The nuclear receptor small heterodimer partner (SHP, Nr0b2) was originally identified in 1996 as a transcriptional repressor based on its protein-protein interactions with several nuclear receptor superfamily members such as retinoid receptors, the thyroid hormone receptor, and the orphan receptor MB67 (Seol et al. 1996). The SHP gene consists of two exons and one intron located at human chromosome 1p36.11, mouse chromosome 4 D2.3, and rat chromosome 5q36. Since no endogenous ligand for SHP has been identified so for, SHP is classified as an orphan nuclear receptor. However, SHP is present in a variety of tissues. In mice, SHP is abundant in the gallbladder and liver while expressed at lower levels in the brainstem, cerebellum, adrenal, pancreas, stomach, duodenum, jejunum, ileum, colon, kidney, ovary, testis, and heart. In humans, SHP expression is detectable in the liver, heart, pancreas, kidney, spleen, small intestine, adrenal gland, and stomach. In humans, the loss of function mutations in the SHP gene increases the morbidity risk of obesity and type 2 diabetes in Japanese populations (Nishigori et al. 2001).
SHP Is a Transcription Repressor
SHP contains the dimerization and ligand-binding domain (LBD) common in other nuclear receptor family members but lacks the conserved DNA binding domain (DBD) (Seol et al. 1996). SHP binds to other nuclear receptors through two functional LXXLL-related motifs (also called nuclear receptor box) and acts as a transcriptional coregulator. To date, many nuclear receptors can interact with SHP proteins including liver receptor homolog-1 (LRH-1), hepatocyte nuclear factor 4 (HNF4), estrogen-related receptors (ERRs), liver X receptors (LXRs), peroxisome proliferator-activated receptors (PPARs), glucocorticoid receptor (GR), estrogen receptors (ERs), thyroid hormone receptor beta (TRβ), retinoic acid receptor α (RARα), farnesoid X receptors (FXRs), pregnane X receptors (PXRs), constitutive androstane receptors (CARs), androgen receptors (AR), nerve growth factor IB (NGFI-B, Nur77), and retinoid X receptors (RXRs) (Zhang et al. 2011).
Three mechanisms appear to explain the inhibitory function of SHP on the transcriptional regulation of nuclear receptor target genes. In the first proposed mechanism, the binding of SHP to nuclear receptors induces a direct competition for coactivators such as p300 and cAMP response element-binding protein (CREB) binding to nuclear receptors. This manner of SHP-mediated transcriptional inhibition appears pronounced in ERs, RXRs, LRH-1, HNF4, ARs, LXRs, ERRs, GRs, Nur77, and FoxO1 induced transcription. In the second proposed mechanism, the binding of SHP to nuclear receptors triggers the recruitment of corepressors to DNA. For example, the SHP protein recruits a common repressor E1A-like inhibitor of differentiation 1 (EID1) to DNA. Recently, a conserved EID1 binding site at the N-terminus of SHP protein was identified, where EID1 mimics helix H1 and becomes an integral part for the SHP protein LBD fold (Zhi et al. 2014). The identification of the SHP-EID1 complex and integral structural motifs is important as it reveals a protein interface that regulates SHP repressive function. The binding of SHP to nuclear receptors in the third proposed mechanism induces the dissociation of the SHP-nuclear receptors complex from the DNA. For instance, the interaction between SHP and nuclear receptor RAR results in the inhibition of RAR-RXR heterodimer and RAR-PXR heterodimer binding to DNA. Interestingly, most studies have revealed SHP as a transcriptional repressor; however, SHP activates nuclear factor-κB (NF-κB) and upregulates the transcriptional activity of PPARγ.
SHP in Bile Acid Synthesis
Bile acids are the end products of cholesterol catabolism in the liver and account for 50% of the daily turnover of cholesterol. The synthesis of bile acids requires the coordinated actions of many enzymes in the hepatocytes including the cytochrome P450 enzyme cholesterol 7 α-hydroxylase (CYP7A1)-initiated neutral pathway in the endoplasmic reticulum and the sterol 27-hydroxylase (CYP27A1)-initiated alternative acidic pathway in the mitochondria. After synthesis, bile acids are secreted into the gallbladder for storage and mixed with phospholipids and cholesterol. Upon ingestion of a meal, cholecystokinin from the small intestine induces gallbladder contraction, releasing micelle bile acids into the intestine to aid food digestion and absorption of fats and lipid-soluble vitamins. Approximately 95% of bile acids are recycled via portal circulation by which bile is reabsorbed from the distal ileum and transported back into the liver; only 5% of bile acid is eliminated in the feces. This small amount of loss is replenished via de novo synthesis of bile acids in the liver.
