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Investigation of the Molecular Mechanisms by Which Endothelin-3 Stimulates Preadipocyte Growth.

ABSTRACT: Endothelins induce many biological responses, and they are composed of three peptides: ET-1, ET-2, and ET-3. Reports have indicated that ET-1 regulates cell proliferation, adipogenesis, and other cell responses and that ET-3 stimulates the growth of gastrointestinal epithelial cells and melanocytes. However, the signalling pathways of ET3 that mediate the growth of fat cells are still unclear. Using 3T3-L1 white preadipocytes, we found that ET-3 induced increases in both cell number and BrdU incorporation. Pretreatment with an ET_AR antagonist (but not an ET_BR antagonist) blocked the ET-3-induced increases in both cell number and BrdU incorporation. Additionally, BQ610 suppressed the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3 proteins, and pretreatment with specific inhibitors of AMPK, JNK/c-JUN, or JAK/STAT3 prevented the ET-3-induced increases in phosphorylation of AMPK, c-JUN, and STAT3, respectively. Neither p38 MAPK inhibitor nor PKC inhibitor altered the effects of ET-3 on cell growth. These data suggest that ET-3 stimulates preadipocyte growth through the ET_AR, AMPK, JNK/c-JUN, and STAT3 pathways. Moreover, ET-3 did not alter HIB1B brown preadipocyte and D12 beige preadipocyte growth, suggesting a preadipocyte type-dependent effect. The results of this study may help explain how endothelin mediates fat cell activity and fat cell-associated diseases.

SUBMITTER: Siao AC

PROVIDER: S-EPMC8176213 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RATIONALE:Large-conductance calcium-activated potassium channels (BK) are composed of pore-forming BK? and auxiliary ?1 subunits in arterial smooth muscle cells (myocytes). Vasoconstrictors, including endothelin-1 (ET-1), inhibit myocyte BK channels, leading to contraction, but mechanisms involved are unclear. Recent evidence indicates that BK? is primarily plasma membrane localized, whereas the cellular location of ?1 can be rapidly altered by Rab11A-positive recycling endosomes. Whether vasoconstrictors regulate the multisubunit composition of surface BK channels to stimulate contraction is unclear. OBJECTIVE:Test the hypothesis that ET-1 inhibits BK channels by altering BK? and ?1 surface trafficking in myocytes, identify mechanisms involved, and determine functional significance in myocytes of small cerebral arteries. METHODS AND RESULTS:ET-1, through activation of PKC (protein kinase C), reduced surface ?1 abundance and the proximity of ?1 to surface BK? in myocytes. In contrast, ET-1 did not alter surface BK?, total ?1, or total BK? proteins. ET-1 stimulated Rab11A phosphorylation, which reduced Rab11A activity. Rab11A serine 177 was identified as a high-probability PKC phosphorylation site. Expression of a phosphorylation-incapable Rab11A construct (Rab11A S177A) blocked the ET-1-induced Rab11A phosphorylation, reduction in Rab11A activity, and decrease in surface ?1 protein. ET-1 inhibited single BK channels and transient BK currents in myocytes and stimulated vasoconstriction via a PKC-dependent mechanism that required Rab11A S177. In contrast, NO-induced Rab11A activation, surface trafficking of ?1 subunits, BK channel and transient BK current activation, and vasodilation did not involve Rab11A S177. CONCLUSIONS:ET-1 stimulates PKC-mediated phosphorylation of Rab11A at serine 177, which inhibits Rab11A and Rab11A-dependent surface trafficking of ?1 subunits. The decrease in surface ?1 subunits leads to a reduction in BK channel calcium-sensitivity, inhibition of transient BK currents, and vasoconstriction. We describe a unique mechanism by which a vasoconstrictor inhibits BK channels and identify Rab11A serine 177 as a modulator of arterial contractility.

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Investigation of the Molecular Mechanisms by Which Endothelin-3 Stimulates Preadipocyte Growth.

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