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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

Expression analyses in Calr overexpressing ATDC5 cells.RT-qPCR expression of Calr, Dio2 and Acan in ATDC5 cells transfected with either a Calr-containing vector for overexpression (Calr+) or 3 unique 27mer siRNA duplexes, specific for Calr, for knockdown (Calr-). Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control samples (dashed line). Differences were analyzed with Student T-Test ((*) P < 0.05).
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pone.0154999.g003: Expression analyses in Calr overexpressing ATDC5 cells.RT-qPCR expression of Calr, Dio2 and Acan in ATDC5 cells transfected with either a Calr-containing vector for overexpression (Calr+) or 3 unique 27mer siRNA duplexes, specific for Calr, for knockdown (Calr-). Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control samples (dashed line). Differences were analyzed with Student T-Test ((*) P < 0.05).

Mentions: Next, we investigated the effect of altered Calr expression on the chondrogenic capacity of ATDC5 cells. As measured by photospectrometry, overexpressing Calr during early 2D chondrogenesis resulted in significant lower Alcian Blue staining, indicating less glycosaminoglycan deposition (Fig 2). Concurrently, RT-qPCR confirmed significant lower mRNA expression levels of Acan (FC = -2,32) (Fig 3). Of note was the 1.8-fold upregulation of Dio2 in the cells overexpressing Calr, albeit that this effect by definition was not significant (P = 0.08). On the other hand, knockdown of Calr, by siRNA duplexes in this model, did not show an effect on the amount of deposited glycosaminoglycans stained by Alcian Blue (Fig 2) nor on the mRNA expression levels of Acan (Fig 3). However, a significant 4.7-fold downregulated effect was observed for Dio2 mRNA expression levels (P = 0,017).


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)

Expression analyses in Calr overexpressing ATDC5 cells.RT-qPCR expression of Calr, Dio2 and Acan in ATDC5 cells transfected with either a Calr-containing vector for overexpression (Calr+) or 3 unique 27mer siRNA duplexes, specific for Calr, for knockdown (Calr-). Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control samples (dashed line). 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.g003: Expression analyses in Calr overexpressing ATDC5 cells.RT-qPCR expression of Calr, Dio2 and Acan in ATDC5 cells transfected with either a Calr-containing vector for overexpression (Calr+) or 3 unique 27mer siRNA duplexes, specific for Calr, for knockdown (Calr-). Values of the RT-qPCR are displayed as the average±SEM, normalized for GAPDH expression and relative to the control samples (dashed line). Differences were analyzed with Student T-Test ((*) P < 0.05).
Mentions: Next, we investigated the effect of altered Calr expression on the chondrogenic capacity of ATDC5 cells. As measured by photospectrometry, overexpressing Calr during early 2D chondrogenesis resulted in significant lower Alcian Blue staining, indicating less glycosaminoglycan deposition (Fig 2). Concurrently, RT-qPCR confirmed significant lower mRNA expression levels of Acan (FC = -2,32) (Fig 3). Of note was the 1.8-fold upregulation of Dio2 in the cells overexpressing Calr, albeit that this effect by definition was not significant (P = 0.08). On the other hand, knockdown of Calr, by siRNA duplexes in this model, did not show an effect on the amount of deposited glycosaminoglycans stained by Alcian Blue (Fig 2) nor on the mRNA expression levels of Acan (Fig 3). However, a significant 4.7-fold downregulated effect was observed for Dio2 mRNA expression levels (P = 0,017).

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