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Activating types 1 and 2 angiotensin II receptors modulate the hypertrophic differentiation of chondrocytes.

Tsukamoto I, Inoue S, Teramura T, Takehara T, Ohtani K, Akagi M - FEBS Open Bio (2013)

Bottom Line: A local tissue-specific renin-angiotensin system (local RAS) has been identified in many organs.To examine the role of a local RAS in the hypertrophic differentiation, we activated angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) separately in the cell line ATDC5, which involves differentiation from mesenchymal stem cells to hypertrophic chondrocytes.Activation of AT1R suppressed and activation of AT2R enhanced the expression of markers of hypertrophic differentiation, including type X collagen, matrix metalloproteinase 13 and runt-related transcription factor 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589-8511, Japan.

ABSTRACT
A local tissue-specific renin-angiotensin system (local RAS) has been identified in many organs. However, no report has described the role of a local RAS in the hypertrophic differentiation of chondrocytes. To examine the role of a local RAS in the hypertrophic differentiation, we activated angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) separately in the cell line ATDC5, which involves differentiation from mesenchymal stem cells to hypertrophic chondrocytes. Activation of AT1R suppressed and activation of AT2R enhanced the expression of markers of hypertrophic differentiation, including type X collagen, matrix metalloproteinase 13 and runt-related transcription factor 2.

No MeSH data available.


Related in: MedlinePlus

Expression of Col.X in the ATDC5 cell line treated with various agents on Day 14. (A) Ang II downregulated the mRNA expression of Col.X in a concentration-dependent manner. (B) When cells were treated with Olmesartan, Ang II upregulated the mRNA expression of Col.X. (C) When cells were treated with PD123319, Ang II downregulated the mRNA expression of Col.X. (D) Western blot analysis showed that Ang II upregulated the expression of Col.X when cells were treated with Olmesartan and that Ang II downregulated the expression of Col.X when cells were treated with PD123319. (E) Western blotting detection of Col.X showed significant differences between treatments. The molar concentration ratios of antagonists to agonist were 2.32 (1.0 μg/ml Olmesartan/1.0 μg/ml AngII) and 1.77 (1.0 μg/ml PD123319/1.0 μg/ml AngII). *P < 0.05 between treatments. Abbreviations: Col.X, type X collagen; Ang II, angiotensin II.
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fig0002: Expression of Col.X in the ATDC5 cell line treated with various agents on Day 14. (A) Ang II downregulated the mRNA expression of Col.X in a concentration-dependent manner. (B) When cells were treated with Olmesartan, Ang II upregulated the mRNA expression of Col.X. (C) When cells were treated with PD123319, Ang II downregulated the mRNA expression of Col.X. (D) Western blot analysis showed that Ang II upregulated the expression of Col.X when cells were treated with Olmesartan and that Ang II downregulated the expression of Col.X when cells were treated with PD123319. (E) Western blotting detection of Col.X showed significant differences between treatments. The molar concentration ratios of antagonists to agonist were 2.32 (1.0 μg/ml Olmesartan/1.0 μg/ml AngII) and 1.77 (1.0 μg/ml PD123319/1.0 μg/ml AngII). *P < 0.05 between treatments. Abbreviations: Col.X, type X collagen; Ang II, angiotensin II.