Bile acids are important signaling molecules in the regulation of lipid, glucose, and energy metabolism. However, because of detergent-like toxic properties, excessive accumulation of bile acids can cause cell damage leading to inflammation and fibrosis in gastrointestinal tract. Therefore, bile acid homeostasis is tightly controlled by a coordinated regulation of genes in bile acid biosynthesis, uptake, and efflux in the liver and ileum. SHP has been implicated as a key repressive regulator in all of these processes. For instance, bile acids bind to the bile acid receptor, FXR, leading to the induction of SHP which in turn represses LRH-1 and HNF4α activation of CYP7A1, resulting in the repression of bile acid synthesis (Goodwin et al. 2000; Lu et al. 2000). In this process, SHP coordinately recruits chromatin-modifying enzymes such as mSin3A-Swi/Snf chromatin remodeling complex, histone deacetylase SIRT1, and G9a methyltransferase to the CYP7A1 promoter, leading to the transcriptional repression of CYP7A1. Additional studies show that both the c-Jun N-terminal kinase (JNK) and PXR pathways are involved in CYP repression by bile acids in Shp-deficient animals (Wang et al. 2002). In addition, bile acid-activated FXR also induces expression of FGF15/19 (FGF15 is the mouse homolog of human FGF19) in the small intestine and the secreted FGF15 represses hepatic CYP7A1 transcription through FGF receptor 4 (FGFR4) (Inagaki et al. 2005). These studies demonstrate that the gut-liver signaling pathway regulated by FXR/FGF15/FGFR4 synergizes with liver FXR/SHP in the maintenance of bile acid homeostasis. Moreover, studies show that bile acids regulate posttranslational modifications of SHP protein, including protein phosphorylation and ubiquitination. Recently, Kim et al. found that the nucleoporin ran-binding protein 2 (RanBP2/Nup358) colocalizes with SHP at the nuclear envelope region and mediates SUMO2 modification of SHP protein at K68 in response to bile acids. Such modification facilitates SHP protein nuclear transportation and interaction with repressive histone modifiers to inhibit BA synthetic genes (Kim et al. 2016). Taken together, these studies indicate that SHP is a critical transcriptional repressor for bile acid synthesis and has an indispensable role in maintaining bile acid homeostasis.
SHP in Lipid Metabolism
Lipid production and clearance are also tightly controlled by a complex network of transcriptional programs regulated by a large number of nuclear receptor family members, many of which SHP interacts with. For instance, SHP augments nuclear receptor PPARγ transactivation and induces hepatic lipid accumulation. Transgenic mice with hepatocyte-specific SHP overexpression display increased hepatic lipid accumulation which is mediated by the increase of fatty acid and triglyceride biosynthesis (Boulias et al. 2005). In line with these observations, deletion of SHP in obese leptin-deficient mice (ob/ob) prevents the development of nonalcoholic fatty liver through both increasing lipid secretion and decreasing de novo fatty acid synthesis and uptake (Huang et al. 2007). On the other hand, a dominant role of SHP in lipid synthesis has been reported as well. For example, bile acids and FXR agonists inhibit lipogenic gene SREBP-1c expression which is mediated through the upregulation of SHP transcription. Another study shows LRH-1 stimulates the transcription of fatty acid synthesis gene, FAS, and this response is completely blocked by SHP overexpression (Matsukuma et al. 2007). The SHP gene promoter contains an E box (CACGTG) element that can be activated by core circadian clock components CLOCK-BMAL1 (circadian locomotor output cycles kaput, brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1) to maintain the rhythmic expression of SHP in the liver. Microsomal triglyceride transfer protein (MTP) is a dedicated chaperone for biosynthesis of apolipoprotein B (apoB) that is required for the secretion of very low-density lipoprotein (VLDL) into the circulation. CLOCK and SHP coordinately regulate circadian changes of MTP that contributes to plasma lipid circadian rhythmicity (Pan et al. 2010). Disruptions in circadian rhythms might interfere with SHP regulation on MTP and, subsequently, induce dyslipidemia. These findings help to explain why night shift workers have increased risk for developing metabolic syndrome. In summary, these studies establish a critical role of SHP in modulating hepatic lipid synthesis and transport.
SHP in Glucose Metabolism
Glucose production and usage is mainly controlled by the balanced secretion and actions of insulin, glucagon, epinephrine, cortisol, and growth hormone. Accumulated evidence indicates SHP has a major function in inhibiting hepatic gluconeogenesis. Glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) are two critical enzymes in hepatic gluconeogenesis. SHP is demonstrated to repress G6Pase and PEPCK gene expression through direct protein interactions with multiple nuclear receptors and transcriptional factors including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), hepatocyte nuclear factor-3 (HNF3), HNF6, and C/EBPα (CCAAT/enhancer-binding protein alpha) (Park et al. 2007). SHP is also involved in the regulation of glucose metabolism by transcription factor forkhead box protein O1 (FOXO1), the basic helix-loop-helix protein BETA2/NeuroD, and the aryl hydrocarbon receptor (AHR)/nuclear translocator (ARNT). In addition, bile acids inhibit the expression of G6Pase, PEPCK, and fructose 1, 6-bis phosphatase (FBP1) through the FXR-SHP nuclear receptor cascade, and the absence of this repression was observed in Fxr−/− and Shp−/− mice. These findings suggest that bile acid-associated FXR-SHP nuclear receptor cascade could be a novel target in treating insulin resistance and type 2 diabetes. Metformin is an antidiabetic drug widely used for the treatment of type 2 diabetes. Metformin induces SHP expression through AMP-activated protein kinase (AMPK) pathway, and the activation of SHP subsequently inhibits PEPCK and G6Pase gene expression, which suggests that SHP is indispensable for anti-diabetes function of metformin.
