MICROARRAY ANALYSIS OF PROLIFERATIVE AND HYPERTROPHIC GROWTH PLATE ZONES IDENTIFIES
DIFFERENTIATION MARKERS AND SIGNAL PATHWAYS
*Wang, Y; *Middleton, F; *Horton, J; **Farnum C E; +*Damron, T A
*SUNY Upstate Medical University, Syracuse, NY and **Cornell University, Ithaca, NY
INTRODUCTION: 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, an
understanding of the factors regulating progression in the growth plate is
not fully explained. Whether the pathways currently implicated in control
of longitudinal growth are the primary ones responsible is unknown.
Combination of laser microdissection and cDNA microarray can enable a
comprehensive investigation of these complex interactions in different
growth plate zones. Our hypothesis was that significant differential gene
expression exists between proliferative (PZ) and hypertrophic (HZ)
chondrocytes that would provide clues to the regulation of this transition
at the transcriptional level.
METHODS: Laser Microdissection and Microarray Analysis: Three 6
week-old and three 7 week male SD rats were sacrificed and the PZ and
HZ chondrocytes were harvested by laser microdissection (Fig 1). The
RAE230A chip was used to evaluate genes by pooled RNA samples
extracted from 3 animals/time point for the PZ and the HZ chondrocytes.
Analyses of Changes in Gene Expression: To identify differentially
expressed genes in the PZ and HZ, Affymetrix MAS and Gene Traffic 2.7
were used to make separate, age-matched comparisons and determine
fold-changes between pooled PZ and HZ specimens using RMA method.
All differentially expressed genes with fold change > 2 at both 6 and 7
weeks for the PZ and HZ from this list were identified as well.
In Situ Hybridization and Real-Time RT-PCR: In situ hybridization with
S35 cRNA riboprobes and Real-Time RT -PCR with SYBER green were
used to confirm selected microarray findings.
RESULTS: Highly Expressed Genes in PZ and HZ: Of the 15,923 probe
sets arrayed on the RAE 230A chips, the 30 genes showing the greatest
absolute expression levels within the PZ and HZ were selected for further
analysis. Fifteen genes from the top 30 genes expressed within the PZ
were also present among the top 30 genes expressed within the HZ, so
only 15 genes from each list were unique to the respective zone (Fig 2).
Fig 1. The growth plate section Fig 2. Thirty genes with the highest
Before and after LMD in the expression in the PZ and HZ
PZ (A, B) and HZ (C, D).
Confirmation of Microarray Results with In Situ Hybridization and
Real-Tme PT-PCR: To confirm the results of microarray, fibromodulin
(Fmod) and proline arginine-rich end leucine-rich repeat protein (Prelp)
were chosen from the PZ and integrin binding sialoprotein ( Ibsp) and
matrix metalloproteinase (MMP-13) were chosen from the HZ (Fig 3).
MMP-13 was also confirmed by Real-Time RT-PCR with the data (4.59
fold at 6 week, 59475.2 fold at 7 week in the HZ compared to the PZ)
consistent with microarray data (3.95 fold at 6 week, 139.5 fold at 7 week
in the HZ compared to the PZ).
Function Categorization of Differentially Expressed Genes: Comparison
of the relative distribution of genes differentially expressed by two fold or
greater within the PZ compared to the HZ and vice-versa shows that a
greater percentage of PZ differentially expressed genes were devoted to
cell cycle (7.5% PZ vs. 0% HZ) and transcription (7.5% PZ vs. 1.9% HZ)
functions. Conversely, a greater percentage of HZ differentially
expressed genes were involved in structure (11.5% HZ vs. 5.0% PZ) and
receptor/transporter function (28.8% HZ vs.10.0% PZ).
Analysis of Established Growth Plate Pathways: TGF-β, PTHrP, BMP,
and IGF pathways previously shown to be important in musculoskeletal
tissues were utilized as a framework with which to display the differential
expression levels of a number of genes. BMP and IGF pathways along
with mediators and differential PZ and HZ expression are shown in Fig 4.
Fmod Prelp Ibsp MMP-13
Fig 3. Localization by in situ hybridization is shown in the PZ (Fmod,
Prelp) and HZ (Ibsp,MMP-13) in 6 week growth plate. Bar = 100µm.
Fig 4. This flow diagram shows HZ differential expression for BMP2 and
mediators of the BMP pathway, compared to the IGF pathways, which
show most components equally expressed in both PZ and HZ.
CONCLUSIONS: Differentiation markers in PZ and HZ: Of the 30
highly expressed genes in each zone, some genes were consistent with
previous studies, such as collagen II and aggrecan in both zones, Fmod in
the PZ, Ibsp and MMP-13 in the HZ. A number of genes never reported
previously in the growth plate were identified as showing differential
expression in the PZ or the HZ, suggesting new potential pathways
regulating the transition from proliferation to hypertrophy. For example,
interleukin-17b was highly expressed in the PZ, and collagen I and Prelp
were highly expressed in the HZ.
Functional categorization of differentially expressed genes: None of the 6
differentially expressed genes involved in cell cycle regulation (cyclin B1,
CDK-4, protein phosphatase-1G) or transcription ( nuclear factor I/X,
thymopoietin, general transcription factor II) in the PZ had been reported
previously in the growth plate, and their differential PZ expression
suggests a role in maintaining the proliferative chondrocyte phenotype.
Collagen I, collagen V, prostaglandin F receptor and 10 membrane
transporters including solute carrier family 12, member 2 and annexin I
of the 21 differentially expressed extracellular structure, receptor genes,
and membrane transporter genes in the HZ were not previously reported
in the chondrocytes, suggesting a potential role for these genes in the
regulation of HZ chondrocyte hypertrophy and mineralization .
Signal pathways involved in growth plate proliferation and hypertrophy:
The presence of equal expression between zones for many of the
components of the TGF-β, PTHrP, and IGF pathways suggest importance
in both zones. Some of the BMPs and their downstream mediators’ high
expression in the HZ indicate that it may be involved in the transition
from proliferation to differentiation.
Acknowledgements: NIH/NCI, David G. Murray Endowment.
PZ chondrocyteHZ chondrocyte
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 0354