A Calcium/Calmodulin-dependent Activation of ERK1/2 Mediates JunD Phosphorylation and Induction of nur77 and20α-hsd Genes by Prostaglandin F2α in Ovarian Cells

Abstract

We have previously demonstrated that prostaglandin F 2␣ (PGF 2␣ ) induces a rapid and transient expression of Nur77 in luteal cells. We have shown that Nur77 plays an important role in ovarian physiology by mediating the PGF 2␣ induction of 20␣-HSD, a steroidogenic enzyme involved in the catabolism of progesterone. In this report we established, using luteinized granulosa cells, that PGF 2␣ stimulates in vitro nur77 expression in a time-and dose-dependent manner. Serial 5-deletion of the nur77 promoter revealed that the necessary and sufficient elements for PGF 2␣ induction of Nur77 promoter activity are located between the nucleotides ؊86 and ؊33 upstream of the transcription start site, this region containing two AP1 elements. JunD binds to these AP1 sites, but its binding is not stimulated by PGF 2␣ . However, mutation of the AP1 sites as well as a dominant-negative JunD abolished nur77 induction by PGF 2␣ . PGF 2␣ induces phosphorylation of JunD bound to the nur77 promoter. Stimulation of nur77 expression and JunD phosphorylation were prevented by inhibitors of calcium, calmodulin, or ERK1/2 kinase. PGF 2␣ -induced ERK1/2 phosphorylation was prevented by calcium/calmodulin inhibitors. We conclude that activation of JunD through a calmodulim-dependent activation of ERK1/2 mediates nur77 induction by PGF 2␣ . Finally, we demonstrated that this molecular mechanism also mediates 20␣-hsd induction.

Menin represses JunD transcriptional activity in protein kinase Cθ-mediated Nur77 expression

“…We confirmed that JunD specifically binds to NAP4 and is activated indirectly by PKC through phosphorylation of JunD by MAP kinases (JNK and ERK) ( Figure 5). In ovarian cells, calcium-dependent activation of ERK mediates JunD phosphorylation and Nur77 induction by prostaglandin (Stocco et al, 2002). In this case, JunD induces the Nur77 through NAP1 and NAP2 elements.…”

JunD is a profibrogenic transcription factor regulated by Jun N-terminal kinase-independent phosphorylation

“…14,20 JunD is phosphorylated at Ser100 in activated HSCs and is necessary for stimulation of TIMP-1 gene transcription. We propose that Ser100 phosphorylated JunD homodimers operate in concert with either RUNX1A or RUNX2 to generate high-level TIMP-1 expression by activated HSCs.…”

“…The Ser100 of JunD is also an efficient target for phosphorylation by ERK1/2. 14,20 Treatment of rat HSCs with the ERK1/2 inhibitor PD98059 resulted in 40% repression of TIMP-1 promoter activity but had no effect on promoter activity in 3T3 cells (Fig. 7C).…”

“…I. Clark (UEA, Norwich, UK). The pSG5JunD Ser100-Ala, 14 was provided by Dr. L.F. Lau (Chicago, IL).The JIP1 Jun binding domain (JIP1 aa 127-282) construct was donated by Dr. A. Whitmarsh (Manchester, UK).…”

“…18,19 The N-terminus of JunD contains 3 MAPK-regulated phospho-acceptor sites (Ser90, Ser100, and Thr117) that are phosphorylated in response to ERK1/2 and JNK activation. 14,20 To determine if HSCs support phosphorylation of JunD, human LX-2 HSCs were transfected with the JunD expression vector. JunD-transfected LX-2 HSCs expressed 2 major protein species (39 and 43 kDa) reactive with both anti-phosphoJun and anti-JunD (Fig.…”