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Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA, Naveiras O, Murthy SK, Ingber DE, Daley GQ, García-Cardeña G, Wenzel PL - J. Exp. Med. (2015)

Bottom Line: Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB).Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution.These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

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Affiliation: Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030.

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WSS stimulates cAMP–PKA signaling and phosphorylation of CREB. (A) Expression of CREB, cAMP–PKA, and calmodulin signaling components believed to lie downstream of noncanonical Wnt/calcium signaling are altered by WSS and inhibition of PGE2 by indomethacin at 6 and 36 h (n = 3 independent experiments at each time point). (B) Phosphorylation of ser133 on CREB (P-CREB) is elevated by 3 h of WSS. (C) P-CREB is reduced in Ncx1−/− PSp (n = 6 mutant, n = 8 wild-type littermates analyzed). Representative data from different gels are separated by white space. (D) Intracellular and circulating (secreted) cAMP is increased by WSS in a PGE2-dependent manner (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). Indomethacin significantly reduced levels of intracellular cAMP (Holm-Šídák comparison: P = 0.009) and circulating cAMP (P = 0.005). Stimulation of cAMP synthesis by adenylyl cyclase with forskolin serves as a positive control. (E) Blocking antibodies against PGE2 or inhibitors of COX1/2 effectively suppress WSS-induced progenitor activity in M3434 methylcellulose assays. PKA inhibition by H89 similarly reduces progenitor activity, whereas stimulation of cAMP levels by forskolin elevates hematopoietic activity above static vehicle controls (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). (F) Interruption of prostaglandin signaling by indomethacin reduces transcription of Ras/MAPK/Akt, CREB, and calcineurin/NFAT pathway components related to calcium transport and calmodulin kinase activity (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Data are represented as mean ± SEM.
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fig7: WSS stimulates cAMP–PKA signaling and phosphorylation of CREB. (A) Expression of CREB, cAMP–PKA, and calmodulin signaling components believed to lie downstream of noncanonical Wnt/calcium signaling are altered by WSS and inhibition of PGE2 by indomethacin at 6 and 36 h (n = 3 independent experiments at each time point). (B) Phosphorylation of ser133 on CREB (P-CREB) is elevated by 3 h of WSS. (C) P-CREB is reduced in Ncx1−/− PSp (n = 6 mutant, n = 8 wild-type littermates analyzed). Representative data from different gels are separated by white space. (D) Intracellular and circulating (secreted) cAMP is increased by WSS in a PGE2-dependent manner (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). Indomethacin significantly reduced levels of intracellular cAMP (Holm-Šídák comparison: P = 0.009) and circulating cAMP (P = 0.005). Stimulation of cAMP synthesis by adenylyl cyclase with forskolin serves as a positive control. (E) Blocking antibodies against PGE2 or inhibitors of COX1/2 effectively suppress WSS-induced progenitor activity in M3434 methylcellulose assays. PKA inhibition by H89 similarly reduces progenitor activity, whereas stimulation of cAMP levels by forskolin elevates hematopoietic activity above static vehicle controls (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). (F) Interruption of prostaglandin signaling by indomethacin reduces transcription of Ras/MAPK/Akt, CREB, and calcineurin/NFAT pathway components related to calcium transport and calmodulin kinase activity (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Data are represented as mean ± SEM.

