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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 ERK1/2 and p38 phosphorylation is GPR55 dependent. (A) Human phospho-kinase array of whole neonatal-ECFC lysates after 15 min treatment with vehicle or 10 μM LPI. Pixel intensity, quantified by ImageJ, revealed an LPI-induced ERK1/2 and p38 phosphorylation. (B) Western blot analysis of total ECFC lysates after 0, 5, 15 and 30 min of 10 μM LPI treatment. Blots were probed with antibody against total or phosphorylated (p) ERK1/2 or p38 or β-actin. (C) Western blot analysis of total and phosphorylated ERK1/2 and p38 phosphorylation in ECFC lysates after 15 min treatment with vehicle, 20 μM CID16020046 (CID), 10 μM LPI or both. Quantification of the ratio of p-ERK1/2 or p-p38 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. (D) Western blot analysis of ERK1/2 and p38 phosphorylation in untreated (−) ECFCs or after transfection with control siRNA (sicontrol) or siRNA against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (15 min). Quantification of the ratio of p-ERK1/2 or p-p38 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from untreated vehicle control; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.
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fig05: LPI-induced ERK1/2 and p38 phosphorylation is GPR55 dependent. (A) Human phospho-kinase array of whole neonatal-ECFC lysates after 15 min treatment with vehicle or 10 μM LPI. Pixel intensity, quantified by ImageJ, revealed an LPI-induced ERK1/2 and p38 phosphorylation. (B) Western blot analysis of total ECFC lysates after 0, 5, 15 and 30 min of 10 μM LPI treatment. Blots were probed with antibody against total or phosphorylated (p) ERK1/2 or p38 or β-actin. (C) Western blot analysis of total and phosphorylated ERK1/2 and p38 phosphorylation in ECFC lysates after 15 min treatment with vehicle, 20 μM CID16020046 (CID), 10 μM LPI or both. Quantification of the ratio of p-ERK1/2 or p-p38 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. (D) Western blot analysis of ERK1/2 and p38 phosphorylation in untreated (−) ECFCs or after transfection with control siRNA (sicontrol) or siRNA against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (15 min). Quantification of the ratio of p-ERK1/2 or p-p38 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from untreated vehicle control; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.

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 ERK1/2 and p38 phosphorylation is GPR55 dependent. (A) Human phospho-kinase array of whole neonatal-ECFC lysates after 15 min treatment with vehicle or 10 μM LPI. Pixel intensity, quantified by ImageJ, revealed an LPI-induced ERK1/2 and p38 phosphorylation. (B) Western blot analysis of total ECFC lysates after 0, 5, 15 and 30 min of 10 μM LPI treatment. Blots were probed with antibody against total or phosphorylated (p) ERK1/2 or p38 or β-actin. (C) Western blot analysis of total and phosphorylated ERK1/2 and p38 phosphorylation in ECFC lysates after 15 min treatment with vehicle, 20 μM CID16020046 (CID), 10 μM LPI or both. Quantification of the ratio of p-ERK1/2 or p-p38 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. (D) Western blot analysis of ERK1/2 and p38 phosphorylation in untreated (−) ECFCs or after transfection with control siRNA (sicontrol) or siRNA against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (15 min). Quantification of the ratio of p-ERK1/2 or p-p38 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from untreated vehicle control; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.
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fig05: LPI-induced ERK1/2 and p38 phosphorylation is GPR55 dependent. (A) Human phospho-kinase array of whole neonatal-ECFC lysates after 15 min treatment with vehicle or 10 μM LPI. Pixel intensity, quantified by ImageJ, revealed an LPI-induced ERK1/2 and p38 phosphorylation. (B) Western blot analysis of total ECFC lysates after 0, 5, 15 and 30 min of 10 μM LPI treatment. Blots were probed with antibody against total or phosphorylated (p) ERK1/2 or p38 or β-actin. (C) Western blot analysis of total and phosphorylated ERK1/2 and p38 phosphorylation in ECFC lysates after 15 min treatment with vehicle, 20 μM CID16020046 (CID), 10 μM LPI or both. Quantification of the ratio of p-ERK1/2 or p-p38 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. (D) Western blot analysis of ERK1/2 and p38 phosphorylation in untreated (−) ECFCs or after transfection with control siRNA (sicontrol) or siRNA against GPR55 (siGPR55) in response to vehicle or 10 μM LPI (15 min). Quantification of the ratio of p-ERK1/2 or p-p38 normalized to β-actin immunostaining. n = 6; ***P < 0.001, significantly different from untreated vehicle control; #P < 0.001, significantly different from LPI-treated sicontrol ECFCs. anova followed by Bonferroni test.
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