s1p receptor agonist 1  (TargetMol)

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Primers for PCR

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Sequencing, Amplification

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Effect of estrogen (E2) and sphingosine‐1‐phosphate (S1P) on human osteoblast cell growth at 24 hours. ( A ) Effect of estradiol, S1P, and an S1PR agonist on osteoblast cell proliferation. At 24 hours, estradiol (10 nM) increased proliferation 20% relative to control. S1P (1 μM) or SEW2871 (1 μM) induced cell growth but less than estradiol, 14% and 10%, respectively. Mean ± SEM, n = 6. *p < 0.05, **p < 0.01 versus control. ( B ) Sphingosine kinase inhibitor (SKi) reduces the estradiol effect on proliferation. SKi alone did not affect proliferation (second bar); SKi with estrogen reduced the effect of estrogen on cell proliferation by two‐thirds. Mean ± SEM, n = 4. ** p < 0.01, ns (not significant), versus control; * p < 0.05 relative to estrogen only.

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Control

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Expression of S1P receptors and osteoblast differentiation‐related mRNAs in human osteoblasts. ( A ) Analysis by real‐time PCR of the expression of S1P receptors 1, 2, and 3 ( A i) in correspondence with osteoblast‐related mRNAs expression ( A ii), including RUNX2, alkaline phosphatase, type I collagen, and osterix in undifferentiated hOB and after 7, 14, and 21 days in differentiation medium. ( B ) Effect of 24‐hour treatment with 10 nM estradiol (E2), or 200 nM S1P, on S1PR1‐3 in undifferentiated hOB. Both E2 and S1P increase S1PR1 mRNA expression, decrease S1PR2, and did not change S1PR3. ( C ) Both S1PR1 and S1PR2 protein production by hOB was increased with S1P treatment at 2 weeks of differentiation. ( D ) In late differentiation (3 weeks), effects of S1P on receptor mRNAs were limited to minor effects on S1PR2. Relative to control, * p < 0.05; ** p < 0.01.

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Short‐term effect of S1P or E2 on osteoblast estrogen receptors, SPHK activity, and MAPK pathways. ( A ) Effect of 24‐hour treatment with 1 μM S1P or 10 nM E2 on ER, S1PR, and SPHK1 mRNA expression. Note the concentration‐dependent increase in ERβ (ii), S1PR1 (iii), and SPHK1 (iv) mRNA expression. Expression of ERα mRNA (i) was unaffected. Effect of estradiol, 10 nM, on mRNAs for SPHK1 at 24 hours (iv). Real‐time PCR normalized to GAPDH, mean ± range, n = 2 (independent experiments with 2 to 4 replicates). * p < 0.05, ** p < 0.01 versus control. ( B ) Effect of (i) 200 nM or 1000 nM S1P or (ii) 10 nM estradiol on sphingosine kinase activity. S1P stimulated the production of fluorescently labeled S1P, reflecting sphingosine kinase activity in osteoblast lysates, after treatment for 15 minutes with unlabeled S1P, pmoles/min/mg protein. Effects of estradiol were similar; separate experiments with separate controls. Mean ± SEM, n = 3 ** p < 0.01 versus control. ( C ) On Western blot, SPHK1 protein production was increased at 24 hours by 10 nM estradiol, or the durable S1P receptor 1 agonist SEW2871, 1 μM. ( D ) Effect of S1P (100 nM) on AKT and ERK phosphorylation in h‐MSC in 5 and 15 minutes. S1P increased phospho‐AKT and phospho‐ERK at 15 minutes relative to AKT and ERK or to β‐actin. ( E ) S1P effect on AKT and ERK phosphorylation in hOB. S1P increased phospho‐AKT at 200 nM and 1000 nM S1P and slightly increase phospho‐ERK at 1000 nM. ( C – E ) Quantification of two ECL blot images normalized to equal total protein to eliminate artifacts of different loading or detection efficiency. * p < 0.05; ** p < 0.01, ns (not significant).

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Activity Assay, Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Control, Labeling, Western Blot, Phospho-proteomics

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Estradiol (10 nM) or S1P (200 nM) promotes osteoblast differentiation. ( A ) (i) Alkaline phosphatase activity after treatment for 3 weeks is shown in whole 9 cm 2 cultures. (ii) Cells photographed after staining with ALP at 20× magnification without phase. (iii) At low power, 10×, in phase, the dense developing tissue and collagen are revealed. (iv) Cells photographed after staining with alizarin red at 20× magnification. ( B ) ALP activity quantified by densitometry analysis and normalized to cell number. Both estradiol and S1P increase ALP activity compared with the control. Results are mean ± SEM, n = 4. ** p < 0.01 versus control.

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Activity Assay, Staining, Control

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Time‐course effect of S1P on characteristic osteoblast, estrogen receptors, and S1P‐related mRNAs. All assays are real‐time PCR in duplicate, shown as mean ± range, and normalized in each case to matched controls = 1.0; * p < 0.05; ** p < 0.01. ( A ) Time‐course effect of 200 nM S1P, with media changed every 3 days, on osteoblast‐related mRNAs. ( B ) Effect of long‐term treatment with 200 nM S1P on estrogen receptors (ERs). In contrast to effects at 24 hours (Fig. ), where only ERβ increased, both ERα and ERβ mRNAs increased after 1 week in 200 nM S1P and dropped after 14 days, in keeping with cell maturation with reduced response to growth and differentiation factors. ( C ) The response to 200 nM S1P supplementation in media of S1P targets SPHK1, SGPP1, and ENPP1 mRNAs at 7, 14, and 21 day of hOB differentiation. The SPHK1, SGPP1 showed variable effects, but each was increased at either 14 or 21 days. ENPP1 was downregulated in osteoblast differentiation.

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Real-time Polymerase Chain Reaction

Journal: JBMR Plus

Article Title: Sphingosine‐1‐Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

doi: 10.1002/jbm4.10037

Figure Lengend Snippet: Effects of estrogen are reversed in many, but not all, cases by inhibiting the sphingosine kinase. Representative estrogen‐sensitive mRNAs in human osteoblasts are shown. For RUNX2 ( A ), OSX ( B ), ALP ( C ), and S1PR1 ( E ), mRNA expressions were induced by 10 nM estradiol at 7 days. A 10‐μM SKi treatment reduced mRNA expression, and the combination of estradiol and SKi mRNA expression remained less than the control. The SPHK1 ( D ) mRNA expression was decreased by the sphingosine kinase inhibitor SKi, but was induced by estradiol with or without SKi. ( F ) Estradiol and S1P have different effects on the RANKL‐OPG pathway. Data shown are for 7‐day treatment. The untreated control is defined as 1.0. RANKL/OPG mRNA expression ratio was decreased 60% to 80% by 5 nM estradiol treatment and further by the sphingosine kinase inhibitor (10 μMSKi) treatment; the combination of SKi and estradiol gave an intermediate result. On the other hand, RANKL/OPG was not reduced in 200 nM S1P. Media were changed at 3‐day intervals. Duplicate results ± range; p values are relative to control = 1.0 except when indicated by bars.

Article Snippet: The S1P receptor‐1 (S1PR1) agonist SEW2871 was from Tocris (Bristol, UK).

Techniques: Expressing, Control