SHP in Inflammation
For the first time, in 2007, Li et al. introduced a link between SHP and anti-inflammation. In the study, FXR ligand, GW4064, was found to increase SHP expression and inhibit interleukin IL-1β-induced production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in vascular smooth muscle cells (Li et al. 2007). Since then, additional studies contributed to elucidate the association of SHP and inflammation suppression. For instance, SHP has been found to inhibit toll-like receptor (TLR)-induced inflammatory responses in innate immune cells through a biphasic interaction with two cytoplasmic partners of TLR4 pathway, TRAF6 and NF-κB subunit p65 (Yuk et al. 2011). Recently, SHP was shown to directly affect the inflammasome, a large multimeric protein complex important for the activation of caspase-1 and maturation of the proinflammatory cytokines interleukin (IL)-1β and IL-18. SHP directly binds to the NLRP3 inflammasome and negatively regulates its activation to prevent excessive inflammatory responses (Yang et al. 2015). These studies indicate that SHP may also play an important role in the inhibition of inflammation.
SHP in Cancer
Accumulating evidence points to the involvement of SHP in the development of cancers, particularly hepatocellular carcinoma (HCC). In human HCC specimens, the expression of SHP is markedly diminished due to the SHP promoter hypermethylation-induced epigenetic silencing (He et al. 2008). Furthermore, an association study on SHP expression and HCC patient survival reveals SHP as a positive prognostic factor. Particularly, HCC patients with low SHP expression combined with a high expression of CDK4, MCM5, EXOCS1, CCNB1, BUB3, or BCL2L2 indicate a poor prognosis with a low chance of survival. Additional studies in Shp−/− mice demonstrate that SHP has a tumor suppressor function. For instance, Shp−/− mice at 12–15 months of age develop spontaneous HCC, which is strongly associated with increases of cyclin D1 expression and cell proliferation (Zhang et al. 2008). Yes-associated protein (YAP) plays a critical role in promoting hepatocyte proliferation and survival during embryonic liver development and hepatocellular carcinogenesis. YAP activation associates with spontaneous liver cancer formation in Fxr−/−Shp−/− double knockout mice. Additional studies show that SHP acts as a pivotal cell death receptor that targets mitochondria and binds to antiapoptotic protein Bcl-2. Such binding, consequently, leads to the disruption of Bcl-2/Bid interaction and cytochrome C release, resulting in the activation of apoptosis. Therefore, loss of SHP in liver cancer results in defective apoptosis that enhances liver cancer development.
DNA methyltransferase 1 (DNMT1) plays a critical role in maintaining CpG methylation. DNMT1 upregulation and its associated aberrant gene silencing of tumor suppressors are frequently observed in human HCC. Recent studies revealed SHP as a potent repressor of DNMT1 transcription either through directly inhibiting nuclear receptor ERRγ-induced DNMT1 transcription or through indirectly inhibiting DNMT1 expression by antagonizing metal-responsive transcription factor-1 (MTF-1) (Zhang et al. 2012). These studies demonstrate that loss of SHP leads to the upregulation of DNMT1 that could further result in aberrant tumor suppressor gene silencing in HCC and drive the HCC development. SHP is also downregulated in many types of cancer, including adrenal cortex, cerebellum, kidney, skin, and thyroid, suggesting that SHP may function as a common tumor suppressor.
SHP is originally identified as a unique nuclear receptor that contains a putative ligand-binding domain but lacks a DNA binding domain. Thus, the transcriptional corepressor relies heavily on protein-protein interactions with multiple nuclear receptors and transcriptional factors to regulate diverse metabolic processes including bile acid synthesis, cholesterol and lipid metabolism, and glucose homeostasis. No endogenous ligand of SHP is known so far. However, bile acid-mediated activation of FXR or FGF15/FGF19 signaling induces SHP expression and increases its protein stability. The loss of function mutation of SHP has been linked to an increased morbidity risk of obesity and diabetes in Japanese populations. Connections between SHP and major regulatory pathways such as inflammation and apoptosis are still being elucidated. Particularly, the antitumor role of SHP in liver cancer development is well addressed in that it is now known that the SHP gene is hypermethylated and silenced in human hepatocellular carcinoma. Such a decrease in SHP leads to upregulation of DNMT1 that further enhances the methylation and silencing of SHP and other tumor suppressor genes. Additionally, the long-term repression of SHP induces cell proliferation through the activation of CyclinD1 and defective apoptosis through loss of inhibition on antiapoptotic protein, BCL2. Furthermore, the loss of SHP in hepatocytes results in increased bile acid levels leading to YAP activation, which also drives liver cancer development. The accumulating insights of SHP and its numerous roles in overall homeostasis reveal SHP may be a promising therapeutic target of several metabolic diseases and liver cancer. However, major challenges for future research such as identifying endogenous SHP ligands and developing specific synthetic SHP ligands for pharmaceutical applications still remain.