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Comparative Study
. 2005 Nov;16(11):5316-33.
doi: 10.1091/mbc.e05-01-0084. Epub 2005 Aug 31.

Microarray analyses of gene expression during chondrocyte differentiation identifies novel regulators of hypertrophy

Claudine G James  1 , C Thomas G Appleton, Veronica Ulici, T Michael Underhill, Frank Beier
Affiliations
Comparative Study

Microarray analyses of gene expression during chondrocyte differentiation identifies novel regulators of hypertrophy

Claudine G James et al. Mol Biol Cell. 2005 Nov.

Abstract

Ordered chondrocyte differentiation and maturation is required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. We used Affymetrix microarrays to examine temporal gene expression patterns during chondrogenic differentiation in a mouse micromass culture system. Robust normalization of the data identified 3300 differentially expressed probe sets, which corresponds to 1772, 481, and 249 probe sets exhibiting minimum 2-, 5-, and 10-fold changes over the time period, respectively. GeneOntology annotations for molecular function show changes in the expression of molecules involved in transcriptional regulation and signal transduction among others. The expression of identified markers was confirmed by RT-PCR, and cluster analysis revealed groups of coexpressed transcripts. One gene that was up-regulated at later stages of chondrocyte differentiation was Rgs2. Overexpression of Rgs2 in the chondrogenic cell line ATDC5 resulted in accelerated hypertrophic differentiation, thus providing functional validation of microarray data. Collectively, these analyses provide novel information on the temporal expression of molecules regulating endochondral bone development.

