Endothelin B Receptors on Primary Chicken Müller Cells and the Human MIO-M1 Müller Cell Line Activate ERK Signaling via Transactivation of Epidermal Growth Factor Receptors
Abstract:Injury to the eye or retina triggers Müller cells, the major glia cell of the retina, to dedifferentiate and proliferate. In some species they attain retinal progenitor properties and have the capacity to generate new neurons. The epidermal growth factor receptor (EGFR) system and extracellular signal-regulated kinase (ERK) signaling are key regulators of these processes in Müller cells. The extracellular signals that modulate and control these processes are not fully understood. In this work we studied whethe… Show more
“…In order to dissect how EDNRA signals to ERK and hence regulates cell growth, we used different kinase inhibitors and found that while a PKC inhibitor did not impact on the protective function of EDN1, inhibiting RAF kinases with RAF265 or blocking RTK activity using the pan RTK inhibitor dovitinib overcame this protection (Fig EV5H and I). This suggests a crosstalk between EDNRA and RTK signalling as it has been described previously (Harada et al , ; Harun‐Or‐Rashid et al , ; Moody et al , ), which ultimately activates ERK through RAF.…”
Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells.
“…In order to dissect how EDNRA signals to ERK and hence regulates cell growth, we used different kinase inhibitors and found that while a PKC inhibitor did not impact on the protective function of EDN1, inhibiting RAF kinases with RAF265 or blocking RTK activity using the pan RTK inhibitor dovitinib overcame this protection (Fig EV5H and I). This suggests a crosstalk between EDNRA and RTK signalling as it has been described previously (Harada et al , ; Harun‐Or‐Rashid et al , ; Moody et al , ), which ultimately activates ERK through RAF.…”
Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells.
“…This overexpression is mainly observed in the GCL where the end feet of MCs reside [ 52 ] and close to the damaged RGCs, as shown in Figure 2 and Figure 4 . Thus, it is tempting to suggest that the activation of the survival MAPK signaling pathway results in neuroprotection through activation of both MCs and RGCs, as has been described in other experimental models such as NMDA excitotoxicity [ 53 , 54 ], or retinal detachment, ischemia-reperfusion, inflammation and glaucoma (reviewed in [ 55 ]). P-MAPK and MAPK was also observed in the outer retina; however, further studies are need to understand this event.…”
We analyze the 7,8-dihydroxyflavone (DHF)/TrkB signaling activation of two main intracellular pathways, mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol 3 kinase (PI3K)/AKT, in the neuroprotection of axotomized retinal ganglion cells (RGCs). Methods: Adult albino Sprague-Dawley rats received left intraorbital optic nerve transection (IONT) and were divided in two groups. One group received daily intraperitoneal DHF (5 mg/kg) and another vehicle (1%DMSO in 0.9%NaCl) from one day before IONT until processing. Additional intact rats were employed as control (n = 4). At 1, 3 or 7 days (d) after IONT, phosphorylated (p)AKT, p-MAPK, and non-phosphorylated AKT and MAPK expression levels were analyzed in the retina by Western blotting (n = 4/group). Radial sections were also immunodetected for the above-mentioned proteins, and for Brn3a and vimentin to identify RGCs and Müller cells (MCs), respectively (n = 3/group). Results: IONT induced increased levels of p-MAPK and MAPK at 3d in DHF- or vehicle-treated retinas and at 7d in DHF-treated retinas. IONT induced a fast decrease in AKT in retinas treated with DHF or vehicle, with higher levels of phosphorylation in DHF-treated retinas at 7d. In intact retinas and vehicle-treated groups, no p-MAPK or MAPK expression in RGCs was observed. In DHF- treated retinas p-MAPK and MAPK were expressed in the ganglion cell layer and in the RGC nuclei 3 and 7d after IONT. AKT was observed in intact and axotomized RGCs, but the signal intensity of p-AKT was stronger in DHF-treated retinas. Finally, MCs expressed higher quantities of both MAPK and AKT at 3d in both DHF- and vehicle-treated retinas, and at 7d the phosphorylation of p-MAPK was higher in DHF-treated groups. Conclusions: Phosphorylation and increased levels of AKT and MAPK through MCs and RGCs in retinas after DHF-treatment may be responsible for the increased and long-lasting RGC protection afforded by DHF after IONT.
“…In a variety of ways, RTK signaling can require mediation by G protein coupled receptors, a mechanisms called transactivation (Kilpatrick & Hill, 2021). Prior in vitro explant studies have implicated a convergence of Ret and Ednrb at the level of protein kinase A activity ( (Barlow et al, 2003;Goto et al, 2013); see also Discussion), although other mechanisms of transactivation have been described for Ednrb to other RTKs (Grantcharova et al, 2006;Harun-Or-Rashid, Konjusha, Galindo-Romero, & Hallbook, 2016), and Ret with other GPCRs (Tsuchioka, Takebayashi, Hisaoka, Maeda, & Nakata, 2008). Because Ret expression was preserved in Wnt1Cre-derived enteric progenitors even as they were nonresponsive to GDNF, we considered that transactivation via Ednrb might be involved.…”
Section: Ret Is Required For Pax2cre-derived Ens Progenitor Cells Mig...mentioning
Two major ligand-receptor signaling axes – endothelin Edn3 and its receptor Ednrb, and glial-derived neurotrophic factor (GDNF) and its receptor Ret – are required for migration of enteric nervous system (ENS) progenitors to the hindgut. Mutations in either component cause colonic aganglionosis, also called Hirschsprung disease. Here, we have used Wnt1Cre and Pax2Cre in mice to show that these driver lines label distinct ENS sublineages during progenitor migration and in their terminal hindgut fates. Both Cre lines result in Hirschsprung disease when combined with conditionalEdnrbor conditionalRetalleles. In vitro explant assays and analysis of lineage-labeled mutant embryos show that GDNF but not Edn3 is a migration cue for cells of both lineages. Instead, Edn3-Ednrb function is required in both for GDNF responsiveness, by expanding the Ret+population in the Pax2Cre lineage and by supporting Ret function in Wnt1Cre-derived cells. Our results demonstrate that two sublineages of progenitors give rise to the ENS, and that these divergently utilize endothelin signaling to support migration to the hindgut.
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