In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/β-catenin activation
Summary (1 min read)
Introduction
- In vascular smooth muscle cells paricalcitol prevents phosphate-induced Wnt/ -catenin activation.
- In conclusion, the differential effect of calcitriol and paricalcitol on vascular calcification appears to be mediated by a distinct regulation of the BMP and Wnt/ -catenin signaling pathways.
- The administration of calcitriol to CKD patients may cause VC by increasing the serum levels of calcium and phosphate.
- Bone morphogenetic proteins (BMPs) and proteins of the Wnt family are extremely potent anabolic regulators of bone formation, and both have been implicated in the regulation of VC (7, 17, 31, 49, 51, 57).
MATERIALS AND METHODS
- HASMCs were obtained from Clonetics (Lonza, Walkersville, MD).
- Cells that were incubated in normal phosphate (0.9 mmol/l) medium were used as controls.
- The protein content was measured using the Bio-Rad protein assay (Bio-Rad Laboratories, Munich, Germany), and the calcium content was normalized for total protein.
- Mander’s coefficient M2 plugin (DAPI vs. green) was used to analyze nuclear translocation of -catenin.
- The difference between means for three or more groups was assessed by one-way ANOVA followed by post hoc Duncan analysis.
RESULTS
- Effect of calcitriol and paricalcitol on VSMC calcification.
- The addition of calcitriol (10 8 M) to cells in HP medium (HP CTR) further increased the degree of calcification observed with HP alone (P 0.05).
- Again calcitriol caused additional increase of Runx2 expression (P 0.05 vs. HP cells), whereas paricalcitol failed to increase the expression of Runx2 mRNA beyond the values observed in HP cells.
- Control cells showed immunofluorescence staining of -catenin only in the cytoplasm, whereas cells cultured in HP showed marked expression of -catenin at the nuclear level.
- Cotreatment with DKK-1 significantly reduced VCAN mRNA levels to control values in all the groups.
DISCUSSION
- The authors found that calcitriol increased calcification, which appeared to be associated with the activation of the Wnt/ -catenin and BMP2 signaling pathways.
- Nonetheless, canonical Wnt/ -catenin signaling acting upstream of BMP signaling has been also reported.
- Wnt signaling may suppress osteogenic differentiation or activate osteogenesis under certain cellular conditions.
- The results show that cells exposed to calcitriol exhibited a significant increase in calcification; by contrast, the addition of paricalcitol to the medium resulted in a significant decrease of calcification.
- In summary, their results suggest differential effects of calcitriol and paricalcitol on VC that appears to be mediated by a distinct regulation of the BMP and Wnt/ -catenin signaling pathways.
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Citations
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Cites background from "In vascular smooth muscle cells par..."
...As a master osteoblast transcription factor and the earliest osteoblastic marker, Runx2 has been generally recognized as an early feature of VOT [24,25,27,28]....
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...Several studies indicate that high-phosphate can activate β-catenin signaling, which is evidenced by the upregulation of βcatenin expression [25,26], the decrease of phosphorylated β-catenin (Ser33/37/Thr41) [26], or the translocation of β-catenin into the nuclei [24,27] in VSMCs....
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References
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"In vascular smooth muscle cells par..." refers background in this paper
...Furthermore, Wnt signaling, which is essential for the commitment of pluripotent mesenchymal cells, has also been shown to be activated during the development of VC in vivo and in vitro (5, 8, 23, 31, 51)....
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204 citations
"In vascular smooth muscle cells par..." refers result in this paper
...As previously demonstrated by us and other researchers (6, 36, 40, 44, 47, 49, 58), VSMCs cultured in high phosphate undergo osteogenic transformation and calcification....
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199 citations
"In vascular smooth muscle cells par..." refers background in this paper
...Following ligand activation, the VDR binds directly to specific sequences located near promoters and recruits a variety of coregulatory complexes that perform the additional functions required to modify transcriptional output (39)....
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195 citations
"In vascular smooth muscle cells par..." refers background in this paper
...Importantly, it has also been shown to be upregulated in VSMCs in response to Wnt signaling (59)....
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Frequently Asked Questions (15)
Q2. What is the role of calcitriol in VC?
Calcitriol increased the calcification, which ap-peared to be associated with the upregulation of the expression of osteoblastic gene markers as BMP2, Runx2, Msx2, and OC and the activation of the Wnt/ -catenin signaling pathway.
Q3. What did the cells show in HP?
Control cells showed immunofluorescence staining of -catenin only in the cytoplasm, whereas cells cultured in HP showed marked expression of -catenin at the nuclear level.
Q4. What is the effect of paricalcitol on VCs?
paricalcitol tended to suppress the calcification induced by phosphate; this is in marked contrast with calcitriol that worsens the calcification induced by phosphate.
Q5. What did the addition of calcitriol do to the HP cells?
Again calcitriol caused additional increase of Runx2 expression (P 0.05 vs. HP cells), whereas paricalcitol failed to increase the expression of Runx2 mRNA beyond the values observed in HP cells.
Q6. What did they show that calcitriol produced in VSMCs?
Jono et al. (19) showed that in VSMCs cultured with high phosphate, the addition of an increasing concentration of calcitriol produced a dose-dependent increase in calcification.
Q7. What is the role of a calcification of the HASMCs?
In the present study, the authors also show that calcification of the HASMCs is also associated with a concomitant activation of the canonical Wnt/ -catenin signaling pathway.
Q8. How long is the incubation of HASMCs in phosphate?
HASMCs are incubated for 9 days in a high (3.3 mmol/l) phosphate (HP) medium (calcification medium) alone or supplemented with either calcitriol 10 8 M (HP CTR) or paricalcitol 3·10 8 M (HP PC).
Q9. What is the effect of calcitriol on VCs?
in a model of uremic rats, the induction of aortic calcifications by calcitriol was partially reversible after discontinuation of calcitriol administration (2).
Q10. What was the cyclin D1 mRNA level in cells treated with HP PC?
In cells treated with HP PC, the cyclin D1 mRNA level was greater than in control (P 0.05) but lower than in HP and HP CTR cells (P 0.05).
Q11. What did the addition of calcitriol do to HP cells?
The addition of calcitriol to HP medium increased the nuclear content of -catenin; however, the addition of paricalcitol caused a reduction in the levels of nuclear -catenin to a level similar to that observed in control cells (Fig. 3A).
Q12. What is the role of the Wnt pathway in regulating growth and development?
Wnt inhibitors have shown early promise; however, given the central role of the Wnt pathway in regulating growth and development in many tissues, considerable work will be needed to ensure the safety of these new therapies (60).
Q13. What is the effect of calcitriol on osteoblasts?
By contrast, paricalcitol decreased calcification, which was accompanied by an inhibition of the Wnt/ -catenin pathway and downregulation of osteoblastic gene expression.
Q14. How was the rabbit anti-mouse antibody stained?
After being washed with PBS (3 5 min), specimens were incubated for 1 h with Alexa Fluor 488 F(ab=)2 fragment of rabbit anti-mouse IgG (1:500; ref. no.
Q15. What is the reason for the lack of uniformity in the literature?
Lack of uniformity in the literature may have resulted from differences in baseline cellular conditions, species employed, or even in the experimental conditions and stimuli applied (26).