Mentions: We first examined the chronological mRNA expression levels of ANG, ACE1, AT1R and AT2R in ATDC5 cells without any agents (group F) using QRT–PCR analysis. The mRNA expression of ANG began to increase in the proliferating phase and maintained expression during the hypertrophic phase with a peak on Day 14 (Fig. 1A). The mRNA expression of AT1R increased intensely in the proliferating phase with a peak on Day 10 (Fig. 1B). Thus, AT1R was still expressed in the early period of hypertrophy. The mRNA expression of ACE1 began to increase on Day 10 and kept increasing (Fig. 1C). The mRNA expression of AT2R began to increase on Day 14 and increased abruptly on Day 21 (Fig. 1D). Thus, AT2R was expressed predominantly in the hypertrophic phase. On Day 14, when both AT1R and AT2R were expressed, the relative mRNA expression of AT1R was about 25 times that of AT2R (Fig. 1E). We also examined the chronological protein synthesis of local RAS components without any agents using western blot analysis (group F). ANG, AT1R and ACE1 were expressed as synthesized proteins in both the proliferating and hypertrophic phases. However, AT2R was not produced during proliferation but was produced intensely in the hypertrophic phase as synthesized protein (Fig. 1F). Thus, we confirmed that ANG, ACE1, AT1R and AT2R were expressed in the early period of the hypertrophic phase. Then, to examine the function of the local RAS in hypertrophic differentiation, we administered specific AT1R and AT2R inhibitors to ATDC5 cells as described above. Because the results of QRT–PCR analysis for the local RAS components suggested that both AT1R and AT2R were expressed on Day 14, we chose this as the day for administration. The mRNA expression of Col.X was downregulated in a concentration-dependent manner with a significant difference from the control cells (group A) when adding Ang II on Day 14 (groups B and C; Fig. 2A). Ang II treatment upregulated the mRNA expression of Col.X with a significant difference from the control cells (group A) treated with Olmesartan (group D; Fig. 2B). On the other hand, Ang II downregulated the expression of Col.X with a significant difference from the control cells (group A) treated with PD123319 (group E; Fig. 2C). We also examined the protein synthesis of Col.X with various agents using western blot analysis on Day 14. Ang II upregulated the expression of Col.X with a significant difference from the control cells (group A) treated with Olmesartan (group D; Fig. 2D and E). On the other hand, Ang II treatment downregulated the expression of Col.X with a significant difference from the control cells (group A) treated with PD123319 (group E; Fig. 2D and E). We also examined the side effects of adding Olmesartan. We administered 0.1, 1.0 and 10 μg/ml Olmesartan on Day 14. Adding the two lower doses did not interfere with the expression of Col.X (groups G and H; Fig. 3). However, treatment with 10 μg/ml Olmesartan without adding AngII upregulated the expression of Col.X with a significant difference from the control cells (group I; Fig. 3). We also administered agents on Days 10 and 21. On Day 10, Ang II treatment downregulated the expression of Col.X with a significant difference from the control cells treated with PD123319 (group J; Fig. 4A). However, Ang II treatment made no significant change to cells treated with Olmesartan on Day 10 (group J; Fig. 4A). On Day 21, Ang II treatment made no significant change to cells treated with PD123319 (group K; Fig. 4B). However, Ang II treatment upregulated the expression of Col.X with a significant difference from the control cells treated with Olmesartan (group K; Fig. 4B). Additionally, we examined the mRNA expression levels of MMP13 and Runx2 using QRT–PCR analysis. Ang II upregulated the mRNA expressions of MMP13 and Runx2 with a significant difference from the control cells (group A) treated with Olmesartan (group D; Fig. 5A and C). On the other hand, Ang II downregulated the expressions of MMP13 and Runx2 with significant differences from the control cells (group A) treated with PD123319 (group E; Fig. 5B and D).


Activating types 1 and 2 angiotensin II receptors modulate the hypertrophic differentiation of chondrocytes.

Tsukamoto I, Inoue S, Teramura T, Takehara T, Ohtani K, Akagi M - FEBS Open Bio (2013)

