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Aberrant Calreticulin Expression in Articular Cartilage of Dio2 Deficient Mice.

Bomer N, Cornelis FM, Ramos YF, den Hollander W, Lakenberg N, van der Breggen R, Storms L, Slagboom PE, Lories RJ, Meulenbelt I - PLoS ONE (2016)

Bottom Line: Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > /1,5/) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044).Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Epidemiology, LUMC, Leiden, Netherlands.

ABSTRACT

Objective: To identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis.

Methods: Previously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition.

Results: Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > /1,5/) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.

Conclusion: We here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

No MeSH data available.


Related in: MedlinePlus

Calreticulin expression in DIO2 overexpressing hBMSCs.qPCR expression of Calr in hBMSCs transduced with either a control virus-vector (eGFP) or a DIO2-eGFP vector. Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control sample at every timepoint. Differences were analyzed with Student T-Test ((*) P < 0.05).
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pone.0154999.g004: Calreticulin expression in DIO2 overexpressing hBMSCs.qPCR expression of Calr in hBMSCs transduced with either a control virus-vector (eGFP) or a DIO2-eGFP vector. Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control sample at every timepoint. Differences were analyzed with Student T-Test ((*) P < 0.05).

Mentions: Finally, to confirm the transcriptional link between of DIO2 and CALR expression, we examined CALR expression in cartilage constructs previously generated from human BMSCs in vitro 3D chondrogenesis and compared them to those generated while applying overexpression of DIO2 by lentiviral transduction[8]. As shown in Fig 4, also in this human BMSCs in vitro 3D chondrogenesis model overexpressing DIO2 coincided with significant higher CALR expression.


Aberrant Calreticulin Expression in Articular Cartilage of Dio2 Deficient Mice.

Bomer N, Cornelis FM, Ramos YF, den Hollander W, Lakenberg N, van der Breggen R, Storms L, Slagboom PE, Lories RJ, Meulenbelt I - PLoS ONE (2016)

Calreticulin expression in DIO2 overexpressing hBMSCs.qPCR expression of Calr in hBMSCs transduced with either a control virus-vector (eGFP) or a DIO2-eGFP vector. Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control sample at every timepoint. Differences were analyzed with Student T-Test ((*) P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862667&req=5

pone.0154999.g004: Calreticulin expression in DIO2 overexpressing hBMSCs.qPCR expression of Calr in hBMSCs transduced with either a control virus-vector (eGFP) or a DIO2-eGFP vector. Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control sample at every timepoint. Differences were analyzed with Student T-Test ((*) P < 0.05).
Mentions: Finally, to confirm the transcriptional link between of DIO2 and CALR expression, we examined CALR expression in cartilage constructs previously generated from human BMSCs in vitro 3D chondrogenesis and compared them to those generated while applying overexpression of DIO2 by lentiviral transduction[8]. As shown in Fig 4, also in this human BMSCs in vitro 3D chondrogenesis model overexpressing DIO2 coincided with significant higher CALR expression.

Bottom Line: Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > /1,5/) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044).Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Epidemiology, LUMC, Leiden, Netherlands.

ABSTRACT

Objective: To identify intrinsic differences in cartilage gene expression profiles between wild-type- and Dio2-/--mice, as a mechanism to investigate factors that contribute to prolonged healthy tissue homeostasis.

Methods: Previously generated microarray-data (Illumina MouseWG-6 v2) of knee cartilage of wild-type and Dio2 -/- -mice were re-analyzed to identify differential expressed genes independent of mechanical loading conditions by forced treadmill-running. RT-qPCR and western blot analyses of overexpression and knockdown of Calr in mouse chondro-progenitor cells (ATDC5) were applied to assess the direct effect of differential Calr expression on cartilage deposition.

Results: Differential expression analyses of articular cartilage of Dio2-/- (N = 9) and wild-type-mice (N = 11) while applying a cutoff threshold (P < 0.05 (FDR) and FC > /1,5/) resulted in 1 probe located in Calreticulin (Calr) that was found significantly downregulated in Dio2-/- mice (FC = -1.731; P = 0.044). Furthermore, overexpression of Calr during early chondrogenesis in ATDC5 cells leads to decreased proteoglycan deposition and corresponding lower Aggrecan expression, whereas knocking down Calr expression does not lead to histological differences of matrix composition.

Conclusion: We here demonstrate that the beneficial homeostatic state of articular cartilage in Dio2-/- mice is accompanied with significant lower expression of Calr. Functional analyses further showed that upregulation of Calr expression could act as an initiator of cartilage destruction. The consistent association between Calr and Dio2 expression suggests that enhanced expression of these genes facilitate detrimental effects on cartilage integrity.

No MeSH data available.


Related in: MedlinePlus