Novel Early Target Genes of Parathyroid Hormone-Related Peptide in Chondrocytes

Hoogendam, Jakomijn; Parlevliet, Edwin T.; Miclea, Razvan L.; Löwik, Clemens W.G.M.; Wit, Jan M.; Karperien, Marcel

doi:10.1210/en.2006-0075

Cited by 18 publications

(26 citation statements)

References 90 publications

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“…In addition, during this process cells become responsive to PTHrP and in agreement with in vivo studies PTHrP inhibits hypertrophic chondrocyte differentiation [16, 17]. Previously, we have identified novel early response genes of PTHrP [18]. In this study, we focus on the identification of late PTHrP response genes regulated 24 or 72 h after PTHrP treatment.…”

Section: Introductionsupporting

confidence: 63%

“…Previously, we have shown that the cells have a pre-hypertrophic phenotype at this stage of culture [18]. PTHrP treatment at day 8 resulted in inhibition of the outgrowth of cartilage matrix 72 h later (fig.…”

Section: Resultsmentioning

confidence: 75%

“…ATDC5 cells were grown as micromasses in Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen, Breda, The Netherlands) as described before [18]. …”

Section: Methodsmentioning

confidence: 99%

“…Next, RNA samples were pooled and time point 0 h was chosen as reference sample. Total RNA (3 µg/reaction) was amplified as described before [19], with slight modifications as described earlier [18]. cRNA (1.2 µg) was reverse transcribed with random hexamer primers, and labeled by incorporation of cyanine 5-dUTP (Cy5) or cyanine 3-dUTP (Cy3) (NEN, Boston, USA) according to the protocols of Ross et al [20] with slight modifications as described previously [18].…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, normalization and gene expression analysis were performed with Rosetta Resolver (Rosetta Biosoftware, Seattle, USA). Due to the overall poor quality of the cDNA spots on the microarray, stringent selection criteria were used for inclusion of spots, to minimize the risk of false positive signals as described previously [18]. In addition, spots were only included if they were not significantly regulated in control arrays, in which vehicle treated cultures were hybridized against 0 h. To identify the selected spots, the PCR-amplified cDNA of each spot (500 ng) was sequenced by the Leiden Genome Technology Center using 12 pmol M13 primers in a total volume of 24 µl.…”

Section: Methodsmentioning

confidence: 99%

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Novel Late Response Genes of PTHrP in Chondrocytes

Hoogendam¹,

Farih-Sips²,

Beek³

et al. 2006

Horm Res Paediatr

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To gain more insight into the downstream effectors of parathyroid hormone (PTH) related peptide (PTHrP) signaling in chondrocytes, we performed microarray analysis to identify late PTHrP response genes using the chondrogenic ATDC5 cell line and studied their response in the osteoblastic KS483 cell line and explanted metatarsals. At day 8 of micromass culture, ATDC5 cells have pre-hypertrophic-like characteristics and at this time point the cells were stimulated with PTHrP for 24 and 72 h and RNA was isolated. PTHrP treatment inhibited outgrowth of cartilage matrix and decreased the expression of Col10a1 mRNA, which is in line with the inhibitory effects of PTHrP on chondrocyte differentiation. Using cDNA microarray analysis, a list of 9 genes (p< 10^–3) was generated, including 3 upregulated (IGFBP4, Csrp2, and Ecm1) and 6 downregulated (Col9a1, Col2a1, Agc, Hmgn2, Calm1, and Mxd4) response genes. Four out of 9 genes are novel PTHrP response genes and 2 out of 9 have not yet been identified in cartilage. Four out of 9 genes are components of the extra-cellular matrix and the remaining genes are involved in signal transduction and transcription regulation. The response to PTHrP was validated by quantitative PCR, using the same RNA samples as labeled in the microarray experiments and RNA samples isolated from a new experiment. In addition, we examined whether these genes also reacted to PTHrP in other PTHrP responsive models, like KS483 osteoblasts and explanted metatarsals. The expression of late PTHrP response genes varied between ATDC5 chondrocytes, KS483 osteoblasts and metatarsals, suggesting that the expression of late response genes is dependent on the cellular context of the PTHrP responsive cells.

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Section: Introductionsupporting

confidence: 63%

Section: Resultsmentioning

confidence: 75%

“…ATDC5 cells were grown as micromasses in Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen, Breda, The Netherlands) as described before [18]. …”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Novel Late Response Genes of PTHrP in Chondrocytes

Hoogendam¹,

Farih-Sips²,

Beek³

et al. 2006

Horm Res Paediatr

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13cRA regulates the differentiation of antler chondrocytes through targeting Runx3

Zhang

Cao

Yang

et al. 2017

Cell Biology International

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Although 13cRA is involved in the regulation of cellular proliferation and differentiation, its physiological roles in chondrocyte proliferation and differentiation still remain unknown. Here, we showed that 13cRA could induce the proliferation of sika deer antler chondrocytes and expression of Ccnd3 and Cdk6. Administration of 13cRA to antler chondrocytes resulted in an obvious increase in the expression of chondrocyte marker Col II and hypertrophic chondrocyte marker Col X. Silencing of Crabp2 expression by specific siRNA could prevent the 13cRA-induced up-regulation of Col X, whereas overexpression of Crabp2 showed the opposite effects. Further study found that Crabp2 mediated the regulation of 13cRA on the expression of Runx3 which was highly expressed in the antler cartilage and inhibited the differentiation of antler chondrocytes. Moreover, attenuation of Runx3 expression greatly raised 13cRA-induced chondrocyte differentiation. Simultaneously, 13cRA could stimulate the expression of Cyp26a1 and Cyp26b1 in the antler chondrocytes. Inhibition of Cyp26a1 and/or Cyp26b1 reinforced the effects of 13cRA on the expression of Col X and Runx3, while overexpression of Cyp26b1 rendered the antler chondrocytes hyposensitive to 13cRA. Collectively, 13cRA may play an important role in the differentiation of antler chondrocytes through targeting Runx3. Crabp2 enhances the effects of 13cRA on chondrocyte differentiation, while Cyp26a1 and Cyp26b1 weaken the sensitivity of antler chondrocytes to 13cRA.

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ATDC5: An excellent in vitro model cell line for skeletal development

Yao

Wang

2013

J of Cellular Biochemistry

158

137

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The ATDC5 cell line is derived from mouse teratocarcinoma cells and characterized as a chondrogenic cell line which goes through a sequential process analogy to chondrocyte differentiation. Thus, it is regarded as a promising in vitro model to study the factors that influence cell behaviors during chondrogenesis. It also provides insights in exploring signaling pathways related to skeletal development as well as interactions with innovative materials. To date, over 200 studies have utilized ATDC5 to obtain lots of significant findings. In this review, we summarized the literature of ATDC5 related studies and emphasized the application of ATDC5 in chondrogenesis. In addition, the general introduction of ATDC5 including its derivation and characterization is covered in this article.

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Novel Early Target Genes of Parathyroid Hormone-Related Peptide in Chondrocytes

Cited by 18 publications

References 90 publications

Novel Late Response Genes of PTHrP in Chondrocytes

Novel Late Response Genes of PTHrP in Chondrocytes

13cRA regulates the differentiation of antler chondrocytes through targeting Runx3

ATDC5: An excellent in vitro model cell line for skeletal development

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