Dual-specificity phosphatase 6 (DUSP6) mRNA and protein abundance is regulated by fibroblast growth factor 2 in sheep granulosa cells and inhibits c-Jun N-terminal kinase (MAPK8) phosphorylation

Regulation of dual specificity phosphatases by fibroblast growth factor signaling pathways in bovine granulosa cells

“…To assess the activation of the MAPK3/1 pathway, the cells were stimulated with 10 ng/mL recombinant human FGF2 (PeproTech, Rocky Hill, NJ, USA; more than 97% similarity with bovine FGF2, NP_776481) for 0, 15, 30, 60, and 240 min and total cell protein was harvested for Western blotting. The ability of FGF2 to regulate abundance of mRNA encoding DUSPs was assessed in dose-response and time-course studies; cells were stimulated with 0, 1, 10, or 50 ng/mL FGF2 for 2 h, or treated with FGF2 at 10 ng/mL for 0, 1, 2, 4, or 8 h. To measure the effect of FGF2 on DUSP protein abundance, cells were stimulated with 10 ng/mL FGF2 for 4, 6, 12, and 24 h. These doses and time periods were based on our previous study with FGF2 in sheep granulosa cells (Relav et al 2021).…”

“…Quantitative PCR (qPCR) was performed on a CFX96 Touch thermocycler (Bio-rad) using 1× Sso Advanced Universal SYBR Green Supermix (Bio-rad) and 375 nM of each specific forward and reverse primer in a total reaction volume of 20 µL. Primers were designed to amplify 14 DUSP mRNA previously detected in sheep (Relav et al 2021), and sequences are presented in Table 1. Common thermocycling conditions were used: 95°C for 3 min, 40 cycles of 95°C for 15 s, and 61°C for 30 s and 72°C for 30 s. Conditions for optimal amplification efficiency were determined with preliminary sample dilution curves.…”

“…Regulation of SPRY1, SPRY2, and SPRY4 mRNA abundance by FGFs has been demonstrated in bovine granulosa cells (Jiang et al 2011, 2013, Han et al 2017, and SPRY proteins have a large spectrum of action downstream of the growth factor receptor and upstream of MAPK activation (Mason et al 2006, Cabrita & Christofori 2008. Many members of the DUSP family are expressed in the ovary, although few appear to be under hormone or growth factor control; abundance of DUSP1, DUSP5, and DUSP6 is stimulated by FSH or growth factors in bovine, sheep, and hen cumulus/granulosa cells (Woods & Johnson 2006, Sen et al 2008, Khan et al 2015, Relav et al 2021, whereas these three DUSPs were inhibited by FSH in rats (Donaubauer et al 2016, Herndon et al 2016.…”

“…The action of DUSPs is the direct control of MAPK phosphorylation (Camps et al 2000), and studies in a variety of cell types show that DUSP1, DUSP5, and DUSP6 dephosphorylate MAPK3/1, MAPK8, and MAPK14 to different extents (Patterson et al 2009, Kidger & Keyse 2016, and DUSP6 appears to target pro-apoptotic MAPK8 in sheep granulosa cells (Relav et al 2021). This suggests that modification of the activity of specific DUSPs may alter cell fate, therefore understanding the mechanisms regulating DUSP expression may lead to strategies to improve ovarian function.…”

Soyasaponin Ag inhibits triple-negative breast cancer progression via targeting the DUSP6/MAPK signaling

“…Dual specificity phosphatase 6 (DUSP6), or MAPK phosphatase 3 (MKP3), encodes proteins that belong to the bispecific specificity protein phosphatase subfamily [23,24]. These phosphatases play biological roles in the negative regulation of the MAPK superfamily, including JNK, MAPK, and p38, and are closely related to cell proliferation and motion [25][26][27]. Emerging evidence has reported that DUSP6 exerts inhibitory function on malignant phenotypes of various cancers [28][29][30][31][32].…”