Bile acid signaling pathways increase stability of Small Heterodimer Partner (SHP) by inhibiting ubiquitin–proteasomal degradation

Abstract

Small Heterodimer Partner (SHP) inhibits activities of numerous transcription factors involved in diverse biological pathways. As an important metabolic regulator, SHP plays a key role in maintaining cholesterol and bile acid homeostasis by inhibiting cholesterol conversion to bile acids. While SHP gene induction by increased bile acids is well established, whether SHP activity is also modulated remains unknown. Here, we report surprising findings that SHP is a rapidly degraded protein via the ubiquitin-proteasomal pathway and that bile acids or bile acid-induced intestinal fibroblast growth factor 19 (FGF19) increases stability of hepatic SHP by inhibiting proteasomal degradation in an extracellular signal-regulated kinase (ERK)-dependent manner. SHP was ubiquitinated at Lys122 and Lys123, and mutation of these sites altered its stability and repression activity. Tandem mass spectrometry revealed that upon bile acid treatment, SHP was phosphorylated at Ser26, within an ERK motif in SHP, and mutation of this site dramatically abolished SHP stability. Surprisingly, SHP stability was abnormally elevated in ob/ob mice and diet-induced obese mice. These results demonstrate an important role for regulation of SHP stability in bile acid signaling in normal conditions, and that abnormal stabilization of SHP may be associated with metabolic disorders, including obesity and diabetes.[Keywords: ERK; FGF19; SHP; bile acids; proteasome; protein stability] Supplemental material is available at http://www.genesdev.org.

Novel Polymorphisms of Nuclear Receptor SHP Associated with Functional and Structural Changes

“…In the absence of MG132, the levels of ubiquitinated FLAG-SHP WT and FLAG-SHP R38H were similar that were enhanced by MG132 treatment (Fig. 4B), consistent with the report in Hepa1c cells (32). On the contrary, the p.K170N mutation robustly increased ubiquitinated FLAG-SHP K170N , as indicated by the ladder of high molecular weight forms of FLAG- , suggesting that the Lys to Asn conversion at 170 position made FLAG-SHP K170N more susceptible to proteasomal degradation, which may contribute to its down-regulation.…”

“…1C). Although Lys-170 was not identified as a major ubiquitination site in a recent study (32), it remains an interesting question if Lys-170 plays a role in SHP protein ubiquitination and if p.K170N mutation alters such process. The antibodies against the FLAG tag produced nonspecific bands in Hepa-1 cells (Fig.…”

“…A recent report has shown that SHP undergoes rapid degradation with a short half-life in HepG2 cells (ϳ30 min) (32). We examined the half-life of each SHP mutant in Hepa-1 cells followed by treatment of cells with protein synthesis inhibitor cycloheximide.…”

Disruption of the Murine Protein Kinase Cβ Gene Promotes Gallstone Formation and Alters Biliary Lipid and Hepatic Cholesterol Metabolism

“…There are many potential mechanisms by which PKC␤ either indirectly or directly can affect hepatic CYP7A1 transcription. First, p42/44 MAPK activation has been linked with CYP7A1 suppression by both SHP-dependent and SHP-independent pathways (44,45). One such study has shown that p42/44 MAPK -induced phosphorylation of hepatic SHP at serine 26 stabilizes this protein by inhibiting ubiquitin-proteasomal degradation (45).…”

Differential regulation of bile acid and cholesterol metabolism by the farnesoid X receptor in Ldlr −/− mice versus hamsters

“…SHP acts as a corepressor of liver receptor homolog 1 (LRH-1/NR5A2)-or hepatocyte nuclear factor 4 ␣ (HNF4 ␣ /NR2A1)-induced transcription of Cyp7a1 and Cyp8b1 ( 14,18,(21)(22)(23). On the other hand, FGF15/19 activates its receptor FGFR4 located on the plasma membrane of hepatocytes, which leads to the inhibition of Cyp7a1 and Cyp8b1 expression either via SHP by substantially increasing the stability of SHP protein or by SHP-independent means ( 19,20,(24)(25)(26)(27). To better defi ne the role of FXR, we determined the effect of three potent and selective, synthetic FXR agonists (RO5186026, the X-Ceptor compound, and GW4064) and one less potent and less selective BA derivative (6-ECDCA, INT-747) on BA and cholesterol metabolism in mice and hamsters.…”