Mentions: To evaluate the contribution of PGE2 to intracellular signaling, we subjected cells to WSS with or without indomethacin and profiled global gene expression as described above. Analysis of differential gene expression revealed that PGE2 contributed to transcription of CREB targets, as well as core components of Wnt/calcium signaling, including Creb1, Ctbp1, Shc1, Calm2, and Camk1 (Fig. 7 A). In chondrocytes of the bone, WSS was recently shown to promote TOP-luciferase (LEF/TCF reporter) and CRE-luciferase (CREB reporter) activity (Ogawa et al., 2014). Whereas addition of exogenous PGE2 was insufficient to drive activation of TOP-luciferase, PGE2 was capable of promoting activity of the CRE-luciferase reporter. Consistent with these findings, we observed that WSS increased phosphorylation of CREB at serine 133, a posttranslational modification required for CREB-mediated transcription (Fig. 7 B). CREB phosphorylation can be mediated by PKA; thus, the increase in PKA transcript Prkaca by WSS at 6 and 36 h (unpaired Student’s t test, P = 0.016 [6 h] and P = 0.004 [36 h]) suggested that modulation of PKA downstream of WSS could contribute to enhanced CREB activity. Furthermore, concomitant reduction in Prkaca transcript, the prohematopoietic CREB target Etv2, and P-CREB levels in uncultured Ncx1−/− PSp independently suggested a role for blood flow in coordinating PKA and CREB activity (Figs. 4 C and 7 C). Of six Ncx1 mutants analyzed in the study, five expressed lower levels of P-CREB relative to wild-type littermates. Total CREB protein was also reduced in four of six mutants, likely indicative of complex signaling inputs known to regulate CREB expression and activity (Shaywitz and Greenberg, 1999). Binding of the EP2 and EP4 receptors by PGE2 stimulates the Gs-adenylyl cyclase pathway, which leads to cAMP production and subsequent PKA activation (Zhang and Daaka, 2011; Yamamizu et al., 2012). As expected, AGM exposed to WSS for 36 h produced higher levels of intracellular and circulating cAMP compared with static cultures (Fig. 7 A). Importantly, antagonists of COX2 or PKA abolished enhancement in hematopoietic colony forming activity with ex vivo exposure to WSS, as did a blocking antibody to PGE2 (Fig. 7 E). The adenylyl cyclase agonist forskolin amplified colony-forming potential in WSS-exposed cultures but not static cultures, suggesting that PGE2 cooperates with additional WSS-responsive signaling in initiation of the hematopoietic program. Consistent with this notion, inhibition of COX2 by indomethacin blocks WSS induction not only of CREB and MAPK signaling but also calcium, calmodulin, calcineurin, and NFAT, primary components of noncanonical Wnt/calcium signaling that function to control cell adhesion, migration, and ventral patterning (Fig. 7 F and Dataset S3; Kohn and Moon, 2005). Based on analyses of BrdU incorporation and Annexin V staining, WSS contributes significantly to survival of the CD144+ CD45− population (Fig. 8). However, PGE2 does not appear to influence cell proliferation or survival, but instead may serve to modulate fate selection through CREB. More detailed analyses of signal transduction will be required to understand the precise nature of how PGE2–PKA interfaces with other developmental signaling pathways to modulate hematopoietic-specific gene expression downstream of WSS in the AGM.


Biomechanical forces promote blood development through prostaglandin E2 and the cAMP-PKA signaling axis.

Diaz MF, Li N, Lee HJ, Adamo L, Evans SM, Willey HE, Arora N, Torisawa YS, Vickers DA, Morris SA, Naveiras O, Murthy SK, Ingber DE, Daley GQ, García-Cardeña G, Wenzel PL - J. Exp. Med. (2015)

WSS stimulates cAMP–PKA signaling and phosphorylation of CREB. (A) Expression of CREB, cAMP–PKA, and calmodulin signaling components believed to lie downstream of noncanonical Wnt/calcium signaling are altered by WSS and inhibition of PGE2 by indomethacin at 6 and 36 h (n = 3 independent experiments at each time point). (B) Phosphorylation of ser133 on CREB (P-CREB) is elevated by 3 h of WSS. (C) P-CREB is reduced in Ncx1−/− PSp (n = 6 mutant, n = 8 wild-type littermates analyzed). Representative data from different gels are separated by white space. (D) Intracellular and circulating (secreted) cAMP is increased by WSS in a PGE2-dependent manner (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). Indomethacin significantly reduced levels of intracellular cAMP (Holm-Šídák comparison: P = 0.009) and circulating cAMP (P = 0.005). Stimulation of cAMP synthesis by adenylyl cyclase with forskolin serves as a positive control. (E) Blocking antibodies against PGE2 or inhibitors of COX1/2 effectively suppress WSS-induced progenitor activity in M3434 methylcellulose assays. PKA inhibition by H89 similarly reduces progenitor activity, whereas stimulation of cAMP levels by forskolin elevates hematopoietic activity above static vehicle controls (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). (F) Interruption of prostaglandin signaling by indomethacin reduces transcription of Ras/MAPK/Akt, CREB, and calcineurin/NFAT pathway components related to calcium transport and calmodulin kinase activity (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Data are represented as mean ± SEM.
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Related In: Results  -  Collection

