate chondrogenic differentiation of mesenchymal cells,29 and it also seems to play a role in the perichondral zone of growth plates 30. Given the observation that GAS6 showed homogenous expression over time in our model, one could assume that the regeneration of localised joint damage might occur through molecular pathways different from whole limb regeneration.…”

Microarray Cluster Analysis of Irradiated Growth Plate Zones Following Laser Microdissection

“…Meaningful differential expression was determined at the 99.9% confidence level. (7) We included genes at the extreme tails of the distribution curve at +/- 3.3 z-scores from the mean, rather than an arbitrary fold difference.…”