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 translationally-controlled 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 chondrocytes and these differentially expressed genes have unique roles to play corresponding to the function of their respective zones.

Figure 1. Top 30 genes showing the greatest expression in PC and RZ

Venn diagram of differential expression according to growth plate zone. A list of genes was designated as “present” in PC and RZ using Affymetrix GCOS/MAS 5 software. Differential gene expressions within PC and RZ were identified by GeneSpring GX and a raw dataset was created using the Robust Multiarray analysis (RMA) method. The top 30 genes by highest expression within both RZ and PC (individually) were identified, excluding unnamed transcribed locus probes and duplicate gene names. Twenty-two genes from the top 30 genes expressed within the PC were also present among the top 30 genes expressed within the RZ. Only 8 genes from each list were unique to the respective zone.

Figure 2. Top 30 genes in PC and RZ by greatest expression compared to 33 genes by two-way ANOVA and 5 times changed

To identify the significant differential gene expression between PC and RZ chondrocytes, the PZ was used as a comparator. Normalized data was filtered on expression using a cutoff of five times changed up or down in PC, RZ and PZ described as in the methods section. Differential expression between zones was analyzed using two-way ANOVA and created a gene list of 33 significant differentially expressed genes. When comparing the gene list of 33 significantly different than the PZ to the top 30 by expression in both PC and RZ, there was one gene in common for each of the zones.