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fig7: WSS stimulates cAMP–PKA signaling and phosphorylation of CREB. (A) Expression of CREB, cAMP–PKA, and calmodulin signaling components believed to lie downstream of noncanonical Wnt/calcium signaling are altered by WSS and inhibition of PGE2 by indomethacin at 6 and 36 h (n = 3 independent experiments at each time point). (B) Phosphorylation of ser133 on CREB (P-CREB) is elevated by 3 h of WSS. (C) P-CREB is reduced in Ncx1−/− PSp (n = 6 mutant, n = 8 wild-type littermates analyzed). Representative data from different gels are separated by white space. (D) Intracellular and circulating (secreted) cAMP is increased by WSS in a PGE2-dependent manner (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). Indomethacin significantly reduced levels of intracellular cAMP (Holm-Šídák comparison: P = 0.009) and circulating cAMP (P = 0.005). Stimulation of cAMP synthesis by adenylyl cyclase with forskolin serves as a positive control. (E) Blocking antibodies against PGE2 or inhibitors of COX1/2 effectively suppress WSS-induced progenitor activity in M3434 methylcellulose assays. PKA inhibition by H89 similarly reduces progenitor activity, whereas stimulation of cAMP levels by forskolin elevates hematopoietic activity above static vehicle controls (n = 3 independent experiments; two-way ANOVA: **, P < 0.001). (F) Interruption of prostaglandin signaling by indomethacin reduces transcription of Ras/MAPK/Akt, CREB, and calcineurin/NFAT pathway components related to calcium transport and calmodulin kinase activity (p-value cutoff 0.05, FDR 0.8, overlap coefficient cutoff 0.5). Data are represented as mean ± SEM.
Mentions: To evaluate the contribution of PGE2 to intracellular signaling, we subjected cells to WSS with or without indomethacin and profiled global gene expression as described above. Analysis of differential gene expression revealed that PGE2 contributed to transcription of CREB targets, as well as core components of Wnt/calcium signaling, including Creb1, Ctbp1, Shc1, Calm2, and Camk1 (Fig. 7 A). In chondrocytes of the bone, WSS was recently shown to promote TOP-luciferase (LEF/TCF reporter) and CRE-luciferase (CREB reporter) activity (Ogawa et al., 2014). Whereas addition of exogenous PGE2 was insufficient to drive activation of TOP-luciferase, PGE2 was capable of promoting activity of the CRE-luciferase reporter. Consistent with these findings, we observed that WSS increased phosphorylation of CREB at serine 133, a posttranslational modification required for CREB-mediated transcription (Fig. 7 B). CREB phosphorylation can be mediated by PKA; thus, the increase in PKA transcript Prkaca by WSS at 6 and 36 h (unpaired Student’s t test, P = 0.016 [6 h] and P = 0.004 [36 h]) suggested that modulation of PKA downstream of WSS could contribute to enhanced CREB activity. Furthermore, concomitant reduction in Prkaca transcript, the prohematopoietic CREB target Etv2, and P-CREB levels in uncultured Ncx1−/− PSp independently suggested a role for blood flow in coordinating PKA and CREB activity (Figs. 4 C and 7 C). Of six Ncx1 mutants analyzed in the study, five expressed lower levels of P-CREB relative to wild-type littermates. Total CREB protein was also reduced in four of six mutants, likely indicative of complex signaling inputs known to regulate CREB expression and activity (Shaywitz and Greenberg, 1999). Binding of the EP2 and EP4 receptors by PGE2 stimulates the Gs-adenylyl cyclase pathway, which leads to cAMP production and subsequent PKA activation (Zhang and Daaka, 2011; Yamamizu et al., 2012). As expected, AGM exposed to WSS for 36 h produced higher levels of intracellular and circulating cAMP compared with static cultures (Fig. 7 A). Importantly, antagonists of COX2 or PKA abolished enhancement in hematopoietic colony forming activity with ex vivo exposure to WSS, as did a blocking antibody to PGE2 (Fig. 7 E). The adenylyl cyclase agonist forskolin amplified colony-forming potential in WSS-exposed cultures but not static cultures, suggesting that PGE2 cooperates with additional WSS-responsive signaling in initiation of the hematopoietic program. Consistent with this notion, inhibition of COX2 by indomethacin blocks WSS induction not only of CREB and MAPK signaling but also calcium, calmodulin, calcineurin, and NFAT, primary components of noncanonical Wnt/calcium signaling that function to control cell adhesion, migration, and ventral patterning (Fig. 7 F and Dataset S3; Kohn and Moon, 2005). Based on analyses of BrdU incorporation and Annexin V staining, WSS contributes significantly to survival of the CD144+ CD45− population (Fig. 8). However, PGE2 does not appear to influence cell proliferation or survival, but instead may serve to modulate fate selection through CREB. More detailed analyses of signal transduction will be required to understand the precise nature of how PGE2–PKA interfaces with other developmental signaling pathways to modulate hematopoietic-specific gene expression downstream of WSS in the AGM.

Bottom Line: Furthermore, Ncx1 heartbeat mutants, as well as static cultures of AGM, exhibit lower levels of expression of prostaglandin synthases and reduced phosphorylation of the cAMP response element-binding protein (CREB).Similar to flow-exposed cultures, transient treatment of AGM with the synthetic analogue 16,16-dimethyl-PGE2 stimulates more robust engraftment of adult recipients and greater lymphoid reconstitution.These data provide one mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030 Program in Children's Regenerative Medicine, Department of Pediatric Surgery, Center for Stem Cell and Regenerative Medicine, The Brown Foundation Institute of Molecular Medicine, and Immunology Program, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, TX 77030.

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