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Figures

Figure 1.
Figure 1.
Qualitative characterization of micromass cultures. Images of micromass cultures undergoing chondrogenesis and chondrocyte differentiation on days 3, 6, 9, 12, and 15 of culture are represented. Cells were stained with Alcian blue to show synthesis of sulfated glycosaminoglycans, for ALP activity and with von Kossa for mineralization. All markers increase throughout the culture period.
Figure 2.
Figure 2.
Affymetrix microarray analysis of chondrogenesis and chondrocyte differentiation. Total RNA from the micromass time course was hybridized to Affymetrix MOE430A chips containing ∼14,000 murine genes. Data from three biological replicates are shown. Preliminary analysis in M.A.S 5.0 and GeneSpring 6.1, in which all probe sets were subject to various filters (see Materials and Methods), resulted in the creation of data set consisting of 3334 probes sets. Expression profiles for Sox9 and Ibsp are highlighted in red and green, respectively (A). Expression patterns for additional characterized cartilage markers such as Aggrecan (Agc1), cartilage-derived retinoic acid-sensitive protein (Cdrap), Sox9, Link protein (Crtl1), and Collagen II (Col2a1) are also shown (Ba). Expression patterns of IGF signaling constituents are shown (Bb). Transcripts involved in extracellular matrix signaling including matrilin (Matn1), perlecan (Hspg2), collagen 11a1 (Col11a1), osteopontin (Opn1), tissue inhibitor of metalloproteinase 3 (Timp3), collagen 9a2 (Col9a2), and osteonectin (Spock1) are shown (Bc). Genes such as insulinlike growth factor binding protein 6 (Igfbp6) and matrix metalloprotease 13 (Mmp13), Ibsp, a disintegrin and metalloprotease domain 23 (Adam23), and cartilage oligomeric protein (Comp) exhibit greater than fivefold changes in gene expression and are not shown for scaling reasons. (B). All values reflect fold changes in gene expression relative to day 3 of micromass culture.
Figure 3.
Figure 3.
Validation of differentially expressed genes by RT-PCR. Temporal expression patterns of Cartilage Link Protein 1 (Crtl1; A), nuclear cap binding protein (Ncbp2; B), and myogenesis differentiation factor 1 (Myod-1; C) were confirmed by RT-PCR using β-actin as a loading control. The microarray expression profile shown as fold change in expression is shown on the left with the corresponding RT-PCR to the right.
Figure 4.
Figure 4.
Distribution of differentially expressed probe sets and corresponding molecular classification according to GO terms. Differential gene expression analyzed in succession shows the number of probe sets exhibiting changes as the developmental time line progresses. Probe sets were filtered according to 2-, 5-, and 10-fold change cutoffs (A). Day 3 versus day 6, and day 12 versus day 15 represent the largest and smallest proportion of changes between culture days, respectively. Genes exhibiting minimum two-fold changes in gene expression between day 3 and day 15 were assigned molecular function and categorized according to GO annotations assigned in the FatiGO program (B). The proportion of genes that are up- or down-regulated in the gene list derived from twofold changes in gene expression between days 3 and 15 of micromass culture (C).
Figure 5.
Figure 5.
Self-organizing maps and k-means clustering of microarray data. SOM analysis of 3334 probe sets from GeneSpring analysis and subsequent k-means clustering of three SOM derived clusters. Cluster 1 contains genes that show up-regulation toward day 15 of the micromass time course (A, left panel). Genes in this cluster include Mmp13, Ibsp, Comp, and fatty acid binding protein 4 (Fabp4; A, right panel). Standard correlation was used to measure similarity between expression profiles. Cluster 2 contains genes following an expression pattern similar to that of Cartilage link protein (Crtl1; B, left panel), such as Angiotensin II receptor type 2 (Atgr2), collagen 14 (Col14a1), and Col2a1 (B, right panel). Cluster 3 represents the expression pattern of genes down-regulated over time (C, left panel), including cardiac actin (Actc1), brain expressed X-linked 2 (Bex2), Tncc, Myod1, troponin (Tnnt1), and alpha actinin (Actn2; C, right panel). Red lines show up-regulation and blue lines show down-regulation of gene expression.
Figure 6.
Figure 6.
Confirmation of Rgs2 expression in murine chondrocyte differentiation. Microarray expression pattern for Rgs2 in micromass cultures show transcript accumulation from days 3 to 12 of culture, with a subsequent drop in expression (A). Rgs2 mRNA up-regulation during micromass differentiation was confirmed by RT-PCR using β-actin as a loading control (B). Rgs2 mRNA expression in the tibia of a E15.5 mouse was analyzed by in situ hybridization (C). Sense probes resulted in no signal, whereas Rgs2 antisense probes demonstrated weak or no expression in resting chondrocytes (R), strong expression of Rgs2 mRNA in proliferating (P) and prehypertrophic chondrocytes, and lower expression in fully hypertrophic chondrocytes (H). Confirmation of RGS2 overexpression in chondrogenic ATDC-5 cells by RT-PCR and Western blotting (C). Ladder (L), pcDNA 3.1+ vector (V), and a 657-bp Rgs2 amplicon are shown (left panel). Cell lysates isolated from ATDC-5 cells overexpressing HA-tagged RGS2 were separated by SDS-PAGE and blotted onto nitrocellulose membrane, which was subsequently probed with an anti-HA antibody (right panel).
Figure 7.
Figure 7.
Functional characterization of RGS2 in chondrocyte differentiation. Differentiating ATDC-5 cells stably overexpressing RGS2 were stained with Alcian blue (A) and ALP on days 3, 12, and 18 of culture (B). ALP activity was subsequently quantified by enzyme assay. Stainings were repeated three times on independent trials, and ALP activity values represent an average of six independent trials. Significant differences from the vector control (***) were determined by p < 0.001. RGS2 overexpression results in accelerated and increased induction of glycosaminoglycan synthesis and ALP activity.
Figure 8.
Figure 8.
Effects of RGS2 overexpression on chondrocyte gene expression. The relative abundance of Sox9 (A), Col2a1 (B), Fgfr3 (C), Ihh (D), and Ibsp (E) transcripts in ATDC-5 cells overexpressing RGS2 was quantified with real-time PCR. Expression of Sox9 and Col2a1 were assessed after 6 d of culture and the expression of all subsequent markers assessed on day 15 of culture. Results represent means and standard deviations from three independent trials completed in triplicate. Expression values are normalized to a Gapdh internal control. Significant differences in expression from control (*) were determined by p < 0.001.
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