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Protein palmitoylation regulates osteoblast differentiation through BMP-induced osterix expression.

Leong WF, Zhou T, Lim GL, Li B - PLoS ONE (2009)

Bottom Line: The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4.However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation.These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Developmental Biology Division, The Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore.

ABSTRACT
Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53(-/-) osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

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Inhibition of protein palmitoylation impeded osteoblast differentiation.A. The effect of 2BP on ALP staining. Primary osteoblasts were treated with increasing amounts of 2BP for 4 days in differentiation medium and then stained for ALP. B. Quantitation of ALP activities that were normalized to the protein levels of the cells. C. The effect of 2BP on the expression of several osteoblast differentiation markers. The experiments were carried out like Fig. 2A and total RNA were isolated from these cells. RT-PCR was carried out to determine the mRNA levels of these markers. The value of control (lane 1) was set at 1.00. D. The effect of 2BP on bone nodule formation. The experiments were carried out like Fig. 2A. After 21 days in culture, the plates were stained using a Von Kossa method.
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pone-0004135-g002: Inhibition of protein palmitoylation impeded osteoblast differentiation.A. The effect of 2BP on ALP staining. Primary osteoblasts were treated with increasing amounts of 2BP for 4 days in differentiation medium and then stained for ALP. B. Quantitation of ALP activities that were normalized to the protein levels of the cells. C. The effect of 2BP on the expression of several osteoblast differentiation markers. The experiments were carried out like Fig. 2A and total RNA were isolated from these cells. RT-PCR was carried out to determine the mRNA levels of these markers. The value of control (lane 1) was set at 1.00. D. The effect of 2BP on bone nodule formation. The experiments were carried out like Fig. 2A. After 21 days in culture, the plates were stained using a Von Kossa method.

Mentions: We first looked at one of the early markers of osteoblast differentiation, alkaline phosphatase (ALP). It was found that 2BP had a dosage dependent inhibitory effect on ALP expression. Fig. 2A shows the ALP staining of osteoblasts cultured in the differentiation medium, while Fig. 2B shows the quantitative ALP activities that were normalized to the total protein levels. It is obvious that inhibition of protein palmitoylation diminished ALP expression. We then tested the expression of a few other markers by RT-PCR. It was found that 2BP mainly down-regulated the mRNA levels of osteocalcin, but not collagen type 1α or osteopontin (Fig. 2C). More significantly, we found that 2BP severely inhibited bone nodule formation, a later marker of osteoblast differentiation and an indicator of osteoblast bone forming activity (Fig. 2D). These results indicate that protein palmitoylation is required for osteoblast differentiation into mature osteocytes. However, treatment of osteoblast with the same amounts of palmitic acid, the substrate of PATs (>150 µM palmitic acid showed cytotoxicity in primary osteoblasts), showed no significant effect on osteoblast differentiation (Fig. 2E). The reason why higher concentrations of palmitic acid did not enhance osteoblast differentiation could be that the serum provides sufficient amount of palmitic acid for protein palmitoylation.


Protein palmitoylation regulates osteoblast differentiation through BMP-induced osterix expression.

Leong WF, Zhou T, Lim GL, Li B - PLoS ONE (2009)

Inhibition of protein palmitoylation impeded osteoblast differentiation.A. The effect of 2BP on ALP staining. Primary osteoblasts were treated with increasing amounts of 2BP for 4 days in differentiation medium and then stained for ALP. B. Quantitation of ALP activities that were normalized to the protein levels of the cells. C. The effect of 2BP on the expression of several osteoblast differentiation markers. The experiments were carried out like Fig. 2A and total RNA were isolated from these cells. RT-PCR was carried out to determine the mRNA levels of these markers. The value of control (lane 1) was set at 1.00. D. The effect of 2BP on bone nodule formation. The experiments were carried out like Fig. 2A. After 21 days in culture, the plates were stained using a Von Kossa method.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2607547&req=5

pone-0004135-g002: Inhibition of protein palmitoylation impeded osteoblast differentiation.A. The effect of 2BP on ALP staining. Primary osteoblasts were treated with increasing amounts of 2BP for 4 days in differentiation medium and then stained for ALP. B. Quantitation of ALP activities that were normalized to the protein levels of the cells. C. The effect of 2BP on the expression of several osteoblast differentiation markers. The experiments were carried out like Fig. 2A and total RNA were isolated from these cells. RT-PCR was carried out to determine the mRNA levels of these markers. The value of control (lane 1) was set at 1.00. D. The effect of 2BP on bone nodule formation. The experiments were carried out like Fig. 2A. After 21 days in culture, the plates were stained using a Von Kossa method.
Mentions: We first looked at one of the early markers of osteoblast differentiation, alkaline phosphatase (ALP). It was found that 2BP had a dosage dependent inhibitory effect on ALP expression. Fig. 2A shows the ALP staining of osteoblasts cultured in the differentiation medium, while Fig. 2B shows the quantitative ALP activities that were normalized to the total protein levels. It is obvious that inhibition of protein palmitoylation diminished ALP expression. We then tested the expression of a few other markers by RT-PCR. It was found that 2BP mainly down-regulated the mRNA levels of osteocalcin, but not collagen type 1α or osteopontin (Fig. 2C). More significantly, we found that 2BP severely inhibited bone nodule formation, a later marker of osteoblast differentiation and an indicator of osteoblast bone forming activity (Fig. 2D). These results indicate that protein palmitoylation is required for osteoblast differentiation into mature osteocytes. However, treatment of osteoblast with the same amounts of palmitic acid, the substrate of PATs (>150 µM palmitic acid showed cytotoxicity in primary osteoblasts), showed no significant effect on osteoblast differentiation (Fig. 2E). The reason why higher concentrations of palmitic acid did not enhance osteoblast differentiation could be that the serum provides sufficient amount of palmitic acid for protein palmitoylation.

Bottom Line: The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4.However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation.These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

View Article: PubMed Central - PubMed

Affiliation: Cancer and Developmental Biology Division, The Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore, Singapore.

ABSTRACT
Osteoporosis is one of the most common diseases and can be treated by either anti-resorption drugs, anabolic drugs, or both. To search for anabolic drug targets for osteoporosis therapy, it is crucial to understand the biology of bone forming cells, osteoblasts, in terms of their proliferation, differentiation, and function. Here we found that protein palmitoylation participates in signaling pathways that control osterix expression and osteoblast differentiation. Mouse calvarial osteoblasts express most of the 24 palmitoyl transferases, with some being up-regulated during differentiation. Inhibition of protein palmitoylation, with a substrate-analog inhibitor, diminished osteoblast differentiation and mineralization, but not proliferation or survival. The decrease in differentiation capacity is associated with a reduction in osterix, but not Runx2 or Atf4. Inhibition of palmitoyl transferases had little effect in p53(-/-) osteoblasts that show accelerated differentiation due to overexpression of osterix, suggesting that osterix, at least partially, mediated the effect of inhibition of palmitoyl transferases on osteoblast differentiation. BMPs are the major driving force of osteoblast differentiation in the differentiation assays. We found that inhibition of palmitoyl transferases also compromised BMP2-induced osteoblast differentiation through down-regulating osterix induction. However, palmitoyl transferases inhibitor did not inhibit Smad1/5/8 activation. Instead, it compromised the activation of p38 MAPK, which are known positive regulators of osterix expression and differentiation. These results indicate that protein palmitoylation plays an important role in BMP-induced MAPK activation, osterix expression, and osteoblast differentiation.

Show MeSH
Related in: MedlinePlus