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The GPR 55 agonist, L-α-lysophosphatidylinositol, mediates ovarian carcinoma cell-induced angiogenesis.

Hofmann NA, Yang J, Trauger SA, Nakayama H, Huang L, Strunk D, Moses MA, Klagsbrun M, Bischoff J, Graier WF - Br. J. Pharmacol. (2015)

Bottom Line: To study underlying signal transduction, Western blot analysis was performed.Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis.These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria.

No MeSH data available.


Related in: MedlinePlus

LPI-induced ECFC proliferation increase is blocked by ERK1/2-inhibitor U0126. (A) Western blot analysis of ERK1/2 phosphorylation in ECFC lysates after 30 min pre-incubation with vehicle or 10 μM U0126 and 15 min exposure to 10 μM LPI. Quantification of the ratio of p-ERK1/2 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (B) Proliferation increase of ECFCs treated with vehicle or U0126 (48 h). n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (C) Proteome profiler human angiogenesis array of neonatal-ECFC supernatants after 24 h treatment with vehicle or 10 μM LPI. Pixel intensity was quantified by ImageJ.
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fig06: LPI-induced ECFC proliferation increase is blocked by ERK1/2-inhibitor U0126. (A) Western blot analysis of ERK1/2 phosphorylation in ECFC lysates after 30 min pre-incubation with vehicle or 10 μM U0126 and 15 min exposure to 10 μM LPI. Quantification of the ratio of p-ERK1/2 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (B) Proliferation increase of ECFCs treated with vehicle or U0126 (48 h). n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (C) Proteome profiler human angiogenesis array of neonatal-ECFC supernatants after 24 h treatment with vehicle or 10 μM LPI. Pixel intensity was quantified by ImageJ.

Mentions: A human phospho-kinase array was used to investigate the molecular mechanisms underlying LPI/GPR55-mediated angiogenesis. Of the various phospho-proteins in the array, LPI significantly induced phosphorylation of only ERK1/2 and p38 in ECFCs (Figure 5A and Supporting Information Table S1). Western blot analysis confirmed a time-dependent activation of ERK1/2 and p38 by 10 μM LPI (Figure 5B) but not the potential involvement of CREB (cAMP response element-binding protein) (data not shown). Pharmacological inhibition of GPR55 by 20 μM CID16020046 significantly reduced LPI-induced ERK1/2 and p38 phosphorylation (Figure 5C). To confirm the GPR55-dependent activation of ERK1/2 and p38 by LPI, GPR55 was silenced by siRNA (Figure 5D). Compared with control siRNA, knock-down of GPR55 suppressed the LPI-stimulated ERK1/2 and p38 phosphorylation (Figure 5D). Moreover, ECFCs pretreated with the well-established and highly selective MEK1/MEK2 inhibitor U0126 (10 μM) (Favata et al., 1998), which blocks downstream activation of ERK1/2, eliminated ERK1/2 basal phosphorylation without altering total ERK1/2 amounts (Figure 6A). However, LPI no longer induced ERK1/2 phosphorylation (Figure 6A). Furthermore, ERK1/2 inhibition blocked normal ECFC proliferation, without reducing the initial cell number, and prevented the LPI-induced ECFC proliferation, indicating a crucial role for ERK1/2 during endothelial cell proliferation (Figure 6B). Together, these results suggest that LPI induces GPR55-dependent activation of ERK1/2 and thus leads to increased angiogenesis.


The GPR 55 agonist, L-α-lysophosphatidylinositol, mediates ovarian carcinoma cell-induced angiogenesis.

Hofmann NA, Yang J, Trauger SA, Nakayama H, Huang L, Strunk D, Moses MA, Klagsbrun M, Bischoff J, Graier WF - Br. J. Pharmacol. (2015)

LPI-induced ECFC proliferation increase is blocked by ERK1/2-inhibitor U0126. (A) Western blot analysis of ERK1/2 phosphorylation in ECFC lysates after 30 min pre-incubation with vehicle or 10 μM U0126 and 15 min exposure to 10 μM LPI. Quantification of the ratio of p-ERK1/2 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (B) Proliferation increase of ECFCs treated with vehicle or U0126 (48 h). n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (C) Proteome profiler human angiogenesis array of neonatal-ECFC supernatants after 24 h treatment with vehicle or 10 μM LPI. Pixel intensity was quantified by ImageJ.
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Related In: Results  -  Collection

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fig06: LPI-induced ECFC proliferation increase is blocked by ERK1/2-inhibitor U0126. (A) Western blot analysis of ERK1/2 phosphorylation in ECFC lysates after 30 min pre-incubation with vehicle or 10 μM U0126 and 15 min exposure to 10 μM LPI. Quantification of the ratio of p-ERK1/2 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (B) Proliferation increase of ECFCs treated with vehicle or U0126 (48 h). n = 6; ***P < 0.001, significantly different from vehicle control; #P < 0.001, significantly different from LPI-treated ECFCs. anova followed by Bonferroni test. (C) Proteome profiler human angiogenesis array of neonatal-ECFC supernatants after 24 h treatment with vehicle or 10 μM LPI. Pixel intensity was quantified by ImageJ.
Mentions: A human phospho-kinase array was used to investigate the molecular mechanisms underlying LPI/GPR55-mediated angiogenesis. Of the various phospho-proteins in the array, LPI significantly induced phosphorylation of only ERK1/2 and p38 in ECFCs (Figure 5A and Supporting Information Table S1). Western blot analysis confirmed a time-dependent activation of ERK1/2 and p38 by 10 μM LPI (Figure 5B) but not the potential involvement of CREB (cAMP response element-binding protein) (data not shown). Pharmacological inhibition of GPR55 by 20 μM CID16020046 significantly reduced LPI-induced ERK1/2 and p38 phosphorylation (Figure 5C). To confirm the GPR55-dependent activation of ERK1/2 and p38 by LPI, GPR55 was silenced by siRNA (Figure 5D). Compared with control siRNA, knock-down of GPR55 suppressed the LPI-stimulated ERK1/2 and p38 phosphorylation (Figure 5D). Moreover, ECFCs pretreated with the well-established and highly selective MEK1/MEK2 inhibitor U0126 (10 μM) (Favata et al., 1998), which blocks downstream activation of ERK1/2, eliminated ERK1/2 basal phosphorylation without altering total ERK1/2 amounts (Figure 6A). However, LPI no longer induced ERK1/2 phosphorylation (Figure 6A). Furthermore, ERK1/2 inhibition blocked normal ECFC proliferation, without reducing the initial cell number, and prevented the LPI-induced ECFC proliferation, indicating a crucial role for ERK1/2 during endothelial cell proliferation (Figure 6B). Together, these results suggest that LPI induces GPR55-dependent activation of ERK1/2 and thus leads to increased angiogenesis.

Bottom Line: To study underlying signal transduction, Western blot analysis was performed.Four target-specific siRNAs against GPR55 prevented these effects of LPI on angiogenesis.These pro-angiogenic effects of LPI were transduced by GPR55-dependent phosphorylation of ERK1/2 and p38 kinase.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Biology and Biochemistry, Medical University Graz, Graz, Austria.

No MeSH data available.


Related in: MedlinePlus