Microarray Analysis of Perichondral and Reserve Growth Plate Zones Identifies Differential Gene Expressions and Signal Pathways

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.