Expression of Col.X in the ATDC5 cell line treated with various agents on Day 14. (A) Ang II downregulated the mRNA expression of Col.X in a concentration-dependent manner. (B) When cells were treated with Olmesartan, Ang II upregulated the mRNA expression of Col.X. (C) When cells were treated with PD123319, Ang II downregulated the mRNA expression of Col.X. (D) Western blot analysis showed that Ang II upregulated the expression of Col.X when cells were treated with Olmesartan and that Ang II downregulated the expression of Col.X when cells were treated with PD123319. (E) Western blotting detection of Col.X showed significant differences between treatments. The molar concentration ratios of antagonists to agonist were 2.32 (1.0 μg/ml Olmesartan/1.0 μg/ml AngII) and 1.77 (1.0 μg/ml PD123319/1.0 μg/ml AngII). *P < 0.05 between treatments. Abbreviations: Col.X, type X collagen; Ang II, angiotensin II.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig0002: Expression of Col.X in the ATDC5 cell line treated with various agents on Day 14. (A) Ang II downregulated the mRNA expression of Col.X in a concentration-dependent manner. (B) When cells were treated with Olmesartan, Ang II upregulated the mRNA expression of Col.X. (C) When cells were treated with PD123319, Ang II downregulated the mRNA expression of Col.X. (D) Western blot analysis showed that Ang II upregulated the expression of Col.X when cells were treated with Olmesartan and that Ang II downregulated the expression of Col.X when cells were treated with PD123319. (E) Western blotting detection of Col.X showed significant differences between treatments. The molar concentration ratios of antagonists to agonist were 2.32 (1.0 μg/ml Olmesartan/1.0 μg/ml AngII) and 1.77 (1.0 μg/ml PD123319/1.0 μg/ml AngII). *P < 0.05 between treatments. Abbreviations: Col.X, type X collagen; Ang II, angiotensin II.
Mentions: We first examined the chronological mRNA expression levels of ANG, ACE1, AT1R and AT2R in ATDC5 cells without any agents (group F) using QRT–PCR analysis. The mRNA expression of ANG began to increase in the proliferating phase and maintained expression during the hypertrophic phase with a peak on Day 14 (Fig. 1A). The mRNA expression of AT1R increased intensely in the proliferating phase with a peak on Day 10 (Fig. 1B). Thus, AT1R was still expressed in the early period of hypertrophy. The mRNA expression of ACE1 began to increase on Day 10 and kept increasing (Fig. 1C). The mRNA expression of AT2R began to increase on Day 14 and increased abruptly on Day 21 (Fig. 1D). Thus, AT2R was expressed predominantly in the hypertrophic phase. On Day 14, when both AT1R and AT2R were expressed, the relative mRNA expression of AT1R was about 25 times that of AT2R (Fig. 1E). We also examined the chronological protein synthesis of local RAS components without any agents using western blot analysis (group F). ANG, AT1R and ACE1 were expressed as synthesized proteins in both the proliferating and hypertrophic phases. However, AT2R was not produced during proliferation but was produced intensely in the hypertrophic phase as synthesized protein (Fig. 1F). Thus, we confirmed that ANG, ACE1, AT1R and AT2R were expressed in the early period of the hypertrophic phase. Then, to examine the function of the local RAS in hypertrophic differentiation, we administered specific AT1R and AT2R inhibitors to ATDC5 cells as described above. Because the results of QRT–PCR analysis for the local RAS components suggested that both AT1R and AT2R were expressed on Day 14, we chose this as the day for administration. The mRNA expression of Col.X was downregulated in a concentration-dependent manner with a significant difference from the control cells (group A) when adding Ang II on Day 14 (groups B and C; Fig. 2A). Ang II treatment upregulated the mRNA expression of Col.X with a significant difference from the control cells (group A) treated with Olmesartan (group D; Fig. 2B). On the other hand, Ang II downregulated the expression of Col.X with a significant difference from the control cells (group A) treated with PD123319 (group E; Fig. 2C). We also examined the protein synthesis of Col.X with various agents using western blot analysis on Day 14. Ang II upregulated the expression of Col.X with a significant difference from the control cells (group A) treated with Olmesartan (group D; Fig. 2D and E). On the other hand, Ang II treatment downregulated the expression of Col.X with a significant difference from the control cells (group A) treated with PD123319 (group E; Fig. 2D and E). We also examined the side effects of adding Olmesartan. We administered 0.1, 1.0 and 10 μg/ml Olmesartan on Day 14. Adding the two lower doses did not interfere with the expression of Col.X (groups G and H; Fig. 3). However, treatment with 10 μg/ml Olmesartan without adding AngII upregulated the expression of Col.X with a significant difference from the control cells (group I; Fig. 3). We also administered agents on Days 10 and 21. On Day 10, Ang II treatment downregulated the expression of Col.X with a significant difference from the control cells treated with PD123319 (group J; Fig. 4A). However, Ang II treatment made no significant change to cells treated with Olmesartan on Day 10 (group J; Fig. 4A). On Day 21, Ang II treatment made no significant change to cells treated with PD123319 (group K; Fig. 4B). However, Ang II treatment upregulated the expression of Col.X with a significant difference from the control cells treated with Olmesartan (group K; Fig. 4B). Additionally, we examined the mRNA expression levels of MMP13 and Runx2 using QRT–PCR analysis. Ang II upregulated the mRNA expressions of MMP13 and Runx2 with a significant difference from the control cells (group A) treated with Olmesartan (group D; Fig. 5A and C). On the other hand, Ang II downregulated the expressions of MMP13 and Runx2 with significant differences from the control cells (group A) treated with PD123319 (group E; Fig. 5B and D).

Bottom Line: A local tissue-specific renin-angiotensin system (local RAS) has been identified in many organs.To examine the role of a local RAS in the hypertrophic differentiation, we activated angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) separately in the cell line ATDC5, which involves differentiation from mesenchymal stem cells to hypertrophic chondrocytes.Activation of AT1R suppressed and activation of AT2R enhanced the expression of markers of hypertrophic differentiation, including type X collagen, matrix metalloproteinase 13 and runt-related transcription factor 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, Kinki University, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589-8511, Japan.

ABSTRACT
A local tissue-specific renin-angiotensin system (local RAS) has been identified in many organs. However, no report has described the role of a local RAS in the hypertrophic differentiation of chondrocytes. To examine the role of a local RAS in the hypertrophic differentiation, we activated angiotensin II type 1 receptor (AT1R) and angiotensin II type 2 receptor (AT2R) separately in the cell line ATDC5, which involves differentiation from mesenchymal stem cells to hypertrophic chondrocytes. Activation of AT1R suppressed and activation of AT2R enhanced the expression of markers of hypertrophic differentiation, including type X collagen, matrix metalloproteinase 13 and runt-related transcription factor 2.

No MeSH data available.


Related in: MedlinePlus